Heterocyclic derivative inhibitor, and preparation thereof and use thereof

Abstract

The present invention belongs to the field of drug synthesis, and specifically relates to a heterocyclic derivative inhibitor, and the preparation thereof and the use thereof. Disclosed in the present invention is a compound having a structure as represented by formula I, or an isomer or pharmaceutically acceptable salt thereof. The compound of formula I provided by the present invention has strong PCSK9 inhibitory activity, and can be used for treating and / or preventing cardiovascular and cerebrovascular diseases.

Description

Heterocyclic derivative inhibitors and preparation and use thereof TECHNICAL FIELD

[0001] The present application belongs to the field of drug synthesis, and particularly relates to a heterocyclic derivative inhibitor and a preparation method and use thereof. BACKGROUND

[0002] Cardiovascular disease (CVD) is a major cause of death worldwide, and high levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor. The accumulation of LDL-C in the inner wall of the artery can cause atherosclerosis, leading to cardiovascular events such as heart attack and stroke. Statins are currently the main lipid-lowering drugs in clinical use, which can lower LDL-C by inhibiting HMG CoA. However, some patients are intolerant to statins, or it is still difficult to achieve therapeutic targets after receiving the maximum tolerated dose.

[0003] PCSK9 (Proprotein convertase subtilisin kexin type 9) is the main regulator of the level of low-density lipoprotein receptor (LDLR) on the surface of hepatocytes. PCSK9 is degraded with LDLR to lysosomes, preventing LDLR from circulating, thereby up-regulating the level of LDL-C. Therefore, inhibiting the function of PCSK9 can effectively reduce the level of LDL-C, providing a new treatment approach for patients with familial hypercholesterolemia.

[0004] Currently marketed PCSK9 inhibitors are large molecule injectable drugs such as Alirocumab, Evolocumab and Inclisiran, which can effectively lower cholesterol and have good safety, but all need to be injected, and the production cost is high and the price is expensive. So far, there is no small molecule PCSK9 inhibitor on the market. Although there have been patent reports, such as: WO2014170786 (Pfizer), WO2014150326 (Shifa), WO2020150473 (AZ) and WO2022133529 (Nyrada) and WO2024078620 (Haosen), but the most advanced AZD-0780 is in clinical phase I, and the others are in preclinical development stage. In addition, there are also polypeptide small molecule inhibitors being developed, but polypeptides have the problem of low bioavailability, which is difficult to meet the clinical needs. Therefore, it is of great clinical significance to develop a small molecule PCSK9 inhibitor with good activity, high bioavailability and oral use. SUMMARY

[0005] In order to achieve the above purpose, the present inventors have designed a new PCSK9 inhibitor with high solubility, good absorption, high activity for PCSK9 inhibitor and effective entry into the body to exert biological effects.

[0006] In one aspect, the present application relates to a compound as shown in Formula I or an isomer, a pharmaceutically acceptable salt thereof:

[0007] wherein,

[0008] R1is selected from deuterium, amino, nitro, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylamino, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted deuterated alkoxy, substituted or unsubstituted deuterated alkyl, substituted or unsubstituted deuterated alkylthio, -SO2NR6R7, -NR6SO2R7, -COSR6, or -CSOR6;

[0009] R2, R3, R4, R5are each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, alkyl, alkenyl, alkynyl, oxo, thioxo, deuterated alkyl, halogenated alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl, cyano-substituted alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -SO2NR6R7, -NR6SO2R, -(CH2) n R6, -(CH2) n OR6, -(CH2) n C(O)R6, -(CH2) n C(O)OR6, -(CH2) n S(O) m R6, -(CH2) n NR7R8, -(CH2) n NR7C(O)OR8, -(CH2) n NR7C(O)(CH2) n1 R8, -(CH2) n NR7C(O)NR7R8, -(CH2) n C(O)NR7(CH2) n1 R8, -OC(R6R7) n (CH2) n1 R8, or -(CH2) n NR7S(O) m R8; optionally, the above amino, alkyl, alkenyl, alkynyl, deuterated alkyl, halogenated alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl, cyano-substituted alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl can be further substituted with one or more of deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thioxo, carboxyl, C 1-3 alkyl, C 1-3 deuterated alkyl, C 1-3 halogenated alkyl, C 1-3 hydroxyalkyl, C 1-3Alkoxy, C 1-3 Deuterated alkoxy, C 1-3 Halogenated alkoxy groups, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 6-10 Aryl, 5-10 member heteroaryl; among which,

[0010] R6, R7, and R8 are each independently selected from hydrogen, deuterium, halogen, nitro, hydroxyl, mercapto, cyano, amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl. The amino, alkyl, deuterated alkyl, haloalkyl, hydroxyalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups mentioned above may be further substituted by substituents.

[0011] n can be 0, 1, 2, 3, or 4;

[0012] n1 is 0, 1, 2, 3 or 4;

[0013] m can be 0, 1, 2, 3, or 4;

[0014] Preferably, R2, R3, R4, and R5 are each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne group, oxo group, thio group, C 1-6 Alkylthio, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 1-6 hydroxyalkyl, cyano-substituted C 1-6 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 6-12 Aryl, 5-14 heteroaryl, -SO2NR6R7, -NR6SO2R7, -(CH2) n R6、-(CH2) n OR6、-(CH2) n C(O)R6、-(CH2) n C(O)OR6、-(CH2) n S(O) m R6、-(CH2) n NR7R8、-(CH2) n NR7C(O)OR8、-(CH2) n NR7C(O)(CH2) n1 R8, -(CH2) nNR7C(O)NR7R8, -(CH2) n C(O)NR7(CH2) n1 R8, -OC(R6R7) n (CH2) n1 R8or -(CH2) n NR7S(O) m R8; optionally, the above-mentioned amino, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkylthio, C 1-6 deuteroalkyl, C 1-6 haloalkyl, C 1-6 alkoxy, haloC 1-6 alkoxy, C 1-6 hydroxyalkyl, cyano-substituted C 1-6 alkyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-12 aryl and 5-14 membered heteroaryl can be further substituted by one or more of deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thioxo, carboxyl, C 1-3 alkyl, C 1-3 deuteroalkyl, C 1-3 haloalkyl, C 1-3 hydroxyalkyl, C 1-3 alkoxy, C 1-3 deuteroalkoxy, C 1-3 haloalkoxy, C 2-4 alkenyl, C 2-4 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl; wherein R6, R7, R8, n, n1, m are as defined above;

[0015] or, any two of R2, R3, R4are linked to form a cycloalkyl, heterocyclyl, aryl or heteroaryl; optionally, the above-mentioned cycloalkyl, heterocyclyl, aryl and heteroaryl can be further substituted by one or more of deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thioxo, carboxyl, C 1-3 alkyl, C 1-3 deuteroalkyl, C 1-3 haloalkyl, C 1-3 hydroxyalkyl, C 1-3 alkoxy, C 1-3 deuteroalkoxy, C 1-3 haloalkoxy, C 2-4 alkenyl, C 2-4 alkynyl, C 3-8cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl;

[0016] X and Y are independently selected from C and N, and X and Y cannot be C at the same time; and when Y is N, R1cannot be cyano, C 4-5 heterocyclyl, C5heteroaryl and C 1-6 alkylamino;

[0017] W is selected from C or N, when W is N and R5is hydrogen, R2, R3, R4are not simultaneously hydrogen or C 1-6 alkyl.

[0018] W is selected from C or N, when W is N and R5is hydrogen, R2, R3, R4are not simultaneously hydrogen or C 1-6 alkyl; and when W is C, W can be further substituted by hydrogen, alkyl, halogen or cyano, preferably halogen or cyano.

[0019] Further, the above R1is selected from deuterium, amino, nitro, alkynyl, substituted or unsubstituted C 1-6 alkylamino, substituted or unsubstituted 3-6 membered heterocyclyl, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted phenyl, substituted or unsubstituted deuterated C 1-6 alkoxy, substituted or unsubstituted deuterated C 1-6 alkyl, substituted or unsubstituted deuterated C 1-6 alkylthio, -SO2NR6R7, -NR6SO2R7, -COSR6or -CSOR6; wherein R6, R7are defined as above.

[0020] Further, the present application provides a compound as shown in formula Ia, formula Ib, formula Ic, formula Id, formula Ie or formula If or an isomer, a pharmaceutically acceptable salt thereof:

[0021] wherein R1to R5are defined as any of the above, and:

[0022] When the above compound has the general formula of formula Ic, formula Id, formula Ie or formula If, R1cannot be heterocyclyl, heteroaryl and alkylamino; or,

[0023] When the above compound has the general formula of formula Ib, formula Id or formula If and R5is hydrogen, R2, R3, R4are not simultaneously hydrogen or alkyl.

[0024] Further, in the above formula Ia, formula Ib, formula Ic, formula Id, formula Ie or formula If:

[0025] When the above compounds have the general formulas Ic, Id, Ie, or If, R1 cannot be C. 4-5 heterocyclic groups, C5 heteroaryl groups and C 1-6 alkylamino; or,

[0026] When the above compounds have the general formula Ib, Id, or If and R5 is hydrogen, R2, R3, and R4 are not simultaneously hydrogen or C. 1-6 alkyl.

[0027] Further, in formula Ia or formula Ib above, R1 is selected from substituted or unsubstituted oxacyclobutyl, substituted or unsubstituted azacyclobutyl, substituted or unsubstituted tetrahydrofuranyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted furanyl, substituted or unsubstituted thiophene, substituted or unsubstituted C 1-6 Alkylamino, deuterated methoxy, deuterated methyl, deuterated methylthio, deuterated difluoromethoxy, or phenyl;

[0028] And / or R2, R3, R4, R5 are each independently selected from hydrogen, deuterium, halogen, nitro, hydroxyl, carboxyl, amide, mercapto, cyano, amino, C 1-6 Alkyl, deuterated C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkenyl, C 1-6 alkynyl group, C 3-6 cycloalkyl, C 2-6 Heterocyclic group, C 5-10 aryl or heteroaryl; optionally, the above-mentioned carboxyl, amide, amino, C 1-6 Alkyl, deuterated C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkenyl, C 1-6 alkynyl group, C 3-6 cycloalkyl, C 2-6 Heterocyclic group, C 5-10 The aryl or heteroaryl group may be further substituted by one or more of the following substituents: deuterium, halogen, nitro, hydroxyl, mercapto, cyano, amino, oxo, thio, carboxyl, C 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Hydroxyalkyl, C 1-3 Alkoxy, C 1-3 Deuterated alkoxy, C 1-3 Halogenated alkoxy groups, C 2-4alkyl, haloC 2-4 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl;

[0029] when the above compound has the general formula Ib and R5 is hydrogen, R2, R3, R4 are not simultaneously hydrogen or C 1-6 alkyl.

[0030] Further, in the above formula Ic, formula Id, formula Ie or formula If, R1 is selected from deuterium, amino, nitro, alkynyl, substituted or unsubstituted oxetanyl, substituted or unsubstituted N-heterocyclobutyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted furanyl, substituted or unsubstituted thienyl, deuterated methoxy, deuterated methyl, deuterated methylthio, deuterated difluoromethoxy, -SO2NR6R7, -NR6SO2R7, -COSR6 or -CSOR6; wherein R6, R7 are as defined above;

[0031] and / or R2, R3, R4, R5 are each independently selected from hydrogen, deuterium, halogen, nitro, hydroxyl, carboxyl, amido, thiol, cyano, amino, C 1-6 alkyl, deuterated C 1-6 haloC 1-6 alkyl, hydroxyC 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkenyl, C 1-6 alkynyl, C 3-6 cycloalkyl, C 2-6 heterocyclyl, C 5-10 aryl or heteroaryl; optionally, the above carboxyl, amido, amino, C 1-6 alkyl, deuterated C 1-6 haloC 1-6 alkyl, hydroxyC 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkenyl, C 1-6 alkynyl, C 3-6 cycloalkyl, C 2-6 heterocyclyl, C 5-10 aryl and heteroaryl can be further substituted with one or more of deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thioxo, carboxyl, C 1-3 alkyl, C 1-3 deuterated alkyl, C 1-3 haloalkyl, C 1-3 hydroxyalkyl, C 1-3 alkoxy, C 1-3 deuterated alkoxy, C 1-3 haloalkoxy, C 2-4 alkenyl, C2-4 alkynyl group, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 6-10 Aryl, 5-10 heteroaryl;

[0032] When the above compounds have the general formula Id or If and R5 is hydrogen, R2, R3, and R4 are not simultaneously hydrogen or C. 1-6 alkyl.

[0033] Furthermore, in the above equation Ia, R1 is selected from... -CD3, -CDF2 or -OCDF2; R2, R3, R4, R5 are each independently selected from hydrogen, fluorine, chlorine, methyl, trifluoromethyl, trifluoromethoxy, methoxy, cyano, -COOCH3 or carboxyl, preferably hydrogen, fluorine, chlorine, methyl, trifluoromethyl, methoxy, cyano, -COOCH3 or carboxyl.

[0034] Furthermore, in the above equation Ia, R1 is selected from... -CD3, -CDF2 or -OCDF2; R2 is selected from fluorine, chlorine, methyl, trifluoromethyl, methoxy, trifluoromethoxy, cyano, -COOCH3 or carboxyl, preferably fluorine, chlorine, methyl, trifluoromethyl, methoxy, cyano, -COOCH3 or carboxyl; R3, R4 and R5 are each independently selected from hydrogen.

[0035] In this invention, "C1 to C6" refers to a number of carbon atoms ranging from 1 to 6. Similar notations are interpreted similarly.

[0036] Furthermore, the above-mentioned compounds or their isomers, pharmaceutically acceptable salts, include the following compounds:

[0037] Furthermore, the hydrogen atoms in the structure of the above-mentioned compounds or their isomers, or pharmaceutically acceptable salts, may be replaced by one or more deuterium atoms.

[0038] On the other hand, the present invention also provides the use of the above-mentioned compounds or their isomers, or pharmaceutically acceptable salts, in the preparation of PCSK9 inhibitors.

[0039] Furthermore, the present invention provides the use of the above-mentioned compound or its isomers, or pharmaceutically acceptable salts, in the preparation of medicaments for lowering LDL-cholesterol levels, VLDL-cholesterol levels, triglyceride levels, or lipoprotein A levels.

[0040] Furthermore, the present invention provides the use of the above-mentioned compounds or their isomers, pharmaceutically acceptable salts, in the preparation of medicaments for treating cardiovascular diseases, cerebrovascular diseases, atherosclerosis and / or related diseases or the like.

[0041] Preferably, the present invention provides the use of the above-mentioned compound or its isomers, or pharmaceutically acceptable salts, in the preparation of medicaments for stroke, hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, dyslipoproteinemia, atherosclerosis, hepatic steatosis, metabolic syndrome, and / or coronary artery disease.

[0042] Compared with the prior art, the present invention has the following beneficial effects: the compound provided by the present invention has strong PCSK9 inhibitory activity, can increase the LDLR concentration of HepG2 cells, and has excellent pharmacokinetic properties. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments and experimental examples. The embodiments and experimental examples of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Any equivalent substitutions made in the art based on the content disclosed in the present invention shall fall within the protection scope of the present invention.

[0044] The structure of the compound is determined by nuclear magnetic resonance (NMR). 1 Determined by ¹H NMR or LC-MS.

[0045] The liquid chromatography-mass spectrometry (LC-MS) system was an Agilent G6120B (compatible with an Agilent 1260 liquid chromatography system); nuclear magnetic resonance (NMR) 1 ¹H NMR shifts (δ) are given in parts per million (ppm), the solvent is DMSO-d6 or CDCl3, the internal standard is tetramethylsilane (TMS), and the chemical shift is expressed in 10⁻¹⁰ ppm. ～6 (ppm) is given as the unit.

[0046] In this invention, the term "room temperature" refers to a temperature between 10 and 25°C.

[0047] Example 1: Preparation of 1-(6-{[(1S,3S)-3-({5-[2-(trifluoromethyl)oxetane-2-yl]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (compound 1)

[0048] Step 1: Preparation of 1-(2-chloropyrimidin-5-yl)-2,2,2-trifluoroethane-1-ol (compound b)

[0049] To compound a (1.80 g, 12.6 mmol) in DMF (22 mL) was added Ruppert-Prakash reagent (2.31 g, 2.40 mL, 16.2 mmol) and potassium carbonate (123 mg, 0.9 mmol). The reaction was stirred at room temperature for about 60 minutes. The reaction was monitored by TLC until the starting material was consumed. The reaction was quenched by adding 3V water and extracted with ethyl acetate three times. The organic phase was washed with water, brine and dried over anhydrous sodium sulfate. The solvent was evaporated to get 3.8 g of crude product which was purified by column chromatography to get the target compound b (2.37 g, 88.4% yield) as a solid.

[0050] ESI-MS: m / z = 213.0 (M+H) + .

[0051] Step 2: Preparation of 1-(2-chloropyrimidin-5-yl)-2,2,2-trifluoroethan-1-one (compound c)

[0052] To compound b (218 mg, 1.03 mmol) in 5 mL of dichloromethane was added Dess Martin reagent (560 mg, 1.32 mmol) at 0 °C. The reaction was stirred for 10 minutes at 0-5 °C and then at room temperature overnight. The reaction was completed and 50 mL of dichloromethane was added followed by the same volume of saturated sodium thiosulfate solution and 50 mL of sodium bicarbonate solution. The reaction mixture was washed with brine and dried over anhydrous sodium sulfate. The solvent was evaporated to get 540 mg of crude product. To the crude product was added 5 mL of ethyl acetate and stirred at room temperature for 2 hours. The reaction mixture was filtered and the filter cake was washed with ethyl acetate. The filter cake was dried under vacuum to get 165 mg of compound c as a white solid in 76.4% yield.

[0053] ESI-MS: m / z = 211.0 (M+H) + .

[0054] Step 3: Preparation of 2-chloro-5-[2-(trifluoromethyl)oxetan-2-yl]pyrimidine (compound d)

[0055] To trimethylsulfoxonium iodide (940 mg, 4.30 mmol), add potassium tert-butoxide (476 mg, 4.30 mmol) dissolved in DMSO (2.5 mL) under N2environment, stir for 15 minutes, then add a solution of compound c (222 mg, 1.05 mmol) in DMSO (7.5 mL) dropwise, stir the reaction at room temperature overnight. The reaction is complete, add ethyl acetate and saturated brine to extract, the organic phase is washed with water, saturated brine, dried over anhydrous sodium sulfate, evaporate the solvent to obtain 280 mg of crude product. Purify by column chromatography, eluent (ethyl acetate: petroleum ether = 1:2), collect the product to obtain 162 mg of white solid compound d. Yield 64.6%, purity 97.20%.

[0056] ESI-MS: m / z = 239.0 (M+H) + .

[0057] Step 4: Preparation of 1-(6-chloropyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (compound g)

[0058] Dissolve 3-methoxy-1,2-dihydropyridin-2-one (compound f, 4.76 g, 38 mmol) in dry DMF (150 mL), add 2-chloro-5-iodopyridine (compound e, 9.9 g, 41.7 mmol), CuI (1.5 g, 7.56 mmol), K2CO3(15.7 g, 114 mmol), and 8-hydroxyquinoline (0.9 g, 7.2 mmol), and stir the mixture at 120 °C for 12 hours. After the starting material is consumed, as monitored by TLC, evaporate the solvent under reduced pressure to obtain the crude product, which is then purified by column chromatography (EA / PE = 3:1 to EA) to obtain the title compound g (6.6 g, yield 73.6%).

[0059] ESI-MS: m / z = 237.1 (M+H) + .

[0060] Step 5: Preparation of tert-butyl {[(1S,3S)-3-{[5-(3-methoxy-2-oxopyridin-1-yl)pyridin-2- yl]amino}cyclopentyl]amino}carboxylate (compound i)

[0061] Compound g (2.36 g, 10 mmol, 1.0 eq), compound h (2.0 g, 10 mmol. 1.0 eq), tBuXphos Pd G3 (795 mg, 1 mmol, 0.1 eq) and tBuOK (224 mg, 2 mmol, 0.2 eq) were dissolved with dioxane (30 mL) and heated to 110 °C, protected with N2 overnight. After TLC monitoring the reaction was complete, the insoluble was filtered, concentrated and separated by column chromatography (EA / PE = 1:2 to 2:1) to obtain compound i (2.55 g, yield 63.8%) as a white solid.

[0062] ESI-MS: m / z = 401.2 (M+H) + .

[0063] Step 6: Preparation of 1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-3- methoxy-1,2-dihydropyridin-2-one (Compound j)

[0064] Compound i (2.55 g, 6.4 mmol, 1.0 eq) was dissolved with HCl solution in dioxane (30 mL) and reacted at room temperature, and solid gradually precipitated. After TLC monitoring the reaction was complete, the white solid compound j (1.84 g, yield 96.2%) was obtained by filtration.

[0065] ESI-MS: m / z = 301.2 (M+H) + .

[0066] Step 7: Preparation of 1-(6-{[(1S,3S)-3-({5-[2-(trifluoromethyl)oxetan-2-yl]pyrimidin-2- yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 1)

[0067] Compound d (144 mg, 0.61 mmol, 1.1 eq), compound j (168 mg, 0.55 mmol, 1.0 eq) were dissolved with DMF (5 mL), cesium carbonate (396 mg, 1.22 mmol, 2.2 eq) was added and reacted at room temperature, and after TLC monitoring the reaction was complete, 3 volumes of water were added to quench, extracted with ethyl acetate, concentrated and separated by column chromatography (EA / PE = 1:1 to 3:1) to obtain compound 1 (236 mg, yield 85.5%).

[0068] ESI-MS: m / z = 503.2 (M+H) + .

[0069] HNMR: (400 MHz, DMSO d6) 8.24 (s, 2H), 7.87 (s, 1H), 7.63-7.61 (d, J = 8 Hz, 1H), 7.35-7.33 (d, J = 8 Hz, 1H), 7.13-7.11 (d, J = 8 Hz, 1H), 6.89-6.81 (m, 2H), 6.50-6.48 (d, J = 8 Hz, 1H), 6.15 (m, 1H), 4.67-4.55 (m, 2H), 4.38-4.27 (m, 2H), 3.68 (s, 3H), 3.11-2.95 (m, 2H), 2.12-2.07 (m, 2H), 1.90-1.83 (m, 2H), 1.54-1.45 (m, 2H).

[0070] Example 2: Preparation of 3-chloro-l-(6-{[(lS,3S)-3-({5-[2- (trifluoromethyl)oxetan-2-yl]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)- 1,2-dihydropyridin-2-one (Compound 2)

[0071] The preparation method is the same as that of Example 1, and the compound f of Step 4 is replaced by 3-chloro-l,2-dihydropyridin-2-one to prepare the title compound 2 with a purity of 97.80%.

[0072] ESI-MS: m / z = 507.2 (M+H) + .

[0073] Example 3: Preparation of 3-cyano-l-(6-{[(lS,3S)-3-({5-[2- (trifluoromethyl)oxetan-2-yl]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)- 1,2-dihydropyridin-2-one (Compound 3)

[0074] The preparation method is the same as that of Example 1, and the compound f of Step 4 is replaced by 3-cyano-l,2-dihydropyridin-2-one to prepare the title compound 3 with a purity of 97.80%.

[0075] ESI-MS: m / z = 498.2 (M+H) + .

[0076] Example 4: Preparation of 3-methyl-l-(6-{[(lS,3S)-3-({5-[2- (trifluoromethyl)oxetan-2-yl]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)- 1,2-dihydropyridin-2-one (Compound 4)

[0077] Preparation method same as the preparation method of example 1, replacing compound f of step 4 with 3-[(trifluoromethyl)oxy]-1,2-dihydropyridin-2-one, to obtain the title compound 5 with purity of 97.52%.

[0078] ESI-MS: m / z = 487.2 (M+H) + .

[0079] Example 5: Preparation of 1-(6-{[(1S,3S)-3-({5-[2-(trifluoromethyl)oxetan-2- yl]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-[(trifluoromethyl)oxy]- 1,2-dihydropyridin-2-one (Compound 5)

[0080] Preparation method same as the preparation method of example 1, replacing compound f of step 4 with 3-[(trifluoromethyl)oxy]-1,2-dihydropyridin-2-one, to obtain the title compound 5 with purity of 97.52%.

[0081] ESI-MS: m / z = 557.2 (M+H) + .

[0082] Example 6: Preparation of 1-(6-{[(1S,3S)-3-({5-[2-(trifluoromethyl)oxetan-2- yl]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-(trifluoromethyl)-1,2- dihydropyridin-2-one (Compound 6)

[0083] Preparation method same as the preparation method of example 1, replacing compound f of step 4 with 3-[(trifluoromethyl)oxy]-1,2-dihydropyridin-2-one, to obtain the title compound 5 with purity of 97.52%.

[0084] ESI-MS: m / z = 541.2 (M+H) + .

[0085] Example 7: Preparation of 1-(6-{[(1S,3S)-3-{[5-(azetidin-1-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 7)

[0086] Step 1: Preparation of tert-butyl {[(1S,3S)-3-[(5-iodopyrimidin-2-yl)amino]cyclopentyl] amino}carboxylate (Compound L)

[0087] Take (1S, 3S)-3-amino cyclopentyl carbamic acid tert-butyl ester (compound h, 2g, 10mmol), add to a solution of 2-chloro-5-iodopyrimidine (2.39g, 10mmol) in DMSO (25ml), add (2.58g, 20mmol) DIPEA, the reaction is magnetically stirred at 60°C, after 2h of reaction, TLC monitoring of raw material is consumed completely. Add 3V water to quench the reaction, then extract with ethyl acetate three times, combine the organic phase, the organic phase is washed with water, saturated brine, dried over anhydrous sodium sulfate, evaporate the solvent to get 5.20g of crude product, column chromatography to separate the target solid compound L (2.39g, yield 59.2%).

[0088] ESI-MS: m / z = 405.1 (M+H) + .

[0089] Step 2: Preparation of {[(1S, 3S)-3-{[5-(azetidin-1-yl)pyrimidin-2-yl]amino} cyclopentyl] amino} carbamic acid tert-butyl ester (compound m)

[0090] Under the condition of nitrogen protection, compound L (2.43g, 6mmol), CuI (0.23g, 1.2mmol), L-Proline (0.27g, 2.4mmol), Cs2CO3 (3.9g, 12.0mmol) are dissolved with dry DMF (30ml), azetidine (513mg, 9mmol) is added, and the reaction is stirred at 100°C for 24h. After the reaction is completed by TLC monitoring, 3V water is added to quench the reaction, then extracted with ethyl acetate three times, combine the organic phase, the organic phase is washed with water, saturated brine, dried over anhydrous sodium sulfate, evaporate the solvent to get the crude product, column chromatography to separate the target solid compound m (1.33g, yield 66.5%).

[0091] ESI-MS: m / z = 334.3 (M+H) + .

[0092] Step 3: Preparation of (1S, 3S)-3-{[5-(azetidin-1-yl)pyrimidin-2-yl]amino} cyclopentan-1-amine (compound n)

[0093] Compound m (1.23g, 3.7mmol, 1.0eq) is dissolved with HCl solution in dioxane (20ml), and the reaction is carried out at room temperature, and solid is gradually precipitated. After the reaction is completed by TLC monitoring, the white solid compound n (761mg, yield 88.3%) is obtained by filtration.

[0094] ESI-MS: m / z = 234.3 (M+H) + .

[0095] Step 4: Preparation of 1-(6-{[(1S,3S)-3-{[5-(azetidin-1-yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 7)

[0096] Compound n (852 mg, 3.6 mmol, 1.1 eq), compound g (779 mg, 3.3 mmol. 1.0 eq), tBuXphos Pd G3 (262 mg, 0.33 mmol, 0.1 eq) and tBuOK (74 mg, 0.66 mmol, 0.2 eq) were dissolved with dioxane (15 mL) and heated to 110 °C, protected by N2 overnight. After TLC monitoring the reaction was complete, the insoluble was filtered, concentrated and separated by column chromatography (EA / PE = 1:2 to 2:1) to obtain white solid compound 7 (985 mg, yield 68.9%).

[0097] ESI-MS: m / z = 434.2 (M+H) + .

[0098] Example 8: Preparation of 3-chloro-1-(6-{[(1S,3S)-3-{[5-(azetidin-1-yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-1,2-dihydropyridin-2-one (Compound 8)

[0099] The preparation method is the same as that of Example 7, and compound g of step 4 is replaced by 3-chloro-1-(6-chloropyridin-3-yl)-1,2-dihydropyridin-2-one to prepare the title compound 8 with a purity of 97.61%.

[0100] ESI-MS: m / z = 438.2 (M+H) + .

[0101] Example 9: Preparation of 1-(6-{[(1S,3S)-3-{[5-(azetidin-1-yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-2-oxopyridine-3-carbonitrile (Compound 9)

[0102] The preparation method is the same as that of Example 7, and compound g of step 4 is replaced by 1-(6-chloropyridin-3-yl)-2-oxopyridine-3-carbonitrile to prepare the title compound 9 with a purity of 97.22%.

[0103] ESI-MS: m / z = 429.2 (M+H) + .

[0104] Example 10: Preparation of 1-(6-{[(1S,3S)-3-{[5-(azetidin-1-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methyl-1,2-dihydropyridin-2-one (Compound 10)

[0105] The title compound 10 was prepared in accordance with the procedure described in Example 7 by replacing compound g of step 4 with 1-(6-chloropyridin-3-yl)-3-methyl-1,2- dihydropyridin-2-one. The purity of the title compound was 97.67%.

[0106] ESI-MS: m / z = 418.2 (M+H) + .

[0107] Example 11: Preparation of 1-(6-{[(1S,3S)-3-{[5-(azetidin-1-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-[(trifluoromethyl)oxy]-1,2- dihydropyridin-2-one (Compound 11)

[0108] The title compound 11 was prepared in accordance with the procedure described in Example 7 by replacing compound g of step 4 with 1-(6-chloropyridin-3-yl)-3-[(trifluoromethyl)oxy]- 1,2-dihydropyridin-2-one. The purity of the title compound was 97.22%.

[0109] ESI-MS: m / z = 488.2 (M+H) + .

[0110] Example 12: Preparation of 1-(6-{[(1S,3S)-3-{[5-(azetidin-1-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-(trifluoromethyl)-1,2-dihydropyridin-2- one (Compound 12)

[0111] The title compound 12 was prepared in accordance with the procedure described in Example 7 by replacing compound g of step 4 with 1-(6-chloropyridin-3-yl)-3-(trifluoromethyl)-1,2- dihydropyridin-2-one. The purity of the title compound was 98.06%.

[0112] ESI-MS: m / z = 472.2 (M+H) + .

[0113] Example 13: Preparation of 1-(6-{[(1S,3S)-3-{[5-(3,3-dimethylazetidin-1- yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2- dihydropyridin-2-one (Compound 13)

[0114] The preparation method is the same as that of Example 7, and the azetidine in Step 2 is replaced by 3,3-dimethylazetidine to prepare the title compound 13 with a purity of 96.81%.

[0115] ESI-MS: m / z = 462.3 (M+H) + .

[0116] 1 H NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 2.7 Hz, 1H), 7.68 (s, 2H), 7.37 (dd, J = 8.9, 2.7 Hz, 1H), 7.16 (dd, J = 7.0, 1.7 Hz, 1H), 6.95 - 6.83 (m, 2H), 6.51 (dd, J = 8.1, 4.6 Hz, 2H), 6.19 (t, J = 7.2 Hz, 1H), 4.26 (dq, J = 17.6, 6.7 Hz, 2H), 3.72 (s, 3H), 3.44 (s, 4H), 2.10 (ddt, J = 19.1, 7.8, 5.3 Hz, 2H), 1.86 (td, J = 14.3, 13.1, 7.7 Hz, 2H), 1.47 (td, J = 12.2, 7.0 Hz, 2H), 1.25 (s, 6H).

[0117] Example 14: Preparation of 3-chloro-1-(6-{[(1S,3S)-3-{[5-(3,3-dimethylazetidin-1- yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-1,2-dihydropyridin-2-one (Compound 14)

[0118] The preparation method is the same as that of Example 7, and the azetidine in Step 2 is replaced by 3,3-dimethylazetidine, and the compound g in Step 4 is replaced by 3-chloro-1-(6-chloropyridin-3-yl)-1,2-dihydropyridin-2-one to prepare the title compound 14 with a purity of 97.22%.

[0119] ESI-MS: m / z = 466.2 (M+H) + .

[0120] Example 15: Preparation of 1-(6-{[(1S,3S)-3-{[5-(3,3-dimethylazetidin-1- yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-2-oxoacetylridine-3- carbonitrile (Compound 15)

[0121] The title compound 15 was prepared in accordance with the preparation method of Example 7 by replacing azetidine in step 2 with 3,3-dimethylazetidine and replacing compound g in step 4 with 1-(6-chloropyridin-3-yl)-2-oxoacetylridine-3- carbonitrile with purity 97.22%.

[0122] ESI-MS: m / z = 457.2 (M+H) + .

[0123] Example 16: Preparation of 1-(6-{[(1S,3S)-3-{[5-(3,3-dimethylazetidin-1- yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methyl-1,2-dihydro- pyridin-2-one (Compound 16)

[0124] The title compound 16 was prepared in accordance with the preparation method of Example 7 by replacing azetidine in step 2 with 3,3-dimethylazetidine and replacing compound g in step 4 with 1-(6-chloropyridin-3-yl)-3-methyl-1,2-dihydro- pyridin-2-one with purity 97.52%.

[0125] ESI-MS: m / z = 446.3 (M+H) + .

[0126] Example 17: Preparation of 1-(6-{[(1S,3S)-3-{[5-(3,3-dimethylazetidin-1- yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-[(trifluoromethyl)oxy]- 1,2-dihydropyridin-2-one (Compound 17)

[0127] The title compound 17 was prepared in accordance with the preparation method of Example 7 by replacing azetidine in step 2 with 3,3-dimethylazetidine and replacing compound g in step 4 with 3-[(trifluoromethyl)oxy]-1,2-dihydropyridin-2- one with purity 97.68%.

[0128] ESI-MS: m / z = 516.2 (M+H) + .

[0129] Example 18: Preparation of 1-(6-{[(1S,3S)-3-{[5-(3,3-dimethylazetidin-1- yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-(trifluoromethyl)-1,2- dihydropyridin-2-one (Compound 18)

[0130] The title compound 18 was prepared in accordance with the procedure described in Example 7 by replacing azetidine in Step 2 with 3,3-dimethylazetidine and replacing compound g in Step 4 with 3-(trifluoromethyl)-1,2-dihydropyridin-2-one. The purity of the title compound 18 was 98.18%.

[0131] ESI-MS: m / z = 500.2 (M+H) + .

[0132] Example 19: Preparation of 1-(6-{[(1S,3S)-3-({5-[(deuteryldifluoromethyl)oxy]pyrimidin- 2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 19)

[0133] Step 1: Preparation of 2-chloro-5-[(deuteryldifluoromethyl)oxy]pyrimidine (Compound p)

[0134] A single-necked flask was charged with 2-chloro-5-hydroxypyrimidine (1 g, 7.66 mmol, 1.0 equiv) and sodium difluorochloroacetate (3.50 g, 22.98 mmol) and dissolved in DMF (25 mL) and heavy water (2 mL) and heated to 90 °C for 24 h under stirring. After completion of the reaction, the reaction mixture was treated with ice water (3x volume), extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to get the crude product which was purified by column chromatography (EA: PE = 1:6) to get compound p (827 mg, 4.57 mmol, 59.7% yield).

[0135] ESI-MS: m / z = 182.1 [M+H] + .

[0136] Step 2: Preparation of 1-(6-{[(1S,3S)-3-({5-[(deuteryldifluoromethyl)oxy]pyrimidin-2- yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 19)

[0137] Compound p (199 mg, 1.1 mmol, 1.0 eq), compound j (330 mg, 1.1 mmol, 1.0 eq) were dissolved in DMF (5 mL), cesium carbonate (745 mg, 2.2 mmol, 2.0 eq) was added and the reaction was allowed to proceed at room temperature. The reaction was monitored by TLC and after completion, the reaction mixture was quenched with 3 volumes of water and extracted with ethyl acetate. The crude compound was purified by column chromatography (EA / PE = 1:1 to 3:1) to get compound 19 (301 mg, yield 61.5%) with 97.72% purity.

[0138] ESI-MS: m / z = 446.2 (M+H) + .

[0139] 1 HNMR: (400 MHz, CDC13) 8.15 (s, 2H), 8.02 (s, 1H), 7.50-7.48 (d, J = 8 Hz, 1H), 6.92-6.90 (d, J = 8 Hz, 1H), 6.65-6.64 (d, J = 4 Hz, 1H), 6.43-6.40 (d, J = 12 Hz, 1H), 6.16-6.14 (d, J = 8 Hz, 1H), 5.29-5.27 (d, J = 8 Hz, 1H), 4.86-4.84 (d, J = 8 Hz, 1H), 4.40-4.37 (d, J = 12 Hz, 1H), 4.24-4.22 (d, J = 8 Hz, 1H), 3.83 (s, 3H), 2.30 (s, 2H), 2.04 (s, 2H), 1.76 (s, 2H).

[0140] Example 20: Preparation of 3-chloro-l-(6-{[(lS,3S)-3-({5-[(deuteriodyl difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-l,2- dihydropyridin-2-one (Compound 20)

[0141] The compound was prepared by following the procedure as mentioned in the preparation of Example 19, replacing compound j of step 2 with 3-chloro-l-(6-{[(lS,3S)-3- amino cyclopentyl]amino}pyridin-3-yl)-l,2-dihydropyridin-2-one to get the title compound 20 with 98.02% purity.

[0142] ESI-MS: m / z = 450.1 (M+H) + .

[0143] 1H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.96 (d, J = 2.7 Hz, 1H), 7.81 (dd, J = 7.3, 1.9 Hz, 1H), 7.66 (dd, J = 6.8, 1.9 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.43 (dd, J = 8.9, 2.7 Hz, 1H), 7.01 (d, J = 6.9 Hz, 1H), 6.54 (d, J = 8.9 Hz, 1H), 6.30 (t, J = 7.0 Hz, 1H), 4.32 (p, J = 6.9 Hz, 2H), 2.21 - 2.04 (m, 2H), 1.89 (tdd, J = 13.2, 10.4, 6.3 Hz, 2H), 1.63 - 1.42 (m, 2H).

[0144] Example 21: Preparation of 1-(6-{[(1S,3S)-3-({5-[(deuteriodyl difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-2- oxopyridine-3-carbonitrile (Compound 21)

[0145] The title compound 21 was prepared by following the same procedure as described in the preparation of Example 19, by replacing the compound j of step 2 with 1-(6-{[(1S,3S)-3- aminocyclopentyl]amino}pyridin-3-yl)-2-oxopyridine-3-carbonitrile, in 97.68% purity.

[0146] ESI-MS: m / z = 441.2 (M+H) + .

[0147] 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.96 (d, J = 2.7 Hz, 1H), 7.81 (dd, J = 7.3, 1.9 Hz, 1H), 7.66 (dd, J = 6.8, 1.9 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.43 (dd, J = 8.9, 2.7 Hz, 1H), 7.01 (d, J = 6.9 Hz, 1H), 6.54 (d, J = 8.9 Hz, 1H), 6.30 (t, J = 7.0 Hz, 1H), 4.32 (p, J = 6.9 Hz, 2H), 2.21 - 2.04 (m, 2H), 1.89 (tdd, J = 13.2, 10.4, 6.3 Hz, 2H), 1.63 - 1.42 (m, 2H).

[0148] Example 22: Preparation of 1-(6-{[(1S,3S)-3-({5-[(deuteryl difluoromethyl)oxy]pyrimidin-2- yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-methyl-1,2-dihydropyridin-2-one (Compound 22)

[0149] The title compound 22 was prepared in accordance with the procedure described in Example 19 by replacing the compound j of step 2 with 1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-3- methyl-1,2-dihydropyridin-2-one in a purity of 98.23%.

[0150] ESI-MS: m / z = 430.2 (M+H) + .

[0151] 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.91 (d, J = 2.7 Hz, 1H), 7.55 - 7.43 (m, 2H), 7.37 (td, J = 8.4, 7.8, 2.2 Hz, 2H), 6.90 (d, J = 6.9 Hz, 1H), 6.52 (d, J = 8.9 Hz, 1H), 6.19 (t, J = 6.8 Hz, 1H), 4.32 (dp, J = 13.7, 6.8 Hz, 2H), 2.21 - 2.06 (m, 2H), 2.02 (s, 3H), 1.96 - 1.82 (m, 2H), 1.60 - 1.42 (m, 2H).

[0152] Example 23: Preparation of 1-(6-{[(1S,3S)-3-({5-[(deuteryl difluoromethyl)oxy]pyrimidin-2- yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-[(trifluoromethyl)oxy]-1,2-dihydropyridin-2-one (Compound 23)

[0153] The title compound 23 was prepared in accordance with the procedure described in Example 19 by replacing the compound j of step 2 with 1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-3- [(trifluoromethyl)oxy]-1,2-dihydropyridin-2-one in a purity of 97.83%.

[0154] ESI-MS: m / z = 450.2 (M+H) + .

[0155] Example 24: Preparation of 1-(6-{[(1S,3S)-3-({5-[(deuteriodyl difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-(trifluoromethyl)-1,2-dihydropyridin-2-one (Compound 24)

[0156] The title compound 24 was prepared in accordance with the procedure described in Example 19 by replacing the compound j of step 2 with 1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-3-(trifluoromethyl)-1,2-dihydropyridin-2-one to afford the title compound 24 with purity 97.65%.

[0157] ESI-MS: m / z = 484.2 (M+H) + .

[0158] 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 8.09 - 7.89 (m, 3H), 7.59 - 7.37 (m, 2H), 7.08 (d, J = 6.8 Hz, 1H), 6.56 (d, J = 8.9 Hz, 1H), 6.43 (t, J = 7.0 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 2.13 (dq, J = 13.6, 6.8 Hz, 2H), 1.90 (hept, J = 6.7 Hz, 2H), 1.52 (tt, J = 13.6, 7.1 Hz, 2H).

[0159] Example 25: Preparation of 1-(6-{[(1S,3S)-3-({5-[(deuteriodyl difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-3-fluoro-1,2-dihydropyridin-2-one (Compound 25)

[0160] The title compound 24 was prepared in accordance with the procedure described in Example 19 by replacing the compound j of step 2 with 1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-3-(trifluoromethyl)-1,2-dihydropyridin-2-one to afford the title compound 24 with purity 97.65%.

[0161] ESI-MS: m / z = 434.2 (M+H) + .

[0162] 1H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 8.00 - 7.92 (m, 1H), 7.52 - 7.40 (m, 4H), 7.00 (d, J = 6.8 Hz, 1H), 6.54 (dd, J = 8.9, 0.7 Hz, 1H), 6.25 (td, J = 7.3, 4.7 Hz, 1H), 4.31 (h, J = 6.7 Hz, 2H), 2.21 - 2.06 (m, 2H), 1.96 - 1.82 (m, 2H), 1.59 - 1.44 (m, 2H).

[0163] Example 26: Preparation of methyl l-(6-{[(lS,3S)-3-({5-[(deuteryl difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-2-oxo-pyridine-3-carboxylate (Compound 26)

[0164] The preparation method is the same as that of Example 19, and the compound j in Step 2 is replaced with methyl l-(6-{[(lS,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-2-oxo-pyridine-3-carboxylate to prepare the title compound 26 with a purity of 98.35%.

[0165] ESI-MS: m / z = 474.2 (M+H) + .

[0166] Example 27: Preparation of l-(6-{[(lS,3S)-3-({5-[(deuteryl difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-2-oxo-pyridine-3-carboxylic acid (Compound 27)

[0167] The preparation method is the same as that of Example 19, and the compound j in Step 2 is replaced with methyl l-(6-{[(lS,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-2-oxo-pyridine-3-carboxylate to prepare the title compound 26 with a purity of 98.35%.

[0168] ESI-MS: m / z = 474.2 (M+H) + .

[0169] 1H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 8.22 (s, 2H), 7.78 (s, 2H), 7.46 (d, J = 7.2 Hz, 1H), 7.01 (s, 1H), 6.89 (s, 1H), 6.53 (s, 1H), 6.28 (s, 1H), 4.28 (dt, J = 14.6, 7.2 Hz, 2H), 2.06 (s, 2H), 1.95 - 1.72 (m, 2H), 1.61 - 1.39 (m, 2H).

[0170] Example 28: Preparation of 5-fluoro-l-(6-{[(lS,3S)-3-({5-[(deuteriodyl difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-l,2- dihydropyridin-2-one (Compound 28)

[0171] The title compound 28 was prepared by following the same procedure of Example 19 using compound j of step 2 was replaced by 5-fluoro-l-(6-{[(lS,3S)-3- aminocyclopentyl]amino}pyridin-3-yl)-l,2-dihydropyridin-2-one in step 1 and step 2 to afford the title compound 28 with purity 97.95%.

[0172] ESI-MS: m / z = 434.2 (M+H) + .

[0173] 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.95 (d, J = 2.7 Hz, 1H), 7.90 (dd, J = 4.8, 3.3 Hz, 1H), 7.64 (ddd, J = 10.4, 7.2, 3.4 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.41 (dd, J = 8.9, 2.7 Hz, 1H), 7.00 (d, J = 6.8 Hz, 1H), 6.53 (d, J = 8.9 Hz, 1H), 6.47 (dd, J = 10.1, 5.5 Hz, 1H), 4.31 (h, J = 6.7 Hz, 2H), 2.19 - 2.05 (m, 2H), 1.97 - 1.81 (m, 2H), 1.60 - 1.41 (m, 2H).

[0174] Example 29: Preparation of 6-fluoro-l-(6-{[(lS,3S)-3-({5-[(deuteriodyl difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-l,2- dihydropyridin-2-one (Compound 29)

[0175] The preparation method is the same as that of Example 19, the compound j of step 2 is replaced with 6-fluoro-1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-1,2- dihydropyridin-2-one, to obtain the title compound 29 with a purity of 98.15%.

[0176] ESI-MS: m / z = 434.2 (M+H) + .

[0177] 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.98 (m, 1H), 7.84 (d, J = 2.9 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.27 (dd, J = 9.0, 2.9 Hz, 1H), 6.88 (dd, J = 8.0, 1.7 Hz, 1H), 6.83 (dd, J = 7.8, 2.4 Hz, 1H), 6.68 (d, J = 6.8 Hz, 1H), 6.52 (d, J = 9.0 Hz, 1H), 4.28 (m, 2H), 2.12 (m, 2H), 1.97 - 1.79 (m, 2H), 1.63 - 1.39 (m, 2H).

[0178] Example 30: Preparation of 2-(6-{[(1S,3S)-3-({5-[(deuteryldifluoromethyl)oxy]pyrimidin-2- yl}amino)cyclopentyl]amino}pyridin-3-yl)-6-methyl-3(2H)-pyridazinone (Compound 30)

[0179] The preparation method is the same as that of Example 19, the compound j of step 2 is replaced with 2-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-6-methyl-3(2H)- pyridazinone, to obtain the title compound 30 with a purity of 98.62%.

[0180] ESI-MS: m / z = 431.2 (M+H) + .

[0181] Example 31: Preparation of 6-fluoro-1-(6-{[(1S,3S)-3-({5-[(deuteryldifluoromethyl)oxy]pyrimidin- 2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-1,2-dihydropyridin-2-one (Compound 31)

[0182] Preparation method same as that of Example 19, replacing compound j of step 2 with 6-chloro-1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-1,2- dihydropyridin-2-one to produce the title compound 31 with a purity of 98.21%.

[0183] ESI-MS: m / z = 450.1 (M+H) + .

[0184] Example 32: Preparation of 1-(6-{[(1S,3S)-3-({5-[(dideuterium difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-6-oxopyridine-2- carbonitrile (Compound 32)

[0185] Preparation method same as that of Example 19, replacing compound j of step 2 with 1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-6-oxopyridine-2- carbonitrile to produce the title compound 32 with a purity of 98.21%.

[0186] ESI-MS: m / z = 441.2 (M+H) + .

[0187] Example 33: Preparation of 4-fluoro-1-(6-{[(1S,3S)-3-({5-[(dideuterium difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-1,2- dihydropyridin-2-one (Compound 33)

[0188] Preparation method same as that of Example 19, replacing compound j of step 2 with 4-fluoro-1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-1,2- dihydropyridin-2-one to produce the title compound 33 with a purity of 98.15%.

[0189] ESI-MS: m / z = 434.2 (M+H) + .

[0190] Example 34: Preparation of 5-chloro-1-(6-{[(1S,3S)-3-({5-[(dideuterium difluoromethyl)oxy]pyrimidin-2-yl}amino)cyclopentyl]amino}pyridin-3-yl)-1,2- dihydropyridin-2-one (Compound 34)

[0191] Preparation method same as the preparation method of example 19, replace compound j of step 2 with 5-chloro-1-(6-{[(1S,3S)-3-aminocyclopentyl]amino}pyridin-3-yl)-1,2- dihydropyridin-2-one to prepare the title compound 34 with a purity of 98.56%.

[0192] ESI-MS: m / z = 450.2 (M+H) + .

[0193] 1 H NMR (400 MHz, DMSO-d6) δ 8.23 (s, 2H), 7.95 (dd, J = 2.7, 0.6 Hz, 1H), 7.90 (dd, J = 2.9, 0.6 Hz, 1H), 7.54 (dd, J = 9.8, 2.9 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.40 (dd, J = 8.9, 2.7 Hz, 1H), 6.97 (d, J = 6.9 Hz, 1H), 6.50 (ddd, J = 11.7, 9.4, 0.7 Hz, 2H), 4.30 (p, J = 6.6 Hz, 2H), 2.21 - 2.04 (m, 2H), 1.88 (tdd, J = 13.2, 10.4, 6.3 Hz, 2H), 1.61 - 1.42 (m, 2H).

[0194] Example 35: Preparation of 1-(6-{[(1S,3S)-3-{[5-(trideuteromethyl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 35)

[0195] Step 1: Preparation of tert-butyl {[(1S,3S)-3-{[5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyrimidin-2-yl]amino}cyclopentyl]amino}carboxylate (Compound r)

[0196] Dissolve compound q (1.2 g, 5 mmol, 1.0 eq), compound h (1.05 g, 5 mmol, 1.05 eq) with DMF (20 mL), add cesium carbonate (3.25 g, 10 mmol, 2.0 eq) and react at room temperature, after TLC monitoring reaction is completed, add 3 times volume of water to quench, extract with ethyl acetate, concentrate and separate by column chromatography (EA / PE = 1:1 to 3:1) to obtain compound r (1.6 g, yield 79.2%).

[0197] ESI-MS: m / z = 405.3 [M+H] + .

[0198] Step 2: Preparation of tert-butyl {[(1S,3S)-3-{[5-(trideuteromethyl)pyrimidin-2- yl]amino}cyclopentyl]amino}carboxylate (Compound s)

[0199] Compound r (1.6 g, 4.0 mmol, 1.0 eq), LiOtBu (352 mg, 4.4 mmol, 1.1 eq), lithium iodide (53 mg, 0.4 mmol, 0.1 eq), copper(I) iodide (3.8 mg, 0.020 mmol, 0.2 eq), 1,3-dimethyl-2-imidazolidinone (DMI, 12 ml), trimethyl phosphate-D9 (656 mg, 4.4 mmol, 1.1 eq) were dissolved in a flask under nitrogen protection and kept at 50 °C for 16 h. After TLC monitoring the reaction was complete, 3 times volume of water was added to quench the reaction, extracted with ethyl acetate, concentrated and separated by column chromatography (EA / PE = 1:1 to 3:1) to give Compound s (708 mg, yield 59.8%).

[0200] ESI-MS: m / z = 296.2 (M+H) + .

[0201] Step 3: Preparation of (1S,3S)-3-{[5-(trideuteromethyl)pyrimidin-2-yl]amino}cyclopentan-1- amine (Compound t)

[0202] Compound s (708 mg, 2.4 mmol, 1.0 eq) was dissolved in dioxane HCl solution (20 mL) and reacted at room temperature for 3 h. After TLC monitoring the reaction was complete, 3 times volume of water was added to quench the reaction, extracted with ethyl acetate, concentrated and separated by column chromatography (EA / PE = 1:1 to 3:1) to give Compound t (410 mg, yield 87.6%).

[0203] ESI-MS: m / z = 196.2 (M+H) + .

[0204] Step 4: Preparation of 1-(6-{[(1S,3S)-3-{[5-(trideuteromethyl)pyrimidin-2-yl]amino}cyclopentyl] amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 35)

[0205] Compound t (410 mg, 2.1 mmol, 1.0 eq), compound g (543 mg, 2.3 mmol. 1.1 eq), tBuXphos Pd G3 (167 mg, 0.21 mmol, 0.1 eq) and tBuOK (46 mg, 0.42 mmol, 0.2 eq) were dissolved with dioxane (15 mL) and heated to 110 °C, protected from N2 overnight. After TLC monitoring the reaction was complete, the insoluble was filtered, concentrated and column chromatography (EA / PE = 1:2 to 2:1) to isolate the white solid compound 35 (567 mg, yield 68.2%) with purity 98.01%.

[0206] ESI-MS: m / z = 396.2 (M+H) + .

[0207] Example 36: Preparation of 3-chloro-l-(6-{[(lS,3S)-3-{[5-(trideuteromethyl)pyrimidin- 2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-l,2-dihydropyridin-2-one (Compound 36)

[0208] The preparation method is the same as that of Example 35, and compound g of step 4 is replaced by 3-chloro-l-(6-chloropyridin-3-yl)-l,2-dihydropyridin-2-one to prepare the title compound 36 with a purity of 97.65%.

[0209] ESI-MS: m / z = 400.2 (M+H) + .

[0210] 1 H NMR (400 MHz, DMSO-d6) d 8.11 (s, 2H), 7.95 (d, J = 2.7 Hz, 1H), 7.81 (dd, J = 7.3, 1.9 Hz, 1H), 7.66 (dd, J = 6.8, 1.9 Hz, 1H), 7.42 (dd, J = 8.9, 2.7 Hz, 1H), 7.04 - 6.94 (m, 2H), 6.54 (d, J = 8.9 Hz, 1H), 6.30 (t, J = 7.1 Hz, 1H), 4.45 - 4.19 (m, 2H), 2.11 (ddd, J = 18.8, 9.2, 5.6 Hz, 2H), 1.92 - 1.81 (m, 2H), 1.49 (td, J = 13.7, 12.9, 7.3 Hz, 2H).

[0211] Example 37: Preparation of l-(6-{[(lS,3S)-3-{[5-(trideuteromethyl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-2-oxoacetonitrile (Compound 37)

[0212] The process was carried out as in the preparation of Example 35, replacing compound g of step 4 with 1-(6-chloropyridin-3-yl)-2-oxopyridine-3-carbonitrile, to give the title compound 37 with a purity of 98.15%.

[0213] ESI-MS: m / z = 391.2 (M+H) + .

[0214] 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (dd, J = 7.2, 2.1 Hz, 1H), 8.11 (s, 2H), 8.05 (dd, J = 6.7, 2.1 Hz, 1H), 7.97 (d, J = 2.7 Hz, 1H), 7.45 (dd, J = 8.9, 2.7 Hz, 1H), 7.05 (d, J = 6.9 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.54 (d, J = 8.9 Hz, 1H), 6.46 (t, J = 7.0 Hz, 1H), 4.32 (q, J = 6.1, 5.3 Hz, 2H), 2.19 - 2.05 (m, 2H), 1.93 - 1.80 (m, 2H), 1.49 (td, J = 14.6, 13.6, 7.3 Hz, 2H).

[0215] Example 38: Preparation of 1-(6-{[(1S,3S)-3-{[5-(trideuteromethyl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methyl-1,2-dihydropyridin-2-one (Compound 38)

[0216] The process was carried out as in the preparation of Example 35, replacing compound g of step 4 with 1-(6-chloropyridin-3-yl)-3-methyl-1,2-dihydropyridin-2-one, to give the title compound 38 with a purity of 97.05%.

[0217] ESI-MS: m / z = 380.2 (M+H) + .

[0218] Example 39: Preparation of 1-(6-{[(1S,3S)-3-{[5-(trideuteromethyl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-[(trifluoromethyl)oxy]-1,2- dihydropyridin-2-one (Compound 39)

[0219] Preparation method same as that of Example 35, replacing compound g of step 4 with 1-(6-chloropyridin-3-yl)-3-[(trifluoromethyl)oxy]-1,2-dihydropyridin-2-one to produce the title compound 39 with a purity of 97.33%.

[0220] ESI-MS: m / z = 450.2 (M+H) + .

[0221] Example 40: Preparation of 1-(6-{[(1S,3S)-3-{[5-(trideuteromethyl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-(trifluoromethyl)-1,2-dihydropyridin-2-one (Compound 40)

[0222] Preparation method same as that of Example 35, replacing compound g of step 4 with 1-(6-chloropyridin-3-yl)-3-(trifluoromethyl)-1,2-dihydropyridin-2-one to produce the title compound 40 with a purity of 97.32%.

[0223] ESI-MS: m / z = 434.2 (M+H) + .

[0224] 1 H NMR (400 MHz, DMSO-d6) δ 8.11 (s, 2H), 8.04 - 7.94 (m, 3H), 7.44 (dd, J = 8.9, 2.7 Hz, 1H), 7.00 (dd, J = 16.9, 7.1 Hz, 2H), 6.54 (d, J = 8.9 Hz, 1H), 6.42 (t, J = 7.0 Hz, 1H), 4.42 - 4.23 (m, 2H), 2.21 - 2.04 (m, 2H), 1.87 (dhept, J = 18.8, 6.7 Hz, 2H), 1.49 (tt, J = 13.1, 7.3 Hz, 2H).

[0225] Example 41: Preparation of 1-(6-{[(1S,3S)-3-{[5-(oxetan-3-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 41)

[0226] Step 1: Preparation of {[(1S,3S)-3-{[5-(oxetan-2-yl)pyrimidin-2-yl]amino}cyclopentyl] amino}methane acid-2-methylprop-2-yl ester

[0227] Compound L (2.02 g, 5 mmol, 1.0 eq), compound u (1.01 g, 5.5 mmol, 1.1 eq) were dissolved in THF (20 mL), H2O (4 ml), Cesium carbonate (3.25 g, 10 mmol, 2.0 eq) was added, followed by Pd(dppf)Cl2.CH2Cl2(410 mg, 0.5 mmol), after replacing the system with N2, the temperature was raised to 80 °C and the reaction was carried out for 8 h. After the reaction was completed by TLC monitoring, 3 times the volume of water was added to quench, extracted with ethyl acetate, concentrated and separated by column chromatography (EA / PE = 1:2 to 1:1) to obtain compound v (1.27 g, yield 76.2%).

[0228] ESI-MS: m / z = 335.2 [M+H] + .

[0229] Step 2: Preparation of (1S,3S)-3-{[5-(oxetan-3-yl)pyrimidin-2-yl]amino}cyclopentan-1- amine (Compound w)

[0230] Compound v (1.27 g, 3.8 mmol, 1.0 eq) was dissolved in a solution of HCl in dioxane (20 mL) and reacted at room temperature, and solid gradually precipitated. After the reaction was completed by TLC monitoring, filtration gave light yellow solid compound w (786 mg, yield 88.5%).

[0231] ESI-MS: m / z = 235.2 (M+H) + .

[0232] Step 3: Preparation of 1-(6-{[(1S,3S)-3-{[5-(oxetan-3-yl)pyrimidin-2-yl]amino}cyclopentyl] amino}pyridin-3-yl)-3-methoxy-1,2-dihydropyridin-2-one (Compound 41)

[0233] Compound w (491 mg, 2.1 mmol, 1.0 eq), compound g (543 mg, 2.3 mmol, 1.1 eq), tBuXphos Pd G3 (167 mg, 0.21 mmol, 0.1 eq) and tBuOK (46 mg, 0.42 mmol, 0.2 eq) were dissolved in dioxane (15 mL) and heated to 110 °C, and the reaction was carried out overnight under N2protection. After the reaction was completed by TLC monitoring, the insoluble matter was filtered, concentrated and separated by column chromatography (EA / PE = 1:2 to 2:1) to obtain white solid compound 41 (603 mg, yield 66.2%), with a purity of 98.03%

[0234] ESI-MS: m / z = 435.2 (M+H) + .

[0235] Example 42: Preparation of 3-chloro-l-(6-{[(lS,3S)-3-{[5-(azetidin-l- yl)pyrimidin-2-yl]amino}cyclopentyl]amino}pyridin-3-yl)-l,2-dihydropyridin-2-one (Compound 42)

[0236] The preparation method is the same as that of Example 41. The compound g in Step 3 is replaced by 3-chloro-l-(6-chloropyridin-3-yl)-l,2-dihydropyridin-2-one to obtain the title compound 42 with a purity of 97.21%.

[0237] ESI-MS: m / z = 439.2 (M+H) + .

[0238] Example 43: Preparation of l-(6-{[(lS,3S)-3-{[5-(oxetan-3-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-2-oxoacetylpyridin-3-carbonitrile (Compound 43)

[0239] The preparation method is the same as that of Example 41. The compound g in Step 3 is replaced by l-(6-chloropyridin-3-yl)-2-oxoacetylpyridin-3-carbonitrile to obtain the title compound 43 with a purity of 97.93%.

[0240] ESI-MS: m / z = 430.2 (M+H) + .

[0241] Example 44: Preparation of l-(6-{[(lS,3S)-3-{[5-(oxetan-3-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-methyl-l,2-dihydropyridin-2-one (Compound 44)

[0242] The preparation method is the same as that of Example 41. The compound g in Step 3 is replaced by l-(6-chloropyridin-3-yl)-3-methyl-l,2-dihydropyridin-2-one to obtain the title compound 44 with a purity of 97.67%.

[0243] ESI-MS: m / z = 419.2 (M+H) + .

[0244] Example 45: Preparation of l-(6-{[(lS,3S)-3-{[5-(oxetan-3-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-[(trifluoromethyl)oxy]-l,2- dihydropyridin-2-one (Compound 45)

[0245] The process was carried out as per the procedure described in Example 41, replacing compound g of step 3 with 1 -(6-chloropyridin-3-yl)-3-(trifluoromethyl)- 1,2-dihydropyridin-2-one to obtain the title compound 45 with purity 96.92%.

[0246] ESI-MS: m / z = 489.2 (M+H) + .

[0247] Example 46: Preparation of 1 -(6-{[(1 S,3S)-3-{[5-(oxetan-3-yl)pyrimidin-2- yl]amino}cyclopentyl]amino}pyridin-3-yl)-3-(trifluoromethyl)-1,2-dihydropyridin- 2-one (Compound 46)

[0248] The process was carried out as per the procedure described in Example 41, replacing compound g of step 3 with 1 -(6-chloropyridin-3-yl)-3-(trifluoromethyl)- 1,2-dihydropyridin-2-one to obtain the title compound 46 with purity 98.06%.

[0249] ESI-MS: m / z = 473.2 (M+H)+.

[0250] Reference Example 1: Positive compound (AZD0780), commercially available, purity 99%.

[0251] Reference Example 2:

[0252] Reference Example 3:

[0253] Reference Example 4:

[0254] Test Example 1: Determination of effect of compounds of the present application on the concentration of PCSK9 secreted by HepG2 cells

[0255] 1. Experimental method

[0256] 1.1 Day-1, cells were seeded in 96 well plates:

[0257] HepG2 cells were seeded with low fat serum medium and incubated at 37°C in a 5% CO2 incubator overnight.

[0258] 1.2 Day 0, preparation of Reference Example 1 compound (positive control), Reference Examples 2-4 and Example compounds:

[0259] a) for Compounds of Examples, final assay concentration: 300, 150, 75, 37, 18.75, 9.375, 4.6875, 2.34375, 1.171875 μM;

[0260] b) for Compounds of Comparative Examples, final assay concentration: 300 μM;

[0261] c) Incubate the cell plate in a 37 °C, 5% CO2 incubator for 2 days;

[0262] d) Final DMSO concentration in all assay systems is 0.5%.

[0263] 1.3 Day 2, Assay:

[0264] a) Collect 100 μL of media supernatant per well;

[0265] b) Dilute the standard samples with lx AlphaLISA Immuno Buffer, add standard samples to the 384-well plate (2ul per well);

[0266] c) Add prepared samples (each compound + standard sample) to the 384-well plate (2ul per well);

[0267] d) Add the mixture of AlphaLISA anti-PCSK9 acceptor beads and biotinylated anti-PCSK9 antibody, incubate at 25 °C for 60 minutes; e) Add streptavidin (SA)-coated donor beads to the plate, incubate at 25 °C (protected from light) for 30 minutes;

[0268] f) Readout using BMG-Alpha Reader.

[0269] 2. Data Analysis

[0270] (1) Measure and record the AlphaLISA counts for each well.

[0271] (2) Calculate the % PCSK9 secretion ratio to Vehicle as follows: % PCSK9 secretion ratio to Vehicle = counts treatment / countsAvg(vehice) * 100%.

[0272] Use GraphPad Prism 8 software to calculate IC50 and plot compound effect dose curve: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope));

[0273] X: Log of cpd concentration;

[0274] Y: % inhibition;

[0275] Top and Bottom: Plateaus in same units as Y;

[0276] logIC50: same log units as X;

[0277] Hill Slope: Slope factor or Hill slope.

[0278] 3. Experimental results

[0279] Test Example 2, PCSK9 protein affinity test

[0280] The effect of the compound on PCSK9 protein binding was detected by using the conventional surface plasmon resonance (SPR) method.

[0281] 1. Reagent preparation:

[0282] 1) Protein: PCSK9, Acro;

[0283] 2) Chip: SA, Cytiva;

[0284] 3) Protein immobilization buffer: 10 mM Hepes, 150 mM NaCl, 0.05% Surfactant P20, pH 7.4 (HBS-P), 0.1 mM CaCl2;

[0285] 4) Sample analysis buffer: 10 mM Hepes, 150 mM NaCl, 0.05% Surfactant P20, pH 7.4 (HBS-P), 0.1 mM CaCl2, 4% DMSO.

[0286] 2. Protein immobilization:

[0287] 1) Prepare protein buffer;

[0288] 2) Dilute PCSK9 protein to the specified concentration: 100 ug / mL with buffer;

[0289] 3) Immobilize PCSK9 protein to SA chip by biotin capture method, the coupling amount is ~6000 RU;

[0290] 4) The reference channel does not perform protein coupling, only activation and blocking treatment;

[0291] 5) The SA chip after immobilizing PCSK9 protein is used for analyte test.

[0292] 3. Sample testing:

[0293] 1) Prepare sample analysis buffer;

[0294] 2) Dilute the compounds of Comparative Examples 1-4 and Example to the specified concentration of 400nM, 3-fold dilution, 8 doses, respectively, with the sample buffer;

[0295] 3) Set the multi-cycle kinetic mode to determine the affinity of each sample to PCSK9;

[0296] 4) Set the dissociation time of binding to: 100s+300s.

[0297] 4. Data analysis:

[0298] 1) At least 5 doses meet the concentration-dependent criteria;

[0299] 2) The experimental results should pass the instrument QC standard;

[0300] 3) Calculate ka, kd and KD values using biacore evaluation software.

[0301] 5. Experimental results:

[0302] Test Example 3, Rat Pharmacokinetic Determination

[0303] 1. Sample preparation method: Take 5mg of the compound of Comparative Example 1, first add 0.05ml of DMSO, ultrasonic to clear and transparent, then add 0.1ml of HS-15, mix and ultrasonic to clear and transparent, finally add appropriate amount of 0.9% sodium chloride injection, dilute to the target concentration, ultrasonic oscillation, mix well. The example compound is given in equimolar dose, and the preparation method is the same as that of Comparative Example 1. Prepare and use before use.

[0304] 2. Dosing: 6 SD rats, male; after overnight fasting, PO dosing, the dose of the compound of Comparative Example 1 is 5mg / kg. The example compound is given in equimolar dose, and the dosing method is the same as that of Comparative Example 1.

[0305] 3. Sample collection: The blood sampling time points after dosing are: 0h, 5min, 15min, 30min, 45min, 1h, 2h, 4h, 8h, 12h, 24h. 0.04mL of orbital blood is collected into an EDTA-K2 test tube, centrifuged at 6000rpm for 6min at 4℃ to separate the plasma, which is stored at -80℃; food is given 4h after dosing.

[0306] 4. Determination results: The final determination results are obtained by using LCMS / MS method.

[0307] 5. Experimental results: The main pharmacokinetic parameters were calculated by WinNonlin 6.1. The results are as follows:

[0308] The above embodiment is only one of the preferred embodiments of the present application, and should not be used to limit the protection scope of the present application, but any modification or polishing without substantial meaning made in the main design idea and spirit of the present application, and the technical problems solved are still consistent with the present application, which should be included in the protection scope of the present application.

Claims

1. A compound of Formula I: ###0001### or an isomer, a pharmaceutically acceptable salt thereof. wherein, R1is selected from deuterium, amino, nitro, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkylamino, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted deuterated alkoxy, substituted or unsubstituted deuterated alkyl, substituted or unsubstituted deuterated alkylthio, -SO2NR6R7, -NR6SO2R7, -COSR6or -CSOR6; R2, R3, R4, and R5 are each independently selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, alkyl, alkenyl, alkynyl, oxo, thio, deuterated alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, cyano-substituted alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, -SO2NR6R7, -NR6SO2R7, and -(CH2). n R6、-(CH2) n OR6、-(CH2) n C(O)R6、-(CH2) n C(O)OR6、-(CH2) n S(O) m R6、-(CH2) n NR7R8、-(CH2) n NR7C(O)OR8、-(CH2) n NR7C(O)(CH2) n1 R8, -(CH2) n NR7C(O)NR7R8、-(CH2) n C(O)NR7(CH2) n1 R8, -OC (R6R7) n (CH2) n1 R8 or -(CH2) n NR7S(O) m R8; Optionally, the amino, alkyl, alkenyl, alkynyl, deuterated alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, cyano-substituted alkyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups may be further substituted by one or more of the following substituents: deuterium, halogen, nitro, hydroxyl, mercapto, cyano, amino, oxo, thio, carboxyl, C 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Hydroxyalkyl, C 1-3 Alkoxy, C 1-3 Deuterated alkoxy, C 1-3 Halogenated alkoxy groups, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 6-10 Aryl, 5-10 member heteroaryl; among which, R6, R7, R8are each independently selected from hydrogen, deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, alkyl, deuterated alkyl, halogenated alkyl, hydroxyalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, which amino, alkyl, deuterated alkyl, halogenated alkyl, hydroxyalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl can be further substituted with substituents; n is 0, 1, 2, 3 or 4; n1is 0, 1, 2, 3 or 4; m is 0, 1, 2, 3 or 4; X and Y are independently selected from C and N, and X and Y cannot both be C; and when Y is N, R1cannot be C 4-5 heterocyclyl, C5heteroaryl, and C 1-6 alkylamino; W is selected from C or N, R2, R3, R4are not simultaneously hydrogen or C 1-6 alkyl.

2. The compound or isomer, pharmaceutically acceptable salt thereof according to claim 1, wherein, R1 is selected from deuterium, amino, nitro, alkynyl, substituted or unsubstituted C. 1-6 Alkylamino, substituted or unsubstituted 3-6 membered heterocyclic groups, substituted or unsubstituted 5-6 membered heteroaryl groups, substituted or unsubstituted phenyl groups, substituted or unsubstituted deuterated C groups 1-6 Alkoxy, substituted or unsubstituted deuterated C 1-6 Alkyl, substituted or unsubstituted deuterated C 1-6 Alkylthio, -SO2NR6R7, -NR6SO2R7, -COSR6 or -CSOR6; wherein R6 and R7 are defined as in claim 1.

3. The compound or isomer, pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein, The compounds have the structure of Formula la, Formula lb, Formula Ic, Formula Id, Formula Ie, or Formula If: wherein, R1to R5are defined in claim 1 or 2, and: R1cannot be C 4-5 heterocyclyl, C5heteroaryl, and C 1-6 alkylamino; or, R2, R3, R4are not simultaneously hydrogen or C1-C4-alkyl when the compound has the general formula Ib, Id or If and R5is hydrogen 1-6 alkyl.

4. The compound or isomer, pharmaceutically acceptable salt thereof according to claim 3, characterized in that, in the formula Ia or Ib, R1is selected from substituted or unsubstituted oxetanyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted tetrahydrofuranyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted furanyl, substituted or unsubstituted thienyl, substituted or unsubstituted C 1-6 alkylamino, deutero-methoxy, deutero-methyl, deutero-methylthio, deutero-difluoromethoxy, or phenyl; and / or R2, R3, R4, R5are each independently selected from hydrogen, deuterium, halogen, nitro, hydroxyl, carboxyl, amido, thiol, cyano, amino, C 1-6 alkyl, deuterated C 1-6 haloalkyl, C 1-6 hydroxyalkyl, C 1-6 alkoxy, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 3-6 cycloalkyl, C 2-6 heterocyclyl, C 5-10 aryl or heteroaryl; optionally, the carboxyl, amido, amino, C 1-6 alkyl, deuterated C 1-6 haloalkyl, C 1-6 hydroxyalkyl, C 1-6 alkoxy, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 3-6 cycloalkyl, C 2-6 heterocyclyl, C 5-10 aryl and heteroaryl can be further substituted with one or more of deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thioxo, carboxyl, C 1-3 alkyl, C 1-3 deuterated alkyl, C 1-3 haloalkyl, C 1-3 hydroxyalkyl, C 1-3 alkoxy, C 1-3 deuterated alkoxy, C 1-3 haloalkoxy, C 2-4 alkenyl, C 2-4 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl; R2, R3, R4are not simultaneously hydrogen or C1-C4-alkyl when the compound has the general formula Ib and R5is hydrogen 1-6 alkyl.

5. The compound of claim 3, or isomer, pharmaceutically acceptable salt thereof, wherein, in the formula Ic, Id, Ie or If, R1is selected from deuterium, amino, nitro, alkynyl, substituted or unsubstituted oxetanyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted furanyl, substituted or unsubstituted thienyl, deuterated methoxy, deuterated methyl, deuterated methylthio, deuterated difluoromethoxy, -SO2NR6R7, -NR6SO2R7, -COSR6or -CSOR6; wherein R6, R7are defined in claim 1; and / or R2, R3, R4, R5are each independently selected from hydrogen, deuterium, halogen, nitro, hydroxyl, carboxyl, amido, thiol, cyano, amino, C 1-6 alkyl, deuterated C 1-6 alkyl, haloC 1-6 alkyl, hydroxyC 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkenyl, C 1-6 alkynyl, C 3-6 cycloalkyl, C 2-6 heterocyclyl, C 5-10 aryl or heteroaryl; optionally, the carboxyl, amido, amino, C 1-6 alkyl, deuterated C 1-6 alkyl, haloC 1-6 alkyl, hydroxyC 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkenyl, C 1-6 alkynyl, C 3-6 cycloalkyl, C 2-6 heterocyclyl, C 5-10 aryl and heteroaryl can be further substituted with one or more of deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thioxo, carboxyl, C 1-3 alkyl, C 1-3 deuterated alkyl, C 1-3 haloalkyl, C 1-3 hydroxyalkyl, C 1-3 alkoxy, C 1-3 deuterated alkoxy, C 1-3 haloalkoxy, C 2-4 alkenyl, C 2-4 alkynyl, C 3-8 cycloalkyl, 3-8 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl; when the compound is of formula Id or If and R5is hydrogen, R2, R3, R4are not simultaneously hydrogen or C 1-6 alkyl.

6. The compound of claim 1, or isomer, pharmaceutically acceptable salt thereof, wherein, The compound is selected from:

7. The compound or isomer, or pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6, wherein the hydrogen in the structure of the compound can be substituted with 1 to multiple deuterium.

8. Use of the compound or isomer, pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the manufacture of a PCSK9 inhibitor.

9. Use of the compound or isomer, pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the manufacture of a medicament for lowering LDL-cholesterol level, VLDL-cholesterol level, triglyceride level or lipoprotein A level.

10. Use of the compound or isomer, pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the manufacture of a medicament for treating cardiovascular disease, cerebrovascular disease, atherosclerosis, sepsis, septic shock and / or their related diseases or symptoms thereof; preferably in the manufacture of a medicament for stroke, hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, dyslipoproteinemia, atherosclerosis, hepatic steatosis, metabolic syndrome, coronary artery disease, sepsis and / or septic shock.

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