Benzenesulfonamide derivative, preparation method thereof and application of benzenesulfonamide derivative in preparation of PARP1 inhibitor and antitumor drug

Abstract

The invention provides a benzenesulfonamide derivative, a preparation method thereof and application of the benzenesulfonamide derivative in preparation of PARP1 inhibitors and antitumor drugs, and belongs to the technical field of medicines. The invention synthesizes a novel benzenesulfonamide derivative, namely a compound N, 4-dimethyl-N-(7H-pyrrolo [2, 3-d] pyrimidine-4-yl) benzenesulfonamide, and the benzenesulfonamide derivative provided by the invention can inhibit PARP1 enzyme activity in a targeted manner so as to inhibit DNA repair, activate a p53-mediated DDR signal channel, inhibit DNA repair and inhibit the expression of a p53-mediated DDR signal channel. The benzenesulfonamide derivative can induce apoptosis of lung cancer tumor cells and influence the cycle of the lung cancer tumor cells, has remarkable antitumor activity, and is simple in synthesis method, easy in raw material obtaining, high in synthesis route yield, environment-friendly and simple and convenient in operation process. The PARP1 inhibitor disclosed by the invention is simple in preparation process, high in drug purity, high in yield, stable in quality and easy for large-scale production. The compound disclosed by the invention has a wide prospect in the aspects of development and application of antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a benzenesulfonamide derivative, its preparation method, and its application in the preparation of PARP1 inhibitors and antitumor drugs. Background Technology

[0002] Lung cancer is one of the leading causes of death and morbidity among malignant tumors worldwide, posing a serious threat to human health. PARP1 (poly-ADP-ribose polymerase 1) is a key DNA damage sensing and repair enzyme, primarily involved in the detection and repair of DNA single-strand breaks (SSBs). When DNA is damaged, PARP1 can rapidly locate the damage site and catalyze ADP-ribose glycosylation, recruiting repair-related proteins to maintain genomic stability. However, in tumor cells, PARP1 is abnormally overexpressed, promoting tumor survival and growth through various mechanisms, such as regulating the transcription of tumor-related genes and promoting the formation of the tumor microenvironment. When PARP1 is inhibited by inhibitory drugs, its repair function is lost, leading to the accumulation of DNA single-strand breaks, which are then converted into more lethal double-strand breaks during DNA replication. This severe DNA damage strongly activates upstream kinases such as ATM / ATR, which phosphorylate p53, leading to significant stabilization and accumulation of the p53 protein. Ultimately, the activated p53 pathway triggers a powerful cell cycle arrest or apoptosis program, thereby inhibiting tumor growth.

[0003] Currently, PARP1 inhibitors, as drugs targeting DNA damage repair pathways, have made significant progress in the treatment research and clinical practice of various tumors, especially in tumors associated with BRCA1 / 2 mutations or homologous recombination repair deficiency (HRD), and have been widely used. They have also received FDA approval for tumors such as ovarian cancer and breast cancer. PARP1 inhibitors work by blocking PARP1-mediated single-strand DNA break repair, causing damage to be converted into double-strand breaks during replication. In tumor cells lacking efficient homologous recombination repair, this cumulative damage leads to cell death, thus achieving a "synthetic lethality" treatment strategy. In the field of lung cancer, especially non-small cell lung cancer (NSCLC), studies have shown that some patients have DNA repair defects or p53 pathway abnormalities, and may be sensitive to PARP1 inhibitors. Currently, several PARP1 inhibitors (such as olaparib, tapazoli, and niraparib) are undergoing clinical trials in lung cancer patients, focusing on exploring their combined use with chemotherapy, radiotherapy, and immune checkpoint inhibitors (ICIs) to improve efficacy and overcome drug resistance. Therefore, PARP1 inhibitors not only have potential value in precision medicine in lung cancer treatment, but may also become an important part of combination therapy, providing more treatment options for patients with specific molecular characteristics.

[0004] The benzenesulfonamide moiety is a fundamental pharmacophore in modern medicinal chemistry. This scaffold, characterized by a benzene ring linked to a sulfonamide group (-SO2NH2), forms the basis of a wide range of therapeutic agents. Its unique chemical properties, including the ability of the sulfonamide group to act as a potent zinc-binding group, enable the development of drugs targeting a broad range of enzymes and receptors. This versatility has led to the discovery of benzenesulfonamide derivatives for use in anticancer, antibacterial, anti-inflammatory, anticonvulsant, and antidiabetic drugs. Summary of the Invention

[0005] The purpose of this invention is to provide a benzenesulfonamide derivative, its preparation method, and its application in the preparation of PARP1 inhibitors and antitumor drugs. This invention synthesizes a novel benzenesulfonamide derivative, namely compound N,4-dimethyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzenesulfonamide (abbreviated as LD-J-9), which belongs to the derivatives containing a benzenesulfonamide structure. This invention demonstrates through systematic experiments that LD-J-9 directly targets PARP1 in NSCLC, causing DNA damage and regulating the p53 signaling pathway to inhibit the growth of non-small cell lung cancer tumors. This invention provides a candidate lead compound for the development of novel PARP1 inhibitors for anti-lung cancer drugs.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] This invention provides a benzenesulfonamide derivative with PARP1 inhibitory activity, the structural formula of which is shown in Formula I;

[0008]

[0009] Formula I.

[0010] This invention provides a method for preparing the aforementioned benzenesulfonamide derivative, the method comprising the following steps:

[0011]

[0012] (1) Weigh compound 1 and dissolve it in dichloromethane solution, then add TrtCl and stir at room temperature. After the reaction is complete, pour in water, extract three times with dichloromethane, separate the organic phase, wash with saturated brine, dry, concentrate by rotary evaporation, and purify by column chromatography to obtain compound 2.

[0013] (2) Weigh the compound 2 and dissolve it in dichloromethane solution. Add CH3NH2 and stir at room temperature. After the reaction is complete, pour in water and extract with dichloromethane three times. Separate the organic phase, wash with saturated brine, dry, concentrate by rotary evaporation, and purify by column chromatography to obtain compound LD-J-7.

[0014] (3) Weigh the compound LD-J-7 and dissolve it in ultra-dry dichloromethane. Slowly add NaH mineral oil mixture and stir. Then add p-toluenesulfonyl chloride and reflux the reaction. After the reaction is complete, slowly pour it into water to quench the reaction. Extract the dichloromethane three times, separate the organic phase, wash with saturated brine, dry, concentrate by rotary evaporation, and purify by column chromatography to obtain compound LD-J-8.

[0015] (4) Weigh the compound LD-J-8 and dissolve it in dichloromethane. Add trifluoroacetic acid and stir at room temperature. After the reaction is complete, adjust the pH to weakly alkaline, pour it into water, extract it three times with dichloromethane, concentrate it by rotary evaporation, and purify it by column chromatography to obtain the benzenesulfonamide derivative.

[0016] The present invention also provides a pharmaceutical composition comprising a compound as shown in Formula I or a pharmaceutically acceptable salt thereof.

[0017] The present invention also provides the use of the benzenesulfonamide derivative or the pharmaceutical composition thereof in the preparation of PARP1 inhibitors.

[0018] Furthermore, the benzenesulfonamide derivative can inhibit PARP1 enzyme activity, induce DNA damage, and activate the DDR pathway.

[0019] The present invention also provides the use of the benzenesulfonamide derivative or the pharmaceutical composition thereof in the preparation of antitumor drugs.

[0020] Furthermore, the tumors include squamous cell carcinoma, lung adenocarcinoma, large cell lung cancer, and non-small cell lung cancer.

[0021] Furthermore, the derivative containing the benzenesulfonamide structure can activate the DDR signaling pathway.

[0022] Furthermore, the application dose of the PARP1 inhibitor is 0.1-50 µM, more preferably 0.1-20 µM.

[0023] Furthermore, the derivative containing the benzenesulfonamide structure can induce cell cycle arrest and apoptosis in lung cancer cells.

[0024] This invention also provides the application of the aforementioned benzenesulfonamide derivative in combination with cisplatin in the preparation of antitumor drugs. This invention has found that the compound of Formula 1 and the aforementioned anti-lung cancer active ingredient, such as cisplatin, exhibit a synergistic sensitizing effect, which helps to further improve anti-lung cancer activity.

[0025] The drug formulation is prepared for administration via a route selected from oral, parenteral, oral, nasal, topical, or rectal administration. The drug formulation includes the following dosage forms: oral formulations (e.g., tablets, capsules, solutions, or suspensions); injectable formulations (e.g., injectable solutions or suspensions, or injectable dry powders that can be used immediately after adding water for injection); and topical formulations (e.g., ointments or solutions).

[0026] More preferably, the anti-lung cancer drug further includes a pharmaceutically acceptable carrier or diluent. "Pharmaceutically acceptable carrier" refers to the inactive ingredients in the drug, including but not limited to calcium carbonate, calcium phosphate, various sugars such as lactose, mannitol, starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic polymers or methacrylic polymers, gels, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated castor oil or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, and peanut oil. "Pharmaceutically acceptable diluent" includes but is not limited to starch (such as corn starch, wheat starch, potato starch, etc.), lactose, dextrin, sucrose, pregelatinized starch, microcrystalline cellulose, inorganic salts (such as calcium hydrogen phosphate, calcium sulfate, residual calcium acid, etc.), and mannitol.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] 1. This invention provides the application of a PARP1 inhibitor in the preparation of drugs for treating lung cancer. PARP1 (poly-ADP-ribose polymerase 1) is a key DNA damage sensing and repair enzyme, abnormally highly expressed in tumor cells, and can promote tumor survival and growth through various mechanisms (such as regulating the transcription of tumor-related genes and promoting the formation of the tumor microenvironment). The PARP1 inhibitor provided by this invention can target and inhibit PARP1, thereby inhibiting DNA repair, activating the p53-mediated DDR signaling pathway, inducing apoptosis in lung cancer tumor cells, and affecting the cell cycle of lung cancer tumor cells. It has significant anti-tumor activity and extremely high application prospects.

[0029] 2. This invention demonstrates through research that the compound of Formula 1 possesses excellent PARP1 inhibition function and pharmacological efficacy. The compound of Formula 1 promotes DNA damage in lung cancer cells and induces apoptosis by inhibiting PARP1 activity. This PARP1 inhibitor can promote lung cancer regression when used alone. Furthermore, it can be synergistically enhanced with the clinical anti-lung cancer drug cisplatin, further synergistically strengthening its anti-cancer activity.

[0030] 3. The synthesis method of the PARP1 inhibitor provided by this invention is simple, the raw materials are readily available, the synthetic route has a high yield, and it is environmentally friendly and easy to operate. The preparation process of the dual-target inhibitor described in this invention is simple, the drug has high purity, high yield, and stable quality, and it is easy to carry out large-scale production. The compounds described in this invention have broad prospects in the development and application of anti-tumor drugs.

[0031] 4. The PARP1 inhibitor in this invention not only has the potential to be developed into a new generation of anti-lung cancer drugs, but also has potential application value in the fields of targeted drugs, probe design and fluorescent biolabeling for lung cancer.

[0032] The PARP1 inhibitor provided by this invention has strong selective PARP1 inhibitory activity. On the one hand, Examples 5 and 6 have shown that it exhibits good tumor-suppressing activity in animal models of lung cancer. On the other hand, the compound has low toxicity as measured by mouse weight, organ indicators and blood system indicators. Its effects on organs and reduction of platelets and white blood cells are not significant. Therefore, the compound provided by this invention is a potential high-activity and low-toxicity anti-lung cancer drug.

[0033] 5. In this invention, the compound of Formula 1 and the aforementioned anti-lung cancer active ingredient cisplatin are used in combination to achieve a synergistic sensitizing effect; it has potential medical value and important medical prospects. Attached Figure Description

[0034] Figure 1 This is the carbon NMR spectrum of N,4-dimethyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzenesulfonamide (LD-J-9) described in this invention;

[0035] Figure 2 This is the 1H NMR spectrum of N,4-dimethyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzenesulfonamide (LD-J-9) described in this invention;

[0036] Figure 3 This is a graph showing the inhibitory effect of different concentrations of LD-J-9 on the proliferation of A549 cells detected by the MTT assay in Example 1 of this invention, where A, B, and C represent the cell proliferation inhibition of LD-J-9 at different treatment times.

[0037] Figure 4 This is a graph showing the inhibitory effect of different concentrations of LD-J-9 on A549 cell clone formation as detected by the crystal violet method in Example 1 of this invention.

[0038] Figure 5This is a graph showing the effect of different concentrations of LD-J-9 on the expression of the DNA damage marker protein γ-H2AX in A549 cells in Example 1 of this invention.

[0039] Figure 6 This is a diagram showing the binding interaction between LD-J-9 and PARP1 protein using surface plasmon resonance (SPR) in Example 1 of this invention.

[0040] Figure 7 This is a diagram illustrating the binding interaction between LD-J-9 and PARP1 proteins in cells, as verified by the Cell Thermal Migration Experiment (CETSA) in Example 1 of this invention.

[0041] Figure 8 This is a graph illustrating the inhibitory effect of LD-J-9 on PARP1 enzyme activity verified by the HT unified chemiluminescent PARP assay in Example 1 of this invention. In the graph, A represents the inhibitory effect of LD-J-9 on PARP1 enzyme activity at a concentration of 10 µM, with 3-ab being the positive pair compound; B represents the IC50 value of LD-J-9 on PARP1 enzyme activity. 50 .

[0042] Figure 9 This is a diagram showing the activation effect of different concentrations of LD-J-9 on the downstream ATM-CHK2 protein in A549 cells after DNA damage, as described in Example 1 of this invention.

[0043] Figure 10 This is a graph showing the effect of different concentrations of LD-J-9 on the expression of P53 protein and its downstream proteins in A549 cells in Example 1 of this invention.

[0044] Figure 11 This is a graph showing the cell cycle arrest effect of different concentrations of LD-J-9 on A549 cells in Example 1 of the invention. Graph A is a flow cytometry graph, and graph B is a statistical graph of cell counts at each cell cycle.

[0045] Figure 12 This is a graph showing the effect of different concentrations of LD-J-9 on the expression of cell cycle-related proteins in A549 cells in Example 1 of the invention.

[0046] Figure 13 This is a graph showing the induction of apoptosis in A549 cells by different concentrations of LD-J-9 in Example 1 of the invention. Graph A is a flow cytometry graph, and graph B is a statistical graph of cell apoptosis.

[0047] Figure 14 This is a graph showing the effect of different concentrations of LD-J-9 on the expression of apoptosis-related proteins in A549 cells in Example 1 of the invention.

[0048] Figure 15This is a graph showing the inhibitory effect of different concentrations of LD-J-9 on the growth of syngeneic xenografts in LLC tumor-bearing mice, as described in Example 1 of the invention. Figure A shows the mouse body weight change curve; Figure B shows the mouse tumor volume change curve; Figure C shows the mouse tumor weight statistics; Figure D shows the mouse spleen index statistics; Figure E shows the mouse blood routine test statistics; and Figure F shows the mouse tumor.

[0049] Figure 16 This is a graph showing the inhibitory effect of 10µM LD-J-9 combined with cisplatin on the proliferation of A549 cells in Example 1 of the invention. In Figure A, cell proliferation inhibition was detected by the MTT assay; and in Figure B, cell clonal changes were detected by crystal violet staining.

[0050] Figure 17 This is a graph illustrating the inhibitory effect of the combination of LD-J-9 and cisplatin on the growth of syngeneic xenografts of Lewis lung cancer cells (LLC) in mice, as described in Example 1 of the invention. Figure A shows the mouse body weight change curve; Figure B shows the mouse tumor volume change curve; Figure C shows the mouse tumor weight statistics; Figure D shows the mouse spleen index statistics; and Figure E shows the mouse tumor. Detailed Implementation

[0051] The technical solution of the present invention will be further described in detail with reference to the following specific examples.

[0052] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0053] Example 1: Synthesis of N,4-dimethyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzenesulfonamide

[0054] 1. N,4-Dimethyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzenesulfonamide

[0055]

[0056] Formula I.

[0057] The synthesis of N,4-dimethyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzenesulfonamide (LD-J-9) includes the following steps:

[0058]

[0059] (1) Synthesis of compound 2: Compound 1 (153 mg, 1 mmol, 1.0 equiv.) was weighed and dissolved in 10 mL of dichloromethane solution, and then TrtCl (361 mg, 1.3 mmol, 1.3 equiv.) was added and stirred at room temperature for 5-8 h. After the reaction was completed, 3 times the volume of water was added, and the mixture was extracted three times with dichloromethane (5 mL). The organic phase was separated, washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by column chromatography to obtain compound 2 (320 mg, yield 81%).

[0060] (2) Synthesis of LD-J-7: Compound 2 (316 mg, 0.8 mmol, 1.0 equiv.) was weighed and dissolved in 10 mL of dichloromethane solution. CH3NH2 (40 mg, 1.2 mmol, 1.5 equiv.) was added and stirred at room temperature for 8 h. After the reaction was completed, 3 times the volume of water was added, and the mixture was extracted three times with dichloromethane (5 mL). The organic phase was separated, washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by column chromatography to obtain compound LD-J-7 (246 mg, yield 78.7%).

[0061] (3) Synthesis of LD-J-8: Compound LD-J-7 (195 mg, 0.5 mmol, 1.0 equiv.) was weighed and dissolved in 10 mL of ultra-dry dichloromethane. 60% NaH mineral oil mixture (40 mg, 1 mmol, 2 equiv.) was slowly added and stirred for 30 min. Then p-toluenesulfonyl chloride (190 mg, 1 mmol, 2 equiv.) was added and the mixture was refluxed for 2-5 h. After the reaction was completed, the reaction was quenched by slowly pouring in 3 times the volume of water. Dichloromethane (5 mL) was extracted three times to separate the organic phase. The organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by column chromatography to obtain compound LD-J-8 (212 mg, yield 78%).

[0062] (4) Synthesis of LD-J-9: Compound LD-J-8 (110 mg, 0.2 mmol, 1.0 quiv.) was weighed and dissolved in 10 mL of dichloromethane. 2 mL of trifluoroacetic acid was added and the mixture was stirred at room temperature for 1 h. After the reaction was completed, the pH was adjusted to weakly alkaline with Na2CO3 solution, poured into water, and extracted three times with dichloromethane (5 mL). The extract was concentrated by rotary evaporation and purified by column chromatography to obtain LD-J-9 (54 mg, yield 90%).

[0063] The NMR data of the prepared N,4-dimethyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzenesulfonamide (hereinafter referred to as LD-J-9) are as follows (e.g.) Figure 1 and2 (as shown)

[0064] 1 H NMR (500 MHz, Chloroform-d) δ 9.83 (s, 1H), 8.60 (s, 1H), 7.64 –7.55 (m, 2H), 7.36 (dd, J = 3.7, 2.1 Hz, 1H), 7.27 (d, J = 8.2 Hz, 3H), 6.94(dd, J = 3.7, 1.8 Hz, 1H), 3.37 (s, 3H), 2.42 (s, 3H).13C NMR (126 MHz, Chloroform-d) δ 153.96, 153.34, 150.39, 144.14, 134.10, 129.53, 128.06,124.76, 102.88, 36.35, 29.70, 21.59.

[0065] Example 2: LD-J-9 inhibits NSCLC cell proliferation

[0066] 1. To investigate the potential antitumor activity of LD-J-9 in non-small cell lung cancer (NSCLC), this invention first verified its effect on the p53 wild-type NSCLC cell line A549. A549 cells were cultured in F-12K medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Cell culture was conducted at 37°C in a 5% CO2 atmosphere. Cell viability was determined using the MTT assay. A549 cells were cultured at a rate of 5 × 10⁶ cells / mL. 4 Cells were seeded at a density of 100 μg / well in 96-well plates and treated with different concentrations of LD-J-9 for 24, 48, or 72 hours. After treatment, MTT solution was added to each well, and the cells were incubated for another 4 hours. Finally, the supernatant was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was measured at 490 nm using a microplate reader. The MTT assay results showed that LD-J-9 significantly inhibited A549 cell viability in a time- and concentration-dependent manner. Figure 3 ).

[0067] 2. Further verification of the effect of LD-J-9 on A549 cell proliferation was conducted using cell cloning experiments. A549 cells were injected at a rate of 1 × 10⁻⁶ cells / mL. 4Cells were seeded at a density of 1000 cells / well in 6-well plates. After treatment with different concentrations of LD-J-9 for 24, 48, or 72 hours, the medium was replaced with fresh, drug-free medium, and the cells were cultured for another 7 days until colony formation was visible. Subsequently, the colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet solution, photographed, and counted. Colony formation assays showed that LD-J-9 significantly reduced the colony-forming ability of A549 cells in a time- and concentration-dependent manner. Figure 4 This indicates that LD-J-9 has a significant inhibitory effect on long-term cell proliferation.

[0068] Example 3: LD-J-9 inhibits PARP1 enzyme activity and induces DNA damage.

[0069] 1. This embodiment first detected γ-H2AX, a core marker of DNA double-strand breaks (DSBs). Validation was performed using Western blot analysis. Total protein was extracted from cells or tissues using RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the BCA method. Equal volumes of protein were separated by SDS-PAGE and transferred to nitrocellulose (NC) membranes. The membranes were blocked with 5% skim milk and then incubated overnight at 4°C with primary antibody (γ-H2AX), followed by incubation at room temperature for 2 hours with secondary antibody. Protein banding was performed using HRP-linked secondary antibody and enhanced chemiluminescence (ECL) reagent, and detected using a chemiluminescence imaging system. Western blot results showed that γ-H2AX levels in A549 cells were significantly upregulated in a concentration-dependent manner after LD-J-9 treatment. Figure 5 This indicates that LD-J-9 treatment led to a significant accumulation of intracellular DNA damage.

[0070] 2. Further analysis of the binding between LD-J-9 and PARP1 was conducted using surface plasmon resonance (SPR). This was performed on a Biacore T200 instrument at a preset temperature of 25°C and a flow rate of 30 µL / min using a CM5 sensor chip. PARP1 was treated with sodium acetate at pH 4.0 and then covalently coupled to the CM5 chip surface. Gradient concentrations of LD-J-9 were prepared using a 2-fold dilution method with buffer solution, and detection was performed after solvent correction. The detection data were fitted to a 1:1 interaction model using Biacore's kinetic evaluation software. At least three measurements were recorded for each interaction, and the KD value was calculated. SPR results showed that LD-J-9 can bind to PARP1 with an affinity of 6.048 µM (…). Figure 6 ).

[0071] 3. Further verification of intracellular binding of LD-J-9 and PARP1 was performed using a cell thermal migration assay (CETSA). Cells treated with different concentrations of LD-J-9 were collected, washed twice with pre-cooled PBS, and resuspended in lysis buffer containing protease inhibitors. After centrifugation, the supernatant was placed in a gradient PCR instrument and heated from 52°C to 64°C for 3 min. After centrifugation, the supernatant was collected. Changes in PARP1 protein expression were detected by Western blot. CETSA results showed that LD-J-9 can bind to and stabilize PARP1 protein intracellularly. Figure 7 ).

[0072] 4. Further, using a highly sensitive fluorescence detection method (HT Unified Chemiluminescent PARP Detection Method, Product No. 4676-096-K), LD-J-9 (final concentration 20 μM) was added to the wells. Then, the PARP enzyme and reaction mixture were added to the system. The reaction was terminated by washing the wells twice with 0.1% Triton X-100 / PBS and twice with PBS. The mixture was then incubated with StrepHRP for 1 hour. Finally, Peroxy-Glow reagent was added to the wells, and chemiluminescence detection was performed immediately on a microplate reader.

[0073] PARP1 activity assay results showed that LD-J-9 could effectively inhibit PARP1 activity, IC50... 50 It is 6.837 µM ( Figure 8 ).

[0074] Example 4: LD-J-9 can activate the DNA damage response (DDR) signaling pathway and induce cell cycle arrest and apoptosis in lung cancer cells.

[0075] 1. This invention further confirms that inhibition of PARP1 enzyme activity leads to DNA damage accumulation and activation of the DNA damage response (DDR) signaling pathway. Western blot analysis was used to examine the phosphorylation activation of proteins in the DDR signaling pathway. The results showed that the phosphorylation levels of ATM and Chk2 significantly increased with increasing LD-J-9 concentration. Figure 9 This indicates that the DDR signal path is activated.

[0076] 2. Further analysis using Western blot was conducted to detect the activation of p53 protein downstream of the DDR signaling pathway and the activation of downstream proteins of p53. The results showed that the expression of p53 protein increased in a dependent manner with increasing LD-J-9 concentration, and the expression of downstream proteins of p53, p21, Puma, Bax, and Noxa, also increased in a dependent manner with increasing LD-J-9 concentration. Figure 10 ).

[0077] 3. To clarify the specific effects of LD-J-9 on the cell cycle of A549 lung cancer cells, flow cytometry was used for analysis in this embodiment. A549 cells were treated with different concentrations of LD-J-9 for 48 hours. Cells were then collected, washed with PBS, and fixed overnight in pre-chilled 70% ethanol at -20°C. After fixation, the cells were washed again and stained with propidium iodide (PI) solution for 30 minutes at room temperature in the dark. The proportions of cells in the G1, S, and G2 / M phases were analyzed using a FACSCalibur™ flow cytometer and analyzed using FlowJO software. Flow cytometry results showed that LD-J-9 treatment significantly arrested A549 cells in the G2 / M phase, significantly increased the proportion of cells in the G2 / M phase, and significantly decreased the proportion of cells in the G1 phase. Figure 11 Furthermore, this invention examined the expression of cycle-related proteins. Western blot analysis showed that, consistent with the G2 / M phase arrest phenotype, the expression level of Cyclin B1, a key protein promoting this phase progression, was cumulatively upregulated, while the expression levels of CDK4 / 6 and Cyclin D1, which are associated with G1 phase, showed no significant changes. Figure 12 This indicates that LD-J-9 can induce cell cycle arrest in A549 lung cancer cells.

[0078] 4. To further clarify the specific effect of LD-J-9 on apoptosis of A549 lung cancer cells, flow cytometry was used for analysis in this embodiment. A549 cells were treated with different concentrations of LD-J-9 for 48 hours. The apoptosis status was quantitatively analyzed using the Annexin V-FITC / PI apoptosis detection kit. Specifically, after collecting cells, they were washed and resuspended in buffer, then stained with Annexin V-FITC and PI, and immediately analyzed using FACSCalibur™ flow cytometry. The percentage of apoptotic cells was calculated using FlowJO software. The flow cytometry results showed that LD-J-9 significantly induced apoptosis in A549 cells (…). Figure 13 Furthermore, this invention detected the expression of apoptosis-related proteins. Western blot analysis showed that after LD-J-9 treatment, the expression level of the pro-apoptotic protein Bax was upregulated, while the expression of the anti-apoptotic proteins Bcl-2 and Mcl-1 was significantly downregulated. The initiator protein Caspase-9 and its downstream core executive protein Caspase-3 in the mitochondrial apoptosis pathway were both cleaved and activated, with the level of their activated forms (cleaved Caspase-9 / 3) increasing in a concentration-dependent manner. Figure 14 This indicates that LD-J-9 can induce apoptosis in A549 lung cancer cells.

[0079] Example 5: Antitumor activity of LD-J-9 in vivo

[0080] To confirm the anti-lung cancer tumor potential of LD-J-9 in an in vivo animal model, this invention constructed a mouse Lewis lung cancer cell (LLC) tumor-bearing mouse syngeneic transplantation model. LLC cells were cultured in high-glucose DMEM medium. Cells were maintained in a humidity-controlled incubator at 37°C and in a 5% carbon dioxide environment. Six-week-old male C57BL / J mice were selected, and 5 × 10⁵ LLC cells were subcutaneously inoculated in their right axilla. When the average tumor volume reached 50-100 mm³, the tumor-bearing mice were randomly divided into five groups (n=6 per group): (1) saline control group; (2) LD-J-9 (20 mg / kg) group; (3) LD-J-9 (10 mg / kg) group; (4) LD-J-9 (5 mg / kg) group. LD-J-9 was administered via intraperitoneal injection (ip) daily. Tumor volume and body weight were measured every two days throughout the treatment period. After 14 consecutive days of treatment, all mice were euthanized. Tumors were removed and mice were weighed. Results showed that, compared to the control group, LD-J-9 effectively inhibited tumor growth in a concentration-dependent manner, with a high-dose inhibition rate of 57%. Mouse weight remained stable throughout the treatment process, showing no difference from the control group. Spleen index and blood routine tests revealed no significant changes in any of the LD-J-9 experimental groups compared to the control group. Figure 15 This indicates that LD-J-9 is safe and effective.

[0081] Example 6: Synergistic effect of LD-J-9 combined with cisplatin on antitumor activity

[0082] 1. To investigate whether LD-J-9 increases the sensitivity of A549 cells to the chemotherapeutic drug cisplatin (DDP), we treated A549 cells with different concentrations of cisplatin alone, different concentrations of LD-J-9 alone, and LD-J-9 in combination with cisplatin. Cell viability was assessed using the MTT assay after 72 hours of treatment. The results showed that the LD-J-9 compound increased the sensitivity of A549 cells to the chemotherapeutic drug cisplatin. Figure 16 (Table 1).

[0083] Table 1. LD-J-9 enhances sensitivity to cisplatin therapy

[0084]

[0085] 2. To confirm the antitumor potential of the combined action of LD-J-9 and cisplatin in an in vivo animal model, this invention used the LLC tumor-bearing mouse syngeneic transplantation model constructed in Example 5. The tumor-bearing mice were randomly divided into five groups (n=6 per group): (1) saline control group; (2) LD-J-9 group (10 mg / kg); (3) low-dose cisplatin group (2.5 mg / kg); (4) high-dose cisplatin group (5 mg / kg); (5) combined treatment group (LD-J-9 10 mg / kg + cisplatin 2.5 mg / kg). LD-J-9 was administered daily, and cisplatin was administered every three days. All treatments were administered via intraperitoneal injection (ip). Tumor volume and body weight were measured every two days throughout the treatment period. After 18 consecutive days of treatment, all mice were euthanized. The tumors were removed and weighed, and the spleens were collected for analysis. Results showed that, compared with the control group, tumor growth curves and final tumor weight analysis indicated that LD-J-9 (10 mg / kg) or low-dose cisplatin (2.5 mg / kg) monotherapy had virtually no inhibitory effect. Combination therapy significantly reduced tumor volume and weight, achieving antitumor efficacy comparable to high-dose cisplatin (5 mg / kg), with an inhibition rate of approximately 80%. Figure 17 This indicates the synergistic antitumor activity of LD-J-9 and cisplatin in vivo.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A benzenesulfonamide derivative, characterized in that, The benzenesulfonamide derivative is N,4-dimethyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzenesulfonamide, and its structural formula is shown in Formula I: ， Formula I.

2. The method for preparing the benzenesulfonamide derivative according to claim 1, characterized in that, The preparation method includes the following steps: ， (1) Weigh compound 1 and dissolve it in dichloromethane solution, then add TrtCl and stir at room temperature. After the reaction is complete, pour in water, extract three times with dichloromethane, separate the organic phase, wash with saturated brine, dry, concentrate by rotary evaporation, and purify by column chromatography to obtain compound 2. (2) Weigh the compound 2 and dissolve it in dichloromethane solution. Add CH3NH2 and stir at room temperature. After the reaction is complete, pour in water and extract with dichloromethane three times. Separate the organic phase, wash with saturated brine, dry, concentrate by rotary evaporation, and purify by column chromatography to obtain compound LD-J-7. (3) Weigh the compound LD-J-7 and dissolve it in ultra-dry dichloromethane. Slowly add NaH mineral oil mixture and stir. Then add p-toluenesulfonyl chloride and reflux the reaction. After the reaction is complete, slowly pour it into water to quench the reaction. Extract the dichloromethane three times, separate the organic phase, wash with saturated brine, dry, concentrate by rotary evaporation, and purify by column chromatography to obtain compound LD-J-8. (4) Weigh the compound LD-J-8 and dissolve it in dichloromethane. Add trifluoroacetic acid and stir at room temperature. After the reaction is complete, adjust the pH to weakly alkaline, pour it into water, extract it three times with dichloromethane, concentrate it by rotary evaporation, and purify it by column chromatography to obtain the benzenesulfonamide derivative.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a compound as shown in Formula I of claim 1 or a pharmaceutically acceptable salt thereof.

4. The use of the benzenesulfonamide derivative of claim 1 or the pharmaceutical composition of claim 3 in the preparation of PARP1 inhibitors.

5. The application according to claim 4, characterized in that, The benzenesulfonamide derivative can inhibit PARP1 enzyme activity, induce DNA damage, and activate the DDR signaling pathway.

6. The use of the benzenesulfonamide derivative of claim 1 or the pharmaceutical composition of claim 3 in the preparation of an antitumor drug.

7. The application according to claim 6, characterized in that, The tumors include squamous cell carcinoma, lung adenocarcinoma, large cell lung cancer, and non-small cell lung cancer.

8. The application according to claim 7, characterized in that, The application dose of the benzenesulfonamide derivative is 0.1-50µM.

9. The application according to claim 7, characterized in that, The derivative containing the benzenesulfonamide structure can induce cell cycle arrest and apoptosis in lung cancer cells.

10. The use of the benzenesulfonamide derivative of claim 1 in combination with cisplatin in the preparation of an antitumor drug.

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