4-piperazinethio urea phenyl sulfonamides-1,8-naphthalimide derivatives, processes for their preparation and use

Abstract

The application discloses a series of 4-piperazine thiourea phenyl sulfonamide-1,8-naphthalimide derivatives with various structures and a preparation method of the derivatives. Test results show that some target compounds have good inhibitory activity on CA IX, and can be used for preparing drugs for inhibiting carbonic anhydrase IX enzyme activity and / or overexpression. Some target compounds have good antitumor activity on various tumor cell strains, and are expected to be used for preparing antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to 4-piperazine thiourea phenyl sulfonamide-1,8-naphthalimide derivatives, their preparation method and application, and belongs to the technical field of medicines. BACKGROUND

[0002] Carbonic anhydrase IX (CA IX) is a transmembrane, hypoxia-induced zinc metalloenzyme that can convert carbon dioxide into bicarbonate and protons. This acid-base regulation function is crucial for maintaining the alkaline intracellular pH that promotes the survival and growth of malignant cells and amplifying the extracellular acidosis that promotes their invasion and metastasis. Cancer cells expressing carbonic anhydrase IX usually represent the most clinically invasive component of heterogeneous tumors, making carbonic anhydrase IX a key biomarker and major therapeutic target. At present, carbonic anhydrase IX inhibitors mainly include sulfonamides and coumarins, among which sulfonamides are the most studied. The invention patent with publication number CN115697981A discloses the compound 2-(3-(2-methyl-6-(p-tolyl)pyridin-3-yl)ureido)benzenesulfonamide as shown below: It is also pointed out that the compound is a small molecule targeting carbonic anhydrase and an inhibitor of carbonic anhydrase activity and overexpression, and can be used as a medicament, especially for treating and / or preventing conditions associated with proliferative diseases, such as cancer.

[0003] Naphthalimides generally contain a coplanar, π-deficient aromatic system and a basic side chain, and are mainly used as DNA and topoisomerase targeting antitumor drugs. Studies have found that naphthalimide derivatives have potential antiproliferative activity against various tumors, such as breast cancer, liver cancer, glioma, melanoma, etc. Some effective antitumor drugs, such as Mitonafide, Amonafide, Ethonafide, Elinafide, etc., are in clinical phase II experiments. However, some factors hinder the further development of naphthalimide derivatives, such as CNS neurotoxicity and hematotoxicity; malignant stem cells may escape treatment with this drug; make tumor cells resistant to therapeutic drugs, and make current anticancer therapy ineffective, ultimately leading to failure. Therefore, the applicant attempts to modify the structure of naphthalimide in order to develop multifunctional, high-selectivity, carbonic anhydrase IX targeting antitumor drugs. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a series of 4-piperazine thiourea phenyl sulfonamide-1,8-naphthalimide derivatives with novel structures and good biological activity, as well as their preparation method and application.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0006] The 4-piperazine thiourea phenyl sulfonamide-1,8-naphthalimide derivative of the present application is a 4-piperazine thiourea phenyl sulfonamide-1,8-naphthalimide derivative having the structure shown in the following formula (I) or a pharmaceutically acceptable salt thereof:

[0007]

[0008] wherein:

[0009] R represents a hydrogen atom, a halogen atom, a hydroxyl group or a mercapto group, or is a C1-C8 alkyl group, an alkenyl group or an alkynyl group, or is a halogenated C1-C8 alkyl group, alkenyl group or alkynyl group, or is a hydroxyl-substituted C1-C8 alkyl group, alkenyl group or alkynyl group, or is an amino-substituted C1-C8 alkyl group, alkenyl group or alkynyl group, or is an amido-substituted C1-C8 alkyl group, alkenyl group or alkynyl group, or is a carboxyl-substituted C1-C8 alkyl group, alkenyl group or alkynyl group, or is a C1-C8 alkoxy group, or is a benzyl group or a substituted derivative thereof, or is a phenyl group or a substituted derivative thereof, or is a five- or six-membered heterocyclic methyl group or a substituted derivative thereof;

[0010] n represents an integer between 0 and 4.

[0011] In the general structure of the above-mentioned 4-piperazine thiourea phenyl sulfonamide-1,8-naphthalimide derivative, each substituent is preferably as follows:

[0012] R represents a C1-C8 alkyl group, or is a hydroxyl-substituted C1-C8 alkyl group, or is an amino-substituted C1-C8 alkyl group, or is a benzyl group or a substituted derivative thereof, or is a phenyl group or a substituted derivative thereof, or is a five- or six-membered heterocyclic methyl group or a substituted derivative thereof;

[0013] n represents 0 or 2.

[0014] Further, each substituent is preferably as follows: R represents a 2-morpholinoethyl group, a 3-morpholinopropyl group, a benzyl group, a 4-fluorobenzyl group, a 4-chlorobenzyl group, a 4-bromobenzyl group, a 4-methylbenzyl group, a 4-methoxybenzyl group, a 3,4,5-trimethoxybenzyl group, a 4-(trifluoromethyl)benzyl group, a 3,4-dihydroxyphenethyl group, a cyclohexyl group, a butyl group, an octyl group, a 2-(dimethylamino)ethyl group, a 2-(diethylamino)ethyl group or a 2-hydroxyethyl group; and n represents 0 or 2.

[0015] The preparation method of the 4-piperazine thiourea phenyl sulfonamide-1,8-naphthalimide derivative of the present application mainly comprises the following steps: placing a compound shown in the following formula (II) and a compound shown in the following formula (III) in an organic solvent to react, recovering the solvent from the obtained reaction material, and obtaining a target compound crude product.

[0016]

[0017] wherein:

[0018] R represents a hydrogen atom, a halogen atom, a hydroxyl group or a mercapto group, or is a C1-C8 alkyl group, a C1-C8 alkenyl group or a C1-C8 alkynyl group, or is a halogenated C1-C8 alkyl group, a halogenated C1-C8 alkenyl group or a halogenated C1-C8 alkynyl group, or is a hydroxyl-substituted C1-C8 alkyl group, a hydroxyl-substituted C1-C8 alkenyl group or a hydroxyl-substituted C1-C8 alkynyl group, or is an amino-substituted C1-C8 alkyl group, an amino-substituted C1-C8 alkenyl group or an amino-substituted C1-C8 alkynyl group, or is an amido-substituted C1-C8 alkyl group, an amido-substituted C1-C8 alkenyl group or an amido-substituted C1-C8 alkynyl group, or is a carboxyl-substituted C1-C8 alkyl group, a carboxyl-substituted C1-C8 alkenyl group or a carboxyl-substituted C1-C8 alkynyl group, or is a C1-C8 alkoxy group, or is a benzyl group or a substituted derivative thereof, or is a phenyl group or a substituted derivative thereof, or is a five- or six-membered heterocyclic ring methyl group or a substituted derivative thereof;

[0019] n represents an integer between 0 and 4.

[0020] In the above preparation method, the organic solvent is preferably one or a combination of two or more selected from the group consisting of acetonitrile, acetone, methanol and dichloromethane. The amount of the organic solvent used is preferably an amount that can dissolve the raw materials involved in the reaction, and in general, 15 to 20 mL of the organic solvent is used to dissolve all the raw materials involved in the reaction, based on 1 mmol of the compound represented by formula (III).

[0021] In the above preparation method, the reaction can be carried out with or without heating, and in particular, the reaction can be carried out at a temperature between room temperature and the boiling point of the organic solvent. When the reaction is carried out with heating, the reaction rate can be accelerated, and therefore, the reaction is further preferably carried out at a temperature of 35 to 60°C. The reaction is monitored by TLC until the reaction is completed. According to the experience of the applicant, when the reaction is carried out at room temperature to 35 to 60°C, the reaction time is preferably controlled to be 12 to 48 h.

[0022] The above method produces a crude product of the target compound, and therefore, the method further includes a step of purifying the crude product of the target compound. In particular, the crude product can be purified by using a conventional purification method to improve the purity of each target compound, such as by using a silica gel column chromatography. The eluent used in the 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 5:1, and further preferably 15:1 to 10:1.

[0023] In the preparation method, the amount of each raw material is stoichiometrically proportional. In actual operation, the molar ratio of the compound represented by formula (II) to the compound represented by formula (III) is usually 1:1 to 1:2. In the preparation method, the compound represented by formula (II) is a 1,8-naphthalimide piperazine derivative, which can be synthesized by referring 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 antitumor evaluation of new 1,8-naphthalimide derivatives targeting nuclear DNA, European Journal of Medicinal Chemistry, 2021, 210, 112951.) or by designing and synthesizing a synthesis route. In this application, the compound represented by formula (II) is preferably prepared according to the following synthesis route (in the synthesis route, BOC in compound S3 and compound S4 represents tert-butyloxy carbonyl):

[0024]

[0025] The specific method for preparing the compound represented by formula (II) includes the following steps:

[0026] 1) Put compound S1 (4-bromo-1,8-naphthalic anhydride) and compound S2 (tert-butyloxy carbonyl piperazine) into solvent A and react under heating. Cool the reaction mixture and collect the precipitate to obtain compound S3.

[0027] 2) Put compound S3 and a primary amine compound (R-NH2, R is selected as described above) into solvent A and react under heating. Cool the reaction mixture and collect the precipitate to obtain compound S4.

[0028] 3) Remove the protecting group (i.e., tert-butyloxy carbonyl) in compound S4 to expose N-H, thereby obtaining the compound represented by formula (II).

[0029] In the steps 1) and 2) of the above method for preparing the compound of formula (II), the solvent A involved can be an alcoholic solvent and / or an aprotic solvent, wherein the alcoholic solvent can be specifically one or more than two selected from methanol, ethanol, propanol and n-butanol; and the aprotic solvent can be specifically one or more than two selected from ethylene glycol methyl ether, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), toluene, carbon tetrachloride and acetone. The amount of the organic solvent used can be determined according to the need, and in general, 10-50 mL of the organic solvent is used to dissolve all the raw materials based on 1 mmol of 4-bromo-1,8-naphthalic anhydride. When the amount of the organic solvent added is large, the reaction is preferably cooled after recovering part of the solvent A (usually 40-50% of the added amount) after the reaction is completed.

[0030] In the steps 1) and 2) of the above method for preparing the compound of formula (II), the reaction is preferably carried out at a temperature between 50°C and the boiling point of the solvent A, and further preferably by refluxing.

[0031] In the step 3) of the above method for preparing the compound of formula (II), the protecting group in the compound S4 is removed by using a conventional method, such as stirring the compound S4 in hydrochloric acid dioxane or a mixed solvent of dichloromethane and trifluoroacetic acid in a volume ratio of 2:1 for a certain period of time to remove the tert-butyloxycarbonyl group on the compound S4. In order to further expose the N-H structure, excess dichloromethane (as the reaction solvent) and excess basic substances (such as inorganic bases (such as sodium hydroxide, potassium hydroxide, etc.) or organic bases (such as triethylamine, diethylamine, etc.)) can be added to the reaction system to neutralize the excess acid.

[0032] The compound obtained in the steps 1) to 3) of the above method for preparing the compound of formula (II) is a crude product, and the crude product is preferably purified according to the conventional method before the next step.

[0033] In the preparation method, the compound of formula (III) involved is 4-isothiocyanatobenzenesulfonamide or its derivative (such as 4-(2-isothiocyanatoethyl)benzenesulfonamide, etc.), wherein the 4-isothiocyanatobenzenesulfonamide can be directly purchased from the market, and the 4-isothiocyanatobenzenesulfonamide derivative (such as 4-(2-isothiocyanatoethyl)benzenesulfonamide, etc.) can be synthesized by referring to the existing literature (Zhang B, Liu Z, Xia S, et al. Design, synthesis and biological evaluation of sulfamoylphenyl-quinazoline derivatives as potential EGFR / CA IX dual inhibitors [J]. Eur J Med Chem, 2021, 216: 113300.).

[0034] The applicant found through experiments that some of the derivatives of the present application have good inhibitory activity on CA IX, therefore, the present application also includes the use of the above-mentioned 4-piperazine thiourea benzene sulfonamide-1,8-naphthalimide derivative or its pharmaceutically acceptable salt in the preparation of a drug for inhibiting the enzyme activity and / or overexpression of carbonic anhydrase IX. In addition, the applicant also found through experiments that some of the derivatives of the present application have good antitumor activity on various tumor cell lines, therefore, the present application also includes the use of the above-mentioned 4-piperazine thiourea benzene sulfonamide-1,8-naphthalimide derivative or its pharmaceutically acceptable salt in the preparation of a drug for treating tumors, further in the preparation of a drug for treating breast cancer, lung cancer or colon cancer.

[0035] Further, the present application also includes a pharmaceutical composition comprising a therapeutically effective dose of the above-mentioned 4-piperazine thiourea benzene sulfonamide-1,8-naphthalimide derivative or its pharmaceutically acceptable salt as an active ingredient, and a pharmaceutically acceptable carrier. The dosage form of the pharmaceutical composition can be the dosage form commonly used in the art, such as tablets, pills, granules, injections, etc. The administration amount of the drug of the present application can vary according to the administration route, the age and weight of the patient, the type and severity of the disease to be treated, etc., and the daily dose can be 0.01-10 mg / kg of body weight, preferably 0.1-5 mg / kg of body weight. It can be administered once or multiple times.

[0036] Compared with the prior art, the present application provides a series of 4-piperazine thiourea benzene sulfonamide-1,8-naphthalimide derivatives with novel structures and their preparation methods. The test results of the applicant show that some of the target compounds of the present application have good inhibitory activity on CA IX, and can be used in drugs for inhibiting the enzyme activity and / or overexpression of carbonic anhydrase IX; some of the target compounds have good antitumor activity on various tumor cell lines, and are expected to be used in the preparation of antitumor drugs. Attached Figure Description

[0037] Figure 1 Subcellular structures of MDA-MB-231 cells treated with different concentrations of compound I32 for 24 hours were observed using transmission electron microscopy.

[0038] Figure 2 The figure shows the effect of different concentrations of compound I32 on ROS levels in MDA-MB-231 cells observed using an inverted fluorescence microscope.

[0039] Figure 3 The image shows the effect of different concentrations of compound I32 on JC-1 staining of MDA-MB-231 cells under an inverted fluorescence microscope.

[0040] Figure 4 A graph showing the apoptosis rate of MDA-MB-231 cells induced by different concentrations of compound I32, as determined by Annexin V / PI double staining.

[0041] Figure 5 The graph shows the ability of different concentrations of compound I32 to inhibit the migration of MDA-MB-231 cells.

[0042] Figure 6 The in vivo antitumor effect of compound I32 in a 4T1 xenograft tumor model (mice were orally administered the carrier or compound I32 (15 or 30 mg / kg) every 3 days for 21 consecutive days); (A) a photograph of tumor tissue isolated after 21 consecutive days of compound I32 administration, (B) the weight of the tumor removed at the end of compound I32 treatment, (C) the change in tumor volume in different treatment groups measured every 3 days, and (D) the effect of compound I32 on the body weight of 4T1 xenograft mouse models.

[0043] Figure 7 H&E staining images of different concentrations of compound I32 inhibiting lung and liver metastasis (magnification, ×200). Detailed Implementation

[0044] To better explain the technical solution of the present invention, the present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0045] The compounds of formula (II) involved in the following examples were prepared according to the following synthetic route:

[0046]

[0047] R represents a hydrogen atom, a halogen atom, a hydroxyl group or a mercapto group, or is a C1-C8 alkyl, alkenyl or alkynyl group, or is a halogenated C1-C8 alkyl, alkenyl or alkynyl group, or is a hydroxyl-substituted C1-C8 alkyl, alkenyl or alkynyl group, or is an amino-substituted C1-C8 alkyl, alkenyl or alkynyl group, or is an amido-substituted C1-C8 alkyl, alkenyl or alkynyl group, or is a carboxyl-substituted C1-C8 alkyl, alkenyl or alkynyl group, or is a C1-C8 alkoxy group, or is a benzyl group or a substituted derivative thereof, or is a phenyl group or a substituted derivative thereof, or is a five- or six-membered heterocyclic methyl group or a substituted derivative thereof.

[0048] The specific preparation method comprises the following steps:

[0049] 1) Put compound S1 (4-bromo-1,8-naphthalic anhydride, 10 mmol) and compound S2 (t-butyloxycarbonylpiperazine, mmol) in a round-bottom flask, add ethylene glycol methyl ether (50 mL), reflux for 3 h (TLC monitoring reaction), after the reaction is completed, filter the reaction solution, recrystallize the filter cake with ethanol overnight, filter, and obtain compound S3 (yellow solid);

[0050] 2) Put compound S3 (1 mmol) and a primary amine compound (R-NH2, 1.2 mmol) in a round-bottom flask, add ethanol (50 mL), reflux for 3 h (TLC monitoring reaction), after the reaction is completed, purify the obtained material on a silica gel column (dichloromethane / methanol=10 / 1, volume ratio), and obtain compound S4 (yellow solid);

[0051] 3) Put compound S4 (1 mmol) in a round-bottom flask, add hydrochloric acid dioxane (10-20 mL) and stir for 1 h, spin dry, then add dichloromethane (20 mL) and triethylamine (10 mL) and react (TLC monitoring), after the reaction is completed, add an appropriate amount of water to extract, collect the organic phase and spin dry, and thus the compound shown in formula (II) is obtained.

[0052] The 4-(2-isothiocyanatoethyl)benzenesulfonamide involved in the following examples is prepared according to the following synthetic route:

[0053]

[0054] In a branch flask containing 50 mL THF (tetrahydrofuran), 10 mmol DCC (dicyclohexyl carbodiimide), 65 mmol CS2 and 10 mmol 4-(2-aminoethyl)benzenesulfonamide are added, and stirred at room temperature overnight (TLC monitoring reaction), after the reaction is completed, the obtained reaction material is filtered and washed with THF, the filtrate is collected, concentrated and purified on a silica gel column (dichloromethane / methanol=100 / 1, volume ratio), and thus 4-(2-isothiocyanatoethyl)benzenesulfonamide is obtained.

[0055] Example 1

[0056] The 4-piperazinethiourea phenyl sulfonamide-1,8-naphthoylimide derivatives of the present application were synthesized according to the following synthetic routes.

[0057]

[0058] I1: R = 2-morpholinoethyl, n = 0; I18: R = 2-morpholinoethyl, n = 2;

[0059] I2: R = 3-morpholinopropyl, n = 0; I19: R = 3-morpholinopropyl, n = 2;

[0060] I3: R = benzyl, n = 0; I20: R = benzyl, n = 2;

[0061] I4: R = 4-fluorobenzyl, n = 0; I21: R = 4-fluorobenzyl, n = 2;

[0062] I5: R = 4-chlorobenzyl, n = 0; I22: R = 4-chlorobenzyl, n = 2;

[0063] I6: R = 4-bromobenzyl, n = 0; I23: R = 4-bromobenzyl, n = 2;

[0064] I7: R = 4-methylbenzyl, n = 0; I24: R = 4-methylbenzyl, n = 2;

[0065] I8: R = 4-methoxybenzyl, n = 0; I25: R = 4-methoxybenzyl, n = 2;

[0066] I9: R = 3,4,5-trimethoxybenzyl, n = 0; I26: R = 3,4,5-trimethoxybenzyl, n = 2;

[0067] I10: R = 4-(trifluoromethyl)benzyl, n = 0; I27: R = 4-(trifluoromethyl)benzyl, n = 2;

[0068] I11: R = 3,4-dihydroxyphenethyl, n = 0; I28: R = 3,4-dihydroxyphenethyl, n = 2;

[0069] I12: R = cyclohexyl, n = 0; I29: R = cyclohexyl, n = 2;

[0070] I13: R = butyl, n = 0; I30: R = butyl, n = 2;

[0071] I14: R = octyl, n = 0; I31: R = octyl, n = 2;

[0072] I15: R = 2-(dimethylamino)ethyl, n = 0; I32: R = 2-(dimethylamino)ethyl, n = 2;

[0073] I16: R = 2-(diethylamino)ethyl, n = 0; I33: R = 2-(diethylamino)ethyl, n = 2;

[0074] I17: R = 2-hydroxyethyl, n = 0; I34: R = 2-hydroxyethyl, n = 2.

[0075] The specific preparation method is as follows: in a round-bottom flask, 1 mmol of the compound shown in formula (II), 1 mmol of the compound shown in formula (III) (4-isothiocyanatobenzenesulfonamide or 4-(2-isothiocyanatoethyl)benzenesulfonamide), and 20 mL of acetonitrile are added, and the reaction is stirred at 50°C for 12-18 h (TLC monitoring of the reaction). After the reaction is completed, the reaction material is sampled, purified by a silica gel column (dichloromethane / methanol = 10 / 1, by volume), and the target compound I is obtained. Different target products and their characterization are as follows:

[0076] 4-(2-(2-morpholinoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenyl)piperazine-1-carbomethylamide (I1): Yield 62%, as a yellow solid. Mp: 223.1-224.5°C. 1 H NMR (400 MHz, DMSO-d6): δ = 9.76 (s, 1H), 8.55 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 7.2 Hz, 1H), 8.42 (d, J = 8.4 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.29 (s, 2H), 4.24 (t, J = 4.0 Hz, 4H), 4.16 (t, J = 6.8 Hz, 2H), 3.52 (t, J = 4.0 Hz, 4H), 2.55 (t, J = 6.8 Hz, 2H), 2.45 ppm (t, J = 4.8 Hz, 4H). 13C NMR (100 MHz, DMSO-d6): δ = 181.64, 163.53, 162.99, 154.97, 144.26, 138.96, 132.15, 130.77, 130.69, 129.10, 126.23, 125.84, 125.34, 123.99, 122.51, 115.90, 115.42, 66.25, 55.66, 53.43, 52.15, 48.33, 36.64 ppm. HR-MS (m / z) (ESI): calcd for C 29 H 32 N6O5S2[M+H] + : 609.1948; found: 609.1941.

[0077] 4-(2-(3-morpholinopropyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenyl)piperazine-l-carboxamide (12): Yield 70%, as a yellow solid. Mp: 211.8-213.2 °C. 1 HNMR (400 MHz, DMSO-d6): δ = 9.79 (s, 1H), 8.52 (d, J = 8.4 Hz, 1H), 8.44 (d, J = 7.2 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.30 (s, 2H), 4.25 (t, J = 4.8 Hz, 4H), 4.06 (t, J = 7.2 Hz, 2H), 3.34 (t, J = 4.8 Hz 4H), 2.35 (t, J = 6.4 Hz, 4H), 2.29 (s, 4H), 1.83-1.71 ppm (m, 2H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.64, 163.53, 162.99, 154.97, 144.26, 138.96, 132.15, 130.77, 130.69, 129.10, 126.23, 125.84, 125.34, 123.99, 122.51, 115.90, 115.42, 66.25, 55.66, 53.43, 52.15, 48.33, 36.64 ppm. HR-MS (m / z) (ESI): calcd for C 30 H 34 N6O5S2[M+H] +: 623.2105; found: 623.2104.

[0078] 4-(2-(4-Fluorobenzyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenyl)piperazine-l-carboxamide (14): Yield 52%, as a yellow solid. Mp: 204.1-206.5 °C. 1 HNMR (400 MHz, DMSO-d6): δ = 9.77, (s, 1H), 8.54 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 6.8 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 7.84-7.74 (m, 3H), 7.54 (d, J = 8.8 Hz, 2H), 7.37-7.20 (m, 8H), 5.22 (s, 2H), 4.25 (s, 4H), 3.36 ppm (s, 4H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.64, 163.56, 163.01, 155.12, 144.20, 138.98, 137.52, 132.36, 130.97, 130.86, 129.15, 128.36, 127.51, 127.02, 126.20, 125.84, 125.29, 124.00, 122.36, 115.65, 115.38, 52.13, 48.32, 42.74 ppm. HR-MS (m / z) (ESI): calcd for C 30 H 27 N5O4S2[M+H] + : 586.1577; found: 586.1580.

[0079] 4-(2-(4-Fluorobenzyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenyl)piperazine-l-carboxamide (14): Yield 52%, as a yellow solid. Mp: 204.1-206.5 °C. 1HNMR (400 MHz, DMSO-d6): δ = 9.78 (s, 1H), 8.56 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 7.2 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.45 - 7.35 (m, 3H), 7.29 (s, 2H), 7.11 (t, J = 8.0 Hz, 2H), 5.20 (s, 2H), 4.25 ppm (s, 4H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.60, 163.62, 163.06, 162.47, 160.06, 155.23, 144.40, 138.91, 133.74, 132.44, 131.03, 130.97, 129.84, 129.76, 129.22, 126.25, 125.84, 125.34, 124.00, 122.40, 115.67, 115.43, 115.22, 115.01, 52.16, 48.31, 42.10 ppm. HR-MS (m / z) (ESI): calcd for C 30 H 26 FN5O4S2[M+H] + : 604.1483; found: 604.1471.

[0080] 4-(2-(4-Chlorobenzyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenyl)piperazine-l-carboxamide (15): Yield 77% as a yellow solid. Mp: 233.5-234.5 °C. 1 HNMR (400 MHz, DMSO-d6): δ = 9.78 (s, 1H), 8.56 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 7.2 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.45 - 7.35 (m, 3H), 7.29 (s, 2H), 7.11 (t, J = 8.0 Hz, 2H), 5.20 (s, 2H), 4.25 (t, J = 4.9 Hz, 4H), 3.37 ppm (t, J = 4.5 Hz, 4H). 13C NMR (100MHz, DMSO-d6): δ=181.64,163.62,163.06,155.25,144.20,138.98,136.57,132.46,131.62,131.06,131.00,129.49,1 29.26,128.34,126.27,125.84,125.35,123.99,122.40,115.67,115.45,52.14,48.32,42.19ppm.HR-MS(m / z)(ESI):calcdfor C 30 H 26 ClN5O4S2[M+H] + :620.1187; found:620.1168.

[0081] 4-(2-(4-bromobenzyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)-N-(4-aminosulfonylphenyl)piperazine-1-methylthioamide (I6): Yield 47%, as a yellow solid. Mp: 267.9-270.2℃. 1 HNMR (400MHz, DMSO-d6): δ = 9.76 (s, 1H), 8.58 (d, J = 8.4Hz, 1H), 8.50 (d, J = 7.2Hz ,1H),8.42(d,J=8.4Hz,1H),7.84(t,J=7.2Hz,1H),7.75(d,J=8.4Hz,2H),7.52( d,J=8.8Hz,2H),7.49(d,J=8.4Hz,2H),7.39(d,J=8.0Hz,1H),7.31(d,J=8.4Hz, 2H), 7.30 (s.2H), 5.20 (s, 2H), 4.25 (t, J = 4.4Hz, 4H), 3.36ppm (t, J = 4.8Hz, 4H). 13 C NMR (100MHz, DMSO-d6): δ=181.63,163.60,163.04,155.23,144.18,138.97,136.98,132.45,131.25,131.04,131.00,129.82 ,129.24,126.25,125.83,125.34,123.98,122.38,120.10,115.65,115.43,52.13,48.30,42.24ppm.HR-MS(m / z)(ESI):calcd for C 30 H 26 BrN5O4S2[M+H] +: 664.0682; found: 664.0672.

[0082] 4-(2-(4-methoxybenzyl)-l,3-dioxo-2,3-dihydro-lH- benzo[de]isoquinolin-6-yl)-N-(4-sulfamoylphenyl)piperazin-l- carboxymide (18): Yield 32%, as a yellow solid. Mp: 205.1-206.7 °C. 1 HNMR (400 MHz, DMSO-d6): δ = 9.76, (s, 1H), 8.55 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 6.8 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.30 (s, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 5.18 (s, 2H), 4.25 (t, J = 4.8 Hz, 4H), 3.40-3.35 (m, 4H), 2.23 ppm (s, 3H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.64, 163.55, 163.00, 155.11, 144.20, 138.99, 136.15, 134.54, 132.35, 130.96, 130.86, 129.14, 128.88, 127.60, 126.22, 125.84, 125.32, 124.00, 122.41, 115.71, 115.41, 52.12, 48.32, 42.47, 20.66 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 29 N5O4S2[M+H] + : 600.1734; found: 600.1729.

[0083] 4-(2-(4-methoxybenzyl)-l,3-dioxo-2,3-dihydro-lH- benzo[de]isoquinolin-6-yl)-N-(4-sulfamoylphenyl)piperazin-l- carboxymide (18): Yield 32%, as a yellow solid. Mp: 205.1-206.7 °C. 1H NMR (400 MHz, DMSO-d6): δ = 9.76 (s, 1H), 8.57 (d, J = 8.4 Hz, 1H), 8.50 (d, J = 7.2 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.30 (s, 2H), 6.85 (d, J = 8.8 Hz, 2H), 5.16 (s, 2H), 4.24 (s, 4H), 3.69 ppm (s, 3H). 13 C NMR (150 MHz, DMSO-d6): δ = 181.63, 163.60, 163.06, 158.38, 155.16, 144.21, 138.99, 132.40, 131.01, 130.92, 129.59, 129.29, 129.18, 126.29, 125.85, 125.36, 124.02, 122.49, 115.79, 115.47, 113.74, 55.06, 52.15, 48.33, 42.17 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 29 N5O5S2[M+H] + : 616.1683; found: 616.1667.

[0084] 4-(1,3-Dioxo-2-(3,4,5-trimethoxybenzyl)-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenyl)piperazine-1-carbothioamide (I9): Yield 76% as a yellow solid. Mp: 250.0-250.2 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 9.76, (s, 1H), 8.54 (t, J = 7.6 Hz, 1H), 8.50 (d, J = 7.6 Hz, 1H), 8.41 (t, J = 8.0 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.36 (t, J = 8.4 Hz, 1H), 7.30 (s, 2H), 6.68 (s, 2H), 5.15 (s, 2H), 4.25 (s, 4H), 3.71 (s, 6H), 3.60 ppm (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ = 181.65, 163.69, 163.13, 155.09, 152.76, 144.20, 138.99, 136.68, 133.29, 132.37, 130.99, 130.84, 129.20, 126.23, 125.85, 125.34, 124.00, 122.48, 115.80, 115.41, 105.30, 59.96, 55.86, 52.13, 48.33, 43.08 ppm. HR-MS (m / z) (ESI): calcd for C 33 H 33 N5O7S2[M+H] + : 676.1894; found: 676.1892.

[0085] 4-(l,3-dioxo-2-(4-(trifluoromethyl)benzyl)-2,3-dihydro-lH- benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenyl)piperazine-l-carboxamide (110): Yield 83%, as a yellow solid. Mp: 265.3-266.5 °C. 1 H NMR (600 MHz, DMSO-d6): δ = 9.77 (s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.51 (d, J = 6.6 Hz, 1H), 8.42 (d, J = 7.8 Hz, 1H), 7.85 (t, J = 8.4 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 7.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 1H), 7.31 (s, 2H), 5.31 (s, 2H), 4.25 (t, J = 4.8 Hz, 4H), 3.37 ppm (t, J = 4.8 Hz, 4H). 13C NMR (150 MHz, DMSO-d6): δ = 181.65, 163.69, 163.12, 155.32, 144.22, 142.36, 139.01, 132.52, 131.12, 131.08, 129.33, 128.11, 128.00, 127.79, 127.58, 127.00, 126.30, 125.87, 125.37, 125.34, 125.31, 125.29, 125.19, 124.02, 123.39, 122.39, 115.64, 115.46, 52.16, 48.32, 42.56 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 26 F3N5O4S2[M+Na] + : 676.1271; found: 676.1282.

[0086] 4-(2-(3,4-Dihydroxyphenethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenyl)piperazine-1-carbothioamide (III): Yield 45%, as a yellow solid. Mp: 191.4-192.5 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 9.77 (br s, 1H), 8.92-8.63 (m, 2H), 8.55 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 7.2 Hz, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.0 Hz 1H), 7.30 (s, 2H), 6.68 (d, J = 2.0 Hz, 1H), 6.63 (d, J = 7.6 Hz, 1H), 6.49 (dd, J = 8.0, 1.6 Hz, 1H), 4.29 (s, 4H), 4.14 (t, J = 8.0 Hz, 2H), 3.34 (s, 4H), 2.71 ppm (t, J = 7.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ = 181.64, 163.40, 162.87, 154.95, 145.20, 144.24, 143.73, 138.98, 132.12, 130.74, 130.67, 129.55, 129.10, 126.24, 125.85, 125.36, 124.02, 122.56, 119.27, 115.99, 115.97, 115.63, 115.45, 52.17, 48.36, 41.29, 33.05 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 29 N5O6S2[M+H] + : 632.1632; found: 632.1637.

[0087] 4-(2-cyclohexyl-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenyl)piperazine-1 -carbothioamide (I12): Yield 46%, as a yellow solid. Mp: 260.7-262.9 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 9.91, (s, 1 H), 8.59 (d, J = 8.4 Hz, 1 H), 8.51 (d, J = 7.2 Hz, 1 H), 8.42 (d, J = 8.0 Hz, 1 H), 7.85 (t, J = 8.0 Hz, 1 H), 7.75 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.0 Hz 1 H), 7.31 (s, 2H), 4.88 (t, J = 6.4 Hz, 1 H), 4.24 (s, 4H), 2.48-2.35 (m, 2H), 1.83 (d, J = 10.7 Hz, 2H), 1.70-1.58 (m, 2H), 1.41 -1.06 ppm (m, 4H). 13 C NMR (101 MHz, DMSO-d6): δ = 181.63, 163.89, 163.40, 154.67, 144.20, 138.96, 132.06, 130.66, 130.34, 129.09, 126.22, 125.83, 125.18, 123.97, 123.07, 116.48, 115.39, 52.59, 52.14, 48.33, 28.69, 26.14, 25.23 ppm. HR-MS (m / z) (ESI): calcd for C 29 H 31N5O4S2[M+H] + :578.1890; found: 578.1891.

[0088] 4-(2-Butyl-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenyl)piperazine-1 -carbothioamide (I13): Yield 46% as a yellow solid. Mp: 192.5-193.7 °C. 1 H NMR (400 MHz, DMSO-d6): d = 9.75, (s, 1 H), 8.56 (dd, J = 7.6, 5.2 Hz, 1 H), 8.51 - 8.45 (m, 1 H), 8.44 - 8.37 (m, 1 H), 7.87 - 7.79 (m, 1 H), 7.75 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.39 (dd, J = 7.7, 4.8 Hz 1 H), 7.30 (s, 2H), 4.24 (t, J = 4.6 Hz, 4H), 4.02 (t, J = 5.2 Hz, 2H), 1.65 - 1.54 (m, 2H), 1.39 - 1.28 (m, 2H), 0.92 ppm (t, J = 7.2 Hz, 3H). 13 C NMR (150 MHz, DMSO-d6): d = 181.64, 163.54, 163.02, 154.94, 144.21, 138.99, 132.13, 130.76, 130.65, 129.10, 126.25, 125.86, 125.37, 124.02, 122.59, 116.02, 115.45, 52.18, 48.35, 40.06, 29.74, 19.85, 13.78 ppm. HR-MS (m / z) (ESI): calcd for C 27 H 29 N5O4S2[M+H] + :552.1734; found: 552.1763.

[0089] 4-(2-Butyl-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenyl)piperazine-1 -carbothioamide (I13): Yield 46% as a yellow solid. Mp: 192.5-193.7 °C. 1H NMR (600 MHz, DMSO-d6): δ = 9.78, (s, 1 H), 8.53 (d, J = 9.6 Hz, 1 H), 8.46 (d, J = 7.2 Hz, 1 H), 8.38 (d, J = 7.8 Hz, 1 H), 7.81 (t, J = 7.8 Hz, 1 H), 7.75 (d, J = 9.0 Hz, 2 H), 7.53 (d, J = 9.0 Hz, 2 H), 7.36 (d, J = 7.8 Hz, 1 H), 7.30 (s, 2 H), 4.24 (s, 4 H), 3.99 (t, J = 7.2 Hz, 2 H), 3.34 (t, J = 3.6 Hz, 4 H), 1.62-1.57 (m, 2 H), 1.26-1.16 (m, 10 H), 0.83 ppm (t, J = 6.6 Hz, 3 H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.65, 163.46, 162.96, 154.88, 144.21, 138.97, 132.08, 130.71, 130.57, 129.04, 126.17, 125.83, 125.34, 123.98, 122.53, 115.96, 115.37, 52.16, 48.34, 31.23, 28.71, 28.59, 27.50, 26.53, 22.08, 13.94 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 37 N5O4S2[M+H] + : 608.2360; found: 608.2361.

[0090] 4-(2-(2-(dimethylamino)ethyl)-1,3-dioxo-2,3-dihydro-1 H- benzo[de]isoquinolin-6-yl)-N-(4-sulfamoylphenyl)piperazine-1 - carboxylic acid amide (115): Yield 62%, as a yellow solid. Mp: 232.2-234.5 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 9.90 (s, 1 H), 8.57 (d, J = 8.4 Hz, 1 H), 8.49 (d, J = 7.2 Hz, 1 H), 8.41 (d, J = 8.0 Hz, 1 H), 7.85 (t, J = 8.0 Hz, 1 H), 7.76 (d, J = 8.4 Hz, 2 H), 7.56 (d, J = 8.6 Hz, 2 H), 7.39 (d, J = 8.0 Hz, 1 H), 7.31 (s, 2 H), 4.30-4.20 (m, 6 H), 3.35 (t, J = 4.2 Hz, 4 H), 2.93 (s, 2 H), 2.52 ppm (s, 6 H).13 C NMR (150 MHz, DMSO-d6): δ = 181.63, 163.80, 163.24, 155.08, 144.28, 138.92, 132.22, 130.82, 130.79, 129.21, 126.24, 125.80, 125.29, 124.00, 122.60, 115.94, 115.38, 55.73, 52.21, 48.38, 44.18, 36.31 ppm. HR-MS (m / z) (ESI): calcd for C 27 H 30 N6O4S2[M+H] + : 567.1843; found: 567.1844.

[0091] 4-(2-(2-(Diethylamino)ethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenyl)piperazine-1-carboxylic acid amide (116): Yield 44% as a yellow solid. Mp: 214.9-215.3 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 9.94 (s, 1H), 8.58 (d, J = 8.4 Hz, 1H), 8.50 (d, J = 6.8 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 7.9 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.4 Hz 1H), 7.31 (s, 2H), 4.31 (s, 2H), 4.27 (s, 4H), 3.16 (s, 4H), 1.26 ppm (t, J = 6.8 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.64, 163.91, 163.32, 155.24, 144.27, 138.93, 132.30, 130.98, 130.89, 129.33, 126.24, 125.79, 125.28, 123.97, 122.59, 115.85, 115.36, 52.21, 48.35, 47.58, 46.59, 34.18, 8.45 ppm. HR-MS (m / z) (ESI): calcd for C 29 H 36 N6O4S2[M+H] + : 595.2156; found: 595.2147.

[0092] 4-(2-(2-hydroxyethyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenyl)piperazine-l-carboxamide (117): Yield 42%, as a yellow solid. Mp: 238.7-241.9 °C. 1 HNMR (400 MHz, DMSO-d6): δ = 9.77 (s, 1H), 8.56 (d, J = 8.0 Hz, 1H), 8.50 (d, J = 7.2 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 4.4 Hz, 1H), 7.30 (s, 2H), 4.81 (t, J = 6.0 Hz, 1H), 4.24 (t, J = 4.4 Hz, 4H), 4.14 (t, J = 6.4 Hz, 2H), 3.61 ppm (dt, J = 12.4, 6.4 Hz, 2H). 13 C NMR (150 MHz, DMSO-d6): δ = 181.64, 163.73, 163.20, 154.91, 144.23, 138.98, 132.09, 130.73, 130.61, 129.21, 126.26, 125.85, 125.38, 124.02, 122.77, 116.21, 115.45, 57.86, 52.20, 48.36, 41.70 ppm. HR-MS (m / z) (ESI): calcd for C 25 H 25 N5O5S2[M+H] + : 540.1370; found: 540.1364.

[0093] 4-(2-(2-morpholinoethyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenethyl)piperazine-l-carboxamide (118): Yield 65%, as a yellow solid. Mp: 239.7-240.9 °C. 1H NMR (400 MHz, DMSO-d6): δ = 8.54 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 6.4 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.01 (s, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.30 (s, 2H), 4.17 (t, J = 6.8 Hz, 2H), 4.09 (t, J = 5.2 Hz, 4H), 3.76 (dt, J = 11.2, 7.2 Hz, 2H), 3.52 (t, J = 4.4 Hz, 4H), 3.26 (t, J = 4.8 Hz, 4H), 2.99 (t, J = 8.0 Hz, 2H), 2.55 (t, J = 6.8 Hz, 2H), 2.46 ppm (t, J = 4.4 Hz, 4H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.74, 163.74, 163.20, 155.28, 143.93, 142.13, 132.38, 130.95, 130.88, 129.31, 129.25, 126.38, 125.92, 125.47, 122.62, 115.94, 115.53, 66.36, 55.78, 53.54, 52.26, 47.32, 46.57, 36.76, 34.58 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 36 N6O5S2[M+H] + : 637.2261; found: 637.2254.

[0094] 4-(2-(3-morpholinopropyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenethyl)piperazine-l-carboxamide (119): Yield 72% as a yellow solid. Mp: 189.7-194.7 °C. 1H NMR (400 MHz, DMSO-d6): δ = 8.51 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 6.8 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.04 (t, J = 5.2 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H), 7.31 (s, 2H), 4.17 - 3.98 (m, 6H), 3.76 (dt, J = 12.8, 6.3 Hz, 2H), 3.25 (t, J = 6.0 Hz, 4H), 2.99 (t, J = 7.2 Hz, 2H), 2.36 (t, J = 6.8 Hz, 2H), 2.29 (s, 4H), 1.81 - 1.73 ppm (m, 2H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.67, 163.66, 163.13, 155.03, 143.80, 142.07, 132.06, 130.66, 130.59, 129.17, 129.17, 126.19, 125.80, 125.34, 122.67, 116.04, 115.35, 66.11, 55.98, 53.19, 52.17, 47.23, 46.47, 38.17, 34.49, 24.09 ppm. HR-MS (m / z) (ESI): calcd for C 32 H 38 N6O5S2[M+H] + : 651.2418; found: 651.2418.

[0095] 4-(2-benzyl-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenethyl)piperazine-1 -carbothioamide (120): Yield 48%, as a yellow solid. Mp: 254.2-256.1 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.57 - 8.46 (m, 2H), 8.46 - 8.36 (m, 1H), 8.04 (s, 1H), 7.87 - 7.71 (m, 3H), 7.43 (d, J = 8.0 Hz, 2H), 7.38 - 7.14 (m, 8H), 5.23 (s, 2H), 4.10 (s, 4H), 3.76 (dt, J = 12.4, 7.6 Hz, 2H), 3.26 (s, 4H), 2.99 ppm (t, J = 6.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ = 181.62, 163.60, 163.04, 155.29, 143.79, 142.08, 137.55, 132.40, 130.98, 130.91, 129.17, 128.39, 127.51, 127.49, 127.04, 126.20, 125.80, 125.31, 122.37, 115.60, 115.37, 52.14, 47.18, 46.48, 42.74, 34.49 ppm. HR-MS (m / z) (ESI): calcd for C 32 H 31 N5O4S2[M+H] + : 614.1890; found: 614.1897.

[0096] 4-(2-(4-Fluorobenzyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenethyl)piperazine-1-carbothioamide (I21): Yield 59% as a yellow solid. Mp: 204.1-206.5 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.53 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 6.8 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.01 (t, J = 4.8 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.47-7.37 (m, 4H), 7.35 (d, J = 8.0 Hz, 1H), 7.31 (s, 2H), 7.11 (d, J = 8.4 Hz, 2H), 5.20 (s, 2H), 4.09 (t, J = 4.4 Hz, 4H), 3.76 (dt, J = 13.4, 6.3 Hz, 2H), 3.26 (t, J = 4.8 Hz, 4H), 2.99 ppm (t, J = 7.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ = 181.65, 163.59, 163.03, 162.45, 160.04, 155.32, 143.76, 142.07, 133.75, 133.72, 132.40, 130.98, 130.93, 129.82, 129.74, 129.19, 129.15, 126.19, 125.78, 125.31, 122.37, 115.58, 115.36, 115.20, 114.99, 52.11, 47.16, 46.45, 42.07, 34.47 ppm. HR-MS (m / z) (ESI): calcd for C 32 H 30 FN5O4S2[M+H] + : 646.1789; found: 632.1782.

[0097] 4-(2-(4-Chlorobenzyl)-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenethyl)piperazine-1 -carbothioamide (I22): Yield 69%, as a yellow solid. Mp: 176.7-178.2 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.55 (d, J = 8.4 Hz, 1 H), 8.49 (d, J = 7.2 Hz, 1 H), 8.41 (d, J = 8.0 Hz, 1 H), 8.02 (t, J = 5.6 Hz, 1 H), 7.83 (t, J = 7.2 Hz, 1 H), 7.77 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.39 - 7.33 (m, 5H), 7.31 (s, 2H), 5.21 (s, 2H), 4.09 (t, J = 4.4 Hz, 4H), 3.76 (dt, J = 12.8, 6.4 Hz, 2H), 3.26 (t, J = 4.8 Hz, 4H), 2.99 ppm (t, J = 7.2 Hz, 2H). 13C NMR (100MHz, DMSO-d6): δ=181.64,163.61,163.05,155.37,143.76,142.07,136.56,132.45,131.62,131.02,130.99,129.48,129.2 4,129.15,128.33,126.22,125.78,125.34,122.37,115.57,115.39,52.12,47.16,46.45,42.18,34.47ppm.HR-MS(m / z)(ESI):calcd forC 31 H 30 ClN5O4S2[M+H] + :648.1501; found:648.1478.

[0098] 4-(2-(4-bromobenzyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)-N-(4-aminosulfonylphenylethyl)piperazine-1-methylthioamide (I23): Yield 45%, as a yellow solid. MPa: 190.9-191.5℃. 1 H NMR (400MHz, DMSO-d6): δ = 8.55 (d, J = 8.4Hz, 1H), 8.49 (d, J = 6.8Hz, 1H), 8.41 (d, J = 8.4Hz, 1 H),8.02(t,J=5.2Hz,1H),7.83(t,J=8.0Hz,1H),7.77(d,J=8.4Hz,2H),7.49(d,J=8.4Hz,2 H),7.43(d,J=8.4Hz,2H),7.36(d,J=8.0Hz,1H),7.33-7.28(m,4H),5.19(s,2H),4.09(t,J =4.6Hz, 4H), 3.75 (dt, J = 14.0, 6.0Hz, 2H), 3.27 (t, J = 4.8Hz, 4H), 2.99ppm (t, J = 7.2Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ = 181.63, 163.61, 163.05, 155.37, 143.76, 142.07, 136.99, 132.45, 131.25, 131.02, 131.00, 129.82, 129.24, 129.15, 126.23, 125.78, 125.34, 122.37, 120.10, 115.57, 115.39, 52.12, 47.16, 46.45, 42.24, 34.47 ppm. HR-MS (m / z) (ESI): calcd for C 32 H 30 BrN5O4S2[M+H] + : 692.0995; found: 692.0997.

[0099] 4-(2-(4-methylbenzyl)-l,3-dioxo-2,3-dihydro-lH-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenethyl)piperazine-l-carbothioamide (I24): Yield 55% as a yellow solid. Mp: 220.1-221.2 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.52 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 7.2 Hz, 1H), 8.42-8.36 (m, 1H), 8.02 (t, J = 4.8 Hz, 1H), 7.84-7.74 (m, 3H), 7.43 (d, J = 8.0 Hz, 2H), 7.37-7.32 (m, 1H), 7.31 (s, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 5.18 (s, 2H), 4.09 (t, J = 3.6 Hz, 4H), 3.76 (dt, J = 13.2, 6.4 Hz, 2H), 3.26 (t, J = 4.8 Hz, 4H), 2.99 (t, J = 6.4 Hz, 2H), 2.23 ppm (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ = 181.64, 163.55, 163.01, 155.25, 143.76, 142.07, 136.13, 134.54, 132.35, 130.94, 130.87, 129.14, 128.87, 127.59, 126.19, 125.78, 125.32, 122.40, 115.64, 115.36, 52.11, 47.16, 46.45, 42.46, 34.47, 20.65 ppm. HR-MS (m / z) (ESI): calcd for C 33 H 33 N5O4S2[M+H] + : 628.2047; found: 628.2049.

[0100] 4-(2-(4-Methoxybenzyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenethyl)piperazine-1-carbothioamide (I25): Yield 82% as a yellow solid. Mp: 167.9-169.8 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.52 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 6.8 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.01 (t, J = 4.8 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.37 - 7.26 (m, 5H), 6.86 - 6.82 (m, 2H), 5.15 (s, 2H), 4.09 (t, J = 4.6 Hz, 4H), 3.76 (dt, J = 14.0, 6.0 Hz, 2H),, 3.69 (s, 3H), 3.26 (t, J = 5.2 Hz, 4H), 2.99 ppm (t, J = 7.6 Hz, 2H). 13CNMR (100 MHz, DMSO-d6): δ = 181.65, 163.54, 163.00, 158.35, 155.24, 143.75, 142.06, 132.33, 130.92, 130.85, 129.57, 129.26, 129.14, 129.12, 126.19, 125.77, 125.31, 122.42, 115.66, 115.36, 113.70, 55.03, 52.10, 47.16, 46.44, 42.13, 34.46 ppm. HR-MS (m / z) (ESI): calcd for C 33 H 33 N5O5S2[M+H] + : 644.1996; found: 644.1982.

[0101] 4-(l,3-dioxo-2-(3,4,5-trimethoxybenzyl)-2,3-dihydro-lH- benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenethyl)piperazine-l- carboxymethanamide (126): Yield 48% as a yellow solid. Mp: 270.9-272.9 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.56 (d, J = 8.8 Hz, 1H), 8.52 (d, J = 7.2 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 8.01 (t, J = 5.2 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.4, 1H), 7.31 (s, 2H), 6.67 (s, 2H), 5.16 (s, 2H), 4.09 (t, J = 5.2 Hz, 4H), 3.76 (dt, J = 12.4, 6.0 Hz, 2H), 3.70 (s, 6H), 3.60 (s, 3H), 3.28 (t, J = 4.8 Hz, 4H), 2.99 ppm (t, J = 7.2 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): δ = 181.63, 163.76, 163.21, 155.29, 152.77, 143.78, 142.08, 136.64, 133.32, 132.44, 131.03, 130.92, 129.27, 129.18, 126.27, 125.80, 125.39, 122.55, 115.79, 115.45, 105.23, 59.98, 55.87, 52.14, 47.19, 46.47, 43.10, 34.49 ppm. HR-MS (m / z) (ESI): calcd for C 35 H 37 N5O7S2[M+H] + : 704.2207; found: 704.2202.

[0102] 4-(l,3-dioxo-2-(4-(trifluoromethyl)benzyl)-2,3-dihydro-lH- benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenethyl)piperazine-l-carboxamide (127): Yield 63%, as a yellow solid. Mp: 206.5-207.4 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.55 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 7.2 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 8.03 (t, J = 5.2 Hz, 1H), 7.86-7.80 (m, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 1H), 7.32 (s, 2H), 5.31 (s, 2H), 4.10 (s, 4H), 3.76 (dt, J = 13.2, 6.4 Hz,, 2H), 3.27 (s, 4H), 2.99 ppm (t, J = 6.8 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): δ = 181.63, 163.66, 163.09, 155.43, 143.79, 142.35, 142.08, 132.50, 131.07, 129.29, 129.18, 128.10, 127.99, 127.78, 127.57, 127.36, 126.99, 126.24, 125.81, 125.34, 125.32, 125.30, 125.27, 125.18, 123.38, 122.34, 121.58, 115.52, 115.39, 52.14, 47.17, 46.48, 42.55, 34.49, 30.73 ppm. HR-MS (m / z) (ESI): calcd for C 33 H 30 F3N5O4S2[M+H] + : 682.1764; found: 682.1787.

[0103] 4-(2-(3,4-Dihydroxyphenethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenethyl)piperazine-1-carbothioamide (I28): Yield 48% as a yellow solid. Mp: 254.1-255.9 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.81 (s, 1H), 8.74 (s, 1H), 8.53 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 7.2 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.01 (s, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.43 (t, J = 8.4 Hz, 2H), 7.36 (d, J = 8.0 Hz, 1H), 7.31 (s, 2H), 6.67 (d, J = 2.0 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 6.49 (dd, J = 8.0, 1.6 Hz, 1H), 4.14 (t, J = 8.0 Hz, 2H), 4.08 (s, 4H), 3.78-3.72 (m, 2H), 3.25 (s, 4H), 2.98 (t, J = 7.6 Hz, 2H), 2.71 ppm (t, J = 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ = 181.72, 163.55, 163.02, 155.20, 145.24, 143.89, 143.78, 142.11, 132.27, 130.85, 130.79, 129.65, 129.26, 129.19, 126.32, 125.88, 125.45, 122.63, 119.37, 116.08, 115.95, 115.70, 115.49, 52.22, 47.29, 46.54, 41.35, 34.55, 33.11 ppm. HR-MS (m / z) (ESI): calcd for C 33 H 33 N5O6S2[M+H] + : 660.1945; found: 660.1946.

[0104] 4-(2-Cyclohexyl-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenethyl)piperazine-1 -carbothioamide (I29): Yield 49%, as a yellow solid. Mp: 287.3-288.9 °C. 1 HNMR (400 MHz, DMSO-d6): δ = 8.50 (d, J = 8.4 Hz, 1 H), 8.45 (d, J = 7.2 Hz, 1 H), 8.37 (d, J = 8.0 Hz, 1 H), 8.02 (t, J = 5.6 Hz, 1 H), 7.84-7.74 (m, 3H), 7.43 (d, J = 8.0 Hz, 2H), 7.38 - 7.28 (m, 3H), 4.88 (t, J = 11.8 Hz, 1 H), 4.08 (s, 4H), 3.76 (dt, J = 12.4, 7.2 Hz, 2H), 3.23 (s, 4H), 2.99 (t, J = 6.8 Hz, 2H), 2.47 - 2.37 (m, 2H), 1.83 (d, J = 12.0 Hz, 2H), 1.71 - 1.57 (m, 2H), 1.42 - 1.13 ppm (m, 4H). 13C NMR (100MHz, DMSO-d6): δ=181.64,163.90,163.40,154.80,143.76,142.07,132.06,130.64,130.34,129.14,129.08,126.18, 125.78,125.17,123.05,116.40,115.35,52.58,52.11,47.18,46.45,34.47,28.69,26.14,25.23ppm.HR-MS(m / z)(ESI):calcd for C 31 H 35 N5O4S2[M+H] + :606.2203; found:606.2203.

[0105] 4-(2-Butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)-N-(4-aminosulfonylphenylethyl)piperazine-1-methylthioamide (I30): Yield 38%, as a yellow solid. Mp: 268.4-269.7℃. 1 H NMR (400MHz, DMSO-d6): δ = 8.50 (d, J = 8.4Hz, 1H), 8.45 (d, J = 7.2Hz, 1H), 8.36 (d, J = 8.0 Hz,1H),8.01(t,J=5.2Hz,1H),7.83-7.75(m,3H),7.43(d,J=8.0Hz,2H),7.36–7.28(m ,3H),4.09(s,4H),4.01(t,J=6.8Hz,2H),3.76(dt,J=13.6,6.0Hz,2H),3.24(s,4H),2 .99(t,J=7.2Hz,2H),1.65-1.62(m,2H),1.40-1.27(m,2H),0.91ppm(t,J=7.2Hz,3H). 13 C NMR (100MHz, DMSO-d6): δ=181.65,163.46,162.94,154.99,143.77,142.07,132.04,130.65,130.54,129.16,129.01,126.12, 125.79,125.29,122.49,115.86,115.31,52.13,47.19,46.46,39.20,34.48,29.71,19.83,13.75ppm.HR-MS(m / z)(ESI):calcd for C 29 H 33N5O4S2[M+H] + : 580.2047; found: 580.2050.

[0106] 4-(2-Octyl-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamoylphenethyl)piperazine-1 -carbothioamide (I31 ): Yield 38%, as a yellow solid. Mp: 142.3-144.5 °C. 1 H NMR (600 MHz, DMSO-d6): d = 8.53 (d, J = 8.4 Hz, 1 H), 8.47 (d, J = 6.6 Hz, 1 H), 8.39 (d, J = 8.4 Hz, 1 H), 8.02 (t, J = 5.4 Hz, 1 H), 7.82 (t, J = 7.8 Hz, 1 H), 7.43 (d, J = 8.4 Hz, 2H), 7.43 (t, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1 H), 7.31 (s, 2H), 4.08 (t, J = 4.8 Hz, 4H), 4.01 (t, J = 7.2 Hz, 2H), 3.76 (dt, J = 13.2, 6.0 Hz, 2H), 3.25 (t, J = 4.8 Hz, 4H), 2.98 (t, J = 7.2 Hz, 2H), 1.63 - 1.57 (m, 2H), 1.36 - 1.20 (m, 10H), 0.83 ppm (t, J = 7.2 Hz, 3H). 13 C NMR (150 MHz, DMSO-d6): d = 181.63, 163.53, 163.00, 155.07, 143.77, 142.08, 132.12, 130.73, 130.64, 129.17, 129.09, 126.21, 125.79, 125.36, 122.57, 115.93, 115.39, 52.15, 47.19, 46.47, 34.48, 31.25, 28.73, 28.60, 27.52, 26.54, 22.09, 13.97 ppm. HR-MS (m / z) (ESI): calcd for C 33 H 41 N5O4S2[M+H] + : 636.2673; found: 636.2674.

[0107] 4-(2-(2-(dimethylamino)ethyl)-l,3-dioxo-2,3-dihydro-lH- benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenethyl)piperazine-l- carboxymide (I32): Yield 76% as a yellow solid. Mp: 263.9-264.6 °C. 1 H NMR (400 MHz, DMSO-d6): δ = 8.53 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 7.2 Hz, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.10 (t, J = 5.2 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.32 (s, 2H), 4.23 (t, J = 6.4 Hz, 2H), 4.10 (t, J = 4.8 Hz, 4H), 3.76 (dt, J = 13.2, 6.4 Hz, 2H), 3.25 (t, J = 4.8 Hz, 4H), 2.99 (t, J = 7.2 Hz, 2H), 2.87 (t, J = 7.2 Hz 2H), 2.48 ppm (s, 6H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.60, 163.91, 163.34, 155.23, 143.79, 142.06, 132.21, 130.79, 129.25, 129.14, 126.18, 125.78, 125.27, 122.61, 115.88, 115.32, 55.28, 52.17, 47.22, 46.45, 43.52, 35.75, 34.45 ppm. HR-MS (m / z) (ESI): calcd for C 29 H 34 N6O4S2[M+H] + : 595.2156; found: 595.2150.

[0108] 4-(2-(2-(dimethylamino)ethyl)-l,3-dioxo-2,3-dihydro-lH- benzo[de]isoquinolin-6-yl)-N-(4-sulfamidophenethyl)piperazine-l- carboxymide (I32): Yield 76% as a yellow solid. Mp: 263.9-264.6 °C. 1H NMR (400 MHz, DMSO-d6): δ = 8.51 (dd, J = 8.4, 2.8 Hz, 1H), 8.46 (dd, J = 7.2, 3.2 Hz, 1H), 8.37 (dd, J = 8.0, 3.2 Hz, 1H), 8.02 (t, J = 5.1 Hz, 1H), 7.81 (dd, J = 8.1, 2.5 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.34 (dd, J = 8.4, 2.4 Hz, 1H), 7.31 (s, 2H), 4.13 - 4.05 (m, 6H), 3.76 (dt, J = 14.4, 6.0 Hz, 2H), 3.24 (t, J = 4.8 Hz, 4H), 2.99 (t, J = 8.0 Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H), 2.53 (q, J = 7.2 Hz, 2H), 0.95 ppm (t, J = 7.2 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.64, 163.52, 162.99, 155.04, 143.77, 142.08, 132.07, 130.68, 130.61, 129.16, 129.07, 126.18, 125.79, 125.34, 122.54, 115.90, 115.37, 52.14, 49.33, 47.19, 47.01, 46.47, 37.42, 34.48, 12.14 ppm. HR-MS (m / z) (ESI): calcd for C 31 H 38 N6O4S2[M+H] + : 623.2469; found: 623.2471.

[0109] 4-(2-(2-Hydroxyethyl)-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl)-N-(4- sulfamidophenethyl)piperazine-1 -carbothioamide (134): Yield 50%, as a yellow solid. Mp: 239.6-241.7 °C. 1H NMR (400 MHz, DMSO-d6): δ = 8.51 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.02 (t, J = 5.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.31 (s, 2H), 4.81 (t, J = 6.0 Hz, 1H), 4.13 (t, J = 6.4 Hz, 2H), 4.09 (t, J = 5.2 Hz, 4H), 3.76 (dt, J = 13.6, 6.4 Hz, 2H), 3.60 (dt, J = 12.4, 6.0 Hz, 2H), 3.24 (t, J = 4.8 Hz, 4H), 2.99 ppm (t, J = 7.2 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6): δ = 181.65, 163.66, 163.14, 154.98, 143.77, 142.07, 132.03, 130.64, 130.53, 129.16, 129.13, 126.15, 125.79, 125.32, 122.67, 116.06, 115.33, 57.85, 52.15, 47.20, 46.45, 41.66, 34.47 ppm. HR-MS (m / z) (ESI): calcd for C 27 H 29 N5O5S2[M+H] + : 568.1683; found: 568.1678.

[0110] The structural formulae of the target compounds I1-I34 obtained in this example are as follows:

[0111]

[0112]

[0113]

[0114] Example 2: Preparation of compounds I15, I32, I33

[0115] Compound I15: Repeat Example 1, except that the reaction was carried out at room temperature throughout (reaction to completion took about 3 days). The final product was obtained as a yellow solid in a yield of 44%. It was characterized by 1H NMR, 13C NMR and mass spectrometry to be compound I15.

[0116] Compound I32: repeat example 1, except that acetone is used instead of acetonitrile, and the reaction is carried out at 55 °C (the reaction is complete in about 12 h). Finally, a yellow solid is obtained, with a yield of 76%. It is characterized by nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum, and is determined to be compound I32.

[0117] Compound I33: repeat example 1, except that methanol is used instead of acetonitrile, and the reaction is carried out at 70 °C (the reaction is complete in about 8 h). Finally, a yellow solid is obtained, with a yield of 74%. It is characterized by nuclear magnetic hydrogen spectrum, carbon spectrum and mass spectrum, and is determined to be compound I33.

[0118] Experimental example 1: in-vitro anti-tumor activity test

[0119] The clinically used drug aminopterin, the CA inhibitor SLC-0111 is used as a positive control drug, the corresponding solvent is used as a negative control, and human breast cancer cells MDA-MB-231, MCF-7, human non-small cell lung cancer cells A549, human colon cancer cells HCT-116, SW480 are used as test cell strains; the MTT method is used to test the in-vitro anti-tumor activity of the target compounds.

[0120] Take well-grown cells and inoculate 2×10 4 ~ 4×10 4 cells per well in a 96-well plate, and incubate in a 37 °C incubator for 24 h, then add 20 μL of the test compound (the initial concentration is 40 μM, and gradient dilution is carried out according to the 2-fold method, and 5 replicate wells are set for each concentration), and place in a cell incubator for 24 h. Add 10 μL of 5 mg / mL MTT working solution to the 96-well plate, incubate in the incubator for 4 h, carefully discard the supernatant, add 150 μL of DMSO to dissolve the reduced formazan violet crystals, and use an enzyme-labeled instrument to measure the absorbance of each well. Calculate the cell proliferation inhibition rate of each dosing well, and the results are shown in Table 1 below.

[0121] Table 1. Inhibition activity of target compounds of the present application on different cell strains

[0122]

[0123]

[0124] a IC 50 The values are the average values ± S.D. of 3 independent experiments.

[0125] As can be seen from Table 1, some compounds show good anti-tumor activity. For human breast cancer cells MDA-MB-231, compounds I15, I32 and I33 have good cytotoxicity, and the IC 508.89±1.80μM, 4.31±0.46μM and 13.51±0.93μM, which are superior to that of amonafide (22.37±0.60μM), wherein the compound I32 is about 5 times stronger than the positive drug amonafide; only the compound I32 has cytotoxicity to the cell MCF-7, IC 50 = 10.84±0.45μM; for the cell HCT-116, the compounds I10 and I13 have moderate cytotoxicity, IC 50 values of 15.78±0.17μM and 14.83±0.21μM, which are superior to that of the positive drug amonafide; for the human colon cancer cell SW480, the compounds I15 and I32 have obvious inhibitory effect, IC 50 values of 16.15±0.68μM and 8.53±0.95μM, and the toxicity of the compound I32 to SW480 is about twice that of amonafide (13.80±0.27μM).

[0126] The above in-vitro anti-tumor activity tests show that the 4-piperazine thiourea phenyl sulfonamide-1,8-naphthalimide derivative described in the present application is expected to be used for the preparation of anti-tumor drugs.

[0127] Experimental Example 2: CA II and CA IX enzyme activity test

[0128] The CA inhibitor SLC-0111 was used as a positive control for the CA II and CA IX enzyme activity test, and the following steps were performed:

[0129] In each well of a 96-well plate, 18μL of CA solution (3.33ng / μL for CA II and 11.1ng / μL for CA IX) was added, 2μL of the test drug (100μM as the initial concentration of the test drug, and the test drug was diluted by 3 times, and 5 replicates were set for each concentration) was added, and after the addition of the drug, it was placed in a constant temperature incubator at 25℃ for 15min; after taking out, 20μL of 1mM 4-NPA solution was added, and the detection of CA II affinity was placed in a constant temperature incubator at 25℃ for 60min (the detection of CA IX was placed in a constant temperature incubator at 25℃ for 90min), and then the absorbance value was measured under an enzyme marker at 405nm.

[0130] The physiological related CA II and CA IX enzyme inhibitory activities were evaluated by esterase method, and the results are shown in Table 2.

[0131] Table 2. Inhibitory activities of some target compounds of the present application to CA II and CA IX

[0132]

[0133] a IC50 The values ​​are the mean ± SD of 3 independent experiments.

[0134] As shown in Table 2, the tested compounds exhibited better enzyme inhibitory activity and selectivity against CA IX than the positive control drug SLC-0111 (IC50). 50 =0.25±0.053μM, SI(CA II / IX)=32). The half-maximal inhibitory concentration (IC50) of compound I32 is... 50 The selectivity was 0.0025±0.0005μM, which is 100 times better than SLC-0111, with a selectivity SI(CA II / IX) = 296; compound I33 showed significant off-target effect on CAII, with SI(CA II / IX) = 665.

[0135] Experiment Example 3: Cell Proliferation Experiment under Hypoxic Environment

[0136] The CA inhibitor SLC-0111 was used as a positive control to simulate hypoxic conditions for cell viability testing, following the steps below:

[0137] MDA-MB-231 cells were used at a rate of 2 × 10⁻⁶ 4 ~4×10 4 The culture medium was seeded into 96-well plates at a density of [number] wells and incubated at 37°C for 24 h. Fresh culture medium was removed and 100 μM CoCl2 was added, and the plates were incubated at 37°C for 24 h, followed by CoCl2 exposure for 48 h. Then, 20 μL of the test compound (starting concentration 40 μM, 2-fold dilution, 5 replicates per concentration) was added, and the plates were incubated at 37°C for 24 h. Finally, 10 μL of 5 mg / mL MTT working solution was added, and the plates were incubated for 4 h. The supernatant was carefully discarded, and 150 μL of DMSO was added to dissolve the formazan purple crystals produced by reduction. The absorbance of each well was measured using a microplate reader.

[0138] Experimental results are expressed in IC 50 The values ​​are shown in Table 3 below, with SLC-0111 (>20 μM) serving as a positive control. The IC50 values ​​of compounds I15, I32, and I33 under hypoxic conditions are also shown. 50 The cytotoxicity levels of the compounds under hypoxic conditions (7.68±0.31, 3.45±0.06, and 7.51±0.48 μM) were all higher than those under normal oxygen conditions (8.89±1.80, 4.31±0.46, and 13.51±0.93 μM). The compounds exhibited higher cytotoxicity against MDA-MB-231 tumor cells under hypoxic conditions, indicating that the compounds further inhibit cell viability by inhibiting CA IX activity.

[0139] Table 3. Anti-proliferative activity of some compounds of the present application against MDA-MB-231 cell line under normoxic and hypoxic conditions

[0140]

[0141] a IC 50 Values are mean ± S.D. of 3 independent experiments

[0142] Experimental Example 4: Transmission electron microscopy imaging

[0143] The transmission electron microscopy imaging experiment of compound I32 was carried out as follows:

[0144] MDA-MB-231 cells in good growth state were inoculated in 6-well plates at about 5 x 10 5 cells per well, and incubated in a 37°C constant temperature incubator for 24 h. The culture medium was removed, and different concentrations of compound I32 were added (3 replicates for each concentration). After 24 h of culture, the cells were washed with PBS buffer solution for 3 times, fixed with 2.5% glutaraldehyde solution for 12 h, and finally observed by transmission electron microscopy.

[0145] After 24 h of treatment of MDA-MB-231 cells with compound I32 at different concentrations, obvious ferroptosis occurred in the mitochondrial structure of the cells under transmission electron microscopy. When the cells were treated with 5 μM compound I32, the mitochondrial structure was obviously smaller and the structure was tighter than that of the blank group. When the cells were treated with 10 μM compound I32, the mitochondrial crinkle was more obvious. Figure 1 The above morphological characteristics showed that compound I32 could effectively induce ferroptosis.

[0146] Experimental Example 5: Reactive oxygen species (ROS) experiment

[0147] The reactive oxygen species (ROS) experiment of compound I32 was carried out as follows:

[0148] MDA-MB-231 cells in good growth state were inoculated in 6-well plates at about 5 x 10 5 cells per well, and incubated in a 37°C constant temperature incubator until use. When the cell growth density reached 60-70%, different concentrations of compound I32 were added, and after 24 h of incubation, the cell culture medium was removed and the cells were washed with PBS solution. 10 μΜ DCFH-DA was added to each well, and after 20-60 min of culture in a 37°C incubator in the dark, the cells were washed with serum-free culture medium for 3 times, and finally observed and photographed by inverted fluorescence microscopy.

[0149] The results of inverted fluorescence microscopy observation are shown in Figure 2 Figure 2 ​It can be seen that the green fluorescence signal is missing or weak in the control group, and the green fluorescence in the experimental group becomes stronger with the increase of the concentration of compound I32, indicating that the level of ROS in the cells is significantly improved compared with the blank group. It shows that compound I32 improves the level of ROS in the cells, thereby leading to cell ferroptosis.

[0150] Experimental Example 6: JC-1 staining experiment

[0151] The process of compound I32 JC-1 staining experiment is as follows:

[0152] Take well-grown MDA-MB-231 cells and inoculate them into a 6-well plate at about 5×105 cells per well, and incubate them in a 37°C constant temperature incubator. When the cells are completely attached and the density reaches 60-70%, replace the fresh culture medium containing different concentrations of compound I32 or carrier, and incubate in the incubator for 24h. Remove the culture medium, wash twice with PBS buffer, and trypsinize the cells. Collect the cells in a 15mL centrifuge tube, centrifuge, discard the supernatant, and wash the cells twice with serum-free culture medium. Add 500μL of JC-1 staining working solution and incubate in the dark for 20min. After incubation, centrifuge, discard the supernatant, and wash twice with JC-1 staining buffer. Add 500μL of serum-free culture medium to suspend the cells, and observe and take pictures under a fluorescence inverted microscope.

[0153] Use different concentrations of compound I32 to treat MDA-MB-231 cells, JC-1 staining, and take pictures under a fluorescence inverted microscope (as shown in Figure 3 The results show that the red fluorescence becomes lighter as the concentration of compound I32 increases, i.e. the proportion of polymers decreases, and the green fluorescent substance increases significantly, indicating that the mitochondrial membrane potential decreases, further indicating that the mitochondria are damaged.

[0154] Experimental Example 7: Compound I32 cell apoptosis test

[0155] The process of compound I32 cell apoptosis test is as follows:

[0156] Inoculate MDA-MB-231 cells into a 6-well plate at 5×10 5 cells per well, and incubate overnight until the density reaches 60-70%. Replace the culture medium with culture medium containing different concentrations of I32 and incubate in the incubator for 24h. Then transfer the cells to a 15mL centrifuge tube by digestion and PBS washing, centrifuge at 1000r / min, add 200μL buffer containing 5μL Annexin V-FITC and incubate at 37°C in the dark for 20min. Then add 300μL buffer and 5μL PI staining agent, mix well, and transfer to a 1.5mL centrifuge tube. Finally, use a flow cytometer for data analysis and collection.

[0157] MDA-MB-231 cells were incubated with different concentrations of compound I32 for 24 h. The results are shown in FIG. 8, which shows that the percentage of cells in Q2 phase in the control group was 6.2%, and the percentages of cells in Q2 phase treated with compound I32 at concentrations of 5 and 10 mM were 66.2% and 92.9%, respectively, indicating that compound I32 can promote late apoptosis of cells in a concentration-dependent manner. Figure 4

[0158] Experimental Example 8: Compound I32 cell scratch test

[0159] The process of the compound I32 cell scratch test is as follows:

[0160] MDA-MB-231 cells were seeded in a 6-well cell culture plate and incubated overnight, and the cells were observed to be just full of the plate holes. The monolayer cells were scraped with a sterile micropipette tip, and the cells were washed with incomplete medium to remove the separated cells. After taking a photo, fresh complete medium containing different concentrations of compound I32 was added and incubated for 24 h. Then the cells were fixed with 4% paraformaldehyde, and photographed under a phase-contrast inverted microscope.

[0161] Based on the fact that I32 can effectively induce ferroptosis and apoptosis of cells, the applicants evaluated the effect of compound I32 on the migration and repair capacity of cells. After MDA-MB-231 cells were incubated with different concentrations of compound I32 for 24 h, the migration potential of MDA-MB-231 cells was analyzed. The width of the scratch was measured before and after drug addition (as shown in FIG. 9), and the scratch area of the untreated control group was significantly narrower compared with the cells incubated with I32, indicating that compound I32 can effectively inhibit the migration of MDA-MB-231 cells. Figure 5

[0162] Experimental Example 9: Compound I32 animal experiment

[0163] The process of the compound I32 animal experiment is as follows:

[0164] A mouse model was established by subcutaneously implanting breast cancer 4T1 cells. When the average tumor volume of the mice reached about 100 mm 3 , the mice were randomly divided into 3 groups, 5 mice in each group, namely the vehicle group, the compound I32 15 mg / kg dose group, and the compound I32 30 mg / kg dose group. Compound I32 was administered intravenously every 3 days, and the tumor volume and body weight were recorded every other day after drug treatment. After 21 days of treatment, the animals were sacrificed by cervical dislocation, and the tumor mass was stripped and weighed. No blood was taken for biochemical parameter detection during the observation period, and the organ tissues (lungs, liver) were taken, fixed with 4% paraformaldehyde, and stained with hematoxylin-eosin.

[0165] ​​Because in vitro antitumor experiments have certain limitations and cannot fully simulate the in vivo survival environment, compound I32 was further evaluated in vivo in vivo. The applicant used a xenograft model of highly metastatic breast cancer 4T1 cells to study the in vivo antitumor activity of compound I32. Twenty-four hours after inoculation, mice were randomly divided into three groups (n=5), and administered compound I32 (15 or 30 mg / kg) or its carrier intravenously every three days for 21 consecutive days. Results are as follows... Figure 6 As shown, compared with the control group, compound I32 can effectively inhibit tumor growth ( Figure 6 A) The tumor growth inhibition rates at doses of 15 mg / kg and 30 mg / kg were 45.5% and 51.2%, respectively. Figure 6 B). Compared with the blank group, the experimental group with added compound I32 significantly reduced tumor volume ( Figure 6 C), but the weight of each group of mice gradually increased, further demonstrating the safety of compound 11o. Figure 6 D). In vivo antitumor activity tests in mice showed that compound I32 could effectively inhibit the growth of breast cancer 4T1 tumors.

[0166] It has been reported that over 90% of deaths from breast cancer are attributed to metastatic complications. Studies have shown that the lungs and liver are the most common sites of metastasis in breast cancer. Therefore, the applicant assessed the infiltration of tumor cells in the lungs and liver by performing liver sections and staining with hematoxylin and eosin (H&E). The results are as follows... Figure 7 As shown, lung and liver metastases occurred 21 days after 4T1 cell engraftment. Treatment with compound I32 significantly inhibited lung and liver metastases of 4T1 cells in a dose-dependent manner. These results indicate that compound I32 has good therapeutic potential.

Claims

1. A 4-piperazinethiourea benzenesulfonamide-1,8-naphthimide derivative of the following structure of formula (I) or a pharmaceutically acceptable salt thereof: ###0001### (I) wherein R and n are selected as follows: R = 4-(trifluoromethyl)benzyl, n = 0, the compound of formula (I) is designated as compound I10; or R = butyl, n = 0, the compound of formula (I) is designated as compound I13; or R = 2-(dimethylamino)ethyl, n = 0, the compound of formula (I) is designated as compound I15; or R = 2-(dimethylamino)ethyl, n = 2, the compound of formula (I) is designated as compound I32; or R = 2-(diethylamino)ethyl, n = 2, the compound of formula (I) is designated as compound I33. (I)； wherein The compound of formula (II) and the compound of formula (III) are reacted in an organic solvent, the reaction mixture is recovered and the solvent is removed to obtain the target compound in crude form. wherein R in the structure of formula (II) and n in the structure of formula (III) are selected as follows: R = 4-(trifluoromethyl)benzyl, n = 0; or R = butyl, n = 0; or R = 2-(dimethylamino)ethyl, n = 0; or R = 2-(dimethylamino)ethyl, n = 2; or R = 2-(diethylamino)ethyl, n = 2. The organic solvent is one or a combination of two or more selected from acetonitrile, acetone, methanol and dichloromethane. The reaction is carried out with or without heating. The reaction is carried out at a temperature between room temperature and the boiling point of the organic solvent. The step of purifying the target compound in crude form is also included.

2. The process for the preparation of 4-piperazinethiourea phenyl sulfonamide- 1,8-naphthimide derivatives according to claim 1, characterized by, 7. Use of the 4-piperazinethiourea benzenesulfonamide-1,8-naphthimide derivative of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the activity and / or overexpression of carbonic anhydrase IX enzyme. (II)、 (III):

8. Use of compound I10 or a pharmaceutically acceptable salt thereof in claim 1 in the preparation of a medicament for treating colon cancer cell HCT-116; compound I13 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating colon cancer cell HCT-116; 3. The preparation method according to claim 2, characterized in that, compound I15 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating breast cancer cell MDA-MB-231 or colon cancer cell SW480; 4. The preparation method according to claim 2, characterized in that, compound I32 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating breast cancer cell MDA-MB-231, breast cancer cell MCF-7 or colon cancer cell SW480; 5. The method of claim 2 wherein the step of forming the first and second layers comprises the step of: compound I33 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating breast cancer cell MDA-MB-231. ​ 6. The production method according to any one of claims 2 to 5, characterized by, 9. A pharmaceutical composition comprising a therapeutically effective dose of the 4-piperazinethiourea benzenesulfonamide-1,8-naphthimide derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier. ​ ​ ​ ​ ​ ​ ​

Images