Debulking surgery followed by chemotherapy are the standard of care for high-grade serous carcinoma. After an initial good response to treatment, the majority of patients relapse with a chemoresistant profile, leading to a poor overall survival. Chemotherapy regimens used in high-grade serous carcinomas are based in a combination of classical chemotherapeutic drugs, namely, Carboplatin and Paclitaxel. The mechanisms underlying drug resistance and new drug discovery are crucial to improve patients’ survival. To uncover the molecular mechanisms of chemoresistance and test drugs capable of overcoming this resistant profile, it is fundamental to use good cellular models capable of mimicking the chemoresistant disease. Herein, we established two high-grade serous carcinoma cell lines with intrinsic resistance to Carboplatin and induced Paclitaxel resistance (OVCAR8 PTX R C and OVCAR8 PTX R P) derived from the OVCAR8 cell line. These two chemoresistant cell line variants acquired an enhanced resistance to Paclitaxel-induced cell death by increasing the drug efflux capacity, and this resistance was stable in long-term culture and following freeze/thaw cycles. The mechanism underlying Paclitaxel resistance resides in a significant increase in P-glycoprotein expression and, when this drug efflux pump was blocked with Verapamil, cells re-acquired Paclitaxel sensitivity. We generated two high-grade serous carcinoma cell lines, with a double-chemoresistant (Carboplatin and Paclitaxel) phenotype that mimics the majority of tumor recurrences in ovarian cancer context. This robust tool is suitable for preliminary drug testing towards the development of therapeutic strategies to overcome chemoresistance. Late diagnosis and resistance to treatment are the main reasons for the high mortality rate of high-grade serous carcinoma (HGSC) patients , 2. TherPlatinum-taxane-based chemotherapy is often used in cancer therapy in order to disrupt different cellular mechanisms involved in the regulation of the cell cycle to induce tumor cell death , 14. UnfIn the majority of established chemoresistant cell lines, Cisplatin is the drug mostly used for the induction of resistance . PTX-resSince HGSC is the most frequent epithelial OC histotype and double-resistant tumors are the major contributors to high mortality rate, we herein present a HGSC cell line model that combines intrinsic resistance to Carboplatin and acquired PTX chemoresistance. We generated two PTX-resistant cell lines derived from OVCAR8, with a stable acquired PTX resistance induced by P-glycoprotein (P-gp) overexpression. The development of these HGSC cell lines, capable of recapitulating the characteristics of double-resistant epithelial ovarian carcinoma, constitutes a key tool for the discovery of therapeutic alternatives to improve patients’ outcome.2. OVCAR8 was authenticated using short tandem repeat profiling and regularly tested for the absence of mycoplasma.OVCAR8 was selected as a HGSC model, particularly since it is described as a Carboplatin-resistant OC cell line retrieved from a HGSC patient after a high-dose Carboplatin treatment . ParentaPTX was purchased from Selleckchem , dissolved in dimethyl sulfoxide  and stored at −80°C, according to the manufacturer’s instructions. Immediately prior to use, an aliquot was diluted at required concentrations.Two OVCAR8 PTX R variants were established in our laboratory from parental OVCAR8 by continuous (C) and pulse (P) exposure to a stepwise increasing PTX concentration (2 to 74.9 nM), for 3 months  cells were exposed to PTX constantly (continuous-selection strategy) and (ii) cells were exposed to PTX, for 4 days followed by recovery in drug-free media for 4 days (pulse-selection strategy) for 2 months. Next, we tested if PTX R phenotype was stable in culture without drug. Hence, after 2 months of freezing cycles, cells were cultured in the absence of PTX medium supplementation  and incubated under the same conditions. After 48 h, 50 μl of PrestoBlue™ Cell Viability Reagent 1× (ThermoFisher Scientific) was added and incubated for 45 min at 37°C and 5% CO2, protected from light. Fluorescence was measured (560 nm excitation/590 nm emission) using a Bio Tek SynergyTM 2 multi-mode microplate reader . Treated cells were compared with control cells (considered 100% viable) containing 1% (v/v) of the vehicle (DMSO). The IC50 for parental OVCAR8 and PTX R variants was used to determine fold resistance, according to the following formula —was performed, according to the manufacturer’s instructions. Briefly, 2 × 10 formula , 39:3 cells/well were seeded into a 96-well plate in complete media and incubated at 37°C and 5% CO2 up to 6 days. At each time point, 500 µg/ml of MTT dye reagent  was added and incubated at 37°C and 5% CO2 for 3 h. Formazan crystals were dissolved in 100 µl of DMSO. Absorbance was measured (570 nm wavelength) using a Bio Tek SynergyTM 2 multi-mode microplate reader.To evaluate cell proliferation, MTT  assay was performed as previously described . BrieflyTo differentiate viable and non-viable cells, trypan blue assay was performed. Cells at 80% confluence were diluted and mixed with a 0.4% trypan blue solution (ThermoFisher Scientific), placed in a Neubauer chamber, and counted in five counting grid squares, under a Leica DMi1 inverted phase contrast microscope , at 50× magnification.5 cells/well were seeded into six-well plates and incubated at 37°C and 5% CO2. After 48 h, cells were treated with 10 nM PTX and incubated under the same conditions. After 48 h, cells were scraped from culture dishes and fixed with 10% (v/v) neutral-buffered formalin (AppliChem). Cells were re-suspended in liquefied HistoGel™ (ThermoFisher Scientific), according to the manufacturer’s instructions, followed by standard histological processing and paraffin embedding. Finally, CMA block was constructed and sectioned with a microtome (All conditions were arrayed in a Cell MicroArray (CMA) block designed and constructed as previously described , 42. Briicrotome . To perfImmunocytochemistry results were evaluated by two independent observers (MN and SR) that register the staining pattern  and the percentage of cells stained .To perform a qualitative assessment of cell ultrastructure, an electron microscopy analysis was performed. Briefly, for transmission electron microscopy and semithin section analysis, cultured cells were grown until 80% confluence, washed three times with phosphate buffer saline , gently scraped from culture dishes and fixed overnight in a glutaraldehyde (2.5%), paraformaldehyde (2%), and 0.1 M sodium cacodylate buffer (1:1) solution, at 4°C. After fixation, cells were pelleted by centrifugation and washed three times in 0.1 M sodium cacodylate buffer for 5 min. A post-fixation in 0.1 M sodium cacodylate buffered 2% osmium tetroxide was performed for 2 h at room temperature (RT). Then, cells were washed three times in distilled water for 5 min. The samples were incubated with 1% uranyl acetate for 30 min at RT, followed by three washes in distilled water for 5 min. Finally, cell pellet was imbedded in Histogel™, dehydrated in ethanol, and embedded in an epoxy resin. Ultrathin sections were stained and observed in a JEOL 100CXII transmission electron microscope  operated at 80 kV, and images were digitally recorded using a CCD digital camera Orius 1100W (JEOL).5 cells/well were seeded into six-well plates and incubated at 37°C and 5% CO2. After 48 h, cells were treated with 10 nM PTX and incubated under the same conditions.To evaluate cell apoptosis and cell cycle distributions, Annexin V-FITC apoptosis detection kit™ (ThermoFisher Scientific) and propidium iodide (PI) staining were used, according to the manufacturer’s instructions. Briefly, 2 × 10For apoptosis analysis, after 48 h, floating and adherent cells were collected using a cell dissociation buffer enzyme-free in PBS, pelleted by centrifugation , filtrated through a 70-μm filter to obtain single-cell suspensions, and suspended in 195 µl of binding buffer. Next, 5 µl of Annexin V-FITC was added and incubated for 10 min, protected from light. Cells were washed, resuspended in 190 µl of binding buffer, and 10 µl of PI (20 μg/ml) was added and cells were incubated for 1 min, protected from light.For cell cycle, following 24 h, floating and adherent cells were collected, pelleted by centrifugation, and resuspended in PBS. Cells were fixed with 70% (v/v) cold ethanol and incubated at 4°C for 30 min. Next, cells were pelleted by centrifugation, resuspended in PBS containing PI (250 µg/ml), and RNase A (100 µg/ml) and incubated at 4°C for 15 min, protected from light.Fluorescence from both assays was assessed by BD FACS Canto™ II flow cytometer and data were analyzed by FlowJo software v10.0.7 .5 cells were seeded into LabTek II chambered cover glass  containing 1 ml of culture medium and incubated at 37°C with 5% CO2. After 24 h, cells were treated with 20 nM PTX in RPMI without phenol red and supplemented with 5% FBS. Images were captured at 5-min intervals up to 72 h under differential interference contrast (DIC) optics, with a 63× objective under an Axio Observer Z.1 SD inverted microscope, equipped with an incubation chamber at 37°C and 5% CO2. Movies were generated from time-lapse images using ImageJ 1.4v software . The mitosis duration and the number of cells arrested at mitosis, dead by apoptosis, or bypassing cytokinesis were scored.Live-cell imaging experiments were performed as previously described . Briefly6 cells/well were seeded into polyHEMA -coated plates and incubated at 37°C and 5% CO2 up to 15 days. At each time point , floating cells were collected and dissociated using trypsin, pelleted by centrifugation, and resuspended in PBS. Cell suspensions were incubated with DAPI (1 µg/ml) for 2 min. Fluorescence was assessed by BD FACS Canto™ II flow cytometer, and data were analyzed by FlowJo software v10.0.7.To evaluate anoikis resistance, an aggregate formation assay was performed as previously described . Briefly3 cells/well were seeded into six-well plates and incubated at 37°C and 5% CO2. Following 24 h, cells were treated with 10 nM PTX and incubated under the same conditions. After 48 h, media was replaced, and cells were allowed to recover for 8 days. Surviving colonies were fixed and stained as previously described , according to the manufacturer’s instructions. cDNA synthesis was performed with iScript™ cDNA Synthesis Kit (Bio-Rad), using total cell RNA as template, following supplier’s instructions. For real-time PCR, cDNA was amplified using iQ™ SYBR Green Supermix Kit (Bio-Rad). Primers for P-gp were as follows: forward: 5’-GCCAAAGCCAAAATATCAGC-3’ and reverse: 5’-TTCCAATGTGTTCGGCATTA-3’; GAPDH: forward: 5’-ACAGTCCAGCCGCATCTTC-3’ and reverse: 5’-GCCCAATACGACCAAATCC-3’; and β-Actin, used as housekeeping gene: forward: 5’-AAT CTG GCA CCA CAC CTT CTA-3’ and reverse 5’-ATA GCA CAG CCT GGA TAG CAA-3’. Data were acquired with CFX Manager™ Software v1.0 (Bio-Rad) and results were analyzed according to ΔCT.Total cell protein extracts and Western blotting were performed as previously described . Membran5 cells of OVCAR8 and OVCAR8 PTX R were seeded into six-well plates and incubated at 37°C and 5% CO2. After 24 h, 1 µM of the fluorescent P-gp substrate Rhodamine 123  was added, in the presence or absence of 20 µM Verapamil (Sigma-Aldrich)—P-gp inhibitor—and incubated under the same conditions. After 1 h, cells were harvested and washed twice with ice-cold PBS, and cell pellet was gently re-suspended in ice-cold PBS for analysis. The mean fluorescence intensity (MFI) was assessed by BD Accuri C6™ II flow cytometer , and data were analyzed by BD Accuri C6 Plus Software, version 1.0.27.1 (BD Biosciences).To evaluate P-gp functional activity, a total of 1 × 106 cells/well were seeded in 96-well plates and incubated at 37°C and 5% CO2. After 24 h, cells were treated with 10-fold serial dilutions of PTX, in the presence or absence of 10 µM Verapamil and incubated under the same conditions. In parallel, cells were treated with equivalent amount of vehicle (DMSO) up to 0.25% concentration. Then, cells were fixed with 50% (m/v) trichloroacetic acid  for 1 h, washed with distilled water, and stained with 0.1% (m/v) SRB in acetic acid (Sigma-Aldrich) for 30 min at RT. After washing with 1% (v/v) acetic acid aquae solution (Merck Millipore), plates were left to dry at RT followed by SRB complex solubilization with 10 mM Tris-Base buffer (Sigma-Aldrich) for 30 min. Absorbance was measured (515 nm wavelength) using a Bio Tek SynergyTM 2 multi-mode microplate reader. The IC50 of PTX, in the presence or absence of Verapamil, was determined as described above in Section 2.3.To determine cell viability to PTX after Verapamil incubation, a Sulforhodamine B (SRB) assay was performed. Briefly, 5 × 10All assays were done in triplicate with at least three independent experiments. Data were expressed as mean ± standard deviation (SD), statistical analysis was carried out in GraphPad Prism Software Inc. v6 using ordinary one-way or two-way ANOVA followed by Tukey’s or Šídák’s multiple comparison test, and values of *<0.05; **<0.001; ***<0.0005; ****<0.0001 were considered statistically significant.p < 0.001) and OVCAR8 PTX R P  variants when compared to OVCAR8 cell line (10.51 ± 1.99 nM), demonstrating a 12.27-fold and 14.54-fold increase in resistance, respectively  when compared to OVCAR8 cell line (127.49 ± 4.76 nM)  and OVCAR8 PTX R P  increase in PTX-induced apoptosis was observed on OVCAR8 cell line (37.01% ± 1.63%) when compared with the nontreated control (7.40 ± 2.48%) (p < 0.0001) in PTX-induced apoptosis in OVCAR8 PTX R C and P variants  when compared to OVCAR8 cell line (37.01% ± 1.63%)  in the percentage of cells in G1 phase (54.61 ± 3.32% vs. 27.84 ± 2.89%) and accumulation of cells in G2/M phase (18.77 ± 0.56% vs. 44.18 ± 4.75%) was observed in the OVCAR8 cell line when compared to control (p < 0.0001) in the percentage of cells in G1 phase was found in OVCAR8 PTX R C and P variants  when compared to parental cell line (27.84% ± 2.89%) (p < 0.0001) in the G2/M population was found in OVCAR8 PTX R C and P variants  when compared to the parental cell line (44.18% ± 4.75%) (p < 0.001) was also observed in sub-G1 fraction (debris and apoptotic cells) for the OVCAR8 cell line (3.11% ± 0.91% vs. 7.60% ± 1.16%) and the OVCAR8 PTX R P variant (2.41% ± 0.79% and 7.15% ± 0.76%) when compared to controls  exhibited by the OVCAR8 cell line (331.29 ± 106.82 min). After 20 nM PTX exposure, the survival fate (outcome of cells delayed in mitosis under PTX exposure) analysis showed that 37.5% of parental OVCAR8 cells died in mitosis after mitotic arrest, and 62.5% of cells underwent normal cycling  resistance, an aggregate formation assay was performed and evaluated by flow cytometry. Our results for anoikis resistance demonstrate no significant differences in cell viability (DAPI negative cells) for OVCAR8 PTX R C (87.88% ± 3.29% and 78.20% ± 3.73%) and OVCAR8 PTX R P variants (89.96% ± 1.74% and 84.03% ± 4.97%) when compared to parental OVCAR8 cell line (94.32% ± 1.15% and 86.47% ± 1.66%), upon 5 and 10 days of culture, respectively (p < 0.0005) occurs in the number of colonies in the OVCAR8 cell line (274.78 ± 49.27 vs. 10.56 ± 8.59) when compared to the untreated control (vs. 254.00 ± 58.68) and OVCAR8 PTX R P variants (241.44 ± 24.44 vs. 182.11 ± 44.29) when compared to untreated control (p < 0.0005) in OVCAR8 PTX R C (85.80% ± 4.80%) and OVCAR8 PTX R P variants (74.92% ± 7.72%) when compared to the parental OVCAR8 cell line (3.98% ± 2.08%)  and OVCAR8 PTX R P variants (25.27% ± 3.55%) when compared to the OVCAR8 cell line (27.63% ± 0.81%) at 48 h  for OVCAR8 PTX R C (89.73% ± 3.22%) and OVCAR8 PTX R P variants (83.14% ± 3.30%) when compared to control, demonstrating an accumulation of RH-123 due to P-gp pump inhibition  and OVCAR8 PTX R P variants (33-fold) when Verapamil was added . This research was supported by European Regional Development Funds (ERDF) funds through the COMPETE 2020–Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação (MCTES), under the project POCI 01-0145-FEDER-029503 (PTDC/MEC-ONC/29503/2017) and CESPU (Cooperativa de Ensino Superior Politécnico e Universitário) under the project ComeTarget_CESPU_2017 (to HB). MN acknowledges FCT/MCTES and UE for financial support through a PhD fellowship (2020.04720.BD) co-sponsored by Fundo Social Europeu (FSE) through Programa Operacional Regional Norte (Norte 2020).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Shc were found in this study. However, the inhibitors of p38 and ERK1/2 significantly reduced the apoptosis of HPdLFs. Interestingly, PTHrP pre-treatment significantly reduced the phosphorylation levels of p38, ERK1/2, and p66Shc. More importantly, MKP1 inhibitor sanguinarine inhibited the dephosphorylation levels of p38, ERK1/2, and p66Shc caused by PTHrP. Altogether, PTHrP can inhibit nitrogen-containing BP-induced apoptosis of HPdLFs by activating MKP1 phosphatase.Massive production of reactive oxygen species (ROS) in human periodontal ligament fibroblasts (HPdLFs) by nitrogen-containing bisphosphonates (BPs) is the main factor causing BP-related osteonecrosis of the jaw. Further, oxidative stress and apoptosis of fibroblasts induced by ROS are closely associated with the activation of MAPK. Parathyroid hormone-related protein (PTHrP) can block the activity of MAPK by regulating the levels of MAPK phosphatase 1 (MKP1). Therefore, it is speculated that PTHrP can inhibit the apoptosis of HPdLFs caused by nitrogen-containing BP via regulating the expression levels of MKP1. Herein, alendronate sodium salt trihydrate  and HPdLFs were co-cultured for 24 h, 48 h, and 72 h, and the levels of ROS and apoptosis were determined, respectively. After 48 h co-culture, FOS significantly increased the levels of ROS and apoptosis, and high phosphorylation levels of p38, ERK1/2 and p66  BPs areShc, an amplifier of ROS, is an important mediator for ROS-induced apoptosis.ROS are produced during oxidative phosphorylation of the mitochondria or may be produced by exogenous substances, such as xenobiotic compounds . When thHormones such as thyroid hormones and melatonin, which are extracellular regulators of oxidative stress, can target various signaling pathways in a variety of tissues ,20,22,23In view of the fact that the oxidative stress and apoptosis of fibroblasts induced by ROS are closely associated with the activation of MAPK and that PTHrP can regulate the activity of MAPK by regulating MKP1, it can be speculated that PTHrP can inhibit ROS-induced apoptosis of fibroblasts by regulating the expression of MKP1. To verify this hypothesis, the levels of ROS and apoptosis were determined after FOS and HPdLFs were co-cultured, and the relationship between PTHrP and the expression level of MKP1 was comprehensively analyzed to disentangle whether and how PTHrP pre-treatment inhibits FOS-induced apoptosis of HPdLFs at the molecular level.2.2.1Shc (Ser36) were acquired from ThermoFisher in China. Propidium iodide was purchased from Solarbio in China.Nitrogen-containing BP, alendronate sodium salt trihydrate (FOS) was purchased from Wako in Japan. SCGMTM BulletKitTM, N-acetyl-cysteine (NAC), fluorescein isothiocyanate (FITC)-conjugated annexin V, and ECL chemiluminescence were acquired from Sigma in China. PTHrP, cell-permeable 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), U0126 (inhibitor of ERK1/2), SB203580 (inhibitor of p38), sanguinarine chloride (inhibitor of MKP1), lipofectamine LTX transfection reagent, Tris HCl, NaCl, EDTA, 1% Triton X-100, 1% sodium hydroxide, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail, cocktail Set II, bovine serum albumin (BSA), Considering bicinchoninic acid (BCA), nitrocellulose membrane were obtained from ThermoFisher in China. In addition, α-Tubulin, peroxidase-conjugated goat anti-rabbit IgG and the antibodies of p-ERK1/2, ERK1/2, p-Akt, Akt, p-p38, p38, p-JNK, JNK, MKP-1, and p-p662.2.2.The normal adult HPdLFs used in this study were purchased from ScienCell (USA). The HPdLFs were cultured with SCGMTM BulletKitTM  at 37°C with 5% COShc were analyzed. In addition, the inhibitors of p38 (SB203580 [5 µM]) and ERK1/2 (U0126 [10 µM]) were used to analyze the roles of p38 and ERK1/2 in apoptosis of HPdLFs.To investigate the effect of FOS on HPdLFs, after the HPdLFs were treated with 30 µM FOS for 24 h, 48 h, and 72 h, the levels of ROS and apoptosis were determined. Based on the result, HPdLFs treated with 30 µM FOS for 48 h were selected as experimental subject, and then the phosphorylation levels of p38, ERK1/2, Akt, JNK and p66To disentangle whether and how PTHrP inhibits FOS-induced apoptosis of HPdLFs, following the treatment with 30 µM FOS, the HPdLFs were treated with 100 nM PTHrP for 30 min, and then with sanguinarine chloride [0.5 µM] for 1 h to analyze the relationship between PTHrP and the expression level of MKP1.2.33) were incubated in a 96-well plate for 24 h, and then treated with 30 µM FOS for 24 h, 48 h, and 72 h, respectively. The cells untreated with FOS in the corresponding time points were considered as controls. Approximately 12 h before the cells were treated with FOS, 90 mM NAC was added to the cell suspension [The HPdLFs (1 × 10spension . To dete2.5.TM (Becton Dickinson) and analyzed with CellQuestTM (Becton Dickinson).To measure apoptosis, the cells were double-stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide for 5 min at room temperature. The number of annexinV(-)/propidium iodide(-) living cells and annexinV(+) apoptotic cells was examined by flow cytometry . The data were collected with FACSCalibur2.6.Shc (Ser36) (1:500) were added, followed by incubation overnight at 4°C. α-Tubulin was used as the loading control. After washing, peroxidase-conjugated goat anti-rabbit IgG was incubated with the membrane, and ECL chemiluminescence was added. The optical density of the protein band was normalized to the corresponding α-tubulin band.Total cellular protein was extracted at pH 7.4 with 50 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium hydroxide, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail, and cocktail Set II. Considering bovine serum albumin (BSA) as the standard, bicinchoninic acid (BCA) was used to determine the content of protein in the cellular extract. The proteins were isolated using 5%-10% polyacrylamide-SDS gels (30–60 μg/Lane). After electrophoresis, the sample was transferred to a nitrocellulose membrane, and blocked with 5% skim milk containing 50 mmol/L Tris-HCl (pH 7.5) and 150 mmol/L NaCl containing 0.05% Tween 20. The corresponding antibodies p-ERK 1/2 (1:1000), ERK 1/2 (1:2000), p-Akt (1:1000), Akt (1:2000), p-p38 (1:1000), p38 (1:2000), p-JNK (1:1000), JNK (1:2000), MKP-1 (1:500), and p-p662.7.P < 0.05 was considered significant.The data are expressed as mean±standard error of mean (SEM). Nonparametric variance analysis  was used to evaluate changes in kinase phosphorylation and apoptosis, followed by post-hoc Dunn’s test. 3.3.1.Previous studies have shown that BPs can induce the generation of ROS in HPdLFs. To verify this conclusion, HPdLFs were treated with 30 μM of FOS for different time points . Subsequently, the ROS content was determined and it was found that the ROS content was significantly increased after FOS and HPdLFs were co-cultured for 48 h . Previou3.2.Shc were significantly increased (Shc. Thus, we analyzed the effect of ERK1/2 and p38 inhibitors on the phosphorylation of p66Shc and found that ERK1/2 and p38 inhibitors can inhibit p66Shc phosphorylation induced by FOS (Shc by regulating ERK1/2 and p38, and subsequently induce apoptosis.It has been reported that ROS can regulate intracellular kinases and activate the related cell signaling pathways ,12. Therncreased , whereasncreased . Phosphod by FOS . This in3.3.2O2-induced ROS in osteoblasts [Shc (Shc through MKP1 [Shc induced by FOS through MKP1. To verify this hypothesis, as described previously [Shc in HPdLFs treated with PTHrP and FOS were significantly higher (Shc.It has been reported that PTHrP can inhibit the phosphorylation of MAPKs induced by Heoblasts . To studsts [Shc . Previouugh MKP1 . Therefoeviously , we usedy higher , indicat3.4.To determine whether PTHrP can inhibit the apoptosis of HPdLFs induced by FOS, HPdLFs cells were co-cultured with PTHrP and FOS for 48 h and the degree of apoptosis was examined. It was found that PTHrP could significantly inhibit FOS-induced apoptosis of HPdLFs . However4.The massive production of ROS can damage HPdLFs, and subsequently cause BRONJ . During It has been reported that BPs can induce the generation of ROS in many types of cells, such as human gingival fibroblasts , osteocl2O2-induced apoptosis of osteoblasts by regulating the phosphorylation of p38 MAPKs and ERK [2O2 [PTHrP regulates various biological processes of various mammalian cells, such as cell differentiation, proliferation, and apoptosis ,45. Prev and ERK . This stERK [2O2 . This stERK [2O2 . TherefoShc at Ser36 can regulate oxidative stress-related apoptotic response and cell senescence [Shc can open the mitochondrial permeability transition pore, leading to the swelling of organelles [Shc can not only regulate apoptotic signals but also continuously enhance the production of ROS through different mechanisms. Therefore, regulation of p66Shc phosphorylation may be the key target for inhibiting apoptosis induced by ROS. Our results show that a part of dephosphorylation of PTHrP to p66Shc is regulated by MKP1 in HPdLFs. In response to the process of oxidative stress, the activation of ERK1/2 and p38 may promote the phosphorylation of p66Shc at Ser36, Ser54, and Thr386 [Shc may be a downstream effector in the ERK1/2 and p38 pro-apoptosis process. We found that the inhibitors of ERK1/2 and p38 can inhibit the FOS-induced phosphorylation of p66Shc, suggesting that p66Shc is a downstream regulator of ERK1/2 and p38 during the process of FOS-induced HPdLF apoptosis.It has been reported that the phosphorylation of p66nescence . The phoganelles . Subsequganelles . Phosphod Thr386 ,51, indi5.In summary, our results suggest that PTHrP inhibits nitrogen-containing BP-induced apoptosis of HPdLFs by activating MKP1 phosphatase.


Aconitum herbs should be processed before clinical application. The processing methods include boiling, steaming, and sand frying. Among these methods, the transformation pathways of diterpenoid alkaloids in the process of sand frying are more complicated. Therefore, crassicauline A, a natural product with two ester bonds, was chosen as the experimental object. Consequently, a known alkaloid, together with three new alkaloids, was derived from crassicauline A. Meanwhile, the cardiotoxicity of converted products was reduced compared with their parent compound. Interestingly, some diterpenoid alkaloids have similar structures but opposite effects, such as arrhythmia and antiarrhythmic. Considering the converted products are structural analogues of crassicauline A, herein, the antiarrhythmic activity of the transformed products was further investigated. In a rat aconitine-induced arrhythmia assay, the three transformed products, which could dose-dependently delay the ventricular premature beat (VPB) incubation period, reduce the incidence of ventricular tachycardia (VT), combined with the increasing arrhythmia inhibition rate, exhibited prominent antiarrhythmic activities. Our experiments speculated that there might be at least two transformation pathways of crassicauline A during sand frying. The structure-activity data established in this paper constructs the critical pharmacophore of diterpenoid alkaloids as antiarrhythmic agents, which could be helpful in searching for the potential drugs that are equal or more active and with lower toxicity, than currently clinical used antiarrhythmic drugs.To ensure safety and efficacy, most  Aconitum L. (Ranunculaceae) is a large herbal medicinal genus, which has 300 species widely distributed in the temperate regions of the northern hemisphere. In China, over 200 species are distributed mainly in western Sichuan, northwestern Yunnan, and eastern Tibet Autonomous Region. Among which 76 species can be extensively employed in traditional Chinese medicine and ethnomedicine  and steaming (steaming for 6∼8 h) methods are diffusely applied in traditional Chinese medicine, of which the structural transformation pathways of DDAs have been elucidated clearly, mainly because DDAs are successively hydrolyzed into less toxic monoester- and amines-diterpenoid alkaloids  of  studies . It was  process . Howeverum Busch . It was Aconitum drugs produced in Yunnan province (Aconitum hemsleyanum E. Pritz. (syn. A. crassicaule W. T. Wang) (Crassicauline A (bulleyaconitine A), the main component of many province , such asT. Wang) , is a coT. Wang) . TherefoNotably, some diterpenoid alkaloids exhibit opposite effects, though their structures are extremely similar. For example, aconitine has serious proarrhythmic effects, while aconitine-like compounds such as 14-benzoyltalatisamine, 14-benzoyldelcosine, and 14-benzoyldictyocarpine  have ant3 with tetramethylsilane (TMS) as internal standard. Mass spectra were carried out on a micrO-TOF-Q-II mass spectrometer . RE-3000B rotary evaporator  was used to evaporate the solvent. Silica gel G (200–300 mesh) for column chromatography and TLC plates (silica gel G) were obtained from Qingdao Sea Chemical Factory .Shimadzu LC-2030C system  and LabSolutions software program were used for chromatographic separation of components and analyzing HPLC data. Preparative-HPLC , equipping with a GL6000-cp1000 high-pressure infusion pump, GL6000-UV3292 ultraviolet detector, and GelaiInst software program. The packing used in the pre-HPLC column  was purchased from Acchrom Co., Ltd. . BL-420F Organism Functional Experimental System  was used to record the lead II electrocardiograms (ECGs). CPA2250 electronic analytical balance  was used to correctly weigh the lab samples. The process of sand frying was simulated by oil bath heating using an HH-SJ heat-collecting magnetic stirrer . Optical rotations were obtained on a Perkin-Elmer 341 polarimeter . Melting points were measured on an X-4 micro-melting point apparatus and uncorrected . NMR spectra were measured on a Bruker Avance 600 spectrometer  in CDCl1–4 (purity ≥95%) were separated and identified from processed crassicauline A, the structures were verified by 1H NMR, 13C NMR, and HR-ESI-MS.Crassicauline A (purity ≥98%) was purchased from Chengdu Desite Bio-Technology Co., Ltd. , aconitine (purity = 99%) from Shaanxi Herbchem Bio-Technology Co., Ltd. . Propafenone hydrochloride (purity = 99.8%) and lidocaine hydrochloride (purity = 93.4%) were purchased from National Institutes for Food and Drug Control , urethane from Chengdu Kelong Chemical Reagent Factory . Compounds HPLC-grade acetonitrile was supplied by Fisher Chemical Co. . Ultrapure water was directly obtained from a ULUP-I-10T water purification system . The remaining reagents were all of analytical grade. Spots on TLC plates were visualized with Dragendorff’s reagent or iodine vapor.SPF grade Sprague Dawley rats  weighing between 180 and 220 g [certificate No. SCXK (CHUAN) 2015-030 and 2020-030] were supplied from Chengdu Dossy experimental animals Co., Ltd.  and maintained under a controlled light/dark cycle and temperature (20 ± 2°C), with free access to food and water. They were left for 2 days for acclimatization to animal room conditions. The animal study was reviewed and approved by the Animal Experimentation Ethics Committee of Chengdu University of Traditional Chinese Medicine (permission No. 2020-15).18 column  maintained at 40°C. The detection wavelength was 260 nm. The mobile phase comprised acetonitrile (A) and 0.03 moL/L NH4HCO3  with a gradient program of 37% A at 0–40 min, 37–70% A at 40–60 min, and 70% A at 60–85 min. The injection volume was 10 μl and the flow rate was 1 ml/min.Quantitative analyses were performed with a Shimadzu LC-2030C system , using a Supersil ODS-B C100.60 mg crassicauline A standard was weighed accurately into a 250 ml volumetric flask and dissolved in dichloromethane to make a concentration of 402.4 μg/ml reaction stock solution. 4 ml of the reaction stock solution was added into a 10 ml volumetric flask, with dichloromethane evaporating, 0.1% (v/v) HCl-methanol was added for dissolution to prepare a final concentration of 160.96 μg/ml. The standard solution was filtered with 0.45 μm syringe filter before injection into the HPLC.Twenty-four 100 ml round-bottomed flasks were divided into four temperature groups , six reaction time points  were set at each temperature. Accurately adding 4 ml of the reaction stock solution into each flask, and the solvent was evaporated. The flasks were subsequently immersed in an oil bath and processing according to set parameters, cooling to room temperature after reaction. The residue was diluted with 0.1% (v/v) HCl-methanol in a 10 ml volumetric flask and subsequently filtered with 0.45 μm syringe filter before injection into the HPLC.1.83 g crassicauline A was dissolved in a 250 ml round-bottomed flask with a proper amount of dichloromethane, and the solvent was evaporated with a rotary evaporator at 40°C to make crassicauline A uniformly adhere to the inner wall of the flask, and processed in an oil bath at 160°C for 30 min, obtaining a crude residue of crassicauline A (1.65 g) for column chromatography.1 (Rf 0.41), Frs T2 (Rf 0.51), and Frs T3 (Rf 0.77). Frs T3 was recrystallized from dichloromethane-methanol to generate compound 3 (100 mg). Frs T1 (200 mg) and Frs T2 (430 mg) were further purified by preparative HPLC, with the chromatographic condition of acetonitrile −0.03 moL/L NH4HCO3 at 42:58 , the collected fractions were evaporated under reduced pressure to no acetonitrile, leaving only the aqueous phase. Following this, the aqueous phase was extracted twice with dichloromethane. The combined extracts were subsequently dried over Na2SO4, the solvent was removed in vacuo to obtain compounds 1 (110 mg) and 2 (310 mg).The residue obtained was purified over silica gel column chromatography  and eluted with petroleum ether-acetone-triethylamine 8:1:0.01 (0.4 L), 6:1:0.01 (3 L), and 3:1:0.01 (1.2 L) to furnish three fractions (A-C). Fraction B (1 g) was further separated by preparative TLC on silica gel G  to obtain Frs T1.11 g crassicauline A was weighed and processed upon above-mentioned processing method, the processing parameters were set at 180°C for 30 min, to obtain a crude residue of crassicauline A (1 g) for column chromatography.4 (Rf 0.55), which was recrystallized from petroleum ether-acetone-methanol to afford compound 4 (300 mg).The residue was subjected to column chromatography  and eluted with dichloromethane-acetone-methanol-triethylamine 60:2:1:0.01 (5.7 L) and 10:2:1:0.01 (0.8 L) to obtain three fractions (D-F). Fraction D (400 mg) was further separated by preparative TLC on silica gel G  to generate fraction Ti.p.) injection of 20% urethane (1.2 g/kg), with their back fixed and four limbs in subcutaneous penetration of needle-electrodes. Lead II ECGs were recorded after the administration of urethane.The rats were anesthetized by intraperitoneal , ventricular tachycardia (VT), and ventricular fibrillation (VF) in rats. Comparing under the same dose, whether the converted products caused arrhythmias can directly reflect the cardiotoxicity pre- and post-processing.in vivo in 233 rats. The rats were randomly divided into the following 15 groups, a minimum of ten animals were used in each group: the blank solvent group , the control group , the positive control groups , the compound 2 groups , and the compound 4 groups . The preparation methods of aconitine, positive drugs, and the experimental compounds were the same as the blank solvent. To confirm whether the blank solvent had an influence on rats, the rats of solvent group were only administered the same volume of blank solvent to observe ECG changes during the recording time.To further investigate the antiarrhythmic activity of the three converted alkaloids, the testing was performed .0 mg/kg  body wei.0 mg/kg  body weii.p.)  . Recordir 10 min , aconitir 10 min  to estabr 10 min . The onsr 10 min  was recor 10 min  after acr 10 min  or arrhyr 10 min , if any,χ2) test. Statistical significance was set at p < 0.05.The data obtained were analysed using statistical package for social sciences (SPSS) version 20 software. Experimental data were expressed as mean ± SD or proportion. Descriptive statistics were examined individually. When the data conformed to the normal distribution, the one-way ANOVA would be used for those with homogeneous variance. If the variance was not homogeneous, Tamhane’s T2 test would be used for comparison between groups. Comparisons of proportions were made with the Pearson’s chi-square , the obtained results showed that crassicauline A exhibited satisfactory linearity within the range of 2–200 μg/ml. The LOD and LOQ of crassicauline A under the chromatographic conditions were 0.18 μg/ml and 0.60 μg/ml, respectively.Analysis of the peak area of the standard sample resulted from HPLC as well as linear regression -8-deacetylcrassicauline A (1) was isolated as an amorphous powder, c = 0.92, CH3OH). It showed a positive reaction with Dragendorff’s reagent. Its molecular formula was deduced to be C32H43NO8 from a protonated molecule ion at m/z 570.3052 (M + H)+  in its HR-ESI-MS. The NMR spectra , three methoxyl groups , one p-methoxyl benzoyloxy group , 114.0 d (2C), 163.9 s, 55.5 q), as well as four quaternary carbons . The aforementioned NMR features suggested an aconitine-type alkaloid for compound 1  was assigned to H-14β  and C-1, 6-OCH3 (δH 3.30) and C-6, 18-OCH3 (δH 3.32) and C-18 in its HMBC spectrum , δ 5.91 , and the 13C NMR signals at δ 130.1 and 134.8 indicated the existence of a disubstituted double bond in the molecule -8-deacetylcrassicauline A, a new diterpenoid alkaloid -8-O-methylcrassicauline A (2) was isolated as a white amorphous powder, c = 0.78, CH3OH). Its molecular formula C33H45NO8 was derived from the HR-ESI-MS spectral data: m/z 584.3203 (M + H)+ (cacld. 584.3218). 1H- and 13C-NMR spectra of 2 , four methoxyl groups , a disubstituted double bond , and a p-methoxyl benzoyloxy group , 113.5 d (2C), 163.4 s, 55.4 q). The 1H-doublet signal at δH 4.95 (J = 4.08 Hz) was assigned to H-14β  and C-1, 6-OCH3 (δH 3.26) and C-6, 8-OCH3 (δH 3.12) and C-8, 18-OCH3 (δH 3.29) and C-18 in the HMBC spectrum of 2 -8-O-methylcrassicauline A, a new diterpenoid alkaloid -crassicauline A (3) was obtained as colorless needles, mp 155–157°C, c = 0.80, CH3OH). A protonated molecular ion at m/z 612.3163 (M + H)+ (cacld. 612.3121) in the HR-ESI-MS spectrum suggested a molecular formula of C34H46NO9. Compound 3 exhibited characteristic NMR features , three methoxyl groups , an acetoxyl group  , 113.7 d (2C), 163.6 s, 55.4 q). The one-proton double signal (J = 4.8 Hz) at δH 4.92 was attributed to H-14β (p-methoxyl benzoyloxy group at C-14 position. The location of three methoxyl groups was based on the long-range correlations between 1-OCH3 (δH 3.24) and C-1, 6-OCH3 (δH 3.15) and C-6, 18-OCH3 (δH 3.27) and C-18 in HMBC spectrum  and δ 6.05  also confirmed this deduction  , as wellto H-14β , implyinspectrum . Furthereduction .3 with crassicauline A -crassicauline A, a new diterpenoid alkaloid  was obtained as colorless needles, mp 129–131°C, c = 0.81, CH3OH). Its molecular formula, determined to be C33H45NO8, was derived from HR-ESI-MS: m/z 584.3222 (M + H)+ . 1H and 13C NMR spectra of 4 , four methoxyl groups , and a p-methoxyl benzoyloxy group , 113.5 d (2C), 163.3 s, 55.4 q). The one-proton double signal (J = 3.0 Hz) at δH 4.93 was attributed to H-14β  and C-1, 6-OCH3 (δH 3.28) and C-6, 16-OCH3 (δH 3.38) and C-16, 18-OCH3 (δH 3.30) and C-18 in the HMBC spectrum of 4  also confirmed this deduction  s. In contrast, the three converted products did not exhibit arrhythmias and behavioral manifestations under the same dose. Through this comparative experiment, it could be concluded that the cardiotoxicity was reduced after processing .VPB is the initial manifestation of aconitine-induced arrhythmia model rats, and the ECG of VPB is characterized by premature and bizarrely shaped QRS complexes that appear wide on the ECG, these complexes are not preceded by a P wave, and a T wave is usually oriented in a direction opposite the major deflection of the QRS . VPB incp < 0.05).In the control group, the typical characteristic ECGs of VPB and VT occurred successively following the injection of aconitine, even VF occurred in parts of the rats, and lasted for more than 30 min, suggesting that the arrhythmia model was successfully replicated. The VPB incubation period of the control group was (116.5 ± 36.4) s, lidocaine and propafenone groups were (280.3 ± 128.7) s and (193.3 ± 39.9) s, respectively. Two positive drugs had a significant difference in comparison with the control group  significantly delayed the emerge of VPB . Similarly, there was a significant difference between 0.40 mg/kg group and lidocaine group (p < 0.05), indicating that a 0.40 mg/kg or more intravenous dose of compound 1 exhibited a marked activity relative to positive drugs.The effects of compounds 1 and 2 is the substituents at C-8 position. To explore the effects of these structural distinctions, the antiarrhythmic effect of compound 2 was further evaluated. In comparison with the control group, different dose groups of compound 2  had a significant delay on the VPB incubation period . Compared with lidocaine, 0.30 and 0.40 mg/kg groups had significant difference (p < 0.05). It was demonstrated that a 0.30 mg/kg or more dose of compound 2 exhibited superior antiarrhythmic activity relative to compound 1 and the positive drugs.As mentioned above, the differentiation between compounds 3, bearing two ester bonds at C-8 and C-14, belonging to DDAs, may exert relatively strong toxicity  significantly delayed the onset time of VPB compared with the control group , but showed an equivalent effect relative to lidocaine (p > 0.05).On the contrary, compound toxicity . Consequ1, 2, and 4 could delay the onset time of VPB in a dose-dependent manner. Additionally, at the dose of 0.40 mg/kg, the VPB incubation periods were compound 2 >compound 1 >compound 4, indicating that compound 2 had the best antiarrhythmic effect among these compounds.In summary, within their own dose ranges, compounds VT is the result of further progression of VPB, as well as an established risk factor for sudden cardiac death . The ECG1 were 0.05–0.20, 0.40, and 0.60 mg/kg. As vividly displayed in χ2 = 14.704, p = 0.001). The pairwise comparison revealed a highly significant drop in the incidence of VT from 85.7% in the 0.05–0.20 mg/kg group to only 18.2% in the 0.60 mg/kg group .In accordance with the data requirements of chi-square test, the adjacent dose groups with similar VT incidence should be combined to carry out chi-square test. The subgroups of compound 2 and 4. Compound 2 was divided into the 0.05, 0.20–0.30, and 0.40 mg/kg groups. As described in χ2 = 13.000, p = 0.002). In comparison with the 0.05 mg/kg (81.8%) and 0.20–0.30 mg/kg (61.9%) groups, the incidence of VT was significantly dropped to 10% in 0.40 mg/kg group .Following the identical research approach as VPB incubation period, we further investigated the incidence of VT in compounds 4 were 0.05–0.20 and 0.40 mg/kg groups. As presented in χ2 = 10.165, p = 0.001). Furthermore, there was a greatly significant reduction in the incidence of the VT from 85.7% in 0.05–0.20 mg/kg group to 36.8% in 0.40 mg/kg group .The subgroups of compound 1, 2, and 4 could reduce the incidence of VT in a dose-dependent manner, suggesting that they could effectively prevent the further progression of VPB.On the basic of aforementioned results, it can be concluded that compounds The incidence of arrhythmia  is definConsidering the number of rats without arrhythmia in dose groups of 0.05 and 0.20 mg/kg was small, the original grouping could not meet the requirements of the chi-square test. Therefore, adopting the similar data analysis method as VT incidence, chi-square analysis was carried out in this paper by combining 0.05 and 0.20 mg/kg groups into 0.05–0.20 mg/kg group in each experimental compound.1  and lidocaine on arrhythmia inhibition rate in rats were compared. Chi-square test showed that the arrhythmia inhibition rates in the four groups were significantly different , as shown in χ2 = 14.258, p = 0.0002; χ2 = 6.820, p = 0.009). Notably, the arrhythmia inhibition rate in 0.60 mg/kg group was higher than lidocaine, exhibiting a better effect, as shown in In this paper, the effects of different doses of compound 2 and 4, the result of chi-square test showed that the arrhythmia inhibition rates in compound 2 subgroups  and lidocaine were statistically different , as shown in χ2 = 11.578, p = 0.001; χ2 = 17.093, p = 0.00004; χ2 = 7.228, p = 0.007, respectively.), indicating that the antiarrhythmic effect of compound 2 was stronger with the increasing dose. Furthermore, a 0.30 mg/kg or more dosage of compound 2 exhibited a superior antiarrhythmic effect than lidocaine, as illustrated in To ascertain the influence of different substituents at C-8 and the location of double bond on drug efficacy, we further analysed the arrhythmia inhibition rate of compounds 4 and lidocaine were significantly different , as shown in χ2 = 6.832, p = 0.009). However, a 0.40 mg/kg intravenously dose of compound 4 exhibited an equal antiarrhythmic activity relative to lidocaine (p > 0.05), as displayed in Chi-square test showed that the arrhythmia inhibition rates in different dose groups (0.05–0.20 and 0.40 mg/kg) of compound 1, 2, and 4 could increase the arrhythmia inhibition rate in a dose-dependent manner. Among them, compound 1 in 0.60 mg/kg group (56%), compound 2 in 0.30 and 0.40 mg/kg groups  had a higher arrhythmia inhibition rate than lidocaine (42.9%), manifesting that these two compounds possessed excellent antiarrhythmic effects.In summary, within their respective dose ranges, compounds 1, 2, 3, and 4, respectively.In this paper, the HPLC method was used to investigate the structural transformation pathway of crassicauline A in sand frying process. Meanwhile, the cardiotoxicity between crassicauline A and its converted products was compared, the antiarrhythmic effect of the products was ultimately investigated. Considering crassicauline A is a monomer compound, readily adhering to the surface of the sand during processing, which is inconvenient to prepare and recover samples. In our previous study , it was 4. (B) a double bond was firstly introduced at C-15/C-16 of crassicauline A by elimination of C-15 hydrogen atom and C-16 methoxyl group, to obtain compound 3. The removed hydrogen atom and methoxyl group might generate methanol. The acetoxyl group at C-8 of compound 3 could undergo transesterification with methanol produced in the previous step, to generate compound 1 and methyl acetate. Finally, compound 2 was obtained by intermolecular dehydration of compound 1 with methanol  the acetoxyl group at C-8 of crassicauline A was firstly hydrolyzed to a hydroxyl group, a double bond was subsequently introduced at C-8/C-15 via further dehydration, which converted into compound methanol . It was methanol , which cCardiotoxicity assays showed that intravenously administration of 0.10 mg/kg crassicauline A caused VPB, VT, and VF in normal rats, but no arrhythmias occurred when injecting the same dose of converted products. The cardiotoxicity of the transformed products was reduced relative to crassicauline A, indicating that the sand frying method could attenuate the cardiotoxicity of the parent compound.+ influx, which accelerates the autonomy of the pacemaker and induces ectopic rhythm points, thus forming a multi-focal ectopic rhythm and shortening the myocardial refractory period, and finally leading to arrhythmia  exhibited superior antiarrhythmic activity. On the other hand, introduction of a double bond at C-8/C-15 (compound 4) rather than at C-15/C-16  displayed a relatively weaker activity. These results indicated that a hydroxyl or a methoxyl group at C-8, elimination of C-16 methoxyl group, and a double bond at C-15/C-16 or C-8/C-15 might be important structural features to the antiarrhythmic activity of this kind of diterpenoid alkaloids. The structures of converted products could be beneficial in searching for the potential antiarrhythmic activity agents that are equal or more active, with lower toxicity, than antiarrhythmic drugs currently in clinical use.The antiarrhythmic structure-activity relationship acquired for the converted products exhibited promising antiarrhythmic effects relative to the parent compound crassicauline A. The activity can be manipulated by the positions of the double bond, as well as the substituents at the C-8 position. For example, compound in vivo experiments that, with the structural transformation of crassicauline A, the converted products displayed lower cardiotoxicity and relatively strong antiarrhythmic effects. However, the converted products were not completely isolated, there might be other structural transformation pathways that remained investigate.In summary, this paper used oil bath heating to simulate the process of sand frying, and screened out the temperature and time parameters for the structural transformation of crassicauline A, which provided a reference for the standardization and quantification of sand frying processing technology. In addition, it was found that the prototype alkaloids, which had different substituents at the C-15 position, might have different transformation pathways. Furthermore, it also demonstrated from the


Different spectroscopic and microscopic techniques were used for the catalyst characterization such XRD, FESEM, TEM, FT-IR, EDX, DLS, VSM, UV-Vis, and ICP analyses. The presence of imidazole as ionic section tags with hydrophilic character on the Co-complex supported on magnetic nanoparticles provides dispersion of the catalyst particles in water, which leads to both higher catalytic performance and also facile catalyst recovery and reuse six times by successive extraction and final magnetic separation. High catalytic activity was found for the catalyst and high to excellent efficiency was obtained for all Suzuki  and Hiyama  derivatives in short reaction times under mild reaction conditions in the absence of any hazardous or expensive materials. There is not any noticeable by-product found whether for Suzuki or Hiyama derivatives, which reflects the high selectivity and also the lower the E factor the more favorable is the process in view of green chemistry. The bi-aryls were achieved from the reaction of various aryl iodides/bromides and even chlorides as the highly challenging substrates, which are more available and cheaper, with triethoxyphenylsilane or phenylboronic acid. To prove the performance of the catalyst components (synergistic of SBL ∼ Co. and IL), its different homologs were incorporated individually and studied for a model reaction. Exclusively, this is an introductory statement on the use of Cobalt binuclear symmetric ionic liquid catalysts in Hiyama reactions.Cellulose, as a green and available phytochemical, was immobilized on the surface of magnetite nanoparticles then doped with imidazole and Co. complex (Fe Green chemistry is the design of chemicals and operations that minimize or remove dangerous substances from the environment. Green chemistry seeks to reduce the adverse effects of chemical experiments on the environment by preventing the contamination of resources . These mToday, nanotechnology is an influential factor in science and industry, and experts and researchers have confirmed that this technology, as an impending revolution, will seriously affect the economic future of countries and their position in the world.. These pIn recent years, many solid substrates have been used in the construction of solid heterogeneous catalysts. One of these widely used substrates is cellulose; ; NanocelAnother important part of catalytic compounds is the presence of ionic liquids. In addition to the low vapor pressure shown by ionic liquids, these compounds can be used as recyclable catalysts due to their high ability to function as environmentally friendly alternatives to organic solvents. Without causing a reduction in its activity . Also, i2, . We have synthesized it as a separable cobalt dual-core magnetic nanocatalyst and evaluated it by various techniques. The nanocatalyst capability has been well investigated due to the Hiyama and Suzuki-Miyaura junction interactions at 70 and 80oC in an aqueous medium . All powder samples were recorded from Bragg angle, 2θ of 10°–80° at scan speed = 1 min−1 and step size = 0.05°. Transmission electron microscopy (TEM) images were obtained using a Philips CM120 microscope. ICP analysis was performed by VARIAN VISTA-PRO CCD simultaneous ICP-OES instrument. The elements in the samples were probed by energy-dispersive X-ray (EDX) spectroscopy accessory to the Philips scanning electron microscopy (SEM). The magnetization behaviour of the NPs was investigated on a Lake Shore vibrating sample magnetometer (VSM) at room temperature. The Zeta-potential is determined by zeta potential analyzer and hydrodynamic diameter is determined by DLS (dynamic light scattering) on a HORIBA-LB550 instrument.The catalyst was characterized by several techniques, including ICP, XRD, EDX, FT-IR, TEM, Fe-SEM, DLS, and VSM. All chemicals were purchased from Sigma and Merck and used without any further purification. All solvents were distilled and dried before use. The progress of the reactions and the purity of the products were determined by TLC on silica-gel Polygram SILG/UV254 plates. Fourier transform infrared (FT-IR) spectra were recorded with a Nicolet system 800 beam splitter KBr SCAL = 800 in the range 400–4,000 cm3O4 and core-shell Fe3O4@CNF nanospheres were prepared according to a previously mentioned (Magnetic nanoparticles Feentioned .3O4@CNF (1.5 g) stirred in dry toluene solution (30 ml) while sonicating. The reaction was exposed for 24 h under N2 gas at reflux conditions. Then the flask content was stirred with Et3N (0.5 ml) for 30 min at room temperature. After finishing the reaction, the solid obtained is separated by an exterior magnet and washed with toluene and diethyl ether. At the end, solid dried in a vacuum oven at 70°C for 24 h.In the first step, imidazole  was dissolved in (5 ml) of dry toluene, and then 3-chloropropyl-trimethoxy silane  was added slowly to the mixture. The mixture was then stirred for 3 h at room temperature. After stirring, the product obtained from imidazole and CPTMS was gently added for 30 min to FeoC). 2-Hydroxy-5- chloromethyl benzaldehyde  was dissolved in 10 ml of CH2Cl2, and then p-Phenylenediamine  was added to the mixture. The contents of the flask were stirred for 4 h at boiling temperature. After reflux, in order to separate the solvent from the obtained precipitate, the desired product Schiff base ligand 6,6'--) bis(methaneylylidene)) bis(3-(chloromethyl) phenol) (SBL: IV) was centrifuged and washed with cold CH2Cl2 and diethyl ether and dried at ambient temperature  cm−1.FT-IR (KBr): = 3,151 (O-H), 3,033, 2,975, 2,865 , 1,665 (C=O), 1,434 (C=C), 673 : δ = 4.80 , 7.56–7.60 , 7.72 , 10.06 , 11.30  ppm.13CNMR : δ = 47.0, 117.8, 124.8, 131.1, 136.1, 136.4, 148.4, 163.2, 197.7 ppm.Spectral information of 6,6'--) bis(methaneylylidene)) bis(3-(chloromethyl) phenol) composition:1HNMR : δ = 4.27 , 6.74 , 6.94 , 7.13–7.15 , 7.91–7.93 , 8.72 , 9.94  ppm.13CNMR : δ = 56.4, 111.9, 116.2, 123.5, 129.5, 134.7, 146.7, 159.2, 162.8 ppm.2 (10 M) in EtOH (25 ml) by stirring at room temperature. Then SBL (1 mmol) was sonicated with a minimum volume of EtOH and added the above solution under stirring. The solid was filtered off, washed with EtOH and finally with diethyl ether and dried in vacuo.After the formation of the desired ligand (V), the next step was dissolving of Co(OAc)3O4@CNF ∼ Im (1.5 g) was added to a flask containing 15 ml of dry toluene in a sonicate for 15 min. The solvent suitable for this stage of the reaction is dry toluene, which SBL ∼ Co. (5 mmol) was dissolved in dry toluene (20 ml) added dropwise to the dispersed mixture and stirred at 75oC for 24 h. The solid obtained was separated by an exterior magnet and was washed with toluene and diethyl ether. In the end, we dried the solid in a vacuum oven at 70°C for 24 h.The synthesized Fe3O4@CNF ∼ ImSBL ∼ Co. (0.3 mol%) in 3 ml of H2O was stirred for 5 min. Then, in a typical run, aryl halide (1.0 mmol) and NaOH (2 mmol), and triethoxyphenylsilane (1.5 mmol) were added, and the resultant mixture was stirred at 70oC in an oil bath. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture cooled to room temperature. The organic layer was extracted with CH2Cl2 (2 × 10 ml) from the aqueous layer. Using anhydrous Mgso4 organic layer was dried, filtered, and the solvent was removed under reduced pressure. The catalyst has remained in the aqueous layer for reuse.A mixture of Fe3O4@CNF ∼ ImSBL ∼ Co. (0.3 mol%) in 3 ml of H2O was stirred for 5 min. Then, in a typical run, aryl halide (1.0 mmol) and K2CO3 (2 mmol), and phenylboronic acid (1 mmol) were added, and the resultant mixture was stirred at 70°C in an oil bath. The reaction progress was monitored by TLC. After the reaction is completed, allowed the mixture to cool. The organic layer was extracted with CH2Cl2 (2 × 10 ml) and dried by anhydrous MgSO4. Then the solvent under reduced pressure was removed. The catalyst is in the aqueous phase and will be separated for another run.A mixture of Fe1HNMR : δ = 7.52–7.47 , 7.67–7.62 , 7.91–7.88  ppm.13CNMR : δ = 128.3, 128.4, 129.3, 141.8 ppm.1HNMR : δ = 7.35–7.40 , 7.47–7.52 , 7.89–7.92 , 8.32–8.37 , 8.43–8.48  ppm.13CNMR : δ = 125.61, 127.4, 127.7, 129.8, 130.2, 140.5, 146.7, 146.8 ppm.1HNMR : δ = 4.01 , 7.07–7.09 , 7.37–7.3 , 7.47–7.49  ppm.13CNMR : δ = 55.3, 114.2, 127.4, 127.7, 129.3, 131.1, 133.4, 141.8 ppm.1HNMR : δ = 7.34–7.39 , 7.46–7.51 , 7.84–7.86 , 7.89–7.91 , 8.03–8.07  ppm.13CNMR : δ = 127.7, 128.0, 129.1, 129.3, 133.4, 138.2, 140.1 ppm.1HNMR : δ = 7.07–7.09 , 7.37–7.45 , 7.83–7.87 , 7.96  ppm.13CNMR : δ = 111.9, 118.1, 127.5, 127.7, 127.8, 129.8, 133.4, 140.2, 144.4 ppm.3O4@CNF ∼ ImSBL ∼ Co. nano catalyst was prepared by immobilization of a novel Co. (II) Schiff base complex on Fe3O4@CNF nanoparticles followed by treatment with imidazole, and was found to be an efficient catalyst for the Suzuki and Hiyama reactions. This catalyst can be a suitable option for organic syntheses as it has features such as simple synthesizing and recovery, non-toxicity, and high catalytic output.Green chemistry, also called sustainable chemistry, is a research philosophy in chemistry that aims to design products and processes, including environmentally friendly catalysts. Also, The ionic liquid section of special importance in green chemistry. Green chemistry seeks to reduce and prevent environmental pollution, As the importance of green chemistry has increased during the last decade, therefore synthesizing new recyclable nano catalyst has been a focus of the scientific community. In order to study the catalysts, a heterogeneous, magnetically recoverable Fe−1 (C–Cl stretching vibration), 1,107 cm−1 (C–O stretching vibration), 1,450–1,580 cm−1 (C=C aromatic stretching vibration), 1,652 cm−1 (C=O stretching vibration), and 2,809 cm−1 , 2,980 cm−1 and 3,100 cm−1 , 3,250 cm−1 (O–H stretching) supports the structure of 2-hydroxy-5-chloromethyl benzaldehyde , 1,520 cm−1 (C=C aromatic stretching vibration), 1,643 cm−1 (C=N stretching vibration) 2,965–3,080 cm−1 , and 3,400 cm−1 (O–H stretching) supports the structure of SBL. 3O4 nanoparticles with two characteristic peaks at 3,402 cm−1 and 578 cm−1 related to O–H and Fe–O stretching bands, sequentially, that confirms the formation of Fe3O4 nanoparticles. In addition to the peak Fe-O, the strong peak at 3,300 cm−1 shows the (O–H stretching vibrations) of cellulose nanofibers , 1,475–1,590 cm−1 (Aromatic Str. C=C), 1,648 cm−1 (C=N stretch), and 2,989 cm−1 (C-H stretching) . These absorption peaks confirm the successful preparation of the catalyst. Complexation of cobalt ions to ligand SBL causes the imine peak absorption  to lower wavenumbers  by about 20 cm−1, indicating the participation of azomethine nitrogen in bonding with metal ion and supporting the coordination of stretch to the metal via a nitrogen atom (−1 (Fe–O), 1,015 cm−1 (Si–O), 1,490 cm−1 (C=C), 1,623 cm−1 (C=N) in the FT-IR spectrum of Fe3O4@CNF ∼ ImSBL ∼ Co., represent the functionalization of Fe3O4@CNF nanoparticles with Co. (II) complex 5  . These agen atom . The maiomplex 5 .3O4@CNF ∼ ImSBL ∼ Co. (The size and morphology of the nanoparticles were observed using transmission electron microscopy (TEM). TEM images show that the particle size has changed after immobilization of complex on modified MNPs . The synBL ∼ Co. .3O4 is paramagnetic in nature the NMR technique was not performed  analysis results for Femposites . These r surface . Neverth3O4 NPs, and content in the Fe3O4@CNF ∼ ImSBL ∼ Co. nanocatalyst. The results are illustrated in 3O4 NPs. TGA spectra of uncoated Fe3O4 NPs show little weight loss, which is about 10% in the range of 25–800°C. This might be due to the loss of residual water in the uncoated Fe3O4 NPs , (311), (400), (422), (511) and (440) reflections of inverse spinel Fe3O4 NPs, respectively. It showed characteristic peaks, and the relative intensity matched well with those of standard Fe3O4 nanoparticles  , (100), (011), (012), (110), (111), (103), and (201) crystallographic phases in XRD pattern are related to Co(OAc)2. . The week broad peak appeared in the range from 2θ = 17.9–20.1 indicates the existence of amorphous cellulose. The crystal size of Fe3O4 NPs and core–shell magnetic Fe3O4@CNF ∼ ImSBL ∼ Co. were determined by using the Debye–Scherrer (d{hkl} = 0.94 λ/βcosθ) equation where ß is the half-width of the highest intensity X-ray diffraction lines, d is the average crystalline diameter, 0.94 is the Scherrer constant, ? is the Bragg angle in degree and ? is the X-ray wavelength. Here, the (311) peak of the highest intensity was selected and d311 obtained 25 nm for Fe3O4 NPs. Comparing between X-ray diffraction pattern of Fe3O4 NPs and Fe3O4@CNF ∼ ImSBL ∼ Co. shows that the coating of cellulose and SBL ∼ Co. complex on Fe3O4 NPs did not significantly affect the structure of NPs.As shown in 334) see  (Ghamari334) see . The XRD2, Fe3O4, Fe3O4@CNF, and Fe3O4@CNF ∼ ImSBL ∼ Co. were carried out in H2O as a solvent at the region of 200–800 nm 2 lmax is in region 500 nm, resulting in UV-Vis spectrum of Schiff base ligand to Co(II) caused the reduction of absorption intensity of n–π* for C=N bond and π–π* transitions for benzene ring, which confirmed the successful chelation of Co(II) to the catalyst. Furthermore, the absence of resonant peak above 450 nm proved the metallic nature of Fe3O4@CNF NPs .Electronic spectrum of Co.(OAc)0–800 nm . The syn@CNF NPs . Accordi3O4 and Fe3O4@CNF are approximately spherical and well dispersed, albeit in some area’s larger structures with non-spherical morphology are perceived , which has an average diameter of 35–43 nm , but also the colloidal dispersion stability. The distinct surface composition of the coated particles was further confirmed by differences in the surface charge of the nanoparticles (ζ) measurements at variable pH. Bare magnetic nanoparticles (Fe3O4) present an isoelectric point (IEP) at ca. 4.5 that is relatively below the usual IEP values reported for magnetite and can indicate oxidation of the particles surface. . Nevertheless, the IEP value here obtained is in agreement with values reported for bare Fe3O4 nanoparticles prepared by oxidative hydrolysis. . At lower pH values the surface is positively charged while at higher pH values the overall charge of the surface is negative. The isoelectric point shifts to high values in the nanoparticles coated with cellulose shells of CNF ( ∼ 5). This shift can be ascribed to hydroxyl groups present on particles surface cellulose. The zeta potential value of Fe3O4@CNF ∼ ImSBL ∼ Co. switched to a positive value after being modified with imidazole and SBL ∼ Co., indicating that imidazole and SBL ∼ Co. molecules were coated on the surface of Fe3O4@CNF. The measured potential value for Fe3O4@CNF ∼ ImSBL ∼ Co. was -20.0 mV.In order to further prove the influence of cellulose polymer and organic compounds  on the property of Fearticles . The sur2 adsorption–desorption isotherm analysis. The specific surface area values, the average pore diameter (according to the BJH method), and the total pore volume are tabulated in 3O4, Fe3O4@CNF, Fe3O4@CNF ∼ Cl, Fe3O4@CNF ∼ Im, and the Fe3O4@CNF ∼ ImSBL ∼ Co. nano catalyst were determined as 480, 460, 415, 400, and 365 m2 g−1, respectively  with triethoxyphenylsilane (1.5 mmol) under different conditions was selected as the model reaction. Selected optimization range achieved for Hiyama cross-coupling reaction including nine solvents, in the presence of four bases, and 0.1–0.4 mol% amount of catalyst, and at 70–98°C. Hence, in general, the optimal points for the Hiyama reaction were investigated. Introductory results demonstrated the maximum yields in water as a green solvent for all two reactions due to the high dispersion of catalyst and strong interaction of active sites with the reactants in this medium , NaOH (2 mmol) as base in the water at 70oC conditions  or any other by-products were observed.After receiving the effective results from the Hiyama reaction in the next part of this research project, the binding reaction leading to the aryl compounds through the Suzuki-Miyavra cross-reaction was considered. For this purpose, the coupling of iodobenzene with phenylboronic acid was selected as the model reaction to optimize the reaction conditions. Afterward, the effect of various parameters including solvent, base, amount of the catalyst, and temperature on the reaction efficiency was studied . Initial at 80°C , entry 4 at 80°C , entry 4 at 80°C . Differe at 80°C . It shouAgain, a variety of aryl halides were served in order to coupling with phenylboronic acid under optimum conditions. A same result was found for the effect of leaving groups and electron-withdrawing groups on efficiency of the coupling products . Based o3O4@CNF, Fe3O4@CNF-imidazole, SBL ∼ Co. and Co. (OAc) 2, some analogs were prepared and tested as a catalyst in the Hiyama and Suzuki model reactions.Because the reaction medium is a water solvent, organic molecules approach the surface of the catalyst to escape water, and the polar nature of the ionic part of the catalyst, which is adjacent to the cobalt ion, causes the components to come together and helping to accelerate the Hiyama and Suzuki–Miyaura cross-coupling reactions. To demonstrate the synergistic effects of different catalyst components  as well 3O4@CNF nanoparticles did not exhibit any catalytic effect as the core and magnetic substrate. On the other hand, homolog Fe3O4@CNF ∼ Im, which only had imidazole coordinated by the core-shell Fe3O4@CNF, produced higher yields than Fe3O4@CNF under identical conditions (3O4@CNF ∼ Im: 60–55%). In the next step, the synergistic effect of the encoded metal and ligand was investigated. Homolog SBL-Co of the main catalyst yielded 68 and 75% of the Hiyama and Suzuki–Miyaura coupling product. In the end, to demonstrate the effect of cobalt and ionic liquid group on the model reactions, the reactions were performed in the presence of both Fe3O4@CNF ∼ Im and SBL ∼ Co.  in the catalyst to Co(I) or Co(0) species under basic conditions  with high catalytic activity as superparamagnetic nature catalyst, non-toxicity, low cost, easy separation of the catalyst from the reaction mixture by external magnetic field and stability for efficient Hiyama and Suzuki–Miyaura cross-coupling reactions. All the reactions were performed via a green process, moderate to high, and excellent conversions in some cases, were achieved for all of entries which this method used is very efficient and useful and tolerates large functional groups with high efficiency. In this way, the catalyst made in the reaction conditions is stable and active for six consecutive times, which is of great importance in terms of environmental issues. We characterized the catalyst from various aspects analyses. The highlight point of the work was of the heterogeneous ionic liquid part, it seems this catalytic system is bi-functional that its imidazole moiety as an ionic liquid shows a useful performance. Because the reaction medium is a water solvent, organic molecules approach the surface of the catalyst to escape the water, and the polar nature of the ionic part of the catalyst, which is adjacent to the cobalt ion, causes the components to come together and helping to accelerate the Hiyama and Suzuki–Miyaura cross-coupling reactions, while on the other hand, the Co(II) complex moiety catalyses the reaction. This methodology is proposed as an affordable alternative to the previous use of noble metal catalysts, especially Pd-based catalytic systems.In continuing the research, a catalytic system with unique properties was designed, synthesize and reported. In summary, we have developed a binucler catalyst on the magnetic cellulose nanofibre with schiff base-Co (denoted as Fe


Error in Figure/TableFigure 10. Spontaneous potentials of SUS304, brazed Ni-11P alloy coating, and brazed Ni-13.4Cr-11.6P alloy coating in 0.06 M/L NaCl aqueous solution at different temperatures. Figure 11. Current density-time curve of galvanic couples of SUS304/brazed Ni-11P alloy coating and SUS304/brazed Ni-13.4Cr-11.6P alloy coating in 0.06 M/L NaCl aqueous solution at different temperatures.” as published. “The 80 °C marked in Figure 10. Spontaneous potentials of SUS304, brazed Ni-11P alloy coating, and brazed Ni-13.4Cr-11.6P alloy coating in 0.06 M/L NaCl aqueous solution at different temperatures. Figure 11. Current density-time curve of galvanic couples of SUS304/brazed Ni-11P alloy coating and SUS304/brazed Ni-13.4Cr-11.6P alloy coating in 0.06 M/L NaCl aqueous solution at different temperatures.” appears below. The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated.In the original article , there wText CorrectionThere was an error in the original article. “The 80 °C in Section 2.5. Electrochemical Analysis and Section 3.5. Corrosion Behaviors should be changed to 60 °C”.Section 2. Materials and Methods and Section 3. Results and Discussion”, “Section 2.5. Electrochemical Analysis and Section 3.5. Corrosion Behaviors”, “Pages 5 of 14, 9 of 14, and 10 of 14”:A correction has been made to ““The spontaneous potential and galvanic current measurements were conducted in a 0.06 M/L NaCl aqueous solution. For spontaneous potential measurement, a saturated silver chloride electrode (Ag/AgCl) was used as the reference electrode. The specimen and the reference electrode were set as positive and negative electrodes, respectively. The volume of the test solution was 200 mL and the distance between the two electrodes was set to 40 mm. The measurement was initially performed at 25 °C for 24 h, then at 60 °C for 72 h, and the solution temperature was subsequently restored to 25 °C for 24 h. For galvanic current measurement, the SUS304 plate and the specimen were set as positive and negative electrodes of a zero-shunt ammeter, respectively. The measurement was performed under the same temperature profile as for spontaneous potential measurement. The microstructure observation before and after the galvanic current measurement was conducted with the EPMA. − ions on the oxide film on the surfaces of the specimens. 2 flowed in the SUS304/brazed Ni-11P alloy coating galvanic couple, indicating that the brazed Ni-11P alloy coating was corroded. At the same temperature, only a current density of up to 0.07 µA/cm2 flowed in the SUS304/brazed Ni-13.4Cr-11.6P alloy coating galvanic couple, suggesting that the brazed Ni-13.4Cr-11.6P alloy coating has a better corrosion resistance than that of the brazed Ni-11P alloy coating. The current flow in the galvanic couples can be attributed to the increase in potential difference between SUS304 and the brazed alloy coatings at 60 °C (as shown in 2.The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated.


We further assessed these molecules using an In-Cell Western assay, which confirmed their potency in the low micromolar range. Among the three molecules, 12 and 25 displayed the most favourable profile of activity and selectivity and were selected as hit compounds for future optimisation studies.Influenza viruses represent a major threat to human health and are responsible for seasonal epidemics, along with pandemics. Currently, few therapeutic options are available, with most drugs being at risk of the insurgence of resistant strains. Hence, novel approaches targeting less explored pathways are urgently needed. In this work, we assayed a library of nitrobenzoxadiazole derivatives against the influenza virus A/Puerto Rico/8/34 H1N1 (PR8) strain. We identified three promising 4-thioether substituted nitrobenzoxadiazoles (  Influenza viruses (IVs) belong to the family of ,,The therapeutic options currently available for IV infections include vaccination and antiviral drugs used to prevent and treat them, respectively,,Given this situation, many lines of research have been directed towards the discovery of novel anti-influenza drugs. To this end, the RdRp has been identified as an interesting target for the inhibition of viral replication because it is an element conserved among different viral strains and is not subjected to great genetic variability50 of the best performing compounds,The enzyme RdRp is a heterotrimeric complex, consisting of three proteins: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). The three subunits form a multi-protein complex, with PB1 as the central core interacting with PB2 on one side and PA on the other side. Previous studies indicated that the PA/PB1 interaction is crucial for IVs replication and that it is rather conserved between the various viral strains. Consequently, the inhibition of protein–protein interactions (PPIs) represents an excellent approach for the development of new antivirals. This strategy is based on the dissociation of viral protein complexes using various types of inhibitors, including small molecules that are able to bind at the interface between the viral subunits PA and PB1. In order to identify novel PA/PB1 interaction inhibitors, Kessler and colleagues screened a large library of compounds through an ELISA-based assay. Then, a plaque reduction assay on MDCK cells infected with IV A/WSN/33 (H1N1) was used to determinate the ICS-transferase P1-1 (GSTP1-1) inhibitorsN-terminal Kinase (JNK1), and the scaffolding protein TNFα-receptor-associated-factor 2 (TRAF2)c]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) 1, initially identified as a potent GSTP1-1 inhibitorPreviously, our group has developed several 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives as glutathione-1 derivatives with an improved pharmacological profile, we developed a library of more than 40 analogues of 1. These include alkyl or alkoxyalkyl derivatives (2–6) possessing a free primary or secondary hydroxyl function, which is also esterified in compounds 7–11. Compounds 12, 14, and 16–19 are propionic acid derivatives, while 13 and 15 possess an ethylamine side chain in which the nitrogen is either acetylated (13) or included in a piperidin-4-one ring (15). The library also includes compounds possessing small alkyl side chains , heteroaromatic groups , as well as aryl moieties variously functionalised at ortho, meta, and mainly para position (24–42). Finally, compounds 43–45 are analogues of 1 in which the sulphur in position 4 is oxidised to sulphinyl (43), sulphonyl (44) or replaced by an amino group (45)In an effort to obtain 1–45 ; reporting chemical shifts in δ (ppm) units relative to the internal reference tetramethylsilane (Me4Si). All compounds were routinely checked by TLC and 1H-NMR. TLC was performed on aluminium-backed silica gel plates  with spots visualised by UV light. Yields of all reactions refer to the purified products. All chemicals were of the highest purity. Mass spectra were recorded on an API-TOF Mariner by Perspective Biosystem; samples were injected by a Harvard pump using a flow rate of 5–10 µL/min, infused in the Electrospray system. Elemental analyses were performed by a PE 2400  analyser and have been used to determine purity of the described compounds, which is >95%. Analytical results are within ±0.40% of the theoretical values.Unless otherwise specified, all chemicals used throughout this work were purchased from Sigma-Aldrich Srl . Melting points were determined on a Buchi 530 melting point apparatus  and are uncorrected.2.1.1.c] oxadiazol-4-yl)thio)hexan-1-ol (5)  and 4-(dimethylamino)pyridine (DMAP)  in dry dichloromethane (DCM) (9 ml) and the resulting mixture was stirred at reflux for 4 h. Upon completion, the reaction mixture was poured into a solution of 2 N HCl (22 ml) and extracted with ethyl acetate (4 × 25 ml). The organic phase was then washed with saturated sodium chloride solution (25 ml), dried over anhydrous sodium sulphate, filtered, and concentrated under vacuum. Finally, the crude residue was purified by silica gel column chromatography eluting with the appropriate mixture CHCl3/MeOH 25:1 to provide the final compound as a yellow powder. Yield: 70%. M.p.: 90–93 °C. 1H-NMR (DMSO) δ 1.809–1.838 , 2.478–2.509 , 3.408 , 4.100 , 7.534 , 8.574 , 12.126–12.266 . MS (ESI), m/z: 368.1 [M – H]-. Anal. (C14H15N3O7S) Calcd. (%): C, 45.53; H, 4.09; -N, 11.38; S, 8.68. Found (%) C, 45.68; H, 4.11; N, 11.30; S, 8.64.Succinic anhydride  was added to a solution of 6-thio)ethoxy)ethan-1-ol  (2) and 4-(dimethylamino)pyridine (DMAP)  in dry dichloromethane (DCM) (9 ml) and the resulting mixture was stirred at reflux for 4 h. Upon completion, the reaction mixture was poured into a solution of 2 N HCl (22 ml) and extracted with ethyl acetate (4 × 30 ml). The organic phase was then washed with saturated sodium chloride solution (30 ml), dried over anhydrous sodium sulphate, filtered, and concentrated under vacuum. Finally, the crude residue was purified by silica gel column chromatography eluting with the mixture CHCl3/MeOH 20:1 to provide the final compound. Yield: 51%. M.p.: 74–76 °C. 1H-NMR (DMSO) δ 2.461–2.509 , 3.598 , 3.673 , 3.835 , 4.148 , 7.583 , 8.579 , 12.170–12.2174 . MS (ESI), m/z: 384.1 [M - H]-. Anal. (C14H15N3O8S) Calcd. (%): C, 43.64; H, 3.92; N, 10.90; S, 8.32. Found (%) C, 43.80; H, 3.94; N, 10.82; S, 8.27.Succinic anhydride  was added to a solution of 2- in a mixture (18 ml) EtOH:H2O (0.3:1 v/v). The mixture was stirred at room temperature for 16 h. Subsequently, the suspension was filtered, and the crude solid was washed on the filter with H2O. The compound obtained was first purified by trituration with CHCl3 and then by column chromatography on SiO2 gel eluting with a mixture CHCl3/MeOH (90:1). Yield: 76%. M.p.: 144–147 °C. 1H-NMR (DMSO): δ 7.416 , 8.288 , 8.683–8.696 , 8.719 . MS (ESI), m/z: 276.0 [M + H]+. Anal. (C10H5N5O3S) Calcd. (%): C, 43.64; H, 1.83; N, 25.44; S, 11.65. Found (%) C, 43.76; H, 1.85; N, 25.37; S, 11.59.Pyridine  and 2-mercaptopyrimidine  were added to a solution of 4-chloro-7-nitrobenzo-. Anal. (C11H10N4O6S) Calcd. (%): C, 40.49; H, 3.09; N, 17.17; S, 9.83. Found (%) C, 40.60; H, 3.11; N, 17.11; S, 9.76.Yield: 78%. M.p.: 166–168 °C. 2.1.3.2.1H-NMR (DMSO): δ 7.136 − 7.417 , 7.430 − 7.716 , 8.582–8.633 , 13.475 . MS (ESI), m/z: 314.0 [M + H]+. Anal. (C13H7N5O3S) Calcd. (%): C, 49.84; H, 2.25; N, 22.35; S, 10.23. Found (%) C, 49.90; H, 2.27; N, 22.28; S, 10.17.Yield: 81%. M.p.: 206–208 °C. 2.2.Madin-Darby canine kidney (MDCK) cells and human lung epithelial cells (A549) were grown in RPMI 1640 and DMEM medium, respectively, supplemented with 10% foetal bovine serum (FBS), 0.3 mg/mL glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin.2.3.4/well in RPMI-1640 without phenol red, supplemented with 10% FBS. After 24 h plating, the compounds dissolved in DMSO (concentrations range 1–30 µM) were added and cells incubated for the following 18 h. Then the medium was replaced with 50 μL of a 1 mg/ml solution of MTT and cells were incubated at 37 °C for 3 h. Following incubation, the remaining water insoluble formazan was solubilised in absolute isopropanol containing 0.1 N HCl. Absorbance of converted dye was measured in an ELISA plate reader at the wavelength of 570 nm. The cytotoxicity of the compounds was calculated as the percentage of reduction of the viable cells compared with the drug-free control cultureThe cytotoxicity of compounds was estimated on MDCK cells by using MTT   assay. Briefly, cells were plated in a 96-well plate at a concentration of 2 × 1050 values for selected compounds. CC50 was defined as the drug concentration required for reducing the viability of A549 cells by 50%.In parallel, cell viability was estimated by Trypan Blue (0.02%) exclusion in MDCK and A549 cells treated or not with the compounds (concentrations range 1–30 µM) and incubated at 37 °C for the following 24 h. Trypan Blue exclusion analysis was also used to calculate CC2.4.Influenza virus A/Puerto Rico/8/34 H1N1 (PR8 virus) was grown in the allantoic cavities of 10-day-old embryonated chicken eggs. After 48 h at 37 °C, the allantoic fluid was harvested and centrifuged at 5000 rpm for 30 min to remove cellular debris. Confluent monolayers of epithelial cells (A549 or MDCK) were challenged for 1 h at 37 °C with PR8 at a multiplicity of infection (m.o.i.) of 0.001 incubated for 1 h at 37 °C, washed with PBS, and then incubated with medium supplemented with 2% FCS. Mock infection was performed with the same dilution of allantoic fluid from uninfected eggs2.5.5/mL were infected with PR8 (0.001 m.o.i.) and, after the adsorption period, washed with PBS and treated with different concentrations (0–30 μM) of each compound. After 24 h post infection (p.i.), viral production was quantified in the supernatants of infected cells by measuring the haemagglutinin units (HAU), using human type 0 Rh + erythrocytes. Compounds were dissolved in DMSO to prepare stock solutions and diluted in RPMI to final concentrations of 0–30 µM. Compounds were added after the adsorption period and maintained in the culture media until the end of the experiments. Control cells were treated with DMSO alone at the same concentration present in the test substance being evaluated. The highest DMSO concentration present in the culture medium was 0.2%. The concentration of compound required to inhibit viral replication of 50% (IC50) was determined by regression analysis using Microsoft Excel software, considering untreated infected cells as control (100%). Experiments were performed in triplicates and IC50 values reported as ± standard deviation (s. d.).MDCK cells plated at concentration of 2 × 102.6.4 cells/well), either infected or mock-infected (Ctr) with PR8, were fixed with 4% formaldehyde, washed, permeabilised with 0.1% Triton X-100 and incubated with PBS containing Odyssey Blocking buffer . The cells were then stained at 4 °C overnight with mouse anti-NP  together with Cell Tag  in PBS containing 5% Odyssey Blocking Buffer. Cells were then washed and stained with goat anti-mouse IRDyeTM 800 antibodies . Protein expression was quantified using the Odyssey Imaging System. For statistical analysis, integrated intensities of fluorescence in wells were determined using software provided with the imager station (LI-COR). The relative amount of NP protein was obtained by normalising to the Cell Tag in all experiments. Data obtained from mock infected A549 cells treated with selected compounds at different concentrations and stained with Cell Tag were used to calculate the CC50 values.The ICW assay was performed using the Odyssey Imaging System 3.3.1.12–34–67–8101213–1820–2124–439 was prepared through a reaction between succinic anhydride and 5 in dry dichloromethane (DCM) in the presence of 4-(dimethylamino)pyridine (DMAP) under reflux conditions  and the proper commercial thiol in a mixture EtOH:H2O (0.3:1 v/v) in the presence of pyridine at room temperature  of each compound and incubated for the following 24 h. The cytotoxicity of each compound was assessed through a MTT assay, as described in the Materials and Methods section. In parallel, we estimated cell viability in the presence of each compound at the tested concentrations by Trypan Blue exclusion. Microscopic examination and Trypan Blue exclusion demonstrated that most compounds exerted a toxic effect at all tested concentrations and were therefore excluded for the antiviral activity screening .In a first set of experiments, we assessed the potential cytotoxicity of the compounds. We plated MDCK at a concentration of 2 × 10creening . Notably9, 11, 12, 17, 19, 25, 40, and 41 were effective against IV . Compounds 6, 8, 14, 21, 23, 27, 28, 30, 31, 32, 33, 37, 38, 39, 43, 44, and 45 did not inhibit viral replication up to the maximum tested dose (30 µM) and are indicated as inactive in Based on cytotoxicity studies, MDCK cells were infected with influenza A/PR8 H1N1 virus strain and, after viral adsorption, cells were treated with different concentrations (ranging from 0.25 to 30 μM) of each compound. After 24 h post infection (p.i.), we quantified viral production through a haemagglutination assay (HAU) on the supernatants of infected cells. Compounds 3.3.12, 17 and 25 were the only ones to display IC50 values below 10 µM, thus being the most effective compounds against IV. Therefore, we decided to evaluate their antiviral activity on a different cell line highly permissive to IVs, the human lung epithelial A549 cells. Viral replication measured by HAU assay demonstrated that all three compounds were effective against IV. In particular, the IC50 for 12 was 5.1 μM, while the CC50 calculated by counting the number of dead cells stained with Trypan Blue compared to the live cells was 54.5 μM. Hence, the Selectivity Index (SI = CC50/IC50) for compound 12 is equal to 11, suggesting that the molecule is active at concentrations that are far below its cytotoxicity threshold. The IC50 values for 17 and 25 were 11.7 and 1.9 μM, respectively, with the SIs equal to 2 and 6, respectively.Among the active molecules, ,12, 17, and 25 and stained with Cell Tag to evaluate their toxicity and to calculate the CC50 using the ICW assay. As reported in 50 values obtained by ICW were in line with what already observed with the previous approach . Moreover, SI values calculated using the ICW IC50s and CC50s  side chain were cytotoxic at all tested concentrations. Esterification of 1 with an acetyl group (7) did not change the toxicity profile; similarly, acylated or alkylated ethylamine derivatives 13 and 15 were cytotoxic.The initial assay devoted to preliminary assessing the cytotoxicity of the compounds revealed that 25 molecules were not toxic in the concentration range we tested (1–30 µM) or toxic only at higher concentrations (≥20 µM) . All 4-t8, the benzoic ester of 1, was not toxic at any tested concentration, but did not display any antiviral activity. Notably, esterification of 1, 2, and 5 with succinic acid yielding compounds 10, 9, and 11, respectively, abolished the cytotoxicity and resulted in mild anti-IV activity. Among the propionic acid derivatives, only two compounds were cytotoxic, namely the piperidin-4-one amide derivative 16 and compound 18, bearing a Cbz-protected amino group in C2. Among the non-toxic molecules, the methyl propionate derivatives 12 and 17 displayed the best antiviral activity, while 19, bearing a free carboxylic acid, showed mild antiviral properties and the hydroxyethyl amide derivative 14 was inactive. Compounds 20 and 21 were toxic and inactive, respectively, suggesting that purely alkyl side chains may be detrimental for antiviral activity. Similarly, compounds bearing an aromatic moiety directly linked to the sulphur were either toxic or substantially inactive. Moreover, compounds 29 and 39, bearing a benzoic or phenylacetic moiety were toxic and inactive, respectively. Remarkably, benzyl derivatives 25, 41 and 42 were not cytotoxic and possessed good anti-IV activity, suggesting that the presence of a methylene spacer between the sulphur and the side-chain aromatic group is essential for activity and selectivity. Finally, 1 analogue in which the sulphur at position 4 was replaced by a 4-sulphinyl (43), 4-sulphonyl (44) or 4-amino (45) were not toxic, but did not possess any antiviral activity at the tested concentrations. These data suggest that the presence of a reduced sulphur is pivotal for antiviral activity.Conversely, compound 9, 10, 11; (ii) the propionic acid derivatives 12, 17, and 19; (iii) the 4-benzylthio derivatives 25, 41, 42. In group (i), it is apparent that increasing the length of the alkyl chain reduces the antiviral activity as demonstrated by the increased IC50 value of 10 compared to 9. In addition, the introduction of polar atoms, such as oxygen, is detrimental for compound activity as indicated by the higher IC50 value of 11 compared to 9. In group (ii), methylation of the carboxylic acid function of N-α protected cysteine residues decreases the IC50 value at least six-fold (compare 17 with 19), and the subsequent removal of the protected amino group at α position leads to a further increase of antiviral potency (compare 12 with 17) suggesting that a small methyl propionate side chain is important for compound activity. In group (iii), the introduction of both carboxy (41) and carbethoxy (42) groups determine a decrease in antiviral activity compared to 25, indicating that substitutions with polar moieties at para position are unfavourable for the 4-benzylthio-NBD derivatives series. Overall, the present data suggest that compact, hydrophobic substitutions are favourable for antiviral activity, alternatively benzyl derivatives are promising anti-IV compounds, although different substitutions in the benzene ring need to be explored.Overall, 10 compounds of the library possessed antiviral activity, all of them being 4-thioether derivatives of NBD. The active molecules can be divided into three subgroups: (i) the emisuccinic esters 12, 17, and 25 also in the A549 cell line. The HAU and ICW assays performed on these cells infected with PR8 confirmed the antiviral activity and selectivity of the three molecules. Overall, compound 25 resulted as the most active in A549 cells, while 12 was the best one in MCDK cells and displayed the best selectivity profile in both cell lines (Given their promising activity in MCDK cells, we assessed compounds ll lines .12 and 25 with their simple structures amenable for modifications represent promising hit compounds for future medicinal chemistry optimisation studies.Despite their specific mode of action has not been clarified yet, the results of this study demonstrate that some 4-thio-NBD derivatives are effective against IV and shed light on structure–activity relationship that could be exploited for the design of new molecules. Indeed, compounds In conclusion, we hope that the present study might drive the search to the identification and characterisation of new potential compounds for fighting IV.