A divalent KEAP1-NRF2 inhibitor, preparation method thereof and pharmaceutical use thereof

Abstract

The present invention discloses a divalent KEAP1-NRF2 inhibitor, a preparation method thereof, and a pharmaceutical use thereof. The present invention provides a divalent KEAP1-NRF2 inhibitor represented by General Formula I. This inhibitor can bind to the dimerized KEAP1 protein, thereby directly dissociating NRF2 from the complex and promoting its nuclear entry, achieving the effect of rapidly activating NRF2; and can directly inhibit the expression of intracellular inflammatory factors, effectively relieve acute inflammatory injury, has potential anti-acute inflammatory activity, and can be prepared into an anti-inflammatory drug for the treatment of numerous acute inflammatory diseases, including acute liver injury, acute lung injury, endotoxemia, sepsis, emphysema, acute respiratory distress syndrome, and novel coronavirus pneumonia, etc.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry and relates to a divalent KEAP1-NRF2 inhibitor, a preparation method thereof and a pharmaceutical use thereof. Background Art

[0002] Oxidative stress and exogenous stimuli exacerbate the occurrence and development of various diseases, including cancer, cardiovascular diseases, inflammation and neurodegenerative diseases, etc. Nuclear factor E2-related factor 2 (NRF2) is a transcription factor that binds to the antioxidant response element (ARE) and is the main regulator of the cellular defense mechanism against oxidative stress and exogenous stimuli. By binding to ARE, NRF2 activates important transcriptional programs in drug detoxification and maintaining metabolic and redox balance. In vivo, the activity of NRF2 is mainly regulated by the substrate regulatory protein Kelch-like epichlorohydrin-associated protein 1 (KEAP1). Two molecules of KEAP1 protein form a dimer through its own BTB region, recognize the high-affinity region ETGE and the low-affinity region DLG in one molecule of NRF2 protein, and form a ternary complex with a stoichiometric coefficient of Keap1:Nrf2 of 2:1. Under non-stress conditions, both the ETGE and DLG regions of NRF2 bind to the KEAP1 dimer, which ubiquitinates NRF2 and transports it to the proteasome for degradation, keeping the protein level of NRF2 low in the cell; under oxidative stress conditions, reactive oxidants covalently bind to the cysteine residues on KEAP1, changing the conformation of KEAP1, so that the newly generated NRF2 protein cannot be ubiquitinated and degraded by KEAP1, and NRF2 accumulates in the cell and enters the nucleus, binds to the ARE promoter region and induces the transcriptional expression of downstream genes.

[0003] Developing KEAP1-NRF2 protein-protein interaction (PPI) inhibitors to inhibit the ubiquitination and degradation of NRF2 and enhance the activity of NRF2 has become a new direction for developing drugs for treating stress- and inflammation-related diseases. At present, multiple KEAP1-NRF2 PPI inhibitors have progressed to the preclinical research stage. However, the existing KEAP1-NRF2 PPI inhibitors are all monovalent inhibitors, which only inhibit one binding site of the KEAP1-NRF2 protein interaction, prevent the ubiquitination and degradation of NRF2 by KEAP1, and promote the accumulation of NRF2 to activate its activity. Such inhibitors cannot directly release the existing NRF2 protein in the cell, and their activation of NRF2 has a lag effect. Therefore, the existing monovalent KEAP1-NRF2 PPI inhibitors show good therapeutic effects in chronic inflammation models in vivo, but lack therapeutic effects on acute inflammation models.

[0004] Based on the deficiencies of existing monovalent KEAP1-NRF2 inhibitors, the present invention realizes the simultaneous inhibition of two sites of the KEAP1-NRF2 interaction by designing a bivalent inhibitor, promotes the direct release of NRF2 mediated by small molecules, and thus achieves the purpose of rapid activation of NRF2; and further uses the bivalent KEAP1-NRF2 inhibitor as a tool to explore the therapeutic effect of NRF2 activation in acute inflammation, especially acute lung injury, so as to provide guidance for the development of therapeutic drugs for acute inflammatory injuries. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a bivalent KEAP1-NRF2 inhibitor, a preparation method thereof and its medical use.

[0006] The above object of the present invention is achieved by the following technical solutions:

[0007] A compound, which is a compound represented by general formula I or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein, the substituents R, n, and m are one of the combinations of the following serial numbers:

[0010]

[0011] A preparation method of the above compound represented by general formula I, comprising the following steps:

[0012] Using 4-nitro-1-naphthylamine as a raw material, reducing the nitro group by hydrogenation to obtain 1,4-diaminonaphthalene, obtaining intermediate 9 through nucleophilic substitution, and then obtaining intermediate 10 through a second nucleophilic substitution; substituting the other amino group of intermediate 10 with 4-nitrobenzenesulfonyl chloride to obtain intermediate 11; reducing the nitro group of intermediate 11 under the action of Pd / C and H2 to obtain intermediate 12, and intermediate 12 is de-methylated to obtain the monomer compound 1 for assembling the bivalent inhibitor; compound 1 reacts with succinic anhydride and glutaric anhydride respectively under the reaction conditions of toluene / 110 °C to obtain intermediate 13 and intermediate 14; subsequently, intermediate 13 and intermediate 14 are respectively condensed with different lengths of diamino linking chains in a stoichiometric ratio of 2:1 to obtain intermediates 15a - 15f, and the target compounds 2 - 7 in general formula I are obtained through hydrolysis reaction to remove the methyl ester; intermediate 14 reacts with N-Boc-1,3-diaminopropane through a condensation reaction to obtain intermediate 19; the other amino group of intermediate 9 obtains intermediate 16 through nucleophilic substitution, and then reduces the nitro group under the action of Pd / C and H2 to obtain intermediate 17, and intermediate 17 reacts with succinic anhydride to obtain intermediate 18; intermediate 18 and intermediate 19 are condensed to obtain intermediate 20, and intermediate 20 is finally hydrolyzed to remove the methyl ester to obtain the target compound 8;

[0013] The synthetic route is as follows:

[0014]

[0015] Reaction parameters in the synthetic route:

[0016] (a) Pd / C, H2, THF, rt, 4 h; (b) 4-Methoxybenzenesulfonyl chloride, Na2CO3, THF, 5 h; (c) DMF, K2CO3, ethyl bromoacetate, rt, 3 h; (d) 4-Nitrobenzenesulfonyl chloride, toluene, pyridine, 100 °C, 2 h; (e) tin dichloride dihydrate, hydrogen chloride, ethylacetate, 70 °C, 2 h; (f) oxane-2,6-dione or succinic anhydride, toluene, 110 °C; (g) ethylenediamine, trimethylenediamine or putrescine, EDCI, HOBT, DIPEA, THF, rt, 4 h; (h) LiOH, MeOH, H2O, rt, 5 h; (i) N-boc-1,3-diaminopropane, EDCI, HOBT, DIPEA, THF, rt, 4 h; (j) HCl / EA, rt, 2 h, 95%; (k) 18, EDCI, HOBT, DIPEA, THF, rt, 4 h.

[0017] Use of the compound shown in the above general formula I for preparing a drug for KEAP1-NRF2 inhibitor.

[0018] Use of the compound shown in the above general formula I for preparing a drug for treating or alleviating a disease treated by inhibiting KEAP1-NRF2.

[0019] Furthermore, the disease is inflammation.

[0020] Even further, the inflammation is acute inflammation.

[0021] Even further, the acute inflammation includes acute liver injury, acute lung injury, endotoxemia, sepsis, emphysema, acute respiratory distress syndrome and novel coronavirus pneumonia.

[0022] Beneficial effects:

[0023] The present invention provides a divalent KEAP1-NRF2 inhibitor represented by general formula I. This inhibitor can bind to the dimerized KEAP1 protein, thereby directly dissociating NRF2 from the complex and promoting its nuclear translocation, achieving the effect of rapidly activating NRF2; and can directly inhibit the expression of intracellular inflammatory factors, effectively relieve acute inflammatory injury, and has potential anti-acute inflammatory activity. It can be prepared into an anti-inflammatory drug for the treatment of numerous acute inflammatory diseases, including acute liver injury, acute lung injury, endotoxemia, sepsis, emphysema, acute respiratory distress syndrome, and novel coronavirus pneumonia, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the ARE luciferase reporter gene activity (induction fold) of the compound to be tested. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following examples specifically introduce the substantial content of the present invention, but do not limit the protection scope of the present invention thereto.

[0026] Example 1: Synthesis and Structure Confirmation of Compounds

[0027] I. General Experimental Rules

[0028] All chemical reagents used in the present invention are commercially available chemical pure or analytical pure. The melting point was determined using an M.P.50 Melting Point System. 1 1H-NMR, 13 13C-NMR nuclear magnetic resonance spectra were measured using a Bruker AV300 type (300 MHz) nuclear magnetic resonance spectrometer (TMS as the internal standard), and mass spectra were measured using an Agilent 1946A-MSD type mass spectrometer (ESI-MS) and a Waters Q-Tof type mass spectrometer (HRMS). The purity was determined by HPLC. The chromatographic column was an Agilent C18 (4.6X150 mm, 3.5 μM) type reversed-phase column, and the mobile phase was methanol: water: trifluoroacetic acid = 85:15:0.1. The concentration of the solvent was carried out using an N-1100 rotary evaporator produced by EYELA Instrument Co., Ltd. (at 40 °C). The silica gel used for column chromatography was 200 - 300 mesh silica gel (Qingdao Marine Chemical Factory Branch), and the eluent was petroleum ether (boiling range 60 - 90 °C) and ethyl acetate. A GF254 thin-layer chromatography silica gel plate with a specification of 0.25X0.75 mm (Yantai Dexin) was used to monitor the reaction, and ultraviolet absorption was detected under a ZF-1 type triple-purpose ultraviolet analyzer (Hangzhou David Science and Education Instrument Co., Ltd.).

[0029] II. Preparation of Intermediates

[0030] Synthesis of 4-methoxy-N-(naphthalen-1-yl)benzenesulfonamide (9):

[0031]

[0032] 4-Nitro-1-naphthylamine (10.00 g, 52.00 mmol) was dissolved in 200.00 mL of THF in a reaction flask, catalytic amount of Pd / C was added, and the reaction mixture was purged with hydrogen and stirred at room temperature for 5 h. After the reaction was completed, the catalyst was removed by suction filtration, and the filtrate was used in the next step without purification. 4-Methoxybenzenesulfonyl chloride (9.90 g, 48.30 mmol) and sodium carbonate (8.46 g, 79.8 mmol) were added to the filtrate obtained in the previous step. The reaction mixture was stirred under nitrogen protection in an ice bath for 5 h. After the reaction was completed, 20.00 mL of petroleum ether was added to the reaction solution for dilution, and a solid was precipitated. The solid was collected by suction filtration to obtain a filter cake. The filter cake was washed with 1 M HCl to adjust the pH to 4 and recrystallized from acetonitrile to obtain a pink solid with a yield of 90%; m.p.: 169 - 171 °C. 1 H-NMR (300 MHz, DMSO-d6) δ 9.39 (s, 1H), 8.05–7.93 (m, 1H), 7.93–7.82 (m, 1H), 7.59–7.48 (m, 2H), 7.37–7.26 (m, 2H), 7.02–6.95 (m, 2H), 6.68 (d, J = 8.0 Hz, 1H), 6.47 (d, J = 8.0 Hz, 1H), 5.80 (s, 2H), 3.78 (s, 3H); HRMS (ESI): found 329.0950 (C 17 H 17 N2O3S, [M+H] + , requires 329.0954).

[0033] Synthesis of methyl N-(4-aminonaphthalen-1-yl)-N-(4-methoxyphenyl)sulfonyl)glycinate (10):

[0034]

[0035] Intermediate 9 (7.16 g, 23.2 mmol) was dissolved in 15.00 mL of DMF in a reaction flask, and then K2CO3 (9.61 g, 696.00 mmol) and ethyl bromoacetate (3.72 g, 24.30 mmol) were added successively. The reaction mixture was stirred at room temperature for 2 h. Subsequently, the reaction mixture was diluted in 200.00 mL of water, and the solid was collected by suction filtration to obtain a filter cake. The filter cake was recrystallized from ethyl acetate / n-hexane to obtain a light pink solid with a yield of 80%; m.p.: 182 - 183 °C. 1H-NMR(300MHz, DMSO-d6) δ 8.00 (dd, J=22.4, 8.3 Hz, 2H), 7.57 (d, J=8.9 Hz, 2H), 7.38 (dt, J=13.9, 7.0 Hz, 2H), 7.06 (d, J=8.4 Hz, 2H), 6.77 (d, J=8.1 Hz, 1H), 6.45 (d, J=8.1 Hz, 1H), 5.98 (s, 2H), 4.37 (s, 2H), 3.83 (s, 3H), 3.55 (s, 3H); HRMS(ESI): found 401.1166 (C 20 H 21 N2O5S, [M+H] + , requires 401.1160).

[0036] Synthesis of methyl N-((4-methoxyphenyl)sulfonyl)-N-(4-((4-nitrophenyl)sulfamoyl)naphthalen-1-yl)glycinate (11):

[0037]

[0038] 4-Nitrobenzenesulfonyl chloride (5.70 g, 25.50 mmol) and pyridine (1.61 g, 20.30 mmol) were added to a solution of 100 mL of toluene and intermediate 10 (6.80 g, 17.00 mmol) in a reaction flask. The reaction mixture was stirred at 100 °C for 2 h under nitrogen protection. After cooling to room temperature, 300 mL of petroleum ether was added to the reaction mixture to dilute and precipitate a solid, which was filtered by suction to obtain a filter cake. The filter cake was recrystallized from acetonitrile to obtain a gray solid with a yield of 72%, m.p.: 213 - 215 °C. 1 H-NMR(300MHz, DMSO-d6) δ 10.25 (s, 1H), 8.37 (d, J=6.6 Hz, 2H), 8.24 - 7.92 (m, 4H), 7.71 - 7.58 (m, 4H), 7.29 - 6.91 (m, 4H), 4.55 (s, 2H), 3.87 (s, 3H), 3.69 (s, 3H); HRMS(ESI): found 586.0948 (C 26 H 24 N3O9S2, [M+H] + , requires 586.0943).

[0039] Synthesis of methyl N-(4-((4-aminophenyl)sulfamoyl)naphthalen-1-yl)-N-((4-methoxyphenyl)sulfonyl)glycinate (12):

[0040]

[0041] 11 (15.00 g, 25.60 mmol) was dissolved in 100 mL of ethyl acetate in a reaction flask. Stannous chloride dihydrate (23.00 g, 102.00 mmol) and HCl (10 mL) were added. After reacting at 70 °C for 2 h, when the reaction was complete as detected by TLC, it was cooled to room temperature. 300 mL of saturated NaHCO3 was added to adjust the pH of the solution to weakly alkaline (7 - 8). The reaction solution was filtered through a sintered funnel, and the filter cake was washed with ethyl acetate until there was no UV absorption. The filtrate was separated into layers, the aqueous layer was discarded, and the organic layer was washed twice with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a gray solid with a yield of 70%, m.p.: 194 - 195 °C. 1 1H-NMR (300 MHz, DMSO-d6) δ 9.76 (s, 1H), 8.04 (d, J = 6.9 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.57 - 7.38 (m, 4H), 7.19 - 6.88 (m, 4H), 6.7 (d, J = 9.3 Hz, 2H), 4.70 (s, 2H), 3.81 (s, 3H), 3.72 (s, 3H); HRMS (ESI): found 556.1201 (C 26 H 26 N3O7S2, [M + H] + , requires 556.1207).

[0042] Synthesis of 4-((4-(N-(4-((4-methoxy-N-(2-methoxy-2-oxoethyl)phenyl)sulfamoyl)naphthalen-1-yl)sulfamoyl)phenyl)amino)-4-oxobutanoic acid (13):

[0043]

[0044] Succinic anhydride (360.00 mg, 3.60 mmol) and intermediate 12 (1.00 g, 1.80 mmol) were dissolved in 30 mL of toluene in a reaction flask. The reaction was stirred at 110 °C for 6 h under nitrogen protection. After the reaction was completed and cooled to room temperature, 100 mL of petroleum ether was added to the reaction solution for dilution, and a solid precipitated. The solid was filtered and the filter cake was retained, rinsed with ether, and dried by rotary evaporation to obtain a white solid with a yield of 65%, m.p.: 209 - 210 °C. 11H NMR (300 MHz, DMSO-d6) δ 12.18 (s, 1H), 10.32 (d, J = 39.8 Hz, 1H), 8.21–8.02 (m, 2H), 7.78–7.59 (m, 4H), 7.52 (t, J = 7.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 7.02–6.89 (m, 4H), 4.49 (s, 2H), 3.84 (s, 3H), 3.56 (s, 3H), 2.63–2.53 (m, 4H); HRMS (ESI): found 656.1361 (C 30 H 30 N3O 10 S2, [M + H] + , requires 656.1367).

[0045] Synthesis of 5-((4-(N-(4-((4-methoxy-N-(2-methoxy-2-oxoethyl)phenyl)sulfamoyl)naphthalen-1-yl)sulfamoyl)phenyl)amino)-5-oxopentanoic acid (14):

[0046]

[0047] In the same way as the synthesis method of intermediate 13, using intermediate 12 (1.00 g, 1.80 mmol), glutaric anhydride (410.40 mg, 3.60 mmol) and toluene (30 mL) as raw materials, a white solid was obtained, with a yield of 65%, m.p.: 214 - 215 °C. 1 1H NMR (300 MHz, DMSO-d6) δ 12.12 (s, 1H), δ 10.29 (d, J = 18.3 Hz, 1H), 8.23–8.04 (m, 2H), 7.85–7.58 (m, 4H), 7.52 (dd, J = 9.8, 4.8 Hz, 4H), 7.06 (d, J = 9.3 Hz, 2H), 6.97 (d, J = 4.0 Hz, 2H), 4.49 (s, 2H), 3.85 (s, 3H), 3.56 (s, 3H), 2.38 (t, J = 7.7 Hz, 2H), 2.29–2.19 (t, J = 7.3 Hz, 2H), 1.86–1.68 (m, 2H); HRMS (ESI): found 670.1520 (C 31 H 32 N3O 10 S2, [M + H] + , requires 670.1524).

[0048] Synthesis of dimethyl 2,2'-((((((4,4'-(ethane-1,2-diylbis(azanediyl))bis(4-oxobutanoyl))bis(azanediyl))bis(4,1-phenylene sulfonyl))bis(azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl)diacetate (15a):

[0049]

[0050] In a reaction flask, EDCI (70.20 mg, 0.366 mmol), HOBT (49.50 mg, 0.366 mmol), DIPEA (79.00 mg, 0.61 mmol) and ethylenediamine (9.20 mg, 0.153 mmol) were dissolved in 20.00 mL of THF. The mixture was stirred for 3 minutes under ice bath conditions, then intermediate 13 (200 mg, 0.305 mmol) was added, and the reaction was continued with stirring for 3 h. The solvent THF was removed by distillation under reduced pressure. The reaction mixture was washed with ethyl acetate until there was no ultraviolet absorption. The filtrate was separated into layers, and the aqueous layer was discarded. The organic layer was washed twice with saturated NaCl solution, dried over anhydrous sodium sulfate, triturated, and purified by column chromatography (DCM:MeOH = 50:1) to obtain a white solid with a yield of 45%, m.p.: 224 - 225 °C. 1 1H NMR (300 MHz, Methanol-d4) δ 8.02 (dd, J = 13.9, 8.3 Hz, 4H), 7.56 (t, J = 8.3 Hz, 8H), 7.45 (dd, J = 8.6, 7.5 Hz, 4H), 7.29 (q, J = 5.2, 4.5 Hz, 4H), 7.05 (p, J = 5.2 Hz, 4H), 6.86 (t, J = 8.9 Hz, 4H), 4.45 (dd, J = 18.7, 8.9 Hz, 2H), 4.27 (dd, J = 17.7, 3.8 Hz, 2H), 3.80 (d, J = 10.8 Hz, 6H), 3.63 (d, J = 9.0 Hz, 6H), 3.26 (d, J = 6.0 Hz, 2H), 3.13 (q, J = 7.0 Hz, 2H), 2.66 (t, J = 7.1 Hz, 4H), 2.45 (t, J = 7.7 Hz, 4H); HRMS (ESI): found 1357.2951 (C 62 1H 62 8NNaO 18 4S4, [M + Na] + , requires 1357.2957).

[0051] Synthesis of Dimethyl 2,2'-((((((4,4'-(Propane-1,3-diylbis(azanediyl))bis(4-oxobutanoyl))bis(azanediyl))bis(4,1-phenylene sulfonyl))bis(azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (15b):

[0052]

[0053] Using the same synthetic method as for intermediate 15a, with EDCI (70.20 mg, 0.366 mmol), HOBT (49.50 mg, 0.366 mmol), DIPEA (79.00 mg, 0.61 mmol), trimethylenediamine (11.32 mg, 0.153 mmol), 20.00 mL of THF, and intermediate 13 (200 mg, 0.305 mmol) as starting materials, a pink solid was obtained. The yield was 45%, m.p.: 119 - 121 °C. 1 H NMR (300 MHz, Methanol-d4) δ 7.92 (dd, J = 20.9, 7.3 Hz, 4H), 7.53 (t, J = 2.3 Hz, 8H), 7.44 (dd, J = 8.6, 6.3 Hz, 4H), 7.32 (q, J = 5.2, 4.8 Hz, 4H), 7.05 (p, J = 8.2 Hz, 4H), 6.88 (t, J = 9.1 Hz, 4H), 4.47 - 4.27 (m, 4H), 3.76 (d, J = 10.8 Hz, 6H), 3.53 (d, J = 8.1 Hz, 6H), 3.23 (t, J = 6.5 Hz, 4H), 2.86 (t, J = 7.2 Hz, 4H), 2.60 (t, J = 7.9 Hz, 4H), 1.76 - 1.43 (m, 2H); HRMS (ESI): found 1371.3110 (C 63 H 64 N8NaO 18 S4, [M+Na] + , requires 1371.3114).

[0054] Synthesis of Dimethyl 2,2'-((((((4,4'-(Butane-1,4-diylbis(azanediyl))bis(4-oxobutanoyl))bis(azanediyl))bis(4,1-phenylene sulfonyl))bis(azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (15c):

[0055]

[0056] Using the same synthetic method as for intermediate 15a, with EDCI (70.20 mg, 0.366 mmol), HOBT (49.50 mg, 0.366 mmol), DIPEA (79.00 mg, 0.61 mmol), putrescine (13.46 mg, 0.153 mmol), 20.00 mL of THF, and intermediate 13 (200 mg, 0.305 mmol) as starting materials, a light pink solid was obtained upon reaction, with a yield of 40%, m.p.: 115 - 117 °C. 1 HNMR (300 MHz, Chloroform - d) δ 8.16 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.1 Hz, 2H), 7.75–7.57 (m, 12H), 7.48 (p, J = 7.0 Hz, 4H), 7.32–7.23 (m, 4H), 7.11 (d, J = 8.1 Hz, 2H), 7.00–6.89 (m, 4H), 4.47 - 4.26 (m, 4H), 3.79 (d, J = 5.2 Hz, 6H), 3.68 (d, J = 4.6 Hz, 6H), 3.18 (q, J = 5.2 Hz, 4H), 2.54 (d, J = 7.1 Hz, 4H), 2.39 (t, J = 7.1 Hz, 4H), 1.84–1.64 (m, 4H); HRMS (ESI): found 1385.3264 (C 64 H 66 N8NaO 18 S4, [M + Na] + , requires 1385.3270).

[0057] Synthesis of dimethyl 2,2'-((((((4,4'-(ethane - 1,2 - diylbis(azanediyl))bis(4 - oxobutanoyl))bis(azanediyl))bis(4,1 - benzenesulfonyl))bis((2 - methoxy - 2 - oxoethyl)azanediyl))bis(naphthalene - 4,1 - diyl))bis(((4 - methoxyphenyl)sulfonyl)azanediyl))diacetate (15d):

[0058]

[0059] Using the same synthetic method as for intermediate 15a, with EDCI (70.20 mg, 0.366 mmol), HOBT (49.50 mg, 0.366 mmol), DIPEA (79.00 mg, 0.61 mmol), ethylenediamine (9.20 mg, 0.153 mmol), 20.00 mL of THF, and intermediate 14 (204 mg, 0.305 mmol) as starting materials, a light pink solid was obtained upon reaction, with a yield of 65%, m.p.: 119 - 120 °C. 11H NMR (300 MHz, DMSO-d6) δ 10.32 (d, J = 20.6 Hz, 4H), 8.16 (dd, J = 9.9, 4.8 Hz, 4H), 7.91 (s, 2H), 7.72 (dd, J = 27.6, 8.8 Hz, 8H), 7.55 (t, J = 7.9 Hz, 8H), 7.09 (d, J = 8.9 Hz, 4H), 7.03 (s, 4H), 4.51 (s, 4H), 3.88 (s, 6H), 3.59 (s, 6H), 3.12 (br, 4H), 2.37 (t, J = 7.2 Hz, 4H), 2.15 (t, J = 7.2 Hz, 4H), 1.92–1.80 (m, 4H); HRMS (ESI): found 1385.3263 (C 64 H 66 N8NaO 18 S4, [M+Na] + , requires 1385.3270).

[0060] Synthesis of dimethyl 2,2'-((((((4,4'-(propane-1,3-diylbis(azanediyl))bis(4-oxobutanoyl))bis(azanediyl))bis(4,1-phenylene)sulfonyl))bis((2-methoxy-2-oxoethyl)azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (15e):

[0061]

[0062] In the same way as the synthesis method of intermediate 15a, using EDCI (70.20 mg, 0.366 mmol), HOBT (49.50 mg, 0.366 mmol), DIPEA (79.00 mg, 0.61 mmol), trimethylenediamine (11.32 mg, 0.153 mmol), 20.00 mL of THF, and intermediate 14 (204 mg, 0.305 mmol) as raw materials, a light pink solid was obtained, with a yield of 55%, m.p.: 117 - 118 °C. 1HNMR(300MHz, DMSO-d6) δ 10.32(s, 2H), 10.15(s, 1H), 8.13(dd, J = 5.1, 2.9Hz, 4H), 7.84(t, J = 5.3Hz, 2H), 7.69(dd, J = 26.9, 8.9Hz, 8H), 7.51(dd, J = 11.8, 6.6Hz, 8H), 7.06(d, J = 8.9Hz, 4H), 6.98(s, 4H), 4.48(s, 4H), 3.84(s, 6H), 3.55(s, 6H), 3.04(q, J = 6.5Hz, 4H), 2.34(t, J = 7.4Hz, 4H), 2.12(t, J = 7.3Hz, 4H), 1.83–1.63(m, 4H), 1.58–1.46(m, 2H); HRMS(ESI): found 1399.3420 (C 65 H 68 N8NaO 18 S4, [M+Na] + , requires 1399.3427).

[0063] Synthesis of dimethyl 2,2'-((((((4,4'-(butane-1,4-diylbis(azanediyl))bis(4-oxobutanoyl))bis(azanediyl))bis(4,1-phenylenesulfonyl))bis((2-methoxy-2-oxoethyl)azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (15f):

[0064]

[0065] In the same synthetic method as intermediate 15a, using EDCI (70.20 mg, 0.366 mmol), HOBT (49.50 mg, 0.366 mmol), DIPEA (79.00 mg, 0.61 mmol), putrescine (13.46 mg, 0.153 mmol), 20.00 mL of THF, and intermediate 14 (204 mg, 0.305 mmol) as raw materials, a light pink solid was obtained, with a yield of 55%, m.p.: 113 - 114 °C. 11H NMR (300 MHz, DMSO-d6) δ 10.45 (s, 2H), 10.28 (s, 2H), 8.14–8.09 (m, 4H), 7.85 (t, J = 5.5 Hz, 2H), 7.76 (d, J = 8.9 Hz, 4H), 7.64 (d, J = 8.9 Hz, 4H), 7.51 (dd, J = 9.1, 7.0 Hz, 8H), 7.06 (d, J = 8.9 Hz, 4H), 6.98 (s, 4H), 4.48 (s, 4H), 3.84 (s, 6H), 3.55 (s, 6H), 3.02 (d, J = 5.2 Hz, 4H), 2.34 (t, J = 7.4 Hz, 4H), 2.11 (t, J = 7.3 Hz, 4H), 1.85–1.57 (m, 4H), 1.36 (s, 4H); HRMS (ESI): found 1413.3580 (C 66 H 70 N8NaO 18 S4, [M+Na] + , requires 1413.3583).

[0066] Synthesis of 4-methoxy-N-(4-(4-nitrophenyl)sulfamoyl)naphthalen-1-yl)benzenesulfonamide (16):

[0067]

[0068] In the same synthetic method as that of intermediate 11, using 4-nitrobenzenesulfonyl chloride (5.70 g, 25.50 mmol), pyridine (1.61 g, 20.30 mmol), 100.00 mL of toluene and intermediate 9 (5.580 g, 17.00 mmol) as raw materials, a gray solid was obtained by reaction, with a yield of 70%, m.p.: 149 - 150 °C. 1 1H NMR (500 MHz, DMSO-d6) δ 8.32–8.28 (m, 2H), 7.98–7.94 (m, 2H), 7.91 (dt, J = 5.6, 3.6 Hz, 2H), 7.66–7.61 (m, 2H), 7.48 (s, 2H), 7.44 (dd, J = 5.6, 3.4 Hz, 2H), 7.01–6.95 (m, 2H), 3.42 (s, 3H); HRMS (ESI): found 531.1006 (C 23 H 20 N3O7S2, [M+NH4] + , requires 531.1003).

[0069] Synthesis of 4-amino-N-(4-((4-methoxyphenyl)sulfamoyl)naphthalen-1-yl)benzenesulfonamide (17):

[0070]

[0071] In the same way as the synthesis method of intermediate 12, using intermediate 16 (5.00 g, 9.74 mmol), 50 mL of ethyl acetate, stannous chloride dihydrate (8.78 g, 38.94 mmol) and HCl (5 mL) as raw materials, a gray solid was obtained by reaction, and the yield was 85%; m.p.: 169 - 170 °C. 1 H NMR (500 MHz, DMSO-d6) δ 8.09 (dd, J = 5.5, 3.5 Hz, 2H), 7.72–7.61 (m, 4H), 7.53–7.46 (m, 2H), 7.44 (dd, J = 5.6, 3.5 Hz, 2H), 7.03–6.94 (m, 2H), 6.57–6.48 (m, 2H), 5.26 (d, J = 5.4 Hz, 1H), 4.90 (d, J = 5.5 Hz, 1H), 3.36 (s, 3H); HRMS (ESI): found 484.0996 (C 23 H 22 N3O5S2, [M + H] + , requires 484.0995).

[0072] Synthesis of 4-((4-(N-(4-((4-methoxyphenyl)sulfamoyl)naphthalen-1-yl)sulfamoyl)phenyl)amino)-4-oxobutanoic acid (18):

[0073]

[0074] In the same way as the synthesis method of intermediate 13, using succinic anhydride (828.00 mg, 8.28 mmol), intermediate 17 (2.00 g, 4.14 mmol) and 30 mL of toluene as raw materials, a light gray solid was obtained by reaction, and the yield was 80%; m.p.: 168 - 169 °C. 1 H NMR (300 MHz, DMSO-d6) δ 12.10 (s, 1H), 8.27 - 8.02 (m, 2H), 7.53 - 7.44 (m, 2H), 7.43 - 7.29 (m, 4H), 7.15 (d, J = 8.8 Hz, 2H), 7.07 - 6.98 (m, 4H), 3.76 (s, 3H), 2.67 - 2.56 (m, 4H); HRMS (ESI): found 584.1150 (C 27 H 26 N3O8S2, [M + H] +, requires 584.1156).

[0075] Synthesis of Methyl N-(4-((4-(4-((3-((tert-Butoxycarbonyl)amino)propyl)amino)-4-oxobutanamido)phenyl)sulfamoyl)naphthalen-1-yl)-N-((4-methoxyphenyl)sulfonyl)glycinate (19):

[0076]

[0077] In a reaction flask, EDCI (70.20 mg, 0.366 mmol), HOBT (49.50 mg, 0.366 mmol), DIPEA (79.00 mg, 0.61 mmol) and N-boc-1,3-diaminopropane (59 mg, 0.335 mmol) were dissolved in 20.00 mL of THF. The mixture was stirred for 3 minutes under an ice bath condition, then intermediate 13 (200 mg, 0.305 mmol) was added, and the reaction was continued with stirring for 3 h. The solvent THF was removed by distillation under reduced pressure. The reaction mixture was washed with ethyl acetate until there was no ultraviolet absorption. The filtrate was separated into layers, and the aqueous layer was discarded. The organic layer was washed twice with saturated NaCl solution, dried over anhydrous sodium sulfate, triturated, and a white solid was obtained by column chromatography with a yield of 70%; m.p.: 157 - 159 °C. 1 H NMR (300 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.21–8.11 (m, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.77–7.55 (m, 7H), 7.48 (dqd, J = 8.2, 6.9, 1.4 Hz, 2H), 7.31 (s, 1H), 7.29–7.10 (m, 2H), 6.99–6.92 (m, 2H), 6.86 (s, 1H), 4.48 (dd, J = 101.0, 17.8 Hz, 2H), 3.90 (s, 3H), 3.68 (s, 3H), 3.27 (d, J = 6.2 Hz, 2H), 3.11 (q, J = 6.3 Hz, 2H), 2.76–2.55 (m, 4H), 1.56 (d, J = 6.1 Hz, 2H), 1.46 (s, 9H); HRMS (ESI): found 812.2624 (C 38 H 46 N5O 11 S2, [M + H] + , requires 812.2630).

[0078] Synthesis of methyl N-(4-((4-(4-((3-(4-((4-(N-(4-((4-methoxyphenyl)sulfamoyl)naphthalen-1-yl)sulfamoyl)phenyl)amino)-4-oxobutanamido)propyl)amino)-4-oxobutanamido)phenyl)sulfamoyl)naphthalen-1-yl)-N-((4-methoxyphenyl)sulfonyl)glycinate (20):

[0079]

[0080] Dissolve 19 (800 mg, 0.985 mmol) in 10 mL of HCl-EA mixed solution in a reaction flask, and stir the reaction at room temperature for 2 h. Remove the solvent by distillation under reduced pressure. Dissolve the reaction mixture in 20 mL of THF, add EDCI (378 mg, 1.97 mmol), HOBT (266 mg, 1.97 mmol), DIPEA (382 mg, 2.96 mmol) and intermediate 18 (575 mg, 0.985 mmol), and stir the reaction under ice bath conditions for 3 h. Remove the solvent THF by distillation under reduced pressure. Wash the reaction mixture with ethyl acetate until there is no ultraviolet absorption. The filtrate is layered, and the aqueous layer is discarded. The organic layer is washed twice with saturated NaCl solution, dried over anhydrous sodium sulfate, triturated, and a light gray solid is obtained by column chromatography with a yield of 40%, m.p.: 127 - 129 °C. 1 H NMR (300 MHz, DMSO-d6) δ 10.35 (d, J = 12.6 Hz, 2H), 10.25 (s, 1H), 10.06 (d, J = 10.9 Hz, 2H), 8.14 (ddd, J = 10.3, 6.1, 3.4 Hz, 2H), 8.06–7.93 (m, 2H), 7.92–7.83 (m, 2H), 7.72 (t, J = 7.8 Hz, 3H), 7.66 (s, 3H), 7.56 (ddd, J = 9.0, 4.6, 2.0 Hz, 8H), 7.42 (dd, J = 7.0, 3.0 Hz, 2H), 7.11–7.04 (m, 2H), 7.00 (dd, J = 8.4, 6.1 Hz, 6H), 4.50 (s, 2H), 3.86 (s, 3H), 3.78 (s, 3H), 3.57 (s, 3H), 3.05 (d, J = 6.4 Hz, 4H), 2.57 (d, J = 7.6 Hz, 4H), 2.40 (t, J = 7.1 Hz, 4H), 1.52 (s, 2H); HRMS (ESI): found 1277.3080 (C 60 H 61 N8O 16 S4, [M + H] + , requires 1277.3083).

[0081] III. Preparation of Compounds 1 - 8

[0082] Synthesis of N - ((4 - aminophenyl)sulfonyl)-N-(4 - ((4 - methoxyphenyl)sulfamoyl)naphthalen - 1 - yl)glycine (1):

[0083]

[0084] Dissolve intermediate 12 (3.90 g, 7.00 mmol) in 10.00 mL of methanol, add 2M aqueous LiOH solution (2.93 g, 70.00 mmol), and stir at room temperature for 1 h. After monitoring the reaction to completion by TLC, evaporate the solvent under reduced pressure. Dissolve the residue in water, filter off the insoluble matter, and then adjust the pH to 4 - 5 with 1M dilute hydrochloric acid. A white precipitate gradually forms, which is filtered to obtain a white solid with a yield of 85%, m.p.: 196 - 197 °C. 1 H NMR (300 MHz, DMSO - d6) δ 12.22 (s, 1H), 10.48 (d, J = 26.8 Hz, 1H), 8.26 (d, J = 45.9 Hz, 2H), 7.79 (dd, J = 30.3, 8.6 Hz, 2H), 7.66–7.50 (m, 6H), 7.17–7.01 (m, 3H), 6.85 (s, 1H), 4.65–4.35 (m, 2H), 3.64–3.48 (m, 3H). 13 C NMR (125 MHz, MEOD) δ 170.81, 163.57, 152.98, 134.50, 134.11, 132.69, 130.37, 129.95, 128.99, 127.57, 126.32, 126.02, 125.33, 124.11, 123.05, 121.50, 113.74, 112.76, 54.91, 52.87. HRMS (ESI): found 564.0873 (C 25 H 23 N3NaO7S2, [M + Na] + , requires 564.0870); HPLC (90:10 methanol:water with 1‰ HAC): t R = 5.97 min, 95.1%.

[0085] Synthesis of 2,2'-((((((4,4'-(ethane - 1,2 - diylbis(azanylylidene))bis(4 - oxobutanoyl))bis(azanylylidene))bis(4,1 - benzenesulfonyl))bis(azanylylidene))bis(naphthalene - 4,1 - diyl))bis(((4 - methoxyphenyl)sulfonyl)azanylylidene))diacetic acid (2):

[0086] In the same synthetic method as that of Compound 1, using intermediate 15a (100.00 mg, 0.075 mmol), 5.00 mL of methanol, and 2 M LiOH aqueous solution (31.40 mg, 0.75 mmol) as raw materials, a light gray solid was obtained through reaction, with a yield of 70%, m.p.: 156 - 158 °C. 1 H NMR (300 MHz, Methanol-d4) δ 8.17–7.97 (m, 4H), 7.69–7.64 (m, 8H), 7.56 (d, J = 8.9 Hz, 4H), 7.46–7.38 (m, 4H), 7.19 (s, 4H), 6.98 (t, J = 9.5 Hz, 4H), 4.57 (dd, J = 17.8, 6.2 Hz, 2H), 4.34 (d, J = 17.8 Hz, 2H), 3.86 (d, J = 12.3 Hz, 6H), 3.53 (q, J = 7.0 Hz, 2H), 3.30 (s, 2H), 2.70–2.65 (m, 4H), 2.53 (t, J = 6.8 Hz, 4H); HRMS (ESI): found 1329.2637 (C 60 H 58 N8NaO 18 S4, [M+Na] + , requires 1329.2644); HPLC (90:10 methanol:water with 1‰ HAC): t R = 4.99 min, 95.6%.

[0087] Synthesis of 2,2'-((((((4,4'-(propane-1,3-diylbis(azanediyl))bis(4-oxobutanoyl))bis(azanediyl))bis(4,1-phenylsulfonyl))bis(azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (3):

[0088]

[0089] In the same synthetic method as that of Compound 1, using intermediate 15b (100.00 mg, 0.074 mmol), 5.00 mL of methanol, and 2 M LiOH aqueous solution (30.98 mg, 0.74 mmol) as raw materials, a light pink solid was obtained through reaction, with a yield of 78%, m.p.: 157 - 158 °C. 11H NMR (300 MHz, Methanol-d4) δ 8.06 (ddd, J = 9.6, 7.6, 3.9 Hz, 4H), 7.66 - 7.51 (m, 8H), 7.56 (d, J = 8.9 Hz, 8H), 7.47–7.41 (m, 4H), 7.19 (s, 4H), 4.57 (d, J = 17.8 Hz, 2H), 4.35 (d, J = 17.8 Hz, 2H), 3.86 (s, 6H), 3.21 (t, J = 6.7 Hz, 4H), 2.70 (t, J = 6.6 Hz, 4H), 2.55 (t, J = 6.8 Hz, 4H), 1.74–1.60 (m, 2H). 13 13C NMR (75 MHz, MeOD) δ 173.24, 171.86, 170.77, 163.54, 142.92, 134.90, 133.87, 133.49, 132.69, 130.33, 129.88, 128.94, 128.05, 127.55, 126.91, 126.40, 126.17, 125.60, 124.21, 122.76, 121.88, 118.70, 113.74, 54.90, 52.82, 36.44, 31.56, 30.17, 28.61. HRMS (ESI): found 1343.2802 (C 61 H 60 N8NaO 18 S4, [M+Na] + , requires 1343.2801); HPLC (90:10 methanol:water with 1‰ HAC): t R = 5.02 min, 95.4%.

[0090] Synthesis of 2,2'-((((((4,4'-(butane-1,4-diylbis(azanediyl))bis(4-oxobutanoyl))bis(azanediyl))bis(4,1-benzenesulfonyl))bis(azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (4):

[0091]

[0092] Using the same synthetic method as for Compound 1, with intermediate 15c (100.00 mg, 0.073 mmol), 5.00 mL of methanol, and 2 M LiOH aqueous solution (30.56 mg, 0.73 mmol) as raw materials, a light pink solid was obtained with a yield of 75%, m.p.: 136 - 138 °C. 11H NMR (300 MHz, Methanol-d4) δ 8.10–7.94 (m, 4H), 7.61 (s, 8H), 7.51 (d, J = 8.7 Hz, 4H), 7.42–7.35 (m, 4H), 7.14 (s, 4H), 6.93 (d, J = 8.7 Hz, 4H), 4.41 (dd, J = 64.6, 17.9 Hz, 4H), 3.80 (s, 6H), 3.12 (br, 4H), 2.64 (t, J = 6.6 Hz, 4H), 2.49 (t, J = 6.7 Hz, 4H), 1.46 (br, 4H). HRMS (ESI): found 1357.2961 (C 62 H 62 N8NaO 18 S4, [M+Na] + , requires 1357.2957); HPLC (90:10 methanol:water with 1‰ HAC): t R = 5.03 min, 97.1%.

[0093] Synthesis of 2,2'-((((((5,5'-(ethane-1,2-diylbis(azanediyl))bis(5-oxopentanoyl))bis(azanediyl))bis(4,1-phenylenesulfonyl))bis(azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (5):

[0094]

[0095] Using the same synthetic method as that of compound 1, with intermediate 15d (100.00 mg, 0.073 mmol), 5.00 mL of methanol, and 2 M LiOH aqueous solution (30.56 mg, 0.73 mmol) as raw materials, a light gray solid was obtained, with a yield of 70%, m.p.: 158 - 159 °C. 1 1H NMR (300 MHz, Methanol-d4) δ 8.03 (ddd, J = 9.6, 7.1, 3.2 Hz, 4H), 7.63 (s, 8H), 7.53 (d, J = 8.9 Hz, 4H), 7.39 (dd, J = 6.5, 3.2 Hz, 4H), 7.17 (s, 4H), 6.94 (d, J = 8.9 Hz, 4H), 4.42 (dd, J = 65.9, 17.8 Hz, 4H), 3.82 (s, 6H), 3.27 (s, 4H), 2.39 (t, J = 7.3 Hz, 4H), 2.25 (t, J = 7.2 Hz, 4H), 2.02–1.87 (m, 4H). HRMS (ESI): found 1357.2952 (C62 H 62 N8NaO 18 S4,[M+Na] + , requires 1357.2957); HPLC(90:10 methanol:water with 1‰ HAC): t R = 5.05 min, 98.5%.

[0096] Synthesis of 2,2'-((((((5,5'-(propane-1,3-diylbis(azanediyl))bis(5-oxopentanoyl))bis(azanediyl))bis(4,1-phenylene sulfonyl))bis(azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (6):

[0097] Using the same synthetic method as for Compound 1, with intermediate 15e (100.00 mg, 0.073 mmol), 5.00 mL of methanol, and 2 M LiOH aqueous solution (30.56 mg, 0.73 mmol) as starting materials, a light gray solid was obtained with a yield of 75%, m.p.: 144 - 145 °C. 1 H NMR(300 MHz, Methanol-d4) δ 8.09–7.97 (m, 4H), 7.70–7.59 (m, 8H), 7.53 (d, J = 8.9 Hz, 4H), 7.40 (dd, J = 6.6, 3.1 Hz, 4H), 7.17 (s, 4H), 6.95 (d, J = 8.9 Hz, 4H), 4.43 (dd, J = 66.1, 17.8 Hz, 4H), 3.82 (s, 6H), 3.19 (t, J = 6.7 Hz, 4H), 2.41 (t, J = 7.2 Hz, 4H), 2.26 (t, J = 7.2 Hz, 4H), 2.00–1.89 (m, 4H), 1.74–1.59 (m, 2H). HRMS(ESI): found 1371.3119 (C 63 H 64 N8NaO 18 S4,[M+Na] + , requires 1371.3114); HPLC(90:10 methanol:water with 1‰ HAC): t R = 5.04 min, 97.1%.

[0098] Synthesis of 2,2'-((((((5,5'-(butane-1,4-diylbis(azanediyl))bis(5-oxopentanoyl))bis(azanediyl))bis(4,1-phenylenesulfonyl))bis(azanediyl))bis(naphthalene-4,1-diyl))bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (7):

[0099] In the same way as the synthesis method of Compound 1, using intermediate 15f (100.00 mg, 0.072 mmol), 5.00 mL of methanol, and 2 M aqueous LiOH solution (30.14 mg, 0.72 mmol) as raw materials, a light gray solid was obtained by reaction, with a yield of 75%, m.p.: 131 - 133 °C. 1 HNMR (300 MHz, Methanol-d4) δ 8.10–7.96 (m, 4H), 7.70–7.59 (m, 4H), 7.53 (d, J = 8.5 Hz, 4H), 7.41 (dd, J = 9.3, 3.1 Hz, 4H), 7.37–7.12 (m, 8H), 6.96 (dd, J = 9.0, 2.7 Hz, 4H), 4.55 (dd, J = 17.8, 5.7 Hz, 2H), 4.34 (dd, J = 17.5, 14.2 Hz, 2H), 3.83 (s, 6H), 3.16 - 3.12 (m, 4H), 2.39 (t, J = 7.2 Hz, 4H), 2.24 (t, J = 7.3 Hz, 4H), 1.98–1.91 (m, 8H). HRMS (ESI): found 1385.3272 (C 64 H 66 N8NaO 18 S4, [M+Na] + , requires 1385.3270); HPLC (90:10 methanol:water with 1‰ HAC): t R = 5.04 min, 98.6%.

[0100] Synthesis of N-(4-((4-(4-((3-(4-((4-(N-(4-((4-methoxyphenyl)sulfamoyl)naphthalen-1-yl)aminosulfonyl)phenyl)amino)-4-oxobutanamido)propyl)amino)-4-oxobutanamido)phenyl)sulfamoyl)naphthalen-1-yl)-N-((4-methoxyphenyl)sulfonyl)glycine (8):

[0101]

[0102] The synthesis method was the same as that of Compound 1. Using intermediate 20 (640.00 mg, 0.50 mmol), 5.00 mL of methanol, and 2 M aqueous LiOH solution (209.30 mg, 5.00 mmol) as raw materials, a light gray solid was obtained with a yield of 54%, m.p.: 114 - 115 °C. 1 H NMR (300 MHz, Methanol - d4) δ 8.14–8.05 (m, 2H), 8.04–7.96 (m, 2H), 7.66 (m, 6H), 7.61–7.51 (m, 4H), 7.50–7.38 (m, 6H), 7.19 (m, 4H), 6.99 (dd, J=9.2, 7.5 Hz, 4H), 4.29 (s, 2H), 3.86 (s, 3H), 3.80 (s, 3H), 3.02 (d, J=6.7 Hz, 4H), 2.70 (t, J=6.7 Hz, 4H), 2.45 (t, J=6.9 Hz, 4H), 1.67 (p, J=6.7 Hz, 2H); HRMS (ESI): found 1263.2920 (C 59 H 59 N8O 16 S4, [M + H] + , requires 1263.2926). HPLC (90:10 methanol:water with 1‰ HAC): t R =5.04 min, 98.5%.

[0103] Example 2: Confirmation of the Activity of the Compound

[0104] I. Experimental Materials and Methods

[0105] 1. Fluorescence Polarization - based Keapl - Nrf2 PPI Competitive Inhibition Assay (FP Assay)

[0106] The FP experiment used a 384-well black plate, model 3676, produced by Corning, with a total reaction volume of 40 μL. The components were added to the wells in the order of 20 μL of compound, 10 μL of 4 nM FITC-labeled NRF29 peptide, and 10 μL of 12 nM KEAP1 Kelch domain protein. For the positive control, 20 μL of 200 nM unlabeled NRF29 peptide, 10 μL of 4 nM FITC-labeled NRF29 peptide, and 10 μL of 12 nM KEAP1 Kelch domain protein were used; for the negative control, 20 μL of HEPES buffer, 10 μL of 4 nM FITC-labeled NRF29 peptide, and 10 μL of 12 nM KEAP1 Kelch domain protein were used; for the blank control, 10 μL of 4 nM FITC-labeled NRF29 peptide and 30 μL of HEPES buffer were used. After adding all the components, the mixture was incubated at room temperature for 30 min. The detection instrument was SpectraMax Multi-Mode Microplate Reader (Molecular Devices). The excitation wavelength was selected as 485 nm and the emission wavelength was 545 nm. The polarization value was calculated by detecting the fluorescence intensity in the horizontal and vertical directions. The formula for the inhibition rate is: inhibition%=1-(P obs -P min ) / (P max -P min ). Among them, P obs is the polarization value of the compound well, P max is the polarization value of the negative control well, and P min is the polarization value of the blank well. Data processing was performed using GraghPad Prism, and the IC 50 of the compound was calculated using the concentration-inhibition rate curve.

[0107] 2. ARE luciferase reporter gene experiment

[0108] HepG2 cells (SCSP-510) were purchased from the Type Culture Collection Committee of the Chinese Academy of Sciences. The ARE-Luc Reporter Lentivirus was constructed by GeneCopoeia Biotechnology (Shanghai) Co., Ltd. A stable HepG2-ARE-Luc cell line was constructed by stably transfecting the ARE-Luc Reporter Lentivirus into HepG2 cells for the ARE luciferase reporter gene experiment.

[0109] HepG2-ARE-Luc cells in the logarithmic growth phase were digested with 0.1% trypsin to prepare a cell suspension. The cell suspension was added to a 96-well microplate at a cell concentration of 4×10 / mL, 100 μL per well. After overnight culture, the DMSO group was used as the negative control, t-BHQ and SFN were used as positive controls, and the cell culture lysis reagent was used as the background value. The compounds were allowed to act for 6 h and 12 h, with three replicates. Carefully aspirate the growth medium from the cells to be tested, add 100 μL of pre-cooled PBS to rinse the cells, remove it, and then add 30 μL of pre-diluted 5X lysis buffer (equilibrate the 1X buffer at room temperature before use) to each well. Lyse the cells on ice for 15 min, aspirate 20 μL of the supernatant and place it in luminoskan ascent (Thermo scientific, USA) for detection. Add 100 μL of luciferase detection reagent to each well and immediately read the value. The light intensity of this reaction can remain stable for nearly 1 minute and then decline slowly, with a half-life of approximately 10 minutes. The typical delay time is 2 seconds, and the typical reading time is 10 seconds. Finally, the measured data is divided by the DMSO group. The larger the ratio obtained, the better the induction ability.

[0110] II. Experimental Results

[0111] The IC 50 values of each compound are shown in the following table. The FP results showed that compared with the monovalent KEAP1-NRF2 inhibitor 1, the KEAP1-NRF2 PPI inhibitory activities of the divalent inhibitors were significantly improved, and the inhibitory activities of compounds 3 and 6 reached the single-digit nanomolar level. Through the fluorescence polarization-based KEAP1-NRF2 PPI competitive inhibition experiment, we confirmed that the divalent inhibitors could effectively competitively bind to KEAP1 at the target level to activate NRF2, and their inhibitory activities were better than those of the monovalent inhibitors.

[0112]

[0113]

[0114] To further verify whether this series of bivalent inhibitors can activate NRF2 more efficiently than monovalent inhibitors at the cellular level, we used the ARE luciferase reporter gene assay to test this series of bivalent inhibitors, with the classical covalent NRF2 activators tBHQ and SFN as positive controls. The basic principle of the ARE luciferase reporter gene assay is to insert the ARE gene into a plasmid in front of the luciferase expression sequence and transfect it into cells to stably express the plasmid. When a small molecule activates NRF2, NRF2 can enter the nucleus and bind to ARE, thereby upregulating the expression of luciferase. When luciferin is present in the substrate, luciferase can oxidize luciferin to emit fluorescence, and the activation ability of the compound on NRF2 can be indirectly reflected by the fluorescence intensity. Figure 1 The results of the luciferase reporter gene assay. Figure 1 It shows that after treating HepG2-ARE-Luc cells with the compound for 6 h, bivalent inhibitor 3 showed significant ARE induction activity at a concentration of 0.05 μM, while other compounds showed no obvious activity; after treating HepG2-ARE-Luc cells with the compound for 12 h, all compounds showed strong ARE induction activity, and among them, compound 3 had the best activity, and its EC 50 value (EC 50 = 55 nM) was approximately 7.5 times that of monovalent inhibitor 1 (EC 50 = 413 nM).

[0115] The function of the above embodiments is to specifically introduce the substantial content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A compound, which is a compound represented by general formula I or a pharmaceutically acceptable salt thereof: Among them, The substituent R and n, m are one of the combinations of the following serial numbers:

2. Use of the compound according to claim 1 for the preparation of a medicament for treating a disease that can be treated or alleviated by inhibiting KEAP1-NRF2.

3. The use according to claim 2, wherein the disease is inflammation.

4. The use according to claim 3, wherein the inflammation is acute inflammation.

5. According to the use of claim 4, the acute inflammation includes acute liver injury, acute lung injury, endotoxemia, sepsis, emphysema, acute respiratory distress syndrome, and novel coronavirus pneumonia.

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