3-Amino-4-(piperazin-1-yl)benzamide derivatives, pharmaceutical compositions containing them, methods of preparation thereof, and applications.

Abstract

This invention provides 3-amino-4-(piperazin-1-yl)benzamide derivatives, pharmaceutical compositions comprising them, methods for their preparation, and applications. These compounds act as PLAGL2 protein inhibitors, playing a significant role in various diseases, including but not limited to pain, inflammation, immune dysfunction, neurological and psychiatric disorders, respiratory diseases, urinary system diseases, reproductive system diseases, embryonic developmental abnormalities, cell metabolic disorders, differentiation problems, and viral infections. PLAGL2 plays a particularly positive role in the occurrence, development, and prognosis of malignant tumors such as liver cancer, lung cancer, and glioma. The compounds of this invention exhibit excellent PLAGL2 inhibitory activity. This invention also provides the compounds comprising the pharmaceutically acceptable salts thereof, pharmaceutically acceptable carriers or excipients, and methods for their preparation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to 3-amino-4-(piperazin-1-yl)benzamide derivatives, pharmaceutical compositions containing them, their preparation methods, and applications. Background Technology

[0002] PLAGL2 (Pleomorphic adenoma gene like 2), like PLAGL1, belongs to the PLAG gene family. As a zinc finger protein transcription factor, it regulates the expression of multiple important genes and participates in various physiological functions, including cell growth, proliferation, and differentiation, playing a crucial role in the occurrence and development of many diseases. Dysregulation of PLAGL2 expression is common in various malignant tumors, such as liver cancer, neuroblastoma, non-small cell lung cancer, prostate cancer, colorectal cancer, and leukemia.

[0003] Currently, most anti-tumor drugs exert their effects primarily by acting on epidermal growth factor receptors (HERs), including HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). The HER family plays a crucial regulatory role in cellular physiological processes, with EGFR being a member, distributed across the surface of various cells and playing a key role in cell growth, proliferation, and differentiation. Although EGFR inhibitors have shown satisfactory efficacy in treating malignant tumors, long-term use may lead to drug insensitivity in cancer cells. The PLAGL2 protein, expressed by the PLAGL2 gene, can act as a transcription factor for EGFR, regulating positive EGFR protein expression, thereby enhancing cancer cell growth, proliferation, and differentiation. Furthermore, PLAGL2 protein can restore the effectiveness of specific anti-tumor drugs and overcome acquired resistance. Combination therapy can synergistically increase apoptosis in non-small cell lung cancer cell lines and inhibit tumor growth in vivo. Therefore, PLAGL2 is considered a potential new target for treating various cancers, and PLAGL2 inhibitors may become novel therapeutic drugs for various tumor diseases, warranting further in-depth research.

[0004] Inventor CN115974766A disclosed formula (IX), but the compound had poor drug-like properties, so this invention made improvements based on it.

[0005] Summary of the Invention

[0006] The purpose of this invention is to provide a 3-amino-4-(piperazin-1-yl)benzamide derivative and its application, and to provide a pharmaceutical composition containing a 3-amino-4-(piperazin-1-yl)benzamide derivative, which can inhibit PLAGL2 activity and has promising application prospects for the treatment of various malignant tumors.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A 3-amino-4-(piperazin-1-yl)benzamide derivative and its pharmaceutically acceptable salt, characterized in that it is a compound with the structure shown in formula (VIII).

[0009]

[0010] in,

[0011] X is selected from carbonyl or sulfone groups;

[0012] A is selected from N-substituted five-membered rings, six-membered rings, seven-membered rings, double rings, or bridge rings;

[0013] R 1 Selected from 2-4 position substituted aryl carboxyl, aryl carboxyl, unsaturated or saturated long chain acetyl, adamantane acetyl, dichloroacetyl, cycloalkyl carboxyl;

[0014] R 2 Substituents selected from mono- or poly-substituted groups at positions 2-4, R 2 Independently selected from halogen, hydroxyl, nitro, cyano, amino, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 1- Cycloalkyl, wherein the substitution is monosubstituted or polysubstituted, and the substituent of R2 is independently halogen, hydroxyl, nitro, cyano or amino.

[0015] The 3-amino-4-(piperazin-1-yl)benzamide derivatives and their pharmaceutically acceptable salts are characterized in that:

[0016] A is selected from

[0017] R 1 Selected from

[0018] Or other adamantane derivatives, conjugated dienes, aromatic ring derivatives;

[0019] R 2Selected from halogen atoms, methoxy groups, or aromatic rings at positions 2, 3, and 4.

[0020] The 3-amino-4-(piperazin-1-yl)benzamide derivatives and their pharmaceutically acceptable salts are characterized in that the compounds are any one of the following structural formulas:

[0021] N-(5-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide;

[0022] (E)-N-(5-(4-(but-2-enyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide;

[0023] (Z)-N-(5-(4-(3,7-dimethyl-5-oxooctane-2,6-dienyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide;

[0024] 2-(4-(4-(-4-isopropylpiperazin-1-yl)-3-(naphthalene-2-sulfonamido)benzoyl)piperazin-1-acyl)-2-oxoacetic acid methyl ester;

[0025] N-(5-(4-(2,2-dichloroacetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide;

[0026] N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide;

[0027] 4-(4-(4-isopropylpiperazin-1-yl)-3-(naphthalene-2-sulfonamido)benzoyl)piperazin-1-carbonylcyanide;

[0028] 5-(4-(4-(-4-isopropylpiperazin-1-yl)-3-(naphthalene-2-sulfonamido)benzoyl)piperazin-1-acyl)-5-oxovaleric acid;

[0029] N-(5-(4-(4-bromobenzoyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide;

[0030] N-(5-(4-(2-fluorobenzoyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide;

[0031] N-(2-(4-isopropylpiperazin-1-yl)-5-(4-(thiophen-2-carbonyl)piperazin-1-carbonyl)phenyl)naphthalene-2-sulfonamide;

[0032] N-(5-(4-(2,3-dimethoxybenzoyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide;

[0033] N-(5-(4-(1H-pyrrolo-3-carbonyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide;

[0034] N-(5-(4-(furan-2-carbonyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide;

[0035] N-(5-(4-(2-(4-(adamantane-1-yl)phenoxy)acetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide;

[0036] N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)-2-fluorobenzamide;

[0037] N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)-4-methoxybenzamide;

[0038] N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)-3-fluorobenzamide;

[0039] N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)-4-fluorobenzamide;

[0040] N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)-2-naphthamide;

[0041] N-(5-(4-((2S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide;

[0042] N-(5-(4-(2-(adamantane-1-yl)acetyl)-1,4-diaza-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide;

[0043] N-(5-(5-(2-(adamantane-1-yl)acetyl)-2,5-diazabicyclo[2.2.2]octane-2-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide;

[0044] Or N-(5-(3-(2-(adamantane-1-yl)acetyl)hexahydropyrimidine-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide.

[0045] The 3-amino-4-(piperazin-1-yl)benzamide derivatives and their pharmaceutically acceptable salts are characterized in that the pharmaceutically acceptable salts include salts formed with the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, acetic acid, trifluoroacetic acid, pyruvic acid, citric acid, tartaric acid, lactic acid, maleic acid, benzenesulfonic acid, or succinic acid.

[0046] This invention discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the 3-amino-4-(piperazin-1-yl)benzamide derivative and its pharmaceutically acceptable salt, and pharmaceutically acceptable excipients.

[0047] Furthermore, the pharmaceutical composition comprises the 3-amino-4-(piperazin-1-yl)benzamide derivative and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable excipients refer to excipients and additives used in the manufacture of pharmaceuticals and formulation, including solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, etc. Pharmaceutically acceptable carriers are systems that can alter the way drugs enter the body and their distribution within the body, control the rate of drug release, and deliver drugs to target organs. These include microcapsules and microspheres, nanoparticles, and liposomes. When a drug composition is used for solid tumor diseases, the preferred composition is an active ingredient plus a carrier.

[0048] Because the 3-amino-4-(piperazin-1-yl)benzamide derivatives have PLAGL2 inhibitory activity, this invention also discloses an application of the 3-amino-4-(piperazin-1-yl)benzamide derivatives, which are used to prepare drugs for the prevention and / or treatment of PLAGL2-mediated diseases.

[0049] As a further improvement to the technical method, the PLAGL2-mediated diseases include pain, inflammation, immune dysfunction, neurological and psychiatric disorders, respiratory diseases, urinary and reproductive disorders, abnormal embryonic development, abnormal cell metabolism, abnormal differentiation, and malignant tumors.

[0050] As a further improvement to the technical method, the malignant tumor is liver cancer, rectal cancer, gastric cancer, lung cancer, or glioma.

[0051] Because the 3-amino-4-(piperazin-1-yl)benzamide derivative has PLAGL2 inhibitory activity, this invention also discloses an application of the 3-amino-4-(piperazin-1-yl)benzamide derivative, which is used to prepare PLAGL2 inhibitors.

[0052] This invention also discloses a method for preparing the aforementioned 3-amino-4-(piperazin-1-yl)benzamide derivative, comprising:

[0053]

[0054] in:

[0055] Step 1: Compound I reacts with a Boc-containing amine to give compound II;

[0056] Step 2: Compound II is refluxed with isopropylpiperazine in the presence of alkaline potassium carbonate to obtain compound III;

[0057] Step 3: Compound III is converted into compound IV under the conditions of palladium on carbon and hydrogen gas;

[0058] Step 4: Compound IV reacts with naphthalenesulfonyl chloride and substituted acyl chloride under pyridine or triethylamine conditions to give compound V;

[0059] Step 5: Compound V removes Boc under the action of trifluoroacetic acid to obtain compound VI;

[0060] Step 6: Compound VI reacts with nucleophiles such as acyl halides and acids to obtain compound VIII of the present invention.

[0061] Compared with CN115974766A (IX), the present invention has the following advantages:

[0062] First, structural optimizations were made. One approach was to replace the alkane linked to A in formula (IX) with a piperazine derivative, increasing the compound's water solubility. Another approach was to replace the aromatic ring structure with an aliphatic alkane structure, increasing the compound's reactivity. Specific data are shown in Table 1.

[0063] Table 1 Comparison of Ic50 activities of different substances

[0064]

[0065] As shown in Table 1, the compound of formula (VIII) of this invention exhibits higher IC50 activity than the compound of formula (IX) in CN115974766A. Furthermore, the compound of formula (VIII) of this invention has a lower LogP (lipid-water partition coefficient), indicating higher water solubility. Secondly, the reaction steps of this invention have been optimized, making the reaction process simpler. Thirdly, it facilitates the synthesis of compounds of formula (VIII) type.

[0066] Beneficial effects

[0067] This invention represents a significant advancement and substantial improvement over existing technologies. Specifically, the compound described in this invention is a novel compound, not previously reported in the literature. It blocks the PLAGL2-EGFR-HIF-1 / 2α signaling pathway by binding to the PLAGL2 protein and inhibiting its transcriptional regulation, thus preventing signal transduction. Furthermore, the compound exhibits high PLAGL2 inhibitory activity at a concentration of 400 nM, demonstrating high application value. Moreover, the preparation method of this compound is simple and highly feasible. Detailed implementation method:

[0068] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0069] In the following examples, "room temperature" refers to approximately 10°C to approximately 35°C. The proportions of the mixed solvents are volume mixing proportions; unless otherwise stated, % refers to wt%.

[0070] In silica gel column chromatography, basic silica gel refers to silica gel bonded with aminopropylsilane. In high-performance liquid chromatography (HPLC), C18 refers to silica gel bonded with octadecyl groups. Elution solvent ratios are volume-mixed ratios unless otherwise specified.

[0071] The following abbreviations are used in the examples and experimental examples below.

[0072] DCM: Dichloromethane

[0073] MeOH: Methanol

[0074] DMSO: dimethyl sulfoxide

[0075] DMF: N,N-dimethylformamide

[0076] DIEA: N,N-diisopropylethylamine

[0077] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate,

[0078] TFA: Trifluoroacetic acid

[0079] M: molar concentration.

[0080] Using Fourier transform type NMR, to determine 1 H-NMR (proton nuclear magnetic resonance spectroscopy). For analysis, use ACD / SpecManager, etc. Peaks of active hydrogens (e.g., hydroxyl, amino, etc.) are not described.

[0081] Example 1 Synthesis of N-(5-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0082]

[0083] Step 1: Synthesis of tert-butyl 4-(4-chloro-3-nitrobenzoyl)piperazine-1-carboxylic acid.

[0084] 4-Chloro-3-nitrobenzoic acid (1.69 g, 1.8 mmol), N-tert-butyloxycarbonylpiperazine (1.7 g, 9.9 mmol), HATU (3.8 g, 9.99 mmol), and DIEA (2.94 g, 22.71 mmol) were dissolved in DCM (5 mL) at room temperature and reacted for three hours at room temperature. After the reaction was complete, water was added for extraction. The product was purified by silica gel column chromatography to give 1.96 g of white solid, yield 56.84%.

[0085] 1 H NMR(300MHz, Methanol-d4)δ6.98(d,J=8.0Hz,1H),6.74(d,J=2.0Hz,1H),6.70–6.63(m ,1H),3.41(s,4H),2.91(s,4H),2.74–2.67(m,4H),1.41(s,10H),1.09(d,J=6.5Hz,6H).

[0086] Step 2, Synthesis of 4-(4-(4-isopropylpiperazin-1-yl)-3-nitrobenzoyl)piperazin-1-carboxylic acid tert-butyl ester.

[0087] (4-(4-chloro-3-nitrobenzoyl)piperazine-1-carboxylic acid tert-butyl ester, 1.0 g, 3.88 mmol) and 1-isopropylpiperazine (0.54 g, 4.27 mmol) were dissolved in CH3CN (5 mL) at room temperature, and then added to K2CO3 (1.34 g, 9.70 mmol). The reaction was carried out at 80 °C for 6 hours. After the reaction was complete, the mixture was cooled to room temperature. The reaction solution was concentrated under vacuum. Water was added for extraction, the organic phases were combined, dried over anhydrous sodium sulfate, and residual solvent was removed under vacuum. The product was purified by silica gel column chromatography to give 1.18 g of orange solid, yield 87.01%.

[0088] Step 3: Synthesis of tert-butyl 4-(3-amino-4-(4-isopropylpiperazin-1-yl)benzoyl)piperazin-1-carboxylic acid.

[0089] At room temperature, 1.0 g (0.42 mmol) of 4-(4-(4-isopropylpiperazin-1-yl)-3-nitrobenzoyl)piperazin-1-carboxylic acid tert-butyl ester was dissolved in 6 mL of methanol, and 10% Pd / C-H2 was added. The reaction was carried out at 40 °C for 5 h. After the reaction was completed, palladium on carbon was removed by filtration, the filtrate was collected, and the solution was concentrated under vacuum to give 0.86 g of a pale yellow solid, with a yield of 91.97%.

[0090] Step four, synthesis of 4-(4-(4-isopropylpiperazin-1-yl)-3-(naphthalene-2-sulfonamido)benzoyl)piperazin-1-carboxylic acid tert-butyl ester.

[0091] At 0 °C, 0.80 g (1.85 mmol) of 4-(3-amino-4-(4-isopropylpiperazin-1-yl)benzoyl)piperazin-1-carboxylic acid tert-butyl ester was dissolved in 5 mL of pyridine, and 0.63 g (2.78 mmol) of 2-naphthalenesulfonyl chloride was added. The reaction was carried out at room temperature for 3 hours. After the reaction was completed, water was added for extraction, and the product was purified by silica gel column chromatography to give 0.68 g of white solid, yield: 59.0%.

[0092] Step 5: Synthesis of 4-fluoro-N-(2-(4-isopropylpiperazin-1-yl)-5-(piperazin-1-carbonyl)phenyl)benzamide.

[0093] At room temperature, 0.5 g (0.80 mmol) of 4-(3-(4-fluorobenzamido)-4-(4-isopropylpiperazin-1-yl)benzoyl)piperazin-1-carboxylic acid tert-butyl ester was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (1 mL, 13.6 mmol) was added. The mixture was stirred at room temperature for 2 h. After the reaction was complete, saturated sodium bicarbonate aqueous solution was added to adjust the pH to 8.00, and the mixture was extracted with DCM. The solvent was concentrated under vacuum to give 0.39 g of a light green solid, with a yield of 94.9%.

[0094] f)N-(5-(4-(cyclopropanecarbonyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide

[0095] At room temperature, N-(2-(4-isopropylpiperazin-1-yl)-5-(piperazin-1-carbonyl)phenyl)naphthalene-2-sulfonamide (0.20 g, 0.38 mmol) and TEA (0.10 g, 1.00 mmol) were dissolved in DCM. At 0 °C, a DCM solution of cyclopropylformyl chloride (0.060 g, 0.58 mmol) was added dropwise to the above solution. The reaction was carried out at 0 °C for 30 min, then transferred to room temperature for three hours. After the reaction was complete, water was added for extraction. The product was then purified by silica gel column chromatography to give 0.15 g of a pale yellow solid, with a yield of 66.34%.

[0096] The experimental data are as follows:

[0097] 1 H NMR(300MHz,Chloroform-d)δ8.45(s,1H),7.89(td,J=10.6,10.0,3.8Hz,3H),7.76 (dd,J=8.7,2.0Hz,1H),7.68–7.59(m,3H),7.13(q,J=9.7,8.9Hz,2H),3.79(d,J=10. 6Hz, 4H), 3.46 (d, J=58.8Hz, 4H), 2.88 (p, J=6.5Hz, 1H), 2.69 (q, J=6.0Hz, 8H), 1.13 (d,J=6.5Hz,6H),1.03(dd,J=4.6,2.8Hz,2H),0.94–0.90(m,1H),0.86–0.80(m,2H).

[0098] Example 2 Synthesis of (E)-N-(5-(4-(but-2-enyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide.

[0099]

[0100] The cyclopropylformyl chloride in step six of Example 1 was replaced with (E)-but-2-enoyl chloride, and the other steps were prepared according to the preparation method in Example 1 to obtain compound (2), yielding 0.15 g of solid, with a yield of 63.03%.

[0101] The experimental data are as follows:

[0102] 1H NMR(300MHz,Chloroform-d)δ8.46(s,1H),7.90(s,3H),7.77(d,J=8.6Hz,1H),7.71–7.48(m,3H),7.29(s,1H),7.17(d,J=7.9Hz,1H),7.11(s,1H ),7.01–6.79(m,1H),6.27(s,1H),4.03–3.48(m,6H),3.36(s,2H),2.93( s,1H),2.73(d,J=15.2Hz,8H),1.99–1.86(m,3H),1.15(d,J=5.4Hz,6H).

[0103] Example 3 Synthesis of (Z)-N-(5-(4-(3,7-dimethyl-5-oxooctane-2,6-dienyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0104]

[0105] The cyclopropylformyl chloride in step six of Example 1 was replaced with methyl crotonyl chloride, and the other steps were prepared according to the preparation method in Example 1. Compound (3) was obtained, yielding 0.13 g of solid, with a yield of 45.19%.

[0106] The experimental data are as follows:

[0107] 1 H NMR(300MHz,Chloroform-d)δ8.73(s,1H),7.99(d,J=25.1Hz,4H),7.61(d,J=30.2Hz,3H),7.28(s,3H),5.80(s,1H),5.35(s,1H) ,3.67(d,J=35.7Hz,8H),3.12(s,4H),2.81(s,1H),2.68(s,4H),2.07(s,3H),1.92(s,2H),1.86(s,3H),1.67(s,3H),1.10(s,6H).

[0108] Example 42: Synthesis of methyl 4-(4-(4-(-4-isopropylpiperazin-1-yl)-3-(naphthalene-2-sulfonamido)benzoyl)piperazin-1-acyl)-2-oxoacetate.

[0109]

[0110] The cyclopropylformyl chloride in step six of Example 1 was replaced with oxaloyl chloride monomethyl ester, and the other steps were prepared according to the preparation method in Example 1. Compound (4) was obtained, yielding 0.14 g of solid, with a yield of 45.89%.

[0111] The experimental data are as follows:

[0112] 1 H NMR (300MHz, Methanol-d4) δ8.42(s,1H),7.92(d,J=8.6Hz,2H),7.86(d,J=7.4Hz,1H),7.74(d,J=8.5Hz,1H),7.63–7.53(m,2H),7.50 (s,1H),7.09(s,2H),3.82(s,3H),3.44(dd,J=57.8,32.0Hz,8H),2.65–2.58(m,1H),2.53(s,4H),2.50(s,4H),0.99(d,J=6.3Hz,6H).

[0113] Example 5 Synthesis of N-(5-(4-(2,2-dichloroacetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0114]

[0115] The cyclopropylformyl chloride in step six of Example 1 was replaced with 2,2-dichloroacetyl chloride, and the other steps were prepared according to the preparation method in Example 1 to obtain compound (5), yielding 0.12 g of solid, with a yield of 57.37%.

[0116] The experimental data are as follows:

[0117] 1 H NMR(300MHz,Chloroform-d)δ8.44(d,J=1.9Hz,1H),7.98–7.83(m,3H),7.76(d,J=6.7Hz,1H),7.64(d,J=5.3Hz ,3H),7.23–7.05(m,2H),6.20(s,1H),4.08–3.24(m,8H),2.86–2.73(m,1H),2.66(s,8H),1.12(d,J=6.5Hz,6H).

[0118] Example 6: Synthesis of N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide

[0119]

[0120] Steps one through seven are described in Example 1.

[0121] Step 7, Synthesis of N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0122] N-(2-(4-isopropylpiperazin-1-yl)-5-(piperazin-1-carbonyl)phenyl)naphthalene-2-sulfonamide (0.20 g, 0.38 mmol), HATU (0.16 g, 0.42 mmol), and DIEA (0.123 g, 0.96 mmol) were dissolved in DCM (5 mL) at room temperature and reacted for three hours at room temperature. After the reaction was complete, water was added for extraction. The product was purified by silica gel column chromatography to give 0.17 g of a white solid, with a yield of 69.09%.

[0123] Example 74 Synthesis of (4-(4-isopropylpiperazin-1-yl)-3-(naphthalene-2-sulfonamido)benzoyl)piperazin-1-carbonyl cyanide.

[0124]

[0125] The 1-adamantaneacetic acid in step six of Example 6 was replaced with cyanoacetic acid, and the other steps were prepared according to the preparation method in Example 6. Compound (7) was obtained, yielding 0.19 g of solid, with a yield of 52.37%.

[0126] The experimental data are as follows:

[0127] 1 H NMR(300MHz,Chloroform-d)δ8.39(s,1H),7.84(q,J=9.7,7.7Hz,3H),7.69(d,J=8.6Hz,1H),7.57(s,3H),7.22 (s,1H),7.06(d,J=8.5Hz,2H),3.51(s,4H),3.42(s,4H),2.79–2.68(m,1H),2.58(s,8H),1.05(d,J=6.3Hz,6H).

[0128] Example 85 Synthesis of 4-(4-(4-(-4-isopropylpiperazin-1-yl)-3-(naphthalene-2-sulfonamido)benzoyl)piperazin-1-acyl)-5-oxovaleric acid.

[0129]

[0130] Replace 1-adamantaneacetic acid in step six of Example 6 with glutaric acid, and follow the preparation method in Example 6 for the other steps to obtain compound (8), yielding 0.15 g of solid, with a yield of 59.35%.

[0131] The experimental data are as follows:

[0132] 1 H NMR(300MHz,Deuterium Oxide)δ8.19(s,1H),7.63(d,J=34.8Hz,4H),7.43–7.16(m,2H),7.05(d,J=24.6Hz,2H),3.26 (d,J=41.1Hz,8H),2.88(d,J=38.8Hz,8H),2.18(s,4H),1.64(d,J=53.9Hz,2H),1.16(s,6H).

[0133] Example 9 Synthesis of N-(5-(4-(4-bromobenzoyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0134]

[0135] The 1-adamantaneacetic acid in step six of Example 6 was replaced with 4-bromobenzoic acid, and the other steps were prepared according to the preparation method in Example 6. Compound (9) was obtained, yielding 0.13 g of solid, with a yield of 62.46%.

[0136] The experimental data are as follows:

[0137] 1 H NMR (300MHz, DMSO-d6) δ8.48(s,1H),8.09(d,J=8.2Hz,2H),7.99(d,J=8.2Hz,1H),7.82(d,J=8.9Hz,1H),7.68(d,J=7.9Hz,3H),7.6 0(s,1H),7.37(d,J=7.9Hz,2H),7.25(s,1H),7.12(s,2H),3.50(s,4H),3.29(d,J=23.2Hz,6H),2.66(s,6H),0.98(d,J=6.1Hz,6H).

[0138] Example 10 Synthesis of N-(5-(4-(2-fluorobenzoyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0139]

[0140] The 1-adamantaneacetic acid in step six of Example 6 was replaced with 2-fluorobenzoic acid, and the other steps were prepared according to the preparation method in Example 6. Compound (10) was obtained, yielding 0.2 g of solid, with a yield of 55.9%.

[0141] The experimental data are as follows:

[0142] 1 H NMR(300MHz,Chloroform-d)δ8.69(s,1H),8.09(ddd,J=23.6,15.9,8.7Hz,4H),7.92–7.74(m,3H),7.66(p,J=7.3Hz,2H),7.52(dd ,J=13.5,6.2Hz,2H),7.34(d,J=8.5Hz,3H),4.33–3.78(m,4H),3.60(s,4H),3.03(q,J=7.1Hz,1H),2.88(s,8H),1.52–1.22(m,6H).

[0143] Example 11 Synthesis of N-(2-(4-isopropylpiperazin-1-yl)-5-(4-(thiophen-2-carbonyl)piperazin-1-carbonyl)phenyl)naphthalene-2-sulfonamide.

[0144]

[0145] The 1-adamantaneacetic acid in step six of Example 6 was replaced with 2-thiophenecarboxylic acid, and the other steps were prepared according to the preparation method in Example 6. Compound (11) was obtained, yielding 0.18 g of solid, with a yield of 58.49%.

[0146] The experimental data are as follows:

[0147] 1 H NMR(300MHz,Chloroform-d)δ8.37(s,1H),7.80(t,J=10.7Hz,3H),7.69(d,J=8.6Hz,1H),7.53(dd,J=16.6,9.3Hz,4H),7.34–7.25( m,1H),7.12(d,J=4.5Hz,1H),7.03(d,J=5.2Hz,2H),3.82–3.16(m,8H),2.81–2.71(m,1H),2.60(d,J=9.7Hz,8H),1.10–1.00(m,6H).

[0148] Example 12 Synthesis of N-(5-(4-(2,3-dimethoxybenzoyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0149]

[0150] The 1-adamantaneacetic acid in step six of Example 6 was replaced with 2,3-dimethoxybenzoic acid, and the other steps were prepared according to the preparation method in Example 6. Compound (12) was obtained, yielding 0.15 g of solid, with a yield of 56.02%.

[0151] The experimental data are as follows:

[0152] 1 H NMR (400MHz, DMSO-d6) δ8.47(s,1H),8.09(d,J=8.8Hz,2H),8.00(d,J=8.3Hz ,1H),7.81(d,J=8.7Hz,1H),7.70–7.57(m,2H),6.79(dd,J=6.9,2.2Hz,1H), 3.84(s,3H),3.74(s,3H),3.36(s,7H),2.67–2.59(m,5H),2.51(q,J=1.9Hz, 5H), 2.48–2.41 (m, 4H), 1.22 (dd, J=21.7, 5.0Hz, 4H), 0.96 (d, J=6.4Hz, 6H).

[0153] Example 13 Synthesis of N-(5-(4-(1H-pyrrole-3-carbonyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0154]

[0155] The 1-adamantaneacetic acid in step six of Example 6 was replaced with 1H-pyrrole-3-carboxylic acid, and the other steps were prepared according to the preparation method in Example 6. Compound (13) was obtained, yielding 0.15 g of solid, with a yield of 56.63%.

[0156] The experimental data are as follows:

[0157] 1 H NMR(300MHz,Chloroform-d)δ9.40(s,1H),8.44(s,1H),7.88(t,J=10.1Hz,3H),7.76(d,J=8.6Hz,1H),7.61(dd,J=14.8,8.5Hz,3H),7.13(t ,J=6.0Hz,3H),6.75(s,1H),6.34(s,1H),3.92–3.55(m,6H),3.40(s,2H),2.77(dt,J=12.9,6.2Hz,1H),2.63(s,8H),1.11(d,J=6.4Hz,6H).

[0158] Example 14 Synthesis of N-(5-(4-(furan-2-carbonyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0159]

[0160] The 1-adamantaneacetic acid in step six of Example 6 was replaced with 2-furanic acid, and the other steps were prepared according to the preparation method in Example 6. Compound (14) was obtained, yielding 0.18 g of solid, with a yield of 56.04%.

[0161] The experimental data are as follows:

[0162] 1 H NMR(300MHz,Chloroform-d)δ8.78(s,1H),8.03(d,J=7.2Hz,3H),7.95(s,1H),7.66(dd,J=15.6,8.4Hz,4H),7.51(s,1H),7.38(s,1H),7.34(s,1H) ,7.07(d,J=3.1Hz,1H),6.50(s,1H),6.25(s,1H),6.23–6.16(m,1H),3.78 (d,J=71.3Hz,8H),3.63(s,1H),3.47–2.75(m,8H),1.37(d,J=6.4Hz,6H).

[0163] Example 15 Synthesis of N-(5-(4-(2-(4-(adamantane-1-yl)phenoxy)acetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0164]

[0165] The 1-adamantaneacetic acid in step six of Example 6 was replaced with 2-(4-(adamantane-1-yl)phenoxy)acetic acid, and the other steps were prepared according to the preparation method in Example 6. Compound (15) was obtained, yielding 0.11 g of solid, with a yield of 48.73%.

[0166] The experimental data are as follows:

[0167] 1 H NMR(300MHz,Chloroform-d)δ8.43(s,1H),7.86(dd,J=15.2,8.0Hz,3H),7.76(d,J=8.6Hz,1H),7.65–7.49(m,

[0168] 3H),7.29(s,2H),7.08(s,2H),6.88(d,J=7.6Hz,2H),4.67(s,2H),3.81–3.28(m,8H),2.92–2.82 (m,1H),2.74(s,4H),2.62(s,4H),2.06(s,3H),1.85(s,6H),1.74(s,6H),1.12(d,J=5.5Hz,6H).

[0169] Example 16 Synthesis of N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)-2-fluorobenzamide.

[0170]

[0171] The 2-naphthalenesulfonyl chloride in step four of Example 1 was replaced with 2-fluorobenzoyl chloride, and the other steps were prepared according to the preparation method in Example 1. Compound (16) was obtained, yielding 0.18 g of solid, with a yield of 54.97%.

[0172] The experimental data are as follows:

[0173] 1 H NMR(300MHz,Chloroform-d)δ8.58(s,1H),8.02(s,3H),7.76(s,3H),7.23(s,2H),4.04–3.45(m,8H), 2.90(s,1H),2.84–2.60(m,8H),2.30(s,2H),2.11(s,3H),1.88–1.67(m,12H),1.22(d,J=3.1Hz,0H).

[0174] Example 17 Synthesis of N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)-4-methoxybenzamide.

[0175]

[0176] The 2-naphthalenesulfonyl chloride in step four of Example 1 was replaced with 4-methoxybenzoyl chloride, and the other steps were prepared according to the preparation method in Example 1. Compound (17) was obtained, yielding 0.15 g of solid, with a yield of 60.45%.

[0177] The experimental data are as follows:

[0178] 1H NMR(300MHz,Chloroform-d)δ9.04(s,1H),8.42(s,1H),7.89(d,J=8.4Hz,2H),7.20(s,2H),7.02(d,J=8.6Hz,

[0179] 2H),3.89(s,3H),3.68(dt,J=12.8,6.5Hz,8H),3.13(t,J=7.6Hz,1H),2.98(s,4H),2 .87(s,4H),2.18(s,2H),1.97(s,3H),1.66(d,J=10.2Hz,12H),1.25(d,J=6.5Hz,6H).

[0180] Example 18 Synthesis of N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)-3-fluorobenzamide.

[0181]

[0182] The 2-naphthalenesulfonyl chloride in step four of Example 1 was replaced with 3-fluorobenzoyl chloride, and the other steps were prepared according to the preparation method in Example 1. Compound (18) was obtained, yielding 0.19 g of solid, with a yield of 58.79%.

[0183] The experimental data are as follows:

[0184] 1 H NMR(300MHz,Chloroform-d)δ9.40–9.34(m,1H),8.55(s,1H),7.70(d,J=7.7Hz,1H) ,7.64(dt,J=9.4,2.3Hz,1H),7.52(td,J=8.0,5.5Hz,1H),7.30(d,J=5.7Hz,1H),7. 26(d,J=5.0Hz,2H),3.72(s,4H),3.61(s,4H),2.99(t,J=4.7Hz,4H),2.92(d,J=6.4 Hz,1H),2.85(s,4H),2.19(s,2H),1.98(s,3H),1.66(s,12H),1.18(d,J=6.5Hz,6H).

[0185] Example 19 Synthesis of N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)-4-fluorobenzamide.

[0186]

[0187] The 2-naphthalenesulfonyl chloride in step four of Example 1 was replaced with 4-fluorobenzoyl chloride, and the other steps were prepared according to the preparation method in Example 1. Compound (19) was obtained, yielding 0.14 g of solid, with a yield of 59.19%.

[0188] The experimental data are as follows:

[0189] 1 H NMR(300MHz,Chloroform-d)δ9.36(s,1H),8.58(s,1H),7.95(s,2H),7.47–7.06(m,5H),3.67(d,J=30 .4Hz,8H),2.99(s,4H),2.78(s,5H),2.19(s,2H),1.98(s,3H),1.66(s,12H),1.15(d,J=3.9Hz,11H).

[0190] Example 20 Synthesis of N-(5-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)-2-naphthamide.

[0191]

[0192] The 2-naphthalenesulfonyl chloride in step four of Example 1 was replaced with 2-naphthaleneyl chloride, and the other steps were prepared according to the preparation method in Example 1. Compound (20) was obtained, yielding 0.11 g of solid, with a yield of 62.74%.

[0193] The experimental data are as follows:

[0194] 1 H NMR(300MHz,Chloroform-d)δ9.07(s,1H),8.76(s,1H),8.47(d,J=5.8Hz,1H),8.02(d,J=10.4Hz,1H),7.94(d,J=9.6Hz,1H),7.77(dd,J=7.5,3.7Hz,1H) ,7.60(s,2H),7.26(s,3H),3.69(d,J=24.7Hz,8H),3.03(s,4H),2.94(s,1H) ,2.68(s,4H),2.19(s,2H),1.98(s,3H),1.66(s,13H),1.08(d,J=6.5Hz,6H).

[0195] Example 21 Synthesis of N-(5-(4-((2S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl)piperazine-1-carbonyl)-2-(4-isopropylpiperazine-1-yl)phenyl)naphthalene-2-sulfonamide.

[0196]

[0197] The cyclopropylformyl chloride in step six of Example 1 was replaced with bicyclo[2.2.1]hept-5-ene-2-carboxylic acid, and the other steps were prepared according to the preparation method in Example 1 to obtain compound (21), yielding 0.12 g of solid, with a yield of 60.85%.

[0198] The experimental data are as follows:

[0199] 1 H NMR(300MHz,DMSO-d6)δ8.52(s,1H),8.15(t,J=8.5Hz,2H),8.07–7.97(m,1H) ,7.85(d,J=8.7Hz,1H),7.77–7.60(m,2H),7.27(s,1H),7.14(s,2H),6.13–6.0 6(m,1H),5.99–5.90(m,1H),3.35(d,J=20.1Hz,8H),3.01(s,1H),2.88–2.84(m ,1H),2.70(s,4H),2.56(s,4H),1.28(d,J=12.3Hz,4H),1.01(d,J=6.4Hz,6H).

[0200] Example 22 Synthesis of N-(5-(4-(2-(adamantane-1-yl)acetyl)-1,4-diaza-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide.

[0201]

[0202] The N-Boc piperazine in step one of Example 6 was replaced with N-Boc piperazine, and the other steps were prepared according to the preparation method in Example 6. Compound (22) was obtained, yielding 0.15 g of solid, with a yield of 65.60%.

[0203] 1H NMR(300MHz,Chloroform-d)δ8.42(s,1H),7.86(t,J=9.4Hz,3H),7.74(d,J=8.6Hz ,1H),7.62(d,J=14.3Hz,3H),7.03(dt,J=19.2,9.8Hz,2H),3.73(d,J=18.0Hz,3H) ,3.53(d,J=15.6Hz,3H),3.32(d,J=17.3Hz,2H),2.82–2.70(m,1H),2.57(d,J=19. 2Hz,8H),2.14(d,J=20.0Hz,2H),1.96(s,3H),1.66(s,12H),1.08(d,J=6.2Hz,6H).

[0204] Example 23 Synthesis of N-(5-(5-(2-(adamantane-1-yl)acetyl)-2,5-diazabicyclo[2.2.2]octane-2-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide.

[0205]

[0206] The N-Boc piperazine in step one of Example 6 was replaced with N-Boc-2,5-diazabicyclo[2.2.2]octane, and the other steps were prepared according to the preparation method in Example 6. Compound (23) was obtained, yielding 0.18 g of solid, with a yield of 68.90%.

[0207] 1H NMR (300MHz, Chloroform-d) δ9.68 (s, 1H), 8.35 (q, J = 0.5Hz, 1H), 8.11 (t, J = 0.5Hz, 1H), 8.00 (dq, J = 1.0, 0.5Hz, 2H), 7.92 (d, J = 2. 0Hz,1H),7.85(d,J=0.5Hz,1H),7.75(dd,J=7.5,2.0Hz,1H),7.62(d,J=0.5Hz,1H),7.52(d,J=0.5Hz,1H),7.27(d,J=7.5Hz,1H),4 .09(s,1H),3.97(s,1H),3.80(d,J=12.4Hz,2H),3.65(d,J=3.0Hz,2H),3.15(d,J=1.6Hz,4H),2.99(s,1H),2.77(s,2H),2.69(s,2 H),2.41(d,J=12.4Hz,1H),2.33(d,J=12.4Hz,1H),1.98(s,3H),1.94–1.81(m,4H),1.65(d,J=9.8Hz,12H),1.16(d,J=15.1Hz,6H).

[0208] Example 24 Synthesis of N-(5-(3-(2-(adamantane-1-yl)acetyl)hexahydropyrimidine-1-carbonyl)-2-(4-isopropylpiperazin-1-yl)phenyl)naphthalene-2-sulfonamide.

[0209]

[0210] The N-Boc piperazine in step one of Example 6 was replaced with N-Boc-hexahydropyrimidine, and the other steps were prepared according to the preparation method in Example 6. Compound (24) was obtained, yielding 0.18 g of solid, with a yield of 65.85%.

[0211] 1H NMR(300MHz,Chloroform-d)δ9.72(s,1H),8.36(q,J=0.6Hz,1H),8.11(t,J=0.5Hz,1H),8.04–7.91(m,3H),7. 84(d,J=0.5Hz,1H),7.76(dd,J=7.5,2.0Hz,1H),7.63(d,J=0.5Hz,1H),7.52(d,J=0.5Hz,1H),7.37(d,J=7.5H z,1H),4.87(s,1H),4.74(s,1H),3.70(d,J=8.1Hz,4H),3.15(s,4H),2.99(s,1H),2.77(s,2H),2.69(s,2H),2 .53(d,J=12.4Hz,1H),2.40(d,J=12.4Hz,1H),1.98(s,3H),1.85(s,2H),1.67(d,J=15.3Hz,12H),1.16(s,6H).

[0212] Example 25: Determination of tumor cell viability using the CCK-8 assay

[0213] Experimental Principle: PLAGL2 inhibitors can suppress the expression of PLAGL2 in liver cancer cells, thereby causing cancer cell death. Therefore, we can indirectly reflect the inhibitory ability of PLAGL2 inhibitors through cytotoxic activity experiments on highly metastatic human liver cancer cells (HCCLM3 / Huh7). The stronger the inhibitory ability of PLAGL2 inhibitors, the lower the survival rate of human liver cancer cells, and the corresponding IC50. 50 The smaller the value.

[0214] Experimental materials and instruments

[0215] (1) Cells: Human hepatocellular carcinoma HCCLM3 and Huh7 cells were purchased from the Chinese Academy of Sciences Center for Type Culture Collection.

[0216] (2) Experimental consumables are shown in Table 2

[0217] Table 2. Experimental Consumables List

[0218] Reagent Name factory Pen Strep Gibco, USA DMEM medium BI Pancreatic enzyme digestive fluid BI FBS (10% fetal bovine serum) Pottery CCK-8 reagent kit Pottery DMSO Pottery PBS buffer Pottery

[0219] Reagent preparation and determination

[0220] (A) Determination of HCCLM3 cell line

[0221] Preparation of HCCLM3 cell culture medium: HCCLM3 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin antibody. The specific preparation process is as follows: Under aseptic conditions, accurately pipette 55 mL of FBS and 5.5 mL of penicillin antibody, add them to 500 mL of bottled DMEM medium, mix well, and store at 4°C for later use.

[0222] Preparation of test compounds: Based on the molecular weight of the test compounds, a 20 mM stock solution was prepared using DMSO and stored in a -80°C freezer.

[0223] Experimental methods:

[0224] (1) Take HCCLM3 cells in the logarithmic growth phase, discard the culture medium, digest the cells with 0.25% Trypsin containing EDTA, and prepare a single-cell suspension.

[0225] (2) Adjust the cell density to 30,000 cells / mL with DMEM medium (containing 10% (V / V) FBS and 1% Pen Strep), seed 100 μL / well into 96-well plates, with approximately 3,000 cells per well, and incubate at 37°C in a 5% CO2 cell culture incubator for 24 h until adherence.

[0226] (3) Preparation of drug-containing culture medium: The test compound was diluted 100-fold to a final concentration of 200 μM, and then serially diluted to final concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 0.313 μM, and 0.156 μM.

[0227] (4) 10 μL of DMEM medium containing 1% DMSO was added to the solvent group to make the final concentration of DMSO 0.01%; 10 μL of different concentrations of the test compound in step (3) were added to the drug administration group to make the final concentrations 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.313 μM and 0.156 μM.

[0228] (5) After 72 hours, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C in a 5% CO2 cell culture incubator for another 2 hours. After incubation, the absorbance (OD) value was measured at 450 nm using a microplate reader. Simultaneously, the IC50 of the test compound was calculated using Graphpad Prism statistical software. 50 value.

[0229] (B) Determination of HCCLM3 cell line

[0230] Replace the MHCC-97H cell line in step (A) of Example 25 with the Huh7 cell line, and keep all other experimental procedures the same. Finally, calculate the IC50 value of the test compound in the Huh7 cell line.

[0231] Cell viability (%) = [A(drug-treated) - A(blank)] / [A(drug-treated) - A(blank)] × 100%

[0232] A (Drug Addition): OD values ​​of wells containing cells, CCK-8 solution, and drug solution.

[0233] A (0 drug added): OD value of wells containing cells and CCK-8 solution but no drug solution.

[0234] A (blank): OD value of pores without cells.

[0235] Cell viability: The results of cell proliferation activity or cytotoxic activity are shown in Table 3.

[0236] Table 3 Results of the effects of different substances on cell viability

[0237]

[0238]

[0239] The test results in Table 2 show that the above compounds exhibit high cell activity against the HCCLM3 / Huh7 cell line, with Examples 5, 6, 16 and 20 showing the best effects, indicating that some of the compounds of the present invention have beneficial effects in inhibiting the proliferation of liver cancer cells.

[0240] Example 26: Clonogenic assay for assessing the invasive ability of tumor cells

[0241] Colony formation experiments were conducted on Examples 5 and 6, which were screened by the CCK-8 assay and showed good anti-proliferative activity, to evaluate their effect on the invasive ability of hepatocellular carcinoma cells.

[0242] Experimental methods:

[0243] Collect healthy HCCLM3 cells in the logarithmic growth phase, wash with PBS, digest with trypsin, collect by centrifugation, and resuspend in the appropriate culture medium. Add 1 μL, 0.5 μL, 0.25 μL, 0.1 μL, and 0.05 μL of a 20 mM compound solution to six-well plates to prepare concentrations of 10 μM, 5 μM, 2.5 μM, 1 μM, and 0.5 μM per well, respectively. For 0 μM, add 1 μL of DMSO. Add 2 mL of culture medium containing 5 × 10³ cells to each well. After mixing, incubate at 37°C with 5% CO2 for 14 days.

[0244] Discard the culture medium, gently wash once with PBS, fix with 4% paraformaldehyde for 40 min, then discard the fixative, wash three more times with PBS, stain with 2.5% crystal violet for 1 h, and gently wash with pure water until no further staining occurs. Then photograph the cells and use ImageJ to count the number of clones. The results are calculated as the percentage of cells at 0 μM, and are shown in Table 4.

[0245] Table 4 Results of the cell invasion ability of different substances

[0246] Example 5 Example 6 0μM 100.00％ 100.00％ 0.5μM 0.05％ 89.55％ 1μM 0.01％ 61.28％ 2.5μM 0.04％ 20.55％ 5μM 0.06％ 0.53％ 10μM 0.09％ 0.07％

[0247] The test results in Table 3 show that Example 5 can significantly inhibit the metastatic and invasive ability of the HCCLM3 cell line, indicating that some of the compounds of the present invention have beneficial effects in inhibiting the invasion of liver cancer cells.

[0248] The above pharmacological data show that the compound of this invention inhibits the proliferation and malignant development of liver cancer cells by acting on the PLAGL2 target, thus exhibiting a strong inhibitory effect on malignant tumors.

[0249] 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 can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A 3-amino-4-(piperazin-1-yl)benzamide derivative and its pharmaceutically acceptable salt, characterized in that, The derivative is a compound with the structure shown in formula (VIII): Formula VIII X is selected from carbonyl or sulfone groups; A is selected from , , or ; R 1 Selected from , , , , , or ; R 2 Selected from halogen atoms at positions 2, 3, and 4, and methoxy groups.

2. A 3-amino-4-(piperazin-1-yl)benzamide derivative and its pharmaceutically acceptable salt, characterized in that, The derivative is any one of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. The 3-amino-4-(piperazin-1-yl)benzamide derivatives and their pharmaceutically acceptable salts according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts include those formed with the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, acetic acid, trifluoroacetic acid, pyruvic acid, citric acid, tartaric acid, lactic acid, maleic acid, benzenesulfonic acid, or succinic acid.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the 3-amino-4-(piperazin-1-yl)benzamide derivatives of claim 1 or 2 and their pharmaceutically acceptable salts, and pharmaceutically acceptable excipients.

5. The use of a 3-amino-4-(piperazin-1-yl)benzamide derivative according to claim 1 or 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 4 in the preparation of a medicament for the prevention or treatment of PLAGL2-mediated diseases, characterized in that, The PLAGL2-mediated diseases mentioned are liver cancer, breast cancer, rectal cancer, stomach cancer, or prostate cancer.

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