Design, Synthesis and Evaluation of Novel 1,4-Disubstituted Piperazine-2,5-dione Derivatives as Antioxidants against H2O2-Induced Oxidative Injury via the IL-6/Nrf2 Loop Pathway

Abstract

Excessive reactive oxygen species (ROS) production leads to oxidative stress in cells, impairing the function of mitochondria and finally inducing cell apoptosis. Considering the essential role of oxidative stress in the pathogenesis of various neurodegenerative diseases and psychiatric disorders, the discovery of novel antioxidants has attracted increasing attention. Herein, a series of novel 1,4-disubstituted piperazine-2,5-dione derivatives were designed, synthesized and evaluated for their antioxidative activity. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay indicated that none of the tested compounds showed significant toxicity to SH-SY5Y cells at concentrations up to 80 μM. Cell counting via flow cytometry revealed that most of the tested compounds could effectively protect SH-SY5Y cells from H₂O₂-induced oxidative damage at 20 μM. Among these compounds, compound 9r exhibited the best antioxidative activity. Further mechanistic investigation indicated that 9r decreased ROS production and stabilized the mitochondrial membrane potential to restrain cell apoptosis, and promoted cell survival via an IL-6/Nrf2 positive-feedback loop. These results suggested the potential of compound 9r as a novel antioxidative candidate for the treatment of diseases caused by oxidative stress.

1. Introduction

Oxidative stress, caused by the unbalanced production of reactive oxygen species (ROS) and antioxidants, is a contributing factor in the onset and progression of many diseases, including neurodegenerative disorders, stroke and inflammation [1]. Under physiological conditions, the level of ROS formation is in equilibrium with the antioxidant capacity. However, supraphysiological levels of ROS lead to oxidative stress and subsequent damage to biomolecules [2]. ROS are produced and result in oxidative stress when cells are subjected to the stimulation of superoxide, hydrogen peroxide and hydroxyl free radicals [3]. Among these species, hydrogen peroxide (H₂O₂) is responsible for redox signaling transmission from the generative site to a target site. Hence, H₂O₂ is considered most suitable for the establishment of the oxidative stress model.

Usually, cells resist oxidative stress via the action of antioxidants as well as the activation of antioxidative enzymes, including catalase, glutathione peroxidase and glutathione peroxidase. The transcription factor nuclear factor erythroid-2 related factor 2 (Nrf2) is one of the crucial antioxidant pathways. Under normal conditions, most Nrf2 is kept in an inactive state in the form of complexes with Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and is constantly targeted for degradation via ubiquitination. However, when cells are undergoing oxidative stress, the complex between Nrf2 and Keap1 depolymerizes and leads to the accumulation of Nrf2 in the cytoplasm. The ARE (the antioxidant response element) is activated when Nrf2 translocates to the nucleus and binds to the small muscular aponeurotic fibrosarcoma (Maf) protein. The activated ARE further leads to the transcriptional activation of various antioxidant enzymes and proteins, including haem oxygenase-1 (HO-1) and prosurvival cytokines (e.g., interleukin (IL)-6) [4]. IL-6, a pleiotropic cytokine, plays an important role in cell survival, proliferation, immunity and inflammation. When cells are under oxidative stress, Nrf2 mediates IL-6 expression by indirectly activating its promoter through the ARE and exerts partial antioxidant activity. In turn, IL-6 reduces cell apoptosis and promotes cell survival by activating downstream signaling, including MAPKs, JAK/Stat3 and PI3K/Akt, and increasing the nuclear translocation or stability of Nrf2 [5,6,7,8]. Hence, the development of antioxidants targeting the IL-6/Nrf2 cross axis may provide a novel therapeutic strategy for the treatment of oxidative stress-related diseases.

Natural bisepoxylignans exhibit diverse biological activities, including antioxidant [9] and anti-inflammatory [10] activities. Furofuran lignans are naturally occurring bisepoxylignans abundant in the plant kingdom with diverse biological activities, including antioxidative properties [11]. Previous studies have indicated that 137 furofuran lignans have been isolated and identified from various plants. Among these compounds, sesamin, syringaresinol, terminaloside K and pinoresinol showed significant antioxidant activities [12]. In our previous study, we also isolated and identified several known bisepoxylignans, including syringaresinol, from the roots of Zea mays L. (maize) and found that they protected SH-SY5Y cells against H₂O₂-induced cell apoptosis [13]. Therefore, bisepoxylignans are considered a good source of natural antioxidants [14]. However, due to their multiple chiral centers (usually more than 4) [15,16], it is difficult to synthesize these antioxidant active compounds. To date, there are few active compounds that can be synthesized in large quantities. Hence, it is still necessary to perform structural modification to retain their antioxidative activity and make them easy to synthesize. 2,5-piperazinedione, prepared via the condensation and cyclization of two molecules of glycine, is the smallest cyclic peptide derivative in nature. It is attracting increasing attention due to its unique chemical properties, including strong rigidity and exceptionally high stability to peptidase, and its biological characteristics, including an extensive range of biological activities. Scaffold hopping, an important and efficient drug-design strategy for molecular-backbone replacements, has been widely used in drug design to develop novel molecules with potent activity [17]. Heterocycle replacements are one of the commonly used methodologies that enable the generation of scaffold hops [18]. Herein, we designed and synthesized a series of novel 1,4-disubstituted piperazine-2,5-diones as derivatives of bisepoxylignans based on a scaffold-hopping strategy (Figure 1) and evaluated their antioxidative activities using a H₂O₂-induced oxidative stress model. The potential antioxidant mechanisms of the best active compound, 9r, were further investigated.

2. Materials and Methods

2.1. General Methods

All reagents and solvents used in the experiment were commercially available and used without further purification. ¹H nuclear magnetic resonance (NMR) data were measured and recorded on a Bruker 600 MHz NMR spectrometer (Bruker; Billerica, MA, USA), and ¹³C NMR spectral data were measured and recorded using a Bruker 151 MHz NMR spectrometer with chloroform-d as the solvent and tetramethylsilane (TMS) as the internal standard. The high-resolution mass spectra (HRMS) of all target compounds were measured using a mass spectrometer (microTOF-Q, Bruker Inc.) using electrospray ionization. The melting points were measured using a melting-point instrument (Shanghai Micro Melting Point Apparatus, Shanghai, China), and the values are uncorrected.

2.2. General Method for the Synthesis of Compounds 9a–9s

2.2.1. Ethyl 2-[(3,5-Dimethoxyphenyl)amino]acetate (2a)

Under a N₂ atmosphere, a mixture of 1a (100 mg, 0.653 mmol) and anhydrous K₂CO₃ (135 mg, 0.977 mmol) in anhydrous acetone (5 mL) was heated to 60 °C and stirred for 1 h. A solution of ethyl bromoacetate (142 mg, 0.850 mmol) in anhydrous acetone (1 mL) was added dropwise, and the mixture was stirred at 60 °C for 8 h (thin-layer chromatography (TLC) monitoring). The K₂CO₃ was filtered off, and the solvent was removed under vacuum. Then, it was purified via column chromatography over silica gel (PE:EA = 30:1) to give ethyl 2-[(3,5-dimethoxyphenyl)amino]acetate(2a) as a yellow oily liquid (134 mg, 0.561 mmol, 86% yield). Electrospray ionization–mass spectrometry (ESI-MS): m/z, 240.1 [M + H]⁺.

2.2.2. Ethyl N-(2-Chloroacetyl)-N-[(3,5-dimethoxyphenyl)amino]acetate (3a)

A solution of chloroacetyl chloride (106 mg, 0.939 mmol) was added to ethyl 2-[(3,5-dimethoxyphenyl)amino]acetate (2a) (150 mg, 0.627 mmol) and a catalytic amount of tetrabutyl ammonium hydrogen sulfate in CH₂Cl₂ (6 mL). A solution of K₂CO₃ (130 mg, 0.941 mmol) in H₂O (2 mL) was slowly added dropwise. The mixture was stirred (TLC monitoring of the reaction). The solvent was removed under vacuum, and the crude product was purified via silica gel column chromatography (PE:EA = 4:1) to give ethyl N-(2-chloroacetyl)-N-[(3,5-dimethoxyphenyl)amino] acetate (3a) as a yellow oily liquid (194 mg, 0.614 mmol, 98% yield). ESI-MS: m/z, 316.2, 318.2 [M + H]⁺.

2.2.3. Ethyl N-(2-Azidoacetyl)-N-[(3,5-dimethoxyphenyl)amino]acetate (4a)

NaN₃ (26 mg, 0.400 mmol) was added to a solution of ethyl N-(2-chloroacetyl)-N-[(3,5-dimethoxyphenyl)amino] acetate (3a) (50 mg, 0.158 mmol) in DMF (3 mL). The mixture was heated at 90 °C for 1 h (completion monitored via TLC). H₂O₂ (5 mL) was added to the mixture. Dimethylformamide (DMF) and H₂O₂ were evaporated at 90 °C. The crude product was subjected to silica gel column chromatography (PE:EA = 2:1) to give N-(2-azidoacetyl)-N-[(3,5-dimethoxyphenyl)amino]ethyl ester acetate (4a) as a colorless oily liquid (50 mg, 0.155 mmol, 98% yield). ESI-MS: m/z, 323.3 [M + H]⁺.

2.2.4. 1-(3,5-Dimethoxyphenyl)piperazine-2,5-dione (7a)

Triphenylphosphine (61 mg, 0.232 mmol) was added to a solution of ethyl N-(2-azidoacetyl)-N-[(3,5-dimethoxyphenyl)amino] acetate (4a) (50 mg, 0.155 mmol) in THF (5 mL) with a few drops of water. The mixture was stirred at room temperature for 4 h (monitoring via TLC for completion). The THF was completely evaporated, and the product was purified via silica gel column chromatography using ethyl acetate (EA) as the eluent to give 1-(3,5-dimethoxyphenyl)piperazine-2,5-dione (7a) as a white solid (23 mg, 0.092 mmol, 59% yield). ESI-MS: m/z, 251.2 [M + H]⁺.

Compounds 7b–7e were synthesized according to the procedure described in 7a.

1-(3,4-Methylenedioxyphenyl)piperazine-2,5-dione (7b): White Solid, 62% Yield: ESI-MS m/z: 235.2 [M + H]⁺.

1-Phenylpiperazine-2,5-dione (7c) [19]: White Solid, 58% Yield: ESI-MS m/z: 191.2 [M + H]⁺.

1-(3-Methoxyphenyl)piperazine-2,5-dione (7d): White Solid, 63% Yield: ESI-MS m/z: 221.1 [M + H]⁺.

1-(4-Methoxyphenyl)piperazine-2,5-dione (7e) [19]: White Solid, 61% Yield: ESI-MS m/z: 221.1 [M + H]⁺.

2.2.5. 1-(3,5-Dimethoxyphenyl)-4-(4-methoxybenzoyl)piperazine-2,5-dione (9a)

4-Methoxybenzoyl chloride (53 mg, 0.312 mmol) in CH₂Cl₂ (1 mL) was added dropwise to 1-(3,5-dimethoxyphenyl)piperazine-2,5-dione (7a) (60 mg, 0.240 mmol), triethylamine (48 mg, 0.474 mmol) and 4-dimethylaminopyridine (3 mg, 0.024 mmol) in CH₂Cl₂ (5 mL) at 0 °C using a dropping funnel. The mixture was stirred for 2 h at room temperature. The completion of the reaction was monitored via TLC. The mixture was poured into 20 mL of saturated brine and extracted with dichloromethane (20 mL × 3). The organic phases were combined and dried with MgSO_4, and the solvent was removed under vacuum. The product was obtained via column chromatography over silica gel using PE-EA (2:1) as the eluent to give 1-(3,5-dimethoxyphenyl)-4-(4-methoxybenzoyl)piperazine-2,5-dione (9a) as a white solid (74 mg, 0.193 mmol, 80% yield). mp: 180–183 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.70 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.48 (d, J = 2.2 Hz, 2H), 6.42 (t, J = 2.2 Hz, 1H), 4.54 (s, 2H), 4.45 (s, 2H), 3.86 (s, 3H), 3.80 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) δ: 170.47, 166.97, 164.84, 163.81, 161.36, 141.02, 131.85, 125.91, 113.81, 103.51, 99.65, 55.68, 55.62, 54.17, 48.77. HRMS (ESI): calcd for C₂₀H₂₁O₆N₂ [M + H]⁺ m/z, 385.13941; found, 385.13763; C₂₀H₂₀O₆N₂Na [M+Na]⁺ m/z, 407.12136; found 407.12033.

Compounds 9b–9s Were Synthesized According to the Procedure Described in 9a

2.2.6. 1-(3,5-Dimethoxyphenyl)-4-(4-methylbenzoyl)piperazine-2,5-dione (9b)

White solid, 82% yield, mp: 131–134 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.59 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 4.57 (s, 2H), 4.46 (s, 2H), 3.81 (s, 6H), 2.41 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.17, 166.88, 164.74, 161.39, 144.09, 141.00, 131.19, 129.26, 129.21, 103.53, 99.69, 55.69, 54.17, 48.58, 21.87. HRMS (ESI): calcd for C₂₀H₂₁O₅N₂ [M + H]⁺ m/z, 369.14450; found, 369.14343; C₂₀H₂₀O₅N₂Na [M + Na]⁺ m/z, 391.12644; found, 391.12540.

2.2.7. 1-(4-Methylbenzoyl)-4-(3,4-methylenedioxyphenyl)piperazine-2,5-dione (9c)

White solid, 88% yield, mp: 174–177 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.58 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.88–6.83 (m, 2H), 6.74 (dd, J = 8.3, 2.2 Hz, 1H), 6.01 (s, 2H), 4.55 (s, 2H), 4.42 (s, 2H), 2.41 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.20, 166.86, 164.93, 148.42, 147.19, 144.07, 133.21, 131.20, 129.23, 129.21, 118.50, 108.65, 107.01, 101.99, 54.59, 48.44, 21.87. HRMS (ESI): calcd for C₁₉H₁₇O₅N₂ [M + H]⁺ m/z, 353.11320; found, 353.11182; C₁₉H₁₆O₅N₂Na [M + Na]⁺ m/z, 375.09514; found, 375.09427.

2.2.8. 1-(4-Methylbenzoyl)-4-phenylpiperazine-2,5-dione (9d)

White solid, 89% yield, mp: 170–173 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.60 (d, J = 8.2 Hz, 2H), 7.46 (t, J = 8.0 Hz, 2H), 7.37–7.31 (m, 3H), 7.25 (d, J = 8.0 Hz, 2H), 4.58 (s, 2H), 4.49 (s, 2H), 2.41 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.17, 166.94, 164.76, 144.07, 139.37, 131.18, 129.56, 129.25, 129.20, 127.63, 124.83, 54.06, 48.53, 21.86. HRMS (ESI): calcd for C₁₈H₁₆O₃N₂Na [M + Na]⁺ m/z, 331.10531; found, 3331.10440.

2.2.9. 1-(3-Methoxyphenyl)-4-(4-methylbenzoyl)piperazine-2,5-dione (9e)

White solid, 86% yield, mp: 142–145 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.59 (d, J = 8.2 Hz, 2H), 7.36 (t, J = 8.1 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 6.93–6.89 (m, 2H), 6.89–6.85 (m, 1H), 4.57 (s, 2H), 4.47 (s, 2H), 3.83 (s, 3H), 2.41 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.16, 166.91, 164.73, 160.40, 144.06, 140.46, 131.18, 130.28, 129.24, 129.19, 116.84, 113.21, 111.15, 55.60, 54.10, 48.55, 21.86. HRMS (ESI): calcd for C₁₉H₁₉O₄N₂ [M + H]⁺ m/z, 339.13393; found, 339.13260; C₁₉H₁₈O₄N₂Na [M + Na]⁺ m/z, 361.11588; found, 361.11490.

2.2.10. 1-(4-Methoxyphenyl)-4-(4-methylbenzoyl)piperazine-2,5-dione (9f)

White solid, 87% yield, mp: 142–145 °C. ¹H NMR (600 MHz, Chloroform-d) δ: 7.59 (d, J = 8.2 Hz, 2H), 7.31–7.18 (m, 4H), 7.01–6.92 (m, 2H), 4.56 (s, 2H), 4.44 (s, 2H), 3.83 (s, 3H), 2.41 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.22, 166.99, 164.85, 158.84, 144.03, 132.15, 131.23, 129.22, 129.19, 126.40, 114.83, 55.66, 54.45, 48.48, 21.86. HRMS (ESI): calcd for C₁₉H₁₉O₄N₂ [M + H]⁺ m/z, 339.13393; found, 339.13303; C₁₉H₁₈O₄N₂Na [M + Na]⁺ m/z, 361.11588; found, 361.11481.

2.2.11. 1-(3,5-Dimethoxyphenyl)-4-benzoylpiperazine-2,5-dione (9g)

White solid, 85% yield, mp: 130–133 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.70–7.65 (m, 2H), 7.61–7.53 (m, 1H), 7.45 (t, J = 7.8 Hz, 2H), 6.49 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 4.59 (s, 2H), 4.47 (s, 2H), 3.81 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.28, 166.82, 164.65, 161.41, 140.97, 134.16, 133.01, 128.94, 128.48, 103.56, 99.73, 55.70, 54.17, 48.47. HRMS (ESI): calcd for C₁₉H₁₉O₅N₂ [M + H]⁺ m/z, 355.12885; found, 355.12750; C₁₉H₁₈O₅N₂Na [M + Na]⁺ m/z, 377.11079; found, 377.10965.

2.2.12. 1-(3,5-Dimethoxyphenyl)-4-(4-fluorobenzoyl)piperazine-2,5-dione (9h)

White solid, 88% yield, mp: 149–152 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.74–7.68 (m, 2H), 7.12 (t, J = 8.5 Hz, 2H), 6.48 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 4.57 (s, 2H), 4.47 (s, 2H), 3.81 (s, 6H). ¹³C NMR (151 MHz, CDCl3) δ: 170.04, 166.89, 165.84 (166.47, 164.78, d, ¹J = 254.9 Hz), 164.52, 161.39, 140.90, 131.76 (131.79, 131.73, d, ³J = 9.3 Hz), 130.19 (130.20, 130.18, d, ⁴J = 3.1 Hz, ), 115.74 (115.82, 115.67, d, ²J = 22.3 Hz), 103.53, 99.65, 55.69, 54.15, 48.55. ¹⁹F NMR (565 MHz, CDCl₃) δ: -104.70. HRMS (ESI): calcd for C₁₉H₁₈O₅N₂F [M + H]⁺ m/z, 373.11943; found, 373.11835; C₁₉H₁₇O₅N₂FNa [M + Na]⁺ m/z, 395.10137; found, 395.10043.

2.2.13. 1-(3,5-Dimethoxyphenyl)-4-(4-(trifluoromethyl)benzoyl)piperazine-2,5-dione (9i)

White solid, 78% yield, mp: 141–144 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.74 (d, J = 8.2 Hz, 2H), 7.71 (d, J = 8.2 Hz, 2H), 6.48 (d, J = 2.2 Hz, 2H), 6.44 (t, J = 2.3 Hz, 1H), 4.63 (s, 2H), 4.48 (s, 2H), 3.81 (s, 6H). ¹³C NMR (151 MHz, CDCl3) δ: 170.02, 166.73, 164.22, 161.45, 140.82, 137.69, 134.05 (134.37, 134.15, 133.94, 133.72, q, ²J = 32.9 Hz), 128.95, 125.53 (125.56, 125.54, 125.51, 125.49, q, ³J = 3.8 Hz), 123.62 (126.32, 124.51, 122.71, 120.90, q, ¹J = 273.0 Hz), 103.57, 99.74, 55.71, 54.14, 48.24. ¹⁹F NMR (565 MHz, CDCl₃) δ: −63.11. HRMS (ESI): calcd for C₂₀H₁₈O₅N₂F₃ [M + H]⁺ m/z, 423.11623; found, 423.11514; C₂₀H₁₇O₅N₂F₃Na [M + Na]⁺ m/z, 445.0918; found, 445.09738.

2.2.14. 1-(3,5-Dimethoxyphenyl)-4-(4-(tert-butyl)benzoyl)piperazine-2,5-dione (9j)

White solid, 70% yield, mp: 193–196 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.64 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 2.0 Hz, 2H), 6.43 (t, J = 2.1 Hz, 1H), 4.57 (s, 2H), 4.47 (s, 2H), 3.81 (s, 6H), 1.34 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.16, 166.91, 164.73, 161.41, 156.99, 141.02, 131.04, 129.14, 125.51, 103.57, 99.75, 55.69, 54.20, 48.62, 35.29, 31.19. HRMS (ESI): calcd for C₂₃H₂₇O₅N₂ [M + H]⁺ m/z, 411.19145; found, 411.18982; C₂₃H₂₆O₅N₂Na [M + Na]⁺ m/z, 433.17339; found, 433.17227.

2.2.15. 1-(3,5-Dimethoxyphenyl)-4-acetylpiperazine-2,5-dione (9k)

White solid, 63% yield, mp: 172–175 °C. ¹H NMR (600 MHz, chloroform-d) δ: 6.42 (d, J = 2.2 Hz, 2H), 6.40 (t, J = 2.2 Hz, 1H), 4.56 (s, 2H), 4.46 (s, 2H), 3.78 (s, 6H), 2.62 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.19, 166.36, 164.27, 161.34, 140.93, 103.46, 99.70, 55.66, 54.74, 46.49, 27.11. HRMS (ESI): calcd for C₁₄H₁₇O₅N₂ [M + H]⁺ m/z, 293.11320; found, 293.11212; C₁₄H₁₆O₅N₂Na [M + Na]⁺ m/z, 315.09514; found, 315.09421.

2.2.16. 1-(3,5-Dimethoxyphenyl)-4-(3-bromobenzoyl)piperazine-2,5-dione (9l)

White solid, 88% yield, mp: 192–195 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.78 (s, 1H), 7.70–7.65 (m, 1H), 7.59–7.54 (m, 1H), 7.32 (t, J = 7.9 Hz, 1H), 6.48 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 4.58 (s, 2H), 4.47 (s, 2H), 3.80 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) δ: 169.78, 166.70, 164.31, 161.41, 140.86, 136.15, 135.71, 131.64, 129.96, 127.31, 122.46, 103.54, 99.73, 55.70, 54.08, 48.34. HRMS (ESI): calcd for C₁₉H₁₈O₅ N₂Br [M + H]⁺ m/z, 433.03936; found, 433.03812.

2.2.17. 1-(3,5-Dimethoxyphenyl)-4-propionylpiperazine-2,5-dione (9m)

White solid, 60% yield, mp: 112–115 °C. ¹H NMR (600 MHz, chloroform-d) δ: 6.42 (d, J = 2.0 Hz, 2H), 6.39 (t, J = 2.0 Hz, 1H), 4.57 (s, 2H), 4.45 (s, 2H), 3.78 (s, 6H), 3.00 (q, J = 7.2 Hz, 2H), 1.19 (t, J = 7.2 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 175.24, 166.24, 164.41, 161.33, 140.94, 103.43, 99.66, 55.65, 54.85, 46.65, 32.81, 8.77. HRMS (ESI): calcd for C₁₅H₁₉O₅N₂ [M + H]⁺ m/z, 307.12885; found, 307.12772; C₁₅H₁₈O₅N₂Na [M + Na]⁺ m/z, 329.11079; found, 329.10980.

2.2.18. 1-(3,5-Dimethoxyphenyl)-4-butyrylpiperazine-2,5-dione (9n)

White solid, 65% yield, mp: 136–139 °C. ¹H NMR (600 MHz, chloroform-d) δ: 6.42 (d, J = 2.2 Hz, 2H), 6.40–6.38 (m, 1H), 4.56 (s, 2H), 4.45 (s, 2H), 3.78 (s, 6H), 2.95 (t, J = 7.4 Hz, 2H), 1.71 (q, J = 7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 174.31, 166.25, 164.43, 161.33, 140.94, 103.43, 99.66, 55.65, 54.86, 46.57, 41.04, 17.95, 13.78. HRMS (ESI): calcd for C₁₆H₂₁O₅N₂ [M + H]⁺ m/z, 321.14450; found, 321.14340; C₁₆H₂₀O₅N₂Na [M + Na]⁺ m/z, 343.12644; found, 343.12558.

2.2.19. 1-(3,5-Difluorobenzoyl)-4-(3,5-dimethoxyphenyl)piperazine-2,5-dione (9o)

White solid, 82% yield, mp: 182–185 °C. ¹H NMR (600 MHz, chloroform-d) δ: 7.19–7.15 (m, 1H), 7.15–7.12 (m, 1H), 7.00 (tt, J = 8.6, 2.3 Hz, 1H), 6.47 (d, J = 2.2 Hz, 2H), 6.43 (t, J = 2.2 Hz, 1H), 4.58 (s, 2H), 4.48 (s, 2H), 3.81 (s, 6H). ¹³C NMR (151 MHz, CDCl3) δ: 168.94 (168.95, 168.93, 168.90, t, ⁴J = 3.3 Hz), 166.68, 164.07, 162.71 (163.58, 163.49, 161.91, 161.83, dd, ¹J = 250.9, 12.1 Hz), 161.45, 140.78, 137.31 (137.37, 137.30, 137.24, t, ³J = 9.2 Hz), 111.83 (111.91, 111.87, 111.76, 111.72, dd, ²J = 21.4, 5.9 Hz), 108.10 (108.26, 108.09, 107.93, t, ²J = 25.2 Hz), 103.57, 99.81, 55.71, 54.06, 48.28. ¹⁹F NMR (565 MHz, CDCl₃) δ: -108.13. HRMS (ESI): calcd for C₁₉H₁₇O₅N₂F₂ [M + H]⁺ m/z, 391.11000; found, 391.10870; C₁₉H₁₆O₅N₂F₂Na [M + Na]⁺ m/z, 413.09195; found, 413.09103.

2.2.20. 1-(3,5-Dimethoxyphenyl)-4-pivaloylpiperazine-2,5-dione (9p)

White solid, 75% yield, mp: 180–183 °C. ¹H NMR (600 MHz, chloroform-d) δ: 6.44 (d, J = 2.2 Hz, 2H), 6.41 (t, J = 2.2 Hz, 1H), 4.42 (s, 2H), 4.37 (s, 2H), 3.79 (s, 6H), 1.36 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ: 184.59, 166.61, 164.93, 161.38, 140.94, 103.48, 99.66, 55.68, 54.29, 49.42, 43.61, 27.32. HRMS (ESI): calcd for C₁₇H₂₃O₅N₂ [M + H]⁺ m/z, 335.16015; found, 335.15897; C₁₇H₂₂O₅N₂Na [M + Na]⁺ m/z, 357.14209; found, 357.14112.

2.2.21. 1-(3,5-Dimethoxyphenyl)-4-(cyclopropanecarbonyl)piperazine-2,5-dione (9q)

White solid, 64% yield, mp: 155–158 °C. ¹H NMR (600 MHz, chloroform-d) δ: 6.44 (d, J = 2.2 Hz, 2H), 6.40 (t, J = 2.2 Hz, 1H), 4.54 (s, 2H), 4.49 (s, 2H), 3.79 (s, 6H), 3.02–2.95 (m, 1H), 1.23–1.19 (m, 2H), 1.09–1.04 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ: 175.86, 166.96, 164.51, 161.34, 140.97, 103.45, 99.70, 55.67, 54.91, 47.14, 16.15, 11.92. HRMS (ESI): calcd for C₁₆H₁₉O₅N₂ [M + H]⁺ m/z, 319.12885; found, 319.16762; C₁₆H₁₈O₅N₂Na [M + Na]⁺ m/z, 341.11079; found, 341.10986.

2.2.22. 1-(3,5-Dimethoxyphenyl)-4-(3-cyclopentylpropanoyl)piperazine-2,5-dione (9r)

White solid, 88% yield, mp: 141–144 °C. ¹H NMR (600 MHz, chloroform-d) δ: 6.42 (d, J = 2.2 Hz, 2H), 6.39 (t, J = 2.2 Hz, 1H), 4.55 (s, 2H), 4.44 (s, 2H), 3.78 (s, 6H), 3.01–2.95 (m, 2H), 1.85–1.74 (m, 3H), 1.72–1.65 (m, 2H), 1.65–1.56 (m, 2H), 1.54–1.48 (m, 2H), 1.14–1.08 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ: 174.64, 166.21, 164.44, 161.31, 140.93, 103.42, 99.63, 55.64, 54.85, 46.62, 39.64, 38.47, 32.62, 30.71, 25.22. HRMS (ESI): calcd for C₂₀H₂₇O₅N₂ [M + H]⁺ m/z, 375.19145; found, 375.18997; C₂₀H₂₆O₅N₂Na [M + Na]⁺ m/z, 397.17339; found, 397.17230.

2.2.23. 1-(3,5-Dimethoxyphenyl)-4-(2-methoxyacetyl)piperazine-2,5-dione (9s)

White solid, 40% yield, mp: 138–141 °C. ¹H NMR (600 MHz, chloroform-d) δ: 6.41 (d, J = 2.2 Hz, 2H), 6.40 (t, J = 2.2 Hz, 1H), 4.62 (s, 2H), 4.60 (s, 2H), 4.46 (s, 2H), 3.78 (s, 6H), 3.49 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ: 171.78, 166.14, 163.81, 161.38, 140.86, 103.51, 99.76, 74.91, 59.62, 55.67, 54.48, 46.28. HRMS (ESI): calcd for C₁₅H₁₉O₆N₂ [M + H]⁺ m/z, 323.12376; found, 323.12238; C₁₅H₁₈O₆N₂Na [M + Na]⁺ m/z, 345.10571; found, 345.10474.

2.3. Biological Antioxidant Assays Used for the Study of Compounds 9a–9s

2.3.1. Cell Culture

SH-SY5Y cells were acquired from the National Cell Culture Collection. Cells were grown at 37 °C with 5% CO₂ in full RPMI-1640 (Gibco, Waltham, MA, USA) media supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Buenos Aires, Argentina).

2.3.2. Measurement of Intracellular ROS Production

An active oxygen analysis kit (Beyotime Biotech, Nantong, China) was used to detect ROS generation according to the instructions. In brief, the treated cells were collected and washed with phosphate-buffered saline (PBS), with the addition of dichlorodihydrofluorescein diacetate (DCFH-DA,10 μM) serum-free Roswell Park Memorial Institute (RPMI)/1640 culture medium. Incubation took place at 37 °C for 30 min, and observation or flow cytometer analysis was performed using a fluorescence microscope.

2.3.3. Determination of Mitochondrial Membrane Potential

To calculate the mitochondrial membrane potential, KGA604 (Key Gen Biotech) was used according to the manufacturer’s instructions. Cells were incubated with JC-1 working liquid for 20 min at 37 °C, washed three times with cold 1 × PBS, and then, the treated cells were observed or detected using flow cytometry under a fluorescence microscope.

2.3.4. Cell Apoptosis

For apoptosis testing, an FITC Annexin V cell apoptosis-detection kit (556547, BD Biosciences, San Jose, CA, USA) was used. Briefly, the treated cells were collected and incubated in a binding solution with 5 μL Annexin V–fluorescein isothiocyanate (FITC) and 5 μL PI. Acea Biosciences (San Diego, CA, USA) measured the rate of apoptosis.

2.3.5. Extraction of Cytoplasmic and Nuclear Proteins

A CW0199S nuclear slurry was used to extract the nuclear slurry separation kit (CW0199S, CWBIOTECH, Beijing, China). Cells were simply harvested, and 1 mL NC Buffer A was added after centrifugation, mixed for 5 s, and incubated for 20 min on ice. Then, 55 μL NC buffer B was stirred in, and the solution was incubated for 1 min on ice. After being personalized for 15 min (4 °C, 12,000 rpm), the liquid was collected and stored. Then, 500 μL of NC Buffer C was added to the precipitate obtained in the preceding procedures, mixed for 5 s, and incubated on ice for 40 min, rotating for 15–30 s every 10 min. After being personalized for 15 min (4 °C, 12,000 rpm), the liquid was collected and preserved.

2.3.6. Immunofluorescence Staining

In brief, the cells were incubated in 4% additional formaldehyde for 15 min before being washed three times with PBS and permeabilized with 0.1% Triton X-100. After three washes with PBS, the membrane was blocked with 4% bovine serum albumin for 30 min before being incubated overnight with the Nrf2 antibody at 4 °C. The next day, PBS was rinsed three times, and Alexa Fisher Science (Thermo Fisher Science, Waltham, MA, USA) was used. The duration of incubation was 5 min. To obtain pictures, a microscope (DMI8, Leica, Wetzlar, Germany) was used.

2.3.7. SiRNA Transfection

China Guangzhou Nuclear Biological Co., Ltd. (Guangzhou, China) designed and synthesized small interfering RNA (siRNA) oligonucleotides for IL-6, Nrf2 and negative-control siRNA (NC-si). The specific operation method can be found in the literature [20]. In short, according to the instructions, the siRNA mixture was introduced into SH-SY5Y cells. After 48 h, the cells were treated with H₂O₂ and 9r, and Western markers were used to detect changes in related proteins. The following siRNA sequences were used: IL-6-siRNA: 5′-GCTACGTGATGAGAGGGA-3′; Nrf2-siRNA: 5′-GCATCTCAGCCCCCTGAA-3′.

2.3.8. Western Blotting

In brief, proteins were collected using lysis buffer (Cat No. P0013B, Beyotime, Shanghai, China), and the protein concentration was measured using a BCA Protein Assay Kit (PC0020, Solarbio Science & Technology, Beijing, China). Before incubation with the antibody, equal quantities of protein samples were separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. A ChemiDoc MP Imaging System was used to image the blots (Bio-Rad, Hercules, CA, USA). p-SAPK/JNK (Thr183/Tyr185; #4685), SAPK/JNK (#9252), p-Src (Ser17; #12432), p-Stat3 (Tyr705; #9145), mTOR (2065AP), p-mTOR (Ser2448; #5536), p-Akt (Ser473; #4060), p-GSK3β (Ser9; #5558), PARP (#9532) and cleaved-PARP (#94885) were provided by Cell Signaling Technology. Src (11097-1-AP), AKT (11097-1-AP), Stat3 (10253-2-AP), ERK1/2 (16443-1-AP), GSK3β (#12456), GAPDH (10494-1-AP), P53 (10442-1-AP), Bcl2 (10068-1-AP), Bax (50599-2-19), IL-6 (21865-1-AP), Nrf2 (16396-1-AP) and HO-1 (10701-1-AP) were purchased from Proteintech. Immobilon^® PVDF membranes were provided by Thermo Fisher Scientific (Waltham, MA, USA) and Merck KGaA (Darmstadt, Germany), respectively.

2.3.9. Statistical Analysis

All experiments were carried out in triplicate and repeated three times. The data are reported as the mean ± standard deviation (SD). Statistical analysis was performed by applying Kruskal–Wallis univariate analysis of variance (ANOVA) in SPSS. The treatment effect was considered significant at p < 0.05.

3. Results and Discussion

3.1. Chemistry

In our present study, precursors 7a–e [19] were synthesized from aromatic amines 1a–e. Compounds 1a–e were allowed to react with ethyl bromoacetate via a nucleophilic substitution reaction to give intermediates 2a–e. The treatment of 2a–e with chloroacetyl chloride under phase-transfer catalysis conditions gave amides 3a–e. Compounds 3a–e were then treated with excess sodium azide in dimethylformamide (DMF) solution at 90 °C to produce azide compounds 4a–e in excellent yields. Subsequently, the intramolecular aza-Wittig reaction was conducted with substrates 4a–e. Intermediates 4a–e were treated with triphenylphosphine in tetrahydrofuran (THF) at room temperature to give compounds 5a–e. Intermediates 6a–e were obtained from compounds 5a–e via the spontaneous cyclization reaction. Next, intermediates 6a–e were stirred in moist THF (not anhydrous), resulting in enol ether cleavage and rearrangement to give 7a–e. Finally, compounds 7a–e reacted with the corresponding chloride (8) to form target compounds 9a-s (Scheme 1). The structures of all the synthetic compounds are summarized in Table 1.

3.2. Biological Evaluation

3.2.1. Cell Viability Assay

Before the examination of the antioxidative activities, the SH-SY5Y cells were treated with the tested compounds at different concentrations to determine their cytotoxicity. As shown in Table S1, almost all the tested compounds at 80 μM showed no obvious cytotoxicity to SH-SY5Y cells. In contrast, most compounds at 20 μM could significantly promote cell proliferation. Therefore, 20 μM of the tested compounds were selected for further evaluation of their potential antioxidant activities. As shown in Table 2, most of the derivatives showed better cytoprotective activity when compared with the lead compound Syringaresinol. In addition, some derivatives (9d, 9e, 9o and 9r) exhibited similar cytoprotective activity or better activity than that of the positive control Neohesperidin. Compound 9r, with the best activity, significantly increased the viability of H₂O₂-treated SH-SY5Y cells from 32.85% (H₂O₂-treated group) to 70.19% (9r-treated group). Therefore, we chose to further explore the possible antioxidative mechanism of compound 9r.

3.2.2. Compound 9r Exhibited Cytoprotective Effects by Partially Restoring Cell Viability and Ameliorating Morphology

The viability of SH-SY5Y cells was further evaluated after treatment with various concentrations of 9r to investigate its dose-dependent antioxidative activity. As shown in Figure 2A, Compound 9r (5–20 μM) dose-dependently protected against H₂O₂-induced oxidative stress and promoted the viability of H₂O₂-treated SH-SY5Y cells. Compound 9r showed better antioxidative activity than the lead compound syringaresinol (Figure 2B). In addition, similar cytoprotective activities against H₂O₂-induced oxidative stress were observed in 9r- and neohesperidin (positive control)-pretreated SH-SY5Y cells (Figure 2B). The results of the morphological alterations showed that H₂O₂ treatment dramatically decreased the number of living cells and resulted in cell contraction and nuclear fragmentation (Figure 2C). However, 9r significantly prevented the cell morphological changes caused by H₂O₂ in a dose-dependent manner. These results suggested that 9r possessed a cytoprotective effect against oxidative damage caused by H₂O₂ treatment in SH-SY5Y cells.

3.2.3. 9r Attenuated H₂O₂-Induced Cell Apoptosis in SH-SY5Y Cells

H₂O₂-induced oxidative stress eventually results in a dramatic increase in cell apoptosis [21]. To investigate whether the cytoprotective effect of 9r was associated with the mediation of cell apoptosis, flow cytometry was used to detect the effect of 9r on H₂O₂-induced apoptosis using an Annexin V–fluorescein isothiocyanate (FITC) apoptosis-detection kit. Figure 3 demonstrates that the apoptosis rate in the H₂O₂-treated group (29.5%) was substantially greater than the apoptosis rate in the control group (1.28%); however, 9r pretreatment for 2 h dose-dependently inhibited H₂O₂-induced apoptosis. To further determine the inhibitory activity of 9r on cell apoptosis, we next examined the influence of 9r on proteins involved in the apoptotic pathways using Western blotting. As indicated in Figure 4, exposure to H₂O₂ dramatically increased the related proapoptotic proteins (p53, Bax, Cl-PARP) while decreasing the antiapoptotic protein Bcl2. However, 9r pretreatment abolished the H₂O₂-induced alterations in the related apoptotic proteins, which was consistent with our prior findings. The data suggested that the cytoprotective activity of 9r was partially attributed to the inhibitory effects of the apoptotic pathway.

3.2.4. Compound 9r Attenuated Mitochondrial Dysfunction Caused by H₂O₂ Treatment

As previously mentioned, mitochondrial dysfunction is one of the major causes of H₂O₂-induced cell apoptosis [22]. Therefore, the effect of 9r on mitochondrial function in H₂O₂-treated SH-SY5Y cells was examined via flow cytometry and inverted fluorescence microscopy using JC-1 dye. As shown in Figure 5, the mitochondrial membrane potential was significantly elevated in the H₂O₂-treated group compared with the control group. However, 9r pretreatment counteracted the effects of H₂O₂ on mitochondrial membrane potential (Figure 5). The results suggested that 9r could resist the mitochondrial dysfunction caused by H₂O₂.

3.2.5. 9r Decreased the Generation of ROS in H₂O₂-Treated SH-SY5Y Cells

Mitochondrial dysfunction often dramatically increases the production of ROS and subsequently promotes cell apoptosis through the mitochondrial apoptosis pathway [23]. Given that 9r can protect the mitochondrial membrane potential, we speculate that 9r can also resist oxidative stress by reducing the production of ROS. Therefore, the effect of 9r on intracellular ROS production after H₂O₂ treatment was examined. As shown in Figure 6, H₂O₂ treatment significantly increased ROS production compared to the control group. Nevertheless, these effects were partly neutralized by 9r pretreatment for 2 h. Accordingly, immunofluorescence analysis also indicated that the increased green fluorescence caused by H₂O₂ was steadily downregulated by 9r pretreatment, suggesting that 9r reduced H₂O₂-induced ROS production in a dose-dependent manner. In summary, 9r may counteract the oxidative injury caused by H₂O₂ by reducing ROS generation.

3.2.6. The Antioxidant Capacity of 9r Is Mediated by the Nrf2/HO-1 Signaling Pathway

In neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and cerebral ischemia, activated Nrf2 is able to ameliorate various pathological processes, such as mitochondrial dysfunction and oxidative stress, thus showing antioxidant benefits [24,25]. Nrf2 has been reported to initiate the transcription of relevant protective genes against oxidative stress after translocation from the cytoplasm to the nucleus by binding to antioxidant response elements. These elements include heme oxygenase-1 (HO-1), an important antioxidant enzyme that is one of the key downstream targets for Nrf2 to exert cytoprotective effects [26]. Therefore, after confirming the protective effect of 9r on mitochondria, we examined its effect on the nuclear translocation of Nrf2. As depicted in Figure 7A–D, Western blot analysis indicated that 9r significantly prevented the reduction in Nrf2 nucleation caused by H₂O₂. This effect of 9r on Nrf2 was also confirmed by immunofluorescence analysis (Figure 7E). These results demonstrated that the Nrf2/HO-1 signaling pathway plays a very important role in the antioxidant activity of 9r.

3.2.7. 9r Exerted Antioxidant Activity Depending on the Activation of Nrf2

Because Nrf2 was confirmed to be associated with the antioxidative activity of 9r, we further determined whether Nrf2 was essential to the inhibitory effect of 9r on oxidative stress. As shown in Figure 8, Nrf2 was knocked down by siRNA transfection. The protein levels of Nrf2 and its downstream proteins, including HO-1 and IL-6, were examined via Western blotting. Accordingly, Nrf2 knockdown reduced the expression of HO-1 and IL-6 (Figure 8). In contrast, these proteins were maintained at relatively high levels after 9r pretreatment, suggesting that the upregulation of Nrf2 would lead to a corresponding increase in IL-6 (Figure 8). More importantly, 9r pretreatment decreased markers of cell apoptosis, including cleaved poly-ADP ribose polymerase (PARP) and p53, but these effects were compromised by Nrf2 knockdown (Figure 8). The results indicated that 9r might resist H₂O₂-induced oxidative stress by targeting the Nrf2/IL-6 positive-feedback loop.

3.2.8. 9r Promotes Cell Survival by Regulating the IL-6/Src/Stat3 Signaling Pathway

In previous cell viability tests, 9r at 20 μM was found to show significant pro-proliferative activity. Therefore, we believe that the antiapoptotic properties of 9r may also be related to the promotion of cell survival, beyond its antioxidant activity. Previous studies have shown that the activation of IL-6 and its downstream signaling pathways could promote cell survival [27]. To verify this hypothesis, we examined the effect of 9r on IL-6 downstream components. In Figure 9, the protein levels of IL-6, p-Stat3 and p-Src were significantly reduced after H₂O₂ treatment. However, the related proteins in H₂O₂-treated SH-SY5Y cells were elevated by 9r pretreatment. These data suggested that 9r can promote cell growth by regulating the IL-6/Src/Stat3 signaling pathway.

3.2.9. 9r Regulates the PI3K and MAPK Signaling Pathways to Promote Cell Survival

IL-6 has also been reported to stimulate the Src-PI3K signaling cascade, which then activates downstream signaling pathways such as PI3K/Akt and mitogen-activated protein kinase (MAPK) [28,29,30,31,32], thereby promoting cell survival. We therefore examined whether the above signaling pathways were involved in the role of 9r in promoting cell growth.

The effect of the PI3K signaling pathway on 9r-promoted cell growth was first explored. Compared to the control group, the phosphorylation levels of PI3K, AKT, mTOR and GSK3β were significantly reduced in the H₂O₂-treated group. However, 9r pretreatment significantly restored the phosphorylation levels of the above proteins (Figure 10). These data implied that PI3K signaling contributed to the prosurvival properties of 9r.

In terms of the MAPK pathway, higher phosphorylation levels of JNK and P38, as well as lower phosphorylation levels of ERK, were observed in the H₂O₂-treated group than in the control group. Instead, 9r reversed the changes in the above proteins induced by H₂O₂ exposure (Figure 11). These results suggested that 9r could also regulate the MAPK signaling pathway to promote cell survival. Taken together, we concluded that 9r exhibited cytoprotective properties against H₂O₂-induced oxidative stress injury to SH-SY5Y cells via the Nrf2 antioxidant pathway and IL-6 prosurvival signaling.

3.2.10. 9r Regulates Oxidative Stress via an IL-6/Nrf2 Positive-Feedback Loop

To further verify the role of the Nrf2/IL-6 positive-feedback loop in the antioxidative activity of 9r, siRNA was employed to knock down IL-6 to confirm the bidirectional regulation between IL-6 and Nrf2 and to explore the effect of 9r on IL-6 and its downstream signaling pathways. Indeed, the results revealed that Nrf2 expression was correspondingly reduced by IL-6 knockdown, suggesting bidirectional regulation between IL-6 and Nrf2. Moreover, the downregulation of IL-6 decreased the levels of the Nrf2 downstream enzymes HO-1 and the IL-6 downstream target molecules p-STAT3, p-Src, p-ERK and p-PI3K (Figure 12). Importantly, the effects of IL-6 knockdown on Nrf2- and IL-6-related downstream pathways were offset, to some extent, by 20 μM 9r pretreatment (Figure 12). Based on the above results, we concluded that 9r significantly suppressed H₂O₂-induced oxidative stress depending on the Nrf2/IL-6 positive-feedback loop.

4. Conclusions

In the present study, a series of 1,4-disubstituted piperazine-2,5-diones were designed and synthesized, and their biological activities were evaluated. Among them, compound 9r exhibited the best antioxidative activity. Further mechanistic studies showed that 9r inhibited apoptosis and promoted cell survival via reducing ROS production and by stabilizing the mitochondrial membrane potential. The cytoprotective properties of 9r against H₂O₂-induced oxidative stress injury in SH-SY5Y cells are dependent on the Nrf2/IL-6 positive-feedback loop (Figure 13). These results implied that compound 9r has the potential to be a novel antioxidant for the treatment of neurodegenerative diseases caused by oxidative stress.

Author Contributions

Conceptualization, L.X. and H.W.; methodology, L.X. and H.W.; software, T.Z.; validation, Q.L., F.L. and M.L.; formal analysis, L.X. and H.W.; investigation, T.Z.; resources, Y.F.; data curation, L.X. and H.W.; writing—original draft preparation, L.X. and H.W.; writing—review and editing, Y.F., L.X. and H.W.; visualization, L.X.; supervision, Y.F.; project administration, Y.F.; funding acquisition, Y.F. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable for studies not involving humans or animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are included within the article and Supplementary.

Acknowledgments

The authors wish to express their gratitude to their Analytical Testing Center for testing the NMR and HR-MS (ESI) data.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures, Scheme and Tables

Table 1

Structures of synthesized piperazine-2,5-dione derivatives 9a–s.

Compound	R1	R2	R3	R4
9a	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9b	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9c	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]		[Image omitted. Please see PDF.]
9d	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9e	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9f	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9g	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9h	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9i	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9j	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9k	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9l	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9m	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9n	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9o	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9p	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9q	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9r	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]
9s	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]

Table 2

The effects of synthetic compounds on H₂O₂-induced oxidative damage in SH-SY5Y cells.

Compound	Viabilities of SH-SY5Y Cells a
	20 (μM)
9a	38.26 ± 1.12
9b	36.09 ± 2.63
9c	49.65 ± 9.64
9d	59.20 ± 6.23
9e	59.12 ± 5.97
9f	47.01 ± 4.87
9g	28.15 ± 1.94
9h	41.24 ± 1.46
9i	23.28 ± 1.75
9j	38.62 ± 1.17
9k	34.41 ± 3.99
9l	25.12 ± 2.96
9m	21.52 ± 1.82
9n	31.55 ± 5.10
9o	61.34 ± 7.52
9p	37.71 ± 3.34
9q	42.41 ± 3.91
9r	70.19 ± 5.64
9s	55.37 ± 5.04
Neohesperidin	65.60 ± 2.72
Syringaresinol	45.39 ± 1.37
H2O2	32.85 ± 10.90

^a Data are representative of three independent experiments, and values are expressed as the mean ± standard deviation (SD). Syringaresinol is the lead compound. Neohesperidin is the positive control.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox11102014/s1, Table S1: the impact of synthetic compounds on SH-SY5Y cytotoxicity.

References

1. Radi, E.; Formichi, P.; Battisti, C.; Federico, A. Apoptosis and Oxidative Stress in Neurodegenerative Diseases. J. Alzheimer’s Dis.; 2014; 42, pp. S125-S152. [DOI: https://dx.doi.org/10.3233/JAD-132738] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25056458]

2. Pizzino, G.; Irrera, N.; Cucinotta, M.; Pallio, G.; Mannino, F.; Arcoraci, V.; Squadrito, F.; Altavilla, D.; Bitto, A. Oxidative Stress: Harms and Benefits for Human Health. Oxidative Med. Cell. Longev.; 2017; 2017, 8416763. [DOI: https://dx.doi.org/10.1155/2017/8416763] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28819546]

3. Bello-Medina, P.C.; Rodríguez-Martínez, E.; Prado-Alcalá, R.A.; Rivas-Arancibia, S. Contaminación por ozono, estrés oxidativo, plasticidad sináptica y neurodegeneración. Neurología; 2022; 37, pp. 277-286. [DOI: https://dx.doi.org/10.1016/j.nrl.2018.10.003]

4. Lee, W.; Lee, C.H.; Lee, J.; Jeong, Y.; Park, J.-H.; Nam, I.-J.; Lee, D.S.; Lee, H.M.; Lee, J.; Yun, N. et al. Botanical Formulation, TADIOS, Alleviates Lipopolysaccharide (LPS)-Induced Acute Lung Injury in Mice via Modulation of the Nrf2-HO-1 Signaling Pathway. J. Ethnopharmacol.; 2021; 270, 113795. [DOI: https://dx.doi.org/10.1016/j.jep.2021.113795]

5. Heinrich, P.C.; Behrmann, I.; Haan, S.; Hermanns, H.M.; Müller-Newen, G.; Schaper, F. Principles of Interleukin (IL)-6-Type Cytokine Signalling and Its Regulation. Biochem. J.; 2003; 374, pp. 1-20. [DOI: https://dx.doi.org/10.1042/bj20030407] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12773095]

6. Kummer, K.K.; Zeidler, M.; Kalpachidou, T.; Kress, M. Role of IL-6 in the Regulation of Neuronal Development, Survival and Function. Cytokine; 2021; 144, 155582. [DOI: https://dx.doi.org/10.1016/j.cyto.2021.155582]

7. Wruck, C.J.; Streetz, K.; Pavic, G.; Götz, M.E.; Tohidnezhad, M.; Brandenburg, L.-O.; Varoga, D.; Eickelberg, O.; Herdegen, T.; Trautwein, C. et al. Nrf2 Induces Interleukin-6 (IL-6) Expression via an Antioxidant Response Element within the IL-6 Promoter. J. Biol. Chem.; 2011; 286, pp. 4493-4499. [DOI: https://dx.doi.org/10.1074/jbc.M110.162008]

8. Li, J.; Calkins, M.J.; Johnson, D.A.; Johnson, J.A. Role of Nrf2-Dependent ARE-Driven Antioxidant Pathway in Neuroprotection. Methods Mol. Biol.; 2007; 399, pp. 67-78. [DOI: https://dx.doi.org/10.1007/978-1-59745-504-6_6]

9. Konishi, T.; Konoshima, T.; Daikonya, A.; Kitanaka, S. Neolignans from Piper Futokadsura and Their Inhibition of Nitric Oxide Production. Chem. Pharm. Bull.; 2005; 53, pp. 121-124. [DOI: https://dx.doi.org/10.1248/cpb.53.121]

10. Li, N.; Wu, J.; Hasegawa, T.; Sakai, J.; Bai, L.; Wang, L.; Kakuta, S.; Furuya, Y.; Ogura, H.; Kataoka, T. et al. Bioactive Lignans from Peperomia Duclouxii. J. Nat. Prod.; 2007; 70, pp. 544-548. [DOI: https://dx.doi.org/10.1021/np0604533]

11. Xu, W.-H.; Zhao, P.; Wang, M.; Liang, Q. Naturally Occurring Furofuran Lignans: Structural Diversity and Biological Activities. Nat. Prod. Res.; 2019; 33, pp. 1357-1373. [DOI: https://dx.doi.org/10.1080/14786419.2018.1474467]

12. Chen, C.-C.; Chen, H.-Y.; Shiao, M.-S.; Lin, Y.-L.; Kuo, Y.-H.; Ou, J.-C. Inhibition of Low Density Lipoprotein Oxidation by Tetrahydrofurofuran Lignans from Forsythia Suspensa and Magnolia Coco. Planta Med.; 1999; 65, pp. 709-711. [DOI: https://dx.doi.org/10.1055/s-1999-14093] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10630110]

13. Wang, A.; Fan, Y.; Ouyang, Q.; Fan, C.; Lin, B.; Liu, J.; Xu, Y. Antiproliferative Ent-Kaurane Diterpenoids Isolated from the Roots of Zea mays L. Fitoterapia; 2019; 134, pp. 44-49. [DOI: https://dx.doi.org/10.1016/j.fitote.2019.02.008] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30763719]

14. Sun, S.; Xu, S.; Yan, Y.; Xie, P.; Lam, W. Optimized High Performance Liquid Chromatography Tandem Chemiluminescent Detector Applied to Assess the Antioxidative Activity of Caulis Stauntoniae Assisted by Chemometrics. Anal. Methods; 2013; 5, 1837. [DOI: https://dx.doi.org/10.1039/c3ay26326f]

15. Tene, M.; Tane, P.; Sondengam, B.L.; Connolly, J.D. Lignans from the Roots of Echinops Giganteus. Phytochemistry; 2004; 65, pp. 2101-2105. [DOI: https://dx.doi.org/10.1016/j.phytochem.2004.05.014]

16. Venkataraman, R.; Gopalakrishnan, S. A Lignan from the Root of Ecbolium Linneanum Kurz. Phytochemistry; 2002; 61, pp. 963-966. [DOI: https://dx.doi.org/10.1016/S0031-9422(02)00387-4]

17. Brown, N. Bioisosteres and Scaffold Hopping in Medicinal Chemistry. Mol. Inform.; 2014; 33, pp. 458-462. [DOI: https://dx.doi.org/10.1002/minf.201400037]

18. Sun, H.; Tawa, G.; Wallqvist, A. Classification of Scaffold-Hopping Approaches. Drug Discov. Today; 2012; 17, pp. 310-324. [DOI: https://dx.doi.org/10.1016/j.drudis.2011.10.024]

19. Majumdar, K.; Ray, K.; Ganai, S. Intramolecular Aza-Wittig Reaction: A New Efficient Tool for the Construction of Piperazine 2,5-Dione Derivatives. Synlett; 2010; 2010, pp. 2122-2124. [DOI: https://dx.doi.org/10.1055/s-0030-1258519]

20. Zhong, T.; Li, M.; Wu, H.; Wang, D.; Liu, J.; Xu, Y.; Fan, Y. Novel Flavan-3,4-Diol Vernicidin B from Toxicodendron Vernicifluum (Anacardiaceae) as Potent Antioxidant via IL-6/Nrf2 Cross-Talks Pathways. Phytomedicine; 2022; 100, 154041. [DOI: https://dx.doi.org/10.1016/j.phymed.2022.154041]

21. Liguori, I.; Russo, G.; Curcio, F.; Bulli, G.; Aran, L.; Della-Morte, D.; Gargiulo, G.; Testa, G.; Cacciatore, F.; Bonaduce, D. et al. Oxidative Stress, Aging, and Diseases. Clin. Interv. Aging; 2018; 13, pp. 757-772. [DOI: https://dx.doi.org/10.2147/CIA.S158513] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29731617]

22. Kudryavtseva, A.V.; Krasnov, G.S.; Dmitriev, A.A.; Alekseev, B.Y.; Kardymon, O.L.; Sadritdinova, A.F.; Fedorova, M.S.; Pokrovsky, A.V.; Melnikova, N.V.; Kaprin, A.D. et al. Mitochondrial Dysfunction and Oxidative Stress in Aging and Cancer. Oncotarget; 2016; 7, pp. 44879-44905. [DOI: https://dx.doi.org/10.18632/oncotarget.9821] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27270647]

23. Kowalczyk, P.; Sulejczak, D.; Kleczkowska, P.; Bukowska-Ośko, I.; Kucia, M.; Popiel, M.; Wietrak, E.; Kramkowski, K.; Wrzosek, K.; Kaczyńska, K. Mitochondrial Oxidative Stress—A Causative Factor and Therapeutic Target in Many Diseases. Int. J. Mol. Sci.; 2021; 22, 13384. [DOI: https://dx.doi.org/10.3390/ijms222413384] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34948180]

24. Ali, T.; Kim, T.; Rehman, S.U.; Khan, M.S.; Amin, F.U.; Khan, M.; Ikram, M.; Kim, M.O. Natural Dietary Supplementation of Anthocyanins via PI3K/Akt/Nrf2/HO-1 Pathways Mitigate Oxidative Stress, Neurodegeneration, and Memory Impairment in a Mouse Model of Alzheimer’s Disease. Mol. Neurobiol.; 2018; 55, pp. 6076-6093. [DOI: https://dx.doi.org/10.1007/s12035-017-0798-6]

25. Kwon, O.Y.; Lee, S.H. Ishige Okamurae Suppresses Trimethyltin-Induced Neurodegeneration and Glutamate-Mediated Excitotoxicity by Regulating MAPKs/Nrf2/HO-1 Antioxidant Pathways. Antioxidants; 2021; 10, 440. [DOI: https://dx.doi.org/10.3390/antiox10030440]

26. Loboda, A.; Damulewicz, M.; Pyza, E.; Jozkowicz, A.; Dulak, J. Role of Nrf2/HO-1 System in Development, Oxidative Stress Response and Diseases: An Evolutionarily Conserved Mechanism. Cell. Mol. Life Sci.; 2016; 73, pp. 3221-3247. [DOI: https://dx.doi.org/10.1007/s00018-016-2223-0]

27. Hirano, T.; Ishihara, K.; Hibi, M. Roles of STAT3 in Mediating the Cell Growth, Differentiation and Survival Signals Relayed through the IL-6 Family of Cytokine Receptors. Oncogene; 2000; 19, pp. 2548-2556. [DOI: https://dx.doi.org/10.1038/sj.onc.1203551] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10851053]

28. Tawfig, N. Proinflammatory Cytokines and Periodontal Disease. J. Dent. Probl. Solut.; 2016; 3, pp. 12-17. [DOI: https://dx.doi.org/10.17352/2394-8418.000026]

29. Chepurnova, D.A.; Samoilova, E.V.; Anisimov, A.A.; Verin, A.D.; Korotaeva, A.A. Compounds of IL-6 Receptor Complex during Acute Lung Injury. Bull. Exp. Biol. Med.; 2018; 164, pp. 609-611. [DOI: https://dx.doi.org/10.1007/s10517-018-4042-9]

30. Mihara, M.; Hashizume, M.; Yoshida, H.; Suzuki, M.; Shiina, M. IL-6/IL-6 Receptor System and Its Role in Physiological and Pathological Conditions. Clin. Sci.; 2012; 122, pp. 143-159. [DOI: https://dx.doi.org/10.1042/CS20110340]

31. Narazaki, M.; Kishimoto, T. The Two-Faced Cytokine IL-6 in Host Defense and Diseases. Int. J. Mol. Sci.; 2018; 19, 3528. [DOI: https://dx.doi.org/10.3390/ijms19113528] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30423923]

32. Yuan, Y.; Shan, N.; Tan, B.; Deng, Q.; Liu, Y.; Wang, H.; Luo, X.; He, C.; Luo, X.; Zhang, H. et al. SRC-3 Plays a Critical Role in Human Umbilical Vein Endothelial Cells by Regulating the PI3K/Akt/MTOR Pathway in Preeclampsia. Reprod. Sci.; 2018; 25, pp. 748-758. [DOI: https://dx.doi.org/10.1177/1933719117725818] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28826365]