A compound, its preparation method and application

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a compound, its preparation method, and its applications. The compound of this invention exhibits strong agonistic activity against ULK1 kinase and strong inhibitory activity against ERK1 / 2 kinase, as well as strong anti-proliferative activity against triple-negative breast cancer cells. Compound UE01, in particular, demonstrates the strongest agonistic activity against ULK1 and the strongest inhibitory activity against ERK1 / 2, and also exhibits the strongest anti-proliferative activity against triple-negative breast cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a compound, its preparation method, and its application. Background Technology

[0002] Breast cancer is the most common malignant tumor among women, and its subtype, triple-negative breast cancer (TNBC), exhibits significant biological heterogeneity. This subtype is characterized by negative immunohistochemical results for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), making it insensitive to endocrine therapy and HER2-targeted therapy. Currently, clinical treatment for TNBC primarily involves surgery combined with chemotherapy. However, due to its aggressive nature and high rate of metastasis and recurrence, clinical treatment outcomes are unsatisfactory, and patient mortality is extremely high. Therefore, identifying new specific targets and developing novel targeted small molecule drugs for TNBC treatment has significant research and clinical value. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a compound, its preparation method, and its application.

[0004] A compound having a structural formula as shown in Formula I or Formula II: Equation I is Equation II is ; Among them, R 1 Selected from at least one of the following groups: , and ;R 2 Selected from at least one of the following groups: , , , , , , , and ;R 3 Selected from at least one of the following groups: and ;R 4 Selected from at least one of the following groups: , , , , , , , , , , , , , , , , and .

[0005] The compounds of this invention are prepared from benzoyl chloride derivatives and exhibit strong agonistic activity against ULK1 kinase while also exhibiting strong inhibitory activity against ERK1 / 2 kinase.

[0006] A salt of the compound described above.

[0007] An isomer of the compound described above.

[0008] The use of the compound, salt, or isomer in the preparation of a therapeutic agent for triple-negative breast cancer.

[0009] Preferably, the therapeutic drug is composed of an active ingredient and excipients, wherein the active ingredient is the compound, the salt, or the isomer.

[0010] Preferably, the excipients include a diluent.

[0011] Preferably, the diluent comprises physiological saline.

[0012] Preferably, the therapeutic agent is used to inhibit the proliferation and metastasis of triple-negative breast cancer.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: The compounds of this invention exhibit strong agonistic activity against ULK1 and strong inhibitory activity against ERK1 / 2, as well as strong anti-proliferative activity against triple-negative breast cancer cells. Compound UE01, in particular, demonstrates the strongest agonistic activity against ULK1 and the strongest inhibitory activity against ERK1 / 2, and also exhibits the strongest anti-proliferative activity against triple-negative breast cancer cells. Attached Figure Description

[0014] Figure 1The results show that UE01 inhibits the proliferation of TNBC cells. In this paper, A is the IC50 value of UE01 in MDA-MB-231 cells and BT-549 cells after 24 h, and B is the effect of treatment with UE01, LYN-1604 (20 μM) and SCH772984 (40 μM) on the colony formation of MDA-MB-231 cells. (1) is the result of the colony formation experiment, and (2) is the statistical graph. The data are expressed as mean ± SEM (n=3).

[0015] Figure 2 The effects of UE01, LYN-1604 (20 μM) and SCH772984 (40 μM) on the proliferation of MDA-MB-231 cells were detected using a 3D tumor spheroid assay. In the figure, A represents the results of the 3D tumor spheroid assay, with a scale bar of 200 μM, and B is a statistical graph. The data are expressed as mean ± SEM (n=3).

[0016] Figure 3 The EdU assay was used to detect the effects of UE01 (20 μM), LYN-1604 (20 μM) and SCH772984 (40 μM) treatments on the proliferation of MDA-MB-231 cells. In the figure, A represents the results of the EdU assay, with a scale bar of 50 μM, and B represents the statistical graph. The data are expressed as mean ± SEM (n=3).

[0017] Figure 4 The results show that UE01 inhibits the growth of TNBC subcutaneous xenograft tumors. In this study, A represents the size of subcutaneous tumors in the UE01, LYN-1604 (50 mg / kg), and SCH772984 (100 mg / kg) administration groups; B represents the change in subcutaneous tumor volume in nude mice over time during administration; C represents the weight of subcutaneous tumors in different administration groups; and D represents the change in mouse body weight over time during administration. Data are expressed as mean ± SEM (n=6).

[0018] Figure 5 This is the result of in vivo imaging of a subcutaneous xenograft tumor.

[0019] Figure 6 The results of Ki-67 immunohistochemical staining of tumor tissues from different drug administration groups are shown. In the figure, A is the staining map with a scale bar of 50 μM, and B is the statistical graph. The data are expressed as mean ± SEM (n=3).

[0020] Figure 7 Immunohistochemical staining results of p-ULK1 and p-ERK1 / 2 in tumor tissues of different drug administration groups. In A, the results of p-ULK1 are shown. (1) is a staining map and (2) is a statistical graph. In B, the results of p-ERK1 / 2 are shown. (1) is a staining map and (2) is a statistical graph. The scale bar is 50 μM. The data are expressed as mean ± SEM (n=3).

[0021] Figure 8 The statistical results of blood biochemical indicators related to liver and kidney function in mice in the drug-treated group are presented as mean ± SEM (n=6).

[0022] Figure 9 HE staining results of heart, liver, spleen, lung and kidney of mice in the drug-treated group, scale bar is 50 μM.

[0023] Figure 10 The results of MDA-MB-231 cells with negative control, ULK1 knockdown and MAPK3 knockdown under no treatment or UE01 (20 μM) treatment are shown. In A, cell clone formation is shown. (1) in A is the result of the clone formation experiment and (2) is a statistical graph. B is the size of 3D tumor microspheres. (1) in B is the tumor microspheres and (2) is a statistical graph. The scale bar is 200 μM. The data are expressed as mean ± SEM (n=3).

[0024] Figure 11 The cumulative fluorescence intensity of the lungs of mouse models with tail vein lung transfer was measured 13 days after administration of different doses of UE01, LYN-1604 (50 mg / kg) and SCH772984 (100 mg / kg). A is a fluorescence graph, and B is a statistical graph. Data are expressed as mean ± SEM (n=5).

[0025] Figure 12 Bioluminescence imaging of the five internal organs of mice with tail vein lung transfer 13 days after drug administration. A is the imaging image, and B is the statistical graph. Data are expressed as mean ± SEM (n=5).

[0026] Note: In the figure, ns represents no significant difference, *p<0.05, **p<0.01, ***p<0.001, #p<0.05, ##p<0.01 Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0028] This invention provides compounds as shown in Formula I and Formula II: Formula I: Where R1 is R2 is , , , , , , or ; Or R1 is R2 is , , , , , , , or ; Or R1 is R2 is , , , , , , , or ; Formula II: Where R3 is R4 is , , , , , , , , , , , , , , , , or ; Or R3 is R4 is , , , , , , , , , , or .

[0029] As a preferred option, in Equation II, R3 is R4 is .

[0030] The present invention also provides pharmaceutically acceptable salts of the compounds described herein. These salts may be nitrates, hydrochlorides, sulfates, or phosphates, etc.

[0031] The present invention also provides the above-mentioned compounds or pharmaceutically acceptable salts thereof in their preparation and their anti-triple-negative breast cancer effects.

[0032] This invention also provides a pharmaceutical composition comprising an effective dose of the above-mentioned compound or a pharmaceutically acceptable salt thereof. Dosage forms include tablets, capsules, aqueous or oily solutions, suspensions, emulsions, and sterile aqueous or oily solutions, suspensions, or sterile emulsions for parenteral (including intravenous, intramuscular, or infusion) administration. Liquid formulations can be prepared using sterile water or a water-propylene glycol solution as a solvent, and the active ingredient can also be formulated in an aqueous polyethylene glycol solution. Aqueous solutions for oral administration can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavoring agents, stabilizers, and thickeners as needed. Aqueous suspensions for oral use can be prepared by dispersing the finely dispersed active ingredient in water along with a viscous substance, such as natural synthetic gums, resins, methylcellulose, carboxymethylcellulose, and other suspending agents known in the pharmaceutical field.

[0033] The pharmaceutical compositions of the present invention may contain only the compounds of the present invention as their active ingredient, or they may be combined with other anti-triple-negative breast cancer compounds as their active ingredient. During treatment, the pharmaceutical compositions of the present invention may be used in combination with other anti-triple-negative breast cancer drugs. Such combination therapy can be achieved by administering various therapeutic ingredients simultaneously, sequentially, or individually. Such combination products utilize compounds of the present invention within the effective dose range and other pharmaceutically active agents within the permissible dose range.

[0034] Example 1: Synthesis of compounds 7a-i and 11a-r.

[0035] Compound 7a-i was synthesized using the following reaction: a) Bromosuccinimide, p-toluenesulfonic acid, acetonitrile, reaction at room temperature; b) tert-Butoxycarbonylpiperazine, potassium carbonate, dichloromethane, reaction at 0°C; c) Sodium borohydride, methanol, reaction at room temperature; d) Benzyl halide, tetrabutylammonium bromide, potassium tert-butoxide, acetonitrile, reaction at 80°C; e) Trifluoroacetic acid, dichloromethane, reaction at 0°C; f) Acyl chloride, triethylamine, dichloromethane, reaction at room temperature.

[0036] 30.0 mmol of 3′,5′-dichloroacetophenone was dissolved in 30 mL of acetonitrile. After complete dissolution, 3.0 mmol of p-toluenesulfonic acid was added. The mixture was cooled in an ice bath, and 30.0 mmol of N-bromosuccinimide was added. After the addition was complete, the ice bath was removed, and the reaction mixture was stirred at room temperature for 48 h. Insoluble solids were removed by filtration, and the filtrate was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether:ethyl acetate = 100:1) to give intermediate 2.

[0037] 9.0 mmol of potassium carbonate was added to an anhydrous dichloromethane solution of 3.6 mmol of tert-butyloxycarbonylpiperazine. The mixture was cooled to 0 °C, and an anhydrous dichloromethane solution of intermediate 2 was added dropwise at 0 °C. The reaction was stirred at 0 °C for 10 min, then heated to room temperature and stirred overnight. The reaction was quenched with a saturated ammonium chloride aqueous solution, extracted with dichloromethane, and the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to give intermediate 3.

[0038] 2.5 mmol of sodium borohydride was added in portions to a 20 mL methanol solution of 5.0 mmol intermediate 3 at room temperature. The resulting mixture was stirred at room temperature for 2 h, and then the solvent was removed under reduced pressure. The reaction solution was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:2) to give intermediate 4.

[0039] 3 mmol of intermediate 4 was dissolved in 15 mL of acetonitrile, followed by the addition of 3.6 mmol of potassium tert-butyloxide. The mixture was heated at 80 °C for 30 min, and then 4.5 mmol of bromides with different substitutions, 0.3 mmol of potassium iodide, and 0.3 mmol of tetrabutylammonium bromide were added. The reaction mixture was reacted under reflux for 12 h. The reaction mixture was extracted with ethyl acetate, washed successively with saturated sodium bicarbonate solution and water, the combined organic layers were dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1) to give intermediate 5.

[0040] 1.5 mmol of intermediate 5 was dissolved in dichloromethane, and then trifluoroacetic acid was added at 0 °C. The mixture was stirred at 0 °C for 2 h, followed by washing with saturated sodium bicarbonate solution and brine successively. The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to give intermediate 6.

[0041] 0.5 mmol of intermediate 6 and 0.5 mmol of triethylamine were added to 5 mL of dichloromethane, followed by 0.55 mmol of benzoyl chloride. The reaction mixture was stirred for 12 h and then washed successively with saturated sodium bicarbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 15:1) to give the final product 7a-i.

[0042] Compound 11a-r was synthesized using the following reaction: a) 1H-imidazolium / 1,2,3-triazole, potassium carbonate, potassium iodide, acetonitrile, reaction at 80°C; b) sodium borohydride, methanol, reaction at room temperature; c) different substituted carboxylic acid compounds, N,N'-dicyclohexylcarbodiimide, dimethylaminopyridine, dichloromethane, reaction at 80°C.

[0043] 10 mmol of 2,2',4'-trichloroacetic acid benzophenone, 15 mmol of 1H-imidazolium / 1,2,3-triazole, potassium carbonate, and potassium iodide were dissolved in 20 mL of acetonitrile. The resulting mixture was stirred at 80 °C for 6–8 h. After the reaction was complete, the mixture was extracted with dichloromethane, washed successively with saturated sodium bicarbonate solution and water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EtOAc, 1:1) to give intermediate 9.

[0044] 5 mmol of intermediate 9 was added to 20 mL of methanol solution, followed by the addition of 2.5 mmol of sodium borohydride in portions at 0 °C. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the mixture was extracted with dichloromethane, washed successively with 1 mol hydrochloric acid and saturated brine, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:4) to give intermediate 10.

[0045] 1.2 mmol of intermediate 10 and 1 mmol of a carboxylic acid compound with different substitutions were dissolved in 3 mL of dichloromethane. Then, at 0 °C, a dichloromethane solution of 1.3 mmol of N,N'-dicyclohexylcarbodiimide and 0.1 mmol of dimethylaminopyridine was added dropwise. The mixture was then heated to room temperature and stirred for 6 h. The precipitate was filtered, the filtrate was dried over anhydrous sodium sulfate, filtered again, and then concentrated under reduced pressure. The crude product was rapidly purified by silica gel column chromatography (PE:EtOAc = 1:1) to give the final product 11a-r.

[0046] Compound 7a, white powder, mp 147-150℃, yield 68%. 1H NMR (600MHz, Chloroform- d ) δ 7.84-7.80(m, 3H), 7.74(s, 1H), 7.57-7.56(m, 1H), 7.50-7.46(m, 3H), 7.41-7.35(m, 6H), 7.33-7.32(m, 1H), 5.08-5.0 6(m, 1H), 4.71-4.69(m, 1H), 4.44-4.42(m, 1H), 3.81-3.75(m, 2H), 3.39(s, 2H), 2.79-2.75(m, 1H), 2.64-2.41(m, 5H). 13 C NMR (150MHz, Chloroform- d )δ170.4, 136.9, 135.9, 135.1, 134.1, 133.7, 133.3, 133.2, 130.0, 129.8, 129.4, 129.0, 128.6, 1 28.3, 128.0, 127.9, 127.2, 127.1, 126.4, 126.2, 126.1, 74.4, 71.2, 63.5, 53.8, 53.2, 47.8, 42.2. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 30 H 29 Cl2N2O2 + 519.1601, measured value 519.1593.

[0047] Compound 7b, white powder, mp 158-161℃, yield 65%. 1 H NMR (600MHz, Chloroform- d ) δ 7.84-7.81(m, 3H), 7.75-7.74(m, 1H), 7.56-7.54(m, 1H), 7.50-7.47(m, 3H), 7.40-7.39(m, 1H), 7.38-7.36(m, 1H), 7.34-7.27(m , 3H), 7.23-7.22(m, 1H), 5.22(s, 1H), 4.67-4.65(m, 1H), 4.50-4.48(m, 1H), 3.99-3.87(m, 2H), 3.34(s, 2H), 2.89-2.51(m, 6H). 13 C NMR (150MHz, Chloroform- d)δ 166.8, 135.4, 134.7, 134.6, 133.7, 133.3, 133.2, 130.5, 130.4, 129.8, 129.6, 128.9, 128.4, 128.0, 12 8.0, 127.9, 127.8, 127.4, 127.3, 127.2, 126.4, 126.3, 126.1, 73.9, 71.3, 62.7, 53.6, 53.6, 53.0, 52.9. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 30 H 28 Cl3N2O2 + 553.1211, measured value 553.1202.

[0048] Compound 7c, white powder, mp 143-146℃, yield 68%. 1 H NMR (600MHz, Chloroform- d ) δ 7.84-7.81(m, 3H), 7.74(s, 1H), 7.57-7.55(m, 1H), 7.50-7.47(m, 3H), 7.40-7.39(m, 1H), 7.35-7.32(m, 3H), 6.89- 6.86 (m, 2H), 5.13 (s, 1H), 4.70-4.68 (m, 1H), 4.46-4.44 (m, 1H), 3.81 (s, 3H), 3.75-3.35 (m, 4H), 2.82-2.59 (m, 6H). 13 C NMR (150MHz, Chloroform- d )δ170.4, 160.9, 135.0, 134.3, 133.7, 133.3, 133.2, 132.1, 132.0, 129.5, 129.3, 128.9, 128.4, 128. 0, 127.9, 127.9, 127.2, 126.4, 126.2, 126.1, 113.8, 74.1, 71.2, 63.2, 55.5, 55.5, 53.5, 47.7, 42.2. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 31 H 31 Cl2N2O3 + 549.1706, measured value 549.1700.

[0049] Compound 7d, white powder, mp 140-143℃, yield 65%. 1H NMR (600MHz, Chloroform- d )δ7.79(s, 1H), 7.77(dd, J =8.1, 4.4Hz, 4H), 7.75-7.72(m, 2H), 7.63(s, 1H), 7.46-7.43(m, 2H), 7.41-7.36(m, 4H), 7.24(t, J= 1.8Hz, 1H), 7.18 (d, J =1.8Hz, 2H), 4.65 (d, J =12.2Hz, 1H), 4.46 (dd, J =8.3, 3.5Hz, 1H), 4.35(d, J =12.2Hz, 1H), 3.75(s, 2H), 3.38(s, 2H), 2.76(dd, J =13.6, 8.4Hz, 1H), 2.61-2.46(m, 3H), 2.42(dd, J =13.6, 3.7Hz, 1H), 2.30 (s, 1H). 13 CNMR (150MHz, Chloroform- d )δ 170.4, 144.5, 135.3, 134.9, 133.7, 133.2, 133.1, 133.0, 132.7, 128.4, 128.4, 128.2, 127.9, 127.8, 127.8 , 127.1, 126.9, 126.8, 126.7, 126.4, 126.2, 125.8, 125.4, 124.3, 77.9, 71.0, 64.7, 54.1, 53.2, 47.7, 42.2. HRMS(ESI-TOF)m / z:[M+H] + Calculated value C 34 H 31 Cl2N2O2 + 569.1757, measured value 569.1742.

[0050] Compound 7e, white powder, mp 141-144℃, yield 62%. 1 H NMR (600MHz, Chloroform- d )δ7.52-7.51(m, 1H), 7.41-7.37(m, 6H), 7.36-7.28(m, 6H), 5.01-5.00(m, 1H), 4.53(d, J =12.0Hz, 1H), 4.28 (d, J=12.0Hz, 1H), 3.80-3.73(m, 2H), 3.40(s, 2H), 2.75-2.71(m, 1H), 2.61-2.41(m, 5H). 13 C NMR (150MHz, Chloroform- d )δ 170.4, 137.7, 137.0, 136.0, 134.1, 133.7, 129.8, 129.4, 128.9, 128.6, 12 8.6, 128.2, 128.1, 127.8, 127.2, 74.4, 71.1, 63.4, 53.9, 53.1, 47.8, 42.3. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 26 H 27 Cl2N2O2 + 469.1444, measured value 469.1444.

[0051] Compound 7f, white powder, mp 147-150℃, yield 63%. 1 H NMR (600MHz, Chloroform- d ) δ 8.57-8.56(m, 1H), 7.83-7.80(m, 3H), 7.78-7.75(m, 1H), 7.74(s, 1H), 7.61-7.59(m, 1H), 7.57-7.56(m, 1H), 7.50-7.46(m, 3H ), 7.38-7.38(m, 1H), 7.33-7.30(m, 2H), 5.06-5.04(m, 1H), 4.71-4.69(m, 1H), 4.44-4.42(m, 1H), 3.84-3.75(m, 2H), 3.56(t, J =4.8Hz, 2H), 2.78-2.74(m, 1H), 2.67-2.58(m, 2H), 2.56-2.45(m, 3H). 13 C NMR (150MHz, Chloroform- d)δ167.6, 154.2, 148.5, 137.2, 137.0, 135.2, 134.1, 133.7, 133.3, 133.1, 129.4, 129.0, 128.3, 128. 0, 127.8, 127.8, 127.0, 126.4, 126.2, 126.1, 124.5, 123.9, 74.4, 71.1, 63.5, 53.8, 53.1, 47.3, 42.5. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 29 H 28 Cl2N3O2 + 520.1553, measured value 520.1553.

[0052] Compound 7g, white powder, mp 161-164℃, yield 65%. 1 H NMR (600MHz, Chloroform- d )δ8.60(d, J =5.7Hz, 2H), 7.79-7.73(m, 3H), 7.64(s, 1H), 7.43(q, J =4.9Hz, 2H), 7.40-7.36(m, 1H), 7.26(s, 1H), 7.19(s, 2H), 7.16(d, J =5.8Hz, 2H), 4.66(d, J =12.1Hz, 1H), 4.47(s, 1H), 4.36(d, J= 11.9Hz, 1H), 3.70(s, 2H), 3.25(s, 2H), 2.81-2.72(m, 1H), 2.62-2.35(m, 4H), 2.29(s, 1H). 13 C NMR (150MHz, Chloroform- d )δ 167.6, 150.3, 135.4, 134.8, 133.2, 133.1, 128.4, 128.3, 127.8, 127.8, 126.9, 126.4 , 126.3, 125.8, 125.4, 123.1, 121.2, 120.9, 77.7, 71.1, 64.4, 53.8, 53.1, 41.9, 29.7. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 29 H 28 Cl2N3O2 + 520.1553, measured value 520.1536.

[0053] Compound 7h, white powder, mp 155-158℃, yield 66%. 1 H NMR (600MHz, Chloroform- d )δ8.62-8.61(m, 2H), 7.45-7.44(m, 1H), 7.31-7.31(m, 1H), 7.29-7.27( m, 2H), 7.25-7.22 (m, 4H), 7.19-7.18 (m, 2H), 4.94-4.93 (m, 1H), 4.47-4. 45(m, 1H), 4.21-4.19(m, 1H), 3.74-3.63(m, 2H), 3.29-3.23(m, 2H), 2.6 8-2.64(m, 1H), 2.56-2.51(m, 2H), 2.46-2.44(m, 2H), 2.36-2.33(m, 1H). 13 C NMR (150MHz, Chloroform- d )δ 167.7, 150.4, 143.6, 137.7, 136.8, 134.2, 133.6, 129.4, 128.9, 128.6 , 128.2, 128.1, 127.8, 121.4, 74.4, 71.1, 63.3, 53.7, 52.9, 47.5, 42.2. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 25 H 26 Cl2N3O2 + 470.1397, measured value 470.1387.

[0054] Compound 7i, white powder, mp 174-177℃, yield 68%. 1 H NMR (600MHz, Chloroform- d )δ8.71(s, 2H), 7.50-7.49(m, 1H), 7.40-7.40(m, 1H), 7.34-7.33(m, 1H), 7.33-7.31(m, 2H), 7.28-7.27(m, 4H), 5.02-5.00(m, 1H), 4.50 -4.48(m, 1H), 4.29-4.27(m, 1H), 3.83-3.77(m, 2H), 3.37(s, 2H), 2.77-2.74(m, 1H), 2.69-2.65(m, 2H), 2.57-2.55(m, 2H), 2.46(s, 1H). 13 CNMR (150MHz, Chloroform- d)δ 167.8, 150.4, 143.5, 136.5, 136.2, 134.3, 133.9, 133.7, 129.5, 129.5, 129.4, 128.8 , 128.8, 127.9, 121.4, 74.8, 70.3, 63.3, 53.8, 53.1, 47.5, 42.1.HRMS (ESI-TOF) m / z: [M+H] + Calculated value C 25 H 25 Cl3N3O2 + 504.1007, measured value 504.0995.

[0055] Compound 11a, white powder, mp 155-158℃, yield 75%. 1 H NMR (600MHz, Chloroform- d )δ8.22(d, J =8.2Hz, 1H), 7.93 (d, J =7.9Hz, 1H), 7.60-7.50(m, 2H), 7.45(s, 1H), 7.40(d, J= 1.6Hz, 1H), 7.20 (s, 1H), 7.17-7.14 (m, 1H), 6.96 (s, 1H), 6.91 (s, 1H), 6.53 (dd, J= 6.2, 3.3Hz, 1H), 4.48-4.40 (m, 2H). 13 C NMR (175MHz, Chloroform- d )δ 158.9, 156.4, 153.3, 137.8, 136.9, 135.7, 132.5, 131.9, 129.8, 129.4, 128.1, 128.1, 128.0, 127.5, 125.8, 122.2, 119.9, 73.6, 49.8. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 19 H 14 Cl2N3O2S + 418.0178, measured value 418.0174.

[0056] Compound 11b, white powder, mp 158-161℃, yield 61%. 1 H NMR (600MHz, Chloroform- d )δ9.84(s, 1H), 7.74(d, J=8.1Hz, 1H), 7.53(s, 1H), 7.47(s, 1H), 7.42-7.36(m, 2H), 7.36-7.32(m, 1H), 7.20(d, J =8.0Hz, 1H), 7.18-7.17(m, 2H), 7.06(s, 1H), 6.88(s, 1H), 6.59-6.55(m, 1H), 4.51(d, J= 14.9Hz, 1H), 4.42 (dd, J= 15.0, 6.0 Hz, 1H). 13 C NMR (151MHz, Chloroform- d )δ160.3, 137.9, 137.6, 135.4, 132.7, 132.3, 129.7, 129.3, 128.1, 127. 9, 127.3, 126.1, 125.8, 122.7, 121.2, 120.0, 112.2, 109.8, 71.5, 49.8. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 20 H 16 Cl2N3O2 + 400.0614, measured value 400.0611.

[0057] Compound 11c, white powder, mp 143-146℃, yield 63%. 1 H NMR (700MHz, Chloroform- d )δ9.15(d, J =2.2Hz, 1H), 8.72 (dd, J =4.9, 1.7Hz, 1H), 8.16(dt, J =7.9, 2.0Hz, 1H), 7.35(d, J =2.1Hz, 1H), 7.33 (dd, J= 8.0, 4.8Hz, 1H), 7.26(s, 1H), 7.09(dd, J =8.4, 2.1Hz, 1H), 6.98(d, J = 8.4Hz, 1H), 6.90 (s, 1H), 6.73 (s, 1H), 6.45 (dd, J =6.3, 3.4Hz, 1H), 4.39(dd, J =15.0, 3.4Hz, 1H), 4.31(dd, J =15.0, 6.3 Hz, 1H).13 C NMR (175MHz, Chloroform- d )δ 163.6,154.3,150.8,137.7,137.3,135.6,132.6,132.4,129.9,129.7,128.0,127.9,125.0,123.7,119.7,72.2,49.7. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 17 H 14 Cl2N3O2 + 362.0458, measured value 362.0462.

[0058] Compound 11d, white powder, mp 155-158℃, yield 66%. 1 H NMR (600MHz, Chloroform- d )δ8.78-8.70(m, 1H), 8.01(d, J =7.8Hz, 1H), 7.80(td, J =7.7, 1.7Hz, 1H), 7.49-7.46(m, 1H), 7.45(s, 1H), 7.38(d, J =2.0Hz, 1H), 7.20(d, J =2.5Hz, 1H), 7.13(dd, J =8.4, 2.0Hz, 1H), 6.92(d, J =12.9Hz, 2H), 6.48 (dd, J =6.3, 3.5Hz, 1H), 4.45-4.35(m, 2H). 13 C NMR (150MHz, Chloroform- d )δ 163.7,150.2,147.0,137.9,137.2,135.4,132.6,132.5,129.7,129.5,128.1,127.9,127.6,125.5,119.9,72.6,49.8. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 17 H 14 Cl2N3O2 + 362.0458, measured value 362.0460.

[0059] Compound 11e, white powder, mp 150-153℃, yield 70%. 1H NMR (600MHz, Chloroform- d )δ7.94(d, J =8.0Hz, 2H), 7.49(t, J =7.4Hz, 1H), 7.36(t, J =7.6Hz, 2H), 7.29(d, J=16.2Hz, 2H), 7.04(d, J =8.5Hz, 1H), 6.98 (d, J =8.4Hz, 1H), 6.89 (s, 1H), 6.72 (s, 1H), 6.44-6.40 (m, 1H), 4.37 (dd, J= 15.0, 3.0Hz, 1H), 4.29(dd, J =14.9, 5.9 Hz, 1H). 13 C NMR (150MHz, Chloroform- d )δ 164.8, 137.8, 135.2, 133.9, 132.9, 132.4, 129.7, 129.6, 129.4, 128.9, 128.8, 128.0, 127.8, 119.9, 71.7, 49.7. HRMS (ESI-TOF) m / z: [M+H] + Calculated value C 18 H 15 Cl2N2O2 + 361.0505, measured value 361.0509.

[0060] Compound 11f, white powder, mp 161-164℃, yield 72%. 1 H NMR (700MHz, Chloroform- d )δ7.83-7.78(m, 2H), 7.56-7.53(m, 2H), 7.37(d, J =2.1Hz, 1H), 7.28(s, 1H), 7.10(dd, J= 8.4, 2.0 Hz, 1H), 6.96 (d, J= 8.4Hz, 1H), 6.92(s, 1H), 6.71(s, 1H), 6.44(dd, J =6.0, 3.4Hz, 1H), 4.41(dd, J =15.0, 3.4Hz, 1H), 4.32(dd, J =15.0, 6.0 Hz, 1H). 13 C NMR (175MHz, Chloroform-d )δ 164.2, 137.7, 135.5, 132.6, 132.5, 132.2, 131.2, 129.8, 129.6, 129.2, 128.0, 127.9, 127.8, 119.8, 71.9, 49.7. HRMS (ESI-TOF) m / z: [M+H] + Calculated value C 18 H 14 BrCl2N2O2 + 438.9610, measured value 438.9604.

[0061] Compound 11g, white powder, mp 170-173℃, yield 68%. 1 H NMR (600MHz, Chloroform- d ) δ 8.07-8.05(m, 2H), 7.45-7.45(m, 1H), 7.38(s, 1H), 7.18-7.14(m, 3H), 7.06-7.05(m, 1 H), 7.00(s, 1H), 6.80(s, 1H), 6.53-6.51(m, 1H), 4.50-4.47(m, 1H), 4.42-4.38(m, 1H). 13 C NMR (150MHz, Chloroform- d )δ 166.4(d, J =255.0Hz), 163.9, 137.9, 135.5, 132.7(d, J =37.5Hz), 132.4 (d, J =10.5Hz), 129.9, 129.7, 128.1, 128.0, 125.3(d, J =3.0Hz), 119.9, 116.2, 116.1, 71,9, 49.8. HRMS (ESI-TOF) m / z: [M+H] + Calculated value C 18 H 14 Cl2FN2O2 + 379.0411, measured value 379.0414.

[0062] Compound 11h, white powder, mp 162-165℃, yield 65%. 1 H NMR (600MHz, Chloroform- d )δ7.77(d, J =8.1Hz, 1H), 7.34 (d, J=2.0Hz, 1H), 7.28(s, 1H), 7.13(d, J= 7.1Hz, 1H), 7.06 (dd, J =8.4, 2.0Hz, 1H), 6.99(d, J =8.4Hz, 1H), 6.91(s, 1H), 6.75(s, 1H), 6.55(t, J= 7.7Hz, 1H), 6.39 (dd, J =5.9, 3.2Hz, 1H), 5.72(s, 1H), 4.36(dd, J= 14.9, 3.2Hz, 1H), 4.28(dd, J =14.9, 6.0Hz, 1H), 2.04(s, 3H). 13 C NMR (150MHz, Chloroform- d )δ 166.6, 149.8, 137.8, 135.7, 135.1, 133.3, 132.3, 129.6, 129.5, 128.6, 128.1, 127.9, 123.4, 119.9, 115.9, 108.5, 71.0, 49.8, 17.4. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 19 H 17 Cl2N2O2 + 375.0662, measured value 375.0660.

[0063] Compound 11i, white powder, mp 166-169℃, yield 70%. 1 H NMR (600MHz, Chloroform- d )δ7.56(d, J=7.7Hz, 1H), 7.47-7.44(m, 1H), 7.36-7.33(m, 2H), 7.30(t, J =8.0Hz, 1H), 7.07 (ddd, J =11.7, 8.4, 2.0Hz, 2H), 6.99(d, J= 8.4Hz, 1H), 6.93(s, 1H), 6.75(s, 1H), 6.43(dd, J =5.9, 3.3Hz, 1H), 4.40(dd, J =14.9, 3.3Hz, 1H), 4.32(dd, J= 14.9, 6.0Hz, 1H), 3.75 (s, 3H). 13CNMR (151MHz, Chloroform- d )δ 164.7, 159.8, 137.8, 135.3, 132.8, 132.4, 130.2, 129.9, 129.7, 129.2, 128.0, 127.9, 121.9, 120.1, 119.9, 114.5, 71.7, 55.5, 49.8. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 19 H 17 Cl2N2O3 + 391.0611, measured value 391.0611.

[0064] Compound 11j, white powder, mp 172-175℃, yield 66%. 1 H NMR (600MHz, Chloroform- d ) δ 8.44-8.42(m, 1H), 7.90-7.86(m, 2H), 7.69-7.62(m, 2H), 7.59-7.57(m, 2H), 7.48-7.48(m, 1H), 7.2 4-7.21 (m, 2H), 7.04 (s, 1H), 6.89 (s, 1H), 6.62-6.60 (m, 1H), 4.54-4.51 (m, 1H), 4.42-4.38 (m, 1H). 13 C NMR (150MHz, Chloroform- d )δ 165.8, 138.2, 135.6, 135.5, 132.7, 132.4, 132.4, 129.9, 129.1, 128.8, 12 8.7, 128.6, 128.5, 128.5, 128.3, 128.1, 125.5, 123.1, 120.2, 72.6, 50.1. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 22 H 16 BrCl2N2O2 + 488.9767, measured value 488.9758.

[0065] Compound 11K, white powder, mp 153-156℃, yield 73%. 1 H NMR (600MHz, Chloroform- d ) δ7.41-7.41(m, 1H), 7.30-7.28(m, 3H), 7.24-7.22(m, 1H), 7.18-7.17(m, 2H), 7.12(dd, J =8.4, 2.4Hz, 1H), 6.99(s, 1H), 6.81-6.79(m, 1H), 6.79(s, 1H), 6.27-6.25(m, 1H), 4.28-4.25(m 1H), 4.21-4.18(m 1H), 2.94(t, J =8.4Hz, 2H), 2.73(t, J =7.8, 2H). 13 C NMR (150MHz, Chloroform- d )δ 171.1, 139.8, 137.6, 135.2, 132.6, 132.4, 129.6, 129.0, 128.7, 128.3, 127.9, 127.8, 126.6, 119.8, 71.2, 49.7, 35.6, 30.7. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 20 H 19 Cl2N2O2 + 389.0818, measured value 389.0816.

[0066] Compound 11, white powder, mp 151-154℃, yield 66%. 1 H NMR (600MHz, Chloroform- d )δ7.43-7.43(m, 1H), 7.41(s, 1H), 7.30-7.27(m, 2H), 7.22-7.19(m, 2H), 7.15-7.13(m, 2H), 7. 03-7.02(m, 2H), 6.79(s, 1H), 6.30-6.28(m, 1H), 4.34-4.31(m, 1H), 4.25-4.21(m, 1H), 2.62(t, J =7.8Hz, 2H), 2.39(td J =7.8, 2.4Hz, 2H), 1.96-1.91(m, 2H). 13 C NMR (150MHz, Chloroform- d)δ 171.8, 141.0, 137.7, 135.4, 133.0, 132.6, 129.8, 129.2, 128.6, 128.6, 128.0, 128.0, 126.3, 119.9, 71.1, 50.0, 35.0, 33.4, 26.2. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 21 H 21 Cl2N2O2 + 403.0975, measured value 403.0973.

[0067] Compound 11m, white powder, mp 171-174℃, yield 69%. 1 H NMR (600MHz, Chloroform- d ) δ 8.19(d, J =12.0Hz, 1H), 7.66-7.65(m, 1H), 7.46-7.44(m, 2H), 7.42(s, 1H), 7.36(td, J =7.8, 1.2Hz 1H), 7.31(td, J =7.2, 1.2Hz 1H), 7.21 (dd, J =8.4, 2.4Hz, 1H), 7.08-7.06 (m, 1H), 7.04 (s, 1H), 6.85 (s, 1H), 6.48 (d, J =12.0Hz, 1H), 6.43-6.42(m, 1H), 4.47-4.44(m, 1H), 4.38-4.34(m, 1H). 13 C NMR (150MHz, Chloroform- d )δ 164.7, 142.7, 137.8, 135.4, 135.4, 132.9, 132.6, 132.2, 131.9, 130.5 , 129.8, 129.4, 128.1, 128.0, 127.9, 127.4, 120.0, 119.1, 71.5, 49.9. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 20 H 16 Cl3N2O2 + 421.0272, measured value 421.0272.

[0068] Compound 11n, white powder, mp 173-176℃, yield 75%. 1H NMR (600MHz, Chloroform- d ) δ 7.67(d, J =16.2Hz, 1H), 7.45-7.45(m, 1H), 7.40-7.37(m, 2H), 7.32-7.31(m, 1H), 7.26-7.24(m 1H), 7.20(dd, J =8.4, 1.8Hz, 1H), 7.12(td, J =8.4, 2.4Hz 1H), 7.09-7.07 (m, 1H), 7.03 (s, 1H), 6.82 (s, 1H), 6.48 (d, J =15.6Hz, 1H), 6.43-6.41(m, 1H), f4.45-4.42(m, 1H), 4.35-4.32(m, 1H). 13 C NMR (150MHz, Chloroform- d )δ 164.9, 163.1(d, J =259.1Hz), 145.6 (d, J =3.0Hz), 137.9, 136.1 (d, J =9.0Hz), 135.4, 132.6 (d, J =48.0Hz), 130.8 (d, J =9.0Hz), 129.8, 129.6, 128.1, 128.0, 124.5(d, J =3.0Hz), 119.9, 118.0, 117.9, 117.9, 114.7(d, J =22.5Hz), 71.5, 49.9. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 20 H 16 Cl2FN2O2 + 405.0567, measured value 405.0563.

[0069] Compound 11o, white powder, mp 163-166℃, yield 59%. 1 H NMR (600MHz, Chloroform- d ) δ 7.45-7.43(m, 1H), 7.41-7.39(m, 1H), 7.31-7.26(m, 2H), 7.25-7.23(m, 1H), 7.23-7.22(m, 1H), 7.13(dd,J =8.4, 1.8Hz, 1H), 6.96(s, 1H), 6.84(d, J =8.4Hz, 1H), 6.59 (s, 1H), 6.30-6.28 (m, 1H), 4.30-4.27 (m, 1H), 4.19-4.16 (m, 1H), 3.85 (s, 1H). 13 C NMR (150MHz, Chloroform- d )δ 168.7, 137.6, 135.4, 134.6, 132.5, 132.4, 131.7, 131.7, 129.9, 129.7, 129.4, 128.8, 127.9, 127.9, 127.4, 119.8, 71.8, 49.8, 39.3. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 19 H 16 Cl3N2O2 + 409.0272, measured value 409.0270.

[0070] Compound 11p, white powder, mp 175-178℃, yield 70%. 1 H NMR (600MHz, Chloroform- d )δ7.41-7.41(m, 1H), 7.31(s, 1H), 7.21-7.17(m, 2H), 7.12(dd, J =8.4, 2.4Hz, 1H), 7.05-7.02(m, 2H), 6.98(s, 2H), 6.8(d, J =8.4Hz, 1H), 6.65(s, 1H), 6.28-6.27(m, 2H), 4.31-4.28(m, 1H), 4.20-4.16(m, 1H), 3.65(s, 1H). 13 C NMR (150MHz, Chloroform- d )δ 169.6, 162.4(d, J =244.5Hz), 137.7, 135.5, 132.6(d, J =7.5Hz), 131.0, 130.0, 129.8, 129.2, 128.8(d, J =3.0Hz), 127.8, (d, J =21.0Hz), 119.7, 115.9 (d, J =21.0Hz), 115.4 (d, J=21.0Hz), 71.6, 49.8, 40.6. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 19 H 16 Cl2FN2O2 + 393.0567, measured value 393.0569.

[0071] Compound 11q, white powder, mp 170-173℃, yield 68%. 1 H NMR (600MHz, Chloroform- d ) δ 7.49-7.47 (m, 2H), 7.41 (d, J =1.8Hz, 1H), 7.30(s, 1H), 7.14(dd, J =8.4, 1.8Hz, 1H), 7.11-7.09(m, 2H), 6.99(s, 1H), 6.81(d, J =8.4Hz, 1H), 6.66(s, 1H), 6.29-6.27(m, 1H), 4.31-4.28(m, 1H), 4.21-4.17(m, 1H), 3.63(s, 2H). 13 C NMR (150MHz, Chloroform- d )δ 169.3, 137.7, 135.5, 132.6, 132.1, 132.0, 131.1, 129.9, 129.4, 127.9, 127.8, 121.8, 119.7, 71.8, 49.8, 40.8. HRMS (ESI-TOF) m / z: [M+H] + Calculated value C 19 H 16 BrCl2N2O2 + 452.9767, measured value 452.9762.

[0072] Compound 11r, white powder, mp 157-160℃, yield 72%. 1 H NMR (600MHz, Chloroform- d )δ8.18(d, J =8.2Hz, 1H), 7.94-7.86 (m, 1H), 7.51 (s, 2H), 7.50 (dd, J =7.6, 1.3Hz, 1H), 7.48-7.44(m, 1H), 7.41(d, J =8.4Hz, 1H), 7.38 (d, J=2.0Hz, 1H), 7.21(dd, J= 8.4, 2.1Hz, 1H), 6.83(dd, J =7.3, 4.4Hz, 1H), 4.97-4.90 (m, 2H). 13 C NMR (150MHz, Chloroform- d )δ 158.7, 156.7, 153.3, 136.8, 135.6, 135.0, 134.53, 133.25, 132.20, 12 9.86, 129.28, 128.58, 127.90, 127.25, 125.72, 122.09, 72.87, 56.87. HRMS(ESI-TOF)m / z: [M+H] + Calculated value C 18 H 13 Cl2N4O2S + 419.0131, measured value 419.0123.

[0073] The results of the agonistic activity of compounds 7a-i and 11a-r on ULK1, the inhibitory activity on ERK1 / 2, and the antiproliferative activity on triple-negative breast cancer cells are shown in Tables 1-5.

[0074] Table 1: Activity of compound 7a-i on ULK1 kinase activation and ERK1 / 2 inhibition and antiproliferative activity Table 2: Continuation of Table 1 Table 3: Activity of compound 11a-r on ULK1 kinase activation and ERK1 / 2 inhibition and antiproliferative activity Table 4: Continuation of Table 3 Table 5: Continuation of Table 4 This invention uses benzoyl chloride derivatives as raw materials to prepare compounds of formula I and formula II, and modifies different parts of the hydrogenated pyrazine compound in formula I, in R 2 When various substituted phenyl groups and naphthyl ring structures are introduced, the cells exhibit lower ERK1 inhibitory activity and ULK1 activating activity, and show poor antiproliferative activity against MDA-MB-231, MCF-7, and BT549 cells; additionally, when R... 2When R is a heterocyclic group such as pyridine, it can significantly enhance the inhibitory activity of ERK1 and the activating activity of ULK1, but has little effect on its anti-proliferative activity. Furthermore, in Formula II, when R... 3 When R is an imidazole group, 4 Introducing heterocyclic aromatic hydrocarbons such as 2-indole, 3-pyridine, and 2-pyridine results in moderate ERK1 inhibition and ULK1 activation activity, but the yields of these compounds are relatively low; R 4 Replacing the phenyl group with an electron-withdrawing substituted phenyl group helps to improve the activity of the kinase, especially the para-substituted bromine atom, which shows excellent performance in ULK1 and ERK1 enzyme activities (ULK1 kinase activation rate of 610.30% and ERK1 kinase inhibition rate of 28.52%).

[0075] The final experimental results showed that the compounds of this invention exhibit strong agonistic activity against ULK1 and strong inhibitory activity against ERK1 / 2, as well as strong anti-proliferative activity against triple-negative breast cancer cells. Compound UE01 exhibited the strongest agonistic activity against ULK1 and the strongest inhibitory activity against ERK1 / 2, and also the strongest anti-proliferative activity against triple-negative breast cancer cells.

[0076] The test compound 11f, namely UE01, exhibited agonistic activity against ULK1, inhibitory activity against ERK1 / 2, and antitumor activity.

[0077] 1. The IC50 values ​​of UE01 in two TNBC cell lines, MDA-MB-231 and BT-549, were detected by MTT assay. The results are as follows: Figure 1 As shown in Figure A, the IC50 of UE01 against MDA-MB-231 cells was 21.86 μM, and the IC50 against BT-549 cells was 24.24 μM. Meanwhile, the IC50 values ​​of the positive control drugs LYN-1604 and SCH772984 in MDA-MB-231 cells were 20.15 μM and 41.29 μM, respectively, indicating that the short-term antiproliferative activity of UE01 against TNBC cells was similar to that of LYN-1604, but significantly superior to that of SCH772984.

[0078] Cloning experiment results as follows Figure 1 As shown in Figure B, the number of cell clones formed decreased significantly with increasing UE01 concentration. Furthermore, the inhibitory effect of medium concentration UE01 on clone formation was not significantly different from that of LYN-1604, but was significantly stronger than that of SCH772984. These results indicate that UE01 can inhibit the proliferation of MDA-MB-231 cells in a concentration-dependent manner, and the long-term inhibitory effect of medium concentration UE01 on proliferation is comparable to that of LYN-1604, but stronger than that of SCH772984.

[0079] 3D tumor sphere experimental results are as follows Figure 2As shown, tumor spheres decreased significantly with increasing UE01 concentration, but there was no significant difference in tumor sphere size between the two positive controls under medium concentration UE01 treatment. This indicates that UE01 can inhibit MDA-MB-231 cell proliferation in a concentration-dependent manner in a three-dimensional environment, but its effect on TNBC cell proliferation may require further analysis using live / dead cell staining. EdU experimental results are shown below. Figure 3 As shown, UE01 treatment significantly reduced the EdU positivity rate, and its EdU positivity rate was not significantly different from that of the LYN-1604 group, but significantly lower than that of the SCH772984 group. This indicates that UE01 can inhibit the proliferation of MDA-MB-231 cells by inhibiting DNA synthesis.

[0080] The above results indicate that UE01 can significantly inhibit the proliferation of triple-negative breast cancer cells, and its short-term and long-term proliferation inhibition effects are superior to the positive control SCH772984 and comparable to LYN-1604.

[0081] 2. To further evaluate the anti-proliferative effect of UE01 in vivo, a subcutaneous tumor-bearing mouse model was established by subcutaneously inoculating nude mice with MDA-MB-231-Luc cells: Three-week-old BALB / c-nude mice were introduced into their cages and acclimatized for one week. Sufficient MDA-MB-231-Luc cells were expanded, and the cells were digested with trypsin, resuspended in PBS, counted, and the cell density was adjusted to 1.2*102. 7 Cell suspension was injected subcutaneously into the axilla of nude mice at a volume of 100 μL per mouse. Tumor growth was observed after inoculation. Two weeks later, mice were randomly divided into groups of 10 and began gavage administration (once daily). Tumor volume was recorded using calipers (V=L*W² / 2), and mouse weight was recorded every three days. Administration was stopped after 13 days. On day 14, the subcutaneous tumor and internal organs were dissected. The tumor volume was considered normal when it reached approximately 100 mm². 3 Mice that successfully developed the model were randomly divided into six groups and administered the drugs via gavage. The dosages were UE01 (low dose: 25 mg / kg; medium dose: 50 mg / kg; high dose: 100 mg / kg), LYN-1604 (50 mg / kg), and SCH772984 (100 mg / kg). The drugs were dissolved in physiological saline before gavage. The administration lasted for 13 days. Changes in subcutaneous tumors were observed using in vivo imaging on days 0, 7, and 14. On day 14, the mice were sacrificed and dissected, and venous blood, internal organs, and tumor fragments were collected for subsequent experiments.

[0082] Tumor volume, tumor weight, and changes in mouse body weight during drug administration are as follows: Figure 4As shown, the tumor volume and weight decreased significantly with increasing UE01 dosage, indicating that UE01 can effectively inhibit TNBC proliferation in vivo. Notably, the in vivo antiproliferative activity of UE01 is close to that of LYN-1604 and stronger than that of SCH772984, which is consistent with the in vitro activity results. Furthermore, changes in mouse body weight revealed that low, medium, and high doses of UE01 and SCH772984 did not cause significant changes in mouse body weight, but LYN-1604 led to a significant decrease in mouse body weight, suggesting that LYN-1604 may have produced significant toxic side effects in vivo.

[0083] In vivo imaging results of subcutaneous xenograft tumors are as follows Figure 5 As shown, compared with the Control group, both UE01 and the positive control group significantly inhibited tumor growth. Furthermore, the trend of cumulative fluorescence intensity of the tumor over time showed that the inhibitory effects of UE01 (medium dose) and LYN-1604 were significantly stronger than those of SCH772984.

[0084] Ki-67 is a biomarker closely related to tumor cell proliferation activity. Its high expression is positively correlated with tumor malignancy, generally indicating stronger tumor cell proliferation. Ki-67 immunohistochemical staining was performed on mouse tumor tissue, and the results were as follows... Figure 6 As shown, compared with the control group, UE01 significantly reduced Ki-67 expression levels. Simultaneously, the Ki-67 protein levels in the positive controls LYN-1604 and SCH772984 were also significantly downregulated. However, the Ki-67 positivity rate of LYN-1604 was not significantly different from that of UE01, while the Ki-67 positivity rate of SCH772984 was significantly higher than that of UE01. These results indicate that UE01 significantly inhibited the malignant proliferation ability of TNBC, with an inhibitory effect comparable to that of LYN-1604, but significantly stronger than that of SCH772984.

[0085] Next, immunohistochemical staining of the tumor tissue with p-ULK1S317 and p-ERK1 / 2 was performed, and the results were as follows: Figure 7 As shown, the p-ULK1 positivity rate of UE01 was significantly higher than that of the model group, but significantly lower than that of LYN-1604; while the p-ERK1 / 2 positivity rate of UE01 was significantly lower than that of the model group, but significantly higher than that of SCH772984. These results indicate that UE01 can effectively activate ULK1 and inhibit ERK1 / 2 activity in vivo, but this activation and inhibition effects are weaker than those of the two positive control groups.

[0086] To further investigate the in vivo toxicity of UE01 and the positive control, blood biochemistry tests were performed on mouse venous blood, and hematoxylin-eosin (HE) staining was performed on the five internal organs of mice. The results of the blood biochemistry tests are shown in Table 6 and... Figure 8 As shown, seven liver and kidney function-related indicators—alanine transaminase (ALT), aspartate transaminase (AST), total protein (TP), albumin (ALB), creatinine (CREA), urea (UREA), and total cholesterol (TC)—did not show significant changes compared to the model group after administration of UE01 and SCH772984, and there were no significant differences between different dose groups of UE01. This indicates that UE01 and SCH772984 did not cause significant liver and kidney toxicity at the experimental doses. However, after administration of LYN-1604, serum ALT levels in mice were significantly upregulated compared to the model group, and AST levels were significantly upregulated compared to the medium-dose UE01 group. The other liver and kidney function indicators did not show significant changes compared to the control group, suggesting that LYN-1604 may have induced drug-induced liver injury. Notably, serum ALT and AST levels after UE01 administration were significantly lower than those after LYN-1604 administration, indicating that the liver and kidney toxicity of UE01 was significantly reduced compared to LYN-1604.

[0087] Table 6: Results of serum biochemical assays in mice Data are expressed as mean ± SD.

[0088] HE staining results are as follows Figure 9 As shown, HE staining of the heart, spleen, lungs, and kidneys in the UE01 and two positive drug treatment groups showed no significant abnormalities. However, HE staining of the liver in the high-dose UE01 and LYN-1604 groups showed significant pathological changes compared to the control group. In HE-stained sections of the high-dose UE01 group, disordered liver lobule structure was observed, hepatocytes around the central vein no longer arranged radially in an orderly manner, and hepatocytes showed significant degeneration, characterized by loose cytoplasm with vacuoles and obvious irregular deformation. In HE-stained sections of LYN-1604, more severe pathological changes were observed. In addition to the above-mentioned manifestations in the high-dose UE01 group, focal hepatocyte necrosis was also clearly observed. The hepatocytes in the necrotic areas lacked normal cell outlines, and the nuclei dissolved and fragmented, appearing as a patch of eosinophilic, unstructured material.

[0089] 3. Knockdown of ULK1 and ERK1 was performed separately, followed by clonogenic assays and 3D tumor sphere assays to observe whether the TNBC proliferation inhibition effect of UE01 could be reversed. Results are as follows: Figure 10As shown, in both the clonogenic and 3D tumor sphere experiments, the anti-proliferative effect of adding UE01 after knocking down ULK1 or ERK1 was significantly weakened compared to the UE01 group. This indicates that UE01 inhibits the proliferation of triple-negative breast cancer by targeting and regulating ULK1 and ERK1 / 2.

[0090] In addition to detecting metastasis-related indicators in the tumor tissue of subcutaneous xenografts, to better evaluate the anti-metastatic effect of UE01 in vivo, a TNBC lung metastasis model was constructed by tail vein injection of MDA-MB-231-Luc cell line with luciferase tagging to observe the effect of drug administration on the degree of lung metastasis. Results are as follows: Figure 11 As shown, with increasing UE01 dosage, the cumulative fluorescence intensity in the mouse lungs significantly decreased, and the fluorescence intensity in the lungs of the two positive control groups was also significantly lower than that of the negative control group. Furthermore, consistent with in vitro experiments, the fluorescence intensity in the lungs of mice treated with a medium dose of UE01 was significantly lower than that of the LYN-1604 and SCH772984 groups, indicating that UE01 has a superior anti-metastatic effect in the TNBC lung metastasis mouse model compared to the positive control.

[0091] In addition, bioluminescence imaging was performed on the mouse's internal organs after dissection. The results are as follows: Figure 12 As shown, significant fluorescence accumulation was observed only in the lungs, and the relative trend of accumulated fluorescence intensity was basically consistent with the in vivo imaging results above, which also indicates that UE01 has excellent anti-TNBC metastasis effect in vivo.

[0092] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0093] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0094] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A compound, characterized in that, The compound has the structural formula shown in Formula I or Formula II: Equation I is Equation II is ; Among them, R 1 Selected from one of the following groups: , and ;R 2 Selected from one of the following groups: , , , , , , , and ;R 3 Selected from one of the following groups: and ;R 4 Selected from one of the following groups: , , , , , , , , , , , , , , , , and .

2. A salt of the compound of claim 1.

3. An isomer of the compound according to claim 1.

4. The use of the compound of claim 1, the salt of claim 2, or the isomer of claim 3 in the preparation of a therapeutic agent for triple-negative breast cancer.

5. The application according to claim 4, characterized in that, The therapeutic drug is composed of an active ingredient and excipients, wherein the active ingredient is the compound, the salt, or the isomer.

6. The application according to claim 5, characterized in that, The excipients include a diluent.

7. The application according to claim 6, characterized in that, The diluent includes physiological saline.

8. The application according to claim 4, characterized in that, The therapeutic drug is used to inhibit the proliferation and metastasis of triple-negative breast cancer.

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