An aminopyrimidine derivative and its application in antitumor therapy

Abstract

This invention discloses an aminopyrimidine derivative and its application in antitumor activity. Experiments show that the aminopyrimidine derivative of this invention can significantly inhibit PAX3 / 7-FOXO1 fusion gene-positive or MYCN-amplified rhabdomyosarcoma cells and neuroblastoma cells. Furthermore, the aminopyrimidine derivative of this invention effectively inhibits tumor growth in mice bearing PAX3 / 7-FOXO1-positive rhabdomyosarcoma cells, and exhibits strong tumor growth inhibition in mouse xenograft models of PAX3 / 7-FOXO1-positive rhabdomyosarcoma and MYCN-amplified neuroblastoma derived from clinically relapsed patients. It can be used to prepare drugs for treating rhabdomyosarcoma and neuroblastoma. The general structural formula I of the aminopyrimidine derivative is as follows:

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to an aminopyrimidine derivative and its application in anti-tumor treatment. It is a compound that inhibits the proliferative activity of PAX3 / 7-FOXO1 fusion gene-positive or MYCN-amplified tumor cells and its pharmaceutical uses. Background Technology

[0002] Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, accounting for more than 50% of all childhood soft tissue sarcomas. The PAX3 / 7-FOXO1 fusion gene is one of the most clinically significant molecular markers in rhabdomyosarcoma, present in approximately 60%–80% of patients, with PAX3-FOXO1 being the most common (approximately 55%–70%) and PAX7-FOXO1 accounting for approximately 10%–20%. The chimeric transcription factor encoded by this fusion gene continuously activates downstream target genes, driving tumorigenesis and progression. Positive PAX3 / 7-FOXO1 fusion gene positivity is typically associated with a high propensity for metastasis, poor response to chemotherapy, and low survival, especially in patients with positive PAX3-FOXO1, whose 5-year event-free survival rate is less than 30%.

[0003] Neuroblastoma (NB) is the most common extracranial solid tumor in children, accounting for approximately 8%–10% of childhood malignancies and about 15% of childhood cancer-related deaths. Among these, MYCN gene amplification, encoding the transcription factor N-MYC, is one of the most clinically significant biomarkers in neuroblastoma. MYCN amplification is present in approximately 20%–30% of neuroblastoma patients, and this proportion can be as high as approximately 40% in high-risk patients. MYCN amplification is typically closely associated with increased tumor aggressiveness, poor response to conventional treatment, and poor prognosis; even with intensive comprehensive therapy, the overall survival rate for these patients is less than 50%.

[0004] Therefore, the development of novel therapeutics targeting PAX3 / 7-FOXO1 fusion gene rhabdomyosarcoma and MYCN-amplified neuroblastoma has significant clinical value. However, the transcription factor proteins encoded by the PAX3 / 7-FOXO1 fusion gene and the MYCN gene have smooth surface structures and lack well-defined drug binding pockets, making it difficult to design direct-targeting inhibitors. This poses a significant obstacle to the development of targeted drugs. Currently, conventional clinical treatments for these fusion-positive rhabdomyosarcomas and MYC-amplified neuroblastomas are still limited to surgical resection, radiotherapy, and traditional chemotherapy regimens, resulting in poor clinical prognosis for patients. There is an urgent clinical need to develop innovative targeted therapeutic strategies. Summary of the Invention

[0005] The purpose of this invention is to provide an aminopyrimidine derivative, which is a compound having the structure shown in general formula I or a pharmaceutically acceptable salt or isotope-labeled compound thereof, as follows: General Formula I Wherein: R1 is a substituted acyl group, and the substituent is an alkenyl, alkynyl, halogen or cyano group; R2 is a substituted amino group, wherein the substituent is a C2-C3 alkyl, a C3-C5 cycloalkyl, an unsubstituted or substituted phenyl, or an aromatic heteroyl group; wherein the phenyl substituent is a cyano, sulfone, carbamoyl, sulfonamide, or phosphine oxide. R3 is methyl or isopropyl; R4 represents hydrogen or methyl.

[0006] Preferably, the compound provided by the present invention, wherein: R1 is , , , , , , ; R2 is , , , , , , , , , , .

[0007] The compounds provided by this invention have one of the following structures or are pharmaceutically acceptable salt or isotope-labeled compounds thereof: Another object of the present invention is to provide the use of the compound of general formula I in the preparation of antitumor drugs, wherein the tumors include neuroblastoma and rhabdomyosarcoma. The compound of the present invention can inhibit the proliferation of PAX3 / 7-FOXO1 fusion gene-positive or MYCN-amplified tumor cells.

[0008] This invention discloses an aminopyrimidine derivative that can significantly inhibit rhabdomyosarcoma cells and neuroblastoma cells that are positive for the PAX3 / 7-FOXO1 fusion gene or have MYCN amplification, and exhibits excellent antitumor activity in various mouse xenograft tumor models. Compared with the existing small molecule targeted inhibitor ceritinib, the aminopyrimidine derivative provided by this invention has a stronger inhibitory effect on the core oncogenic transcriptional program, and can exert superior in vitro and in vivo antitumor activity by regulating the MAPK / ERK and PI3K / AKT / mTOR signaling pathways and MYC-related signaling pathways. The aminopyrimidine derivative provided by this invention can also be used to prepare drugs against rhabdomyosarcoma and neuroblastoma. Attached Figure Description

[0009] Figure 1 Tumor-suppressive effect of compound AF-2 in RH30 xenograft tumor model.

[0010] Figure 2 The tumor-suppressive effect of compound AF-2 in a patient-derived xenograft model of rhabdomyosarcoma.

[0011] Figure 3 The tumor-suppressive effect of compound AF-2 in a neuroblastoma patient-derived xenograft tumor model.

[0012] Figure 4 RNA sequencing results of RH30 cells treated with compounds AF-2 and ceritinib. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the embodiments of the present invention are not limited thereto. Various substitutions and modifications can be made based on ordinary technical knowledge and conventional means in the art without departing from the above-described technical concept of the present invention, and all such substitutions and modifications should be included within the scope of the present invention.

[0014] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified can be purchased from reputable suppliers as standard products. The structures of the compounds were determined by nuclear magnetic resonance and high-resolution mass spectrometry. 1 H-NMR and 13 C-NMR was performed using a Bruker 500MHz NMR spectrometer with TMS as an internal standard. LC-HRMS was performed using an Agilent 1290-HPLC-6224 system. Column chromatography was performed using 200-300 mesh silica gel.

[0015] Example 1 .

[0016] 4-(3-methoxy-4-nitrophenyl)piperazine-1-carboxylic acid tert-butyl ester (1-2): Intermediate 1-1 (5.13 g, 30 mmol) and 1-Boc-piperazine (5.87 g, 31.5 mmol) were dissolved in 40 mL of N,N-dimethylformamide, and potassium carbonate (8.29 g, 60 mmol) was added. The system was heated to 75 °C and stirred overnight. Thin-layer chromatography was used to monitor the completeness of the reaction. The reaction solution was slowly added dropwise to 400 mL of vigorously stirred water, and stirred until the solid completely precipitated. The mixture was filtered. The filter cake was washed three times with 20 mL of water each time and dried at 55 °C to obtain a yellow solid intermediate 1-2 (9.30 g, 92% yield). 1 H NMR (500 MHz, CDCl3) δ 7.99 (d, J = 9.3 Hz, 1H), 6.40 (dd, J = 9.4, 2.5 Hz, 1H), 6.30 (d, J = 2.4 Hz, 1H), 3.94 (s, 3H), 3.64-3.54 (m, 4H), 3.44-3.32 (m, 4H), 1.48 (s, 9H); HRMS (ESI) m / z: theoretical value [M+H] + 338.1711, measured value 338.1709.

[0017] 4-(4-amino-3-methoxyphenyl)piperazine-1-carboxylic acid tert-butyl ester (1-3): Intermediate 1-2 (2.36 g, 7 mmol) was dissolved in 30 mL of methanol, and then 5% palladium on carbon (944 mg) was added. Hydrogen gas was introduced under ice-water bath conditions, and the mixture was allowed to naturally warm to room temperature. The reaction was stirred overnight, and the reaction was monitored for completeness by thin-layer chromatography. The reaction solution was filtered through a diatomaceous earth filter, and the filtrate was concentrated under reduced pressure. The residue was purified by dichloromethane / methanol (120:1 v / v) column chromatography to give a purple solid intermediate 1-3 (1.98 g, 92% yield). 1 H NMR (500 MHz, DMSO- d 6) δ 6.59 –6.44 (m, 2H), 6.30 (dd, J= 8.4, 2.2 Hz, 1H), 4.27 (s, 2H), 3.74 (s, 3H), 3.43 (s, 4H), 3.03 – 2.73 (m, 4H), 1.41 (s, 9H); HRMS (ESI) m / z: theoretical value [M+H] + 308.1969, measured value 308.1965.

[0018] 5-Chloro-N 4 -(2-Isopropysylphenyl)-N 2 -(2-methoxy-4-(piperazin-1-yl)phenyl)pyrimidine-2,4-diamine (1-4): In a sealed reaction tube, add 1,3,5-trichloro-N 4 -(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (1 equivalent) and intermediate 1-3 (1.05 equivalent) were dissolved in isopropanol (5 mL isopropanol per 1 mmol solute), followed by the addition of trifluoroacetic acid (1 mL trifluoroacetic acid per 1 mmol solute). The system was stirred overnight at 100 °C. After the reaction, the solvent was removed under reduced pressure, and the residue was dissolved in water. A 1 mol / L sodium hydroxide aqueous solution was slowly added dropwise to adjust the pH to 12. The mixture was extracted three times with ethyl acetate (20 mL per 1 mmol solute each time), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography using dichloromethane / methanol (20:1) as the eluent to give a yellow-green solid intermediate 1-4 (395 mg, 42% yield). 1 H NMR (500 MHz, CDCl3) δ 9.53 (s, 1H), 8.59(d, J = 8.4 Hz, 1H), 8.11 (s, 1H), 8.01 (d, J = 8.7 Hz, 1H), 7.90 (dd, J =8.0, 1.7 Hz, 1H), 7.61 (ddd, J = 8.7, 7.3, 1.7 Hz, 1H), 7.30 (s, 1H), 7.23(d, J = 7.3 Hz, 1H), 6.55 (d, J = 2.6 Hz, 1H), 6.47 (dd, J = 8.8, 2.6 Hz,1H), 3.87 (s, 3H), 3.23 (septet, J= 6.9 Hz, 1H), 3.14 – 3.08 (m, 4H), 3.06 –3.04 (m, 4H), 1.30 (d, J = 6.9 Hz, 6H); HRMS (ESI) m / z: theoretical value [M+H] + 517.1783, measured value 517.1783.

[0019] Compound AF-1: Intermediate 1-4 (1 equivalent) was dissolved in 7.5 mL of dichloromethane, and N,N-diisopropylethylamine (1.5 equivalent) was added with stirring. Chloroacetyl chloride (1.05 equivalent) dissolved in 7.5 mL of dichloromethane was slowly added dropwise under ice-water bath conditions. After the addition was complete, the mixture was allowed to warm to room temperature and stirred for 2 h. After the reaction was complete, dichloromethane was added to dilute the mixture, and it was washed three times with saturated ammonium chloride aqueous solution. The organic phase was separated, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography in a dichloromethane / methanol system to give a white solid, AF-1 (53 mg, yield 59%). 1 H NMR (500 MHz, CDCl3) δ10.57 (s, 1H), 10.47 (s, 1H), 8.32 (d, J = 8.2 Hz, 1H), 7.96 (s, 1H), 7.93 –7.86 (m, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 7.3 Hz, 1H), 6.63 – 6.56 (m, 1H), 6.56 – 6.46 (m, 1H), 4.14 (s, 2H), 3.86(s, 2H), 3.80 (s, 3H), 3.76 (s, 2H), 3.30 (s, 2H), 3.28 – 3.22 (m, 2H), 3.21 – 3.16 (m, 1H), 1.29 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 165.4, 157.0, 153.9, 153.7, 149.9, 143.2, 136.6, 134.7, 131.6, 126.5, 125.4, 125.3, 123.6, 119.2, 115.0, 108.4, 105.6, 101.2, 56.6, 55.9, 50.4, 50.1, 46.1, 42.0, 40.8, 15.4. HRMS (ESI) m / z: Theoretical value [M+H] + 593.1499, measured value 593.1494.

[0020] Example 2 Compound AF-2: .

[0021] At room temperature, cyanoacetic acid (1.1 equivalents) and HBTU (1.25 equivalents) were dissolved in dichloromethane (5 mL dichloromethane per 1 mmol solute). N,N-diisopropylethylamine (1.5 equivalents) was added with stirring, and stirring was continued for 15 min. Intermediates 1-4 (1 equivalent) were added in portions, and the reaction was carried out with stirring at room temperature for 2 h. After the reaction was complete, the mixture was diluted with dichloromethane and washed three times with saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography using dichloromethane / methanol as the eluent to give the target compound AF-2 (480 mg, 53% yield) as a yellow solid. 1 H NMR (500MHz, CDCl3) δ 9.46 (s, 1H), 8.49 (d, J = 8.2 Hz, 1H), 8.03 (s, 1H), 8.00 (d, J = 8.6 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.26 (s,1H), 7.19 – 7.14 (m, 1H), 6.44 (s, 1H), 6.37 (d, J = 8.3 Hz, 1H), 3.79 (s,3H), 3.72 (s, 2H), 3.55 (s, 2H), 3.46 (s, 2H), 3.25 – 2.97 (m, 5H), 1.21 (d, J = 6.7 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 162.0, 159.3, 155.8, 155.1, 149.3, 138.7, 135.3, 131.4, 125.8, 123.9, 123.5, 123.3, 120.9, 107.7, 101.2, 56.0, 55.4, 49.5, 49.2, 45.7, 42.1, 38.7, 25.3, 15.3. HRMS (ESI) m / z: Theoretical value [M+H] + 584.1842, measured value 584.1839.

[0022] Example 3 Compound AF-3: .

[0023] The reaction was carried out in accordance with Example 2, except that cyanoacetic acid was replaced with 2-fluoroacrylic acid, to give the target compound AF-3 (43 mg, yield 24%) as a white solid. 1 H NMR (500 MHz, CDCl3) δ 9.54 (s, 1H), 8.59 (d, J = 8.3 Hz, 1H), 8.13 (s, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.90 (dd, J = 7.9, 1.3Hz, 1H), 7.61 (dd, J = 11.4, 4.2 Hz, 1H), 7.34 (s, 1H), 7.26 (dd, J = 9.0, 6.2 Hz, 1H), 6.54 (d, J = 2.3 Hz, 1H), 6.47 (dd, J = 8.7, 2.3 Hz, 1H), 5.24(ddd, J = 32.2, 20.4, 3.5 Hz, 2H), 3.88 (s, 3H), 3.79 (s, 4H), 3.23 (dt, J =13.7, 6.8 Hz, 1H), 3.20 – 3.07 (m, 4H), 1.30 (d, J = 6.9 Hz, 6H). 13 C NMR (125MHz, CDCl3) δ 161.3, 161.1, 157.6 (d,J = 135 Hz), 156.5, 155.4, 155.4,149.5, 147.0, 138.6, 134.5, 131.3, 124.8, 123.8, 123.2, 123.0, 120.5, 108.8,105.9, 101.3, 100.1 (d, J = 16.2 Hz), 55.8, 55.6, 15.5. HRMS (ESI) m / z: Theoretical value [M+H] + 589.1795, measured value 589.1793.

[0024] Example 4 Compound AF-4: .

[0025] The reaction was carried out in accordance with Example 1, except that chloroacetyl chloride was replaced with acryloyl chloride, to give the target compound AF-4 (38 mg, yield 22%) as a white solid. 1 H NMR (500 MHz, CDCl3) δ 10.52 (d, J = 18.2 Hz, 2H), 8.31 (d, J = 8.2 Hz, 1H), 7.99 (s, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 6.66 (s,1H), 6.61 (dd, J = 16.9, 10.8 Hz, 1H), 6.56 (d, J = 8.6 Hz, 1H), 6.36 (d, J =16.8 Hz, 1H), 5.81 (d, J = 10.6 Hz, 1H), 3.96 (s, 2H), 3.84 (s, 2H), 3.80 (s, 3H), 3.29 (s, 4H), 3.19 (septet, J = 6.8 Hz, 1H), 1.30 (d, J = 6.8 Hz, 6H). 13CNMR (125 MHz, CDCl3) δ 165.9, 157.0, 153.8, 153.4, 149.0, 142.9, 136.4, 134.6, 131.6, 129.3, 126.8, 126.3, 125.4, 125.4, 123.56, 119.6, 108.6, 105.6, 101.3, 56.5, 55.8, 50.8, 50.4, 45.3, 41.6, 15.3. HRMS (ESI) m / z: Theoretical value [M+H] + 571.1889, measured value 571.1890.

[0026] Example 5 Compound AF-5: .

[0027] The reaction was carried out in accordance with Example 2, except that cyanoacetic acid was replaced with 2-butynedic acid, to obtain the target compound AF-5 (150 mg, yield 86%) as a yellow solid. 1 H NMR (500 MHz, DMSO-d6) δ 9.35 (s, 1H), 8.40 (s,1H), 8.12 (s, 1H), 7.99 (s, 1H), 7.61 (dd, J = 8.0, 1.5 Hz, 1H), 7.41 (t, J =7.5 Hz, 1H), 7.22 (d, J = 8.5 Hz, 1H), 7.18 – 7.07 (m, 1H), 6.51 (d, J = 2.5Hz, 1H), 6.32 (dd, J = 8.7, 2.5 Hz, 1H), 3.68 – 3.62 (m, 2H), 3.58 (s, 3H), 3.49 – 3.43 (m, 2H), 3.24 (septet, J = 6.8 Hz, 1H), 3.04 – 2.98 (m, 2H), 2.97– 2.92 (m, 2H), 1.87 (s, 3H), 0.97 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 164.4, 158.8, 155.3, 154.6, 152.6, 151.9, 148.8, 138.2, 134.8, 130.8, 125.2, 123.4, 122.8, 120.5, 107.5, 103.8, 100.9, 89.6, 72.9, 55.4, 54.9, 49.5, 49.0, 46.1, 40.8, 38.2, 14.8, 3.3. HRMS (ESI) m / z: Theoretical value [M+H] + 583.1889, measured value 583.1888.

[0028] Example 6 .

[0029] ( E )-2-Cyano-3-cyclopropylacrylic acid (1-5): Cyanoacetic acid (851 mg, 10 mmol) and cyclopropylformaldehyde (771 mg, 11 mmol) were dissolved in 10 mL of pyridine, and tetrahydropyrrole (171 mg, 2.4 mmol) was added. The mixture was stirred at room temperature for 4 h, and the reaction was monitored by TLC until complete. The solution was diluted with 30 mL of ethyl acetate and washed successively with 30 mL of 6 mol / L hydrochloric acid solution and 30 mL of water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. No purification was required to give a white solid intermediate 1-5 (1.02 g, yield 74%). 1 H NMR (500MHz, DMSO- d 6) δ 13.48 (s, 1H), 7.17 (d, J = 11.4 Hz, 1H), 1.96 – 1.89 (m, 1H), 1.30 – 1.25 (m, 2H), 1.08 – 1.04 (m, 2H); HRMS (ESI) m / z: theoretical value [M+H] + 138.0550, measured value 138.0549.

[0030] Compound AF-6: .

[0031] The reaction was carried out in accordance with Example 2, except that cyanoacetic acid was replaced with intermediates 1-5 to obtain the target compound AF-6 (42 mg, yield 39%) as a yellow solid. 1H NMR (500 MHz, CDCl3) δ 9.53 (s, 1H), 8.57 (d, J =8.4 Hz, 1H), 8.12 (s, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.93 – 7.86 (m, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.26 (t, J = 7.6 Hz, 1H), 6.65 (d, J = 11.2 Hz, 1H), 6.54 (d, J = 2.2 Hz, 1H), 6.46 (dd, J = 8.8, 2.2 Hz, 1H), 3.87 (s, 3H), 3.81 (s, 2H), 3.23 (septet, J = 6.9 Hz, 1H), 3.18 – 3.17 (m,2H), 2.79 (s, 8H), 2.12 – 2.05(m, 1H), 1.30 (d, J = 6.9 Hz, 6H). 13 C NMR (125MHz, CDCl3) δ 166.5, 162.3, 157.8, 155.43, 155.38, 149.5, 147.0, 138.6, 134.5, 131.3, 124.8, 123.8, 123.3, 123.0, 120.5, 115.7, 108.8, 107.4, 105.9, 101.3, 55.8, 55.7, 50.6, 38.7, 15.8, 15.5, 10.9. HRMS (ESI) m / z: Theoretical value [M+H] + 636.2155, measured value 636.2155.

[0032] Example 7 .

[0033] 2,5-Dichloro-N-cyclopropylpyrimidine-4-amine (2-1A): Under ice-water bath conditions, 2,4,5-trichloropyrimidine (1.82 g, 10 mmol) was dissolved in 40 mL of ethanol, and cyclopropylamine (856 mg, 15 mmol) was slowly added dropwise. After the addition was complete, the system was allowed to warm naturally from 0 °C to room temperature, and the reaction was stirred overnight. The reaction was monitored by TLC until complete. The solvent was removed by vacuum distillation, and the residue was mixed with 10 mL of petroleum ether and stirred. The solid was collected by suction filtration and dried at 45 °C to give a white solid intermediate 2-1A (1.34 g, 66% yield). 1 ¹H NMR (500 MHz, CDCl₃) δ 8.00 (s, 1H), 5.64 (s, 1H), 2.96 – 2.85 (m, 1H), 0.95 – 0.90 (m, 2H), 0.63 – 0.59 (m, 2H); HRMS (ESI) m / z: theoretical value [M+H] + 204.0090, measured value 204.0090.

[0034] 5-Chloro-N 4 -Cyclopropyl-N 2 -(2-methoxy-4-(piperazin-1-yl)phenyl)pyrimidine-2,4-diamine (2-1B): Referring to the reactions of intermediates 1-4, except that 1,3,5-trichloro-N 4 The 2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine was replaced with intermediate 2-1A to give a yellow-green solid, 187 mg, in 50% yield. 1 H NMR (500 MHz, CDCl3)δ 8.57 – 8.50 (m, 1H), 7.87 (s, 1H), 7.41 (s, 1H), 6.53 (dd, J = 5.3, 2.5 Hz, 2H), 5.41 (d, J = 2.5 Hz, 1H), 3.87 (s, 3H), 3.24 (s, 8H), 3.18 – 3.16 (m,1H), 2.86 (tq, J = 6.8, 3.7 Hz, 1H), 0.93 – 0.88 (m, 2H), 0.67 – 0.62 (m, 2H). HRMS (ESI) m / z: Theoretical value [M+H] + 375.1695, measured value 375.1694.

[0035] Compound AF-7: The reaction was carried out in accordance with Example 2, except that intermediates 1-4 were replaced with intermediates 2-1B, to obtain the brown oily target compound AF-7 (150 mg, yield 85%). 1 H NMR (500 MHz, CDCl3) δ 8.54 (d, J = 8.5 Hz,1H), 7.88 (s, 1H), 7.42 (s, 1H), 6.55 – 6.51 (m, 2H), 5.41 (s, 1H), 3.88 (s,3H), 3.80 (t, J = 5.2 Hz, 2H), 3.64 (t, J = 5.1 Hz, 2H), 3.53 (s, 2H), 3.18(t, J = 5.1 Hz, 2H), 3.12 (t, J = 5.2 Hz, 2H), 2.88 – 2.84 (m, 1H), 0.93 –0.88 (m, 2H), 0.67 – 0.63 (m, 2H). 13 C NMR (125 MHz, CDCl3) δ 160.1, 159.1, 158.2, 153.2, 148.8, 124.6, 119.2, 109.3, 104.4, 101.7, 55.9, 50.9, 50.8, 46.8, 42.8, 33.5, 25.0, 24.1, 7.4. HRMS (ESI) m / z: Theoretical value [M+H] + 442.1753, measured value 442.1753.

[0036] Example 8 .

[0037] 2,5-Dichloro-N-phenylpyrimidine-4-amine (2-2A): 2,4,5-Trichloropyrimidine (1.82 g, 10 mmol) and aniline (935 mg, 10 mmol) were dissolved in 20 mL of N,N-dimethylformamide, and potassium carbonate (4.14 g, 30 mmol) was added. The mixture was stirred overnight at 75 °C, and the reaction was monitored by TLC until complete. The reaction solution was added dropwise to 200 mL of water, stirred to induce crystallization, and then filtered. The filter cake was washed three times with 20 mL of water each time and dried at 55 °C to give an off-white solid intermediate 2-2A (2.09 g, yield 87%). 1 H NMR (500 MHz, DMSO-d 6) δ 9.52(s, 1H), 8.38 (s, 1H), 7.57 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.0 Hz, 2H), 7.19 (t, J = 7.0 Hz, 1H); HRMS (ESI) m / z: theoretical value [M+H] + 240.0090, measured value 240.0088.

[0038] 5-Chloro-N 2 -(2-methoxy-4-(piperazin-1-yl)phenyl)-N 4 -Phenylenidine-2,4-diamine (2-2B): Following the reaction of intermediate 2-1B, except that intermediate 2-1A was replaced with intermediate 2-2A, a yellow solid of 255 mg was obtained, with a yield of 42%. 1 H NMR (500 MHz, CDCl3) δ 8.08 (d, J = 9.0 Hz, 1H), 8.04 (s,1H), 7.61 (d, J = 7.5 Hz, 2H), 7.37 (t, J = 8.0 Hz, 2H), 7.30 (s, 1H), 7.16(t, J = 7.5 Hz, 1H), 7.03 (s, 1H), 6.53 (d, J = 2.5 Hz, 1H), 6.45 (dd, J =9.0, 2.5 Hz, 1H), 3.86 (s, 3H), 3.16 – 3.10 (m, 8H), 2.77 – 2.70 (m, 1H). HRMS (ESI) m / z: Theoretical value [M+H] + 411.1695, measured value 411.1692.

[0039] Compound AF-8: The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-2B, to obtain the target compound AF-8 (44 mg, yield 31%) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ 8.12 (d, J= 8.7 Hz,1H), 8.05 (s, 1H), 7.61 (s, 1H), 7.59 (s, 1H), 7.42 – 7.34 (m, 3H), 7.17 (t, J = 7.4 Hz, 1H), 7.05 (s, 1H), 6.52 (d, J = 2.5 Hz, 1H), 6.44 (dd, J = 8.8,2.4 Hz, 1H), 3.87 (s, 3H), 3.83 – 3.79 (m, 2H), 3.66 – 3.62 (m, 2H), 3.54 (s,2H), 3.20 – 3.16 (m, 2H), 3.15 – 3.11 (m, 2H). 13 C NMR (125 MHz, CDCl3) δ 160.0, 157.9, 155.8, 154.2, 149.2, 146.3, 137.9, 132.3, 130.9, 128.9, 124.4, 123.5, 121.9, 120.3, 113.8, 109.1, 104.7, 101.4, 55.7, 50.7, 50.5, 46.6, 42.6, 24.9. HRMS (ESI) m / z: Theoretical value [M+H] + 478.1753, measured value 478.1749.

[0040] Example 9 .

[0041] 2,5-Dichloro-N-cyclopentylpyrimidine-4-amine (2-3A): In an ice-water bath, 2,4,5-trichloropyrimidine (1.82 g, 10 mmol) was dissolved in 40 mL of tetrahydrofuran, and cyclopentanamine (856 mg, 15 mmol) was slowly added dropwise. The system was heated from 0 °C to room temperature and stirred overnight. The reaction was monitored by TLC until complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was added to 20 mL of petroleum ether and stirred, filtered, and dried at 45 °C to give intermediate 2-3A (2.05 g, 89% yield), which was used directly in the next reaction. HRMS (ESI) m / z: theoretical value [M+H] + 232.0403, measured value 232.0411.

[0042] 5-Chloro-N 4 -cyclopentyl-N 2-(2-methoxy-4-(piperazin-1-yl)phenyl)pyrimidine-2,4-diamine (2-3B): Following the reaction of intermediate 2-1B, but replacing intermediate 2-1A with intermediate 2-3A, a yellow solid of 242 mg was obtained, with a yield of 60%. 1 H NMR (500 MHz, CDCl3) δ 8.31 (d, J = 9.4 Hz, 1H), 7.85 (s,1H), 7.29 (s, 1H), 6.53 (dq, J = 4.1, 2.5 Hz, 2H), 5.14 (d, J = 6.8 Hz, 1H), 4.40 (h, J = 6.8 Hz, 1H), 3.87 (s, 3H), 3.18 (q, J = 5.8 Hz, 9H), 2.12 (dq, J = 12.4, 7.2, 6.7 Hz, 2H), 1.81 – 1.73 (m, 2H), 1.71 – 1.65 (m, 2H), 1.53 (dq, J = 14.4, 7.7, 7.1 Hz, 2H). HRMS (ESI) m / z: Theoretical value [M+H] + 403.2008, measured value 403.2009.

[0043] Compound AF-9 The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-3B, to obtain the target compound AF-9 (44 mg, yield 31%) as a yellow solid. 1 H NMR (500 MHz, DMSO-d6) δ 7.93 (d, J = 8.8 Hz,1H), 7.85 (s, 1H), 7.44 (s, 1H), 6.75 (d, J = 7.2 Hz, 1H), 6.66 (d, J = 2.6Hz, 1H), 6.48 (dd, J = 8.9, 2.6 Hz, 1H), 4.31 (h, J = 7.2 Hz, 1H), 4.10 (s,2H), 3.83 (s, 3H), 3.61 (t, J= 5.2 Hz, 2H), 3.49 (t, J = 5.1 Hz, 2H), 3.13(t, J = 5.1 Hz, 2H), 3.07 (t, J = 5.3 Hz, 2H), 1.96 – 1.89 (m, 2H), 1.71 –1.66 (m, 2H), 1.61 – 1.50 (m, 4H). 13 C NMR (125 MHz, DMSO-d6) δ 162.0, 158.6, 157.7, 153.4, 150.2, 147.3, 122.3, 121.4, 116.6, 107.9, 103.8, 101.3, 56.2, 52.6, 49.8, 49.5, 45.8, 42.1, 38.7, 32.3, 25.2, 24.0. HRMS (ESI) m / z: Theoretical value [M+H] + 470.2066, measured value 470.2070.

[0044] Example 10 .

[0045] 2,5-Dichloro-N-(2-methoxyethyl)pyrimidin-4-amine (2-4A): Under ice-water bath conditions, 2,4,5-trichloropyrimidine (1.82 g, 10 mmol) was dissolved in 7 mL of methanol, and 8 mol / L 2-methoxyethylamine ethanol solution (1.935 mL, 22 mmol) was slowly added dropwise. The mixture was allowed to warm naturally to room temperature and stirred overnight. The reaction was monitored by TLC until complete. The solvent was removed under reduced pressure, and the residue was slurried with 20 mL of petroleum ether. After filtration, the residue was dried at 45 °C to give a white solid intermediate 2-4A (1.62 g, 73% yield). 1 H NMR (500 MHz, DMSO- d 6) δ 8.13 (s, 1H), 7.86 (t, J = 5.6 Hz, 1H), 3.53 (q, J = 6.0, 5.5 Hz, 2H), 3.47 (t, J = 5.4 Hz, 2H), 3.25 (s, 3H); HRMS (ESI) m / z: theoretical value [M+H] + 222.0196, measured value 222.0198.

[0046] 5-Chloro-N 2 -(2-methoxy-4-(piperazin-1-yl)phenyl)-N 4 -(2-methylthioethyl)pyrimidine-2,4-diamine (2-4B): Following the reaction of intermediate 2-1B, but replacing intermediate 2-1A with intermediate 2-4A, a dark purple solid of 171 mg was obtained, with a yield of 44%. 1 H NMR (500 MHz, CDCl3) δ 8.19 (d, J = 8.5 Hz, 1H), 7.88 (s,1H), 7.24 (s, 1H), 6.53 (d, J = 7.5 Hz, 2H), 5.56 (s, 1H), 3.86 (s, 3H), 3.70(dd, J = 10.5, 5.0 Hz, 2H), 3.60 (t, J = 5.0 Hz, 2H), 3.40 (s, 3H), 3.09 –3.03 (m, 8H). HRMS (ESI) m / z: Theoretical value [M+H] + 393.1801, measured value 393.1805.

[0047] Compound AF-10: The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-4B, to obtain the target compound AF-10 (43 mg, yield 37%), which was an off-white solid. 1 H NMR (500 MHz, DMSO-d6) δ 7.98 – 7.79 (m,2H), 7.50 (s, 1H), 7.04 (t, J = 5.5 Hz, 1H), 6.66 (d, J = 2.5 Hz, 1H), 6.47(dd, J = 8.8, 2.6 Hz, 1H), 4.10 (s, 2H), 3.82 (s, 3H), 3.61 (t, J = 5.2 Hz, 2H), 3.55 – 3.45 (m, 6H), 3.25 (s, 3H), 3.13 (t, J = 5.2 Hz, 2H), 3.08 (t, J = 5.2 Hz, 2H). 13C NMR (125 MHz, DMSO-d6) δ 161.5, 158.2, 157.5, 153.0, 150.0, 146.9, 121.8, 121.2, 116.2, 107.4, 103.3, 100.8, 70.0, 57.9, 55.7, 54.9, 49.3, 49.0, 45.3, 41.6, 24.8. HRMS (ESI) m / z: Theoretical value [M+H] + 460.1859, measured value 460.1851.

[0048] Example 11 .

[0049] 2,5-Dichloro-N-(pyridin-2-yl)pyrimidin-4-amine (2-5A): At -5°C, 60% sodium hydride (1.0 g, 25 mmol) was dispersed in 10 mL of anhydrous tetrahydrofuran, and 5 mL of anhydrous tetrahydrofuran solution containing 2-aminopyridine (941 mg, 10 mmol) was added dropwise. The mixture was stirred at this temperature for 30 min. Then, 5 mL of anhydrous tetrahydrofuran solution containing 2,4,5-trichloropyrimidine (1.82 g, 10 mmol) was added dropwise, and the mixture was heated to room temperature and stirred for 1.5 h. The reaction was monitored by TLC until complete. The reaction mixture was poured into 200 mL of water, extracted with dichloromethane, and the organic phase was concentrated under reduced pressure. The residue was purified by column chromatography with petroleum ether / ethyl acetate (20:1 v / v) to give a white solid intermediate 2-5A (252 mg, 11% yield). 1 H NMR (500 MHz, CDCl3) δ 8.41 (dt, J = 8.4, 1.0 Hz, 1H), 8.34(ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.27 (s, 1H), 8.01 (s, 1H), 7.80 (ddd, J =8.4, 7.3, 1.9 Hz, 1H), 7.09 (ddd, J = 7.3, 4.9, 1.0 Hz, 1H); HRMS (ESI) m / z: theoretical value [M+H] + 241.0043, measured value 241.0042.

[0050] 5-Chloro-N 2 -(2-methoxy-4-(piperazin-1-yl)phenyl)-N4 -(pyridin-2-yl)pyrimidin-2,4-diamine (2-5B): Following the reaction of intermediate 2-1B, but replacing intermediate 2-1A with intermediate 2-5A, a dark purple solid of 164 mg was obtained, with a yield of 40%. 1 H NMR (500 MHz, CDCl3) δ 8.36 (dt, J = 8.4, 1.1 Hz, 1H), 8.31(ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.11 (s, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.80(s, 1H), 7.67 (ddd, J = 8.7, 7.3, 1.9 Hz, 1H), 7.23 (s, 1H), 7.01 (ddd, J =7.3, 4.9, 1.0 Hz, 1H), 6.57 – 6.52 (m, 2H), 3.88 (s, 3H), 3.24 – 3.21 (m, 4H), 3.20 – 3.17 (m, 4H). HRMS (ESI) m / z: Theoretical value [M+H] + 412.1647, measured value 412.1648.

[0051] Compound AF-11: The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-5B, to obtain the target compound AF-11 (34 mg, yield 36%) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ 8.34 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 4.1 Hz, 1H), 8.09 (s, 1H), 8.06 (d, J = 8.7 Hz, 1H), 7.80(s, 1H), 7.71 – 7.62 (m, 1H), 7.28 (s, 1H), 7.01 (dd, J = 7.1, 5.0 Hz, 1H), 6.54 (d, J = 2.1 Hz, 1H), 6.51 (d, J= 8.7 Hz, 1H), 3.87 (s, 3H), 3.83 – 3.77(m, 2H), 3.66 – 3.61 (m, 2H), 3.57 (s, 2H), 3.25 – 3.17 (m, 2H), 3.16 – 3.11(m, 2H). 13 C NMR (125 MHz, CDCl3) δ 160.3, 158.0, 155.0, 154.8, 151.6, 149.9, 148.2, 147.0, 137.9, 123.0, 121.3, 119.1, 114.5, 114.1, 108.8, 105.4, 101.4, 55.8, 55.6, 50.5, 50.4, 46.5, 42.6, 25.0. HRMS (ESI) m / z: Theoretical value [M+H] + 479.1705, measured value 479.1696.

[0052] Example 12 .

[0053] 2-(2,5-Dichloropyrimidin-4-yl)amino)benzonitrile (2-6A): In an ice-water bath, 60% sodium hydride (1.0 g, 25 mmol) was dispersed in 10 mL of anhydrous N,N-dimethylformamide, and 5 mL of anhydrous N,N-dimethylformamide solution containing 1.18 g, 10 mmol of 2-aminobenzonitrile was added dropwise. The mixture was stirred at 0 °C for 30 min. Then, 5 mL of tetrahydrofuran solution containing 1.82 g, 10 mmol of 2,4,5-trichloropyrimidine was added dropwise, and the mixture was heated to room temperature and stirred for 3 h. The reaction was monitored by TLC until complete. The reaction mixture was added to 200 mL of water, stirred to induce crystallization, and then filtered. The crude intermediate 2-6A was used directly in the next reaction. HRMS (ESI) m / z: theoretical value [M+H] + 265.0043, measured value 265.0045.

[0054] 2-(5-chloro-2-(2-methoxy-4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)benzonitrile (2-6B): Following the reaction of intermediate 2-1B, but replacing intermediate 2-1A with intermediate 2-6A, a dark yellow solid of 412 mg was obtained, with a yield of 47%. 1H NMR (500 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.12 (s, 1H), 7.86(dd, J = 7.5, 1.0 Hz, 1H), 7.72 (s, 1H), 7.69 (dd, J = 7.5, 1.5 Hz, 1H), 7.66(d, J = 7.5 Hz, 1H), 7.44 – 7.41 (m, 1H), 7.40 (dd, J = 7.5, 1.0 Hz, 1H), 6.59 (d, J = 2.5 Hz, 1H), 6.26 (d, J = 7.5 Hz, 1H), 3.76 (s, 3H), 3.20 – 3.11 (m, 8H). HRMS (ESI) m / z: Theoretical value [M+H] + 426.1647, measured value 436.1646.

[0055] Compound AF-12: The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-6B, to obtain the target compound AF-12 (58 mg, yield 67%) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ 8.46 (d, J = 8.4 Hz,1H), 8.12 (s, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 6.3 Hz, 2H), 7.57(t, J = 7.7 Hz, 1H), 7.33 (s, 1H), 7.18 (t, J = 7.5 Hz, 1H), 6.60 – 6.50 (m,1H), 6.44 (d, J = 8.4 Hz, 1H), 3.87 (s, 3H), 3.80 (s, 2H), 3.63 (s, 2H), 3.18(s, 2H), 3.13 (s, 2H), 2.79 (s, 2H). 13C NMR (125 MHz, CDCl3) δ 160.23,157.83, 155.28, 155.17, 149.64, 146.89, 141.08, 133.65, 132.59, 123.71,122.95, 122.23, 120.78, 116.6, 114.03, 108.82, 105.51, 103.43, 101.43, 55.82,50.56, 46.55, 42.64, 38.69, 25.01. HRMS (ESI) m / z: Theoretical value [M+H] + 503.1705, measured value 503.1698.

[0056] Example 13 .

[0057] 2-((2,5-dichloropyrimidin-4-yl)amino)-N-methylbenzamide (2-7A): 2,4,5-Trichloropyrimidine (910 mg, 5 mmol) and 2-amino-N-methylbenzamide (750 mg, 5 mmol) were dissolved in 20 mL of acetonitrile, and N,N-diisopropylethylamine (1.74 mL, 10 mmol) was added. The mixture was refluxed and stirred overnight, and the reaction was monitored by TLC until complete. The reaction solution was filtered, and the filter cake was washed with water (20 mL) and dried at 45 °C to give a pale yellow solid intermediate 2-7A (1.27 g, yield 86%). 1 H NMR (500 MHz, CDCl3) δ 11.68 (s, 1H), 8.71(d, J = 8.5 Hz, 1H), 8.21 (s, 1H), 7.59 – 7.53 (m, 1H), 7.50 (dd, J = 8.0,1.5 Hz, 1H), 7.17 – 7.10 (m, 1H), 6.32 (s, 1H), 3.04 (d, J = 5.0 Hz, 3H); HRMS(ESI) m / z: theoretical value [M+H] + 297.0305, measured value 297.0306.

[0058] 2-((5-chloro-2-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide (2-7B): Following the reaction with intermediate 2-1B, but replacing intermediate 2-1A with intermediate 2-7A, a dark purple solid was obtained, 272 mg, in 58% yield. The crude product was used directly in the next reaction. HRMS (ESI) m / z: theoretical value [M+H] + 468.1910, measured value 468.1915.

[0059] Compound AF-13: The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-7B, to obtain the pale yellow solid target compound AF-13 (56 mg, yield 58%). 1 H NMR (500 MHz, DMSO- d 6) δ 11.61 (s, 1H), 8.72 (s, 1H), 8.61 (s, 1H), 8.14 (s, 1H), 8.10 (s, 1H), 7.71 (d, J = 7.0 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.33 (s, 1H), 7.08 (t, J = 6.0 Hz, 1H), 6.69 (s,1H), 6.51 (d, J = 7.5 Hz, 1H), 4.12 (s, 2H), 3.77 (s, 3H), 3.64 (s, 2H), 3.52(s, 2H), 3.19 (s, 2H), 3.14 (s, 2H), 2.80 (s, 3H). 13 C NMR (125 MHz, DMSO- d 6) δ 168.9, 161.5, 158.9, 154.9, 154.6, 152.5, 148.6, 139.5, 131.4, 127.8, 125.1, 121.5, 121.0, 120.7, 116.1, 107.2, 104.13, 104.11, 100.8, 55.5, 49.1, 48.78, 45.2, 41.6, 26.3, 24.8. HRMS (ESI) m / z: Theoretical value [M+H] + 535.1968, measured value 535.1973.

[0060] Example 14 .

[0061] (2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (2-8A): 2,4,5-Trichloropyrimidine (550 mg, 3 mmol) and (2-aminophenyl)dimethylphosphine oxide (338 mg, 2 mmol) were dissolved in 4 mL of N,N-dimethylformamide, and potassium carbonate (552 mg, 4 mmol) was added. The mixture was stirred overnight at 75 °C, and the reaction was monitored by TLC until complete. The reaction solution was added dropwise to 200 mL of water, extracted with dichloromethane, and the organic phase was concentrated under reduced pressure. The residue was purified by dichloromethane / methanol (100:1 v / v) column chromatography to give an off-white solid intermediate 2-8A (518 mg, 82% yield). 1 H NMR (500 MHz, CDCl3) δ 11.54 (s, 1H), 8.66 (dd, J = 8.4, 4.4 Hz, 1H),8.21 (s, 1H), 7.77 – 7.46 (m, 1H), 7.32 – 7.26 (m, 1H), 7.17 (tdd, J = 7.6,2.3, 1.0 Hz, 1H), 1.84 (d, J = 13.2 Hz, 6H); HRMS (ESI) m / z: theoretical value [M+H] + 316.0168, measured value 316.0173.

[0062] (2-((5-chloro-2-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (2-8B) Following the reaction of intermediate 2-1B, but replacing intermediate 2-1A with intermediate 2-8A, a dark purple solid of 216 mg was obtained, with a yield of 44%. 1 H NMR (500 MHz, CDCl3) δ 10.80 (s, 1H), 8.62 (dd, J = 8.5,4.5 Hz, 1H), 8.14 – 8.06 (m, 2H), 7.49 (t, J = 8.0 Hz, 1H), 7.32 – 7.26 (m,2H), 7.12 (td, J = 7.5, 1.5 Hz, 1H), 6.55 (d, J= 2.5 Hz, 1H), 6.52 – 6.46(m, 1H), 3.11 – 3.05 (s, 3H), 3.08 m,, 8H), 1.83 (d, J = 13.5 Hz, 6H). HRMS(ESI) m / z: Theoretical value [M+H] + 487.1773, measured value 487.1772.

[0063] Compound AF-14: The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-8B, to obtain the pale yellow solid target compound AF-14 (55 mg, yield 60%). 1 H NMR (500 MHz, DMSO- d 6) δ 11.17 (s, 1H), 8.48 (s, 1H), 8.09 (s, 1H), 8.07 (s, 1H), 7.53 (dd, J = 14.0, 7.0 Hz, 1H), 7.46(d, J = 8.5 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.10 (t, J = 7.0 Hz, 1H), 6.68(d, J = 12.0 Hz, 1H), 6.49 (dd, J = 8.5, 2.0 Hz, 1H), 4.12 (s, 2H), 3.78 (s,3H), 3.64 (t, J = 4.0 Hz, 2H), 3.52 (t, J = 4.5 Hz, 2H), 3.18 (t, J = 4.0 Hz,2H), 3.16 – 3.11 (m, 2H), 1.76 (d, J = 13.5 Hz, 6H). 13 C NMR (125 MHz, DMSO- d 6) δ 161.5, 158.8, 155.2, 154.9, 152.5, 148.5, 143.3, 131.8, 130.5 (d, J =11.2 Hz), 127.3, 125.0, 122.2 (d, J= 12.5 Hz), 121.7 (d, J = 5.0 Hz), 120.8, 120.5 (d, J = 91.2 Hz), 107.3, 104.1, 100.8, 55.5, 49.1, 48.8, 45.3, 41.6, 24.8, 18.5, 17.9. HRMS (ESI) m / z: Theoretical value [M+H] + 554.1831, measured value 554.1830.

[0064] Example 15 .

[0065] 3-(2,5-Dichloropyrimidin-4-yl)-1H-indole (2-9A): Under ice-water bath conditions, 2,4,5-trichloropyrimidine (819 mg, 4.5 mmol) was dissolved in 15 mL of 1,2-dichloroethane, and aluminum trichloride (600 mg, 4.5 mmol) was added in portions, with stirring for 30 min. A solution of indole (351 mg, 3 mmol) in 15 mL of 1,2-dichloroethane was added dropwise, and the mixture was slowly heated to 80 °C and stirred overnight. The reaction was monitored by TLC until complete. The system was quenched with ice water, extracted three times with dichloromethane, and the organic phases were combined and concentrated under reduced pressure. The residue was slurried with petroleum ether and filtered to give a brown solid intermediate 2-9A (480 mg, 61% yield). 1 H NMR (500 MHz, CDCl3) δ 8.75 (s,1H), 8.70 (dd, J = 5.7, 3.2 Hz, 1H), 8.53 (d, J = 3.1 Hz, 1H), 8.51 (s, 1H), 7.47 (dd, J = 5.7, 3.2 Hz, 1H), 7.38 – 7.34 (m, 2H); HRMS (ESI) m / z: theoretical value [M+H] + 264.0090, measured value 264.0088.

[0066] 5-Chloro-4-(1H-indol-3-yl)-N-(2-methoxy-4-(piperazin-1-yl)phenyl)pyrimidin-2-amine (2-9B) Following the reaction of intermediate 2-1B, but replacing intermediate 2-1A with intermediate 2-9A, a dark purple solid was obtained, 97 mg, in 72% yield. The crude product was used directly in the next reaction. HRMS (ESI) m / z: theoretical value [M+H] + 435.1695, measured value 435.1692.

[0067] Compound AF-15: The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-9B, to obtain the pale yellow solid target compound AF-15 (129 mg, yield 51%). 1 H NMR (500 MHz, DMSO- d 6) δ 11.91 – 11.78 (m,1H), 8.47 (d, J = 3.0 Hz, 1H), 8.32 (s, 1H), 8.29 (s, 1H), 8.26 (s, 1H), 7.47(dd, J = 17.7, 8.4 Hz, 2H), 7.20 – 7.14 (m, 1H), 6.98 (t, J = 7.5 Hz, 1H), 6.72 (d, J = 2.5 Hz, 1H), 6.53 (dd, J = 8.6, 2.5 Hz, 1H), 4.13 (s, 2H), 3.77(s, 3H), 3.69 – 3.61 (m, 2H), 3.58 – 3.49 (m, 2H), 3.25 – 3.19 (m, 2H), 3.18– 3.15 (m, 2H). 13 C NMR (125 MHz, DMSO- d 6) δ 164.5, 161.6, 159.5, 157.8,157.2, 149.0, 153.3, 135.9, 130.8, 126.2, 123.3, 122.3, 120.8, 120.5, 116.2,114.1, 111.6, 110.9, 107.3, 101.0, 55.5, 49.0 (d, J = 41.2 Hz), 45.5 (d, J =61.2 Hz), 41.6, 24.8, 8.6. HRMS (ESI) m / z: Theoretical value [M+H] +502.1753, measured value 502.1752.

[0068] Example 16 .

[0069] 2-((2,5-dichloropyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamide (2-10A): Following the preparation process of intermediate 2-6A, 2,4,5-trichloropyrimidine and 2-amino-N,N-dimethylbenzenesulfonamide were used as raw materials. The crude product was subjected to petroleum ether / dichloromethane (volume ratio 2:1) column chromatography to obtain white solid intermediate 2-10A (215 mg, yield 21%). 1 H NMR (500 MHz, CDCl3) δ 9.86 (s, 1H), 8.59 (d, J = 8.5 Hz, 1H), 8.28 (s, 1H), 7.86 (dd, J = 8.0, 1.5 Hz, 1H), 7.71 – 7.64 (m, 1H), 7.30 – 7.27 (m, 1H), 2.74 (s, 6H); HRMS (ESI) m / z: theoretical value [M+H] + 347.0131, measured value 347.0133.

[0070] 2-((5-chloro-2-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzenesulfonamide (2-10B): The reaction was carried out according to Example 7, except that intermediate 2-1B was replaced with intermediate 2-10B, yielding a yellow solid, 127 mg, with a yield of 49%. 1 H NMR (500 MHz, DMSO- d 6) δ 9.39 (s, 1H), 8.57 (s, 1H), 8.28 (s, 1H), 8.16 (s, 1H), 7.75 (dd, J = 8.0, 1.3 Hz, 1H), 7.52 (t, J = 6.8 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 6.46 (dd,J = 8.7, 2.5 Hz, 1H), 3.90 (s, 1H), 3.75 (s, 3H), 3.10 – 3.05 (m, 4H), 2.89 – 2.85 (m, 4H), 2.64 (s, 6H). HRMS (ESI) m / z: Theoretical value [M+H] + 518.1736, measured value 518.1735.

[0071] Compound AF-16: The reaction was carried out in accordance with Example 7, except that intermediate 2-1B was replaced with intermediate 2-10B, to obtain the target compound AF-16 (46 mg, yield 66%) as a white solid. 1 H NMR (500 MHz, CDCl3) δ 9.38 (s, 1H), 8.55(d, J = 8.5 Hz, 1H), 8.10 – 8.08 (m, 2H), 7.85 (dd, J = 8.0, 1.0 Hz, 1H), 7.58 (dd, J = 11.5, 4.0 Hz, 1H), 7.36 (s, 1H), 7.23 (t, J = 7.5 Hz, 1H), 6.53(d, J = 2.0 Hz, 1H), 6.45 (d, J = 8.0 Hz, 1H), 3.87 (s, 3H), 3.84 – 3.78 (m,2H), 3.67 – 3.60 (m, 2H), 3.56 (s, 2H), 3.16 (d, J = 28.0 Hz, 4H), 2.73 (s, 6H). 13 C NMR (125 MHz, CDCl3) δ 160.2, 157.8, 155.4, 155.2, 149.4, 146.6, 137.2, 133.5, 130.4, 124.5, 124.1, 123.2, 120.4, 114.0, 108.9, 105.8, 101.5, 55.8, 50.5, 50.5, 46.6, 42.6, 37.7, 25.0. HRMS (ESI) m / z: Theoretical value [M+H] + 585.1794, measured value 585.1794.

[0072] Example 17 Compound AF-17: .

[0073] The reaction was carried out according to Example 14, except that cyanoacetic acid was replaced with ( E )-2-cyano-4,4-dimethyl-2-enpentanoic acid was used to give the target compound AF-17 (63 mg, 78% yield) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ10.81 (s, 1H), 8.60 (dd, J = 8.4, 4.3 Hz, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.09(s, 1H), 7.50 (dd, J = 8.2, 7.6 Hz, 1H), 7.31 (s, 1H), 7.30 – 7.26 (m, 1H), 7.16 – 7.12 (m, 1H), 7.01 (s, 1H), 6.54 (d, J = 2.5 Hz, 1H), 6.48 (dd, J =8.8, 2.4 Hz, 1H), 3.88 (s, 3H), 3.77 (s, 4H), 3.17 (d, J = 3.8 Hz, 4H), 1.83(d, J = 13.1 Hz, 6H), 1.32 (s, 9H). 13 C NMR (125 MHz, CDCl3) δ 167.6, 163.2,157.8, 156.1, 155.1, 149.4, 146.6, 144.0 (d, J = 1.2 Hz), 132.5 (d, J = 2.5Hz), 129.7 (d, J = 11.2 Hz), 123.6, 123.24 (d, J = 1.2 Hz), 123.20 (d, J =1.2 Hz), 122.7 (d, J= 12.5 Hz), 120.5, 115.1, 109.1, 108.2, 106.4, 101.5, 77.4, 55.8, 50.7, 29.2, 19.0, 18.4. HRMS (ESI) m / z: Theoretical value [M+H] + 622.2457, measured value 622.2458.

[0074] Example 18 Compound AF-18: .

[0075] The reaction was carried out according to Example 14, except that cyanoacetic acid was replaced with intermediates 1-5 to obtain the target compound AF-18 (60 mg, yield 62%) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ 11.90 (s, 1H), 10.62 (s,1H), 8.37 – 8.33 (m, 1H), 7.87 (s, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.32 – 7.26(m, 1H), 7.25 – 7.18 (m, 2H), 6.68 (d, J = 11.2 Hz, 1H), 6.59 (d, J = 2.3 Hz, 1H), 6.52 (dd, J = 8.6, 2.4 Hz, 1H), 3.87 (s, 4H), 3.80 (s, 3H), 3.30 (d, J =4.3 Hz, 4H), 2.16 – 2.04 (m, 1H), 1.87 (d, J = 13.2 Hz, 6H), 1.32 – 1.24 (m, 2H), 0.98 – 0.89 (m, 2H). 13 C NMR (125 MHz, CDCl3) δ 167.1, 162.5, 162.2 (d, J = 37.5 Hz), 157.3, 154.2, 153.1, 150.1, 141.9, 141.4, 132.8, 130.0 (d, J =11.2 Hz), 127.0, 125.1 (d, J = 12.5 Hz), 125.0, 123.5 (d,J = 6.2 Hz), 120.5(d, 47.5 Hz), 119.10, 116.0 (d, J = 143.8 Hz), 115.7, 108.4, 107.0, 106.0, 101.2, 55.9, 50.1, 19.0, 18.5, 15.9, 11.0. HRMS (ESI) m / z: Theoretical value [M+H] + 606.2144, measured value 606.2141.

[0076] Example 19 .

[0077] 2-Fluoro-4-isopropoxy-1-toluene (3-2): 3-Fluoro-4-methylphenol (1.26 g, 10 mmol) and potassium carbonate (2.76 g, 20 mmol) were dissolved in 20 mL of N,N-dimethylformamide, and 2-bromopropane (3.69 g, 30 mmol) was slowly added. The mixture was stirred overnight at 105 °C under nitrogen protection. The reaction mixture was added dropwise to 200 mL of ice water, and extracted three times with diethyl ether (100 mL each time). The organic phase was concentrated under reduced pressure. The residue was subjected to column chromatography with petroleum ether / ethyl acetate (100:1 v / v) to give a colorless oily intermediate 3-2 (407 mg, 24% yield). 1 H NMR (500 MHz, CDCl3) δ 7.03 (t, J = 9.0 Hz, 1H), 6.62 – 6.52(m, 2H), 4.47 (sept, J = 6.0 Hz, 1H), 2.18 (d, J = 1.5 Hz, 3H), 1.32 (d, J =6.0 Hz, 6H).

[0078] 1-Fluoro-5-isopropoxy-2-methyl-4-nitrobenzene (3-3): Under ice-water bath conditions, intermediate 3-2 (407 mg, 2.4 mmol) was dissolved in 20 mL of dichloromethane, and fuming nitric acid (483 mg) was slowly added dropwise. After the addition, the mixture was heated to room temperature and stirred for 0.5 h. The reaction was monitored by TLC until it was complete.

[0079] The solvent was removed by vacuum distillation, and the residue was subjected to column chromatography with pure petroleum ether as the eluent to give a colorless oily intermediate 3-3 (208 mg, yield 40%).1 H NMR (500 MHz, CDCl3) δ 7.74 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 11.0 Hz, 1H), 4.57 (sept, J = 6.0 Hz, 1H), 2.24 (d, J = 1.5 Hz, 3H), 1.39(d, J = 6.0 Hz, 6H).

[0080] 4-(5-isopropoxy-2-methyl-4-nitrophenyl)piperazine-1-carboxylic acid tert-butyl ester (3-4): Following the preparation process of intermediate 1-2, intermediate 3-3 and 1-Boc-piperazine were reacted as raw materials to obtain yellow solid intermediate 3-4 (243 mg, yield 66%). 1 H NMR (500 MHz, CDCl3) δ 7.74 (s, 1H), 6.56 (s, 1H), 4.57 (sept, J = 6.0 Hz, 1H), 3.64 – 3.57 (m, 4H), 2.94 – 2.88 (m,4H), 2.25 (s, 3H), 1.48 (s, 9H), 1.38 (d, J = 6.0 Hz, 6H); HRMS (ESI) m / z: theoretical value [M+H] + 380.2180, measured value 380.2187.

[0081] 4-(4-amino-5-isopropoxy-2-methylphenyl)piperazine-1-carboxylic acid tert-butyl ester (3-5): Following the preparation process of intermediates 1-3, intermediates 3-4 were used as raw materials to react and obtain purple solid intermediates 3-5 (716 mg, yield 92%). 1 H NMR (500 MHz, CDCl3) δ 6.57 (s, 1H), 6.55 (s, 1H), 4.46– 4.39 (m, 1H), 3.63 (s, 2H), 3.54 (s, 4H), 2.78 – 2.71 (m, 4H), 2.18 (s,3H), 1.48 (s, 9H), 1.33 (d, J = 6.1 Hz, 6H); HRMS (ESI) m / z: theoretical value [M+H]+ 350.2438, measured value 350.2437.

[0082] (2-(5-chloro-2-(2-isopropoxy-5-methyl-4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)dimethylphosphine oxide (3-6): Following the synthesis process of intermediate 2-8B, intermediates 3-5 and 2-8A were used as raw materials to react and obtain purple solid intermediate 3-6 (200 mg, yield 63%). 1 H NMR (500 MHz, DMSO- d 6) δ 12.01 (s, 1H), 9.39 (s, 2H), 8.41 (s, 1H), 8.31 (d, J = 8.7 Hz, 1H), 7.68 (dd, J = 13.4, 7.6 Hz,1H), 7.44 (dd, J = 17.7, 11.4 Hz, 2H), 7.29 (t, J = 7.2 Hz, 1H), 6.75 (s,1H), 4.64 (p, J = 6.0 Hz, 1H), 3.56 (s, 3H), 3.22 (s, 4H), 3.08 (t, J = 4.9Hz, 4H), 1.80 (d, J = 13.6 Hz, 6H), 1.23 (d, J = 5.9 Hz, 6H). HRMS (ESI) m / z: Theoretical value [M+H] + 529.2242, measured value 529.2240.

[0083] Example 19 Compound AF-19: The reaction was carried out in accordance with Example 17, except that intermediates 2-8B were replaced with intermediates 3-6, to obtain the target compound AF-19 (43 mg, yield 59%) as a yellow solid. 1 H NMR (500 MHz, CDCl3) δ 10.79 (s, 1H), 8.61(dd, J = 8.5, 4.5 Hz, 1H), 8.11 (s, 1H), 8.09 (s, 1H), 7.49 (t, J = 8.0 Hz,1H), 7.43 (s, 1H), 7.32 – 7.27 (m, 1H), 7.12 (t, J= 7.0 Hz, 1H), 6.97 (s,1H), 6.60 (s, 1H), 4.55 – 4.46 (m, 1H), 3.73 (s, 4H), 2.90 (s, 4H), 2.19 (s,3H), 1.83 (d, J = 13.0 Hz, 6H), 1.35 (d, J = 6.0 Hz, 6H), 1.30 (s, 9H). 13 CNMR (125 MHz, CDCl3) δ 163.1, 161.1, 156.3, 155.5, 150.7, 150.0, 148.5,147.4, 135.7 (d, J = 3.4 Hz), 129.3 (d, J = 8.1 Hz), 128.7, 121.8, 115.5, 115.3, 113.7, 111.2, 104.8, 79.0, 68.6, 56.0, 46.0, 45.2, 39.2, 29.4, 28.7, 23.1, 11.4. HRMS (ESI) m / z: Theoretical value [M+H] + 664.2926, measured value 664.2927.

[0084] Example 20 Compound AF-20 .

[0085] The reaction was carried out in accordance with Example 19, except that ( E The target compound AF-20 (41 mg, 46% yield) was obtained by replacing 2-cyano-4,4-dimethyl-2-envalonic acid with intermediates 1-5. 1 H NMR (500 MHz, CDCl3) δ12.01 (s, 1H), 10.60 (s, 1H), 8.45 (dd, J = 7.5, 3.5 Hz, 1H), 7.86 (s, 1H), 7.40 (s, 1H), 7.37 – 7.29 (m, 1H), 7.24 – 7.17 (m, 2H), 6.65 (d, J = 11.5 Hz,1H), 6.63 (s, 1H), 4.45 (dt, J= 12.0, 6.0 Hz, 1H), 3.82 (s, 4H), 2.95 (s,4H), 2.20 (s, 3H), 2.14 – 2.06 (m, 1H), 1.85 (d, J = 13.0 Hz, 6H), 1.27 –1.26 (m, 8H), 0.94 – 0.89 (m, 2H). 13 C NMR (125 MHz, CDCl3) δ 166.4, 162.5,157.2, 153.2, 149.9, 149.2, 142.2, 132.7, 130.0 (d, J = 11.0 Hz), 128.8 (d, J = 16.9 Hz), 127.8, 124.6 (d, J = 10.2 Hz), 123.3 (d, J = 6.9 Hz), 122.7, 121.2, 120.4, 115.8, 107.5, 106.4, 105.9, 72.0, 51.9, 22.0, 19.2, 18.7, 17.2, 15.8, 10.9. HRMS (ESI) m / z: Theoretical value [M+H] + 648.2614, measured value 648.2623.

[0086] Example 21: The compound provided by the present invention exhibits inhibitory activity against the proliferation of PAX3 / 7-FOXO1 fusion gene-positive rhabdomyosarcoma cells.

[0087] SRB experiment: PAX3 / 7-FOXO1 fusion gene-positive rhabdomyosarcoma RH30 and RH28 cells, and MYCN-amplified neuroblastoma IMR-32 cells were cultured at 2×10⁻⁶ cells per cell line. 3Cells were seeded at a density of 50 μL / well in 96-well plates. After overnight cell adhesion, the test compound was added the following day (day 0). On day 3, the culture medium was discarded, and the cells were washed once with phosphate-buffered saline. 10% trichloroacetic acid was added, and the plates were fixed at 4 °C for 1 h. The plates were then washed five times with water and dried in an oven for 1 h. After the plates returned to room temperature, sulfonylrhodamine B was added for staining at room temperature for 25 min. The free dye was washed away sequentially with 1% acetic acid and pure water. The plates were dried again in an oven for 1 h and then cooled to room temperature. 50 μL of tris(hydroxymethyl)aminomethane solution was added to each well to dissolve the dye bound to the protein. The absorbance was measured at 515 nm using a microplate reader. The cell proliferation inhibition at different concentrations was calculated, and an IC50 curve was plotted to calculate the IC50. 50 The specific results are shown in the table below.

[0088] Table 1. Inhibitory activity of the compounds provided in this invention in different tumor cells .

[0089] Example 22 Pharmacokinetic study of the compound provided by the present invention in rats.

[0090] Six SD rats were randomly divided into two groups and administered compound AF-2 via intravenous injection (iv) at a dose of 5 mg / kg or oral administration (po), respectively. Blood samples (approximately 0.25 mL) were collected via tail vein at the following time points: 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after intravenous administration. The concentration of compound AF-2 in rat plasma samples was determined using LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNolin software. Specific results are shown in Table 3 below, including the time to peak concentration of AF-2 in rats. max The shorter (h) indicates that the AF-2 compound has a faster onset of action, and the oral half-life t is relatively short. 1 / 2 The (h) duration is relatively long and can reach the therapeutic concentration of cells, indicating that the drug has a relatively long efficacy after oral administration.

[0091] Table 2. Pharmacokinetic parameters of compound AF-2 provided by this invention .

[0092] Example 23: Antitumor activity of the compounds provided by the present invention in a RH30 xenograft mouse model.

[0093] Female nude mice aged 4-6 weeks were used in the experiment. First, subcutaneous inoculation was performed in the axillary region of the mice. One RH30 cell; until the tumor volume grows to approximately [number missing] cells. At that time, the tumor was dissected and cut into pieces of approximately [volume missing]. The tumor fragments were inoculated into the axillae of a new generation of nude mice using the tumor fragment transplantation method. When the tumor volume reached... Mice were then randomly divided into groups and administered saline, AF-2 (25 mg / kg), AF-2 (50 mg / kg), and the positive control ceritinib (25 mg / kg) by gavage, respectively. Mouse body weight and tumor volume were monitored daily; once the tumor volume in the saline group reached a certain level... The experiment was terminated at the designated time. Mice were then euthanized, tumors were removed, and each mouse was weighed and photographed. The relative tumor volume (RTV) was calculated based on the measurements using the formula: RTV = Vt / V0. Where V0 is the tumor volume measured at the time of administration (d0), and Vt is the tumor volume at each measurement. The evaluation index for antitumor activity was the relative tumor proliferation rate (T / C) (%), calculated using the formula: T / C = T RTV / C RTV ×100% (T) RTV Treatment group RTV; C RTV (Negative control group RTV) The specific results are shown in Table 3 and Appendix below. Figure 1 The growth inhibition of compound AF-2 on RH30 xenograft tumors was dose-dependent, and the growth inhibition rate of AF-2 at both doses was better than that of the ceritinib group, showing significant efficacy.

[0094] Table 3. Growth inhibitory effect of compound AF-2 on RH30 xenograft tumors Example 24: Antitumor activity of the compounds provided by the present invention in a mouse model of clinically recurrent rhabdomyosarcoma patient cell xenograft tumors.

[0095] Another clinically recurrent rhabdomyosarcoma specimen was taken and cut into approximately [number missing] pieces. Tissue blocks were subcutaneously injected into the axillae of nude mice. The tumors were allowed to grow to approximately [size missing]. Then remove it and divide it again into approximately The tumor fragments were transplanted into the axillae of nude mice. When the tumor volume reached... Animals were randomly divided into groups and administered saline, AF-2 (50 mg / kg), and ceritinib (25 mg / kg) by gavage, respectively. Tumor volume and mouse weight were measured daily. The treatment continued until the tumor volume in the saline group increased to [a certain value]. The experiment was then concluded. Mice were euthanized, tumors were removed, and photographs and weights were taken. The same testing indicators were performed as above. Specific results are shown in Table 3 and Appendix. Figure 2 After administration of compound AF-2, the growth of patient-derived xenograft tumors of rhabdomyosarcoma was significantly inhibited, which was superior to positive ceritinib.

[0096] Table 3. Growth inhibitory effect of compound AF-2 on patient-derived xenograft tumors of rhabdomyosarcoma Example 25: Antitumor activity of the compounds provided by the present invention in a mouse model of clinically recurrent neuroblastoma patient cell xenograft tumors.

[0097] Another clinically recurrent neuroblastoma specimen was taken and cut into approximately [number missing] pieces. Tissue blocks were subcutaneously injected into the axillae of nude mice. The tumors were allowed to grow to approximately [size missing]. Then remove it and divide it again into approximately The tumor fragments were transplanted into the axillae of nude mice. When the tumor volume reached... Animals were randomly divided into groups and administered saline, AF-2 (50 mg / kg), and cisplatin (3 mg / kg) by gavage, respectively. Tumor volume and mouse body weight were measured daily. The treatment continued until the tumor volume in the saline group increased to [a certain value]. The experiment was then concluded. Mice were euthanized, tumors were removed, and photographs and weights were taken. The same testing indicators were performed as above. Specific results are shown in Table 4 and Appendix. Figure 3 After administration of compound AF-2, the growth of xenograft tumors derived from neuroblastoma patients was significantly inhibited, which was superior to the first-line positive cisplatin in clinical trials. Table 4. Growth inhibitory effect of compound AF-2 on neuroblastoma patient-derived xenograft tumors Example 26. Transcriptome sequencing results of RH30 cells treated with the compounds provided in this invention.

[0098] RH30 cells were used Cells were seeded at a density of 1 cell / well, with 2 mL of culture medium added to each well. The following day, cells were treated with either AF-2 or ceritinib, and samples were collected 48 h after treatment. Total RNA was extracted and sequenced. Results are available in […]. Figure 4 .

[0099] Differential expression analysis showed that both AF-2 and ceritinib altered gene expression patterns in cells compared to the control group (Figure 4A). This study further conducted gene set enrichment analysis (GSEA) based on the above data to explore molecular responses at the pathway level. Notably, AF-2 downregulated the expression of more gene sets, while ceritinib treatment upregulated the expression of multiple pathways, suggesting a possible feedback response that could weaken the drug's efficacy (Figure 4B). Since the MAPK / ERK and PI3K / AKT / mTOR signaling cascades are common downstream pathways of ALK and FGFR4, we specifically analyzed gene sets related to these pathways from transcriptome sequencing data. Comparing the effects of the two drugs, AF-2 showed a more significant inhibitory effect on MAPK and PI3K-related gene sets (Figures 4C and 4D). The malignant progression of fusion gene-positive rhabdomyosarcoma (RMS) is closely related to MYC pathway activation; therefore, this study further investigated the effects of the two drugs on MYC-related transcriptional programs. The results showed that, compared with the control group, AF-2 significantly downregulated multiple MYC-related gene sets (Figure 4E); and for MYC characteristic gene sets such as MYC_TARGETS_REPRESSED_BY_SERUM and MYC_AMPLIFICATION_TARGETS_UP, AF-2's inhibitory effect was stronger than that of ceritinib (Figure 4F). In summary, transcriptome sequencing analysis confirmed that in RH30 cells, AF-2 has a broader and stronger inhibitory range on core oncogenic transcriptional programs.

Claims

1. An aminopyrimidine derivative, characterized in that, It has the structure shown in general formula I: ； in: R1 is a substituted acyl group, wherein the substituent is an alkenyl, alkynyl, halogen, or cyano group; R2 is a substituted amino group, wherein the substituent is a C2-C3 alkyl, a C3-C5 cycloalkyl, an unsubstituted or substituted phenyl, or an aromatic heteroyl group; wherein the phenyl substituent is a cyano, sulfone, carbamoyl, sulfonamide, or phosphine oxide. R3 is methyl or isopropyl; R4 represents hydrogen or methyl; The compounds include pharmaceutically acceptable salts and isotope-labeled compounds.

2. An aminopyrimidine derivative according to claim 1, characterized in that, R1 is , , , , , , ; R2 is , , , , , , , , , , .

3. An aminopyrimidine derivative according to any one of claims 1 to 2, characterized in that, Having one of the following structures or pharmaceutically acceptable salts or isotope-labeled compounds:

4. The use of an aminopyrimidine derivative according to any one of claims 1 to 3 in the preparation of an antitumor drug, characterized in that, The tumors mentioned include neuroblastoma and rhabdomyosarcoma.

5. The application according to claim 4, characterized in that, The compound is used to inhibit the proliferation of tumor cells that are positive for the PAX3 / 7-FOXO1 fusion gene or have amplified MYCN.

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