A coumarin-pyrazole ring hybrid derivative, its preparation method and application

Abstract

This invention discloses a coumarin-pyrazole ring hybrid derivative, its preparation method, and its applications, belonging to the field of pharmaceutical synthesis technology. Based on coumarin as the backbone, the derivative is synthesized through a three-step reaction. The key steps include the preparation of intermediates 1 and 2 and the synthesis of the target compound. The method is simple, efficient, and uses readily available raw materials. The target compound exhibits strong antitumor activity, significantly inhibiting tumor cells and showing low toxicity to normal cells. This class of compounds conforms to drug-like drug rules and can be formulated into various dosage forms, providing lead compounds for antitumor drug development and possessing broad application prospects. The compounds are those shown in the general formula and their pharmaceutically acceptable salts, deuterated derivatives, or optical isomers. Experiments have confirmed that this series of compounds is simple to prepare, has high synthetic efficiency, and can be used to prepare antitumor drug candidates.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, and in particular to a coumarin-pyrazole ring hybrid derivative, its preparation method, and its application. Background Technology

[0002] Recent global epidemiological studies show that cancer is exhibiting an unprecedented trend of development, with a continuous rise in the number of cancer deaths worldwide and a significant increase in mortality rates. It is projected that cancer will replace cardiovascular and cerebrovascular diseases as the leading threat to human health in the future. Tumors are classified into two main categories: malignant and benign. Malignant tumors, due to their invasiveness, metastasis, and difficulty in eradication, seriously threaten people's lives and health. Currently, in clinical practice, mainstream cancer treatment options include surgery, drug therapy (chemotherapy), and radiotherapy. While these methods have shown some progress in improving patient survival rates, traditional chemotherapy drugs still face multiple clinical challenges, including widespread drug resistance, significant off-target toxicity, a narrow therapeutic window, and severe adverse reactions caused by invasive treatment methods. Therefore, developing highly effective and low-toxicity novel drugs is extremely urgent and important for cancer treatment, and natural products can provide a practical approach to meeting this requirement.

[0003] Coumarins, as natural compounds with broad application prospects, exhibit great development potential in their natural forms and derivatives. Coumarin derivatives possess a wide range of pharmacological activities, including antiarrhythmic, anti-osteoporosis, anticoagulant, antioxidant, anti-HIV, antibacterial, and antitumor effects, demonstrating potential medicinal value. Coumarin compounds are also highly promising anticancer drugs, with anticancer mechanisms involving multiple aspects of cancer pathways, effective against most cancers with few adverse reactions. Furthermore, coumarin compounds have relatively simple structures, making them easy to synthesize and modify. Therefore, there is a need to provide a compound based on the coumarin skeleton as a highly effective and low-toxicity antitumor candidate drug. Summary of the Invention

[0004] The purpose of this invention is to provide a coumarin-pyrazole ring hybrid derivative, its preparation method, and its application, in order to solve the above-mentioned problems.

[0005] This invention provides a coumarin-pyrazole ring hybrid derivative, the general structural formula of which is:

[0006]

[0007] Where R is

[0008] Its structure includes a 3-hybridized pyrazole ring at the coumarin position, with a 2,4-dinitrobenzenesulfonyl chloride group attached to the pyrazole ring; coumarin-pyrazole ring hybrid derivatives also include pharmaceutically acceptable salts, deuterated derivatives or optical isomers of the compounds shown in the general formula.

[0009] A method for preparing the coumarin-pyrazole ring hybrid derivative as described above includes the following steps:

[0010] S1. Synthesize intermediate 1 by fully dissolving 2-hydroxy-4-diethylaminobenzaldehyde and 4-hydroxy-6-methyl-2-pyranone in anhydrous ethanol, then slowly adding a catalytic amount of anhydrous piperidine to the reaction system, and refluxing the reaction under nitrogen protection. After the reaction is completed, remove the anhydrous ethanol under reduced pressure, extract with ethyl acetate, wash with saturated brine, remove water with anhydrous sodium sulfate, and purify the crude product to obtain intermediate 1.

[0011] S2. Synthesize intermediate 2. After fully dissolving intermediate 1 in anhydrous ethanol, slowly add hydrazine monohydrate dropwise to the reaction system. React at 82°C for 1 hour under nitrogen protection. After the reaction is complete, cool to room temperature and add saturated brine. After standing, a large amount of yellow flocculent precipitate appears. Filter under reduced pressure and wash with a large amount of distilled water to remove sodium chloride to obtain a yellow solid, which is intermediate 2.

[0012] S3. To synthesize the compound, intermediate 2 was dissolved in anhydrous tetrahydrofuran, and then NaH was weighed and poured into the reaction flask. The mixture was stirred at room temperature for 1 hour under nitrogen protection. Then 2,4-dinitrobenzenesulfonyl chloride was added, and the mixture was stirred for another 2 hours. The reaction was stopped, NaH was quenched with water, extracted three times, dried, filtered, concentrated under reduced pressure, and purified to obtain the target compound.

[0013] The reaction formula is as follows:

[0014]

[0015] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S1, the equivalent ratio of 2-hydroxy-4-diethylaminobenzaldehyde: 4-hydroxy-6-methyl-2-pyranone: anhydrous ethanol: anhydrous piperidine is 1.2:1.2:25:1.5; the reaction conditions are 80-82°C for 4 hours.

[0016] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S1, the crude product is purified by silica gel column chromatography with an eluent ratio of CH2Cl2:EtOAC = 6:1.

[0017] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S2, the equivalent ratio of intermediate 1: anhydrous ethanol: hydrazine monohydrate is 1:15:7.8.

[0018] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S3, the equivalent ratio of intermediate 2: anhydrous tetrahydrofuran: NaH: 2,4-dinitrobenzenesulfonyl chloride is 1:10:4:1.2; the extraction is carried out using a mixture of water and DCM, with a water:DCM volume ratio of 1:3.

[0019] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S3, drying is performed using anhydrous sodium sulfate; purification is performed using silica gel column purification, with the eluent being a mixture of petroleum ether and ethyl acetate, PE:EtOAc = 2:1.

[0020] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, the compound is added to a pharmaceutically acceptable salt excipient to prepare any pharmaceutically acceptable dosage form.

[0021] An application of a coumarin-pyrazole ring hybrid derivative as described above, wherein the deuterated or optical isomer of the compound is used in the preparation of antitumor and anti-inflammatory drugs.

[0022] Preferably, in the application of the above-mentioned coumarin-pyrazole ring hybrid derivative, the compound is used to inhibit the growth activity of cancer cells.

[0023] Therefore, the present invention, employing the above-mentioned coumarin-pyrazole ring hybrid derivative and its preparation method and application, has the following beneficial effects:

[0024] (1) The coumarin-pyrazole hybrid derivative provided by the present invention can be used to incubate the compound, different cancer cells and culture medium in a 96-well plate, and then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value of each well at a wavelength of 450 nm. It can be observed that the derivative has a significant inhibitory effect on the growth of cancer cells.

[0025] (2) The compounds obtained conform to the five principles of drug-likeness (Lipinski) and are drug lead molecules with good application prospects, providing high-quality candidate compounds and theoretical basis for the development of anti-tumor drugs.

[0026] (3) The preparation method provided by the present invention can successfully synthesize coumarin-pyrazole hybrid derivatives. The synthesis method is simple and efficient, the raw materials are inexpensive and readily available, which is conducive to large-scale industrial production and has broad market application prospects.

[0027] (4) The obtained compounds can be used to prepare drugs for various cancers, including but not limited to lung cancer, gastric cancer, liver cancer, bladder cancer, breast cancer, etc.; they can be formulated into tablets, capsules, injections and other dosage forms with pharmaceutically acceptable excipients to meet different drug administration needs; their pharmaceutically acceptable salts, deuterated compounds and optical isomers can be extended to anti-tumor, anti-inflammatory and other drug applications.

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0029] Figure 1 This is the 1H NMR spectrum of the target compound (coumarin-pyrazole hybrid derivative) prepared in Example 2 of this invention;

[0030] Figure 2 This is the 1H NMR spectrum of compound 8 (coumarin-pyrazole hybrid derivative) prepared in Example 2 of this invention;

[0031] Figure 3 This is the 1H NMR spectrum of compound 10 (coumarin-pyrazole hybrid derivative) prepared in Example 2 of this invention;

[0032] Figure 4 This is the 1H NMR spectrum of compound 20 (coumarin-pyrazole hybrid derivative) prepared in Example 2 of this invention;

[0033] Figure 5 This is the 1H NMR spectrum of compound 24 (coumarin-pyrazole hybrid derivative) prepared in Example 2 of this invention;

[0034] Figure 6 This refers to the inhibitory rate of the 25 target compounds in Example 2 of this invention on the proliferation of A549 cells;

[0035] Figure 7 This refers to the inhibitory rate of the 25 target compounds in Example 2 of this invention on the proliferation of H1299 cells;

[0036] Figure 8 This refers to the inhibitory rate of the 25 target compounds in Example 2 of this invention on the proliferation of H1975 cells;

[0037] Figure 9 This refers to the inhibitory rate of the 25 target compounds in Example 2 of this invention on the proliferation of BGC-823 cells;

[0038] Figure 10 This refers to the inhibitory rate of the 25 target compounds in Example 2 of this invention on the proliferation of SGC-7901 cells;

[0039] Figure 11 This refers to the inhibitory rate of the 25 target compounds in Example 2 of this invention on the proliferation of HepG-2 cells;

[0040] Figure 12 This refers to the inhibition rate of UMUC2 cell proliferation by 25 target compounds in Example 2 of this invention. Detailed Implementation

[0041] To better understand the above technical solutions, a detailed description of the solutions will be provided below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms, and “multiple” generally includes at least two unless the context clearly indicates otherwise.

[0043] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the article or device that includes said element.

[0044] This invention provides a coumarin-pyrazole ring hybrid derivative, the general structural formula of which is:

[0045]

[0046] Where R is

[0047] Its structure includes a 3-hybridized pyrazole ring at the coumarin position, with a 2,4-dinitrobenzenesulfonyl chloride group attached to the pyrazole ring; the coumarin-pyrazole ring hybrid derivatives also include pharmaceutically acceptable salts, deuterated derivatives or optical isomers of the compounds shown in the general formula.

[0048] A method for preparing the coumarin-pyrazole ring hybrid derivative as described above includes the following steps:

[0049] S1. Synthesize intermediate 1 by fully dissolving 2-hydroxy-4-diethylaminobenzaldehyde and 4-hydroxy-6-methyl-2-pyranone in anhydrous ethanol, then slowly adding a catalytic amount of anhydrous piperidine to the reaction system, and refluxing the reaction under nitrogen protection. After the reaction is completed, remove the anhydrous ethanol under reduced pressure, extract with ethyl acetate, wash with saturated brine, remove water with anhydrous sodium sulfate, and purify the crude product to obtain intermediate 1.

[0050] S2. Synthesize intermediate 2. After fully dissolving intermediate 1 in anhydrous ethanol, slowly add hydrazine monohydrate dropwise to the reaction system. React at 82°C for 1 hour under nitrogen protection. After the reaction is complete, cool to room temperature and add saturated brine. After standing, a large amount of yellow flocculent precipitate appears. Filter under reduced pressure and wash with a large amount of distilled water to remove sodium chloride to obtain a yellow solid, which is intermediate 2.

[0051] S3. To synthesize the compound, intermediate 2 was dissolved in anhydrous tetrahydrofuran, and then NaH was weighed and poured into the reaction flask. The mixture was stirred at room temperature for 1 hour under nitrogen protection. Then 2,4-dinitrobenzenesulfonyl chloride was added, and the mixture was stirred for another 2 hours. The reaction was stopped, NaH was quenched with water, extracted three times, dried, filtered, concentrated under reduced pressure, and purified to obtain the target compound.

[0052] The reaction formula is as follows:

[0053]

[0054] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S1, the equivalent ratio of 2-hydroxy-4-diethylaminobenzaldehyde: 4-hydroxy-6-methyl-2-pyranone: anhydrous ethanol: anhydrous piperidine is 1.2:1.2:25:1.5; the reaction conditions are 80-82°C for 4 hours.

[0055] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S1, the crude product is purified by silica gel column chromatography with an eluent ratio of CH2Cl2:EtOAC = 6:1.

[0056] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S2, the equivalent ratio of intermediate 1: anhydrous ethanol: hydrazine monohydrate is 1:15:7.8.

[0057] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S3, the equivalent ratio of intermediate 2: anhydrous tetrahydrofuran: NaH: 2,4-dinitrobenzenesulfonyl chloride is 1:10:4:1.2; the extraction is carried out using a mixture of water and DCM, with a water:DCM volume ratio of 1:3.

[0058] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, in step S3, drying is performed using anhydrous sodium sulfate; purification is performed using silica gel column purification, with the eluent being a mixture of petroleum ether and ethyl acetate, PE:EtOAc = 2:1.

[0059] Preferably, in the above-mentioned method for preparing a coumarin-pyrazole ring hybrid derivative, the compound is added to a pharmaceutically acceptable salt excipient to prepare any pharmaceutically acceptable dosage form.

[0060] An application of a coumarin-pyrazole ring hybrid derivative as described above, wherein the deuterated or optical isomer of the compound is used in the preparation of antitumor and anti-inflammatory drugs.

[0061] Preferably, in the application of the above-mentioned coumarin-pyrazole ring hybrid derivative, the compound is used to inhibit the growth activity of cancer cells.

[0062] It should be noted that "pharmaceutically acceptable salts" include salts formed by compounds and their hydrates with acids and basic salts formed with inorganic bases. The acids include, but are not limited to, hydrochloric acid, hydrobromic acid, salicylic acid, sulfuric acid, citric acid, succinic acid, pyruvic acid, phosphoric acid, lactic acid, acetic acid, tartaric acid, mandelic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid. The inorganic bases include, but are not limited to, potassium salts, magnesium salts, lithium salts, sodium salts, calcium salts, and aminobutanetriol salts.

[0063] Coumarin-pyrazole hybrid derivatives are used in the preparation of antitumor drugs, including but not limited to skin cancer, breast cancer, lymphoma, leukemia, colon cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, intestinal cancer, pancreatic cancer, oral cancer, melanoma, head and neck cancer, kidney cancer, esophageal cancer, liver cancer, bile duct cancer, and bladder cancer.

[0064] Any compound may be added to a pharmaceutically acceptable salt excipient to form any pharmaceutically acceptable dosage form, including but not limited to tablets, capsules, suspensions, suppositories, injections, granules, ointments, and sprays.

[0065] To more clearly and in detail introduce the coumarin-pyrazole ring hybrid derivative, its preparation method, and its application provided by the embodiments of the present invention, the following description will be based on specific embodiments.

[0066] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial means; the methods and steps not described in detail are all conventional techniques in the field.

[0067] Example 1

[0068] Preparation of intermediates 1-2:

[0069]

[0070] Different substituents in the target compound:

[0071]

[0072] The specific steps are as follows:

[0073] (1) Synthetic intermediate 1.

[0074] Accurately weigh 3.00 g (15.50 mmol, 1.2 equiv.) of 2-hydroxy-4-diethylaminobenzaldehyde and 1.60 g (12.90 mmol, 1.2 equiv.) of 4-hydroxy-6-methyl-2-pyranone into a dry 100.0 mL round-bottom flask. Add 25.0 mL of anhydrous ethanol as a solvent to the system to fully dissolve the mixture. Then, slowly add a catalytic amount of anhydrous piperidine to the reaction system. React at 82 °C for 4 h under nitrogen protection. The reaction progress was monitored by thin-layer chromatography (TLC). Heating was stopped when the starting materials were almost completely eliminated. Anhydrous ethanol was removed under reduced pressure, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, and the organic phases were combined and the water was removed with anhydrous sodium sulfate. The mixture was filtered at room temperature, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (CH2Cl2:EtOAC = 6:1, v / v). The purified product was then concentrated under reduced pressure to give an orange-yellow needle-like solid (yield 89.17%). f =0.71, developing solvent is PE:EtOAC=2:1).

[0075] (2) Synthesize intermediate 2.

[0076] Compound 1 (400.0 mg, 1.33 mmol, 1.0 equiv.) was accurately weighed into a 25.0 mL dry round-bottom flask. 15.0 mL of anhydrous ethanol was added to the system to fully dissolve the reactants. Then, 80% hydrazine monohydrate (515.0 mg, 10.29 mmol, 7.8 equiv.) was accurately measured and slowly added dropwise to the reaction system. The reaction was carried out at 82 °C for 1 h under nitrogen protection. The reaction progress was monitored by thin-layer chromatography (TLC). Heating was stopped when the starting material was almost completely eliminated. After the reaction cooled to room temperature, saturated brine was added. Upon standing, a large amount of yellow flocculent precipitate appeared. The precipitate was filtered under reduced pressure and washed with plenty of distilled water to remove a large amount of sodium chloride, yielding a yellow solid (yield 82.01%, R). f =0.31, developing solvent is PE:EtOAc=2:1).

[0077] Example 2

[0078] Synthesis of target compound 1: Intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) was accurately weighed into a 25.00 mL dry round-bottom flask. Anhydrous pyridine was slowly added under nitrogen protection to dissolve it (anhydrous pyridine served as both solvent and acid-binding agent). Then, dimethylcarbamoyl chloride (86.46 mg, 0.80 mmol, 1.2 equiv.) was slowly added dropwise into the reaction system, and the reaction was allowed to proceed overnight at room temperature. The reaction was monitored by thin-layer chromatography (TLC). When the starting material was almost completely eliminated, the reaction was stopped. The reaction solution was poured into pre-prepared 4N hydrochloric acid to remove excess pyridine. Extraction was performed with dichloromethane (30 mL × 3). The organic phases were combined, and the water in the dichloromethane organic layer was removed using anhydrous sodium sulfate. The mixture was filtered, concentrated under reduced pressure to obtain the crude product, and the residue was purified by silica gel column chromatography (EA:PE:DCM = 1:4:1) to obtain the target compound as a bright yellow solid. (Yield 39.70%, R f =0.47, developing solvent is PE:EtOAc = 2:1).

[0079] Synthesis of target compound 2: The synthesis procedure of compound 2 is the same as that of compound 1. The reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and benzoyl chloride (113.0 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a pure yellow solid (yield 57.10%). f =0.73, developing solvent is PE:EtOAc=2:1).

[0080] Synthesis of target compound 3: The synthesis procedure of compound 3 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 4-nitrobenzyl chloride (150.00 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a pure yellow solid (yield 34.66%). f =0.64, developing solvent is PE:EtOAc=2:1).

[0081] Synthesis of target compound 4: The synthesis process of compound 4 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 2-thiophenecarboxyl chloride (117.86 mg, 0.80 mmol, 1.2 equiv.), respectively, yielding the target compound as a golden yellow solid (yield 25.72%, R). f =0.57, developing solvent is PE:EtOAc=2:1).

[0082] Synthesis of target compound 5: The synthesis procedure of compound 5 is the same as that of compound 1. The reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and furfural chloride (104.01 mg, 0.80 mmol, 1.2 equiv.), yielding the target compound as a golden yellow solid (yield 34.60%). f =0.48, developing solvent is PE:EtOAc=2:1).

[0083] Synthesis of target compound 6: Intermediate 2 (200.00 mg, 0.66 mmol, 1.0 equiv.) was accurately weighed into a dry 15 mL round-bottom flask and dissolved in a small amount of anhydrous dichloromethane. Then, 4-nitrophenylhydrazine (121.00 mg, 0.81 mmol, 1.2 equiv.) was rapidly added to the reaction system. Under nitrogen protection, the mixture was refluxed at 80 °C and stirred for 2 hours. The reaction was confirmed by TLC. The mixture was diluted with dichloromethane and extracted with saturated sodium chloride solution. The organic layer was collected, dried over anhydrous sodium sulfate, and the solvent was recovered by vacuum distillation to obtain the crude product. Finally, the target product was purified by column chromatography (DCM:MeOH = 100:1–20:1) to obtain a yellow solid powder (yield 89.10%, R...). f =0.37, developing solvent is PE:EtOAc = 2:1).

[0084] Synthesis of target compound 7: The synthesis procedure of compound 7 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 3-nitrobenzenesulfonyl chloride (179.01 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a yellow solid (yield 37.50%). f =0.45, developing solvent is PE:EtOAc=2:1).

[0085] Synthesis of target compound 8: Intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) was accurately weighed into a dry 25.0 mL round-bottom flask. The compound was first dissolved in anhydrous tetrahydrofuran. Then, NaH (64.31 mg, 2.68 mmol, 4.0 equiv.) was weighed and added to the reaction flask. The mixture was stirred at room temperature for 1 h under nitrogen protection. Then, 2,4-dinitrobenzenesulfonyl chloride (214.12 mg, 0.80 mmol, 1.2 equiv.) was added, and stirring continued for 2 h. The reaction progress was monitored by thin-layer chromatography (TLC). When the starting material was almost completely eliminated, the reaction was stopped. NaH was quenched with water, and the mixture was extracted three times with DCM. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EA:PE:DCM = 1:4:1) to obtain the target compound as a red solid (yield 49.70%, R). f =0.73, developing solvent is PE:EtOAc=2:1).

[0086] Synthesis of target compound 9: The synthesis procedure of compound 9 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 4-nitrobenzenesulfonyl chloride (179.01 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a yellow solid (yield 34.60%). f =0.48, developing solvent is PE:EtOAc=2:1).

[0087] Synthesis of target compound 10: The synthesis procedure of compound 10 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 2-nitrobenzenesulfonyl chloride (179.01 mg, 0.80 mmol, 1.2 equiv.), respectively, yielding the target compound as a yellow solid (yield 35.10%, R). f =0.45, developing solvent is PE:EtOAc=2:1).

[0088] Synthesis of target compound 11: The synthesis procedure of compound 11 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 2-methyl-5-nitrobenzenesulfonyl chloride (197.94 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a yellow solid (yield 66.60%). f =0.75, developing solvent is PE:EtOAc=2:1).

[0089] Synthesis of target compound 12: The synthesis procedure of compound 12 is the same as that of compound 1. The reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 3,5-bis(trifluoromethyl)benzenesulfonyl chloride (252.01 mg, 0.80 mmol, 1.2 equiv.), yielding the target compound as a yellow solid (yield 47.12%). f =0.76, developing solvent is PE:EtOAc=2:1).

[0090] Synthesis of target compound 13: The synthesis procedure of compound 13 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 4-trifluoromethylbenzenesulfonyl chloride (196.01 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a yellow solid (yield 69.21%). f =0.56, developing solvent is PE:EtOAc=2:1).

[0091] Synthesis of target compound 14: The synthesis procedure of compound 14 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 2-trifluoromethylbenzenesulfonyl chloride (196.01 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a yellow solid (yield 40.12%). f =0.62, developing solvent is PE:EtOAc=2:1).

[0092] Synthesis of target compound 15: The synthesis procedure of compound 15 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 3-trifluoromethylbenzenesulfonyl chloride (196.01 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a yellow solid (yield 66.45%). f =0.65, developing solvent is PE:EtOAc=2:1).

[0093] Synthesis of target compound 16: The synthesis procedure of compound 16 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 2-nitro-4-trifluoromethylbenzenesulfonyl chloride (196.01 mg, 0.80 mmol, 1.2 equiv.), yielding the target compound as a yellow solid (yield 76.21%, R). f =0.59, developing solvent is PE:EtOAc=2:1).

[0094] Synthesis of target compound 17: The synthesis of compound 17 was the same as that of compound 1. The reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 3,5-dichlorobenzenesulfonyl chloride (206.23 mg, 0.80 mmol, 1.2 equiv.), yielding the target compound as a yellow solid (yield 82.16%, R). f =0.42, developing solvent is PE:EtOAc=2:1).

[0095] Synthesis of target compound 18: The synthesis procedure of compound 18 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 4-cyanobenzenesulfonyl chloride (161.02 mg, 0.80 mmol, 1.2 equiv.), yielding the target compound as a yellow solid (yield 34.65%). f =0.72, developing solvent is PE:EtOAc=2:1).

[0096] Synthesis of target compound 19: The synthesis procedure of compound 19 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and benzenesulfonyl chloride (148.36 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a yellow solid (yield 75.66%). f =0.38, developing solvent is PE:EtOAc=2:1).

[0097] Synthesis of target compound 20: The synthesis procedure of compound 20 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 4-methylbenzenesulfonyl chloride (152.18 mg, 0.80 mmol, 1.2 equiv.), respectively, yielding the target compound as a yellow solid (yield 31.75%). f =0.58, developing solvent is PE:EtOAc=2:1).

[0098] Synthesis of target compound 21: The synthesis procedure of compound 21 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 4-methoxybenzenesulfonyl chloride (165.18 mg, 0.80 mmol, 1.2 equiv.), respectively. The target compound was obtained as a yellow solid (yield 25.66%). f =0.39, developing solvent is PE:EtOAc=2:1).

[0099] Synthesis of target compound 22: The synthesis of compound 22 was the same as that of compound 1. The reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (226.59 mg, 0.80 mmol, 1.2 equiv.), yielding the target compound as a yellow solid (yield 55.96%). f =0.64, developing solvent is PE:EtOAc=2:1).

[0100] Synthesis of target compound 23: The synthesis procedure of compound 23 was the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and 1-naphthalenesulfonyl chloride (190.41 mg, 0.80 mmol, 1.2 equiv.), yielding the target compound as a yellow solid (yield 62.45%). f =0.68, developing solvent is PE:EtOAc=2:1).

[0101] Synthesis of target compound 24: The synthesis procedure of compound 24 is the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and quinoline-8-sulfonyl chloride (182.09 mg, 0.80 mmol, 1.2 equiv.), respectively, yielding the target compound as a yellow solid (yield 62.46%, R). f =0.34, developing solvent is PE:EtOAc = 2:1).

[0102] Synthesis of target compound 25: The synthesis procedure of compound 25 was the same as that of compound 1. The amounts of reactants were intermediate 2 (200.00 mg, 0.67 mmol, 1.0 equiv.) and pyridine-3-sulfonyl chloride (149.19 mg, 0.80 mmol, 1.2 equiv.), yielding the target compound as a yellow solid (yield 65.86%). f =0.52, developing solvent is PE:EtOAc=2:1).

[0103] Example 3

[0104] The 25 coumarin derivatives prepared in Examples 1 and 2 were initially screened for anticancer activity, as detailed below:

[0105] This study aimed to systematically evaluate the in vitro antitumor activity of 25 novel coumarin derivatives against seven human tumor cell lines (including non-small cell lung cancer cell lines A549, H1299, and H1975; gastric cancer cell lines BGC-823 and SGC-7901; hepatocellular carcinoma cell line HepG-2; and bladder cancer cell line UMUC2). The inhibition rate of tumor cell proliferation after 48 hours of treatment with the compounds was determined by the CCK-8 colorimetric assay, and the half-maximal inhibitory concentration (IC50) was calculated based on the absorbance (OD) at 450 nm using a microplate reader. 50 The study used a cytotoxicity index (CQI) to screen for candidate compounds with significant cytotoxic activity. Simultaneously, by analyzing the influence of different substituents (such as hydroxyl, methoxy, nitro, trifluoromethyl, etc.) and their different positions on activity, the structure-activity relationship (SAR) was preliminarily explored, providing a theoretical basis for subsequent drug molecule design.

[0106] The inhibitory effects of the synthesized compounds on seven human tumor cell lines were first assessed using two concentrations (100 μM and 10 μM). Cell viability was detected using the CCK-8 assay. Each group was configured with three replicates, and the experiment was repeated three times. The inhibition rates of the 25 compounds on the seven cancer cell lines at high and low concentrations are shown in the figure below. Figures 6-12 As shown.

[0107] like Figure 6 As shown, compounds 2, 3, 5, 6, 8, 10, 16, 20, and 24 all inhibited the growth of the non-small cell lung cancer cell line A549 at low concentrations, with inhibition rates greater than 75.02%. Compound 8, in particular, showed an inhibition rate of up to 90.01% against A549 at low concentrations. At higher concentrations, the inhibition rates of some compounds decreased. Compounds 1, 11, 21, and 22 had almost no inhibitory effect on A549 cells.

[0108] like Figure 7 As shown, compounds 1, 2, 8, 10, and 17, regardless of their concentration (high or low), showed significant inhibitory effects on the non-small cell lung cancer cell line H1299. Most of the other compounds had no significant inhibitory effect on H1299, with inhibition rates below 10.02%.

[0109] like Figure 8 As shown, compounds 2, 8, 10, 14, 16, 23, and 24 showed good inhibitory effects on the non-small cell lung cancer cell line H1975 at both high and low concentrations, with inhibition rates greater than 50.78%. Compounds 1, 5, 6, 12, 18, 19, 20, and 25 showed inhibition rates greater than 70.38% against H1975 cells at high concentrations, while the remaining compounds showed extremely low inhibitory effects on H1975 cells.

[0110] like Figure 9As shown, compounds 2, 3, 5, 6, 8 and 10 showed good inhibitory effects on the gastric cancer cell line BGC-823 at both high and low concentrations, with inhibition rates greater than 60.57%. Most of the other compounds showed weak inhibitory effects on BGC-823, below 20.02%.

[0111] like Figure 10 As shown, some compounds showed poor inhibitory effects on the gastric cancer cell line SGC-7901 at low concentrations, with inhibition rates of less than 10%. Compounds 8, 9, 10, 18, and 20, however, exhibited significant inhibitory effects on SGC-7901 at high concentrations, with inhibition rates ranging from 60.65% to 80.25%.

[0112] like Figure 11 As shown, all compounds exhibited inhibitory effects on the HepG-2 liver cancer cell line. Compounds 2, 3, 8, 10, 14, 16, and 20 showed significant inhibitory effects on HepG-2 cells at high concentrations, with inhibition rates all greater than 60.68%. The remaining compounds showed weak inhibitory effects on HepG-2 regardless of whether the concentration was high or low.

[0113] like Figure 12 As shown, most compounds did not have a significant inhibitory effect on the bladder cancer cell line UMUC2 at low concentrations, with inhibition rates all below 10.25%. Compounds 1, 2, 6, 8, 12, and 16, however, showed inhibitory effects on cells of over 60.02% at high concentrations.

[0114] Experimental data showed that the five target compounds exhibited significant inhibitory effects on seven types of human tumor cell lines, namely 2, 8, 10, 20, and 24. Further fine-tuning of these five compounds and determination of their IC50 values ​​were then conducted. 50 value.

[0115] Example 4

[0116] The five coumarin derivatives screened in Example 3 were subjected to IC50 analysis. 50 The value was determined as follows:

[0117] A gradient concentration method was used, with test compounds (2, 8, 10, 20, and 24) prepared at concentrations of 100, 10, 1, 0.1, and 0.01 μM. Doxorubicin (dox) was prepared at five concentrations as a positive control. These compounds were applied to in vitro proliferation inhibition experiments on seven human tumor cell lines. The toxicity to normal tissues was determined using normal rat hepatocytes (BRL-3A). OD values ​​were measured using the CCK-8 assay to calculate cell viability. Experimental data were analyzed using GraphPad Prism 8.0 software for nonlinear regression analysis to calculate IC50. 50 Value, IC 50 The values ​​are shown in Table 1-2.

[0118] Table 1 shows the effects of compounds 2, 8, 10, 20, and 24 on three non-small cell lung cancer IC50 strains. 50 Summary table of (μM) values

[0119]

[0120]

[0121] Table 2 shows the IC50 values ​​of compounds 2, 8, 10, 20, and 24 against five other cell types. 50 Summary table of (μM) values

[0122]

[0123]

[0124] Compounds 2 and 10 showed good inhibitory effects on A549, SGC-7901, and HepG-2, with significantly better inhibitory effects on SGC-7901 and HepG-2 than the positive control drug dox (IC50). 50 The values ​​were 0.87±1.05 and 0.53±0.27 μM, respectively. Compound 2 showed IC50 values ​​for SGC-7901 and HepG-2 cells. 50 The values ​​were 0.31±0.25 and 0.15±0.17 μM, respectively, indicating that compound 10 had an IC50 value of 0.31±0.25 and 0.15±0.17 μM against SGC-7901 and HepG-2 cells. 50 The values ​​were 0.37±0.48 and 0.39±0.48 μM, respectively; compound 8 showed significant inhibitory effects on six cell types (except BGC-823 cells), with IC50 values ​​of 0.37±0.48 μM and 0.39±0.48 μM, respectively. 50 The values ​​were 0.36±0.12, 3.14±0.79, 0.43±0.32, 0.52±0.81, 0.12±0.14, and 0.12±0.17 μM, respectively. The inhibitory effect on SGC-7901 and HepG-2 was superior to the positive control dox (IC50). 50 The values ​​were 0.87±1.05 and 0.53±0.27 μM, respectively. Compounds 20 and 24 showed significantly greater inhibitory effects on SGC-7901 and HepG-2 cells than on other cell types. Most importantly, none of the five compounds showed significant anti-proliferative activity against normal BRL-3A cells, indicating their strong safety profile. In conclusion, compound 8 exhibited excellent antitumor activity, comparable to the inhibitory activity of the positive control drug doxorubicin (dox).

[0125] As can be seen, this invention uses 25 synthesized coumarin-pyrazole hybrid compounds as research objects, and evaluates the in vitro antiproliferative activity of the compounds against seven tumor cell lines using the CCK-8 assay. Five compounds with relatively excellent antitumor effects were screened for IC50 analysis.50 The determination of the values ​​showed that compounds 2 and 10 had good inhibitory effects on A549, SGC-7901, and HepG-2, with the inhibitory effect on SGC-7901 and HepG-2 being significantly better than that on the positive control drug dox (IC50). 50 The values ​​were 0.87±1.05 and 0.53±0.27 μM, respectively. Compound 2 showed IC50 values ​​for SGC-7901 and HepG-2 cells. 50 The values ​​were 0.31±0.25 and 0.15±0.17 μM, respectively; compound 8 showed significant inhibitory effects on six cell types (except BGC-823 cells), with IC50 values ​​of 0.36±0.12, 3.14±0.79, 0.43±0.32, 0.52±0.81, 0.12±0.14, and 0.12±0.17 μM, respectively. The inhibitory effect on SGC-7901 and HepG-2 was superior to the positive control dox (IC50). 50 The values ​​were 0.87±1.05 and 0.53±0.27 μM, respectively; the IC50 values ​​of compound 10 for SGC-7901 and HepG-2 cells were 0.87±1.05 and 0.53±0.27 μM, respectively; 50 The values ​​were 0.37±0.48 and 0.39±0.48 μM, respectively; compounds 20 and 24 showed significantly greater inhibitory effects on SGC-7901 and HepG-2 cells than on other cell types. Furthermore, none of the five compounds showed significant anti-proliferative activity against normal cells. In conclusion, compound 8 exhibited excellent antitumor activity against six cancer cell types, comparable to the inhibitory activity of the positive control drug doxorubicin (Dox).

[0126] Therefore, this invention, using the aforementioned coumarin-pyrazole hybrid derivative and its preparation method and application, yields the following conclusions: The coumarin-pyrazole hybrid derivative provided by this invention, after mixing and incubating the compound, different cancer cells, and culture medium in a 96-well plate, and detecting the OD value of each well at a wavelength of 450 nm using an ELISA reader, demonstrates its excellent inhibitory effect on cancer cell growth; Most of the compounds in the coumarin-pyrazole hybrid derivative provided by this invention conform to the Lipinski principle of drug-like compounds, among which compound 8 is a promising drug lead molecule, exhibiting inhibitory activity against cancer cells comparable to that of the positive control drug doxorubicin (Dox); The preparation method provided by this invention can successfully synthesize coumarin-pyrazole hybrid derivatives; the synthesis method is simple, efficient, and uses inexpensive and readily available raw materials, which is conducive to large-scale industrial production and has broad market application prospects.

[0127] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A coumarin-pyrazole ring hybrid derivative, characterized in that, The general structural formula of coumarin-pyrazole ring hybrid derivatives is: wherein R is Its structure includes a 3-hybridized pyrazole ring at the coumarin position, with a 2,4-dinitrobenzenesulfonyl group attached to the pyrazole ring; coumarin-pyrazole ring hybrid derivatives also include pharmaceutically acceptable salts and deuterated derivatives of the compounds shown in the general formula.

2. A method for preparing the coumarin-pyrazole ring hybrid derivative as described in claim 1, characterized in that, Includes the following steps: S1. Synthesize intermediate 1 by fully dissolving 2-hydroxy-4-diethylaminobenzaldehyde and 4-hydroxy-6-methyl-2-pyranone in anhydrous ethanol, then slowly adding a catalytic amount of anhydrous piperidine to the reaction system, and refluxing at 80-82°C for 4 hours under nitrogen protection. After the reaction is completed, remove the anhydrous ethanol under reduced pressure, extract with ethyl acetate, wash with saturated brine, remove water with anhydrous sodium sulfate, and purify the crude product to obtain intermediate 1. S2. Synthesize intermediate 2. After fully dissolving intermediate 1 in anhydrous ethanol, slowly add hydrazine monohydrate dropwise to the reaction system. Reflux at 82°C for 1 hour under nitrogen protection. After the reaction is complete, cool to room temperature and add saturated brine. After standing, a large amount of yellow flocculent precipitate appears. Filter under reduced pressure and wash with a large amount of distilled water to remove sodium chloride to obtain a yellow solid, which is intermediate 2. S3. To synthesize the compound, intermediate 2 was dissolved in anhydrous tetrahydrofuran, and then NaH was weighed and poured into the reaction flask. The mixture was stirred at room temperature for 1 hour under nitrogen protection. Then 2,4-dinitrobenzenesulfonyl chloride was added, and the mixture was stirred for another 2 hours. The reaction was stopped, NaH was quenched with water, extracted three times, dried, filtered, concentrated under reduced pressure, and purified to obtain the target compound. The reaction formula is as follows: 。 3. The method for preparing a coumarin-pyrazole ring hybrid derivative according to claim 2, characterized in that, In step S1, the equivalent ratio of 2-hydroxy-4-diethylaminobenzaldehyde: 4-hydroxy-6-methyl-2-pyranone: anhydrous ethanol: anhydrous piperidine is 1.2:1.2:25:1.

5.

4. The method for preparing a coumarin-pyrazole ring hybrid derivative according to claim 2, characterized in that, In step S1, the crude product is purified by silica gel column chromatography with an eluent ratio of CH2Cl2:EtOAC=6:

1.

5. The method for preparing a coumarin-pyrazole ring hybrid derivative according to claim 2, characterized in that, In step S2, the equivalent ratio of intermediate 1: anhydrous ethanol: hydrazine monohydrate is 1:15:7.

8.

6. The method for preparing a coumarin-pyrazole ring hybrid derivative according to claim 2, characterized in that, In step S3, the equivalent ratio of intermediate 2: anhydrous tetrahydrofuran: NaH: 2,4-dinitrobenzenesulfonyl chloride is 1:10:4:1.2; the extraction is carried out using a mixture of water and DCM, with a water:DCM volume ratio of 1:

3.

7. The method for preparing a coumarin-pyrazole ring hybrid derivative according to claim 2, characterized in that, In step S3, drying is performed using anhydrous sodium sulfate; purification is performed using silica gel column purification, with the eluent being a mixture of petroleum ether and ethyl acetate, PE:EtOAc = 2:

1.

8. An application of the coumarin-pyrazole ring hybrid derivative as described in claim 1, characterized in that, The application of the compounds and their pharmaceutically acceptable salts and deuterated derivatives in the preparation of antitumor drugs that inhibit the growth of non-small cell lung cancer, gastric cancer, liver cancer, and bladder cancer cells.

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