4-Piperazineamide benzenesulfonamide-1,8-naphthalimide derivatives and their preparation methods and applications

Abstract

The present invention discloses a series of novel 4-piperazinamide benzenesulfonamide-1,8-naphthalimide derivatives and their preparation methods. The applicant's experimental results indicate that some of the target compounds of the present invention exhibit good inhibitory activity against CA IX and can be used in drugs that inhibit the activity and / or overexpression of carbonic anhydrase IX. Some of the target compounds also exhibit good anti-tumor activity against various tumor cell lines and are expected to be used in the preparation of anti-tumor drugs.

Description

Technical Field

[0001] The invention relates to a 4-piperazineamide benzenesulfonamide-1,8-naphthalimide derivative and a preparation method and application thereof, belonging to the technical field of medicine. Background Art

[0002] Carbonic anhydrase IX (CA IX) has been found in various tumor cell types, and its overexpression is often associated with poor cell proliferation. Studies have shown that CA IX plays an important role in tumor proliferation, metastasis, and maintaining the cellular microenvironment. Therefore, CA IX holds significant promise as a target for tumor diagnosis and novel small molecule tumor inhibitors. Currently, CA IX inhibitors primarily fall into the sulfonamide and coumarin classes, with sulfonamides receiving the most research. In 2009, Supuran's group first resolved the crystal structure of CA IX (Alterio V, Hilvo M, Di Fiore A, et al. Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX. Proc Natl Acad Sci USA, 2009, 106, 16233-16238). This provides a structural basis for the rational development of CA IX inhibitors.

[0003] Naphthalimides typically contain a coplanar, π-defective aromatic system and a basic side chain, and are primarily used as DNA- and topoisomerase-targeting antitumor drugs. Studies have shown that naphthalimide derivatives have potential antiproliferative activity against a variety of tumors, such as breast cancer, liver cancer, glioma, and melanoma. Several effective antitumor drugs, such as mitonafide, amonafide, ethonafide, and elinafide, are currently in Phase II clinical trials. However, several factors have hindered the further development of naphthalimide derivatives. For example, while amonafide, the first compound to enter clinical trials, exhibits excellent antitumor activity, its CNS neurotoxicity and hematotoxicity limit its further application and development. Malignant stem cells may escape treatment with this drug, leading to drug resistance in tumor cells and the ineffectiveness of current anticancer therapies, ultimately leading to their failure. Therefore, the applicants have attempted to structurally modify naphthalimides in order to develop multifunctional, highly selective antitumor drugs targeting carbonic anhydrase IX. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a series of 4-piperazineamide benzenesulfonamide-1,8-naphthalimide derivatives with novel structures and good biological activity, as well as preparation methods and applications thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The 4-piperazineamide benzenesulfonamide-1,8-naphthalimide derivative of the present invention is a compound having a structure represented by the following formula (I) or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Wherein, R represents a hydrogen atom, a halogen atom, a hydroxyl group or a mercapto group, or a C1-C7 alkyl, alkenyl or alkynyl group, or a halogenated C1-C7 alkyl, alkenyl or alkynyl group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with a hydroxyl group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with an amino group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with an amide group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with a carboxyl group, or a C1-C7 alkyl, alkenyl or alkynyl group, or a C1-C7 alkoxy group, or a benzyl group or a substituted derivative thereof, or a phenyl group or a substituted derivative thereof, or a five-membered or six-membered heterocyclic methyl group or a substituted derivative thereof.

[0009] In the general structure of the above-mentioned 4-piperazineamidebenzenesulfonamide-1,8-naphthalimide derivative, R is preferably a C1-C7 alkyl group, or a C1-C7 alkyl group substituted with a hydroxyl group, or a C1-C7 alkyl group substituted with an amino group, or a benzyl group or a substituted derivative thereof, or a phenyl group or a substituted derivative thereof, or a five-membered or six-membered heterocyclic methyl group or a substituted derivative thereof.

[0010] In some specific embodiments, R represents 2-(methylamino)ethyl, 2-(dimethylamino)ethyl, benzyl, 2-(2-aminoethyl)amino)ethyl, 2-morpholinylethyl, 3-morpholinylpropyl, 4-aminophenethyl, 2-hydroxyethyl, 4-chlorobenzyl, butyl, phenylamino, 2-(piperazin-1-yl)ethyl, pyridin-2-ylamino, cyclohexyl, 2-(diethylamino)ethyl, 4-bromobenzyl, heptyl or 4-methylbenzyl.

[0011] The preparation method of the 4-piperazineamide benzenesulfonamide-1,8-naphthalimide derivative of the present invention mainly comprises the following steps: placing a compound represented by the following formula (II) and a compound represented by the following formula (III) in an organic solvent, reacting them under heating or non-heating conditions, recovering the solvent from the obtained reaction material, and obtaining a crude target compound;

[0012]

[0013] Wherein, R represents a hydrogen atom, a halogen atom, a hydroxyl group or a mercapto group, or a C1-C7 alkyl, alkenyl or alkynyl group, or a halogenated C1-C7 alkyl, alkenyl or alkynyl group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with a hydroxyl group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with an amino group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with an amide group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with a carboxyl group, or a C1-C7 alkyl, alkenyl or alkynyl group, or a C1-C7 alkoxy group, or a benzyl group or a substituted derivative thereof, or a phenyl group or a substituted derivative thereof, or a five-membered or six-membered heterocyclic methyl group or a substituted derivative thereof.

[0014] In the above preparation method, the organic solvent is preferably selected from one or a combination of two or more of N,N-dimethylformamide (DMF), dichloromethane, acetone, and ethanol. The organic solvent is preferably dehydrated with molecular sieves before use. The amount of organic solvent used is preferably sufficient to dissolve the raw materials involved in the reaction. Typically, 20 to 40 mL of organic solvent is used to dissolve all the raw materials involved in the reaction, based on 1 mmol of the compound represented by formula (II).

[0015] In the above preparation method, when the reaction is carried out under heating conditions, the reaction rate can be accelerated. Therefore, the present application preferably carries out the reaction under heating conditions, generally at a temperature between ≥35°C and the boiling point of the organic solvent, and more preferably at ≥40°C. The reaction is monitored by TLC tracking until the reaction is complete. According to the applicant's experience, when N,N-dimethylformamide is used as the organic solvent and the reaction is carried out at 90-120°C, the reaction time is preferably controlled within 6-10 hours.

[0016] The above method produces a crude target compound. Therefore, the method of the present invention further includes a step of purifying the crude target compound. Specifically, conventional purification methods can be used to improve the purity of the target compound, such as silica gel column chromatography. The eluent used for column chromatography is preferably a mixed solvent of dichloromethane and methanol. The volume ratio of dichloromethane to methanol in the mixed solvent is preferably 20:1 to 10:1, more preferably 15:1 to 10:1.

[0017] In the preparation method of the present invention, the dosage ratio of each raw material is a stoichiometric ratio. In actual operation, the molar ratio of the compound represented by formula (II) to the compound represented by formula (III) is usually 1:1-2.

[0018] In the preparation method of the present invention, the compound shown in the raw material formula (II) is a 1,8-naphthalimide piperazine derivative, which can be synthesized with reference to the existing literature (Gui-Bin Liang, Jian-Hua Wei, Hong Jiang, Ri-Zhen Huang, Jing-Ting Qin, Hui-Ling Wang, Heng-Shan Wang, Ye Zhang, Design, synthesis and antitumorevaluation of new 1,8-naphthalimide derivatives targeting nuclear DNA, European Journal of Medicinal Chemistry, 2021, 210, 112951.), or a self-designed synthesis route can be synthesized. In this application, the compound shown in formula (II) is preferably prepared according to the following synthetic route (BOC in compound S3 and compound S4 in the synthetic route represents tert-butyloxycarbonyl):

[0019]

[0020] The specific method for preparing the compound represented by formula (II) comprises the following steps:

[0021] 1) Compound S1 (4-bromo-1,8-naphthalene dicarboxylic anhydride) and Compound S2 (tert-butyloxycarbonylpiperazine) are placed in solvent A and reacted under heating conditions. The reactants are cooled and the precipitate is collected to obtain Compound S3;

[0022] 2) Compound S3 and a primary amine compound (R-NH2, where R is selected as described above) are placed in solvent A and reacted under heating conditions. The reactants are cooled and the precipitate is collected to obtain compound S4;

[0023] 3) Removing the protecting group (ie, tert-butyloxycarbonyl) in compound S4 to expose the NH group, thereby obtaining the compound represented by formula (II).

[0024] In steps 1) and 2) of the above-mentioned method for preparing the compound represented by formula (II), the solvent A involved can be an alcoholic solvent and / or an aprotic solvent. Specifically, the alcoholic solvent can be one or a combination of two or more selected from methanol, ethanol, propanol, and n-butanol; and the aprotic solvent can be one or a combination of two or more selected from ethylene glycol monomethyl ether, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), toluene, carbon tetrachloride, and acetone. The amount of the organic solvent used can be determined as needed. Typically, all the reaction raw materials are dissolved in 25-50 mL of organic solvent, based on 1 mmol of 4-bromo-1,8-naphthalenedicarboxylic anhydride. When a large amount of organic solvent is added, it is preferred to recover some of the organic solvent after the reaction is complete (typically removing 40-50% of the solvent A added) before cooling the reactants.

[0025] In steps 1) and 2) of the above method for preparing the compound represented by formula (II), the reaction is preferably carried out at a temperature between 50° C. and the boiling point of solvent A, and more preferably, a reflux reaction is used.

[0026] In step 3) of the above-described method for preparing the compound represented by formula (II), the protecting group in compound S4 is removed using conventional methods. For example, compound S4 is stirred in dioxane hydrochloride or a mixed solvent consisting of dichloromethane and trifluoroacetic acid in a volume ratio of 2:1 for a predetermined period of time to remove the tert-butyloxycarbonyl group. To further expose the NH structure, an excess of dichloromethane (as the reaction solvent) and an excess of a basic substance (e.g., an inorganic base (e.g., sodium hydroxide, potassium hydroxide, etc.) or an organic base (e.g., triethylamine, diethylamine, etc.)) can be added to the reaction system to neutralize the excess acid.

[0027] The crude products obtained in steps 1) to 3) of the above-mentioned method for preparing the compound represented by formula (II) are all crude products of the compound, which can be purified according to conventional methods, usually by recrystallization or silica gel column purification.

[0028] In the preparation method of the present invention, the raw material represented by formula (III) is 2-chloro-N-(4-sulfamoylphenyl)acetamide, which can be synthesized with reference to the existing literature (CB Mishra, S. Kumari, A. Angeli, S. Maria Monti, M. Buonanno, M. Tiwari, CT Supuran, Discovery of benzenesulfonamides with potent human carbonic anhydrase inhibitory and effective anticonvulsant action: design, dynthesis, and pharmacological assessment, J. Med. Chem. 2017, 60, 2456-2469.). In this application, the compound represented by formula (III) is preferably prepared according to the following synthetic route:

[0029]

[0030] The specific method for preparing the compound represented by formula (III) is: compound 1 (sulfonamide) and compound 2 (chloroacetyl chloride) are placed in solvent B, and reacted with or without heating to obtain the compound.

[0031] In the above method for preparing the compound represented by formula (III), solvent B is preferably acetone, and the reaction is preferably carried out at room temperature. The resulting liquid is preferably first extracted with ethyl acetate, washed sequentially with water and brine, the organic phase is collected, dried over anhydrous sodium sulfate, and the resulting residue is purified on a silica gel column (eluent: dichloromethane) to obtain a high-purity compound represented by formula (III).

[0032] The "solvent A" and "solvent B" appearing in the above preparation methods are names given to indicate the selection of different solvents.

[0033] The applicants have discovered through experiments that some of the derivatives of the present invention have good inhibitory activity against CA IX. Therefore, the present invention also includes the use of the aforementioned 4-piperazinamide benzenesulfonamide-1,8-naphthalimide derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for inhibiting the activity and / or overexpression of carbonic anhydrase IX. Furthermore, the applicants have also discovered through experiments that some of the derivatives of the present invention have good anti-tumor activity against various tumor cell lines. Therefore, the present invention also includes the use of the aforementioned 4-piperazinamide benzenesulfonamide-1,8-naphthalimide derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating tumors, and further, in the preparation of medicaments for treating breast cancer or colon cancer.

[0034] Furthermore, the present invention also includes a pharmaceutical composition comprising a therapeutically effective dose of the above-mentioned 4-piperazinamide benzenesulfonamide-1,8-naphthalimide derivative or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be in the dosage form commonly used in the art, such as tablets, pills, granules, injections, etc. The dosage of the drug of the present invention can vary depending on the route of administration, the patient's age, weight, the type and severity of the disease being treated, etc. The daily dose can be 0.01 to 10 mg / kg body weight, preferably 0.1 to 5 mg / kg body weight. It can be administered once or multiple times.

[0035] Compared to existing technologies, the present invention provides a series of novel 4-piperazinamide benzenesulfonamide-1,8-naphthalimide derivatives and their preparation methods. The applicant's experimental results indicate that some of the target compounds of the present invention exhibit excellent inhibitory activity against CA IX and can be used as drugs to inhibit the activity and / or overexpression of carbonic anhydrase IX. Some of the target compounds also exhibit significant anti-tumor activity against various tumor cell lines and are expected to be used in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a graph showing the effects of different concentrations of compound Io on ROS levels in MDA-MB-231 cells after 24 hours of action, as observed using an inverted fluorescence microscope.

[0037] Figure 2 This is a graph showing the effect of different concentrations of compound Io on JC-1 staining of MDA-MB-231 cells observed under an inverted fluorescence microscope.

[0038] Figure 3 This is a graph showing the apoptosis rate of MDA-MB-231 cells induced by different concentrations of compound Io detected by Annexin V / PI method.

[0039] Figure 4 This is a flow cytometry PI staining experiment to detect the cell cycle diagram of MDA-MB-231 cells after the action of different concentrations of compound Io for 48 hours.

[0040] Figure 5 The graph shows the ability of compound Io at different concentrations to inhibit the migration of MDA-MB-231 cells.

[0041] Figure 6Figure 2 shows the in vivo antitumor effect of compound Io in a 4T1 xenograft tumor model (mice were orally administered vehicle or compound Io (20 or 40 mg / kg) every 3 days for 21 consecutive days); (A) photographs of tumor tissue isolated after 21 consecutive days of compound Io administration, (B) weight of tumors excised at the end of compound Io treatment, (C) changes in tumor volume in different treatment groups measured every 3 days, and (D) effect of compound Io on body weight in a 4T1 xenograft mouse model.

[0042] Figure 7 H&E staining images of the inhibition of lung and liver metastasis by compound Io at different concentrations (magnification, ×200). DETAILED DESCRIPTION

[0043] In order to better explain the technical solution of the present invention, the present invention is further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0044] The compounds represented by formula (II) involved in the following examples were prepared according to the following synthetic route:

[0045]

[0046] R represents a hydrogen atom, a halogen atom, a hydroxyl group or a mercapto group, or a C1-C7 alkyl, alkenyl or alkynyl group, or a halogenated C1-C7 alkyl, alkenyl or alkynyl group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with a hydroxyl group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with an amino group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with an amide group, or a C1-C7 alkyl, alkenyl or alkynyl group substituted with a carboxyl group, or a C1-C7 alkyl, alkenyl or alkynyl group, or a C1-C7 alkoxy group, or a benzyl group or a substituted derivative thereof, or a phenyl group or a substituted derivative thereof, or a five-membered or six-membered heterocyclic methyl group or a substituted derivative thereof.

[0047] The specific preparation method comprises the following steps:

[0048] 1) Compound S1 (4-bromo-1,8-naphthalene dicarboxylic anhydride, 10 mmol) and compound S2 (tert-butyloxycarbonylpiperazine, mmol) were placed in a round-bottom flask, ethylene glycol methyl ether (50 mL) was added, and the mixture was refluxed for 3 h (TLC monitoring the reaction). After the reaction was completed, the reaction solution was filtered, and the filter cake was recrystallized from ethanol overnight and filtered to obtain compound S3 (yellow solid);

[0049] 2) Compound S3 (1 mmol) and a primary amine compound (R-NH2, 1.2 mmol) were placed in a round-bottom flask, ethanol (50 mL) was added, and the mixture was refluxed for 3 h (TLC monitoring the reaction). After completion of the reaction, the resulting material was purified on a silica gel column (dichloromethane / methanol = 10 / 1, volume ratio) to obtain compound S4 (yellow solid);

[0050] 3) Compound S4 (1 mmol) was placed in a round-bottom flask, dioxane hydrochloride (10 mL) was added and stirred for 1 h, and then the mixture was spin-dried to dryness. Then, excess dichloromethane (20 mL) and excess triethylamine (20 mL) were added to react (TLC monitoring). After the reaction was completed, an appropriate amount of water was added for extraction, and the organic phase was collected and spin-dried to obtain the compound represented by formula (II).

[0051] The compound represented by formula (II) involved in the following examples (i.e., 2-chloro-N-(4-sulfamoylphenyl)acetamide) was prepared according to the following synthetic route:

[0052]

[0053] The specific preparation method is as follows: 2 g (1 mmol) of sulfonamide is dissolved in acetone (25 mL), 2 ml (25 mmol) of chloroacetyl chloride is added dropwise, and potassium carbonate (3.2 g, 23 mmol) is added. After reacting at room temperature for 8 hours, the reaction is quenched with water, and the reaction mixture is extracted with ethyl acetate, washed with water and brine in sequence, the organic phase is collected, dried over anhydrous sodium sulfate, and then spin-dried. The resulting residue is purified by silica gel chromatography (dichloromethane) to obtain the compound represented by formula (II), 2-chloro-N-(4-sulfamoylphenyl)acetamide.

[0054] Example 1

[0055] The 4-piperazineamide benzenesulfonamide-1,8-naphthalimide derivative of the present invention was synthesized according to the following synthetic route.

[0056]

[0057] The specific preparation method is as follows: 5 mmol of the compound represented by formula (II), 5 mmol of the compound represented by formula (III), and 30 mL of DMF (dehydrated with molecular sieves before use) are added to a round-bottom flask, and the reaction is stirred at 120°C for 6-8 hours (TLC monitoring the reaction). After the reaction is completed, the solvent is recovered, and the resulting residue is purified on a silica gel column (dichloromethane / methanol = 10 / 1, volume ratio) to obtain the target compound I. The different target products and their characterizations are as follows:

[0058]

[0059] 2-(4-(2-(2-(methylamino)ethyl)-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ia); yellow solid; yield approximately 55.43%; 1 H NMR(400MHz,DMSO-d6)δ10.17(s,1H),8.46(s,2H),8.36(s,1H),7.65(s,2H),7.60(s,2H),7.33(s,1H),7.27(s,3 H), 4.24 (d, J = 7.1Hz, 2H), 3.42 (dd, J = 7.1, 5.0Hz, 2H), 3.29 (d, J = 25.4Hz, 7H), 2.90 (s, 6H), 1.05 (t, J = 7.0Hz, 1H). 13 C NMR (126MHz, DMSO-d6) δ170.04,169.40,164.29,163.76,156.16,142.05,141.81,139.04,138.79,132.75,131.0 6,127.15,125.74,123.03,119.52,115.99,115.52,61.99,55.28,53.14,53.01,43.34,38.03.HR-MS(m / z):calcd for C 27 H 30 N6O5S[M+H] + :551.2071;found:551.2053.

[0060]

[0061] 2-(4-(2-(2-(dimethylamino)ethyl)-1,3-dioxy-2,3-dihydro-1H-benzo[generation]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ib); yellow solid; yield: 39.81%; 1 H NMR (400MHz, DMSO-d6) δ10.17(s,1H),8.48-8.39(m,3H),7.87-7.77(m,5H),7.36(d,J=8.2Hz,1H),7.2 7(s,2H),4.17(t,J=6.7Hz,2H),3.33(s,5H),2.89(t,J=4.5Hz,4H),2.64(t,J=6.7Hz,2H),2.31(s,6H). 13C NMR(101MHz,DMSO-d6)δ169.35,164.10,163.56,156.16,142.03,139.01,132.72,131.18,131.07,127.1 0,126.54,125.75,119.48,115.99,115.55,61.95,56.80,53.08,52.99,45.50,37.53.HR-MS(m / z):calcd for C 28 H 32 N6O5S[M+H] + :565.2228; found:565.2224.

[0062]

[0063] 2-(4-(2-Benzyl-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ic); yellow solid; yield 40.37%; 1 H NMR (400MHz, DMSO-d6) δ10.17(s,1H),8.54-8.37(m,3H),7.88-7.77(m,5H),7.36(d,J=8.1Hz,2H),7.34(d,J=1.2Hz,1H) ,7.32-7.26(m,4H),7.26-7.21(m,1H),5.24(s,2H),3.36(d,J=2.9Hz,2H),3.33(d,J=6.3Hz,4H),2.90(d,J=4.7Hz,4H). 13 C NMR(101MHz,DMSO-d6)δ169.36,164.09,163.55,156.33,142.03,139.00,138.02,132.97,131.40,131.27,129.70,128.83 ,127.94,127.47,127.10,126.58,125.78,122.90,119.48,115.79,115.61,61.95,53.08,52.99,43.18.HR-MS(m / z):calcd for C 31 H 29 N5O5S[M+H] + :584.1962; found:584.1969.

[0064]

[0065] 2-(4-(2-(2-(2-aminoethyl)amino)ethyl)-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Id); yellow solid; yield: 39.64%; 1 H NMR (400MHz, DMSO-d6) δ10.20(d,J=6.1Hz,1H),8.37(d,J=3.1Hz,3H),7.86(d,J=6.9Hz,2H),7.79(d,J=7.8Hz,3H),7.35(d,J= 8.1Hz,1H),7.28(s,2H),3.33(d,J=20.4Hz,12H),2.89(t,J=4.6Hz,4H),2.45(dd,J=12.5,3.5Hz,2H),1.65(d,J=12.5Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ169.36,163.89,155.83,142.04,132.61,131.04,130.70,127.09,126.52,1 23.52,119.48,116.52,115.53,61.94,53.08,53.02,52.95,29.16,26.60,25.69.HR-MS(m / z):calcd for C 28 H 34 N7O5S[M+H] + :580.2337; found:580.2321.

[0066]

[0067] 2-(4-(2-(2-morpholinylethyl)-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ie); yellow solid; yield: 38.62%; 1H NMR (400MHz, DMSO-d6) δ10.22(s,1H),8.44(d,J=2.8Hz,1H),8.43-8.36(m,2H),7.87(s,1H),7.86(d,J=2.1Hz,1H),7.80(s,2H),7.78(s,1H) ,7.34(d,J=8.2Hz,1H),7.29(s,2H),4.16(t,J=7.0Hz,2H),3.53(t,J=4.5Hz,5H),3.32(s,5H),2.90(d,J=4.5Hz,5H),2.46(d,J=4.6Hz,5H). 13 C NMR(101MHz,DMSO-d6)δ174.11,168.73,168.20,160.85,146.80,143.73,137.49,135.83,134.31,131.85 ,131.27,130.48,127.70,124.22,120.69,120.30,71.44,66.69,60.86,58.63,41.81.HR-MS(m / z):calcd for C 30 H 34 N6O6S[M+H] + :607.2333; found:607.2330.

[0068]

[0069] 2-(4-(2-(3-morpholinopropyl)-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (If); yellow solid; yield: 33.89%; 1 H NMR (400MHz, DMSO-d6) δ10.27(s,1H),8.32-8.24(m,4H),7.86(s,2H),7.79(d,J=8.9Hz,3H),7.22(d,J=8.2Hz,2 H), 4.00 (d, J = 3.8Hz, 2H), 3.25 (t, J = 4.7Hz, 4H), 2.86 (s, 6H), 2.28 (dt, J = 18.7, 5.3Hz, 10H), 1.73-1.68 (m, 2H). 13C NMR(101MHz,DMSO-d6)δ169.35,163.41,155.87,142.09,139.00,132.41,130.86,130.71,129.42,127.10,126.2 9,125.60,122.95,119.47,116.01,115.33,66.55,61.91,56.39,53.60,53.09,52.95,24.56.HR-MS(m / z):calcd forC 31 H 36 N6O6S[M+H] + :621.2490; found:621.2489.

[0070]

[0071] 2-(4-(2-(4-aminophenethyl)-1,3-dioxy-2,3-dihydro-1H-benzo[generation]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (1 g); yellow solid; yield: 41.18%; 1 H NMR (400MHz, DMSO-d6) δ10.18(s,1H),8.42(s,3H),7.78(d,J=8.1Hz,6H),7.34(d,J=6. 9Hz, 4H), 7.28 (s, 2H), 5.24 (d, J = 2.8Hz, 2H), 3.35-3.30 (m, 8H), 2.89 (t, J = 4.7Hz, 4H). 13 C NMR(101MHz,DMSO-d6)δ169.36,164.10,163.56,156.33,142.04,139.01,138.02,132.97,131.40,131.28,129 .69,128.83,127.94,127.47,127.10,125.80,122.91,119.49,61.94,53.08,52.99,43.18.HR-MS(m / z):calcd for C 31 H 36 N6O6S[M+H] + :613.2228; found:613.2206.

[0072]

[0073] 2-(4-(2-Hydroxyethyl)-1,3-dioxy-2,3-dihydro-1H-benzo[geno]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ih); yellow solid; yield: 35.58%; 1 H NMR (400MHz, DMSO-d6) δ10.20 (s, 1H), 8.46-8.34 (m, 3H), 7.89-7.76 (m, 5H), 7.31 (d, J = 25.3Hz, 3H), 4 .81(t,J=6.0Hz,1H),4.12(s,2H),3.60(d,J=6.3Hz,2H),3.30(t,J=4.9Hz,4H),2.88(t,J=4.7Hz,4H). 13 C NMR(101MHz,DMSO-d6)δ168.92,163.68,163.15,155.54,141.60,138.54,132.10,130.57,130.41,129.15,12 6.64,126.02,125.27,122.68,119.03,115.72,115.04,61.49,57.86,52.64,52.53,41.64.HR-MS(m / z):calcd for C 25 H 28 N5O6S[M+H] + :538.1755; found:538.1741.

[0074]

[0075] 2-(4-(2-(4-chlorobenzyl)-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ii); yellow solid; yield: 34.53%; 1 H NMR (400MHz, DMSO-d6) δ10.18 (s, 1H), 8.48-8.39 (m, 3H), 7.87 (d, J = 9.0Hz, 2H), 7.82-7.78 (m, 3H), 7.38-7.32(m,5H),7.29(s,2H),5.21(s,2H),3.37(s,2H),3.35-3.31(m,4H),2.90(t,J=4.7Hz,4H). 13CNMR(101MHz,DMSO-d6)δ169.35,164.06,163.49,156.35,142.03,139.00,137.02,132.96,132.08,131.39,131.29 ,129.97,128.78,127.11,126.53,122.81,119.49,115.68,115.56,61.95,53.07,52.97,42.61.HR-MS(m / z):calcd for C 31 H 28 ClN5O5S[M+H] + :618.1577; found:618.1567.

[0076]

[0077] 2-(4-(2-butyl-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ij); yellow solid; yield: 45.10%; 1 H NMR (400MHz, DMSO-d6) δ10.16(s,1H),8.50-8.40(m,3H),7.86-7.76(m,5H),7.37(d,J=8.2Hz,1H),7.27(s,2H),4.04(t,J=7.4Hz ,2H),3.35(s,4H),3.31(s,2H),2.89(t,J=4.6Hz,4H),1.60(tt,J=7.9,6.4Hz,2H),1.34(q,J=7.5Hz,2H),0.92(t,J=7.3Hz,3H). 13 C NMR (126MHz, DMSO-d6) δ169.39,164.05,163.53,156.12,142.07,139.03,132.70,131.16,129.62,127.13,12 6.58,125.82,123.09,119.52,116.10,115.61,62.00,53.13,53.02,30.23,20.32,14.25.HR-MS(m / z):calcd for C 28 H 31 N5O5S[M+H] + :550.2119;found:550.2121.

[0078]

[0079] 2-(4-(1,3-dioxy-2-(phenylamino)-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl(piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ik); yellow solid; yield: 31.76%; 1 H NMR (400MHz, DMSO-d6) δ10.18(s,1H),8.60(s,1H),8.54(s,1H),8.52(s,1H),7.86(d,J=1.8Hz,1H),7.85(d,J=2.1Hz,2H),7.78(d,J=8.8Hz,2H), 7.41(d,J=8.2Hz,1H),7.28(s,2H),7.14(dd,J=8.5,7.3Hz,2H),6.79-6. 75(m,1H),6.73(d,J=1.2Hz,1H),6.71(s,1H),3.37(s,6H),2.91(s,4H). 13 C NMR (101MHz, DMSO-d6) δ169.36,163.51,162.98,156.57,147.93,142.04,139.00,133.25,131.72,131.56,129. 62,129.31,127.10,126.70,123.29,119.78,119.49,116.08,115.75,112.93,61.95,53.07.HR-MS(m / z):calcd for C 30 H 28 N5O5S[M+H] + :585.1915;found:585.1915.

[0080]

[0081] 2-(4-(1,3-dioxy-2-(2-(piperazin-1-yl)ethyl)-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (I1); yellow solid; yield: 30.09%; 1H NMR (500MHz, DMSO-d6) δ10.19(s,1H),8.49(dd,J=11.7,7.9Hz,2H),8.44(d,J=8.0Hz,1H),7.80(s,3H),7.39(d, J=8.1Hz,1H),7.29(d,J=6.0Hz,4H),4.19(d,J=7.2Hz,2H),3.35(s,4H),3.16(s,2H),2.91(s,5H),2.60(s,9H). 13 C NMR (126MHz, DMSO-d6) δ169.40,164.08,163.55,156.20,142.06,141.99,139.03,138.98,132.78,131.23,129 .66,127.13,127.11,125.82,123.06,119.53,119.47,116.03,115.65,61.98,53.14,53.02.HR-MS(m / z):calcd forC 30 H 35 N7O5S[M+H] + :607.2527; found:607.2512.

[0082]

[0083] 2-(4-(1,3-dioxy-2-(2-pyridinylamino)-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Im); yellow solid; yield: 37.71%; 1 H NMR (400MHz, DMSO-d6) δ10.18(s,1H),8.53-8.50(m,2H),8.44(d,J=8.1Hz,1H),7.95-7.92(m,1H),7.88-7.84(m,3H),7.80-7.77(m,2H),7.61-7 .56(m,1H),7.41(d,J=8.2Hz,1H),7.28(s,2H),6.83(dd,J=8.5,1.2Hz, 1H),6.76-6.72(m,1H),3.37(s,4H),3.35(s,2H),2.91(t,J=4.7Hz,4H). 13C NMR(101MHz,DMSO-d6)δ169.35,163.37,162.86,157.83,156.51,147.87,142.03,139.00,137.93,133.18,131.66,131 .52,129.62,127.10,126.69,125.97,123.31,119.50,116.13,115.73,108.37,61.95,53.08,53.00.HR-MS(m / z):calcd for C 29 H 27 N7O5S[M+H] + :586.1867; found:586.1853.

[0084]

[0085] 2-(4-(1,3-dioxy-2-(cyclohexyl)-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (In); yellow solid; yield: 28.75%; 1 H NMR (400MHz, DMSO-d6) δ10.58 (s, 1H), 8.87-8.78 (m, 3H), 8.27 (d, J = 9.0Hz, 2H), 8.23-8.16 (m, 4H), 7.69 (s, 2H), 4.45-4. 39(m,2H),3.80(s,1H),3.74(d,J=5.0Hz,4H),3.31(t,J=4.7Hz,4H),2.06-1.98(m,2H),1.71-1.62(m,6H),1.25(s,2H). 13 C NMR(101MHz,DMSO-d6)δ169.77,156.47,142.45,139.43,133.04,131.50,129.97,127.52,126.91,126.17,123.4 3,119.91,116.45,115.95,62.38,53.52,53.40,32.11,28.38,27.41,22.95,20.70,14.82.HR-MS(m / z):calcdfor C 30 H 33 N5O5S[M+H] + :576.2275; found:576.2265.

[0086]

[0087] 2-(4-(2-(2-(diethylamino)ethyl)-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Io); yellow solid; yield: 29.78%; 1 H NMR (400MHz, DMSO-d6) δ10.17(s,1H),8.47-8.35(m,3H),7.86(d,J=8.7Hz,2H),7.79(d,J=9.0Hz,3H),7.34(t,J=7.8Hz,1H),7.28(s,2H),4.0 8(dt,J=11.0,5.3Hz,2H),3.36(s,2H),3.31(d,J=6.4Hz,4H),2.89(s,4H),2.62(d,J=7.7Hz,2H),2.53(d,J=9.8Hz,4H),0.96(t,J=7.0Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ169.34,163.97,163.45,142.04,139.01,132.59,131.05,130.93,129.53,1 27.10,119.48,116.03,115.54,61.97,53.10,52.97,49.80,47.47,37.85,12.60.HR-MS(m / z):calcd for C 31 H 39 N5O5S[M+H] + :593.2545; found:593.2535.

[0088]

[0089] 2-(4-(2-(4-bromobenzyl)-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ip); yellow solid; yield: 31.67%; 1 H NMR(400MHz,DMSO-d6)δ10.17(s,1H),8.52-8.37(m,4H),7.81(d,J=19.2Hz,6H),7 .38(dd,J=8.2,5.6Hz,1H),7.34-7.26(m,4H),3.35(d,J=6.3Hz,8H),2.89(s,4H). 13C NMR(101MHz,DMSO-d6)δ131.72,130.28,127.10,125.81,119.48,28.89,26.96,22.52.HR-MS(m / z):calcd forC 31 H 28 BrN5O5S[M+H] + :664.1047; found:664.1052.

[0090]

[0091] 2-(4-(2-heptyl-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (1q); yellow solid; yield: 43.53%; 1 H NMR (400MHz, DMSO-d6) δ10.17 (s, 1H), 8.47 (d, J = 1.2Hz, 2H), 7.84 (s, 4H), 7.79 (s ,2H),7.40(s,2H),3.35(s,8H),2.90(s,4H),2.89(d,J=5.1Hz,10H),0.00(s,3H). 13 C NMR (126MHz, DMSO-d6) δ169.40,142.06,139.03,131.76,130.31,127.13,125.85,119. 52,61.99,53.12,53.03,31.68,28.93,28.04,27.00,22.56,14.46.HR-MS(m / z):calcd for C 31 H 37 N5O5S[M+H] + :592.2594; found:592.2560.

[0092]

[0093] 2-(4-(2-(4-methylbenzyl)-1,3-dioxy-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)-N-(4-sulfamoylphenyl)acetamide (Ir); yellow solid; yield: 43.51%; 1H NMR (400MHz, DMSO-d6) δ8.50(ddd,J=11.8,7.9,1.2Hz,2H),8.40(d,J=8.1Hz,1H),8.15(s,1H),7.82(dd,J=8.5,7.3Hz,1H),7.34( d,J=8.1Hz,1H),7.26-7.22(m,2H),7.09(d,J=7.9Hz,2H),5.18(s,2H),3.72(s,4H),3.36(s,2H),3.26-3.15(m,4H),2.24(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.00,163.46,161.60,155.86,136.60,134.98,132.79,131.41,131.13,129.53,12 9.33,128.05,126.79,125.92,122.89,116.40,116.18,53.61,52.80,45.37,42.92,21.11.HR-MS(m / z):calcd for C 32 H 31 N5O5S[M+H] + :598.2119; found:598.2107.

[0094] Example 2: Preparation of Compounds Ib, Ie, Io, and Ip

[0095] Compound Ib: Example 1 was repeated, except that dichloromethane was used instead of DMF. The reaction was carried out at 39°C (reaction was allowed to complete in approximately 10 hours). A yellow solid was obtained with a yield of 39.81%. Characterization by H NMR, C NMR, and MS confirmed it to be Compound Ib.

[0096] Compound Ie: Example 1 was repeated, except that the reaction was carried out entirely at room temperature (the reaction took approximately 1 day to complete). A yellow solid was obtained with a yield of 38.62%. Characterization by H NMR, C NMR, and MS confirmed it to be Compound Ie.

[0097] Compound Io: Example 1 was repeated, except that acetone was used instead of DMF. The reaction was carried out at 55°C (reaction was completed in approximately 12 hours). A yellow solid was obtained in a yield of 29.78%. Characterization by H NMR, C NMR, and MS confirmed it to be Compound Io.

[0098] Compound Ip: Example 1 was repeated, except that anhydrous ethanol was used instead of DMF. The reaction was carried out at 75°C (reaction was completed in approximately 14 hours). A yellow solid was obtained with a yield of 31.67%. Characterization by H NMR, C NMR, and MS confirmed it to be Compound Ip.

[0099] Experimental Example 1: In vitro antitumor activity test

[0100] The CA inhibitor SLC-0111 and mitonadine were used as positive drugs, and the MTT method was used to determine the anti-proliferative activity of the target compound of the present invention in MCF-7 (human breast cancer cells), MDA-MB-231 (human breast cancer cells), HCT-116 (human colon cancer cells), SW480 (human colon cancer cells), and LO2 (human normal liver cells).

[0101] Take cells in good growth state and use 4×10 4 Cells were seeded into 96-well plates at 40 μM per well and incubated in a 37°C incubator for 24 hours. After that, 20 μL of the test compound was added (starting at 40 μM, serially diluted by 2-fold dilutions, with four replicates per well) and incubated in a cell culture incubator for 48 hours. 10 μL of 5 mg / mL MTT working solution was added to the 96-well plate and incubated in the incubator for 4 hours. The supernatant was carefully discarded, and 150 μL of DMSO was added to dissolve the reduced formazan purple crystals. The absorbance of each well was measured using a microplate reader. The cell proliferation inhibition rate was calculated for each dosing well, and the results are shown in Table 1 below.

[0102] Table 1. Inhibitory activity of the compounds of the present invention against different cell lines

[0103]

[0104] The above in vitro anti-tumor cell proliferation activity screening results showed that only Ib and Io had moderate inhibitory activity in HCT-116 cell line, IC 50 The values ​​were: 16.31±4.46μM, 29.33±8.51μM; in SW480 cell line, Ib, If, Io, and Ip had moderate inhibitory activity, IC 50 The values ​​were: 21.67±2.89μM, 17.59±1.71μM, 32.57±6.27μM, 24.98±6.79μM; Ie, If, Ij, Io, and Ip had moderate inhibitory activity in MDA-MB-231 cells, IC 50 The values ​​were 34.95±3.81μM, 35.60±2.66μM, 30.45±2.75μM, 14.63±7.63μM, and 20.73±0.028μM, respectively; Ib, Ij, Io, and Ip had moderate inhibitory activity in MCF-7, with IC 50The values ​​were 16.30±2.10μM, 30.11±8.54μM, 13.23±0.45μM, and 34.32±3.41μM, respectively. The compounds of the present invention have low toxicity to normal human cells, and most of the compounds have stronger inhibitory activity than SLC-0111.

[0105] The above in vitro antitumor activity test shows that the novel naphthalene imide compound of the present invention can be used for the preparation of antitumor drugs.

[0106] Experimental Example 2: CA II and CA IX enzyme activity test

[0107] Use SLC-0111 as a positive control to test CAII and CAIX enzyme activities. Follow the steps below:

[0108] Add 18 μL of CA solution (CA II concentration of 3.33 ng / μL, CAIX concentration of 11.1 ng / μL) to each well of a 96-well plate, add 2 μL of the drug to be tested (100 μM is the initial concentration of the drug to be tested, and the test drug is diluted in a 3-fold gradient, and 5 replicate wells are set for each concentration). After adding the drug, place it in a constant temperature incubator at 25°C and culture for 15 minutes; after taking it out, add 20 μL of 1 mM 4-NPA solution, and place the plate for detecting CA II affinity in a constant temperature incubator at 25°C and culture for 60 minutes (place the plate for detecting CA IX in a constant temperature incubator at 25°C and culture for 90 minutes), and then measure the absorbance at 405 nm using a microplate reader.

[0109] The physiologically relevant CA II and CA IX enzyme inhibitory activities were assessed by the esterase method, and the results are shown in Table 2 .

[0110] Table 2. Inhibitory activity of some compounds of the present invention against CA II and CA IX

[0111]

[0112] As shown in Table 2, SLC-0111 (IC 50 =0.46±0.0072μM, SI(CA II / IX)=17.22). The half inhibitory concentration (IC 50 ) was 0.0057±0.0047 μM, 100-fold better than SLC-0111, with selectivity SI (CA II / IX) = 75.40.

[0113] Experimental Example 3: Cell proliferation experiment under hypoxic environment

[0114] SLC-0111 was used as a positive control to simulate hypoxia in the cell (HCT-116, MDA-MB-231, MCF-7) activity test, as follows:

[0115] Taking MDA-MB-231 cells as an example: MDA-MB-231 cells were cultured at a rate of 4×10 4 / well were seeded into 96-well plates and placed in a 37°C incubator for 24 hours. The culture medium was removed and a fresh culture medium containing 100 μM CoCl2 was added and cultured in a 37°C incubator for 24 hours. The CoCl2 exposure was then maintained for 48 hours. After that, 20 μL of the test compound was added (the starting concentration was 40 μM, 2-fold dilution, and 5 replicates were set for each concentration), and the cells were placed in a cell culture incubator for 24 hours at 37°C. Finally, 10 μL of 5 mg / mL MTT working solution was added and incubated in an incubator for 4 hours. The supernatant was carefully discarded and 150 μL of DMSO was added to dissolve the reduced formazan purple crystals. The absorbance of each well was measured using a microplate reader.

[0116] The experimental results are shown in IC 50 The IC values ​​of compounds Ib, Ie, If, Ij, Io, and Ip under hypoxic conditions were as shown in Table 3 below, where SLC-0111 (>40 μM) was used as a positive reference. 50 The compounds showed higher cytotoxicity against tumor cells HCT-116, MDA-MB-231, and MCF-7 under hypoxic conditions, indicating that the compounds further inhibited cell activity by inhibiting the activity of CA IX.

[0117] Table 3 Antiproliferative activity of some compounds of the present invention on MDA-MB-231 and other cell lines under normal oxygen and hypoxic conditions

[0118]

[0119] Experimental Example 4: Reactive Oxygen Species (ROS) Experiment

[0120] The experimental process of compound Io reactive oxygen species is as follows:

[0121] MDA-MB-231 cells with good growth status were seeded in 6-well plates, with approximately 5×10 cells per well. 5 Cells were cultured in a 37°C incubator until ready to use. When the cell density reached 60-70%, various concentrations of compound Io were added. After incubation for 24 hours, the cell culture medium was removed and the cells were washed with PBS. 10 μM DCFH-DA was added to each well and the cells were cultured in a dark, 37°C incubator for 20-60 minutes. Afterwards, the cells were washed three times with serum-free medium and observed and photographed using an inverted fluorescence microscope.

[0122] like Figure 1 As shown, when the stained cells were examined under an inverted fluorescence microscope, no obvious green fluorescence was observed in the control group MDA-MB-231 cells. However, after the MDA-MB-231 cells were treated with different concentrations of compound Io for 24 hours, the brightness of the green fluorescence increased significantly with the increase in drug concentration, confirming that compound Io can cause overexpression of reactive oxygen species in MDA-MB-231 cells in a concentration-dependent manner, thereby directly leading to cell apoptosis.

[0123] Experimental Example 5: JC-1 staining experiment

[0124] The experimental procedure for compound Io JC-1 staining is as follows:

[0125] MDA-MB-231 cells with good growth status were seeded into 6-well plates, with about 5×10 cells per well. 5 Cells were cultured in a constant temperature incubator at 37°C until use. After the cells were completely attached and the density reached 60-70%, fresh culture medium containing different concentrations of compound Io or vector was replaced and cultured in the incubator for 24 hours. The culture medium was removed, washed twice with PBS buffer, digested with trypsin, and the cells were collected in a 15 mL centrifuge tube. After centrifugation, the supernatant was discarded and the cells were washed twice with serum-free culture medium. 500 μL JC-1 staining working solution was added and incubated in the dark for 20 minutes. After the incubation, centrifugation was completed, the supernatant was discarded, and the cells were washed twice with JC-1 staining buffer. 500 μL serum-free culture medium was added to suspend the cells and observed and photographed using a fluorescence inverted microscope.

[0126] like Figure 2 As shown, when the stained cells were observed under a fluorescence microscope, the control group MDA-MB-231 cells exhibited strong red fluorescence but no significant strong green fluorescence. However, after 24 hours of treatment with different compound Io, the red fluorescence of the MDA-MB-231 cells gradually decreased and the green fluorescence gradually increased due to increasing drug concentrations. In other words, the cell fluorescence gradually transitioned from red to green. Simultaneously, the mitochondrial membrane potential of the cells gradually decreased, indicating that compound Io can cause a concentration-dependent decrease in the mitochondrial membrane potential of MDA-MB-231 cells, thereby causing cell apoptosis.

[0127] Experimental Example 6: Compound Io Cell Apoptosis Test

[0128] The experimental process of compound Io cell apoptosis test is as follows:

[0129] MDA-MB-231 cells were cultured at 5 × 10 5Cells were seeded into 6-well plates at a density of 100 cells / well and incubated overnight to a density of 60-70%. The culture medium was replaced with culture medium containing different concentrations of Io and incubated in an incubator for 24 hours. Following digestion and PBS washing, the cells were transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm. 200 μL of buffer containing 5 μL of Annexin V-FITC was added and incubated at 37°C in the dark for 20 minutes. Subsequently, 300 μL of buffer and 5 μL of PI stain were added, mixed thoroughly, and the cells were transferred to a 1.5 mL centrifuge tube. Data were analyzed and collected using a flow cytometer.

[0130] like Figure 3 Flow cytometry apoptosis assays revealed that compound Io's apoptosis profiles increased in the Q2 and Q3 regions with increasing drug concentration compared to the control group, demonstrating that compound Io was able to induce apoptosis or necrosis in MDA-MB-231 cells. After 24 hours of treatment with compound Io at concentrations of 0, 5, 10, and 20 μM, the survival rates of MDA-MB-231 cells were 94.5%, 89.0%, 76.1%, and 66.1%, respectively, and the total apoptosis rates were 5.51%, 10.86%, 23.66%, and 33.40%, respectively. These results demonstrate that the compound significantly induced apoptosis in MDA-MB-231 cells in a concentration-dependent manner.

[0131] Experimental Example 7: Cell cycle test of compound Io

[0132] The experimental process of the cell cycle test of compound Io is as follows:

[0133] MDA-MB-231 cells were cultured at 5 × 10 5 Cells were inoculated into 6-well plates at a density of 60-70% per well and incubated overnight until the density reached 60-70%. The culture medium was replaced with a culture medium containing different concentrations of Io and incubated in an incubator for 24 hours. The cells were then digested and washed with PBS and transferred to a 15 mL centrifuge tube. The tubes were centrifuged at 1000 r / min, the supernatant was discarded, and 2 mL of 70% ice ethanol was added dropwise to solidify the cells in a vortex mixer. The cells were then placed in a refrigerator and stored at -20°C. When loading the tube, the solidified cells were first taken out of the refrigerator at -20°C and centrifuged at 2000 r / min for 10 minutes. After discarding the supernatant, 2 mL of ice PBS was added to resuspend the cells and centrifuged at 2000 r / min for 5 minutes. This operation was repeated 3 times. Discard the supernatant and resuspend the cells in 0.5 mL of 100 μg / mL RNaseA (1×) solution. Place the cells in a 37°C water bath for 30 minutes. Then, add 25 μL of 1 mg / mL PI solution and incubate the cells in the dark for 30 minutes. Finally, analyze and collect data using a flow cytometer.

[0134] like Figure 4 As shown in the results of cell cycle arrest experiments, after 48 hours of treatment with compound Io at different concentrations, compared to the control group, the percentage of cells in the G1 phase increased significantly, ranging from 51% to 73%, with an increase in concentration. The percentage in the S phase decreased, ranging from 32.96% to 13.66%, and the percentage in the G2 phase remained unchanged, ranging from 11% to 15.97%. The most significant changes in the percentages of cells in the G1 and S phases were observed at different drug concentrations, with the G1 phase percentages at 51.07%, 63.81%, 69.13%, and 73.66%, respectively, and the S phase percentages at 32.96%, 24.27%, 19.87%, and 13.66%, respectively, decreasing with increasing concentration. Therefore, compound Io primarily induces cell death by arresting the G1 / S phase of the cell cycle.

[0135] Experimental Example 8: Cell scratch test with compound Io

[0136] The process of the cell scratch assay with compound Io is as follows:

[0137] MDA-MB-231 cells were seeded in 6-well cell culture plates and cultured overnight until the cells were confluent. The monolayer was scraped with a sterile micropipette tip and washed with incomplete culture medium to remove detached cells. After imaging, the cells were supplemented with fresh complete culture medium containing various concentrations of Compound Io and incubated for 24 hours. The cells were then fixed with 4% paraformaldehyde and photographed using a phase-contrast inverted microscope.

[0138] Based on the fact that Io can effectively induce cell ferroptosis and apoptosis, we evaluated the effect of compound Io on cell migration and repair ability. After incubating MDA-MB-231 cells with different concentrations of compound Io for 24 hours, the migration potential of MDA-MB-231 cells was analyzed. The width of the scratch was measured before and after drug addition (such as Figure 5 ), compared with the cells incubated with Io, the scratch area of ​​the cells in the untreated control group was significantly narrowed, indicating that compound Io can effectively inhibit the migration of MDA-MB-231 cells.

[0139] Experimental Example 9: Tumor transplantation inhibition experiment in nude mice with compound Io

[0140] The experimental process of compound Io tumor inhibition in nude mice is as follows:

[0141] The nude mice were divided into four groups (a low-concentration group and a high-concentration group) with blank control and experimental groups, with 4 nude mice in each group. The breast cancer 4T1 cells were digested and centrifuged, collected into a centrifuge tube, and serum-free culture medium and matrix gel were added in the same proportion. The cells were injected subcutaneously into the left or right armpit of the nude mice. The cells were then injected into the nude mice until the tumor grew to 100-150 mm.3 1. Before administration, wipe and disinfect the injection site with 75% alcohol, turn the mouse head downward, insert the needle 1 cm to the left or right of the abdomen, pierce the epidermis first, insert the needle 0.5 cm at a 30-45° angle, slowly inject the drug into the abdominal cavity of the nude mouse, and then rotate it at a certain angle to slowly withdraw the needle. 21 days after administration, kill the nude mouse by dislocation. First, inject 0.2 mL of 1% sodium barbital solution and perfuse anesthetics into the abdominal cavity of the nude mouse. Then, use the thumb and index finger of your left hand to press the mouse head hard on the ground, grab the mouse tail with your right hand, and slowly pull it outward to dislocate the animal's head and neck, causing the animal to die. Then dissect the nude mouse, cut the abdomen horizontally, then cut it vertically, and remove tissues such as the heart, liver, spleen, lungs, kidneys, and brain, as well as tumors.

[0142] like Figure 6 As shown in Figure 2, we used a xenograft model of highly metastatic breast cancer 4T1 cells to study the in vivo antitumor activity of compound Io. 24 hours after inoculation, mice were randomly divided into three groups (5 = 10) and intravenously injected with compound Io (20 or 40 mg / kg) or vehicle every 3 days for 21 consecutive days. The results are shown in Figure 2. Figure 6 As shown, compared with the control group, compound Io can effectively inhibit tumor growth after 21 days of administration ( Figure 6 A), the tumor growth inhibition rates of compound Io at 20 mg / kg and 40 mg / kg were 42.7% and 47.9%, respectively ( Figure 6 B). After 21 days of administration, Io significantly reduced the tumor volume ( Figure 6 C in the figure). The body weight of mice in each group gradually increased, confirming the safety of Io ( Figure 6 D). The results of the anti-tumor activity test in mice showed that compound Io could effectively inhibit the growth of breast cancer 4T1 tumors.

[0143] Experimental Example 10: Compound Io liver and lung slice experiments

[0144] It is reported that more than 90% of deaths caused by breast cancer are attributed to complications related to metastasis. Studies have shown that the lungs and liver are the most common sites of breast cancer metastasis. Therefore, the applicant evaluated the infiltration of tumor cells in the lungs and liver by sectioning the lungs and liver and staining them with hematoxylin-eosin (H&E). The results are as follows Figure 7 As shown, 4T1 cells metastasized to the lungs and liver 21 days after implantation. Compound Io treatment significantly inhibited the lung and liver metastasis of 4T1 cells in a dose-dependent manner.

Claims

1. A 4-piperazineamide benzenesulfonamide-1,8-naphthalimide derivative having the structure represented by the following formula (I) or a pharmaceutically acceptable salt thereof: in, R represents 2-(dimethylamino)ethyl, and the compound represented by formula (I) is referred to as compound Ib; Or it represents 2-morpholinylethyl, in which case the compound represented by formula (I) is referred to as compound Ie; Or it represents 3-morpholinopropyl, in which case the compound represented by formula (I) is referred to as compound If; Or it represents a butyl group, in which case the compound represented by formula (I) is referred to as compound Ij; Alternatively, it represents 2-(diethylamino)ethyl, in which case the compound having the structure represented by formula (I) is referred to as compound Io; Alternatively, it represents 4-bromobenzyl, in which case the compound having the structure represented by formula (I) is referred to as compound Ip.

2. The method for preparing the 4-piperazineamide benzenesulfonamide-1,8-naphthalimide derivative according to claim 1, characterized in that: The compound represented by the following formula (II) and the compound represented by the following formula (III) are placed in an organic solvent and reacted with or without heating. The solvent is recovered from the obtained reaction mass to obtain a crude target compound; wherein R represents a 2-(dimethylamino)ethyl group, a 2-morpholinoethyl group, a 3-morpholinopropyl group, a butyl group, a 2-(diethylamino)ethyl group, or a 4-bromobenzyl group.

3. The preparation method according to claim 2, characterized in that: The organic solvent is one or a combination of two or more selected from N,N-dimethylformamide (DMF), dichloromethane, acetone and ethanol.

4. The preparation method according to claim 2, wherein The reaction is carried out at ≥35°C.

5. The preparation method according to claim 2 or 3, characterized in that: The method also includes the step of purifying the obtained crude target compound.

6. Use of the 4-piperazinamide benzenesulfonamide-1,8-naphthalimide derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for inhibiting carbonic anhydrase IX enzyme activity and / or overexpression.

7. Use of compound Ib or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating colon cancer cells HCT-116, colon cancer cells SW480 or breast cancer cells MCF-7; Use of compound 1e or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating breast cancer cells MDA-MB-231; Use of compound If or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating colon cancer cells SW480 or breast cancer cells MDA-MB-231; Use of compound Ij or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating colon cancer cells SW480, breast cancer cells MDA-MB-231 or breast cancer cells MCF-7; Use of compound Io or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating colon cancer cells HCT-116, colon cancer cells SW480, breast cancer cells MDA-MB-231, or breast cancer cells MCF-7; Use of compound Ip or a pharmaceutically acceptable salt thereof in preparing a drug for treating breast cancer cells MDA-MB-231 or breast cancer cells MCF-7.

8. A pharmaceutical composition comprising as an active ingredient a therapeutically effective dose of the 4-piperazinamide benzenesulfonamide-1,8-naphthalimide derivative according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Images