A bis(naphthalenesulfonamide) derivative and its application

Abstract

This invention discloses a bis(naphthyl)sulfonamide derivative with the following general structural formula: The bis(naphthyl)sulfonamide derivative provided by this invention can achieve the self-degradation of Keap1 protein. This bis(naphthyl)sulfonamide derivative can serve as a new tool for the chemical knockout of Keap1 protein, and also broadens the application of E3 ubiquitin ligase tools in PROTAC research. This bis(naphthyl)sulfonamide derivative can serve as a homodivalent Keap1 degradation-targeting chimera for the prevention or treatment of oxidative stress-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a bis(naphthyl)sulfonamide derivative and its homo-PROTAC as a Keap1-targeted degradation chimera. Keap1 It is used for the prevention or treatment of diseases related to oxidative stress. Background Technology

[0002] Activating Nrf2 by targeting the Keap1 (Kelch like ECH-associated protein 1)-Nrf2 (nuclear factor E2-related factor 2)-ARE (antioxidant redox element) signaling pathway can treat oxidative stress-related diseases. On the one hand, Nrf2 covalent activators lack selectivity for Keap1 and other cysteine-rich targets ubiquitous in cells, and this non-specific binding may increase risks in clinical development [Med Chem Res, 2020, 29:846-867.]. On the other hand, blocking the protein-protein interaction between Keap1 and Nrf2 avoids the potential side effects of covalent modification activating Nrf2 [Medchemcomm, 2016, 8:286-294.]. Among these, naphthalenesulfonamide derivatives are the most widely studied structural type, with the most effective inhibitors reaching nanomolar levels. Furthermore, several compounds have been shown in animal models to activate Nrf2 in vivo, exerting therapeutic effects on various oxidative stress-related diseases [Bioorg Chem, 2020, 103:104172; Eur J Med Chem, 2021, 222:113599; J Med Chem, 2022, 65:8289-8302; J Med...]. [Chem, 2023, 66: 8267-8280; Redox Biol, 2023, 64: 102793.] However, while these inhibitors have made breakthrough progress, they have also faced many challenges in terms of targeting, drug-likeness, and biological function.

[0003] With the discovery in recent years that Keap1 is an important component of the Cullin3 E3 ligase complex, protein degradation-targeting chimeras based on the Keap1E3 ubiquitination system (PROTAC) have achieved the degradation of proteins such as Tau and BRD4 [Eur J MedChem, 2018, 146:251-259; Chin Chem Lett, 2021, 32:1197-1201; Sci Rep, 2020, 10:15543; J Am Chem Soc, 2021, 143:15073-15083.], showing promising research prospects. Therefore, PROTAC... Keap1 Theoretically, it can also be used to degrade anti-inflammatory and antioxidant stress targets. As an E3 ligase and a target protein that binds to small molecules of PPI inhibitor ligands, Keap1 can be considered a very suitable protein system for designing homo-PROTACs, where the two ligands of the E3 ligase are directly linked to form a ternary complex. Small molecules bind to the Keap1 target protein while recognizing and recruiting the Keap1 E3 ligase, thus achieving the self-degradation of Keap1. Summary of the Invention

[0004] The first objective of this invention is to provide a bis(naphthalenesulfonamide) derivative.

[0005] A second objective of this invention is to provide the application of the aforementioned bis(naphthalenesulfonamide) derivative in the preparation of isomorphic divalent Keap1 degradation-targeting chimeras.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a bis(naphthalenesulfonamide) derivative with the following general structural formula:

[0008]

[0009] Wherein, L is selected from one of the following groups:

[0010]

[0011] n is an integer selected from 1 to 30 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15);

[0012] m is selected from an integer from 1 to 30 (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15).

[0013] Preferably, in the bis(naphthalenesulfonamide) derivative, L is selected from one of the following groups:

[0014]

[0015] Most preferably, the bis(naphthalenesulfonamide) derivative is selected from one of the following compounds:

[0016]

[0017]

[0018]

[0019] In a second aspect, the present invention provides the application of the aforementioned bis(naphthalenesulfonamide) derivative in the preparation of isomorphic divalent Keap1 degradation-targeting chimeras.

[0020] The bis(naphthalene)sulfonamide derivatives exhibit good affinity for the Keap1 target and can serve as homo-bivalent Keap1 degradation-targeting chimeras (homo-PROTAC). Keap1 It enables the self-degradation of Keap1 protein in human breast cancer cells.

[0021] The human breast cancer cells mentioned are MDA-MB-231 cells.

[0022] A third aspect of the present invention provides the use of the aforementioned bis(naphthyl)sulfonamide derivative in the preparation of a medicament for the prevention or treatment of oxidative stress-related diseases.

[0023] The bis(naphthyl)sulfonamide derivatives, as isotype-divalent Keap1 degradation-targeting chimeras, have a protective effect on LPS-induced macrophages.

[0024] The macrophages were RAW264.7 cells.

[0025] The oxidative stress-related diseases are selected from diseases caused by inflammation, infectious diseases, malignant tumors, degenerative diseases of the central nervous system, immune diseases, cardiovascular diseases, autoimmune diseases, skin diseases, kidney-related diseases, gastrointestinal-related diseases, respiratory diseases, liver-related diseases, metabolic diseases such as diabetes, endocrine-related diseases, lymphatic system diseases, eye diseases, mental disorders, pain-related diseases, and aging-related diseases (the 11th edition of the WHO International Classification of Diseases classifies aging as a disease, coded MG2A; with the continuous deepening of research on aging, oxidative stress is considered to be one of the main drivers of cellular and tissue aging (Ref: Ageing Res Rev. 2024, 13:102582. Phytomedicine. 2024; 135:156239.).).

[0026] In a fourth aspect, the present invention provides the use of the aforementioned bis(naphthyl)sulfonamide derivative in the preparation of an anti-inflammatory medicament.

[0027] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0028] The bis(naphthyl)sulfonamide derivatives provided by this invention can achieve the self-degradation of Keap1 protein. These bis(naphthyl)sulfonamide derivatives can serve as a novel tool for the chemical knockout of Keap1 protein, and also broaden the application of E3 ubiquitin ligase tools in PROTAC research. Furthermore, these bis(naphthyl)sulfonamide derivatives can serve as homodivalent Keap1 degradation-targeting chimeras for the prevention or treatment of oxidative stress-related diseases. Attached Figure Description

[0029] Figure 1 This is a schematic diagram showing the detection results of the degradation efficiency of the compounds prepared in Examples 1-2 and 5-10 and the compound NXPZ-2 (3, 1, 0.3, 0.1 μM) in the patent application with publication number CN108752245A.

[0030] Figure 2 This is a schematic diagram illustrating the anti-inflammatory effects of the compound prepared in Example 8 and compound NXPZ-2 (10 μM) from the patent application with publication number CN108752245A in RAW264.7 cells. Detailed Implementation

[0031] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions or as recommended by the manufacturer. Reagents used in the synthesis experiments were purchased from Aladdin, Exploration Platform, Adamas, Bid Chemical, and Energie, and all reagents were of analytical or chemical purity. The nuclear magnetic resonance spectrometer was a Bruker Advance 500MHz or 600MHz (manufactured by Bruker GmbH, Germany), using tetramethylsilane (TMS) as an internal standard and DMSO-d6 as the solvent. Chemical shift (δ) and coupling constant (J) are expressed in ppm and Hz, respectively. Column chromatography used 200-300 mesh silica gel (manufactured by Qingdao Marine Chemical, China), and thin-layer chromatography (TLC) analysis used GF254 silica gel plates (manufactured by Qingdao Marine Chemical, China). ESI mass spectrometry was performed using an API-3000LC-MS mass spectrometer. High performance liquid chromatography (HPLC) was performed using a Shimadzu CTO-20A model column (YMC-Pack ODS-A, S-5μm, 250×10.0 mm l.D), with a mobile phase of 90% methanol / 10% water, a flow rate of 2 mL / min, and a purity greater than 95%.

[0033] The synthetic route of intermediate 4-amino-N-(4-((4-methoxyphenyl)sulfonamido)naphth-1-yl)benzenesulfonamide (P7) is shown below. Refer to patent application with publication number CN115093444A, entitled: A small molecule compound of naphthalenesulfonamide phosphate and its application.

[0034]

[0035] Example 1

[0036] 2-({[4-({5-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio}phenyl)amino]-1,5-dioxylidenepentyl}amino)phenyl]dioxylidene-λ 6 ⇌ thioyl}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthal-1-yl}amino)acetamide (compound 1)

[0037]

[0038] Step 1, Preparation of compound 1a

[0039] Compound P7 (500 mg, 1.03 mmol) was dissolved in DMF (5 mL), and glutaric acid (273 mg, 2.07 mmol), HATU (1.180 g, 3.10 mmol), and DIPEA (426 μL, 3.10 mmol) were added at room temperature. After the addition was complete, the reaction was carried out at 60 °C for 3 h. TLC (DCM / MeOH = 20:1, R f =0.4) Monitoring showed that the reaction was complete. The reaction solution was washed with dilute hydrochloric acid (1 mol / L, 30 mL × 3) and extracted with ethyl acetate (× 3). The organic phase was dried with Na2SO4, filtered, the solvent was removed under reduced pressure, and dried to give compound 1a (213 mg, 0.20 mmol, yield 19%), which was used directly in the next step.

[0040] The second step is the preparation of compound 1.

[0041] Compound 1a (213 mg, 0.20 mmol), 2-bromoacetamide (221 mg, 1.60 mmol), K₂CO₃ (222 mg, 1.60 mmol), and KI (33 mg, 0.20 mmol) were dissolved in DMF (5 mL). After addition, the mixture was reacted at 65 °C for 2 h. TLC (DCM / MeOH = 10:1, R) was performed. f =0.4) Monitoring showed that the reaction was complete. The reaction solution was washed with dilute hydrochloric acid (1 mol / L, 30 mL × 3) and extracted with ethyl acetate (× 3). The organic phase was dried with Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH = 5:1) to give target compound 1, a white solid (52 mg, 0.04 mmol, yield 20%). 11H NMR (600 MHz, DMSO-d6) δ 10.69 (d, J = 13.38 Hz, 1H), 10.60 (d, J = 13.20 Hz, 1H), 8.34 - 8.31 (m, 2H), 8.21 - 8.20 (m, 2H), 7.91 - 7.88 (t, J = 15.60 Hz, 2H), 7.82 - 7.79 (t, J = 7.50 Hz, 2H), 7.59 - 7.54 (m, 12H), 7.37 (s, 2H), 7.32 (d, J = 9.72 Hz, 2H), 7.13 (d, J = 8.64 Hz, 2H), 7.05 (d, J = 8.46 Hz, 2H), 7.01 (d, J = 9.42 Hz, 2H), 6.94 (d, J = 10.44 Hz, 2H), 6.84 - 6.80 (dd, J = 7.98 Hz, J = 15.24 Hz, 2H), 4.32 - 4.20 (m, 7H), 4.15 (d, J = 16.38 Hz, 1H), 3.88 (s, 3H), 3.82 (s, 3H), 2.55 - 2.52 (t, J = 7.14 Hz, 2H), 2.47 - 2.45 (m, 2H), 2.00 - 1.94 (m, 2H). 13 13C NMR (151 MHz, DMSO-d6) δ 171.92, 168.78, 168.73, 162.93, 162.79, 143.75, 143.62, 137.09, 137.03, 136.94, 133.19, 133.10, 132.97, 131.30, 130.70, 130.21, 130.00, 129.46, 129.20, 129.01, 128.94, 126.52, 126.31, 125.91, 125.71, 124.85, 124.77, 124.66, 118.49, 118.29, 114.21, 55.86, 55.75, 54.10, 53.99, 39.91, 39.77, 39.64, 39.50, 39.36, 39.22, 39.08, 35.69, 20.67. MS (ESI, positive) m / z calcd for C 59 H 58 N 10 O 16 S4[M + H] + : 1291.3; found 1291.3. HPLC analysis: retention time = 7.2 min; peak area, >95% (210, 254 nm).

[0042] Example 2

[0043] 2-({[4-({6-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio}phenyl]amino]-1,6-dioxane-hexyl}amino]phenyl]dioxane-λ6-thio}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthalene-1-yl}amino)acetamide (compound 2)

[0044]

[0045] In Example 1, glutaric acid was replaced with adipic acid, and all other steps were the same as in Example 1, to obtain target compound 2, a white solid (38 mg, 0.03 mmol, yield 26%). 1 H NMR (600MHz, DMSO-d6) δ10.43(d,J=8.10Hz,1H),10.35(d,J=7.74Hz,1H),8.33-8.30(m,2H),8.21-8.18 (dd,J=5.34Hz,J=9.84Hz,2H),7.86-7.83(m,2H),7.77-7.75(m,2H),7.60-7.55(m,12H),7.33(s,2H),7. 2(d,J=6.42Hz,2H),7.12(d,J=8.94Hz,2H),7.06-7.01(m,5H),6.94(d,J=10.62Hz,2H),6.87-6.81(m,2H ),4.31-4.21(m,7H),4.17-4.15(m,1H),3.88(s,3H),3.82(s,3H),2.46-2.40(m,4H),1.72-1.65(m,4H). 13C NMR (151MHz, DMSO-d6) δ172.57,172.50,169.39,169.34,169.28,163.37,163.26,144.02,143.89,137.50,137. 34,133.59,133.49,133.39,131.94,131.29,130.66,130.45,129.83,129.72,129.47,129.41,127.05,126.89, 126.43,126.23,125.20,125.09,124.98,118.96,118.75,114.69,114.62,56.22,56.15,54.54,54.45,54.41,4 1.92,41.78,41.64,40.19,40.06,39.92,39.78,39.64,39.50,39.36,36.80,25.05.MS(ESI,positive)m / zcalcd for C 60 H 60 N 10 O 16 S4[M+Na] + :1327.3; found 1327.3. HPLC analysis: retention time=6.5min; peak area,>95%(210,254nm).

[0046] Example 3

[0047] 2-({[4-({7-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio}phenyl]amino]-1,7-dioxane-heptyl}amino]phenyl]dioxane-λ 6 ⇌ thioyl}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthalene-1-yl}amino)acetamide (compound 3)

[0048]

[0049] In Example 1, glutaric acid was replaced with pimelic acid, and all other steps were the same as in Example 1, to obtain target compound 3, a white solid (93 mg, 0.07 mmol, yield 32%). 11H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 10.31 (s, 1H), 8.33 - 8.30 (m, 2H), 8.20 - 8.18 (m, 2H), 7.84 (d, J = 10.14 Hz, 2H), 7.75 (d, J = 9.96 Hz, 2H), 7.60 - 7.54 (m, 12H), 7.33 - 7.28 (m, 4H), 7.12 (d, J = 10.14 Hz, 2H), 7.06 - 7.02 (m, 5H), 6.95 (d, J = 10.38 Hz, 2H), 6.86 - 6.81 (dd, J = 9.66, J = 22.92 Hz, 2H), 4.31 - 4.14 (m, 8H), 3.89 (s, 3H), 3.83 (s, 3H), 2.43 - 2.37 (m, 4H), 1.67 - 1.65 (m, 4H), 1.41 - 1.39 (m, 2H). 13 13C NMR (151 MHz, DMSO-d6) δ 172.70, 172.61, 169.32, 169.25, 163.36, 163.25, 144.04, 143.91, 137.49, 137.33, 133.58, 133.47, 133.38, 131.93, 131.27, 130.66, 130.44, 129.85, 129.71, 129.45, 127.03, 126.89, 126.45, 126.24, 125.18, 124.96, 118.91, 118.70, 114.68, 114.62, 56.22, 56.15, 54.52, 54.40, 42.06, 41.93, 41.79, 41.65, 40.22, 40.08, 39.94, 39.80, 39.66, 39.52, 39.38, 36.97, 36.81, 29.48, 29.36, 29.24, 29.09, 28.73, 25.35, 25.14. MS (ESI, positive) m / z calcd for C 61 H 62 N 10 O 16 S4[M + H] + : 1319.3; found 1319.3. HPLC analysis: retention time = 7.6 min; peak area, >95% (210, 254 nm).

[0050] Example 4

[0051] 2-({[4-({16-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thioyl}phenyl)amino]-1,16-dioxane-hexadecyl}amino)phenyl]dioxane-λ 6 ⇌ thioyl}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthalene-1-yl}amino)acetamide (compound 4)

[0052]

[0053] In Example 1, glutaric acid was replaced with hexadecanoic acid, and everything else was the same as in Example 1, to obtain target compound 4, a white solid (158 mg, 0.11 mmol, yield 46%). 1 H NMR (600MHz, DMSO-d6) δ10.36(s,1H),10.27(s,1H),8.33-8.30(m,2H),8.21-8.18(m,2H),7.83(d,J=8. 76Hz,2H),7.74(d,J=8.58Hz,2H),7.59-7.54(m,12H),7.32(s,2H),7.28(s,2H),7.13-7.10(m,2H),7.06 -7.01(m,5H),6.94(d,J=10.2Hz,2H),6.86-6.81(dd,J=8.04Hz,J=21.78Hz,2H),4.31-4.20(m,7H),4.15 (d,J=16.32Hz,1H),3.88(s,3H),3.83(s,3H),2.39-2.33(m,4H),1.63-1.57(m,4H),1.31-1.25(m,22H). 13C NMR (151MHz, DMSO-d6) δ172.79,172.71,169.32,169.26,163.36,163.25,144.04,143.91,137.49,137. 33,133.58,133.47,133.37,131.86,131.20,130.65,130.44,129.84,129.69,129.43,127.03,126.87,1 26.41,126.20,125.16,118.91,118.70,114.68,114.61,56.20,56.15,54.52,54.41,40.20,40.06,39. 92,39.78,39.64,39.50,39.36,36.96,29.48,29.36,29.24,29.09,25.35,25.14.MS(ESI,positive)m / z calcd forC 70 H 80 N 10 O 16 S4[M+Na] + :1467.4; found 1467.4. HPLC analysis: retention time=6.7min; peak area,>95%(210,254nm).

[0054] Example 5

[0055] 2-{[(4-{[3-({3-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio}phenyl]amino]-3-oxylidenepropyl}oxy)propionyl]amino}phenyl]dioxylidene-λ 6 [-thio]{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ] 6 [Thio]amino]naphth-1-yl}amino}acetamide (compound 5)

[0056]

[0057] In Example 1, glutaric acid was replaced with propionic acid-polyethylene glycol-propionic acid, and the rest was the same as in Example 1, to obtain target compound 5, a white solid (228 mg, 0.17 mmol, yield 33%). 1H NMR (600MHz, DMSO-d6) δ10.47(d,J=5.04Hz,1H),10.38(d,J=4.92Hz,1H),8.32-8.30(m,2H),8.21-8.18( m,2H),7.84(d,J=8.34Hz,2H),7.75(d,J=8.22Hz,2H),7.61-7.55(m,12H),7.33(s,2H),7.28(s,2H),7.12 (d,J=8.82Hz,2H),7.06-7.01(m,5H),6.94(d,J=8.82Hz,2H),6.88-6.82(dd,J=7.98Hz,J=22.8Hz,2H),4. 32-4.21(m,7H),4.17(d,J=16.32Hz,1H),3.88(s,3H),3.82(s,3H),3.79-3.71(m,4H),2.68-2.60(m,4H). 13 C NMR(151MHz,DMSO-d6)δ170.66,169.32,163.38,163.27,143.85,137.51 ,137.30,133.58,131.49,130.65,130.44,129.73,129.47,127.06,125. 19,124.94,118.99,118.80,114.62,66.64,56.23,56.15,54.41,40.17,40.03,39.89,39.75,39.61,39.47,39.33,37.55.MS(ESI,positive)m / z calcd for C 60 H 61 N 10 O 17 S4[M+H] + :1321.3; found 1321.3. HPLC analysis: retention time=7.2min; peak area,>95%(210,254nm).

[0058] Example 6

[0059] 2-({[4-({10-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio}phenyl]amino]-1,10-dioxane-4,7-dioxanedec-1-yl}amino]phenyl]dioxane-λ 6⇌ thioyl}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthalene-1-yl}amino)acetamide (compound 6)

[0060]

[0061] In Example 1, glutaric acid was replaced with propionic acid-diethylene glycol-propionic acid, and the rest was the same as in Example 1, to obtain target compound 6, a white solid (194 mg, 0.14 mmol, yield 28%). 1 H NMR (600MHz, DMSO-d6) δ10.48(s,1H),10.39(s,1H),8.33-8.30(m,2H),8.21-8.18(m,2H),7.84(d,J=8.5 2Hz,2H),7.76(d,J=8.52Hz,2H),7.60-7.53(m,12H),7.33(s,2H),7.29(s,2H),7.12(d,J=8.94Hz,2H),7. 05-7.02(m,5H),6.95(d,J=9.96Hz,2H),6.86-6.81(dd,J=7.98Hz,J=21.78Hz,2H),4.30-4.20(m,7H),4. 15(d,J=16.38Hz,1H),3.88(s,3H),3.82(s,3H),3.75-3.69(m,4H),3.57-3.52(m,4H),2.65-2.60(m,4H). 13 C NMR(151MHz,DMSO-d6)δ170.70,170.63,169.36,169.30,169.26,163.38,163.26,143.87,143.74,137.50,137.32,13 3.58,133.47,133.38,133.32,132.05,131.43,130.65,130.43,129.82,129.71,129.46,129.40,127.03,126.89,126. 62,126.43,126.23,125.19,125.10,124.95,118.98,118.79,114.69,114.62,70.04,70.01,66.84,56.24,56.15,54. 51,54.44,54.39,41.92,41.79,41.65,40.20,40.06,39.93,39.79,39.65,39.51,39.37,37.67.MS(ESI,positive)m / z calcd for C62 H 64 N 10 O 18 S4[M+H] + :1365.3; found 1365.3. HPLC analysis: retention time=6.5min; peak area,>95%(210,254nm).

[0062] Example 7

[0063] 2-({[4-({13-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio[]phenyl]amino-1,13-dioxane-4,7,10-trioxatridecane-1-yl]amino]phenyl]dioxane-λ 6 ⇌ thioyl}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthalene-1-yl}amino)acetamide (compound 7)

[0064]

[0065] In Example 1, glutaric acid was replaced with propionic acid-polyethylene glycol-propionic acid, and the rest was the same as in Example 1, to obtain target compound 7, a white solid (44 mg, 0.03 mmol, yield 25%). 1 H NMR (600MHz, DMSO-d6) δ10.44(s,1H),10.36(s,1H),8.33-8.30(m,2H),8.21-8.18(m,2H),7.84(d,J=8.8 8Hz,2H),7.75(d,J=8.58Hz,2H),7.60-7.54(m,12H),7.32(s,2H),7.28(s,2H),7.12(d,J=8.94Hz,2H),7. 06-7.01(m,5H),6.94(d,J=10.14Hz,2H),6.87-6.82(dd,J=8.04Hz,J=22.2Hz,2H),4.30-4.20(m,7H),4. 16(d,J=16.38Hz,1H),3.88(s,3H),3.83(s,3H),3.74-3.68(m,4H),3.53-3.50(m,8H),2.64-2.58(m,4H), 13C NMR(151MHz,DMSO-d6)δ170.75,170.68,169.40,169.34,169.30,163.39,163.26,143.85,143.73,137.50,137.31,133.57,133.46 ,133.38,133.31,132.04,131.42,130.65,130.44,129.79,129.71,129.46,129.36,127.56,127.05,126.88,126.60,126.42,126.2 3,125.18,125.09,124.95,118.99,118.80,118.55,114.69,114.62,113.30,70.14,70.10,66.81,56.24,56.14,54.51,54.44,54.4 0,42.06,41.92,41.78,41.64,41.50,40.16,40.02,39.88,39.74,39.60,39.46,39.32,37.70,37.67,29.41.MS(ESI,positive)m / z calcd for C 64 H 68 N 10 O 19 S4[M+H] + :1409.4; found1409.4. HPLC analysis: retention time=6.5min; peak area,>95%(210,254nm).

[0066] Example 8

[0067] 2-({[4-({16-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio[]phenyl]amino]-1,16-dioxane-4,7,10,13-tetraoxahexadecane-1-yl]amino]phenyl]dioxane-λ 6 ⇌ thioyl}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthalene-1-yl}amino)acetamide (compound 8)

[0068]

[0069] In Example 1, glutaric acid was replaced with propionic acid-tetraethylene glycol-propionic acid, and the rest was the same as in Example 1, to obtain target compound 8, a white solid (95 mg, 0.07 mmol, yield 27%). 1 H NMR(600MHz,DMSO-d6)δ10.45(s,1H),10.36(s,1H),8.34-8.32(m,2H),8.22-8.19(m,2H),7.85(d ,J=8.88Hz,2H),7.76(d,J=8.52Hz,2H),7.61-7.55(m,12H),7.33-7.29(m,4H),7.13(d,J=8.88Hz ,2H),7.06-7.02(m,5H),6.95(d,J=10.08Hz,2H),6.87-6.82(dd,J=7.98Hz,J=22.08Hz,2H),4.31 -4.15(m,8H),3.89(s,3H),3.83(s,3H),3.74-3.69(m,4H),3.54-3.48(m,12H),2.65-2.60(m,4H), 13 CNMR(151MHz,DMSO-d6)δ170.73,170.66,169.36,169.30,169.26,163.38,163.26,143.87,143.75,137.51,137.32,133.58, 133.48,133.39,133.32,132.04,131.41,130.66,130.44,129.82,129.71,129.46,129.39,127.03,126.88,126.41,126.22,1 25.20,125.10,124.95,118.97,118.78,114.68,114.62,70.19,70.14,70.10,67.17,66.83,56.24,56.15,54.52,54.44,54. 41,41.92,41.78,41.64,40.20,40.06,39.92,39.78,39.64,39.51,39.37,37.71,33.33,29.43,29.11.MS(ESI,positive)m / z calcd for C 66 H 72 N 10 O 20 S4[M+H] +:1453.4; found 1453.4. HPLC analysis: retention time=6.4min; peak area,>95%(210,254nm).

[0070] Example 9

[0071] 2-({[4-({19-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio[]phenyl]amino]-1,19-dioxane-4,7,10,13,16-pentaoxane-1-yl]amino]phenyl]dioxane-λ 6 ⇌ thioyl}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthalene-1-yl}amino)acetamide (compound 9)

[0072]

[0073] In Example 1, glutaric acid was replaced with propionic acid-pentaethylene glycol-propionic acid, and all other steps were the same as in Example 1, to obtain target compound 9, a white solid (47 mg, 0.03 mmol, yield 20%). 1 H NMR (600MHz, DMSO-d6) δ10.57(s,1H),10.49(s,3H),8.34-8.31(m,2H),8.21-8.19(m,2H),7.87(d,J=8. 82Hz,2H),7.78(d,J=8.58Hz,2H),7.59-7.54(m,12H),7.34-7.29(m,4H),7.13(d,J=8.94Hz,2H),7.06-7 .00(m,5H),6.94(d,J=10.02Hz,2H),6.85-6.81(dd,J=7.98Hz,J=19.08Hz,2H),4.31-4.19(m,7H),4.15 (d,J=16.38Hz,1H),3.89(s,3H),3.83(s,3H),3.74-3.69(m,4H),3.54-3.47(m,16H),2.66-2.60(m,4H). 13C NMR(151MHz,DMSO-d6)δ175.44,173.98,168.07,167.94,148.56,148.44,142.19,142.00,1 38.28,138.17,136.70,136.06,135.36,135.14,134.42,134.16,134.03,131.74,131.55,13 1.07,130.87,129.90,123.65,123.46,119.38,119.31,74.87,74.79,71.52,60.94,60.84,5 9.21,59.11,44.95,44.78,44.61,44.45,44.28,44.11,43.94,42.38.MS(ESI,positive)m / z calcd for C 68 H 76 N 10 O 21 S4[M+H] + :1497.4; found 1497.4. HPLC analysis: retention time=7.1min; peak area,>95%(210,254nm).

[0074] Example 10

[0075] 2-({[4-({31-[(4-{[(formamidomethyl){4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-thio]amino]naphthal-1-yl}amino]dioxane-λ 6 [-thio[]phenyl]amino]-1,31-dioxane-4,7,10,13,16,19,22,25,28-nonoxatrione-1-yl]amino]phenyl]dioxane-λ 6 ⇌ thioyl}{4-[(formamidomethyl)[(4-methoxyphenyl)dioxane-λ 6 [-Thio]amino]naphthalene-1-yl}amino)acetamide (compound 10)

[0076]

[0077] In Example 1, glutaric acid was replaced with propionic acid-non-polyethylene glycol-propionic acid, and all other steps were the same as in Example 1, to obtain target compound 10, a white solid (62 mg, 0.04 mmol, yield 21%). 11H NMR (600 MHz, DMSO-d6) δ 10.45 (s, 1H), 10.37 (s, 1H), 8.33 - 8.31 (m, 2H), 8.21 - 8.18 (m, 2H), 7.84 (d, J = 8.82 Hz, 2H), 7.75 (d, J = 8.64 Hz, 2H), 7.60 - 7.54 (m, 11H), 7.32 - 7.24 (m, 4H), 7.12 (d, J = 8.88 Hz, 2H), 7.06 - 7.00 (m, 5H), 6.94 (d, J = 10.08 Hz, 2H), 6.86 - 6.81 (dd, J = 8.04 Hz, J = 21.06 Hz, 2H), 4.30 - 4.18 (m, 7H), 4.15 (d, J = 16.38 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 3.75 - 3.69 (m, 4H), 3.55 - 3.46 (m, 30H), 2.65 - 2.59 (m, 4H), 1.23 (s, 2H). 13 13C NMR (151 MHz, DMSO-d6) δ 170.72, 170.65, 169.33, 169.27, 169.23, 163.38, 163.25, 143.88, 143.76, 137.51, 137.32, 133.59, 133.48, 133.39, 132.00, 131.38, 130.65, 130.43, 129.81, 129.70, 129.45, 129.38, 127.01, 126.85, 126.38, 126.18, 125.20, 125.10, 124.96, 118.95, 118.76, 114.68, 114.61, 113.27, 72.71, 70.18, 70.11, 66.84, 60.66, 56.25, 56.15, 54.51, 54.43, 41.92, 41.78, 41.64, 40.21, 40.07, 39.93, 39.80, 39.66, 39.52, 39.38, 37.71, 29.42. MS (ESI, positive) m / z calcd for C 76 H 92 N 10 O 25 S4 [M + H] + : 1673.5; found 1673.5. HPLC analysis: retention time = 7.1 min; peak area, >95% (210, 254 nm).

[0078] Example 11

[0079] Determination of Keap1-Nrf2 PPI inhibitory activity by fluorescence polarization method

[0080] The equilibrium dissociation constant K of each compound was determined using fluorescence anisotropy. D2 FITC-βAla-DEETGEF-OH was used as a competitive fluorescent probe. It was co-incubated with different concentrations of Keap1 (8 semi-serial dilutions starting from 10 μM) at room temperature for 60 min in a buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl, and 1 mM 1,4-dithionitro alcohol (DTT) for 60 min. The binding affinity was characterized as K... D1 The protein concentration required for 75% binding to the fluorescent probe was determined based on the binding curve. Compounds of different concentration gradients were added to a buffer containing the fluorescent probe and Keap1, and incubated at room temperature for 60 min. Excitation wavelength was 485 nm, and emission wavelength was 535 nm. The anisotropy values ​​of the compounds and Keap1 were read using SpectraMax M5 microplates. The binding constant of the compounds to Keap1 protein was obtained by fitting with Mathematica 7 (Wolfram Research Inc.). D2 The results are shown in Table 1. The final protein concentration was 600 nM, the final probe concentration was 10 nM, and the compound concentration was serially diluted starting from 50 μM. Three replicates were set for each concentration point, and the DMSO content was less than 2%.

[0081] The structural formula of compound NXPZ-2 is shown below:

[0082]

[0083] Table 1. PPI inhibitory activity of the compounds (Keap1-Nrf2).

[0084]

[0085]

[0086] a The target affinity K for each compound was determined using fluorescence anisotropy assay. D2 The value indicates that the lower the value, the higher the activity of the compound.

[0087] Fluorescence polarization experiments showed that, except for compound 4 which was inactive, compound 3 exhibited low activity (K0). D2 (At the micromolar level), the remaining compounds all exhibited similar or better inhibitory activity to the parent compound NXPZ-2, and their inhibitory activity was at the nanomolar level. Ki of compounds 1, 5, 6, 8, and 10 D2All values ​​were <100 nM. The affinities of compounds 7 and 9 were 136 nM and 111 nM, respectively. Among them, compound 8, with the best activity, showed a Keap1-Nrf2 PPI inhibitory activity of 41 nM, a significant improvement compared to the parent compound NXPZ-2 (which had a Keap1-Nrf2 PPI inhibitory activity of 53 nM). This method validated the biomolecular interaction between these bis(naphthyl)sulfonamide derivatives and the Keap1 protein, preliminarily demonstrating that they may act as a homodivalent Keap1 degradation-targeting chimera, forming a strong binding mode with Keap1, laying a theoretical foundation for subsequent research.

[0088] Example 12

[0089] Detection of compound degradation efficiency

[0090] The degradation efficiency of the compounds prepared in the embodiments of the present invention was tested.

[0091] Human breast cancer MDA-MB-231 cells were seeded in 6-well plates using DMEM + 10% FBS medium and incubated at 37°C with 5% CO2 for 24 h to allow complete adhesion. The supernatant was then removed. 2 mL of fresh medium containing the compounds prepared in Examples 1-2 and 5-10 (DMSO content less than 0.1%) was added to each well, and the plates were incubated for 24 h. A blank control group was prepared using complete medium. The supernatant was aspirated, and the cells were washed with ice-cold PBS. Cell proteins were then extracted, and NP40 lysis buffer (Beyotime, ST2045) containing 1% v / v protease, phosphatase, and PMSF inhibitor (weiaoobio, WB0122) was added and the plates were placed on ice for 15 min. The cell lysates were collected and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant protein concentration was determined using a BCA protein quantification kit (Biosharp, BL521A), and then 5× loading buffer was added to the sample. Protein extracts were separated by SDS-PAGE on a 10-15% polyacrylamide gel and transferred to nitrocellulose (NC) membranes (Millipore) via electrotransfer. The NC membranes containing proteins were blocked with 5% skim milk at room temperature for 1 h. The milk blocking solution was then aspirated, and the membranes were washed three times with PBS (PBST) containing 0.1% Tween-20 for 5 min each time. Immediately afterward, diluted primary antibody (Keap1, Santa Cruz, sc-365626; GAPDH, Proteintech, 60004-1-Ig) was added, and the membranes were incubated overnight at 4°C. The primary antibody dilution was aspirated, and the membranes were washed three times with PBST for 5 min each time. Immediately afterwards, diluted goat anti-mouse secondary antibody (…) was added… 800CW (ab216772), the membrane was incubated at room temperature for 1 h, the secondary antibody dilution buffer was aspirated, and the membrane was washed three times with PBST for 5 min each time. Images were captured using an Odyssey system (Li-CorBiosciences) and then analyzed using ImageJ software. The results are as follows: Figure 1 As shown. Figure 1 This is a schematic diagram showing the detection results of the degradation efficiency of the compounds prepared in Examples 1-2 and 5-10 and the compound NXPZ-2 (3, 1, 0.3, 0.1 μM) in the patent application with publication number CN108752245A. In the diagram, A is a schematic diagram of the degradation of Keap1 protein in MDA-MB-231, B is a schematic diagram of grayscale analysis statistics at a concentration of 3 μM, and C is a schematic diagram of grayscale analysis statistics at a concentration of 0.1 μM. *p<0.05, ***p<0.001 are compared with the NXPZ-2 group.

[0092] Figure 1 Immunoblot experiments in protein A showed that the compounds in this invention could degrade Keap1 to varying degrees in human breast cancer cells, while the parent compound NXPZ-2 did not significantly degrade the protein. This preliminarily verifies that the bis(naphthyl)sulfonamide derivative in this invention can serve as a homodivalent Keap1 degradation-targeting chimera. Figure 1 B shows that at 3 μM, all compounds except those in Example 6 degraded Keap1 to varying degrees. Among them, compounds 5, 7, 9 and 10 degraded more than 50% of the Keap1 protein, with compound 5 showing the most significant degradation at 3 μM, reaching 95%. Figure 1 C shows that at a lower concentration of 0.1 μM, most compounds still retained the ability to reduce Keap1 protein levels, while compound 8 showed an 89% degradation rate at 0.1 μM. In summary, Figure 1 The high degradation efficiency of this type of bis(naphthalene)sulfonamide derivatives on Keap1 at four different concentrations was verified and analyzed.

[0093] Example 13

[0094] In vitro anti-inflammatory activity of compound 8

[0095] The in vitro anti-inflammatory activity of the compound prepared in Example 8 of this invention was tested. Mouse mononuclear macrophages RAW264.7 were seeded in 96-well plates using DMEM + 10% FBS medium and incubated at 37°C with 5% CO2 for 24 h to allow complete adhesion. The supernatant was then removed. 200 μL of fresh medium was added to each well. The blank group used complete medium, the model group used medium containing lipopolysaccharide (LPS, 1 μg / mL), and the drug-treated group used medium containing LPS (1 μg / mL) and the compound prepared in Example 8 (10 μM) or the compound NXPZ-2 (10 μM) from the patent application published in CN108752245A. The DMSO content was less than 0.1%. The plates were incubated for 2 / 4 h. Cell-free supernatants were collected for analysis of IL-6 (2 h) and TNF-α (4 h) levels. Chemiluminescence values ​​were recorded at 450 nm using a SpectraMax M5 microplate reader (Molecular Devices, California). The results are as follows: Figure 2 As shown. Figure 2 This is a schematic diagram showing the anti-inflammatory effects of the compound prepared in Example 8 and the compound NXPZ-2 (10 μM) in patent application CN108752245A in RAW264.7 cells. In the diagram, A is a schematic diagram of the concentration change of IL-6, and B is a schematic diagram of the concentration change of TNF-α. *p<0.05, ***p<0.001 are compared with the NXPZ-2 group.

[0096] As can be seen from the figure, compound 8 can significantly inhibit the expression of IL-6 and TNF-α in the LPS-induced inflammation model, and has a better anti-inflammatory effect than the parent compound NXPZ-2.

[0097] The above experimental results show that the bis(naphthalene)sulfonamide derivatives prepared in this invention have good Keap1-Nrf2PPI inhibitory activity, as well as high Keap1 self-degradation activity and in vitro anti-inflammatory and antioxidant activating properties, laying the foundation for subsequent research.

[0098] This invention provides a new small molecule chemical tool for the study of the key signaling pathway Keap1-Nrf2 in oxidative stress and for the targeted degradation of Keap1. It has better activity and a novel structure, enabling precise chemical regulation of Keap1 and exhibiting good anti-inflammatory activity, thus providing a treatment strategy for oxidative stress-related diseases.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A bis(naphthalenesulfonamide) derivative, characterized in that, The general structural formula is as follows: Wherein, L is selected from one of the following groups: n is an integer selected from 1 to 30; m is selected from integers from 1 to 30.

2. The bis(naphthalenesulfonamide) derivative according to claim 1, characterized in that, In the bis(naphthalene)sulfonamide derivatives, L is selected from one of the following groups:

3. The bis(naphthalene)sulfonamide derivative according to claim 2, characterized in that, The bis(naphthyl)sulfonamide derivatives are selected from one of the following compounds:

4. The use of a bis(naphthalene)sulfonamide derivative according to any one of claims 1 to 3 in the preparation of isomorphic divalent Keap1 degradation-targeting chimeras.

5. The application according to claim 4, characterized in that, The bis(naphthyl)sulfonamide derivatives, as homodivalent Keap1 degradation-targeting chimeras, enable the self-degradation of Keap1 protein in human breast cancer cells.

6. The application according to claim 5, characterized in that, The human breast cancer cells mentioned are MDA-MB-231 cells.

7. The use of a bis(naphthyl)sulfonamide derivative according to any one of claims 1 to 3 in the preparation of a medicament for the prevention or treatment of oxidative stress-related diseases.

8. The use of a bis(naphthyl)sulfonamide derivative according to any one of claims 1 to 3 in the preparation of an anti-inflammatory medicament.

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