Substituted 2-(piperazine-1-yl) acetamide derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a substituted 2-(piperazine-1-yl) acetamide derivative as well as a preparation method and application thereof. The structure of the compound is shown as a formula I. Experiments prove that the compound has good anti-hepatoma activity and lays a foundation for preparation of anti-hepatoma drugs. Formula I

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a substituted 2-(piperazin-1-yl)acetamide derivative, its preparation method, and its application. Background Technology

[0002] Liver cancer is the sixth most common malignant tumor worldwide, with approximately 870,000 new cases in 2022. It is also the third leading cause of cancer-related deaths, causing approximately 760,000 deaths. Surgical resection is the primary treatment for liver cancer, but only 15% of patients meet the criteria for tumor resection, and the five-year recurrence rate is as high as 60%–70%. The emergence of molecular targeted therapy has improved the treatment of liver cancer, but due to the inevitable development of drug resistance during treatment, the prognosis for patients with advanced stages remains poor.

[0003] Since sorafenib was approved for the treatment of advanced liver cancer in 2007, the field of multi-target tyrosine kinase inhibitors (TKIs) has continued to develop. Lenvatinib (approved in 2018) has become a new first-line treatment option, while regorafenib (2017), cabozantinib (2019), and ramucirumab (2019) have prolonged patient survival in second-line treatment. Meanwhile, although immunotherapy for hepatocellular carcinoma (HCC) has shown some promise, its efficacy still needs further improvement. In monotherapy, the efficacy of the first-line drug lenvatinib is comparable to that of sorafenib, with a median overall survival (mOS) of 13.6 months, slightly higher than sorafenib's 12.3 months. Furthermore, the combination therapy of atezolizumab and bevacizumab (approved in 2020) achieved a median overall survival of 19.2 months. Hepatocellular carcinoma (HCC) heterogeneity is a key factor contributing to resistance to tyrosine kinase inhibitors and immunotherapy, as well as poor prognosis. HCC exhibits significant genetic diversity, making it difficult for monotherapy to target all cancer cell subpopulations, especially treatment-resistant tumor-initiating cells with stem cell-like characteristics. While combination therapies using immune checkpoint inhibitors (ICIs) with tyrosine kinase inhibitors have shown promise, complex immune escape mechanisms driven by tumor heterogeneity continue to limit treatment efficacy. Therefore, cellular heterogeneity and the complexity of the microenvironment jointly contribute to the limitations of current treatment methods, highlighting the urgent need for further research into the mechanisms of HCC development and progression, and for expanding treatment options to improve patient survival and prognosis.

[0004] Oxidative stress is a pathological state characterized by elevated levels of intracellular reactive oxygen species, leading to redox signaling dysregulation and macromolecular oxidative damage. As a key marker of cancer, oxidative stress significantly promotes the occurrence and progression of hepatocellular carcinoma (HCC) through multiple mechanisms, including increasing cell damage and gene mutations, influencing specific gene variations, activating related signaling pathways, and also participating in the regulation of tumor cell growth, microenvironment remodeling, inducing immunosuppression, and promoting angiogenesis. Ketohexokinase (KHK) is the first rate-limiting enzyme in fructose metabolism. KHK-A is one of the isoforms produced by alternative splicing of the KHK gene, mainly expressed in various cancer tissues, and exhibits extremely low fructose phosphorylation activity. Studies have shown that KHK-A plays an important role in various cancers: in colorectal cancer, it is associated with fructose metabolism, promoting cancer cell proliferation and metastasis; in gastric cancer, under high glucose conditions, it can be activated by fructose produced by the polyol pathway, promoting epithelial-mesenchymal transition (EMT) and enhancing cell migration and invasion; in HCC, it can phosphorylate p62 to activate the Nrf2 signaling pathway, helping cancer cells adapt to metabolic stress.

[0005] Studies have shown that elevated KHK-A phosphorylation levels are associated with decreased patient survival, highlighting the importance of KHK-A overexpression in the severity of HCC. Considering the association between the oxidative stress protein KHK-A and the malignant progression of HCC, and the scarcity of therapeutic targets, this application aims to provide a compound that targets KHK-A and exhibits good anti-hepatocellular carcinoma efficacy. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a substituted 2-(piperazin-1-yl)acetamide derivative, its preparation method and application, and this type of compound has good anti-hepatocellular carcinoma cell activity.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows: Technical Topic 1 A substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutically acceptable salt thereof, having the structure shown in Formula I; Formula I Wherein, R1 is hydrogen or a benzene ring substituted or unsubstituted by one or more independent R4s; R2 is a benzene ring that is substituted or unsubstituted by one or more independent R4s; R4 is independently selected from C1-C5 alkyl, halogen, and hydroxyl groups; R3 is selected from hydrogen and C1-C5 alkyl groups.

[0008] As a further improvement of the present invention, the C1-C5 alkyl groups are selected from -CH3, -CH2CH3, -CH(CH3)2, -(CH2)2CH3, -(CH2)3CH3, -CH2CH(CH3)2, -C(CH3)3, -CH(CH3)CH2CH3, -(CH2)4CH3, -CH(CH3)(CH2)2CH3, -CH(CH2CH3)2, -CH2C(CH3)3, -CH2CH(CH3)CH2CH3, -C(CH3)2CH2CH3, -CH(CH3)CH(CH3)2, -(CH2)2CH(CH3)2 The halogen is selected from F, Cl, Br, and I.

[0009] As a further improvement of the present invention, the The structure is selected from one of the following: .

[0010] As a further improvement of the present invention, the structure of the compound is shown below: .

[0011] Technical Theme Two A method for preparing a compound as described in Technical Subject 1, characterized in that the preparation route of the compound is selected from one of the following: Route 1 ; Route 2 .

[0012] Technical Theme 3 A pharmaceutical composition comprising the substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutically acceptable salt thereof as described in Technical Subject 1, and optionally, one or more pharmaceutically acceptable carriers or excipients.

[0013] As used herein, a "pharmaceutical composition" comprising a therapeutically effective amount of a substituted 2-(piperazin-1-yl)acetamide derivative of Formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, is prepared in the form of tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, tinctures, decoctions, lozenges, mixtures, suppositories, injections, inhalants, or sprays. Preferably, the pharmaceutical composition contains 0.1% to 99.5% by weight of the substituted 2-(piperazin-1-yl)acetamide derivative of the present invention or a pharmaceutically acceptable salt thereof as the active ingredient, more preferably 0.5% to 99.5% by weight of the active ingredient.

[0014] As used herein, “pharmaceuticalally acceptable carriers or excipients” include: diluents, fillers, binders, disintegrants, lubricants, flow aids, granulators, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. Those skilled in the art will understand that some pharmaceutically acceptable excipients may be used for more than one function and for alternative functions, depending on the amount of said excipient present in the formulation and what other ingredients are present in the formulation. For example, when used orally, it can be formulated into oral preparations such as tablets, capsules, granules, and pills, containing fillers (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., gelatin, polyvinylpyrrolidone, and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as sodium carboxymethyl cellulose and polyvinylpyrrolidone), lubricants (e.g., talc, calcium stearate, magnesium stearate, cetyl, boric acid, sodium benzoate, and leucine), stabilizers (e.g., methylparaben, propylparaben, etc.), and flavoring agents (e.g., commonly used sweeteners, acidulants, and flavorings, etc.). When used parenterally, the drug can be formulated as an injection, comprising a sterile powder for injection and a solvent for injection. The carrier or excipient used may contain sterile water, Ringer's solution, and isotonic sodium chloride solution. Suitable adjuvants such as antioxidants, buffers, and antibacterial agents may also be added depending on the properties of the drug. When used for rectal administration, the drug can be formulated as suppositories, etc. When used for pulmonary administration, the drug can be formulated as an inhaler or spray, etc. Many resources are available to those skilled in the art describing pharmaceutically acceptable excipients and which can be used to select suitable pharmaceutically acceptable excipients, such as the Remington Pharmacy Encyclopedia, the Chinese Pharmaceutical Yearbook, and Pharmaceutics.

[0015] This invention can be administered by any suitable method known in the art, such as oral, intravenous, intraperitoneal, intramuscular, local, transdermal, ocular, nasal, inhalation, subcutaneous, intramuscular, oral, sublingual, or rectal administration. The compound described above can be administered at any amount from 1 μg to 2000 mg / kg of the subject's body weight, for example, at doses of 1 μg to 1000 mg / kg body weight / day, 50 μg to 1000 mg / kg body weight / day, 100 μg to 1000 mg / kg body weight / day, 1 to 500 mg / kg body weight / day, 2 to 200 mg / kg body weight / day, or 5 to 100 mg / kg body weight / day. In some embodiments of this invention, the compound described above can be administered four times daily, three times daily, twice daily, once daily, once every two days, once weekly, or at other intervals, optionally repeating the dosing regimen as appropriate weekly or monthly. In this invention, the dosage of the compound can be adjusted according to factors such as the severity of the patient's or subject's condition, age, weight, gender, administration method, and course of treatment.

[0016] The compounds of the present invention can be used alone or in combination with one or more other active ingredients for the treatment, prevention, inhibition, or improvement of diseases or symptoms, wherein the combined use of the drugs is safer or more effective than the use of any one drug alone. Such other drugs can be administered simultaneously or sequentially with the compounds of the present invention in the manner and amount normally used therein. When the compounds of the present invention are used simultaneously with one or more other drugs, pharmaceutical compositions containing the other drug and the compounds of the present invention in a unit dosage form are preferred, particularly in combination with a pharmaceutically acceptable carrier. However, combination therapy may also include treatment with the compounds of the present invention and one or more other drugs administered on different overlapping schedules. It is also contemplated that when used in combination with one or more other active ingredients, the compounds of the present invention and the other active ingredients can be used at lower doses than when used individually. Therefore, in addition to the compounds of the present invention, the pharmaceutical compositions of the present invention also include those compositions containing one or more other active ingredients.

[0017] Technical Theme 4 The use of a substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutical salt thereof as described in Technical Subject 1 in the preparation of a medicament for inhibiting KHK-A protein.

[0018] Technology Theme 5 The use of a substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutically acceptable salt thereof as described in Technical Subject 1 in an antitumor drug.

[0019] As a further improvement of the present invention, the tumor is hepatocellular carcinoma.

[0020] The beneficial effects of adopting the above technical solution are as follows: This invention provides a substituted 2-(piperazin-1-yl)acetamide derivative. Experimental verification has shown that this type of compound has good anti-tumor effects, laying the foundation for the development of drugs against hepatocellular carcinoma. Attached Figure Description

[0021] Figure 1 This is a diagram illustrating the effect of the compound HK-4 provided by this invention on apoptosis in HepG2 cells; Figure 2 This is a flow cytometry result of the effect of compound HK-4 provided by this invention on apoptosis in HepG2 cells; Figure 3 This is a diagram showing the cell cycle effect of the compound HK-4 provided by this invention on HepG2 cells; Figure 4 The image shows the results of Western blotting analysis of the effect of the compound HK-4 provided in this invention on HepG2 cells. Figure 5 The figure shows the cell migration results of hepatocellular carcinoma cells after treatment with the compound HK-4 and / or sorafenib provided in this invention, where A represents HepG2 cells and B represents PLC / PRF / 5 cells. Figure 6 The figure shows the results of an invasion experiment of the compound HK-4 and / or sorafenib provided by this invention against HCCLM3 and PLC / PRF / 5 cells, where A represents HCCLM3 cells and B represents PLC / PRF / 5 cells. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0023] The HepG2, HCCLM3, HuH7, and PLC / PRF / 5 cell lines used in this invention were purchased from the China Center for Type Culture Collection (CCTCC). All cell lines were identified by short tandem repeat (STR) analysis (Promega) and confirmed to be free of mycoplasma contamination.

[0024] The preparation route of the compound described in this invention is selected from one of the following: Route 1 ; Route 2 .

[0025] Example 1 Preparation of compound A-3 Step 1: Preparation of ethyl 2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)acetate (3) 1-Chloro-4-(chloro(phenyl)methyl)benzene (500.0 mg, 2.119 mmol) and ethyl 2-(piperazin-1-yl)acetate (437.4 mg, 2.5428 mmol) were dissolved in DMF solution (15 ml) and stirred. K2CO3 (878.6 mg, 6.357 mmol) and KI (351.8 mg, 2.119 mmol) were added sequentially, and the reaction was stirred at 90°C for 3 hours. TLC analysis was performed. After the reaction was complete, the reaction solution was extracted with ethyl acetate (40 ml × 3) and water, washed with distilled water and saturated brine, and the ethyl acetate layer was separated. The layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness. The resulting crude, pale yellow oily substance was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to give 500 mg of oily liquid 3, with an overall yield of 63%.

[0026] Step 2: Preparation of 2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)acetic acid (4) 500.0 mg (1.344 mmol) of ethyl 2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl) was dissolved in a mixture of tetrahydrofuran (10 ml) and water (10 ml). 96.8 mg (4.032 mmol) of solid LiOH was added, and the reaction was stirred at 50°C for 5 hours. TLC was used for monitoring. After the reaction was complete, acetic acid was added to quench the reaction mixture, and the pH was adjusted to approximately 4-5. The reaction mixture was then evaporated to dryness. The product was subsequently extracted with ethyl acetate (40 ml × 3) and water, washed with distilled water and saturated brine, and the ethyl acetate layer was separated. The layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness, yielding 400 mg of a white solid crude product with a purity of 92%. No further purification was required, and the product was directly used for the next reaction, with an overall yield of 79.7%.

[0027] Step 3: Preparation of N-((1-benzyl-5-oxopyrrolidone-3-yl)methyl)-2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)acetamide (A-3) 2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)acetic acid (400 mg, 1.163 mmol) was dissolved in 1,4-dioxane, and HATU (884.4 mg, 2.326 mmol) and NaH (111.6 mg, 4.652 mmol) were added sequentially. The mixture was activated at room temperature for half an hour, and then 4-(aminomethyl)-1-benzylpyrrolidone-2-one (284.7 mg, 1.3956 mmol) was added to the reaction flask. The reaction was carried out at room temperature for 3 hours. TLC analysis was performed. After the reaction was completed, water was added to quench the reaction solution, and the reaction solution was evaporated to dryness. Subsequently, it was extracted with ethyl acetate (40 ml × 3) and water, washed with distilled water and saturated brine, and the ethyl acetate layer was separated. The layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness. The crude yellow oily substance was separated by silica gel column chromatography (dichloromethane:methanol = 30:1) to obtain 300 mg of yellow oily liquid A-3, with an overall yield of 48.7%. 1 H NMR (600 MHz, Methanol- d 4) δ 7.46 (dd, J =12.5, 8.1 Hz, 4H), 7.36 – 7.29 (m, 6H), 7.27 – 7.21 (m, 4H), 4.47 – 4.38 (m,2H), 3.85 (s, 2H), 3.72 (p, J = 6.6 Hz, 5H), 3.41 (dd, J = 10.3, 8.0 Hz, 1H), 3.27 (d, J = 6.6 Hz, 2H), 3.22 (q, J = 7.4 Hz, 4H), 3.04 (dd, J = 10.2, 5.3 Hz,1H), 2.63 – 2.53 (m, 2H), 2.23 (q, J = 9.8 Hz, 1H). 13 C NMR (151 MHz, Methanol- d 4) δ 176.19, 137.52, 134.45, 130.48,130.07,130.01, 129.82, 129.09, 128.99, 128.91, 128.76, 75.42, 53.81, 51.38,47.40, 43.78, 36.05, 32.74. Example 2 Preparation of compound HK-4 The preparation of compound HK-4 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1H NMR (600 MHz, Methanol-d4)δ 7.58 (dd, J = 8.6, 2.2 Hz, 2H), 7.46(dd, J = 12.5, 8.1 Hz, 2H), 7.36 – 7.29 (m, 6H), 7.27 – 7.21 (m, 3H), 4.47 –4.38 (m, 2H), 3.85 (s, 2H), 3.72 (p, J = 6.6 Hz, 5H), 3.41 (dd, J = 10.3, 8.0Hz, 1H), 3.27 (d, J = 6.6 Hz, 2H), 3.22 (q, J = 7.4 Hz, 4H), 3.04 (dd, J =10.2, 5.3 Hz, 1H), 2.63 – 2.53 (m, 2H), 2.23 (q, J = 9.8 Hz, 1H) 13 C NMR (151 MHz, Methanol- d 4) δ 176.19, 142.31, 134.45, 133.14,130.07, 130.01, 129.82, 129.09, 128.99, 128.91, 128.76, 75.42, 53.81, 51.38,47.40, 43.78, 36.05, 32.74 Example 3 Preparation of compound A-1 The preparation of compound A-1 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 8.42 (t, J = 6.1 Hz, 1H), 8.17 (s, 2H), 7.48 (d, J=8.2 Hz, 3H), 7.36 – 7.30 (m, 4H), 7.30 – 7.27 (m, 2H), 7.26 – 7.21 (m, 2H), 7.20 – 7.15 (m, 2H), 4.54 (s, 1H), 4.47 (d, J = 14.7 Hz, 1H), 4.31 (d, J = 14.8Hz, 1H), 3.74 – 3.64 (m, 2H), 3.47 (s, 1H), 3.40 – 3.17 (m, 7H), 2.98 (dd, J =10.3, 5.0 Hz, 1H), 2.87 (d, J = 54.8 Hz, 3H), 2.61 – 2.50 (m, 2H), 2.28 – 2.18 (m, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 164.44, 138.94, 135.93, 129.38,128.94, 128.48, 128.19, 127.92, 127.89, 58.17, 51.80, 50.30, 49.01, 46.78,42.86, 35.25, 31.21. Example 4 Preparation of compound A-2 The preparation of compound A-2 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Methanol-) d 4) δ 7.47 (dd, J = 8.8, 5.5 Hz, 4H), 7.31 (t, J = 7.8 Hz, 2H), 7.24 (t, J = 6.3 Hz, 3H), 7.06 (t, J = 8.7 Hz, 4H), 4.53 (s, 1H), 4.47 –4.38 (m, 2H), 3.86 (s, 2H), 3.42 (dd, J = 10.3, 7.3 Hz, 2H), 3.27 (d, J = 6.4 Hz, 2H), 3.04 (dd, J= 10.0, 5.1 Hz, 2H), 2.63 – 2.33 (m, 4H), 2.23 (q, J = 9.7 Hz, 1H). 13 C NMR (151 MHz, Methanol- d 4) δ 176.18, 165.86, 162.81, 138.20,137.52,130.70, 130.65, 129.83, 129.10, 128.77, 116.74, 116.59, 74.45, 57.84,53.93, 51.38, 49.59, 47.40, 43.38, 36.04, 32.74. Example 5 Preparation of compound A-4 The preparation of compound A-4 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 7.74 (d, J = 7.3 Hz, 1H), 7.37 (d, J = 7.9 Hz, 2H), 7.28 (t, J = 7.3 Hz, 3H), 7.25 – 7.17 (m, 6H), 7.12 – 7.05 (m, 2H), 4.49 – 4.43(m, 2H), 4.37 (d, J = 14.7 Hz, 1H), 3.48 (s, 2H), 3.37 – 3.29 (m, 2H), 3.20(dt, J = 12.5, 5.5 Hz, 1H), 2.96 (dd, J = 10.0, 5.2 Hz, 3H), 2.61 – 2.44 (m, 7H), 2.35 (s, 4H), 2.18 (q, J = 9.9 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d) δ 173.44, 136.33, 135.95, 130.79,128.87, 128.76, 128.60, 128.26, 127.81, 127.23, 127.13, 126.75, 126.43,71.11, 61.32, 53.91, 50.94,50.01, 46.69, 42.23, 35.34, 31.70, 20.01. Example 6 Preparation of compound A-5 The preparation of compound A-5 followed the same route as that of compound A-3, and the resulting NMR results are as follows: 1 H NMR (500 MHz, Chloroform- d ) δ 7.71 (d, J = 7.5 Hz, 1H), 7.43 (s, 1H), 7.30 (dd, J =16.1, 8.9 Hz, 6H), 7.24 – 7.13 (m, 6H), 4.81 (s, 1H), 4.51 (d, J = 51.5 Hz, 2H), 4.36 (d, J = 14.9 Hz, 1H), 3.38 – 3.15 (m, 8H), 2.99 (dd, J = 10.5, 4.7 Hz, 1H), 2.56 (d, J = 14.9 Hz, 3H), 2.36 (d, J = 5.7 Hz, 4H), 2.19 (d, J = 11.2 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 174.21, 164.63, 155.29, 137.86,135.97, 129.68, 129.62, 129.51, 128.99, 128.74, 128.34, 128.17, 127.94,123.19, 120.54, 117.25, 58.14,51.88, 50.92, 50.25, 48.98, 46.75, 42.80,35.23, 31.20. Example 7 Preparation of compound B-1 The preparation of compound B-1 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 7.46 – 7.38 (m, 5H), 7.35 – 7.30 (m, 2H), 7.29 –7.27 (m, 1H), 7.22 – 7.17 (m, 2H), 4.48 (d, J = 14.7 Hz, 1H), 4.31 (d, J = 14.7Hz, 1H), 4.17 (s, 2H), 3.47 (s, 1H), 3.39 (dd, J = 10.4, 7.9 Hz, 1H), 3.33 –3.21 (m, 7H), 3.08 (s, 4H), 3.02 (dd, J = 10.3, 4.6 Hz, 1H), 2.64 – 2.56 (m,2H), 2.27 (dq, J = 14.8, 9.0, 7.0 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 174.39, 167.79, 135.76, 131.08,130.48, 129.60, 129.03, 128.25, 128.09, 60.80, 59.48, 50.46, 50.26, 49.88,46.91, 42.62, 35.14, 31.22. Example 8 Preparation of compound B-2 The preparation of compound B-2 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 8.03 (d, J = 25.2 Hz, 1H), 7.34 (t, J = 1.9 Hz, 1H),7.33 – 7.29 (m, 2H), 7.28 – 7.26(m, 2H), 7.19 (d, J = 6.8 Hz, 2H), 4.49 (d, J =14.7 Hz, 1H), 4.31 (d, J= 14.7 Hz, 1H), 3.78 (s, 2H), 3.46 (s, 2H), 3.37 (dd, J = 10.2, 7.8 Hz, 1H), 3.31 (dt, J = 11.8, 6.1 Hz, 1H), 3.25 (dt, J = 13.3, 6.3 Hz, 1H), 3.12 (dt, J = 9.9, 5.0 Hz, 4H), 3.00 (dd, J = 10.2, 4.8 Hz, 1H), 2.98 – 2.85(m, 4H),2.59 (t, J = 9.8 Hz, 2H), 2.23 (q, J = 9.1 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 173.90, 136.12, 135.74, 129.20,128.95, 128.28, 128.19, 127.91, 60.15, 59.16, 51.51, 50.31, 46.73, 45.90,42.87, 35.30, 31.17. Example 9 Preparation of compound B-3 The preparation of compound B-3 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 7.39 (d, J = 7.0 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.32 (tt, J = 8.1, 1.8 Hz, 2H), 7.29 – 7.26 (m, 1H), 7.24 – 7.20 (m, 3H), 5.29(s, 1H), 4.49 (d, J = 14.6 Hz, 1H), 4.38 (d, J = 14.6 Hz, 1H), 3.58 (s, 2H), 3.38– 3.31 (m, 2H), 3.24 (dt, J = 13.4, 6.3 Hz, 1H), 2.98 (dd,J = 9.9, 5.2 Hz, 3H),2.64 – 2.55 (m, 3H), 2.51 (s, 7H), 2.20 (q, J = 9.9 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 173.43, 136.38, 135.00, 131.58,129.39, 128.90, 128.29, 127.86, 127.15, 61.45, 58.56, 53.67, 53.56, 53.14,50.05, 46.73, 42.26, 35.34,31.72. Example 10 Preparation of compound B-4 The preparation of compound B-4 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Methanol-) d 4) δ 7.46 (d, J = 8.1 Hz, 2H), 7.37 – 7.31 (m, 3H), 7.29 –7.22 (m, 3H), 4.86 (d, J = 1.8 Hz, 4H), 4.44 (q, J = 14.8 Hz, 2H), 4.12 (s, 2H), 3.70 (s, 2H), 3.43 (dd, J = 10.3, 7.9 Hz, 1H), 3.19 (s, 2H), 3.08 – 3.04 (m,4H), 2.63 – 2.55 (m, 2H), 2.25 (q, J = 9.7 Hz, 1H). 13 C NMR (151 MHz, Methanol- d 4) δ 167.22, 138.30, 137.55, 131.90,130.02,129.85, 129.11, 128.80, 58.50, 56.43, 53.14, 51.41, 51.37, 47.42,43.36, 36.05, 32.74. Example 11 Preparation of compound B-5 The preparation of compound B-5 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 7.46 (d, J = 2.8 Hz, 1H), 7.33 (td, J = 5.9, 5.4, 3.0Hz, 2H), 7.28 (dd, J = 8.0, 2.5 Hz, 2H), 7.24 – 7.20 (m, 2H),7.17 (dd, J = 8.5, 2.7 Hz, 1H), 4.49 (d, J = 14.7 Hz, 1H), 4.38 (d, J = 14.7 Hz, 1H), 3.58 (s, 2H), 3.38 – 3.31 (m, 2H), 3.24 (dt, J = 13.4, 6.4 Hz, 1H), 3.01 – 2.96 (m, 3H), 2.56(ddd, J = 29.0, 12.8, 8.3 Hz, 10H), 2.20 (q, J = 9.9 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 173.46, 170.68, 136.38, 132.87,132.45, 130.67, 130.34, 128.91, 128.40, 128.30, 127.86, 61.44, 58.81, 53.65,53.21, 50.03, 46.71,42.21, 35.33, 31.72. Example 12 Preparation of compound B-6 The preparation of compound B-6 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 7.33 (dd, J = 8.0, 6.3 Hz, 2H), 7.31 – 7.27 (m, 1H),7.22 (ddd, J = 7.9, 5.7, 1.8 Hz, 4H), 7.00 (dq,J = 9.2, 4.8, 3.8 Hz, 1H), 4.50(d, J = 14.7 Hz, 1H), 4.38 (d, J = 14.7 Hz, 1H), 3.72 (s, 2H), 3.49 (s, 1H), 3.37– 3.31(m, 2H), 3.23 (dt, J = 13.4, 6.4 Hz, 1H), 2.98 (dd, J = 10.2, 5.2 Hz, 3H),2.67 – 2.45 (m, 9H), 2.20 (q, J = 9.8 Hz, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 136.42, 128.92, 128.32, 127.86,125.68, 50.07, 46.73, 35.37, 31.70. Example 13 Preparation of compound B-7 The preparation of compound B-7 followed route 1 and the preparation of compound A-3. The NMR results of the obtained products are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 7.45 (dd, J = 8.6, 6.2 Hz, 1H), 7.36 (t, J = 7.3 Hz, 3H), 7.33 – 7.29 (m, 1H), 7.25 (s, 1H), 7.14 (dd, J = 8.5, 2.7 Hz, 1H), 6.99(td, J = 8.3, 2.7 Hz, 1H), 4.52 (d, J = 14.7 Hz, 1H), 4.41 (d, J = 14.7 Hz, 1H), 3.61 (s, 2H), 3.41 – 3.35 (m, 2H), 3.27 (dt, J = 13.4, 6.4 Hz, 1H), 3.02 (d, J =12.8 Hz, 3H), 2.66 – 2.44 (m, 10H), 2.29 – 2.19 (m, 1H). 13 C NMR (151 MHz, Chloroform- d ) δ 173.43, 170.73, 162.43, 160.78,136.35, 134.91, 134.84, 131.80, 131.74, 131.59, 128.88, 128.25, 127.83,116.92, 116.75, 114.07, 113.93,61.44, 58.48, 53.66, 53.10, 50.01, 46.69,42.20, 35.30, 31.69. Example 14 Preparation of compound C-1 The preparation of compound C-1 followed route 2, and the NMR results of the obtained product are as follows: 1 H NMR (600 MHz, Methanol-) d 4) δ 7.45 (dd, J = 11.8, 8.1 Hz, 4H), 7.34 – 7.32 (m, 4H), 7.26 – 7.23 (m, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.06 – 7.03 (m, 2H), 7.00 (d, J = 7.7 Hz, 1H), 4.51 (s, 1H), 4.38 (d, J = 3.9 Hz, 2H), 3.85 (s, 2H), 3.40 (dd, J = 10.3, 8.0 Hz, 2H), 3.27 (d, J = 6.6 Hz, 2H), 3.02 (dd, J = 10.3, 5.3 Hz, 2H), 2.63 – 2.49 (m,4H),2.29 (s, 3H), 2.22 (q, J = 9.6 Hz, 1H). 13 C NMR (151 MHz, Methanol- d4) δ 176.12, 139.68, 137.40, 134.44,130.46,130.07, 130.00, 129.76, 129.72, 129.43, 128.98, 128.90, 126.14, 75.41,57.88, 53.86, 51.34, 47.37, 43.35, 36.06, 32.72. Example 15 Preparation of compound C-2 The preparation of compound C-2 followed route 2, and the NMR results of the obtained product are as follows: 1 H NMR (600 MHz, Methanol-) d 4) δ 7.48 – 7.43 (m, 4H), 7.35 – 7.31 (m, 4H), 7.25 (t, J = 6.7 Hz, 1H), 7.14 – 7.10 (m, 4H), 4.52 (s, 1H), 4.41 – 4.34 (m, 2H), 3.86 (s, 2H), 3.40 (dd, J = 10.2, 7.8 Hz, 2H), 3.28 – 3.25 (m, 2H), 3.02 (dd, J = 10.2, 5.1 Hz,2H), 2.73 (s, 4H), 2.65 – 2.34 (m, 4H),2.29 (s, 3H), 2.22 (q, J = 9.6 Hz, 1H). 13 C NMR (151 MHz, Methanol- d 4) δ 138.63, 134.46, 134.40, 130.45,130.07,130.03, 130.01, 129.10, 128.99, 128.90, 53.89, 47.12, 32.68. Example 1 Hepatocellular carcinoma cells HepG2, HuH7, and PLC / PRF / 5 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, respectively, and incubated in a constant temperature incubator at 37°C and 5% carbon dioxide. The medium and additives were purchased from Gibco.

[0028] In the CCK-8 cell counting assay, cells were seeded at a density of 3000 cells per well in 96-well plates and cultured for 24 hours. Cells were then treated for 48 hours with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at different concentrations (0.78-100 μmol / L) of HK-4. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance was then measured at 450 nm using a microplate reader. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 9.0 software through nonlinear regression analysis. 50 The specific result is the half-maximal inhibitory concentration (IC50) of compound HK-4 on HepG2 cells. 50 =22.54 μM; IC50 of compound HK-4 against PLC / PRF / 5 cells 50 =23.91 μM; IC50 of compound HK-4 against HuH7 cells 50 =23.38μM, the above results indicate that HK-4 has a general inhibitory effect on hepatocellular carcinoma cells.

[0029] Example 2 1. Flow cytometry assay for apoptosis rate The apoptosis-inducing effect of compound HK-4 on tumor cells was evaluated using the Annexin V / PI double staining method. HepG2 cells were cultured at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and treated with 0, 25, or 50 μmol / L HK-4 at 37°C for 24 hours. After treatment, cells were digested with trypsin, washed with PBS, and resuspended in 100 μL binding buffer. 5 μL Annexin V was added, and the cells were incubated in the dark for 30 minutes, followed by 5 μL PI and another 30 minutes in the dark. Then, 400 μL binding buffer was added, filtered, and the proportion of apoptotic cells was quantitatively analyzed by flow cytometry. The results are shown below. Figure 1 and Figure 2 As shown, the results indicate that 50 μM HK-4 can significantly increase the proportion of early and late apoptosis in HepG2 cells, meaning that high concentrations of HK-4 have a strong activity in inducing tumor cell apoptosis.

[0030] 2. Flow cytometry for cell cycle assays The effect of HK-4 on tumor cell cycle arrest was detected using PI staining. HepG2 cells were cultured at 5 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells / well in 6-well plates and treated with different doses (0, 25, or 50 μmol / L) of HK-4 at 37°C for 24 hours. After treatment, cells were collected with trypsin, fixed with 70% ethanol, and stored at -20°C. The fixed cells were washed with PBS, resuspended in 400 μL PBS, and 4 μL of RNase was added. The cells were incubated at 37°C for 30 minutes to digest RNA, followed by 4 μL of PI staining solution and staining in the dark for 60 minutes. Cell cycle distribution was analyzed by flow cytometry. Results are as follows: Figure 3 As shown, with increasing HK-4 concentration, the proportion of cells in G1 phase in HepG2 cells increased significantly, while the proportion of cells in S phase and G2 / M phase decreased, indicating that HK-4 treatment for 24 hours can induce G1 phase arrest in HepG2 cells in a dose-dependent manner.

[0031] 3. Western blotting analysis assay HepG2 cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and treated with different doses (0, 25, or 50 μmol / L) of HK-4 compound at 37°C for 24 hours. After incubation, cells were collected, lysed with 100 μL LRIPA buffer, and protein concentration was determined using a BCA protein quantification kit. For Western blot analysis, samples were denatured by heating at 100°C for 15 minutes in a metal bath, followed by gel electrophoresis, transfer, and detection.

[0032] The results showed that after 24 hours of HK-4 treatment, the protein levels of p-AKT and p-mTOR were significantly reduced. This indicates that HK-4 exerts its anti-tumor effect by inhibiting KHK-A protein and further reducing the activation state of the PI3K-AKT signaling pathway in tumor cells (Figure 4).

[0033] Example 3 1. Scratch healing experiment Cell migration ability was assessed using a scratch healing assay. Cells (HepG2 and PLC / PRF / 5) were sputter-crossed at 100°C. 6Cells were seeded in 6-well plates and cultured for 24 hours. Clear scratches were then made on the cell monolayer using a 200 μL pipette tip, followed by 3 washes with PBS to remove cell debris. Cells were incubated in serum-free DMEM medium with either HK-4 (10 μmol / L) or sorafenib (5 μmol / L). After 24 hours of treatment, images of the scratched areas were taken using an inverted microscope from Olympus Tokyo, Japan. The distance between cell leading edges was analyzed using ImageJ software to assess scratch healing.

[0034] The results are as follows Figure 5 As shown, treatment with 10 μmol / L HK-4 or 5 μmol / L sorafenib significantly reduced the migration ability of HepG2 and PLC / PRF / 5 cells.

[0035] 2. Invasion Experiment Cell invasion ability was assessed using 24-well Transwell chambers (Millipore, SCWP04700, USA) containing an 8.0 μm polycarbonate filter membrane. The upper chamber was lined with 60 μL of Matrigel (BD, Matrigel Matrix, 354230, 40 μL per well). 2 × 10⁶ cells were then placed in each chamber. 5 HCCLM3 or PLC / PRF / 5 cells were suspended in serum-free medium containing HK-4 (10 μmol / L) or sorafenib (5 μmol / L) and added to the upper chamber. DMEM medium containing 10% fetal bovine serum was added to the lower chamber. After culturing for 24 hours, uninvaded cells from the upper chamber were removed, washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and stained with 0.1% crystal violet for 20 minutes. Finally, images were taken, and four fields of view were randomly selected for cell counting to assess cell invasion ability.

[0036] The results showed that 10 μmol / L HK-4 significantly reduced the number of invasive cells, suggesting that even at low concentrations, HK-4 can weaken the invasive ability of HCCLM3 and PLC / PRF / 5 cells (Figure 6). In summary, these results indicate that HK-4 can exert potent anti-hepatocellular carcinoma activity by inhibiting the migration and invasion of hepatocellular carcinoma cells.

[0037] 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; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A substituted 2-(piperazine-1)acetamide derivative or a pharmaceutically acceptable salt thereof, characterized in that: Its structure is shown in Equation I; Formula I Wherein, R1 is hydrogen or a benzene ring substituted or unsubstituted by one or more independent R4s; R2 is a benzene ring that is substituted or unsubstituted by one or more independent R4s; R4 is independently selected from C1-C5 alkyl, halogen, and hydroxyl groups; R3 is selected from hydrogen and C1-C5 alkyl groups.

2. The substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The C1-C5 alkyl groups are selected from —CH3, —CH2CH3, —CH(CH3)2, —(CH2)2CH3, —(CH2)3CH3, —CH2CH(CH3)2, —C(CH3)3, —CH(CH3)CH2CH3, —(CH2)4CH3, —CH(CH3)(CH2)2CH3, —CH(CH2CH3)2, —CH2C(CH3)3, —CH2CH(CH3)CH2CH3, —C(CH3)2CH2CH3, —CH(CH3)CH(CH3)2, —(CH2)2CH(CH3)2; The halogens are F, Cl, Br, and I.

3. The substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The The structure is selected from one of the following: 。 4. The substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The structure of the compound is shown below: 。 5. A method for preparing the compound according to any one of claims 1-4, characterized in that, The preparation route of the compound is selected from one of the following: Route 1 ； Route 2 。 6. A pharmaceutical composition comprising a substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4, and optionally, one or more pharmaceutically acceptable carriers or excipients.

7. The use of a substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutical salt thereof as described in any one of claims 1-4 in the preparation of a medicament for inhibiting KHK-A protein.

8. The use of a substituted 2-(piperazin-1-yl)acetamide derivative or a pharmaceutical salt thereof as described in any one of claims 1-4 in the preparation of an antitumor drug.

9. The application according to claim 8, characterized in that, The tumor is hepatocellular carcinoma.

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