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

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.


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 (H 2 O 2 ) is responsible for redox signaling transmission from the generative site to a target site. Hence, H 2 O 2 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

General Methods
All reagents and solvents used in the experiment were commercially available and used without further purification. 1 H nuclear magnetic resonance (NMR) data were measured and recorded on a Bruker 600 MHz NMR spectrometer (Bruker; Billerica, MA, USA), and 13 C 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.

General Methods
All reagents and solvents used in the experiment were commercially available and used without further purification. 1 H nuclear magnetic resonance (NMR) data were measured and recorded on a Bruker 600 MHz NMR spectrometer (Bruker; Billerica, MA, USA), and 13 C 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. Under a N 2 atmosphere, a mixture of 1a (100 mg, 0.653 mmol) and anhydrous K 2 CO 3 (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 K 2 CO 3 was filtered off, and the solvent was removed under vacuum. Then, it was purified via column chromatography over silica 4-Methoxybenzoyl chloride (53 mg, 0.312 mmol) in CH 2 Cl 2 (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 CH 2 Cl 2 (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. 1

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.

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.

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.

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.

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.

SiRNA Transfection
China Guangzhou Nuclear Biological Co., Ltd. (Guangzhou, China) designed and synthesized small interfering RNA (siRNA) oligonucleotides for IL-6, Nrf2 and negativecontrol 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 H 2 O 2 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 .

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.

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.                          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 H 2 O 2 -treated SH-SY5Y cells from 32.85% (H 2 O 2 -treated group) to 70.19% (9r-treated group). Therefore, we chose to further explore the possible antioxidative mechanism of compound 9r.

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 H 2 O 2 -induced oxidative stress and promoted the viability of H 2 O 2 -treated SH-SY5Y cells. Compound 9r showed better antioxidative activity than the lead compound syringaresinol ( Figure 2B). In addition, similar cytoprotective activities against H 2 O 2 -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 H 2 O 2 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 H 2 O 2 in a dose-dependent manner. These results suggested that 9r possessed a cytoprotective effect against oxidative damage caused by H 2 O 2 treatment in SH-SY5Y cells.

9r Attenuated H 2 O 2 -Induced Cell Apoptosis in SH-SY5Y Cells
H 2 O 2 -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 H 2 O 2 -induced apoptosis using an Annexin V-fluorescein isothiocyanate (FITC) apoptosisdetection kit. Figure 3 demonstrates that the apoptosis rate in the H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 dramatically increased the related proapoptotic proteins (p53, Bax, Cl-PARP) while decreasing the antiapoptotic protein Bcl2. However, 9r pretreatment abolished the H 2 O 2 -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.
Antioxidants 2022, 11, x FOR PEER REVIEW 13 The results of the morphological alterations showed that H2O2 treatment dramatical creased the number of living cells and resulted in cell contraction and nuclear fragm tion ( Figure 2C). However, 9r significantly prevented the cell morphological cha caused by H2O2 in a dose-dependent manner. These results suggested that 9r posses cytoprotective effect against oxidative damage caused by H2O2 treatment in SHcells. Data are provided as the mean ± standard deviation of three independent e ments. Compared to the described control group, *** p < 0.001. Compared to the H2O2-treated g & p < 0.05, && p < 0.01 and &&& p < 0.001.

9r Attenuated H2O2-Induced Cell Apoptosis in SH-SY5Y Cells
H2O2-induced oxidative stress eventually results in a dramatic increase in cell a tosis [21]. To investigate whether the cytoprotective effect of 9r was associated wit mediation of cell apoptosis, flow cytometry was used to detect the effect of 9r on H induced apoptosis using an Annexin V-fluorescein isothiocyanate (FITC) apoptos tection kit. Figure 3 demonstrates that the apoptosis rate in the H2O2-treated group (2 was substantially greater than the apoptosis rate in the control group (1.28%); how 9r pretreatment for 2 h dose-dependently inhibited H2O2-induced apoptosis. To fu determine the inhibitory activity of 9r on cell apoptosis, we next examined the influ of 9r on proteins involved in the apoptotic pathways using Western blotting. As indi in Figure 4, exposure to H2O2 dramatically increased the related proapoptotic pro (p53, Bax, Cl-PARP) while decreasing the antiapoptotic protein Bcl2. However, 9r treatment abolished the H2O2-induced alterations in the related apoptotic proteins, w was consistent with our prior findings. The data suggested that the cytoprotective ac of 9r was partially attributed to the inhibitory effects of the apoptotic pathway.    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

Compound 9r Attenuated Mitochondrial Dysfunction Caused by H 2 O 2 Treatment
As previously mentioned, mitochondrial dysfunction is one of the major causes of H 2 O 2 -induced cell apoptosis [22]. Therefore, the effect of 9r on mitochondrial function in H 2 O 2 -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 H 2 O 2 -treated group compared with the control group. However, 9r pretreatment counteracted the effects of H 2 O 2 on mitochondrial membrane potential ( Figure 5). The results suggested that 9r could resist the mitochondrial dysfunction caused by H 2 O 2 . The results are shown as the mean ± SD of three independent tests. Compared to the described control group, *** p < 0.001. compared to the H2O2−treated group, & p < 0.05 and && p < 0.01.

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 re- The results are shown as the mean ± SD of three independent tests. Compared to the described control group, *** p < 0.001. compared to the H 2 O 2 −treated group, & p < 0.05 and && p < 0.01.

9r Decreased the Generation of ROS in H 2 O 2 -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 H 2 O 2 treatment was examined. As shown in Figure 6, H 2 O 2 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 H 2 O 2 was steadily downregulated by 9r pretreatment, suggesting that 9r reduced H 2 O 2induced ROS production in a dose-dependent manner. In summary, 9r may counteract the oxidative injury caused by H 2 O 2 by reducing ROS generation. 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. 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 H 2 O 2 . 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.

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 H 2 O 2 -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 H 2 O 2 treatment. However, the related proteins in H 2 O 2 -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.

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 H 2 O 2 -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.  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  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.

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. 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 H 2 O 2 -treated group than in the control group. Instead, 9r reversed the changes in the above proteins induced by H 2 O 2 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 H 2 O 2 -induced oxidative stress injury to SH-SY5Y cells via the Nrf2 antioxidant pathway and IL-6 prosurvival signaling. 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. 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 H 2 O 2 -induced oxidative stress depending on the Nrf2/IL-6 positive-feedback loop.

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.

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 H 2 O 2 -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. Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: the impact of synthetic compounds on SH-SY5Y cytotoxicity.