A 3,4-dihydroquinoxalin-2-one / acetophenone hybrid compound, a preparation method and application thereof

Abstract

The application discloses a 3,4-dihydroquinoxalin-2-one / acetophenone hybrid compound and a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The compound is shown as formula (I) or formula (II). The application further provides a pharmaceutical composition containing the compound, application of the compound in preparation of a medicine for preventing and / or treating tumors, and a preparation method of the compound. The compound of the application shows excellent anti-proliferation activity (IC 50 =0.50±0.01 μM) on triple-negative breast cancer cell strain MDA-MB-468, and low toxicity on normal cells. Further research shows that the compound plays an anti-tumor role by targeting HSP70 protein, blocking the interaction between HSP70 and EGFR, and then inhibiting the PI3K-AKT signal pathway. In addition, the compound and 12-hydroxyamoorastatin (HAR) have a synergistic anti-tumor effect. The application provides a new candidate compound and treatment strategy for triple-negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a novel class of 3,4-dihydroquinoxalin-2-one / acetophenone hybrid compounds, pharmaceutical compositions comprising these compounds, methods for their preparation, and their use in the preparation of medicaments for the prevention and / or treatment of tumors (particularly triple-negative breast cancer). Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women. Triple-negative breast cancer (TNBC) is characterized by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), making it insensitive to endocrine and targeted therapies, limiting clinical treatment options, and resulting in a very poor prognosis. Therefore, developing novel therapeutics for TNBC is of significant clinical importance.

[0003] Heat shock protein 70 (HSP70) is a highly conserved molecular chaperone protein that plays a crucial role in cell protection, protein folding, assembly, and degradation. Studies have shown that HSP70 is highly expressed in various tumor cell lines and, through interactions with multiple client proteins (such as EGFR), promotes tumor cell proliferation, invasion, and metastasis while inhibiting apoptosis. Therefore, HSP70 has become a promising target for anti-tumor drugs. However, no drugs targeting HSP70 have yet been successfully marketed, and the development of highly effective and low-toxicity HSP70 inhibitors remains a hot topic in drug development.

[0004] Therefore, designing and synthesizing novel, efficient, and low-toxicity HSP70 inhibitors to develop new anti-tumor drugs, especially drugs against triple-negative breast cancer, has important clinical significance and scientific value. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a novel class of 3,4-dihydroquinoxalin-2-one / acetophenone hybrid compounds.

[0006] Another object of the present invention is to provide a pharmaceutical composition comprising the said compound.

[0007] Another object of the present invention is to provide a method for preparing the said compound.

[0008] Another object of the present invention is to provide the use of the compound in the preparation of medicaments for the prevention and / or treatment of tumors, particularly in the preparation of medicaments for the treatment of triple-negative breast cancer.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a 3,4-dihydroquinoxalin-2-one / acetophenone hybrid compound as shown in formula (I) or (II), or a pharmaceutically acceptable salt thereof: .

[0010] Preferably, the pharmaceutically acceptable salt may be a hydrochloride, sulfate, p-toluenesulfonate, phosphate, maleate, or fumarate, which will not be listed here.

[0011] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0012] Preferably, the pharmaceutical composition further comprises 12-hydroxyamoorastatin (hereinafter referred to as HAR). HAR is a limonene triterpenoid compound with CAS number 71590-47-1 and its chemical structural formula is as follows:

[0013] Thirdly, the present invention provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof, or the above-mentioned pharmaceutical compositions, in the preparation of medicaments for the prevention and / or treatment of tumors.

[0014] Preferably, the tumor is breast cancer; more preferably, the breast cancer is triple-negative breast cancer.

[0015] Preferably, the drug exerts its effect through at least one of the following mechanisms: (a) Inhibits tumor cell proliferation; (b) Targeting the HSP70 protein; (c) Inhibits the interaction between HSP70 and EGFR; (d) Inhibit phosphorylation activation of the PI3K-AKT signaling pathway.

[0016] Fourthly, the present invention provides the use of the compound of formula (I) above or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating triple-negative breast cancer, said medicament further comprising HAR.

[0017] Preferably, the molar ratio of compound (I) to HAR is 0.31 - 22:0.04.

[0018] Preferably, the drug further includes pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, and lubricants. Examples of combinations of at least two include, for instance, a combination of binders and excipients, a combination of binders and flavoring agents, a combination of binders and fillers, etc. Other combinations are also acceptable and will not be elaborated upon here.

[0019] Preferably, the dosage form of the drug may be tablets, capsules, granules, injections, oral liquids, pills, ointments, suspensions, dispersants, syrups, or patches.

[0020] Fifthly, the present invention provides a method for preparing compounds of formula (I) or formula (II) as described above, comprising the following steps: (1) The acetophenone derivative was reacted with dimethyl oxalate under alkaline conditions to obtain enol intermediate B; (2) The enol intermediate B obtained in step (1) is reacted with the o-diaminobenzene derivative C in an organic solvent to obtain the compound of formula (I) or formula (II).

[0021] Preferably, in step (1), the acetophenone derivative A is selected from 4-fluoroacetophenone or 3-cyclopentoxyacetophenone; the alkaline condition is the use of sodium methoxide; preferably, the concentration of sodium methoxide is 0.5-2 mol / L, more preferably 1 mol / L; the molar ratio of the acetophenone derivative A to sodium methoxide is 1:2.

[0022] The molar ratio of acetophenone derivative A to dimethyl oxalate is 1:1-5, more preferably 1:2; the reaction is carried out under argon protection at 70-100℃ for 1-5 hours, more preferably under reflux at 70℃.

[0023] Preferably, in step (2), the o-diaminobenzene derivative C is selected from o-phenylenediamine or 4,5-difluoroo-phenylenediamine; the organic solvent is selected from benzene, toluene or methanol; the reaction is carried out under argon protection at 60-100℃ for 12-24h; more preferably, the reaction is carried out at 80℃ for 3-12h; the molar ratio of the enol intermediate B to the o-diaminobenzene derivative C is 1:1-3, more preferably 1:1.

[0024] Compared with the prior art, the present invention has the following significant advantages: Novel compound entities: This study provides a new class of 3,4-dihydroquinoxalin-2-one / acetophenone hybrid compounds, enriching the library of antitumor drug compounds.

[0025] Excellent antitumor activity: The compounds of this invention exhibit excellent antiproliferative activity (IC50) against the triple-negative breast cancer cell line MDA-MB-468. 50 = 0.50±0.01μM), and has low toxicity to normal cells and a high safety factor.

[0026] Clear target and mechanism of action: For the first time, it has been confirmed that the compound of this invention exerts its anti-tumor effect by targeting the HSP70 protein, blocking the interaction between HSP70 and EGFR, and thereby inhibiting the downstream PI3K-AKT signaling pathway, providing a new target and strategy for the treatment of TNBC.

[0027] Synergistic combination therapy: When the compound of this invention is used in combination with HAR, it exhibits a strong synergistic antitumor effect both in vitro and in vivo, providing a new option for clinical combination therapy.

[0028] The preparation method is simple: the raw materials for the preparation of the compounds of this invention are readily available, the reaction conditions are mild, the operation is simple, and the yield is high, making it suitable for industrial production. Attached Figure Description

[0029] Figure 1 The results show the in vitro antiproliferative activity of 3,4-dihydroquinoxalin-2-one / acetophenone hybrids against different breast cancer cells and normal cells, where A represents the results of hybrid (I) and B represents the results of hybrid (II). Figure 2 The effect of 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (I) on the colony formation of triple-negative breast cancer cells, where A represents the results of the plate colony assay and B represents the results of Image J quantification (*** P < 0.001, **** P < 0.0001). Figure 3 The results confirm the HSP70 target of the heterozygote (Ⅰ), where A is the result of the cell thermal displacement experiment, B is the result of the LiP-MS experiment, and C is the result of the SPR experiment. Figure 4 The results are for verifying the mechanism of action of the heterozygote (Ⅰ), where A is the result of the CO-IP experiment, B is the expression of related proteins, and C is the relative quantitative analysis of expression levels performed by Image J.

[0030] Figure 5 The in vitro evaluation of the combination of heterozygote (Ⅰ) and triterpenoid compound HAR is shown in A, which represents the combined effect on MDA-MB-468 cells, and B represents the combined effect on 4T1 cells.

[0031] Figure 6The in vivo anti-triple-negative breast cancer activity of the heterozygote (Ⅰ) alone or in combination with the triterpenoid compound HAR is evaluated. In the figure, A represents tumor morphology, B represents the statistical change in tumor volume during the dosing cycle, C represents the statistical change in tumor weight of mice after the dosing cycle, and D represents the statistical change in body weight of mice (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 5). Figure 7 Liver and kidney tissue sections of experimental mice 18 days after treatment with heterozygote (I); Figure 8 The serum ALT (A), AST (B), CREA-S (C), and UREA (D) levels in experimental mice were measured 18 days after treatment with heterozygote (I). (* P < 0.05, **** P < 0.0001, n = 5). Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0033] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0034] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0035] The features and performance of the present invention will be further described in detail below with reference to the embodiments. Table 1 shows the source and specifications of the main raw materials and reagents in the following embodiments.

[0036] Table 1. Sources of main raw materials and reagents

[0037] Example 13, Preparation of 4-dihydroquinoxalin-2-one / acetophenone hybrids (I) or (II) Weigh the desired acetophenone derivative (4-fluoroacetophenone or 3-cyclopentoxyacetophenone) and dimethyl oxalate into a round-bottom flask, add sodium methoxide (1 M in MeOH) to dissolve them completely, and stir the mixture under reflux at 70°C for 5 min to 5 h. Monitor the reaction process using TLC until the reactants are completely consumed. Quench with distilled water, thoroughly dissolve the solid, adjust the pH to acidic 3-4 with 1N HCl, filter with filter paper, wash with methanol, and collect the white solid on the filter paper to obtain pure enol intermediate B.

[0038] Enol intermediate B and o-diaminobenzene derivative C were dissolved in benzene. The reaction was carried out under argon protection at 80°C for 3-12 h with stirring. The reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (I) or (II).

[0039] Characterization of heterozygote (I) 1 H NMR and 13 The C NMR data are shown in Table 2.

[0040]

[0041] Table 2. 3,4-Dihydroquinoxalin-2-one heterozygotes (I) 1 H and 13 C NMR (DMSO-) d6 )data

[0042] Characterization of hybrid compound (II) 1 H NMR and 13 The C NMR data are shown in Table 3.

[0043]

[0044] Table 3. 3,4-Dihydroquinoxalin-2-one heterozygotes (II) 1 H and 13 C NMR (DMSO-) d6 )data

[0045] Example 2 Preparation of Heteropolymer (I) Enol Intermediate B1

[0046] Weigh the required 4-fluoroacetophenone A1 (100 mg, 0.7 mmol) and dimethyl oxalate (171 mg, 1.4 mmol) into a round-bottom flask, add sodium methoxide (1 M in MeOH, 1.4 ml, 1.4 mmol) and dissolve thoroughly. Stir the mixture for 5 min under reflux at 70 °C under argon protection. Monitor the reaction process using TLC until the reactants are completely consumed. Quench with distilled water, thoroughly dissolve the solid, adjust the pH to acidic 3-4 with 1N HCl, filter with filter paper, wash with methanol, and collect the white solid on the filter paper to obtain pure enol intermediate B1 (yield: 95%).

[0047] Weigh the required amounts of 4-fluoroacetophenone A1 (300 mg, 2.2 mmol) and dimethyl oxalate (513 mg, 4.3 mmol) into a round-bottom flask. Add sodium methoxide (1 M in MeOH, 4.3 ml, 4.3 mmol) and dissolve thoroughly. Stir the mixture for 5 min under reflux at 70°C with argon protection. Monitor the reaction process using TLC until the reactants are completely consumed. Quench with distilled water, thoroughly dissolve the solid, and adjust the pH to acidic 3-4 with 1N HCl. Filter the mixture through filter paper, wash with methanol, and collect the white solid on the filter paper to obtain pure enol intermediate B1 (yield: 90%).

[0048] Example 3 Preparation of heterozygous (II) enol intermediate B2

[0049] Weigh the required amount of 3-cyclopentoxyacetophenone A2 (227 mg, 1.1 mmol) and dimethyl oxalate (262 mg, 2.2 mmol) into a round-bottom flask, add sodium methoxide (1 M in MeOH, 2.2 ml, 2.2 mmol) and dissolve thoroughly. Stir the mixture at 70°C under reflux argon protection for 3 h. Monitor the reaction process using TLC until the reactants are completely consumed. Quench with distilled water, thoroughly dissolve the solid, adjust the pH to acidic 3-4 with 1N HCl, filter with filter paper, wash with methanol, and collect the white solid on the filter paper to obtain pure enol intermediate B2 (yield: 99%).

[0050] Weigh the required amount of 3-cyclopentoxyacetophenone A2 (454 mg, 2.2 mmol) and dimethyl oxalate (524 mg, 4.4 mmol) into a round-bottom flask, add sodium methoxide (1 M in MeOH, 11 ml, 4.4 mmol) and dissolve thoroughly. Stir the mixture for 5 min under reflux at 70 °C for argon protection. Monitor the reaction process using TLC until the reactants are completely consumed. Quench with distilled water, thoroughly dissolve the solid, adjust the pH to acidic 3-4 with 1N HCl, filter with filter paper, wash with methanol, and collect the white solid on the filter paper to obtain pure enol intermediate B2 (yield: 97%).

[0051] Example 4 Preparation of hybrid (Ⅰ)

[0052] Enol intermediate B1 (100 mg, 0.4 mmol) and o-diphenylamine C1 (48 mg, 0.4 mmol) were dissolved in benzene (1.5 ml). The reaction was stirred for 3 h under argon protection at 80 °C, and the reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (Ⅰ) (yield: 90%).

[0053] Enol intermediate B1 (300 mg, 1.3 mmol) and o-diphenylamine C1 (144 mg, 1.3 mmol) were dissolved in benzene (4.5 ml). The reaction was stirred for 3 h under argon protection at 80 °C, and the reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (Ⅰ) (yield: 95%).

[0054] Example 5 Preparation of hybrid (Ⅰ)

[0055] Enol intermediate B1 (300 mg, 1.3 mmol) and o-diphenylamine C1 (144 mg, 1.3 mmol) were dissolved in toluene (4.5 ml). The reaction was stirred at 80 °C under argon protection for 3 h, and the reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (Ⅰ) (yield: 85%).

[0056] Example 6 Preparation of hybrid (Ⅰ)

[0057] Enol intermediate B1 (300 mg, 1.3 mmol) and o-diphenylamine C1 (144 mg, 1.3 mmol) were dissolved in methanol (4.5 ml). The reaction mixture was stirred at 80 °C under argon protection for 3 h, and the reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (Ⅰ) (yield: 80%).

[0058] Example 7 Preparation of Heterozyme (II)

[0059] Enol intermediate B2 (100 mg, 0.34 mmol) and 4,5-difluoro-o-phenylenediamine C2 (49 mg, 0.34 mmol) were dissolved in benzene (3.4 ml). The reaction was stirred for 12 h under argon protection at 80 °C, and the reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (2) (yield: 98%).

[0060] Enol intermediate B2 (300 mg, 1.03 mmol) and 4,5-difluoro-o-phenylenediamine C2 (148 mg, 1.03 mmol) were dissolved in benzene (10 ml). The reaction mixture was stirred at 80 °C under argon protection for 12 h, and the reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (II) (yield: 90%).

[0061] Example 8 Preparation of hybrid (II)

[0062] Enol intermediate B2 (300 mg, 1.03 mmol) and 4,5-difluoro-o-phenylenediamine C2 (148 mg, 1.03 mmol) were dissolved in toluene (10 ml). The reaction was stirred at 80 °C under argon protection for 12 h, and the reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (II) (yield: 70%).

[0063] Example 9 Preparation of Heterozyme (II)

[0064] Enol intermediate B2 (300 mg, 1.03 mmol) and 4,5-difluoro-o-phenylenediamine C2 (148 mg, 1.03 mmol) were dissolved in methanol (10 ml). The reaction mixture was stirred at 80 °C under argon protection for 12 h, and the reaction was monitored by TLC until the reactants were completely consumed. The mixture was quenched with distilled water and concentrated under vacuum to obtain a yellow solid. The yellow solid was filtered through filter paper and washed successively with distilled water, methanol, and dichloromethane. The solid on the filter paper was collected to obtain the pure novel 3,4-dihydroquinoxalin-2-one / acetophenone hybrid (II) (yield: 75%).

[0065] Experimental Example 1: In vitro activity test The experimental cases tested the antiproliferative activity of heterozygotes (I) or (II) against various human tumor cell lines.

[0066] 1. Experimental Methods Dissolve the heterozygote (Ⅰ) or (Ⅱ) to be tested in DMSO to prepare a working solution with a concentration of 10 μM, and store it for later use.

[0067] The working solution concentration started at 10 μM and was diluted twofold to create five concentration gradients. MDA-MB-468 (3650 cells / well), MDA-MB-231 (4000 cells / well), MCF10A (2100 cells / well), and HCC1937 (4000 cells / well) cells in logarithmic growth phase were cultured overnight for 24 h in 96-well plates. The serially diluted working solutions of the compounds were added for treatment, with each concentration set up in triplicate. Control and blank control groups were also included. Cell viability was assessed using the MTT assay.

[0068] Cell viability = (OD in drug-treated group - OD in blank group) / (OD in DMSO group - OD in blank group).

[0069] IC 50 Assay: Cell lines in logarithmic growth phase were cultured in 96-well plates. After 24 h, 10 μL of the initially screened compound was added to each well for rescreening (starting at 10 μM concentration, with five 2-fold serial dilutions) for 72 h. Three replicates were performed for each concentration. The cells were stained with 5 mg / mL MTT at 37℃ for 2.5 h. After removing the culture medium, 100 μL of DMSO was added, and absorbance was measured at 490 nm. Nonlinear regression analysis was performed using GraphPad Prism 10 software to determine the IC50 of the tested compounds. 50 value.

[0070] 2. Experimental Results The experimental results are shown in Figure 1 The results showed that the hybrid (Ⅰ) of the present invention exhibited the best inhibitory effect on MDA-MB-468 (IC). 50 =0.50±0.01μM). The low sensitivity of MCF-10A cells, as normal mammary epithelial cells, to the drug suggests that heterozygote (Ⅰ) has certain selective toxicity, which can reduce damage to normal cells, with a safety factor of 17.22. The heterozygote (Ⅱ) of this invention has certain inhibitory activity against MDA-MB-468, MDA-MB-231, and HCC1937 cells, with an IC50 of 0.50±0.01μM. 50 The values ​​were 6.44±0.517μM, 0.8±0.053μM, and 0.81±0.024μM, respectively.

[0071] Experimental Example 2: Antiproliferative Activity Experiment Based on the in vitro experimental data from Example 1, heterozygote (I) was found to have the best activity against MDA-MB-468 cells. Therefore, MDA-MB-468 cells were selected to further observe whether their colony formation could be affected by heterozygote (I). MDA-MB-468 cells were seeded into 6-well plates and treated with heterozygote (I) (0, 0.5, 1.0, 2.0 μM) for 48 hours. The culture medium containing the reagent was replaced with fresh medium, allowing the cells to regrow for 12 days. The cells were then fixed with paraformaldehyde and stained with crystal violet.

[0072] See results Figure 2 The heterozygote (Ⅰ) inhibited the clone formation of MDA-MB-468 cells in a concentration-dependent manner and had good in vitro anti-breast cancer activity.

[0073] Experimental Example 3: Validation of the target site of the hybrid (I) 1. Target validation 1.1 Experimental Methods 1.1.1 Cell thermal displacement experiment Logarithmically growing MDA-MB-468 cells were digested with trypsin, resuspended, and counted. Cells were aliquoted into EP tubes (1.0 × 10⁶ cells / tube) according to experimental groups and temperature gradients. Cells were washed twice with PBS containing 1 mM PMSF and subjected to three freeze-thaw cycles (4 minutes each) in liquid nitrogen to lyse the cells. Subsequently, DMSO (control group) or 10 μM heterozygote (I) (experimental group) was added and incubated for 5 hours to ensure sufficient drug binding to the target protein. The protein was heated for 3 minutes at preset temperature gradients (40, 45, 50, 55, 60, 65°C), then rapidly transferred to a 25°C water bath for 3 minutes, and then placed on ice. After all samples were processed, the samples were centrifuged at 4°C and 16,000 rpm for 30 minutes, and the supernatant was collected. Protein concentrations were determined using a BCA protein assay kit and uniformly adjusted. After boiling denaturation, SDS-PAGE electrophoresis was performed. Proteins were transferred to PVDF membranes, blocked, and incubated with primary and secondary antibodies. Target proteins were detected using a fully automated chemiluminescence image analysis system. The obtained images were quantitatively analyzed for grayscale values ​​using ImageJ, and statistical charts were generated using GraphPad Prism 10.

[0074] 1.1.2 Lip-MS enzyme digestion experiment Take 30 μg of recombinant protein and add PBS solution (pH 7.4) to a final volume of 200 μl. Take six groups of 100 μl protein solutions (15 μg each) and divide them into drug treatment groups and control groups (solvent treatment), with three replicates per group. Incubate with an equal volume of DMSO and 50 μM drug at 25°C for 10 minutes. Add 1% proteinase K to each group of samples and incubate at 25°C for 5 minutes. After incubation, heat at 95°C for 3 minutes, add 1% SDC, and heat again at 95°C for 3 minutes to stop the digestion. Add TCEP (tris(2-carboxyethyl)phosphine) and CAA (chloroacetamide) to each group of samples and incubate at 37°C with shaking for 1 hour to perform the reductive alkylation reaction. Add 1 / 10 volume of 1M Tris-HCl solution to the reductively alkylated samples to adjust the pH of the solution, add trypsin at a mass ratio of enzyme to protein of 1:50, and incubate at 37°C with shaking overnight to perform the digestion. The next day, 1% TFA was added to stop the enzyme digestion. The supernatant was then desalted using an SDB-RPS desalting column, vacuum dried, and stored at -20°C.

[0075] 1.1.3 Surface Plasmon Resonance (SPR) Experiment The affinity between heterozygous compound 1 and HSP70 was determined using the Biacore T200 molecular interaction analysis system. Before the experiment, the HSP70 receptor was immobilized on the CM5 chip according to the standard operating procedure (immobilization amount 426.6 RU). 10 mM Glycine-HCl (pH 2.0) with high binding capacity and stable regeneration effect in previous experiments was directly selected as the regeneration buffer. For sample preparation, the ligand protein was diluted with 10 mM NaAc (pH 4.5), and the heterozygote (I) was serially diluted with 0.05% Tween-PBS (pH 7.4) to 17 concentration gradients (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625, 0.48828125, 0.244140625, 0.122070312, 0.0610351562, 0 nM). During operation, samples were loaded at a flow rate of 30 µl per minute. The binding time of the heterozygote (I) to the receptor protein was 180 seconds, followed by natural dissociation in 300 seconds. Experimental data were analyzed using the Kinetics kinetic model with Biacore T200 Evaluation Software 3.2.1 to fit and calculate the binding kinetic parameters of the hybrid (Ⅰ) and HSP70.

[0076] 1.2 Experimental Results See Figure 3Using MDA-MB-468 TNBC cells as a model, this study, combined with CETSA and Western blotting, verified that the heterozygote (I) can directly bind to HSP70 and significantly enhance its thermostability. LiP-MS was used to analyze the drug-binding region, and after high-coverage identification of the HSP70 protein, 12 differentially expressed peptides were screened. Amino acid residues 317–327 were identified as the key drug-binding site, and this binding did not affect ATPase activity. SPR analysis of binding kinetic parameters showed that the heterozygote (I) binds to HSP70 with strong and specific affinity at the nanomolar level, and the fitting data were reliable, clearly indicating that HSP70 is the high-affinity target of the heterozygote (I).

[0077] 2. Verification of the mechanism of action 2.1 Experimental Methods 2.1.1 CO-IP Experiment Take MDA-MB-468 cells in logarithmic growth phase, at 4 × 10⁻⁶ 6 Cells were seeded in 10 cm culture dishes and cultured overnight. The next day, 1, 2, and 5 μM heterozygote (I) were added for 48 hours, respectively. Cells were collected and lysed with RIPA lysis buffer, and protein concentration was determined by BCA method. Magnetic beads were incubated with HSP70 antibody at 4°C overnight, followed by the addition of approximately 500 μg of protein sample for binding at room temperature for 3 hours. After washing, 1× loading buffer was added and the mixture was boiled at 95°C for 10 minutes. Denatured proteins were separated by SDS-PAGE electrophoresis, and PVDF membranes were transferred at 110 V for 60 minutes. After blocking with 5% skim milk, the membranes were incubated sequentially with EGFR, HSP70, and GAPDH primary antibodies at 4°C overnight. Rabbit secondary antibody was added and incubated at room temperature for 1 hour. Chemiluminescence detection was performed, quantification was performed using ImageJ, and plotting was done using GraphPad Prism 10.

[0078] 2.1.2 Western Blot Experiment Log-phase MDA-MB-468 cells were harvested at a concentration of 3.5 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and incubated overnight at 37 °C with 5% CO2. The next day, 1, 2, 5, and 10 μM of heterozygote (I) were added and co-incubated with the cells for 48 hours. Total protein was then extracted, and subsequent Western blot operations were performed according to the method described in Section 1.2.6. The primary antibodies used in the experiment and their dilution ratios were as follows: EGFR (1:1000), HSP70 (1:1000), p-AKT (1:1000), AKT (1:1000), p-PI3K (1:1000), PI3K (1:1000), and GAPDH (1:10000, internal control).

[0079] 2.2 Experimental Results See Figure 4Using HSP70 as an IP to bind EGFR in MDA-MB-468 cells, the interaction between HSP70 and EGFR was demonstrated. After co-precipitation of HSP70 and EGFR, the high concentration of heterozygote (Ⅰ) significantly weakened the interaction between HSP70 and EGFR, indicating that heterozygote (Ⅰ) can inhibit the interaction between HSP70 and EGFR.

[0080] After treatment with the hybrid (Ⅰ), HSP70, EGFR, p-AKT, and p-PI3K showed a downregulation trend, while AKT and PI3K did not show a change trend. This suggests that the compound may inhibit the proliferation of triple-negative breast cancer by targeting HSP70, preventing the interaction between HSP70 and EGFR, inhibiting the activation of PI3K-AKT phosphorylation.

[0081] Experimental Example 3: In vitro combined drug administration activity test Heterozygotes (I) combined with HAR enhance therapeutic efficacy 1. Experimental Methods This study used the SynergyFinder online analysis platform to evaluate the combined antitumor effects of heterozygotes (I) and HAR in MDA-MB-468 and 4T1 cells. MDA-MB-468 cells were cultured at 4 × 10⁻⁶ cells / year. 3 4T1 cells / well were seeded at a density of 1 × 10⁶ cells / well in 96-well plates. 3 (cells / well), after culturing for 24 hours, based on the half-inhibitory concentration (IC50) determined in Experimental Example 1. 50 Six concentration gradients were set up for heterozygote (Ⅰ) (0.078, 0.156, 0.312, 0.625, 1.25, 2.5 μM) and HAR (0.041, 0.123, 0.37, 1.11, 3.33, 10 μM). The concentrations of heterozygote (Ⅰ) in 4T1 cells were (1.25, 2.5, 5, 10, 20, 40 μM), and the HAR concentrations were consistent with those used in MDA-MB-468 cells. Cell viability was assessed using the MTT assay, and absorbance at 490 nm was measured using a microplate reader. The raw data was imported into SynergyFinder software (https: / / synergyfinder.fimm.fi), and the Response Surface Model and Zero Interaction Potency (ZIP) algorithm were selected. The ZIP synergy score was calculated using the "inhibition index" (inhibition index = 100 - cell viability %). A score > 0 was considered synergistic (marked in red), and a score > 10 was considered strong synergistic. At the same time, a drug combination response heatmap was generated to visually assess the therapeutic potential and concentration-response relationship of the combined drugs.

[0082] 2. Experimental Results See Figure 5 In vitro experiments showed that low-dose HAR may synergistically enhance the cytotoxicity of heterozygote (I) against MDA-MB-468 and 4T1 cells. Based on the novel concentration gradient and corresponding inhibition index, the synergistic score of the drugs was calculated using the online SynergyFinder software. The results showed that the maximum proportion of antitumor response due to drug interaction was 18.61 in MDA-MB-468 cells and 27.146 in 4T1 cells (synergistic score >10). We also validated the synergistic effect using CompuSyn software, which showed that a HAR concentration of 0.04 μM was the lowest concentration containing the highest synergistic region. Therefore, 0.04 μM was selected as the optimal combined concentration of HAR. To further confirm the optimal dose of heterozygote (I), we tested cell viability at different concentrations. The results showed that a heterozygote (I) concentration of 0.3125 μM was the optimal concentration, and a 4T1 concentration of 22 μM was the optimal concentration.

[0083] Experimental Example 4: In vivo activity test Based on in vitro experimental data, heterozygote (I) was found to have the best activity against 4T1 cells. Therefore, we selected 4T1 cells to construct a nude mouse xenograft model to investigate the in vivo antitumor activity of heterozygote (I). The experimental steps are as follows: After passage culture of an appropriate amount of 4T1 cell line, well-grown cells were collected and a tumor cell suspension was prepared. 1×10 6 Cells were inoculated subcutaneously into mice, and the tumors were allowed to grow to 100 mm. 3 Mice were randomly divided into a saline group, a heterozygous (I) treatment group (50 mg / kg), a HAR control group (1 mg / kg), and a combined drug administration group. The drugs were administered intraperitoneally every two days for 24 consecutive days, and tumor volume changes over time were observed. Eighteen days after treatment, blood was collected from the tail vein, and the mice were euthanized. The harvested tumor tissue was rapidly frozen in liquid nitrogen. Mice kidney and liver tissues, preserved in 4% paraformaldehyde, were dehydrated and embedded in paraffin. The embedded and cooled tissue sections were 5 μm thick. The sections were stained with hematoxylin and eosin, sealed, and observed under an optical microscope.

[0084] See results Figure 6 In mice in the saline control group, the heterozygous (I) group alone, and the HAR group alone, tumor volume gradually increased, while the tumor volume in the combined treatment group was significantly smaller than that in the other three groups, with a TGI of 68.7%. Additionally... Figure 7 Pathological examination of the lungs, liver, and kidneys showed no obvious pathological damage to these organs in heterozygotes (I) and HAR. Figure 8 Serum biochemical analysis showed that neither heterozygote (I) nor combined administration caused changes in the levels of CREA-S, UREA, AST and ALT.

[0085] This further demonstrates that the hybrid compound has low hepatotoxicity and nephrotoxicity in vivo.

[0086] Application Example 1: A pharmaceutical composition for treating triple-negative breast cancer A pharmaceutical composition for treating triple-negative breast cancer includes the hybrid (I) of the present invention, wherein the hybrid (I) and an excipient are added in a weight ratio of 1:1, and then granulated and compressed into tablets. The excipient is specifically a commonly used excipient such as hydroxypropyl cellulose or gelatin.

[0087] Application Example 2: A pharmaceutical composition for treating triple-negative breast cancer A pharmaceutical composition for treating triple-negative breast cancer, comprising the heterozygote (I) of the present invention, wherein the heterozygote (I) is formulated into a capsule.

[0088] Application Example 3: A pharmaceutical composition for treating triple-negative breast cancer A pharmaceutical composition for treating triple-negative breast cancer, comprising the hybrid (I) of the present invention, starch, corn syrup and magnesium stearate, combined into a tablet.

[0089] Each tablet contains 10 mg of heterozygote (I).

[0090] The preparation method involves mixing the hybrid (I) or its pharmaceutical composition with an adjuvant, granulating and compressing it into tablets.

[0091] Application Example 4: A pharmaceutical composition for treating triple-negative breast cancer A pharmaceutical composition for treating triple-negative breast cancer includes the hybrid (I) of the present invention, and further includes adjuvants starch and magnesium stearate. It is prepared as a capsule.

[0092] The preparation method is as follows: Mix the hybrid (Ⅰ) with the auxiliary agents starch and magnesium stearate, sieve, mix evenly in a suitable container, and fill the resulting mixture into hard gelatin capsules.

[0093] Each capsule contains 10 mg of heterozygote (Ⅰ).

[0094] Application Example 5: A pharmaceutical composition for treating triple-negative breast cancer A pharmaceutical composition for treating triple-negative breast cancer, comprising 2 mg of the heterozygote (I) of the present invention and 10 mg of sodium chloride. Prepared as an ampoule.

[0095] Preparation method: Dissolve the hybrid (Ⅰ) and sodium chloride in an appropriate amount of water for injection, filter the resulting solution, and fill it into an ampoule under aseptic conditions to prepare the ampoule.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A 3,4-dihydroquinoxalin-2-one / acetophenone hybrid compound as shown in formula (I) or (II), or a pharmaceutically acceptable salt thereof: 。 2. A pharmaceutical composition, characterized in that, It comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition also contains 12-hydroxyamoorastatin.

4. The use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in any one of claims 2-3, in the preparation of a medicament for treating tumors.

5. The application according to claim 4, characterized in that, The tumor is breast cancer; Preferably, the breast cancer is triple-negative breast cancer.

6. The application according to claim 4 or 5, characterized in that, The drug works through at least one of the following mechanisms: (a) Inhibits tumor cell proliferation; (b) Targeting the HSP70 protein; (c) Inhibits the interaction between HSP70 and EGFR; (d) Inhibit phosphorylation activation of the PI3K-AKT signaling pathway.

7. The use of a compound of formula (I) as described in claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating triple-negative breast cancer, characterized in that, The drug also includes 12-hydroxyamoorastatin; Preferably, the molar ratio of compound (I) to 12-hydroxyamoorastatin is 0.31 - 22:0.

04.

8. A method for preparing the compound of formula (I) or formula (II) as described in claim 1, characterized in that, Includes the following steps: (1) The acetophenone derivative was reacted with dimethyl oxalate under alkaline conditions to obtain enol intermediate B; (2) The enol intermediate B obtained in step (1) is reacted with the o-diaminobenzene derivative C in an organic solvent to obtain the compound of formula (I) or formula (II).

9. The method according to claim 8, characterized in that, In step (1), the acetophenone derivative A is selected from 4-fluoroacetophenone or 3-cyclopentoxyacetophenone; Preferably, the alkaline condition is the use of sodium methoxide; Preferably, the molar ratio of acetophenone derivative A to dimethyl oxalate is 1:1-5; Preferably, the reaction is carried out at 70-100°C for 1-5 hours under argon protection.

10. The method according to claim 8, characterized in that, In step (2), the o-diaminobenzene derivative C is selected from o-phenylenediamine and 4,5-difluoro-o-phenylenediamine, and the alkaline condition is the use of sodium methoxide; Preferably, the organic solvent is selected from benzene, toluene, or methanol; Preferably, the reaction is carried out under argon protection at 60-100°C for 12-24 hours; Preferably, the molar ratio of enol intermediate B to o-diaminobenzene derivative C is 1:1-3.

Images