Anilinopyrimidine and heterocyclic compounds, preparation method, pharmaceutical composition and application thereof

Abstract

This invention discloses an aniline pyrimidine heterocyclic compound, its preparation method, pharmaceutical composition, and applications. The compound has the structure of Formula I and also contains a pharmaceutically acceptable salt. It effectively inhibits WEE1 and HDAC with an optimal inhibition rate exceeding 90%, and also effectively inhibits tumor cell proliferation with an inhibition rate exceeding 50%. It has wide applications, exerting pharmacological effects at both the molecular and cellular levels, with optimal nanomolar concentrations. Furthermore, its preparation method is easily expandable and has good versatility.

Description

Technical Field

[0001] This invention relates to an aniline pyrimidine heterocyclic compound, its preparation method, pharmaceutical composition, and application, and more particularly to an aniline pyrimidine heterocyclic compound with dual target inhibitory activities of WEE1 and HDAC, its preparation method, pharmaceutical composition, and application. Background Technology

[0002] The primary mechanism of action of WEE1 inhibitors is through synthetic lethality against the p53 gene. Since most tumors have mutations or deletions in the p53 gene, they rely on the WEE1 kinase-mediated G2 / M phase cell cycle checkpoint to arrest cells with DNA damage in the G2 / M phase, providing time for DNA repair. Inhibition of WEE1 kinase causes these DNA-damaged cells to prematurely enter mitosis, triggering mitotic catastrophe and ultimately leading to apoptosis. Several WEE1 inhibitors are currently in clinical trials, such as Zentalis' ZN-C3 (phase II), Debio's Debio-0123 (phase I / II), Shanghai Yingpai Pharmaceutical's IMP7068 (phase I), and SC0191 (phase I) jointly developed by Zhikang Hongren and WuXi AppTec. However, tumor pathogenesis is complex, often involving multiple signaling pathways, and single-target drugs are often insufficient. Literature reports that acute myeloid leukemia cells treated with WEE1 inhibitors may produce a compensatory pathway that may be activated by CHK1-mediated, thereby reducing the antiproliferative effect of WEE1 inhibitors.

[0003] HDAC is classified into four classes (I, II, III, and IV) with 18 subtypes. Overexpression of types I, II, and IV leads to alterations in normal cell cycle and metabolic behavior, thereby inducing tumors. Based on this, several HDAC inhibitors have been approved for marketing, such as Voronostat, Romidepsin, Belinostat, and Panobinostat approved by the US FDA. Chidamide, independently developed in my country, was also launched domestically in 2015. These drugs have played a significant role in the treatment of hematological malignancies. Studies have shown that HDAC participates in regulating DNA damage repair pathways and has a significant synergistic effect with WEE1 inhibitors. Inhibiting HDAC in various tumor cells can suppress the compensatory activation pathway induced by WEE1 inhibitors, thereby resensitizing tumor cells to WEE1 inhibitors.

[0004] Considering that combination therapy often has problems such as toxic side effects due to drug-drug interactions and heterogeneous pharmacokinetic properties, the development of WEE1 / HDAC dual-target drugs is of great significance and potential application value. Summary of the Invention

[0005] Objectives of the invention: The first objective of this invention is to provide an aniline pyrimidine heterocyclic compound; the second objective is to provide a method for preparing the compound; the third objective is to provide a pharmaceutical composition comprising the compound; and the fourth objective is to provide an application of the compound and the pharmaceutical composition thereof.

[0006] Technical solution: The aniline pyrimidine heterocyclic compound of the present invention has the structure of Formula I and further comprises its pharmaceutically acceptable salt:

[0007]

[0008] in:

[0009] R1 is selected from substituted or unsubstituted C. 1-4 Alkyl, C 2-4 Alkenyl, 5-7 aryl, 5-7 heteroaryl containing 1-3 N, O, S, wherein the substituents are selected from one or more hydrogens, halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl, cyano, nitro, hydroxyl, amino;

[0010] R2 is selected from one or more hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl, cyano, nitro, hydroxyl, amino;

[0011] R3 is selected from hydroxyl, substituted or unsubstituted 5-7 aryl groups, and 5-7 heteroaryl groups containing 1-3 N, O, or S atoms, wherein the substituent is selected from one or more hydrogens, halogens, or C atoms. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl, cyano, nitro, hydroxyl, amino;

[0012] X is selected from Where R a Selected from hydrogen, halogens, C 1-4 Alkoxy, 3-7 membered cycloalkoxy, C 1-4 Haloalkyl, C 1-4 Hydroxyalkyl, C 1-4 Alkoxy-substituted C 1-4 Alkyl, cyano, nitro, amino, hydroxyl, Q is selected from CH, N, R b Selected from substituted or unsubstituted C 1-4 Alkyl, C 2-4 Alkenyl, 5-7 aryl, 5-7 aromatic heterogroups containing 1-3 N, O, S, wherein the substituents are selected from one or more hydrogens, halogens, C1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkyl, cyano, nitro, hydroxyl, amino;

[0013] Y is selected from -(CH2) n -、-(CH2) n O-, -(CH2) n S-, -(CH2) n CONH-, -(CH2) n NHCO-, -(CH2) n SO2NH-、-(CH2) n NHSO2-, Where n = 0-7.

[0014] Preferably, in the structure:

[0015] R1 is selected from substituted or unsubstituted C. 1-4 Alkyl, C 2-4 Alkenyl, phenyl, wherein the substituent is selected from one or more hydrogens, halogens, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy;

[0016] R2 is selected from one or more hydrogen, halogen, C 1-4 alkyl;

[0017] R3 is selected from hydroxyl groups or substituted phenyl groups, and the substituents are selected from halogens or amino groups;

[0018] X is selected from Where R a Selected from hydrogen, halogens, C 1-4 Alkoxy, 3-5 membered cycloalkoxy, C 1-4 Haloalkyl, C 1-4 Hydroxyalkyl, C 1-4 Alkoxy-substituted C 1-4 Alkyl group, Q is selected from CH, N, R b Selected from C 1-4 alkyl;

[0019] Y is selected from -(CH2) n -、-(CH2) n O-, -(CH2) n CONH-、 Where n = 0-7.

[0020] Preferably, in the structure:

[0021] R1 is selected from

[0022] R2 is selected from one or more hydrogen, methyl, or halogen elements;

[0023] R3 is selected from hydroxyl, o-aminophenyl, and 2-amino-4-fluorophenyl;

[0024] X is selected from

[0025] Y is selected from -(CH2) n -、-(CH2) n O-, -(CH2) n CONH-、 Where n = 0-7.

[0026] Preferably, in the structure:

[0027] R1 is selected from

[0028] R2 is selected from hydrogen;

[0029] R3 is selected from hydroxyl groups;

[0030] X is selected from

[0031] Y is selected from -(CH2) n O-, -(CH2) n CONH-、 Where n = 6, 7.

[0032] Specifically, the aniline pyrimidine heterocyclic compound is selected from any of the following compounds:

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] Preferably, the pharmaceutically acceptable salt is a salt formed by the aniline pyrimidine heterocyclic compound and an acid selected from any of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

[0039] The method for preparing the compound described in this invention is selected from any of the following methods:

[0040] (1) When R3 is a hydroxyl group, the methimazole compound is coupled and ammonolyzed to obtain compound I:

[0041]

[0042] Compound IV is prepared by reacting compound V with mCPBA, using a solvent selected from chloroform, dichloromethane, toluene, tetrahydrofuran, 1,4-dioxane, ethyl acetate, acetone, N,N-dimethylformamide, or any mixture thereof, with dichloromethane being preferred.

[0043] Compound II is prepared by reacting compound IV with compound III. The base used is selected from triethylamine, N,N-diisopropylethylamine, preferably N,N-diisopropylethylamine; the solvent used is selected from chloroform, dichloromethane, toluene, tetrahydrofuran, 1,4-dioxane, ethyl acetate, acetone, N,N-dimethylformamide or any mixture of two solvents, preferably toluene.

[0044] Compound IA is prepared by reacting compound II with hydroxylamine hydrochloride. The base used is selected from sodium ethoxide, potassium acetate, sodium hydroxide, potassium hydroxide, potassium carbonate, and sodium carbonate, with potassium hydroxide being preferred. The solvent used is selected from methanol, ethanol, n-propanol, isopropanol, tert-butanol, n-butanol, and isobutanol, with methanol being preferred.

[0045] (2) When R3 is not a hydroxyl group, the meththiolated compound is coupled, hydrolyzed, and acylated to obtain compound I:

[0046]

[0047] Compound IIB is prepared by hydrolysis of compound II. The base used is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, and sodium carbonate, with sodium hydroxide being preferred. The solvent used is selected from tetrahydrofuran, 1,4-dioxane, methanol, and a mixture of ethanol and water, with a mixture of tetrahydrofuran and water being preferred.

[0048] Compound IB is prepared by reacting compound IIB with R3-NH2. The condensing agent used is selected from carbonyl diimidazole (CDI), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) or benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (PyBOP), with HATU being preferred. The acid-binding agent is selected from N,N-diisopropylethylamine or triethylamine, with N,N-diisopropylethylamine being preferred. The solvent used is selected from dichloromethane, tetrahydrofuran, 1,4-dioxane, ethyl acetate, acetone, and N,N-dimethylformamide, with N,N-dimethylformamide being preferred.

[0049] The definitions of R1, R2, R3, X, and Y are as described above, R c It is methyl or ethyl;

[0050] The corresponding acid is used to form a salt with compound I prepared by the above method to obtain a pharmaceutically acceptable salt of compound I.

[0051] The pharmaceutical composition of the present invention comprises the aniline pyrimidine heterocyclic compound and a pharmaceutically acceptable carrier, specifically by adding common pharmaceutical excipients such as flavorings, sweeteners, liquid / solid fillers, and diluents to prepare common pharmaceutical preparations, such as tablets, capsules, syrups, suspensions, or injections.

[0052] The aniline pyrimidine heterocyclic compounds and their drug combinations described in this invention are used to prepare dual-target inhibitors of WEE1 and HDAC, specifically for the preparation of antitumor drugs.

[0053] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0054] These compounds can effectively inhibit WEE1 and HDAC, with an optimal inhibition rate of over 90%, and can also effectively inhibit tumor cell proliferation, with an inhibition rate of over 50%. They have a wide range of applications, exerting their effects at both the molecular and cellular levels, with the optimal concentration reaching nanomolar levels. Their preparation methods are easy to extend structurally and have good versatility. Detailed Implementation

[0055] The technical solution of the present invention will be further described below with reference to the embodiments.

[0056] Example 1: Preparation of N 1 -(4-((2-Allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)-N 8 Hydroxyoctanediamide (Compound I-1)

[0057]

[0058] Step 1: Synthesis of Intermediate 3

[0059] Starting materials 1 (1.66 g, 7.97 mmol), 2 (1.50 g, 7.97 mmol), HATU (3.64 g, 9.56 mmol), DIEA (1.54 g, 11.95 mmol), and DCM (20 mL) were added to a round-bottom flask and dissolved. The mixture was stirred at room temperature for 3 hours, and the reaction was monitored for completion by TLC (DCM:MeOH = 35:1). The reaction solution was diluted with water, extracted three times with DCM, and the organic layers were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was then subjected to silica gel column chromatography to give 2.84 g of a white solid, which was intermediate 3, with a yield of 94.2%. 1 H NMR(400MHz,Chloroform-d)δ7.45(d,J=8.9Hz,2H),7.32(d,J=8.9Hz,2H),3.69(s,3H),2.3 9-2.29(m,4H),1.74(p,J=7.3Hz,2H),1.65(p,J=7.4Hz,2H),1.53(s,9H),1.46-1.33(m,4H).

[0060] Step 2: Synthesis of Intermediate 4. Intermediate 3 (2.80 g, 7.40 mmol), TFA (19 mL), and DCM (19 mL) were added to a round-bottom flask. The mixture was stirred at room temperature for 12 hours, and the reaction was monitored for completion by TLC (dichloromethane:methanol = 20:1). The reaction solution was concentrated under reduced pressure, quenched with saturated sodium bicarbonate, extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 1.91 g of a pink solid, which was Intermediate 4, with a yield of 92.7%. 1 H NMR (300MHz, DMSO-d6) δ10.49-9.94(m,3H),7.70(d,J=8.9Hz,2H),7.30(d,J=8. 8Hz,2H),3.58(s,3H),2.37-2.24(m,4H),1.64-1.45(m,4H),1.35-1.23(m,4H).

[0061] Step 3: Synthesis of Intermediate 7

[0062] Add starting materials 5 (25.74 g, 110.60 mmol), 6 (20.00 g, 116.14 mmol), DIEA (73.00 mL, 442.40 mmol), and THF (300 mL) to a round-bottom flask. Reflux at 75 °C for 12 hours. Monitor the reaction for completeness by TLC (petroleum ether:ethyl acetate = 10:1). Concentrate the reaction solution under reduced pressure, add diethyl ether, stir, filter, and concentrate the filtrate under reduced pressure. Add trifluoroacetic acid (100 mL), stir at room temperature for 1 hour, then stir at 70 °C for 1 hour, concentrate under reduced pressure, add sodium hydroxide (6 M, 200 mL) and ethanol (100 mL), stir at room temperature for 15 minutes, monitor the reaction for completeness by TLC (dichloromethane:methanol = 20:1), adjust the pH to acidic with concentrated hydrochloric acid, and concentrate under reduced pressure. The solution was dissolved in a mixed solvent of DCM and MeOH, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 14.60 g of yellow solid, which was intermediate 7, with a yield of 59.4%. 1 H NMR (400MHz, DMSO-d6) δ8.18(s,1H),5.86(ddt,J=16.1,10.5,5.4Hz,1H),5.07-4.95(m,2H),4.32(d,J=5.4Hz,2H),2.36(s,3H).

[0063] Step 4: Synthesis of Intermediate 9

[0064] Starting material 8 (25.00 g, 115.72 mmol) and diethyl ether (200 mL) were added to a three-necked flask. The mixture was purged with nitrogen, and methylmagnesium iodide (3 M diethyl ether solution, 100 mL) was added using a syringe at 0 °C. The mixture was slowly brought to room temperature, and the reaction was monitored by TLC (petroleum ether:ethyl acetate = 10:1) to ensure complete reaction. The reaction was quenched with 1 M hydrochloric acid, extracted three times with EA, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 25.00 g of a yellow oily substance, with a yield of 100%. 1 H NMR (300MHz, Chloroform-d) δ7.57(t,J=7.7Hz,1H),7.41-7.38(m,1H),7.37(dd,J=4.2,0.9Hz,1H),3.98(s,1H),1.55(s,6H).

[0065] Step 5: Synthesis of Intermediate 10

[0066] Intermediates 7 (14.60 g, 65.69 mmol), 9 (14.19 g, 65.69 mmol), cuprous iodide (13.76 g, 72.25 mmol), potassium carbonate (13.98 g, 101.15 mmol), and 1,4-dioxane (120 mL) were added to a three-necked flask. The mixture was purged with argon, and trans-N,N'-dimethyl-1,2-cyclohexanediamine (10.28 g, 72.25 mmol) was added using a syringe. The reaction was carried out at 85 °C for 16 hours, and the reaction was monitored for completion by TLC (dichloromethane:methane = 20:1). The reaction solution was cooled to room temperature, quenched with ammonia, extracted three times with EA, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was then subjected to silica gel column chromatography to obtain 12.30 g of a white solid, which was intermediate 10, in 52.4% yield. 1 HNMR(300MHz,Chloroform-d)δ8.96(s,1H),7.93(t,J=7.9Hz,1H),7.78(d,J=8.1Hz,1H),7.42(d,J=6.8Hz,1H),5.71(ddt,J=16. 5,10.2,6.2Hz,1H),5.07(d,J=10.2Hz,1H),4.94(d,J=17.1Hz,1H),4.82(d,J=6.2Hz,2H),3.84(s,1H),2.60(s,3H),1.60(s,6H).

[0067] Step 6: Synthesis of Intermediate 11

[0068] Intermediate 10 (600 mg, 1.68 mmol), mCPBA (1.04 g, 6.04 mmol), and toluene (10 mL) were added to a round-bottom flask. The reaction was carried out at room temperature for 1 hour, and the reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:1) to confirm completion. Intermediate 4 (708 mg, 2.01 mmol) and DIEA (2.00 mL, 11.75 mmol) were added, and the reaction was carried out at room temperature for 2 hours, and the reaction was monitored by TLC (dichloromethane: methanol = 25:1) to confirm completion. The reaction solution was quenched with saturated sodium bicarbonate solution, extracted three times with EA, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was then subjected to silica gel column chromatography to obtain 366 mg of a yellow solid, which was intermediate 11, with a yield of 37.1%. 1H NMR(300MHz,Chloroform-d)δ8.85(s,1H),7.92(t,J=7.9Hz,1H),7.75(d,J=8.0 Hz,1H),7.61-7.51(m,4H),7.47(s,1H),7.38(d,J=7.6Hz,1H),5.72(ddt,J=16. 5,10.2,6.1Hz,1H),5.06(d,J=10.2Hz,1H),4.94(d,J=18.3Hz,1H),4.77(d,J=6 .2Hz,2H),3.69(s,3H),2.43-2.28(m,4H),1.83-1.62(m,4H),1.45-1.37(m,4H).

[0069] Step 7: Synthesis of Target Compound I-1. Hydroxylamine hydrochloride (710 mg, 10.21 mmol), methanol (6 mL), and potassium hydroxide (860 mg, 15.31 mmol) were added to a round-bottom flask. The mixture was reacted at 40 °C for 10 minutes, filtered, and intermediate 11 (300 mg, 0.51 mmol) was added to the filtrate. The mixture was reacted at room temperature for 1 hour, and the reaction was monitored for completeness by TLC (dichloromethane:methanol = 10:1). The pH of the reaction mixture was adjusted to neutral by adding 1 M hydrochloric acid. The mixture was extracted three times with DCM, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. PTLC yielded 62 mg of a yellow solid, which was the target compound I-1, with a yield of 20.6%. 1 H NMR (300MHz, DMSO-d6) δ10.44(s,1H),10.19(s,1H),9.96(s,1H),8.87(s,1H),7.99(t,J=7. 9Hz,1H),7.75(d,J=7.9Hz,1H),7.69-7.61(m,3H),7.56(d,J=8.8Hz,2H),5.67(ddt,J=16.4, 10.2,5.9Hz,1H),5.39(s,1H),5.00(d,J=10.2Hz,1H),4.82(d,J=17.1Hz,1H),4.68(d,J=5.9 Hz,2H),2.30(t,J=7.3Hz,2H),1.96(t,J=7.3Hz,2H),1.64-1.40(m,10H),1.36-1.21(m,4H).

[0070] Example 2: Preparation of 2-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)-N-hydroxyacetamide (I-2)

[0071]

[0072] Step 1: Synthesis of Intermediate 13

[0073] Following the method of Example 1, intermediate 13 (264 mg) was prepared from intermediates 10 (600 mg, 1.68 mmol) and 12 (277 mg, 1.68 mmol) as raw materials, with a yield of 33.1%. 1 H NMR(400MHz,Chloroform-d)δ8.86(s,1H),7.90(t,J=7.9Hz,1H),7.77(d,J=8.1Hz,1H),7.59(d,J=8.6Hz,2H),7.40(d,J=7.6Hz,1H),7.27(d,J=8.4Hz, 2H),5.71(ddt,J=16.6,10.2,6.2Hz,1H),5.04(d,J=10.1Hz,1H),4.93(d,J =17.1Hz,1H),4.77(d,J=6.2Hz,2H),3.71(s,3H),3.63(s,2H),1.60(s,6H).

[0074] Step 2: Synthesis of target compound I-2

[0075] Following the method of Example 1, compound I-2 (102 mg) was prepared from intermediate 13 (264 mg, 0.56 mmol) with a yield of 38.6%. 1 H NMR (400MHz, DMSO-d6+D2O) δ8.82(s,1H),8.00(t,J=7.9Hz,1H),7.67(d,J=8.0Hz,1H),7.62-7.53(m,3H),7.20(d,J=8.4Hz,2H),5. 61(ddt,J=16.3,10.1,5.7Hz,1H),4.98(d,J=11.2Hz,1H),4.77(d,J=17.0Hz,1H),4.64(d,J=5.8Hz,2H),3.25(s,2H),1.43(s,6H).

[0076] Example 3: Preparation of 3-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)-N-hydroxypropionamide (I-3)

[0077]

[0078] Step 1: Synthesis of Intermediate 15

[0079] Starting material 14 (2.70 g, 16.42 mmol), tetrabutylammonium nitrate (10.00 g, 32.84 mmol), and DCM (40 mL) were added to a round-bottom flask. Trifluoromethanesulfonic anhydride (4.63 g, 16.41 mmol) was slowly added at -30 °C, and the reaction was maintained at this temperature for 30 minutes. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 10:1) to ensure complete reaction. Sodium bicarbonate was added to the reaction solution, and the mixture was filtered. The filtrate was concentrated under reduced pressure and subjected to silica gel column chromatography to obtain 940 mg of a yellow solid, which was intermediate 15, with a yield of 27.4%. 1 H NMR (300MHz, Chloroform-d) δ8.17(d,J=8.7Hz,2H),7.39(d,J=8.7Hz,2H),3.69(s,3H),3.08(t,J=7.5Hz,2H),2.70(t,J=7.5Hz,2H).

[0080] Step 2: Synthesis of Intermediate 16

[0081] Intermediate 15 (940 mg, 4.49 mmol), Pd / C (100 mg), methanol (10 mL), and ethyl acetate (1 mL) were added to a round-bottom flask. The mixture was reacted at room temperature for 12 hours, and the reaction was monitored for completeness by TLC (petroleum ether:ethyl acetate = 10:1). The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure and subjected to silica gel column chromatography to obtain 572 mg of a yellow solid, which was intermediate 16, with a yield of 71.0%. 1 H NMR (300MHz, DMSO-d6) δ6.98(d,J=8.3Hz,2H),6.62(d,J=8.3Hz,2H),3.66(s,3H),3.58(s,2H),2.84(t,J=7.5Hz,2H),2.57(t,J=7.5Hz,2H).

[0082] Step 3: Synthesis of Intermediate 17

[0083] Following the method of Example 1, intermediate 17 (242 mg) was prepared from intermediates 10 (600 mg, 1.68 mmol) and 16 (333 mg, 2.01 mmol) as raw materials, with a yield of 29.5%. 1H NMR(400MHz,Chloroform-d)δ8.87(s,1H),7.90(d,J=11.7Hz,1H),7.77(d,J=1 2.0Hz,1H),7.55(d,J=4.3Hz,2H),7.39(s,1H),7.28(s,1H),7.21(d,J=12.3Hz ,2H),5.77-5.66(m,1H),5.07(d,J=10.3Hz,1H),4.96(d,J=17.0Hz,1H),4.78( s,2H),3.70(s,3H),2.97(t,J=9.5Hz,2H),2.66(t,J=9.7Hz,2H),1.61(s,6H).

[0084] Step 4: Synthesis of target compound I-3

[0085] Following the method of Example 1, the target compound I-3 (152 mg) was prepared from intermediate 17 (242 mg, mmol) with a yield of 62.8%. 1 H NMR(400MHz,DMSO-d6)δ10.39(s,1H),10.23(s,1H),8.88(s,1H),8.72(s,1H),8.05(t ,J=7.9Hz,1H),7.76(d,J=8.0Hz,1H),7.67-7.56(m,3H),7.16(d,J=8.1Hz,2H),5.67( ddt,J=16.4,10.2,5.9Hz,1H),5.35(s,1H),5.00(d,J=8.7Hz,1H),4.83(d,J=17.1Hz, 1H), 4.69 (d, J = 5.8Hz, 2H), 2.79 (t, J = 7.6Hz, 2H), 2.26 (t, J = 7.6Hz, 2H), 1.47 (s, 6H).

[0086] Example 4: Preparation of 2-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyacetamide (I-4)

[0087]

[0088] Step 1: Synthesis of Intermediate 20

[0089] Intermediates 18 (5.00 g, 23.90 mmol), 19 (3.66 g, 23.90 mmol), cesium carbonate (9.34 g, 28.68 mmol), and DMF (50 mL) were added to a round-bottom flask. The mixture was stirred at room temperature for 12 hours, and the reaction was monitored for completeness by TLC (dichloromethane:methanol = 35:1). 200 mL of water was added to the reaction mixture, and the mixture was filtered. The filter cake was dried under infrared spectroscopy to give 6.05 g of a white solid, which was intermediate 20, in 90.0% yield. 1 H NMR (400MHz, Chloroform-d) δ7.28 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 4.62 (s, 2H), 3.82 (s, 3H), 1.53 (s, 9H).

[0090] Step 2: Synthesis of intermediate 21

[0091] Add 20 (6.00 g, 21.33 mmol), trifluoroacetic acid (20 mL), and dichloromethane (20 mL) to a round-bottom flask. Stir at room temperature for 3 hours. Monitor the reaction for completeness by TLC (dichloromethane:methanol = 35:1). Concentrate the reaction solution under reduced pressure, add saturated sodium bicarbonate solution, extract three times with EA, combine the organic layers, wash with saturated sodium chloride solution, dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give 3.81 g of yellow liquid, which is intermediate 21, with a yield of 98.6%. 1 H NMR (400MHz, Chloroform-d) δ6.79 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 8.9 Hz, 2H), 4.58 (s, 2H), 3.82 (s, 3H), 3.23 (s, 2H).

[0092] Step 3: Synthesis of Intermediate 22

[0093] Following the method of Example 1, intermediate 22 (477 mg) was prepared from intermediates 10 (600 mg, 1.68 mmol) and 21 (365 mg, 2.01 mmol) as raw materials, with a yield of 57.9%. 1H NMR (300MHz, Chloroform-d) δ8.85 (s, 1H), 7.87 (t, J = 7.9 Hz, 1H), 7.74 (d, J = 7. 2Hz,1H),7.52(d,J=9.0Hz,2H),7.37(d,J=8.4Hz,1H),6.93(d,J=9.0Hz,2H),5 .71(ddt,J=16.5,10.1,6.1Hz,1H),5.05(d,J=10.2Hz,1H),4.95(d,J=17.0Hz, 1H), 4.76 (d, J = 6.5Hz, 2H), 4.67 (s, 2H), 4.04 (s, 1H), 3.84 (s, 3H), 1.60 (s, 6H).

[0094] Step 4: Synthesis of target compound I-4

[0095] Following the method of Example 1, the target compound I-4 (192 mg) was prepared from intermediate 22 (466 mg, 0.95 mmol) with a yield of 41.1%. 1 H NMR(400MHz,DMSO-d6)δ10.94(s,1H),10.24(s,1H),9.03(s,1H),8.86(s,1H) ,8.05(t,J=7.9Hz,1H),7.75(d,J=8.1Hz,1H),7.67-7.57(m,3H),6.96(d,J=8. 7Hz,2H),5.67(ddt,J=16.5,10.2,5.9Hz,1H),5.38(s,1H),4.99(d,J=10.3Hz ,1H),4.82(d,J=15.6Hz,1H),4.69(d,J=6.0Hz,2H),4.47(s,2H),1.47(s,6H).

[0096] Example 5: Preparation of 3-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxypropionamide (I-5)

[0097]

[0098] Step 1: Synthesis of intermediate 24

[0099] Starting materials 23 (8.05 g, 52.62 mmol), 19 (7.32 g, 52.62 mmol), KOH (7.38 g, 131.55 mmol), and water (73 mL) were added to a flask. The mixture was refluxed at 105 °C for 12 hours, and the reaction was monitored for completion by TLC (dichloromethane:methanol = 10:1). The reaction solution was cooled to room temperature, and the pH was adjusted to acidic with 6 M hydrochloric acid. The mixture was extracted three times with EA, and the organic layers were combined. The mixture was then extracted three times with saturated sodium bicarbonate solution, and the aqueous layers were combined. The pH was adjusted to acidic with 6 M hydrochloric acid, and the mixture was filtered. The filter cake was dried under vacuum to give 2.74 g of a white solid, which was intermediate 24, with a yield of 24.7%. 1 H NMR (300MHz, DMSO-d6) δ12.49(s,1H),8.21(d,J=9.2Hz,2H),7.16(d,J=9.2Hz,2H),4.32(t,J=5.9Hz,2H),2.76(t,J=6.0Hz,2H).

[0100] Step 2: Synthesis of Intermediate 25

[0101] Intermediate 24 (2.74 g, 12.98 mmol) and anhydrous methanol (30 mL) were added to a three-necked flask. Acetyl chloride (0.51 g, 6.49 mmol) was added using a syringe at -4 °C. The reaction was maintained at this temperature for 10 hours. The reaction was monitored by TLC (dichloromethane:methanol = 10:1) to ensure complete reaction. The mixture was filtered, and the filter cake was dried under vacuum to give 2.62 g of gray solid, which was intermediate 25, with a yield of 89.7%. 1 H NMR (300MHz, Chloroform-d) δ8.22(d,J=9.2Hz,2H),6.99(d,J=9.3Hz,2H),4.37(t,J=6.3Hz,2H),3.77(s,3H),2.88(t,J=6.3Hz,2H).

[0102] Step 3: Synthesis of Intermediate 26

[0103] Referring to Example 3, intermediate 26 (2.24 g) was prepared from intermediate 25 (2.6 g, 11.55 mmol) with a yield of 99.3%. 1 HNMR (300MHz, DMSO-d6) δ6.64(d,J=8.8Hz,2H),6.49(d,J=8.8Hz,2H),4.64(s,2H),4.04(t,J=6.0Hz,2H),3.63(s,3H),2.71(t,J=6.0Hz,2H).

[0104] Step 4: Synthesis of target compound I-5

[0105] Following the method of Example 1, the target compound I-5 was prepared using intermediates 26 and 10 as raw materials. 1 H NMR(400MHz,DMSO-d6)δ10.57(s,1H),10.20(s,1H),8.87(s,1H),8.85(s,1H),8.05(t ,J=8.1Hz,1H),7.75(d,J=8.0Hz,1H),7.68-7.58(m,3H),6.92(d,J=8.8Hz,2H),5.67( ddt,J=16.5,10.2,6.0Hz,1H),5.34(s,1H),5.00(d,J=10.3Hz,1H),4.83(d,J=18.6Hz ,1H),4.69(d,J=5.9Hz,2H),4.18(t,J=6.0Hz,2H),2.44(t,J=6.0Hz,2H),1.47(s,6H).

[0106] Example 6: Preparation of 4-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxybutyramide (I-6)

[0107]

[0108] Following the method of Example 4, the target compound 6 was prepared using the intermediate methyl 4-bromobutyrate as the starting material. 1 HNMR(300MHz,DMSO-d6)δ10.45(s,1H),10.25(s,1H),8.85(s,1H),8.75(s,1H),8.05(t,J=7 .9Hz,1H),7.75(d,J=8.0Hz,1H),7.68-7.57(m,3H),6.92(d,J=9.0Hz,2H),5.67(ddt,J=16. 4,10.2,5.9Hz,1H),5.36(s,1H),5.00(d,J=10.3Hz,1H),4.82(d,J=17.1Hz,1H),4.69(d,J= 6.0Hz, 2H), 3.95 (t, J = 6.3Hz, 2H), 2.14 (t, J = 7.4Hz, 2H), 1.93 (p, J = 6.7Hz, 2H), 1.46 (s, 6H).

[0109] Example 7: Preparation of 5-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxypentanamide (I-7)

[0110]

[0111] Following the method of Example 4, compound I-7 was prepared using intermediate methyl 5-bromopentanoate as the starting material. 1 H NMR(300MHz,DMSO-d6)δ10.41(s,1H),10.22(s,1H),8.85(s,1H),8.73(s,1H),8.06( t,J=7.9Hz,1H),7.75(d,J=8.0Hz,1H),7.68-7.55(m,3H),6.92(d,J=9.0Hz,2H),5.67 (ddt,J=16.1,10.2,5.9Hz,1H),5.36(s,1H),5.00(d,J=11.7Hz,1H),4.82(d,J=18.7 Hz,1H),4.69(d,J=6.0Hz,2H),3.94(d,J=6.0Hz,2H),1.76-1.59(m,4H),1.46(s,6H).

[0112] Example 8: Preparation of 6-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyhexanoamide (I-8)

[0113]

[0114] Following the method of Example 4, compound I-8 was prepared using methyl 6-bromohexanoate as the starting material. 1H NMR (300MHz, DMSO-d6) δ10.39(s,1H),10.22(s,1H),8.85(s,1H),8.71(s,1H),8.05(t,J=7.8Hz,1 H),7.74(d,J=8.2Hz,1H),7.66-7.56(m,3H),6.92(d,J=9.1Hz,2H),5.76-5.57(m,1H),5.36(s,1H) ,4.99(d,J=10.3Hz,1H),4.82(d,J=17.1Hz,1H),4.69(d,J=5.9Hz,2H),3.94(t,J=6.3Hz,2H),1.98 (t,J=7.2Hz,2H),1.69(p,J=6.9Hz,2H),1.55(p,J=7.0Hz,2H),1.46(s,6H),1.38(p,J=7.0Hz,2H).

[0115] Example 9: Preparation of 7-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyheptanamide (I-9)

[0116]

[0117] Following the method of Example 4, compound I-9 was prepared using methyl 7-bromoheptanoate as the starting material. 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),10.22(s,1H),8.85(s,1H),8.66(s,1H),8.05(t,J=7.7Hz,1 H),7.74(d,J=8.0Hz,1H),7.65-7.58(m,3H),6.92(d,J=9.1Hz,2H),5.67(ddt,J=16.2,10.2,6.0Hz ,1H),5.33(s,1H),5.00(d,J=10.3Hz,1H),4.83(d,J=18.6Hz,1H),4.69(d,J=6.0Hz,2H),3.94(t, J=6.4Hz,2H),1.96(p,J=6.7Hz,2H),1.70(p,J=6.7Hz,2H),1.58-1.37(m,10H),1.35-1.29(m,2H).

[0118] Example 10: Preparation of 8-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyoctamide (I-10)

[0119]

[0120] Following the method of Example 4, compound I-10 was prepared using ethyl 8-bromooctanoate as the starting material. 1 H NMR (400MHz, DMSO-d6) δ10.36(s,1H),10.23(s,1H),8.85(s,1H),8.68(s,1H),8.05(t,J=8.1 Hz,1H),7.75(d,J=7.7Hz,1H),7.67-7.55(m,3H),6.91(d,J=8.5Hz,2H),5.67(ddt,J=16.3,10 .2,5.9Hz,1H),5.34(s,1H),4.99(d,J=10.1Hz,1H),4.83(d,J=15.6Hz,1H),4.69(d,J=5.8Hz ,2H),3.94(t,J=6.4Hz,2H),1.96(t,J=7.3Hz,2H),1.70(p,J=6.7Hz,2H),1.58-1.22(m,14H).

[0121] Example 11: Preparation of 2-(4-(4-(2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyacetamide (I-11)

[0122]

[0123] Step 1: Synthesis of Intermediate 29

[0124] Starting materials 18 (4.43 g, 28.95 mmol), 28 (5 g, 24.13 mmol), DIEA (7.80 g, 60.32 mmol), and DMF (50 mL) were added to a flask and stirred overnight at room temperature. The reaction was monitored by TLC (dichloromethane:methanol = 15:1) to ensure complete reaction. 200 mL of water was added, and the mixture was filtered. The filter cake was dried under infrared radiation to give 6.04 g of yellow solid, which was intermediate 29, with a yield of 89.6%. 1HNMR(400MHz,Chloroform-d)δ8.14(d,J=9.4Hz,2H),6.84(d,J=9.4Hz,2H),3.76(s,3H),3.53-3.46(m,4H),3.32(s,2H),2.79-2.72(m,4H).

[0125] Step 2: Synthesis of Intermediate 30

[0126] Intermediate 29 (6.00 g, 24.07 mmol), Pd / C (300 mg), and methanol (50 mL) were added to a gai-shaped flask. The mixture was purged with hydrogen and reacted at room temperature for 12 hours. The reaction was monitored for completeness by TLC (dichloromethane:methanol = 15:1). The reaction solution was filtered through diatomaceous earth, concentrated under reduced pressure, and purified by silica gel column chromatography to give 5.24 g of a brown solid, which was intermediate 30, in 87.3% yield. 1 H NMR (300MHz, Chloroform-d) δ6.82(d,J=8.8Hz,2H),6.65(d,J=8.8Hz,2H),3.75(s,3H),3.29(s,2H),3.16-3.03(m,4H),2.79-2.70(m,4H).

[0127] Step 3: Synthesis of target compound I-11

[0128] Following the method of Example 1, the target compound I-11 was prepared using intermediates 30 and 10 as raw materials. 1 H NMR (400MHz, DMSO-d6) δ10.48(s,1H),10.12(s,1H),8.83(s,1H),8.81(s,1H),8.05(t,J= 7.9Hz,1H),7.75(d,J=8.1Hz,1H),7.66-7.49(m,3H),6.93(d,J=9.0Hz,2H),5.67(ddt,J= 16.4,10.2,6.0Hz,1H),5.34(s,1H),5.00(d,J=10.2Hz,1H),4.83(d,J=17.0Hz,1H),4.68 (d,J=6.0Hz,2H),3.12(t,J=4.9Hz,4H),2.95(s,2H),2.60(t,J=4.3Hz,4H),1.47(s,6H).

[0129] Example 12: Preparation of 3-(4-(4-((2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxypropionamide (I-12)

[0130]

[0131] Following the method of Example 11, methyl 3-bromopropionate was used as the starting material to prepare the target compound I-12. 1 H NMR (400MHz, DMSO-d6) δ10.43(s,1H),10.15(s,1H),8.83(s,1H),8.77(s,1H),8.05(t,J=8.2Hz, 1H),7.76(d,J=7.9Hz,1H),7.66-7.49(m,3H),6.92(d,J=8.7Hz,2H),5.67(ddt,J=16.4,10.2,6. 0Hz,1H),5.33(s,1H),4.99(d,J=10.2Hz,1H),4.83(d,J=17.1Hz,1H),4.69(d,J=6.0Hz,2H),3.0 9(t,J=5.0Hz,4H),2.58(t,J=7.0Hz,2H),2.54-2.47(m,4H),2.18(t,J=7.0Hz,2H),1.47(s,6H).

[0132] Example 13: Preparation of 4-(4-(4-(2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxybutyramide (I-13)

[0133]

[0134] Following the method of Example 11, methyl 4-bromobutyrate was used as the starting material to prepare the target compound I-13. 1H NMR (300MHz, DMSO-d6) δ10.28(s,2H),8.88(s,1H),8.06(t,J=7.8Hz,1H),7.76(d,J=8.0Hz,1H ),7.65-7.46(m,3H),6.92(d,J=8.6Hz,2H),5.67(ddt,J=16.4,10.1,5.9Hz,1H),5.35(s,1H), 4.99(d,J=11.7Hz,1H),4.83(d,J=16.6Hz,1H),4.69(d,J=6.0Hz,2H),3.19-2.99(m,4H),2.49 -2.43(m,4H),2.30(t,J=7.1Hz,2H),2.00(t,J=7.2Hz,2H),1.69(p,J=7.8Hz,2H),1.47(s,6H).

[0135] Example 14: Preparation of 5-(4-(4-(4-(2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxypentanamide (I-14)

[0136]

[0137] Following the method of Example 11, methyl 5-bromopentanoate was used as the starting material to prepare the target compound I-14. 1 H NMR (400MHz, DMSO-d6) δ10.37(s,1H),10.15(s,1H),8.83(s,1H),8.68(s,1H),8.05(t,J=6.3 Hz,1H),7.75(d,J=8.1Hz,1H),7.67-7.50(m,3H),6.93(d,J=8.7Hz,2H),5.67(ddt,J=16.5,1 0.2,5.9Hz,1H),5.00(d,J=8.7Hz,1H),4.83(d,J=18.7Hz,1H),4.68(d,J=6.0Hz,2H),3.18-3 .03(m,4H),2.58-2.52(m,4H),2.38-2.27(m,2H),1.98(t,J=7.0Hz,2H),1.60-1.39(m,10H).

[0138] Example 15: Preparation of 6-(4-(4-(2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyhexanoamide (I-15)

[0139]

[0140] Following the method of Example 11, methyl 6-bromohexanoate was used as the starting material to prepare the target compound I-15. 1 H NMR (300MHz, DMSO-d6) δ10.38(s,1H),10.17(s,1H),8.83(s,1H),8.71(s,1H),8.04(t,J=8.7Hz,1H),7 .76(d,J=7.8Hz,1H),7.67-7.46(m,3H),6.92(d,J=8.5Hz,2H),5.67(ddt,J=16.4,11.0,5.9Hz,1H),5.3 6(s,1H),4.99(d,J=10.1Hz,2H),4.82(d,J=17.1Hz,1H),4.69(d,J=6.0Hz,2H),3.57-3.26(m,4H),3.1 9-2.97(m,4H),2.31(t,J=8.4Hz,2H),1.96(t,J=7.0Hz,2H),1.64-1.37(m,10H),1.25(p,J=8.1Hz,2H).

[0141] Example 16: Preparation of 7-(4-(4-(2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyheptamide (I-16)

[0142]

[0143] Following the method of Example 11, methyl 7-bromoheptanoate was used as the starting material to prepare the target compound I-16. 1H NMR (300MHz, DMSO-d6) δ10.43(s,1H),10.19(s,1H),8.84(s,1H),8.05(t,J=7.9Hz,1H),7.76(d,J =7.9Hz,1H),7.64-7.51(m,3H),6.90(d,J=8.6Hz,2H),5.67(ddt,J=16.5,10.1,6.0Hz,1H),5.38(s ,1H),4.99(d,J=9.5Hz,1H),4.82(d,J=18.7Hz,1H),4.70(d,J=6.0Hz,2H),3.15-2.98(m,4H),2.56 -2.46(m,4H),2.30(t,J=6.6Hz,2H),1.96(t,J=7.3Hz,2H),1.56-1.35(m,10H),1.32-1.15(m,4H).

[0144] Example 17: Preparation of 8-(4-(4-(4-(2-allyl-1-(6-(2-hydroxypropyl-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (I-17)

[0145]

[0146] Following the method of Example 11, the target compound I-17 was prepared using methyl 8-bromooctanoate as the starting material. 1 H NMR (300MHz, DMSO-d6) δ10.43(s,1H),10.19(s,1H),8.83(s,1H),8.07(t,J=13.5Hz,1H),7.76(d,J =7.4Hz,1H),7.67-7.47(m,3H),6.90(d,J=8.5Hz,2H),5.67(ddt,J=16.4,10.2,5.9Hz,1H),5.38(s ,1H),4.99(d,J=10.1Hz,1H),4.82(d,J=18.0Hz,1H),4.70(d,J=5.7Hz,2H),3.13-2.98(m,4H),2.5 0-2.40(m,4H),2.27(t,J=7.3Hz,2H),1.95(t,J=7.3Hz,2H),1.56-1.34(m,10H),1.31-1.15(m,6H).

[0147] Example 18: Preparation of 8-(4-(2-allyl-1-(3-(2-hydroxypropyl-2-yl)phenyl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyoctamide (I-18)

[0148]

[0149] Step 1: Following the method of Example 4, intermediate 37 was prepared using intermediates 35 and 19 as starting materials. 1 H NMR (300MHz, DMSO-d6) δ6.67(d,J=8.9Hz,2H),6.58(d,J=8.9Hz,2H),4.04(q,J=7.1Hz,2H),3.80(t,J=6.4Hz,2H ),2.27(t,J=7.4Hz,2H),1.63(p,J=6.6Hz,1H),1.52(p,J=7.5Hz,1H),1.43-1.22(m,6H),1.17(t,J=7.1Hz,3H).

[0150] Step 2: Following the method of Example 1, intermediate 34 is prepared using intermediate 32 as the starting material. 1 H NMR(400MHz,Chloroform-d)δ8.92(s,1H),7.58(t,J=1.9Hz,1H),7.54(t,J=1.6Hz,1H),7.50(t,J=7.7Hz,1H),7.30(dd,J=2.2,1.3Hz,1 H),5.77-5.66(m,1H),5.14(dq,J=10.1,1.1Hz,1H),4.99(dq,J=17.0,1.3Hz,1H),4.46(dt,J=6.0,1.4Hz,2H),2.52(s,3H),1.64(s,6H).

[0151] Step 3: Following the method of Example 1, using intermediates 34 and 37 as raw materials, the target compound I-18 was prepared. 1HNMR(300MHz,DMSO-d6)δ10.37(s,1H),10.14(s,1H),8.83(s,1H),8.70(s,1H),7.7 4-7.42(m,5H),7.31(s,1H),6.85(d,J=8.9Hz,2H),5.68(ddt,J=16.1,10.7,5.6Hz, 1H),5.23(s,1H),5.08(d,J=11.9Hz,1H),4.91(d,J=17.1Hz,1H),4.23(s,2H),3.90 (t,J=6.4Hz,2H),1.95(t,J=7.3Hz,2H),1.67(p,J=6.9Hz,2H),1.56-1.17(m,14H).

[0152] Example 19: Preparation of 8-(4-(4-(2-allyl-1-(3-(2-hydroxypropyl-2-yl)phenyl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (I-19)

[0153]

[0154] Following the method of Example 11, intermediate 40 was prepared using intermediates 35 and 28 as raw materials. 1 H NMR(300MHz,Chloroform-d)δ6.83(d,J=8.8Hz,2H),6.66(d,J=8.7Hz,2H),4.14(q,J=7.1Hz,2H),3.46(s,2H),3.10(t,J =5.0Hz,4H),2.64(t,J=5.0Hz,4H),2.41(t,J=8.1Hz,2H),2.30(t,J=7.5Hz,1H),1.72-1.48(m,4H),1.41-1.21(m,12H).

[0155] Following the method of Example 1, the target compound I-19 was prepared using intermediates 34 and 40 as raw materials. 1H NMR(300MHz,DMSO-d6)δ10.35(s,1H),10.10(s,1H),8.81(s,1H),8.71(s,1H),7.68-7.42(m,5 H),7.35-7.25(m,1H),6.86(d,J=9.1Hz,2H),5.68(ddt,J=16.2,10.7,5.6Hz,1H),5.22(s,1H), 5.08(dd,J=10.3,1.5Hz,1H),4.91(dd,J=17.1,1.6Hz,1H),4.41-4.17(m,2H),3.06(t,J=5.0H z,4H),2.52(t,J=5.0Hz,4H),2.33(t,J=6.2Hz,2H),1.94(t,J=7.3Hz,2H),1.55-1.18(m,16H).

[0156] Example 20: Preparation of N-hydroxy-8-(4-(1-(6-(2-hydroxypropane-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)octamide (I-20)

[0157]

[0158] Step 1: Synthesis of Intermediate 42

[0159] Starting material 5 (10.00 g, 42.98 mmol) and ethanol (200 mL) were added to a round-bottom flask. Hydrazine hydrate (5.24 mL, 86.96 mmol) was added at 0 °C, and the reaction was maintained at this temperature for 1 hour. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 5:1) to ensure complete reaction. The reaction solution was concentrated under reduced pressure, and methyl tert-butyl ether was added and stirred. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give 5.19 g of a white solid, intermediate 42, with a yield of 52.9%. 1 H NMR (300MHz, DMSO-d6) δ9.03 (s, 1H), 8.50 (s, 1H), 4.80 (s, 2H), 4.28 (q, J = 7.1Hz, 2H), 2.53 (s, 3H), 1.30 (t, J = 7.1Hz, 3H).

[0160] Step 2: Synthesis of intermediate 43

[0161] Intermediate 42 (5.00 g, 21.92 mmol) and acetone (100 mL) were added to a round-bottom flask and reacted at 70 °C for 12 hours. The mixture was concentrated under reduced pressure, dissolved in methanol (100 mL), and then sodium cyanoborohydride (2.07 g, 32.89 mmol) and concentrated hydrochloric acid (2 mL) were added at 0 °C. The reaction was continued at room temperature for 12 hours, and the reaction was monitored by TLC (petroleum ether:ethyl acetate = 3:1) to ensure complete reaction. The reaction was quenched with saturated sodium bicarbonate solution, extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to give 4.15 g of a white solid, which was intermediate 43, in 70.0% yield. 1 H NMR(300MHz,DMSO-d6)δ9.20(d,J=6.2Hz,1H),8.53(s,1H),5.37-5.27(m,1H),4.29(q,J=7.1Hz ,2H),3.35(s,1H),3.28-3.15(m,1H),2.52(s,3H),1.30(t,J=7.1Hz,3H),1.02(d,J=6.2Hz,6H).

[0162] Step 3: Synthesis of intermediate 44

[0163] Intermediate 43 (4.10 g, 15.17 mmol), sodium hydroxide solution (5 M, 50 mL), and methanol (25 mL) were added to a round-bottom flask. The mixture was stirred at room temperature for 12 hours, and the reaction was monitored for completeness by TLC (dichloromethane:methanol = 3:1). The pH of the reaction solution was adjusted to acidic by adding concentrated hydrochloric acid, and the solution was concentrated under reduced pressure. The solution was dissolved in a mixed solvent of DCM and MeOH, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was then subjected to silica gel column chromatography to give 1.73 g of a white solid, which was intermediate 44, with a yield of 50.8%. 1 H NMR (300MHz, DMSO-d6) δ8.22(s,1H),4.57(hept,J=6.6Hz,1H),2.39(s,3H),1.22(d,J=6.7Hz,6H).

[0164] Step 4: Synthesis of target compound I-20

[0165] Following the method of Example 1, compound I-20 was prepared using intermediate 44 as a raw material. 1H NMR (300MHz, DMSO-d6) δ10.42(s,1H),10.18(s,1H),8.85-8.58(m,2H),8.07(t,J=6.9Hz,1H),7.75-7.57(m,4H),6.89(d,J=8.4 Hz,2H),5.37(s,1H),4.28-4.07(m,1H),3.91(t,J=6.6Hz,2H),1.96(t,J=6.9Hz,2H),1.67(p,J=6.9Hz,2H),1.56-1.07(m,22H).

[0166] Example 21: Preparation of N-hydroxy-8-(4-(1-(6-(2-hydroxypropane-2-yl)pyridin-2-yl)-2-isopropyl-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)octamide (I-21)

[0167]

[0168] Compound I-21 was prepared using intermediates 45 and 40 as raw materials, following the method of Example 1. 1 H NMR(300MHz,DMSO-d6)δ10.37(s,1H),10.14(s,1H),8.85-8.63(m,2H),8.08(t,J =7.8Hz,1H),7.72(d,J=8.0Hz,1H),7.65(d,J=7.7Hz,1H),7.56(d,J=8.1Hz,2H),6 .89(d,J=8.8Hz,2H),5.35(s,1H),4.22-4.07(m,1H),3.08(t,J=4.9Hz,4H),2.48( t,J=4.9Hz,4H),2.30(t,J=7.3Hz,2H),1.94(t,J=7.3Hz,2H),1.56-1.12(m,22H).

[0169] Example 22: Preparation of 8-(4-(2-allyl-1-(6-(2-methoxypropane-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyoctamide (I-22)

[0170]

[0171] Step 1: Synthesis of Intermediate 48

[0172] Intermediate 9 (5.50 g, 25.45 mmol), methyl iodide (10.84 g, 76.36 mmol), and anhydrous tetrahydrofuran (100 mL) were added to a pear-shaped flask. Sodium hydride (2.93 g, 76.36 mmol) was added, and the mixture was stirred at room temperature for 12 hours. The reaction was monitored for completeness by TLC (petroleum ether:ethyl acetate = 5:1). The reaction was quenched with water, extracted three times with EA, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 5.42 g of a yellow oil, which was intermediate 48, with a yield of 92.6%. 1 H NMR (400MHz, Chloroform-d) δ7.61-7.49 (m, 2H), 7.36 (dd, J = 6.8, 1.9Hz, 1H), 3.20 (s, 3H), 1.55 (s, 6H).

[0173] Step 2: Synthesis of target compound I-22

[0174] Following the method of Example 1, the target compound I-22 was prepared using intermediates 48 and 37 as raw materials. 1 H NMR (300MHz, DMSO-d6) δ10.38(s,1H),10.23(s,1H),8.86(s,1H),8.70(s,1H),8.08(t,J=7.9Hz,1H),7 .81(d,J=7.9Hz,1H),7.62(d,J=8.4Hz,2H),7.46(d,J=7.5Hz,1H),6.92(d,J=8.5Hz,2H),5.67(ddt,J=1 6.4,10.1,5.9Hz,1H),4.99(dd,J=10.3,1.5Hz,1H),4.82(dd,J=17.0,1.5Hz,1H),4.71(d,J=5.9Hz,2H ),3.93(t,J=6.4Hz,2H),3.09(s,3H),1.96(t,J=7.3Hz,2H),1.68(p,J=6.9Hz,2H),1.57-1.17(m,14H).

[0175] Example 23: Preparation of 8-(4-(4-(2-allyl-1-(6-(2-methoxypropane-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (I-23)

[0176]

[0177] Following the method of Example 1, the target compound I-23 was prepared using intermediates 49 and 40 as raw materials.1 H NMR (300MHz, DMSO-d6) δ10.38(s,1H),10.20(s,1H),8.84(s,1H),8.08(t,J=7.9Hz,1H),7.82(d,J=8 .0Hz,1H),7.59(d,J=8.4Hz,2H),7.46(d,J=7.6Hz,1H),6.92(d,J=8.7Hz,2H),5.67(ddt,J=16.4,10. 2,6.0Hz,1H),4.99(d,J=10.2Hz,1H),4.82(d,J=18.7Hz,1H),4.71(d,J=6.0Hz,2H),3.19-3.09(m,4 H),2.66-2.52(m,4H),2.33(t,J=7.4Hz,2H),1.95(t,J=7.3Hz,2H),1.60-1.36(m,10H),1.26(s,6H).

[0178] Example 24: Preparation of N-hydroxy-8-(4-(1-(6-(2-hydroxypropane-2-yl)pyridin-2-yl)-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)octamide (I-24)

[0179]

[0180] Step 1: Synthesis of intermediate 51

[0181] Starting material 5 (5.00 g, 21.49 mmol), phenylhydrazine (2.32 g, 21.49 mmol), triethylamine (8.70 g, 85.96 mmol), and tetrahydrofuran (100 mL) were added to a flask and stirred overnight at room temperature. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 3:1) until complete. The mixture was diluted with water, extracted three times with EA, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was then subjected to silica gel column chromatography to give 2.85 g of a white solid, which was intermediate 51, with a yield of 43.6%. 1 H NMR(400MHz,Chloroform-d)δ9.65(s,1H),8.70(s,1H),7.28-7.21(m,2H),6.96-6 .87(m,3H),6.35(s,1H),4.41(q,J=7.1Hz,2H),2.29(s,3H),1.43(t,J=7.1Hz,3H).

[0182] Step 2: Synthesis of target compound I-24

[0183] Compound I-24 was prepared using intermediate 51 as a raw material, following the method of Example 20. 1 H NMR (300MHz, DMSO-d6) δ10.44-10.29(m,2H),8.93(s,1H),8.69(s,1H),8.02(t,J=7.8 Hz,1H),7.88(s,1H),7.67(s,1H),7.45(d,J=7.8Hz,1H),7.35(d,J=4.3Hz,4H),7.22- 7.14(m,1H),6.95(d,J=8.5Hz,2H),5.08(s,1H),3.96(t,J=6.4Hz,2H),1.96(t,J=7.2 Hz,2H),1.71(p,J=7.2Hz,2H),1.51(p,J=7.3Hz,2H),1.44-1.20(m,6H),0.98(s,6H).

[0184] Example 25: Preparation of N-hydroxy-8-(4-(1-(6-(2-hydroxypropane-2-yl)pyridin-2-yl)-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)octamide (I-25)

[0185]

[0186] Following the method described in Example 1, the target compound I-25 was prepared using intermediates 53 and 40 as starting materials. LC / MS: m / z = 680.37 [M+H] + .

[0187] Example 26: Preparation of 8-(4-(2-allyl-3-oxo-1-(pyridin-2-yl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyoctamide (I-26)

[0188]

[0189] Following the method of Example 1, the target compound I-26 was prepared using intermediates 56 and 7 as raw materials. 1H NMR (400MHz, DMSO-d6) δ10.38(s,1H),10.24(s,1H),8.85(s,1H),8.70(s,1H),8.54(d,J=4.6Hz,1H ),8.09(s,1H),7.88(d,J=8.1Hz,1H),7.61(s,2H),7.43-7.35(m,1H),6.91(d,J=8.3Hz,2H),5.68(d dt,J=16.4,10.2,5.9Hz,1H),5.01(d,J=10.2Hz,1H),4.87(d,J=18.8Hz,1H),4.60(s,2H),3.93(t,J =6.5Hz,2H),1.95(t,J=7.3Hz,2H),1.69(p,J=6.7Hz,2H),1.50(p,J=7.3Hz,2H),1.43-1.20(m,6H).

[0190] Example 27: Preparation of 8-(4-(4-(2-allyl-3-oxo-1-(pyridin-2-yl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (I-27)

[0191]

[0192] Following the method of Example 1, the target compound I-27 was prepared using intermediates 57 and 40 as raw materials. 1 H NMR (400MHz, DMSO-d6) δ10.38(s,1H),10.20(s,1H),8.84(s,1H),8.54(d,J=6.8Hz,1H),8.12- 8.07(m,2H),7.89(d,J=8.1Hz,2H),7.67-7.55(m,2H),7.43-7.35(m,1H),6.92(d,J=6.1Hz,2H) ,5.75-5.61(m,1H),5.02(d,J=10.2Hz,2H),4.87(d,J=17.1Hz,1H),4.61(s,2H),3.11(s,4H),2 .53(s,4H),2.33(t,J=7.3Hz,2H),1.94(t,J=7.3Hz,2H),1.55-1.40(m,4H),1.31-1.20(m,6H).

[0193] Example 28: Preparation of 8-(4-(2-allyl-3-oxo-1-(6-(trifluoromethyl)pyridin-2-yl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyoctamide (I-28)

[0194]

[0195] Following the method described in Example 1, the target compound I-28 was prepared using intermediates 60 and 7 as starting materials. LC / MS: m / z = 586.24 [M+H] + .

[0196] Example 29: Preparation of 8-(4-(4-(2-allyl-3-oxo-1-(6-(trifluoromethyl)pyridin-2-yl)-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (I-29)

[0197]

[0198] Following the method described in Example 1, the target compound I-29 was prepared using intermediates 61 and 40 as starting materials. LC / MS: m / z = 654.30 [M+H] + .

[0199] Example 30: Preparation of 8-(4-(2-allyl-1-(6-methoxypyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyoctamide (I-30)

[0200]

[0201] Following the method of Example 1, compound I-30 was prepared using intermediates 64 and 7 as raw materials. 1H NMR (400MHz, DMSO-d6) δ10.36(s,1H),10.23(s,1H),8.85(s,1H),8.68(s,1H),7.96(t,J=8.0Hz,1H),7 .62(s,2H),7.44(d,J=6.8Hz,1H),6.90(d,J=8.7Hz,2H),6.80(d,J=8.1Hz,1H),5.70(ddt,J=16.4,10.2 ,5.9Hz,1H),5.04(d,J=11.9Hz,1H),4.93(d,J=18.7Hz,1H),4.66(d,J=5.7Hz,2H),3.93(t,J=6.5Hz,2 H),3.89(s,3H),1.95(t,J=7.3Hz,2H),1.69(p,J=6.7Hz,2H),1.50(p,J=7.2Hz,2H),1.42-1.20(m,8H).

[0202] Example 31: Preparation of 8-(4-(4-(2-allyl-1-(6-methoxypyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (I-31)

[0203]

[0204] Compound I-31 was prepared using an intermediate as a raw material, following the method described in Example 1. 1 H NMR(400MHz,DMSO-d6)δ10.36(s,1H),10.19(s,1H),8.83(s,1H),8.70(s,1H),7.98(s,1H),7.59(s,2H) ,7.45(d,J=7.7Hz,1H),6.91(d,J=8.8Hz,2H),6.80(d,J=8.1Hz,1H),5.69(ddt,J=16.4,10.3,5.9Hz,1H ),5.04(dd,J=10.3,1.5Hz,1H),4.93(dd,J=17.2,1.6Hz,1H),4.66(d,J=6.0Hz,2H),3.89(s,3H),3.09( s,4H),2.53(s,4H),2.31(t,J=7.5Hz,2H),1.94(t,J=7.3Hz,2H),1.54-1.38(m,4H),1.35-1.19(m,6H).

[0205] Example 32: Preparation of 8-(4-(2-allyl-1-(6-ethoxypyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyoctamide (I-32)

[0206]

[0207] Step 1: Synthesis of Intermediate 69

[0208] Anhydrous ethanol (5.8 g, 126.64 mmol) and tetrahydrofuran (50 mL) were added to a flask. Sodium hydride was added at 0 °C, and a tetrahydrofuran solution (50 mL) of starting material 68 (10 g, 42.21 mmol) was added dropwise. The reaction was allowed to proceed at room temperature for 12 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 10:1) until complete. The reaction was quenched with water, extracted three times with EA, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Silica gel column chromatography yielded 7.11 g of a transparent oil, which was intermediate 69, with a yield of 83.4%. 1 H NMR (300MHz, Chloroform-d) δ7.42(t,J=6.0Hz,1H),7.04(dd,J=7.5,0.7Hz,1H),6.67(dd,J=8.2,0.7Hz,1H),4.36(q,J=7.1Hz,2H),1.39(t,J=7.1Hz,3H).

[0209] Step 2: Synthesis of target compound I-32

[0210] Compound I-32 was prepared using intermediates 69 and 7 as raw materials, following the method of Example 30. 1H NMR (400MHz, DMSO-d6) δ10.35(s,1H),10.22(s,1H),8.84(s,1H),8.68(s,1H),7.96(t,J=8.0Hz,1H),7.63(d,J =8.5Hz,2H),7.41(d,J=7.7Hz,1H),6.90(d,J=8.7Hz,2H),6.78(d,J=8.1Hz,1H),5.69(ddt,J=16.4,10.2,5.8H z,1H),5.04(dd,J=10.3,1.5Hz,1H),4.92(dd,J=17.1,1.7Hz,1H),4.63(d,J=5.8Hz,2H),4.30(q,J=7.0Hz,2H) ,3.93(t,J=6.5Hz,2H),1.95(t,J=7.4Hz,2H),1.69(p,J=6.7Hz,2H),1.50(p,J=7.3Hz,2H),1.44-1.18(m,9H).

[0211] Example 33: Preparation of 8-(4-(4-(2-allyl-1-(6-ethoxypyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (I-33)

[0212]

[0213] Following the method of Example 1, compound I-33 was prepared using intermediates 70 and 40 as raw materials. 1 H NMR (300MHz, DMSO-d6) δ10.36(s,1H),10.18(s,1H),8.83(s,1H),8.69(s,1H),7.96(t,J=8.0Hz,1H),7.60(d,J =8.8Hz,2H),7.42(d,J=7.7Hz,1H),6.91(d,J=8.7Hz,2H),6.78(d,J=8.1Hz,1H),5.69(ddt,J=16.4,10.3,5.8H z,1H),5.04(dd,J=10.3,1.5Hz,1H),4.92(dd,J=17.2,1.6Hz,1H),4.63(d,J=5.9Hz,2H),4.31(q,J=7.0Hz,2H) ,3.10(t,J=4.9Hz,4H),2.55(t,J=5.1Hz,4H),2.35(t,J=7.3Hz,2H),1.94(t,J=7.3Hz,2H),1.57-1.15(m,9H).

[0214] Example 34: Preparation of 8-(4-(2-allyl-1-(6-cyclopropoxypyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenoxy)-N-hydroxyoctamide (I-34)

[0215]

[0216] Compound I-34 was prepared using intermediates 68 and 74 as raw materials, following the method of Example 32. 1 H NMR (400MHz, DMSO-d6) δ10.35(s,1H),10.24(s,1H),8.85(s,1H),8.68(s,1H),7.99(t,J=8.2Hz,1H),7.68-7. 46(m,3H),6.92(d,J=8.7Hz,2H),6.83(d,J=8.1Hz,1H),5.69(ddt,J=16.5,10.2,6.0Hz,1H),5.03(dd,J=10.3, 1.5Hz, 1H), 4.91 (dd, J=17.1, 1.5Hz, 1H), 4.74 (d, J=5.9Hz, 2H), 4.21 (tt, J=6.3, 3.0Hz, 1H), 3.94 (t, J=6.5Hz, 2H),1.95(t,J=7.3Hz,2H),1.70(p,J=6.7Hz,2H),1.50(p,J=7.4Hz,2H),1.44-1.19(m,6H),0.88-0.68(m,4H).

[0217] Example 35: Preparation of 8-(4-(4-(2-allyl-1-(6-cyclopropoxypyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (I-35)

[0218]

[0219] Following the method of Example 1, compound I-35 was prepared using intermediates 76 and 40 as raw materials. 1H NMR(400MHz,DMSO-d6)δ10.36(s,1H),10.20(s,1H),8.83(s,1H),8.69(s,1H),7.99(s,1H),7.67-7.44(m,3 H),6.92(d,J=8.7Hz,2H),6.83(d,J=8.1Hz,1H),5.69(ddt,J=16.5,10.2,6.0Hz,1H),5.03(dd,J=10.3,1.5H z,1H),4.91(dd,J=17.2,1.6Hz,1H),4.74(d,J=6.0Hz,2H),4.22(tt,J=6.3,3.1Hz,1H),3.11(s,4H),2.53(s ,4H),2.34(t,J=7.4Hz,2H),1.94(t,J=7.3Hz,2H),1.54-1.38(m,4H),1.36-1.12(m,6H),0.85-0.67(m,4H).

[0220] Example 36: Preparation of 8-(4-(4-(6-(2,6-dichlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidin[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (Compound II-1)

[0221]

[0222] Step 1: Synthesis of Intermediate 78

[0223] Starting material 5 (10 g, 42.98 mmol), methylamine hydrochloride (6.67 g, 98.85 mmol), DIEA (18.39 g, 141.83 mmol), and tetrahydrofuran (100 mL) were added to a pear-shaped flask. The mixture was reacted at 70 °C for 12 hours, and the reaction was monitored for completeness by TLC (petroleum ether:ethyl acetate = 5:1). The mixture was filtered, and the filtrate was concentrated under reduced pressure to give 9.77 g of a white solid, which was intermediate 78, in 100% yield. 1 HNMR (300MHz, Chloroform-d) δ8.63(s,1H),8.22(s,1H),4.33(q,J=7.0Hz,2H),3.10(d,J=4.4Hz,3H),2.57(s,3H),1.38(t,J=7.0Hz,3H).

[0224] Step 2: Synthesis of Intermediate 79

[0225] Intermediate 78 (9.77 g, 42.98 mmol), sodium hydroxide (2.06 g, 51.58 mmol), tetrahydrofuran (100 mL), and water (100 mL) were added to a round-bottom flask. The mixture was stirred at 50 °C for 12 hours, and the reaction was monitored by TLC (dichloromethane:methanol = 10:1) to ensure complete reaction. The pH was adjusted to acidic with concentrated hydrochloric acid, and the tetrahydrofuran was removed by concentration under reduced pressure. The mixture was filtered, and the filter cake was dried under vacuum to give 8.56 g of a white solid, which was intermediate 79, in 100% yield. 1 H NMR (300MHz, DMSO-d6) δ8.96 (s, 1H), 8.51 (s, 1H), 3.05 (d, J = 4.8Hz, 3H), 2.57 (s, 3H).

[0226] Step 3: Synthesis of Intermediate 80

[0227] Intermediate 79 (8.56 g, 42.98 mmol), DMF (5 d), and thionyl chloride (100 mL) were added to a round-bottom flask, and the mixture was reacted at 90 °C for 12 h. The reaction solution was concentrated under reduced pressure, stirred with toluene, filtered, and the filtrate was concentrated under reduced pressure and transferred to a three-necked flask. 2,6-Dichloroaniline (6.96 g, 42.98 mmol), pyridine (4.25 g, 53.73 mmol), and dichloromethane (100 mL) were added to the three-necked flask, and the mixture was stirred at room temperature for 12 h. The reaction was monitored for completeness by TLC (petroleum ether:ethyl acetate = 5:1). The reaction solution was concentrated under reduced pressure, and silica gel column chromatography was performed to give 6.67 g of a yellow solid, which was intermediate 80, in 45.2% yield. 1 H NMR (300MHz, DMSO-d6) δ10.33(s,1H),8.74(s,1H),8.62(s,1H),7.60(d,J=7.9Hz,2H),7.41(t,J=8.1Hz,1H),3.57(s,3H),2.95(d,J=3.6Hz,3H).

[0228] Step 4: Synthesis of intermediate 81

[0229] Intermediate 80 (6.67 g, 19.43 mmol), cesium carbonate (25.32 g, 77.71 mmol), dibromomethane (10.13 g, 58.28 mmol), and acetonitrile (100 mL) were added to a round-bottom flask. The mixture was reacted at 80 °C for 7 days, and the reaction was monitored for completion by TLC (petroleum ether:ethyl acetate = 5:1). The reaction solution was concentrated under reduced pressure, diluted with water, extracted three times with EA, and the organic layers were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Silica gel column chromatography yielded 2.31 g of a yellow solid, which was intermediate 81, with a yield of 33.4%. 1H NMR (300MHz, DMSO-d6) δ8.83(s,1H),7.47(s,1H),7.10(t,J=8.1Hz,1H),5.21(s,2H),3.10(s,3H),2.55(s,3H).

[0230] Step 5: Synthesis of target compound II-1

[0231] Following the method of Example 1, the target compound II-1 was prepared using intermediates 81 and 40 as raw materials. 1 H NMR (400MHz, DMSO-d6) δ10.36(s,1H),9.74(s,1H),8.45(s,1H),7.90(s,1H),7.70-7.57(m,4H),7.48(t,J=7.6Hz,1H),6.91(d,J=8.8Hz,2 H),4.95(s,2H),3.12(s,4H),3.09(s,3H),2.60(s,4H),2.41(t,J=7.6Hz,2H),1.94(t,J=7.3Hz,2H),1.54-1.41(m,4H),1.29-1.21(m,6H).

[0232] Example 37: Preparation of 8-(4-(6-(2,6-dichlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidine[4,5-d]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-2)

[0233]

[0234] Following the method of Example 1, the target compound II-2 was prepared using intermediates 81 and 37 as raw materials. 1 H NMR(300MHz,DMSO-d6)δ10.36(s,1H),9.77(s,1H),8.69(s,1H),8.46(s,1H),7.71-7.59(m,4H),7.54-7.43(m,1H),6.89(d,J =9.1Hz,2H),4.96(s,2H),3.93(t,J=6.5Hz,2H),3.09(s,3H),1.95(t,J=7.3Hz,2H),1.69(p,J=6.6Hz,2H),1.53-1.15(m,8H).

[0235] Example 38: Preparation of 8-(4-(6-(2-chlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidine[4,5-d]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-3)

[0236]

[0237] Compound II-3 was prepared using intermediate 79 and 2-chloroaniline as raw materials, following the method of Example 36. 1 H NMR (400MHz, DMSO-d6) δ10.39(s,1H),9.74(s,1H),8.69(s,1H),8.45(s,1H),7.66(d,J=8.5Hz,2H),7 .62(dd,J=7.2,2.1Hz,1H),7.52(dd,J=7.4,2.2Hz,1H),7.44(pd,J=7.3,1.7Hz,4H),6.89(d,J=9.0Hz ,2H),5.06(d,J=9.0Hz,1H),4.89(d,J=9.2Hz,1H),3.92(t,J=6.5Hz,2H),3.18(d,J=5.1Hz,3H),1.95 (t,J=7.3Hz,2H),1.69(p,J=6.7Hz,2H),1.50(p,J=7.3Hz,2H),1.44-1.36(m,2H),1.35-1.20(m,4H).

[0238] Example 39: Preparation of 8-(4-(4-(6-(2-chlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidin[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamido(II-4)

[0239]

[0240] Following the method of Example 37, the target compound II-4 was prepared using intermediates 85 and 40 as raw materials. 1 H NMR(300MHz,DMSO-d6)δ10.35(s,1H),9.68(s,1H),8.68(s,1H),8.44(s,1H) ,7.67-7.58(m,3H),7.55-7.38(m,3H),6.90(d,J=9.1Hz,2H),4.98(d,J=42.8 Hz,2H),3.07(s,3H),3.07(t,J=3.7Hz,4H),2.48(t,J=3.7Hz,1H),2.30(t,J =7.3Hz,2H),1.94(t,J=7.3Hz,2H),1.57-1.37(m,4H),1.25(d,J=8.4Hz,6H).

[0241] Example 40: Preparation of 8-(4-(6-(2-fluorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidine[4,5-d]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-5)

[0242]

[0243] Following the method of Example 36, the target compound II-5 was prepared using intermediate 5, 2-fluoroaniline and 37 as raw materials. 1 HNMR (300MHz, DMSO-d6) δ10.40(s,1H),9.75(s,1H),8.69(s,1H),8.46(s,1H),7.66(d,J=8.5Hz,2H),7.53-7.23(m,4H),6.89(d,J=9.0Hz,2 H),5.04(s,2H),3.93(t,J=6.4Hz,2H),3.09(s,3H),1.95(t,J=7.3Hz,2H),1.69(p,J=6.5Hz,2H),1.50(p,J=7.4Hz,2H),1.43-1.18(m,6H).

[0244] Example 41: Preparation of 8-(4-(6-(2-fluorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidine[4,5-d]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-6)

[0245]

[0246] Following the method of Example 36, the target compound II-6 was prepared using intermediate 5, 2-fluoroaniline, and 40 as raw materials. 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),9.69(s,1H),8.68(s,1H),8.45(s,1H),7.62(d,J=8.5Hz,2H),7.51-7.24(m,4H),6.90(d,J=8.7Hz,2H),5. 03(s,2H),3.15-2.99(m,J=7.3Hz,7H),2.48(t,J=5.0Hz,4H),2.30(t,J= 7.4Hz,2H),1.94(t,J=7.4Hz,2H),1.54-1.37(m,4H),1.36-1.25(m,6H).

[0247] Example 42: Preparation of N-hydroxy-8-(4-(8-methyl-5-oxo-6-(o-tolyl)-5,6,7,8-tetrahydropyrimidin[4,5-d]pyrimidin-2-yl)amino)phenoxy)octamide (II-7)

[0248]

[0249] Following the method described in Example 36, the target compound II-7 was prepared using intermediates 5, 2-methylaniline, and 37 as starting materials. LC / MS: m / z = 519.28 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ10.40(s,1H),9.70(s,1H),8.68(s,1H),8.44(s,1H),7.66(d ,J=8.5Hz,2H),7.41-7.21(m,4H),6.89(d,J=9.0Hz,2H),5.09(d,J=9.2Hz,1H),4.82( d,J=9.4Hz,1H),3.92(t,J=6.5Hz,2H),3.08(s,3H),2.19(s,3H),1.95(t,J=7.3Hz,2H ),1.69(p,J=6.7Hz,2H),1.50(p,J=7.3Hz,2H),1.44-1.36(m,2H),1.35-1.19(m,4H).

[0250] Example 43: Preparation of N-hydroxy-8-(4-((8-methyl-5-oxo-6-(o-tolyl)-5,6,7,8-tetrahydropyrimidin[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)octamide (II-8)

[0251]

[0252] Following the method of Example 36, the target compound II-8 was prepared using intermediate 5, 2-methylaniline and 40 as raw materials. 1H NMR(400MHz,DMSO-d6)δ10.34(s,1H),9.63(s,1H),8.68(s,1H),8.43(s,1H),7.6 2(d,J=8.3Hz,3H),7.37-7.23(m,4H),6.90(d,J=9.1Hz,2H),5.09(d,J=9.4Hz,1H ),4.81(d,J=9.3Hz,1H),3.13-2.99(m,7H),2.49(t,J=5.0Hz4H),2.30(t,J=7.4H z,2H),2.19(s,3H),1.94(t,J=7.3Hz,2H),1.54-1.39(m,4H),1.32-1.21(m,6H).

[0253] Example 44: Preparation of 8-(4-(6-(3-chlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidine[4,5-d]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-9)

[0254]

[0255] Following the method of Example 36, the target compound II-9 was prepared using intermediates 5, 3-chloroaniline, and 37 as raw materials. 1 HNMR(400MHz,DMSO-d6)δ10.36(s,1H),9.75(s,2H),8.68(s,1H),8.48(s,1H), 7.66(d,J=8.4Hz,2H),7.50(t,J=2.0Hz,1H),7.46(t,J=8.0Hz,1H),7.39-7.31( m,2H),6.89(d,J=9.1Hz,2H),5.12(s,2H),3.92(t,J=6.5Hz,2H),3.11(s,3H),1 .95(t,J=7.3Hz,2H),1.69(p,J=6.6Hz,2H),1.50(p,J=7.4Hz,2H),1.32(m,6H).

[0256] Example 45: Preparation of 8-(4-(4-(6-(3-chlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidin[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (II-10)

[0257]

[0258] Following the method of Example 36, the target compound II-10 was prepared using intermediates 5, 3-chloroaniline, and 40 as raw materials.1 H NMR(400MHz,DMSO-d6)δ10.34(s,1H),9.71(s,1H),8.66(s,1H),8.47(s,1H),7.62(d ,J=6.6Hz,2H),7.49(t,J=1.9Hz,1H),7.46(t,J=8.0Hz,1H),7.39-7.30(m,2H),6.90( d,J=9.0Hz,2H),5.12(s,2H),3.10(s,3H),3.08(t,J=4.8Hz,4H),2.49(t,J=4.8Hz,4H ),2.31(t,J=7.4Hz,2H),1.94(t,J=7.3Hz,2H),1.54-1.38(m,4H),1.34-1.17(m,6H).

[0259] Example 46: Preparation of 8-(4-(6-(4-chlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidin[4,5-d]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-11)

[0260]

[0261] Following the method of Example 36, the target compound II-11 was prepared using intermediates 5, 4-chloroaniline and 37 as raw materials. 1 H NMR(300MHz,DMSO-d6)δ10.40(s,1H),9.75(s,1H),8.68(s,1H),8.48(s,1H ),7.66(d,J=8.7Hz,2H),7.49(d,J=8.8Hz,2H),7.41(d,J=8.9Hz,2H),6.89( d,J=9.1Hz,2H),5.10(s,2H),3.92(t,J=6.5Hz,2H),3.10(s,3H),1.95(t,J= 7.3Hz,2H),1.69(p,J=7.4Hz,2H),1.50(p,J=7.4Hz,2H),1.41-1.18(m,6H).

[0262] Example 47: Preparation of 8-(4-(4-(6-(4-chlorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidin[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamidoamide (II-12)

[0263]

[0264] Following the method of Example 36, the target compound II-12 was prepared using intermediates 5, 4-chloroaniline and 40 as raw materials. 1 H NMR(300MHz,DMSO-d6)δ10.35(s,1H),9.69(s,1H),8.69(s,1H),8.46(s,1H),7. 61(d,J=8.0Hz,2H),7.49(d,J=8.7Hz,2H),7.40(d,J=8.7Hz,2H),6.90(d,J=8.9H z,2H),5.09(s,2H),3.10(s,3H),3.07(t,J=4.2Hz,4H),2.49(t,J=4.2Hz,4H),2. 30(t,J=7.3Hz,2H),1.94(t,J=7.2Hz,2H),1.59-1.37(m,4H),1.16-1.16(m,6H).

[0265] Example 48: Preparation of 8-(4-(6-(2-chloro-6-fluorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidine[4,5-d]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-13)

[0266]

[0267] Following the method of Example 36, the target compound II-13 was prepared using intermediate 5, 2-chloro-6-fluoroaniline and 37 as raw materials. 1 H NMR (400MHz, DMSO-d6) δ10.38(s,1H),9.79(s,1H),8.68(s,1H),8.47(s,1H),7. 66(d,J=8.5Hz,2H),7.55-7.47(m,2H),7.42(ddd,J=9.6,6.2,3.6Hz,1H),6.89(d ,J=9.0Hz,2H),4.97(q,J=9.6Hz,2H),3.93(t,J=6.5Hz,2H),3.09(s,3H),1.95( t,J=7.3Hz,2H),1.69(p,J=6.6Hz,2H),1.50(p,J=7.4Hz,2H),1.42-1.21(m,6H).

[0268] Example 49: Preparation of 8-(4-(4-(6-(2-chloro-6-fluorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidine[4,5-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (II-14)

[0269]

[0270] Following the method of Example 36, the target compound II-14 was prepared using intermediate 5, 2-chloro-6-fluoroaniline and 40 as raw materials. 1 H NMR(400MHz,DMSO-d6)δ10.35(s,1H),9.74(s,1H),8.68(s,1H),8.46(s,1H) ,7.62(d,J=7.8Hz,2H),7.54-7.49(m,2H),7.49-7.37(m,1H),6.90(d,J=9.0 Hz,2H),4.96(q,J=9.6Hz,2H),3.16-3.02(m,7H),2.51-2.45(m,4H),2.31(t ,J=7.4Hz,2H),1.94(t,J=7.3Hz,2H),1.56-1.39(m,4H),1.34-1.18(m,6H).

[0271] Example 50: Preparation of 8-(4-(6-(2,6-dichlorophenyl)-5-oxo-5,6,8,9-tetrahydroimidazo[1,2-a]pyrimidino[5,4-e]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-15)

[0272]

[0273] Step 1: Synthesis of Intermediate 88

[0274] Triphosgene (9.16 g, 30.87 mmol) and DCM (50 mL) were added to a flask. Intermediate 87 (5 g, 30.87 mmol) was slowly added, followed by dropwise addition of triethylamine (9.01 mL, 6.48 mmol) at -4 °C. After the addition was complete, the reaction was allowed to proceed at room temperature for 12 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 10:1) until complete. The mixture was concentrated under reduced pressure to obtain 5.80 g of a yellow solid, which was intermediate 88, with a yield of 99%. This intermediate was added directly to the next step without further purification.

[0275] Step 2: Synthesis of Intermediate 90

[0276] A solution of intermediate 88 (5.80 g, 30.86 mmol) in diethyl ether (90 mL) was added to a round-bottom flask. A solution of intermediate 89 (15.27 g, 95.28 mmol) in diethyl ether (90 mL) was added dropwise at 0 °C. The reaction was carried out at room temperature for 4 hours. The reaction was monitored by TLC (dichloromethane:methanol = 20:1) to ensure complete reaction. The mixture was filtered, and the filter cake was subjected to silica gel column chromatography to obtain 8.86 g of white solid, which was intermediate 90, with a yield of 82.4%. 1HNMR(300MHz,DMSO-d6)δ8.09(s,1H),7.48(d,J=8.1Hz,2H),7.26(dd,J=8.6,7.6Hz ,1H),6.80(t,J=5.3Hz,1H),6.38(t,J=5.7Hz,1H),3.21-2.96(m,4H),1.39(s,9H).

[0277] Step 3: Synthesis of Intermediate 91

[0278] Intermediate 90 (8.86 g, 25.36 mmol), DCM (100 mL), and a 1,4-dioxane solution of hydrogen chloride (4 M, 100 mL) were added to a round-bottom flask. The mixture was reacted at room temperature for 12 hours, and the reaction was monitored for completeness by TLC (dichloromethane:methanol = 20:1). The reaction solution was filtered, and the filter cake was dried under vacuum to give 7.22 g of a white solid, which was intermediate 91, in 100% yield. This was added directly to the next step without further purification. 1 H NMR (300MHz, DMSO-d6) δ8.43(s,1H),8.09(s,2H),7.49(d,J=8.1Hz,2H),7.27(dd,J= 8.6,7.6Hz,1H),6.85(d,J=5.1Hz,1H),3.31(q,J=6.5Hz,2H),2.86(t,J=6.7Hz,2H).

[0279] Step 4: Synthesis of Intermediate 92

[0280] Intermediate 91 (7.22 g, 25.36 mmol), 5 (8.85 g, 38.04 mmol), DIEA (13.11 g, 101.43 mmol), and acetonitrile (125 mL) were added to a flask. The mixture was purged with nitrogen and reacted at 80 °C for 3 hours. The reaction was monitored by TLC (dichloromethane:methanol = 20:1) to ensure complete reaction. The mixture was then filtered and dried under infrared light to give 9.77 g of a white solid, which was intermediate 92, with a yield of 86.7%. 1 H NMR (400MHz, DMSO-d6) δ8.55(s,1H),8.39(t,J=5.8Hz,1H),8.05(s,1H),7.47(d,J=8.1Hz,2H),7. 28-7.22(m,1H),6.48(t,J=5.8Hz,1H),4.27(q,J=7.1Hz,2H),2.48(s,3H),1.30(t,J=7.1Hz,3H).

[0281] Step 5: Synthesis of Intermediate 93

[0282] Intermediate 92 (9.77 g, 21.99 mmol) and phosphorus oxychloride (100 mL) were added to a round-bottom flask and reacted at 110 °C for 12 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:1) to ensure complete reaction. The reaction solution was concentrated under reduced pressure, and a saturated sodium bicarbonate solution was added at 0 °C. The mixture was filtered, and the filter cake was slurried with ethyl acetate, filtered again, and dried under infrared radiation to give 5.86 g of a white solid, which was intermediate 93, with a yield of 70.1%. 1 H NMR (400MHz, DMSO-d6) δ8.77(s,1H),7.70(d,J=8.0Hz,2H),7.57(dd,J=8.8,7.5Hz,1H),4.18(t,J=8.7Hz,2H),3.83(t,J=8.7Hz,2H),2.59(s,3H).

[0283] Step 6: Synthesis of target compound II-15

[0284] Following the method of Example 1, the target compound II-15 was prepared using intermediates 93 and 37 as raw materials. 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),10.19(s,1H),8.72-8.59(m,2H),7.79-7.48(m,5H),6.92(d,J=9.0Hz,2H),4.17(t,J=8.2Hz, 2H), 3.94 (t, J = 6.5Hz, 2H), 3.82 (t, J = 8.8Hz, 2H), 1.95 (t, J = 7.3Hz, 2H), 1.70 (p, J = 6.5Hz, 2H), 1.50 (p, J = 7.4Hz, 2H), 1.33 (m, 6H).

[0285] Example 51: Preparation of 8-(4-(4-(6-(2,6-dichlorophenyl)-5-oxo-5,6,8,9-tetrahydroimidazol[1,2-a]pyrimidino[5,4-e]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamide (II-16)

[0286]

[0287] Following the method of Example 50, the target compound II-16 was prepared using intermediates 40 and 93 as raw materials. 1H NMR(400MHz,DMSO-d6)δ10.34(s,1H),10.30(s,1H),8.65(s,1H),7.89(s,1H) ,7.74-7.62(m,3H),7.59-7.47(m,2H),6.92(d,J=8.6Hz,2H),4.17(t,J=7.0H z,2H),3.81(t,J=8.8Hz,2H),3.11(t,J=5.0Hz,4H),2.60-2.50(m,4H),2.33( t,J=7.4Hz,2H),1.94(t,J=7.3Hz,2H),1.54-1.39(m,4H),1.35-1.15(m,6H).

[0288] Example 52: Preparation of 8-(4-(4-(6-(o-tolyl)-8-methyl-5-oxo-5,6-dihydropyridin[4,3-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)-N-hydroxyoctamidoamide (II-17)

[0289]

[0290] Step 1: Synthesis of Intermediate 98

[0291] Starting materials 95 (95.00 g, 658.94 mmol) and 96 (94.21 g, 790.73 mmol) were added to a round-bottom flask and reacted at 80 °C for 10 min. Then, intermediate 97 (59.40 g, 658.94 mmol) was added, and the reaction was carried out at 110 °C for 15 h. The reaction was monitored by TLC until complete (petroleum ether:ethyl acetate = 10:1). The reaction solution was cooled to room temperature, diluted with ethyl acetate, washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Silica gel column chromatography yielded 93.04 g of a white solid, intermediate 98, in 62.4% yield. 1 H NMR (300MHz, Chloroform-d) δ8.94(s,1H),4.39(q,J=7.1Hz,2H),3.15(q,J=7.5Hz,2H),2.62(s,3H),1.41(t,J=7.1Hz,3H),1.31(t,J=7.5Hz,3H).

[0292] Step 2: Synthesis of Intermediate 99. Intermediate 98 (93.04 g, 411.15 mmol), lithium hydroxide (19.69 g, 822.31 mmol), tetrahydrofuran (600 mL), and water (600 mL) were added to a round-bottom flask. The mixture was reacted at room temperature for 3 hours, and the reaction was monitored for completion by TLC (dichloromethane:methanol = 10:1). The reaction solution was concentrated under reduced pressure, and the pH was adjusted to acidic with 1 M hydrochloric acid. The mixture was filtered, and the filter cake was dried under vacuum to give 44.86 g of a white solid, which was Intermediate 99, with a yield of 100%. 1 H NMR (300MHz, DMSO-d6) δ8.90 (s, 1H), 3.08 (q, J = 7.4Hz, 2H), 2.56 (s, 3H), 1.21 (t, J = 7.4Hz, 3H).

[0293] Step 3: Synthesis of Intermediate 100

[0294] Intermediate 99 (5.00 g, 25.22 mmol), o-methylaniline (2.70 g, 25.22 mmol), HATU (11.51 g, 30.27 mmol), DIEA (4.89 g, 37.83 mmol), and DMF (100 mL) were added to a round-bottom flask. The mixture was reacted at room temperature for 3 hours, and the reaction was monitored by TLC (petroleum ether:ethyl acetate = 3:1) to ensure complete reaction. 300 mL of water was added to the reaction mixture, and the mixture was filtered. The filter cake was dried under vacuum to give 6.63 g of a yellow solid, which was intermediate 100, with a yield of 91.5%. 1 H NMR(400MHz,Chloroform-d)δ8.68(s,1H),8.47(d,J=8.3Hz,1H),8.00(s,1H),7.45(d,J=9.5Hz,1H ),7.41-7.32(m,1H),7.20-7.11(m,1H),3.05(q,J=7.5Hz,2H),2.64(s,3H),1.37(t,J=7.5Hz,3H).

[0295] Step 4: Synthesis of Intermediate 101

[0296] Intermediate 100 (5.50 g, 19.41 mmol), (chloromethylene)dimethylammonium chloride (7.45 g, 58.23 mmol), and DMF (30 mL) were added to a gai-shaped flask. The mixture was purged with nitrogen and reacted at 50 °C. The reaction was monitored by TLC (dichloromethane:methanol = 35:1) to ensure complete reaction. The reaction was quenched with saturated sodium bicarbonate solution, followed by the addition of 100 mL of water. The mixture was filtered, and the filter cake was dried under vacuum and precipitated by silica gel column chromatography to obtain 4.00 g of white solid, which was intermediate 101, with a yield of 69.3%. 1H NMR (300MHz, Chloroform-d) δ9.42 (s, 1H), 7.67-7.56 (m, 1H), 7.53-7.37 (m, 2H), 7.23 (q, J = 1.2Hz, 1H), 2.70 (s, 3H), 2.34 (d, J = 1.2Hz, 3H).

[0297] Step 5: Synthesis of target compound II-17

[0298] Following the method of Example 1, the target compound II-17 was prepared using intermediates 101 and 40 as raw materials. 1 H NMR(400MHz,DMSO-d6)δ10.34(s,1H),10.10(s,1H),9.12(s,1H),8.68(s,1H ),7.92-7.67(m,2H),7.59(s,1H),7.45-7.27(m,4H),6.94(d,J=8.9Hz,2H),3 .09(t,J=4.4Hz,4H),2.49(t,J=3.6Hz,4H),2.30(t,J=7.4Hz,2H),2.21(s,3 H),2.10(s,3H),1.94(t,J=7.3Hz,2H),1.53-1.41(m,4H),1.31-1.21(m,6H).

[0299] Example 53: Preparation of 8-(4-(6-(o-tolyl)-8-methyl-5-oxo-5,6-dihydropyridine[4,3-d]pyrimidin-2-yl)amino)phenoxy)-N-hydroxyoctamide (II-18)

[0300]

[0301] Following the method of Example 1, the target compound II-18 was prepared using intermediates 101 and 37 as raw materials. 1 H NMR (400MHz, DMSO-d6) δ10.32(s,1H),10.15(s,1H),9.14(s,1H),8.67(s,1H),7.93-7.72(m,2H),7.60(s,1H),7.44-7.25(m,4H),6.93(d,J=9.1 Hz,2H),3.94(t,J=6.5Hz,2H),2.21(s,3H),2.10(s,3H),1.95(t,J=7.3 Hz,2H),1.70(p,J=6.6Hz,2H),1.50(p,J=7.4Hz,2H),1.45-1.21(m,2H).

[0302] Example 54: Preparation of N-(2-aminophenyl)-8-(4-(8-methyl-5-oxo-6-(o-tolyl)-5,6-dihydropyridine[4,3-d]pyrimidin-2-yl)amino)phenoxy)octamide (II-19)

[0303]

[0304] Step 1: Synthesis of intermediate 103

[0305] Following the method in step 2 of Example 52, intermediate 103 (80 mg) was prepared from intermediate 102 (100 mg, 0.19 mmol) with a yield of 84.6%. 1 H NMR (300MHz, DMSO-d6) δ10.15(s,1H),9.14(s,1H),7.83(d,J=7.2Hz,2H),7.60(s,1H),7.46-7.26(m,4H),6.93(d,J=9.1Hz, 2H),3.95(t,J=6.5Hz,2H),2.24-2.18(m,5H),2.10(s,3H),1.70(p,J=6.4Hz,2H),1.51(p,J=7.2Hz,2H),1.44-1.21(m,6H).

[0306] Step 2: Synthesis of target compound II-19

[0307] Following the method in step 3 of Example 52, the target compound II-19 (38 mg) was prepared from intermediate 103 (80 mg, 0.16 mmol) and o-phenylenediamine (15 mg, 0.16 mmol) with a yield of 40.2%. 1 H NMR(400MHz,Chloroform-d)δ9.34(s,1H),8.82(s,1H),7.69(d,J=8.0Hz,2H),7.46-7.14(m,7H),7.08-6.98(m,2H),6.91(d,J=9.0Hz,2H),3.94( t,J=6.6Hz,2H),2.43(t,J=7.7Hz,2H),2.28(s,3H),2.19(s,3H),1.76(p ,J=6.5Hz,2H),1.73-1.63(m,2H),1.50-1.40(m,2H),1.39-1.31(m,4H).

[0308] Example 55: Preparation of N-(2-amino-4-fluorophenyl)-8-(4-(8-methyl-5-oxo-6-(o-tolyl)-5,6-dihydropyridin[4,3-d]pyrimidin-2-yl)amino)phenoxy)octamide (II-20)

[0309]

[0310] Following the method in step 3 of Example 52, the target compound II-20 (92 mg) was prepared from intermediate 103 (100 mg, 0.20 mmol) and p-fluoro-o-phenylenediamine (26 mg, 0.20 mmol) with a yield of 75.6%. 1 H NMR(400MHz,Chloroform-d)δ9.35(s,1H),7.77-7.62(m,3H),7.41-7.30(m,3H),7.24(d,J=7.4Hz,1H),7.20(d,J=1.3Hz,1H),7.10-7.02(m,1H),6. 95(d,J=9.0Hz,2H),6.54-6.43(m,2H),4.00(t,J=6.4Hz,2H),2.42(t,J=7 .6Hz,2H),2.30(s,3H),2.21(s,3H),1.86-1.73(m,4H),1.54-1.42(m,6H).

[0311] Example 56: Preparation of N 1 -(4-(6-(2-chloro-6-fluorophenyl)-8-methyl-5-oxo-5,6,7,8-tetrahydropyrimidin[4,5-d]pyrimidin-2-yl)amino)phenyl)-N 8 1-Hydroxyoctadiamide (II-21)

[0312]

[0313] Following the method of Example 36, the target compound II-21 was prepared using intermediate 5, 2-chloro-6-fluoroaniline and 4 as raw materials. 1 H NMR (400MHz, DMSO-d6) δ10.35(s,1H),9.74(s,1H),9.17(s,1H),8.68(s,1H),8.46(s,1H),7.66-7.58(m,4H),7.38-7.27(m, 3H), 4.96 (q, J = 9.6Hz, 2H), 3.19 (s, 3H), 2.31 (t, J = 7.4Hz, 2H), 1.94 (t, J = 7.3Hz, 2H), 1.69-1.50 (m, 4H), 1.39-1.31 (m, 4H).

[0314] Example 57: Determination of the inhibitory activity of the compound on in vitro cell proliferation (H1299)

[0315] 1. Experimental materials

[0316] The H1299 non-small cell lung cancer cell line was purchased from Guangzhou Genio Biotechnology Co., Ltd., and cultured with RPMI-1640 and 10% fetal bovine serum (Gibcol) at 37°C, 5% CO2, and 95% humidity.

[0317] cell lines Cell types Cell count / well culture medium H1299 Floating 10000 RPMI-1640 + 10% FBS

[0318] 2. Experimental Procedure

[0319] Cells in the logarithmic growth phase were harvested, and cell viability was assessed using trypan blue staining. Cells were counted using a Thermo Countness II cell counter. Appropriate cell volumes were obtained, and cell concentrations were adjusted. 50 μL of cell suspension was added to each well of a 96-well plate, with 5000 cells per well, and the plates were cultured overnight. Test compound solutions were prepared by adding 50 μL of the test compound solution to each well, with a final concentration of 5 μM for each compound. Three replicates were set for each compound concentration, and the plates were incubated for another 72 hours. After 72 hours of incubation following drug administration, 10 μL of CCK-8 solution was added to each well. The 96-well plate was then placed in an incubator and incubated for another 2 hours. The absorbance was read at 450 nm, and the relative inhibition rate was calculated.

[0320] 3. Experimental Results

[0321] The antiproliferative effects of some compounds on the H1299 cell line are shown in Table 1 below.

[0322] Table 1. Inhibitory activity of some compounds of the present invention against H1299 cell proliferation

[0323]

[0324]

[0325] Inhibition rate: 50%–100% (denoted as A), 20%–50% (denoted as B), <20% (denoted as C).

[0326] Example 58: In vitro WEE1 kinase activity inhibition assay

[0327] All test compounds were dissolved in 100% DMSO to prepare a 10 mM stock solution. Store at -20°C protected from light. Prepare 1x kinase buffer: 1x kinase buffer, 50 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM DTT, 0.01% Tween-20, 0.01% BSA. Dilute the compounds with 100% DMSO to 100 times the final maximum inhibitor concentration required for the reaction. For 0.1 μM compounds, prepare a 10 μM DMSO solution in this step. For 0.01 μM compounds, prepare a 1 μM DMSO solution in this step. Add 100 μL of 100% DMSO to two empty wells in the same 96-well plate for a compound-free control and an enzyme-free control, labeling the plate as the source plate. Transfer 40 μL of the compound from the source plate to a new 384-well Echo plate as an intermediate plate. Transfer 200 nL from each well of the 384-well Echo plate to the 384-well assay plate. Prepare a WEE1 inhibitor solution in 1x kinase buffer at twice the final concentration of each reagent in the experiment. Add 10 μL of kinase solution to each well of the assay plate (replace with 10 μL of 1x kinase buffer) except for the control wells which do not contain the enzyme. Shake the plate. Incubate at room temperature for 10 min. Prepare substrate solutions of fluorescein-polyGT and ATP in 1x kinase reaction buffer at twice the final concentration of each reagent required in the experiment. Add 10 μL of substrate solution to each well of the assay plate to start the reaction. Shake the plate. Cover the assay plate and incubate at room temperature for a period of time. Prepare a detection solution at twice the final concentration in antibody dilution buffer. Add 20 μL of detection solution to each well of the assay plate to stop the reaction. Briefly mix with a centrifuge and incubate at room temperature for 60 min, then read the fluorescence on a microplate reader. Collect data at excitation at 340 nm, emission at 520 nm, and 495 nm on Envision.

[0328] Copy the RFU values ​​from the Envision program. Calculate the RFU 520nm / RFU 495nm ratio. Convert the ratio values ​​to inhibition percentage values. Inhibition percentage = (max - sample ratio) / (max - min) * 100. "min" refers to the ratio of the enzyme-free control, and "max" refers to the ratio of the DMSO control. Data are presented in MS Excel, and curves are fitted using the XLFit Excel add-in version 5.4.0.8. IC 50 Calculation formula: Y = Bottom + (Top - Bottom) / (1 + (IC) 50 / X)^HillSlope.

[0329] Example 59: In vitro HDAC1 enzyme activity inhibition assay

[0330] Prepare 1x assay buffer (modified Tris buffer). Serial dilution of compounds: Transfer compounds to the assay plate in 100% DMSO via Echo. The final DMSO fraction is 1%. Prepare enzyme solution in 1x assay buffer. Add trypsin and Ac-peptide substrate to 1x assay buffer to prepare substrate solution. Transfer 15 μL of enzyme solution to the assay plate, or for a low concentration control, transfer 15 μL of 1x assay buffer. Incubate at room temperature for 15 min. Add 10 μL of substrate solution to each well to start the reaction. Read the plate on Envision, showing excitation at 355 nm and emission at 460 nm. Curve fitting: Fit data in Excel and calculate inhibition rate: Inh% = (Max-Signal) / (Max-Min)*100; fit data in XL-Fit and calculate IC50. 50 Value: Y=Bottom+(Top-Bottom) / (1+(IC 50 / X)*HillSlope), where Y is the inhibition rate and X is the compound concentration. The in vitro enzyme activity inhibition results are shown in Table 2 below.

[0331] Table 2. Results of percentage inhibition tests of some compounds of the present invention against WEE1 kinase and HDAC1 enzyme.

[0332]

[0333]

[0334] As can be seen from the above activity results, most of the compounds designed in this invention have a strong inhibitory effect on WEE1 kinase and HDAC1 enzyme, and have considerable anti-tumor cell proliferation activity. They can be used to prepare drugs for treating diseases caused by overexpression of WEE1 kinase and / or HDAC enzyme.

Claims

1. An aniline-pyrimidine heterocyclic compound, characterized in that, It has the structure of Formula I and also contains its pharmaceutically acceptable salt: in: R1 is selected from substituted or unsubstituted C. 1-4 Alkyl, C 2-4 alkenyl, phenyl, wherein the substituents are selected from one or more halogens; R2 is selected from one or more hydrogen atoms; R3 is selected from hydroxyl groups; X is selected from Where R a Selected from hydrogen, C 1-4 Alkoxy, 3-7 membered cycloalkoxy, C 1-4 Hydroxyalkyl, C 1-4 Alkoxy-substituted C 1-4 Alkyl group, Q is selected from CH, N, R b Selected from unsubstituted C 1-4 alkyl; Y is selected from -(CH2) n O-, -(CH2) n CONH-、 Where n = 6, 7.

2. The aniline pyrimidine heterocyclic compound according to claim 1, characterized in that, In the structure: R1 is selected from substituted or unsubstituted C. 1-4 Alkyl, C 2-4 alkenyl, phenyl, wherein the substituents are selected from one or more halogens; R2 is selected from one or more hydrogen atoms; R3 is selected from hydroxyl groups; X is selected from Where R a Selected from hydrogen, C 1-4 Alkoxy, 3-5 membered cycloalkoxy, C 1-4 Hydroxyalkyl, C 1-4 Alkoxy-substituted C 1-4 Alkyl group, Q is selected from CH, N, R b Selected from C 1-4 alkyl; Y is selected from -(CH2) n O-, -(CH2) n CONH-、 Where n = 6, 7.

3. The aniline-pyrimidine heterocyclic compound according to claim 1, characterized in that, In the structure: R1 is selected from R2 is selected from one or more hydrogen atoms; R3 is selected from hydroxyl groups; X is selected from Y is selected from -(CH2) n O-, -(CH2) n CONH-、 Where n = 6, 7.

4. The aniline pyrimidine heterocyclic compound according to claim 1, characterized in that, In the structure: R1 is selected from R2 is selected from hydrogen; R3 is selected from hydroxyl groups; X is selected from Y is selected from -(CH2) n O-, -(CH2) n CONH-、 Where n = 6, 7.

5. An aniline pyrimidine heterocyclic compound, characterized in that, Selected from any of the following compounds:

6. The aniline pyrimidine heterocyclic compound according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from any of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

7. A method for preparing the aniline pyrimidine heterocyclic compound of claim 1, characterized in that, Choose from the following methods: Compound I was prepared by coupling and ammonolysis of the methylthio group: Wherein, R1, R2, X, and Y are defined as described in claim 1, R c It is methyl or ethyl; The corresponding acid is used to form a salt with compound I prepared by the above method to obtain a pharmaceutically acceptable salt of compound I.

8. A pharmaceutical composition, characterized in that, It comprises the aniline pyrimidine heterocyclic compound of claim 1 or 5 and a pharmaceutically acceptable carrier.

9. The use of an aniline pyrimidine heterocyclic compound of claim 1 or 5, or the pharmaceutical composition of claim 8, in the preparation of a dual-target inhibitor of WEE1 and HDAC.

10. The application according to claim 9, characterized in that, The drug in question is an anti-tumor drug.

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