A phenylisoindolinone and zidovudine splicing compound, and a preparation method and application thereof

Abstract

The application discloses a phenyl isoindolinone and zidovudine splicing compound and a preparation method and application thereof. The compound has potential biological activity, provides a compound source for biological activity screening, has important application value for drug screening and the pharmaceutical industry, and through in-vitro anti-tumor activity screening, it is found that the compound can selectively inhibit human lung cancer NCI-H1975 cells, and the selectivity index of the compound to normal cells BEAS-2B is high (the inhibition rate is only 12.51 %), the safety is about 7.2 times of that of osimertinib, and the compound exhibits good tumor cell selectivity and low potential side effects. In combination with the changes of NCI-H1975 cell mitochondrial ROS and membrane potential, it is indicated that the phenyl isoindolinone and zidovudine splicing compound derived from an EGFR allosteric inhibitor can play an anti-tumor role by affecting mitochondrial function.

Description

Technical Field

[0001] This invention relates to the field of chemical technology, and in particular to a compound made by splicing phenyl isoindoleone and zidovudine and its application in the research of anti-lung cancer related drugs. Background Technology

[0002] According to the latest statistics, lung cancer is one of the malignant tumors with the highest incidence and mortality rates worldwide, with 2.5 million new cases in 2022 alone, accounting for 12.4% of all new cancer cases globally. Based on histopathological characteristics, lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with approximately 85% being NSCLC. Currently, clinical treatments for NSCLC mainly include surgery, radiotherapy, chemotherapy, and targeted therapy. Among these, a series of drugs, represented by EGFR inhibitors, are often used as targeted therapy for NSCLC. However, patients using these EGFR inhibitors are very prone to acquired resistance, and subsequent drug use is very limited. Therefore, actively searching for safe, effective, and low-toxicity novel compounds is of significant clinical importance for the treatment of lung cancer.

[0003] Phenylene isoindole is an important structural component of alkaloids in traditional Chinese medicine. Many of its derivative compounds often exhibit broad pharmacological activities in antitumor, antiviral, and anti-inflammatory aspects. For example, EAI045 and JBJ-02-112-05, which have an indole structure, still have significant activity against non-small cell lung cancer patients who are resistant to drugs due to epidermal growth factor receptor (EGFR) mutations. However, due to defects such as poor oral bioavailability and easy accumulation in the blood, they have not entered clinical use.

[0004] Zidovudine (AZT), commonly used clinically as an anti-HIV drug, has recently been found to exert anti-tumor and radiosensitizing effects by inhibiting Pol γ interference with mtDNA replication, reducing mtDNA content in lung cancer cells by 40% and decreasing mitochondrial membrane potential by 25%, ultimately activating the caspase-9-mediated apoptosis pathway. Its anti-tumor research has covered various malignant tumors, including lung cancer, colon cancer, parathyroid cancer, breast cancer, cervical cancer, liver cancer, and gastric cancer. Its efficacy studies in parathyroid cancer, colon cancer, and gastrointestinal cancers have entered the clinical trial stage.

[0005] Molecular combination refers to the covalent bonding of two different drug molecules into a single molecule, or the compatibility of their pharmacophores within a single molecule. The newly formed molecule often combines the properties of both original drugs, enhancing pharmacological effects and reducing the corresponding toxic side effects; or it allows both drugs to complement each other, exerting their respective pharmacological activities synergistically to complete the therapeutic process. Because new drugs are created by combining drugs with known therapeutic effects, the pharmacological activity of the combined drug can be easily predicted based on the pharmacological effects of the active pharmaceutical ingredients. This gives new drug development a strong sense of purpose and foundation, thus shortening the development process and saving significant human, material, and financial resources. Currently, many pharmaceutical companies and research institutes both domestically and internationally are also dedicated to developing new drugs using the principle of combination. Therefore, drug combination has become a rapid and effective means of discovering new drugs and has been successfully applied in the synthesis of various drugs.

[0006] Based on the above facts, we hypothesized that compounds synthesized by splicing phenylisoindolone with zidovudine should also possess good antitumor activity. Furthermore, the splice products of the EGFR small molecule inhibitor lapatinib and perphenazine / fluphenazine also showed better inhibitory effects on breast cancer, indirectly providing feasibility evidence for our hypothesis. Therefore, we spliced ​​indole with zidovudine to synthesize the modified product, and through activity testing, we found that the obtained modified product has good antitumor activity and can be further developed and studied. It can also provide a source of compounds for bioactivity screening, which has important application value for drug screening and the pharmaceutical industry. Summary of the Invention

[0007] The purpose of this invention is to provide a phenylisoindolone and zidovudine compound, its preparation method and application. It is an important class of drug molecule analogs and pharmaceutical intermediate analogs, and at the same time provides a compound source for bioactivity screening. It has important application value for drug screening and the pharmaceutical industry, and its synthesis method is also economical and simple.

[0008] This invention also discovers the application of this type of compound in the preparation of lung cancer drugs.

[0009] This invention is achieved as follows: the compound of phenyl isoindolone and zidovudine has the structure shown below:

[0010]

[0011] In the formula: R is H, OH or cyclobutoxy; R1 is H or Br; R2 is H or Br.

[0012] The preparation method of the compound made by splicing phenyl isoindolone and zidovudine is as follows:

[0013] Substituted aminophenylacetic acid 1 and substituted 2-bromomethylbenzoate 2 are dissolved in a solvent and reacted under alkaline conditions to generate intermediate 3. This intermediate is then hydrolyzed under alkaline conditions to give intermediate 4. Further substitution with propargyl bromide under alkaline conditions yields intermediate 5. Finally, a Click reaction with zidovudine under copper catalysis is performed to give the target compound 7. The reaction route is as follows:

[0014] ;

[0015] Alternatively, intermediate 3 can be first alkylated to obtain intermediate 8, then hydrolyzed under basic conditions to obtain intermediate 9, which is then substituted with propargyl bromide under basic conditions to obtain intermediate 10. Finally, it undergoes a Click reaction with zidovudine under copper catalysis to obtain the target compound 11. The reaction route is as follows:

[0016] .

[0017] The solvent is one or a combination of N,N'-dimethylformamide, tetrahydrofuran, methanol, and water.

[0018] The alkaline reagents are N,N-diisopropylethylamine, lithium hydroxide, and potassium carbonate.

[0019] The reaction temperature is 0℃~80℃, and the reaction time is 1~72 hours.

[0020] The phenylisoindolone and zidovudine compound obtained by the above-described technical method possesses potential biological activity and provides a compound source for biological activity screening. This has significant application value for drug screening and the pharmaceutical industry. Furthermore, in vitro antitumor activity screening revealed that this type of compound exhibits good inhibitory effects on human lung adenocarcinoma cells PC-9, NCI-H1975, and SK-MES-1, and also shows good safety against normal human BEAS-2B cells. It is highly likely to be further developed into a new drug for the prevention and treatment of tumors. The method of this invention is economical and simple, with high product yield and inexpensive and readily available raw materials, allowing for further research and exploration of its antitumor activity. Attached Figure Description

[0021] Appendix Figure 1 To detect the effect of compound 7b on mitochondrial membrane potential in NCI-H1975 cells by flow cytometry. Detailed Implementation

[0022] Example 1

[0023] A chemical synthesis method for a compound composed of phenyl isoindole and zidovudine is described below, but it should be emphasized that the compounds of the present invention are not limited to those represented by the following route.

[0024]

[0025] Compound 1a (1.0 g, 6.05 mmol), 20 mL of N,N-dimethylformamide, and methyl 2-bromomethylbenzoate esters 2a-2c (6.05 mmol) with different substituents were added to a 50 mL globular flask. N,N-diisopropylethylamine (2.1 mL, 12.10 mmol) was added under stirring in an oil bath at 40 °C. The mixture was heated to reflux at 40 °C for 4 h, then heated to 80 °C and refluxed again. The reaction was monitored by TLC until complete. After the reaction was complete, the system was cooled to room temperature, and the reaction was quenched slowly with appropriate amounts of water. Extraction was performed with ethyl acetate (100 mL × 3), followed by washing with saturated brine, drying over anhydrous sodium sulfate, and concentration under reduced pressure to obtain 3a-3c. Compounds 3a-3c, tetrahydrofuran (10 mL), distilled water (10 mL), and methanol (10 mL) were added to a 100 mL pear-shaped reaction flask. Lithium hydroxide monohydrate (1.27 g, 30.25 mmol / L) was added with stirring. After reacting for 1 h, the reaction was confirmed to be complete by TLC. The solvent was removed by vacuum concentration, and the residue was diluted with ice water. Concentrated hydrochloric acid was slowly added dropwise with stirring to adjust the pH to 3-4. The suspension was stirred and filtered through a Buchner funnel for separation. The solids were dried at room temperature to obtain compounds 4a-4c. The products were used directly in the next reaction without further purification.

[0026] Compound 4a-4c (1.12 mmol), anhydrous potassium carbonate (6.72 mmol), and 20 mL of N,N-dimethylformamide were added to a 50 mL pomegranate-shaped reaction flask and stirred in the dark for 30 min. Then, bromopropyne (2.24 mmol) was added, and the reaction was continued with stirring in the dark. The reaction was monitored by TLC until complete, and the reaction was quenched with an appropriate amount of water. Extraction was performed with ethyl acetate (70 mL × 3), washing with saturated brine, drying over anhydrous sodium sulfate, and concentrating under reduced pressure to obtain 5a-5c. 5a-5c, cuprous iodide (0.37 mmol), sodium ascorbate (0.37 mmol), zidovudine (0.56 mmol), 20 mL of N,N-dimethylformamide, and 4 mL of distilled water were added to a 50 mL pomegranate-shaped reaction flask. N,N-diisopropylethylamine (2.24 mmol) was added with stirring, and the reaction was monitored by TLC until complete. After the reaction was complete, the reaction was quenched with an appropriate amount of water. Extracted with ethyl acetate (70 mL × 3), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The products were separated by column chromatography using petroleum ether / ethyl acetate (10 / 1) as the eluent to give the target compounds 7a-7c.

[0027] Compound 7a: 42.5% yield, yellow solid, melting point 93.6–95.8 °C. 1 H NMR (600 MHz, DMSO-d6) δ: 11.35 (s, 1H, NH), 8.35 (s, 1H), 7.95 (s, 1H), 7.81 (s, 1H), 7.74 (d,J = 7.6 Hz, 1H), 7.63 – 7.59 (m, 1H), 7.57 – 7.54 (m, 1H), 7.53 – 7.48 (m,1H), 7.44 – 7.38 (m, 2H), 7.36 – 7.32 (m, 2H), 6.41 (t, J = 6.6 Hz, 1H), 6.10(s, 1H), 5.40 – 5.35 (m, 1H), 5.33 (s, 2H), 5.31 – 5.21 (bs, 1H, OH), 4.59 (d, J = 17.3 Hz, 1H), 4.22 – 4.17 (m, 1H), 3.93 (d, J = 17.2 Hz, 1H), 3.71 –3.66 (m, 1H), 3.64 – 3.58 (m, 1H), 2.74 – 2.68 (m, 1H), 2.67 – 2.60 (m, 1H), 1.81 (d, J = 1.3 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ: 169.6, 167.6, 163.8,162.3, 150.5, 142.1, 141.5, 136.3, 133.9, 131.9, 131.1, 129.1 (C×2), 128.8(C×2), 128.1, 124.8, 123.8, 123.1, 109.6, 84.4, 83.9, 60.7, 59.3, 58.3,57.8, 47.7, 37.2, 12.3. HR-MS(m / z): C 29 H 29 N6O7([M+H)) + Calculated value: 573.2098, Measured value: 573.2101.

[0028] Compound 7b: 46.7% yield, white solid, melting point 138.3–140.9 °C. 1H NMR (600 MHz, DMSO-d6) δ: 11.37 (s, 1H), 8.36 (s, 1H), 7.85 – 7.81 (m, 2H), 7.79 – 7.76 (m,1H), 7.51 (d, J = 7.1 Hz, 1H), 7.45 – 7.41 (m, 3H), 7.40 – 7.36 (m, 2H), 6.41(t, J = 6.6 Hz, 1H), 6.08 (s, 1H), 5.40 – 5.34 (m, 1H), 5.33 (s, 2H), 5.28(bs, 1H, OH), 4.52 (d, J = 17.5 Hz, 1H), 4.22 – 4.17 (m, 1H), 3.94 (d, J =17.6 Hz, 1H), 3.72 – 3.67 (m, 1H), 3.63 – 3.58 (m, 1H), 2.74 – 2.69 (m, 1H), 2.67 – 2.61 (m, 1H), 1.81 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ: 169.4, 166.8,163.8, 162.3, 150.5, 141.9, 141.5, 136.6, 134.9, 133.6, 130.7, 129.1 (C×2),128.9, 128.8 (C×2), 124.8, 122.7, 117.4, 109.7, 84.5, 83.9, 60.7, 59.3,58.4, 57.9, 48.3, 37.2, 12.3. HR-MS(m / z): C 29 H 28 N6O7Br([M+H)) + Calculated value: 651.1203, Measured value: 651.1205.

[0029] Compound 7c: 45.4% yield, white solid, melting point 119.4–121.8 °C. 1H NMR (600 MHz, DMSO-d6) δ: 11.36 (s, 1H), 8.35 (s, 1H), 7.95 (s, 1H), 7.82 (s, 1H), 7.81 (s,1H), 7.72 – 7.65 (m, 2H), 7.42 – 7.41 (m, 2H), 7.34 – 7.32 (m, 2H), 6.41 (t,J = 6.6 Hz, 1H), 6.08 (s, 1H), 5.42 – 5.30 (m, 1H), 5.33 (s, 2H), 5.28 (bs,1H, OH), 4.58 (d, J = 17.6 Hz, 1H), 4.21 – 4.16 (m, 1H), 3.69 (d, J = 17.2Hz, 1H), 3.72 – 3.66 (m, 1H), 3.63 – 3.58 (m, 1H), 2.74 – 2.68 (m, 1H), 2.66– 2.60 (m, 1H), 1.81 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ: 169.5, 166.8,163.7, 162.3, 150.5, 144.3, 141.5, 136.2, 133.7, 131.4, 130.4, 129.1 (C×2),128.9, 128.8 (C×2), 127.0, 125.6, 125.0, 109.7, 84.4, 83.9, 60.7, 59.3,58.3, 57.9, 47.5, 37.1, 12.3. HR-MS(m / z): C 29 H 28 N6O7Br([M+H)) + Calculated value: 651.1203, Measured value: 651.1205.

[0030] Example 2

[0031]

[0032] In a 50 mL pear-shaped reaction flask, intermediate 1b-1c (1.0 g, 6.05 mmol), 20 mL of N,N-dimethylformamide, and 2d-2 g (6.05 mmol) of methyl 2-bromomethylbenzoate with different substituents were added. N,N-diisopropylethylamine (2.1 mL, 12.10 mmol) was added with stirring in an oil bath at 40 °C. The mixture was heated to 40 °C and refluxed for 4 h. The temperature was then raised to 80 °C and refluxed again. TLC was used to monitor the reaction until complete. After the reaction was complete, the system was cooled to room temperature, and the reaction was quenched slowly with an appropriate amount of water. Extraction was performed with ethyl acetate (100 mL × 3), followed by washing with saturated brine, drying over anhydrous sodium sulfate, and concentration under reduced pressure to obtain 3d-3 g. 3d-3g, tetrahydrofuran (10 mL), distilled water (10 mL), and methanol (10 mL) were added to a 100 mL pear-shaped reaction flask. Lithium hydroxide monohydrate (1.27 g, 30.25 mmol / L) was added with stirring. After reacting for 1 h, the reaction was confirmed to be complete by TLC. The solvent was removed by vacuum concentration, and the residue was diluted with ice water. Concentrated hydrochloric acid was slowly added dropwise with stirring to adjust the pH to 3-4. The suspension was stirred and filtered through a Buchner funnel for separation. The solid was dried at room temperature to obtain compound 4d-4f. The product was used directly in the next reaction without further purification.

[0033] Compound 4d-4f (1.12 mmol), anhydrous K₂CO₃ (6.72 mmol), and 20 mL of N,N-dimethylformamide were added to a 50 mL pomegranate-shaped reaction flask and stirred in the dark for 30 min. Then, bromopropyne (2.24 mmol) was added, and the reaction was continued with stirring in the dark. The reaction was monitored by TLC until complete, and the reaction was quenched with an appropriate amount of water. Extraction was performed with ethyl acetate (70 mL × 3), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 5d-5f. 5d-5f, cuprous iodide (0.37 mmol), sodium ascorbate (0.37 mmol), zidovudine (0.56 mmol), 20 mL of N,N-dimethylformamide, and 4 mL of distilled water were added to a 50 mL pomegranate-shaped reaction flask. N,N-diisopropylethylamine (2.24 mmol) was added with stirring, and the reaction was monitored by TLC until complete. After the reaction was complete, the reaction was quenched with an appropriate amount of water. Extracted with ethyl acetate (70 mL × 3), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was separated by column chromatography using petroleum ether / ethyl acetate (10 / 1) as the eluent to give the target compound 7d-7f.

[0034] Compound 7d: 24.7% yield, brownish-red oil. 1H NMR (600 MHz, DMSO-d6) δ: 11.36(s, 1H), 8.36 (s, 1H), 7.96 – 7.76 (m, 3H), 7.52 – 7.48 (m, 1H), 7.45 – 7.36(m, 5H), 6.41 (t, J = 6.6 Hz, 1H), 6.12 (s, 1H), 5.41 – 5.36 (m, 1H), 5.34(s, 2H), 5.29 – 5.20 (m, 1H), 4.54 – 4.37 (m, 1H), 4.22 – 4.17 (m, 1H), 3.88– 3.78 (m, 1H), 3.72 – 3.67 (m, 1H), 3.64 – 3.58 (m, 1H), 2.74 – 2.68 (m,1H), 2.67 – 2.61 (m, 1H), 1.81 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ: 169.4,166.8, 163.7, 162.3, 150.5, 141.9, 141.5, 136.3, 134.9, 133.5, 130.7, 129.1(C×2), 128.9, 128.8 (C×2), 124.8, 122.7, 117.4, 109.6, 84.4, 83.9, 60.7,59.3, 58.4, 57.9, 48.3, 37.2, 12.3. HR-MS(m / z): C 29 H 28 N6O7Br([M+H)) + Calculated value: 651.1203, Measured value: 651.1206.

[0035] Compound 7e: 25.3% yield, brown solid, melting point 144.7–146.2 °C. 1H NMR (600 MHz, DMSO-d6) δ: 11.39 (s, 1H), 8.36 (s, 1H), 7.84 – 7.79 (m, 1H), 7.74 (d, J =7.6 Hz, 1H), 7.61 (t, J = 7.4 Hz, 1H), 7.51 (t, J = 7.4 Hz, 1H), 7.44 – 7.39(m, 3H), 7.36 – 7.33 (m, 2H), 6.41 (t, J = 6.7 Hz, 1H), 6.10 (d, J = 7.1 Hz,1H), 5.42 – 5.38 (m, 1H), 5.33 (s, 2H), 5.31 – 5.26 (m, 1H), 4.59 (d, J =17.3 Hz, 1H), 4.21 – 4.15 (m, 1H), 3.93 (d, J = 17.3 Hz, 1H), 3.73 – 3.65 (m,1H), 3.63 – 3.56 (m, 1H), 2.74 – 2.68 (m, 1H), 2.67 – 2.60 (m, 1H), 1.81 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ: 169.6, 167.6, 163.8, 162.3, 150.5, 142.1,141.5, 136.6, 133.9, 131.9, 131.1, 129.0 (C×2), 128.8 (C×2), 128.1, 124.8,123.8, 123.1, 109.6, 84.5, 83.9, 60.7, 59.3, 58.3, 57.8, 47.7, 37.1, 12.3.HR-MS(m / z): C 29 H 28 N6O7Br([M+H)) + Calculated value: 651.1203, Measured value: 651.1203.

[0036] Compound 7f: 34.8% yield, white solid, melting point 102.2–106.5 °C. 1H NMR (600 MHz, DMSO-d6) δ: 11.33 (s, 1H), 8.44 (s, 1H, OH), 7.81 (s, 1H), 7.76 – 7.70 (m,1H), 7.64 – 7.48 (m, 3H), 7.37 – 7.27 (m, 2H), 7.15 – 7.09 (m, 1H), 7.05 –6.99 (m, 1H), 6.42 (t, J = 6.6 Hz, 1H), 6.07 (s, 1H), 5.43 – 5.35 (m, 1H), 5.34 – 5.24 (m, 2H), 5.20 (s, 1H), 4.55 (d, J = 17.7 Hz, 1H), 4.27 – 4.17 (m,1H), 3.96 (d, J = 17.5 Hz, 1H), 3.74 – 3.57 (m, 2H), 2.79 – 2.61 (m, 2H),1.81 (s, 3H). HR-MS(m / z): C 29 H 28 N6O8Br([M+H)) + Calculated value: 667.1152, Measured value: 667.1147.

[0037] Example 3

[0038]

[0039] 3 g (1.0 g, 2.66 mmol) of intermediate, 40 mL of N,N-dimethylformamide, 1.84 g (13.29 mmol) of anhydrous K₂CO₃, and 896 μL (7.97 mmol) of (bromomethyl)cyclobutane were added to a 100 mL pear-shaped reaction flask. The mixture was heated to 40 °C under reflux and the reaction was monitored by TLC until complete. After the reaction was complete, the system was cooled to room temperature and the reaction was quenched by slowly adding an appropriate amount of water. The mixture was extracted with ethyl acetate (100 mL × 3), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 4 g of product. This product was used directly in the next reaction without further purification. 4 g of the product, 10 mL of tetrahydrofuran, 10 mL of distilled water, and 10 mL of methanol were added to a 100 mL pear-shaped reaction flask. Lithium hydroxide monohydrate (557.70 mg, 13.29 mmol) was added with stirring, and the reaction was allowed to proceed for 1 h. The reaction was then monitored by TLC until complete. The solvent was removed by vacuum concentration, and the residue was diluted with ice water. Concentrated hydrochloric acid was slowly added dropwise with stirring to adjust the pH to 3-4. The suspension was stirred and filtered through a Buchner funnel for separation. The solid was dried at room temperature to obtain 5g of the compound.

[0040] In a 50 mL g-shaped reaction flask, 5 g (300 mg, 0.70 mmol) of the compound, 483.7 mg (3.50 mmol) of anhydrous K₂CO₃, and 20 mL of N,N-dimethylformamide were added and stirred in the dark for 30 min. Then, bromopropyne (181 μL, 2.10 mmol) was added, and the reaction was continued in the dark with stirring. The reaction was monitored by TLC until complete, and the reaction was quenched with an appropriate amount of water. Extraction was performed with ethyl acetate (50 mL × 3), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 8. Add 8, cuprous iodide (44.4 mg, 0.23 mmol), sodium ascorbate (46.7 mg, 0.23 mmol), zidovudine (99.1 mg, 0.35 mmol), 20 mL of N,N-dimethylformamide, and 4 mL of distilled water to a 50 mL pear-shaped reaction flask. Stir and add N,N-diisopropylethylamine (243 μL, 1.40 mmol). Monitor the reaction by TLC until complete. After the reaction is complete, quench the reaction with appropriate amount of water. Extract with ethyl acetate (50 mL × 3), wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Separate the product by column chromatography using petroleum ether / ethyl acetate (10 / 1) as the eluent to obtain the target compound 9.

[0041] Compound 9: 20.3% yield, brown oil. 1 H NMR (400 MHz, DMSO-d6) δ: 11.36 (s,1H), 8.34 (s, 1H), 7.95 (s, 1H), 7.81 (s, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.24(d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 6.41 (t, J = 6.5 Hz, 1H), 5.99(s, 1H), 5.44 – 5.35 (m, 1H), 5.32 (s, 2H), 5.28 (bs, 1H, OH), 4.55 (d, J =17.7 Hz, 1H), 4.22 – 4.17 (m, 1H), 4.04 – 3.89 (m, 3H), 3.74 – 3.58 (m, 2H), 2.72 – 2.61 (m, 3H), 2.11-2.03 (m, 2H), 1.91-1.85 (m, 2H), 1.84-1.77 (m, 2H),1.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ: 169.7, 166.7, 163.7, 162.3, 159.0,150.5, 144.3, 141.5, 136.2, 131.3, 130.5, 130.1 (C×2), 127.0, 125.6, 125.3,124.9, 114.9 (C×2), 109.7, 84.4, 83.9, 71.5, 60.7, 59.3, 58.2, 57.3, 47.5,37.1, 33.9, 24.3 (C×2), 18.1, 12.3. HR-MS(m / z): C 34 H 36 N6O8Br([M+H)) + Calculated value: 735.1778, Measured value: 735.1778.

[0042] Example 4

[0043] Using EAI045 and osimertinib as positive controls, the MTT assay was used to test the inhibitory activity of compounds 7a-7f and 9 against human lung adenocarcinoma cells PC-9, human lung adenocarcinoma cells NCI-H1975, human lung squamous cell carcinoma cells SK-MES-1, and human bronchial epithelial cells BEAS-2B. However, it should be emphasized that the compounds of this invention are not limited to the cytotoxicity expressed in human lung cancer cells (PC-9, NCI-H1975, SK-MES-1).

[0044] Table 1. Inhibitory effects of compounds 7a-7f and 9 on four cell types (concentration: 40 μM)

[0045]

[0046] The test results are shown in Table 1. All compounds showed better inhibitory effects on NCI-H1975 cells than on PC-9 and SK-MES-1 cells. For NCI-H1975 cells, four compounds (7a, 7b, 7d, and 7f) exhibited inhibition rates exceeding 50%, with compound 7b being particularly outstanding, achieving an inhibition rate of 89.91%, far superior to EAI045 and comparable to the positive control osimertinib. For PC-9 and SK-MES-1 cells, the inhibition rates of all compounds were below 50%.

[0047] Compound 7b exhibited good inhibitory activity against NCI-H1975 cells while also demonstrating excellent safety against normal BEAS-2B cells. Its inhibition rate against BEAS-2B cells was only 12.51%, approximately 7.2 times that of the positive control osimertinib (which inhibited BEAS-2B cells by 90.05%). This demonstrates that compound 7b can selectively target NCI-H1975 cells and also exhibits good safety against normal BEAS-2B cells.

[0048] In addition, we set compound concentration gradients of 40, 10, 2.5, 0.625, 0.15625, and 0.039 μM, and used the above method to further study the half-maximal inhibitory concentrations (IC50) of compounds 7b, 7d, and 7f on NCI-H1975 cells. 50 The results are shown in Table 2. The IC50 of compound 7b... 50 It was also similar to the positive control osimertinib, showing a strong inhibitory effect.

[0049] Table 2. IC50 of compounds 7b, 7d, and 7f on NCI-H1975 cells 50 value

[0050]

[0051] Example 5

[0052] Using osimertinib as a positive control, the HTRF method was used to determine compounds 7a-7f and 9 against EGFR. wt EGFR L858R / T790M / C797S The specific steps for inhibiting kinase activity are as follows:

[0053] (1) The compound was serially diluted with DMSO in a 384-well PP compound dilution plate (Beckman, 001-14555). 0.1 μL of the compound was transferred to a 384-well reaction plate (Grenier, 784075) using an Echo655 liquid workstation to make the final concentration of DMSO in the reaction system 1% (double replicate).

[0054] (2) Kinase reactions were performed in 1× kinase reaction buffer (Revvity, 62EZBFDD). 5 mM MgCl2 and 1 mM DTT were added to the SRC reaction system; 1 mM MnCl2 was added to the EGFR and EGFR mutant reaction systems. Then, 5 μL of 2× kinase solution was added to each well and incubated at 25°C for 60 min.

[0055] (3) Add 5 μL of a mixed solution containing ATP and TK substrate (Revvity, 61TK0BLC) to each well and continue incubation at 25°C for 60 min to initiate the kinase reaction.

[0056] (4) Add 10 μL of Sa-XL665 (Revvity, 610SAXLG) and TK-antibody-Cryptate (HTRF KinEASE-TK kit) to each well and incubate at 25°C for 60 min.

[0057] (5) Use the PHERAstar FSX microplate reader (BMG Labtech) to detect fluorescence signals and read the fluorescence intensity at 620 nm (Cryptate) and 665 nm (XL665) wavelengths.

[0058] (6) Wells containing 1% DMSO, enzyme, substrate, and ATP were designated as High Control, and wells containing 1% DMSO, substrate, and ATP were designated as Low Control. The compound inhibition rate was calculated using the following formula:

[0059]

[0060] Conditions of each kinase reaction system

[0061]

[0062] All compounds at a concentration of 500 nM showed effects on EGFR. wt EGFR L858R / T790M / C797S The inhibitory activities of the kinases are shown in Table 3. Overall, none of the compounds showed inhibitory effects on either EGFR kinase; some even promoted kinase activity. This indicates that at this concentration, the series of compounds do not inhibit EGFR kinase activity. wt EGFR L858R / T790M / C797S There was no inhibitory activity. Based on the in vitro cell activity results, the synthesized compound does not act on EGFR kinase to exert its anti-lung cancer effect, but may exert its anti-tumor activity through other pathways.

[0063] Table 3. Effects of compounds on EGFR at 500 nM concentration wt EGFR L858R / T790M / C797S Kinase inhibition rate (%)

[0064]

[0065] Example 6

[0066] Based on the results of in vitro cell antiproliferative activity and kinase activity, the effect of the preferred compound 7b on intracellular ROS levels in NCI-H1975 cells was investigated using fluorescence staining. Because the DCFH-DA (2', 7'-dichlorofluorescein diacetate) probe is lipid-soluble and non-fluorescein-producing, it is oxidized by ROS in vivo to form the highly fluorescent DCF (2', 7'-dichlorofluorescein). The fluorescence intensity is positively correlated with the intracellular level, and the amount of ROS generated in the cell can be detected by observing the fluorescence intensity. The procedure is as follows:

[0067] (1) Grouping: blank control group, positive control group, and different concentration drug groups (10 μM, 20 μM, 30 μM, 40 μM)

[0068] (2) Cell drug administration: NCI-H1975 cells were administered at a dose of 1×10⁻⁶. 6 The culture medium was seeded at a density of 100 mg / dL in 6-well plates and cultured for 24 hours. The next day, the old culture medium was discarded, and complete culture medium containing different drug concentrations, blank control group, and positive control group was added. The plates were then incubated at 37°C and 5% CO2 for 4 hours.

[0069] (3) Probe dilution: Following the principle of preparing and using immediately, after thawing the DCFH-DA probe, dilute it to 5 μM with serum-free culture medium and keep it in the dark throughout the process.

[0070] (4) Probe loading and removal: Remove the culture medium from all groups of cells, wash once with PBS, and add probe mixture. For 6-well plates, add 1 mL per well. Incubate the cells in a CO2 incubator for 20 min, and then wash three times with serum-free complete culture medium.

[0071] (5) Data processing: Cell samples were photographed using a fluorescence microscope and the images were analyzed using ImageJ software.

[0072] The results of mitochondrial reactive oxygen species (ROS) assays are shown in Table 4. With increasing compound 7b concentration, the fluorescence intensity in NCI-H1975 cells significantly increased, exhibiting a dose-dependent relationship. The fluorescence intensity peaked at a compound 7b concentration of 20 μM, comparable to the positive control group (Rosup). The bright-field and fluorescence field overlay plots (Merge) further confirmed that the fluorescence signal was mainly concentrated in the cytoplasm, and with high concentration treatment, some cells showed significant morphological shrinkage, consistent with early apoptosis. This evidence strongly supports the conclusion that compound 7b, under different concentration gradients, significantly increased mitochondrial ROS levels in NCI-H1975 cells, exhibiting a clear dose-dependent upward trend.

[0073] Table 4. Effects of compound 7b on intracellular reactive oxygen species (ROS) levels in H1975 cells.

[0074]

[0075] Example 7

[0076] Based on the results of its in vitro antiproliferative activity in cells, compound 7b was selected. Using zidovudine as a positive control, the effects of the selected compound on the mitochondrial membrane potential in NCI-H1975 cells were investigated to explore whether the selected compound exerts its antitumor effect by influencing the mitochondrial membrane potential. The specific procedures are as follows:

[0077] (1) Grouping: blank control group, positive control group (CCCP), positive control group, and drug treatment group

[0078] (2) Setting up the positive control CCCP: CCCP is 10mM, diluted to 10 μM at a ratio of 1 / 1000 and added to 2mL of culture medium.

[0079] (3) Cell drug administration: H1975 cells were administered at a dose of 1×10⁻⁶. 6 The cells were seeded at a density of 10 μM in 6-well plates and cultured for 24 h. The next day, the old culture medium was discarded, and NCI-H1975 cells were incubated in drug-containing medium containing 40 μM zidovudine, 0.1% DMSO, and different concentrations of 7b solution (10 μM, 20, 30 μM, 40 μM).

[0080] (4) Preparation of JC-1 dye working solution: Follow the principle of preparing and using immediately. The amount required for each well of a 6-well plate is 1 mL. Take an appropriate amount of thawed JC-1 (200×) and dilute it with 8 mL of ultrapure water for every 50 μL (200×). Vortex or sonicate to dissolve it completely. Let it stand or bathe in a water bath at 37℃ for 1-2 min. Add 2 mL of JC-1 staining buffer (5×) and store it in the dark for later use.

[0081] (5) Staining: After 4 hours, remove the drug-containing culture medium and wash away any remaining drug solution with PBS. Add trypsin solution to digest the adherent cells and collect the cell suspension. Centrifuge at 1000 rpm for 5 minutes, remove the supernatant, wash twice with PBS, and then add JC-1 staining working solution. Stain at 37°C in the dark for 15 minutes, taking care to avoid light during staining. After incubation, remove the 6-well plate, aspirate the supernatant, wash twice with JC-1 staining buffer, and then wash three times with PBS.

[0082] (6) The changes in mitochondrial membrane potential after NCI-H1975 cells were treated with different concentrations of compound 7b were analyzed by flow cytometry.

[0083] The results are attached. Figure 1As shown, after administration of zidovudine or compound 7b, the red fluorescence (J-aggregates) of JC-1 cells in NCI-H1975 cells was enhanced. Furthermore, the mitochondrial membrane potential increased in a dose-dependent manner after administration of compound 7b, and the mitochondrial membrane potential was reversed, suggesting that the cells may be in the early hyperpolarization stage of apoptosis.

[0084] Research Conclusions: The inhibitory effect of the phenylisoindolone / zidovudine compound on NCI-H1975 cells was significantly better than that on PC-9 and SK-MES-1 cells, with compound 7b showing the most outstanding performance. It exhibited a high selectivity index against normal BEAS-2B cells (inhibition rate of only 12.51%) and a safety profile approximately 7.2 times that of osimertinib, demonstrating good tumor cell selectivity and low potential toxicity. Combined with changes in mitochondrial ROS and membrane potential in NCI-H1975 cells, this suggests that compound 7b may exert its antitumor effect by influencing mitochondrial function. Further experimental studies on its antitumor mechanism suggest that compound 7b has the potential to be developed into a clinically applicable antitumor drug. Therefore, these compounds have the potential to be developed into antitumor drugs and possess certain economic and clinical value, warranting further in-depth research.

Claims

1. A compound composed of phenyl isoindolone and zidovudine, characterized in that: These compounds have the following structures: ， In the formula: R is H, OH or cyclobutoxy; R1 is H or Br; R2 is H or Br.

2. A method for preparing the compound of phenyl isoindolone and zidovudine as described in claim 1, characterized in that, The preparation method is as follows: Substituted aminophenylacetic acid 1 and substituted 2-bromomethylbenzoate 2 are dissolved in N,N-dimethylformamide and reacted in the presence of N,N-diisopropylethylamine to generate intermediate 3. This intermediate is then hydrolyzed under lithium hydroxide conditions to give intermediate 4, which further undergoes a substitution reaction with propargyl bromide under potassium carbonate conditions to give intermediate 5. Finally, it undergoes a Click reaction with zidovudine under cuprous iodide catalysis to give the target compound 7. The reaction route is as follows: ； Alternatively, intermediate 3 can be alkylated to obtain intermediate 8, then hydrolyzed under lithium hydroxide conditions to obtain intermediate 9, which is then substituted with propargyl bromide under potassium carbonate conditions to obtain intermediate 10. Finally, it undergoes a Click reaction with zidovudine under cuprous iodide catalysis to obtain the target compound 11. The reaction route is as follows: 。 3. The preparation method according to claim 2, characterized in that: The reaction temperature is 0℃~100℃, and the reaction time is 1~72 hours.

4. A compound of phenyl isoindolone and zidovudine as described in claim 1, characterized in that: Application in the preparation of anti-lung cancer drugs, wherein the lung cancer is human lung adenocarcinoma PC-9, human lung adenocarcinoma NCI-H1975 or human lung squamous cell carcinoma SK-MES-1.

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