Further investigation revealed that compound 9c had versatile properties against tumors, including inhibition of cell migration and invasion as well as inducing apoptosis. Reactive oxygen species (ROS) assay and western blotting analysis suggested that compound 9c promoted cancer cell apoptosis by increasing ROS levels and upregulating the expression of caspase-3 in MDA-MB-231 cells. These results indicated that compound 9c could be promising lead compound for further antitumor drug research.Coumarins possesses immeasurable antitumor potential with minimum side effects depending on the substitutions on the basic nucleus, which exhibits great prospects for antitumor drug development. In an attempt to develop novel antitumor candidates, a series of coumarin sulfonamides and amides derivatives were designed and synthetized. The majority of these derivatives showed good cytotoxic activity against MDA-MB-231 and KB cell lines, among which compound  Cancer is a widespread and lethal disease characterized by uncontrolled growth of abnormal cells. It has been expected to be the leading cause of death in the future ,2. AlthoCoumarins are a wide family of secondary metabolites found in various species of plants but also fungi and microorganisms ,4, whichI  salicylaldehyde 8 in the same condition afforded the desired compound 9. The coumarin amides derivatives 12a–12c were synthesized as described in 11 was prepared by the condensation of 4-(diethylamino) salicylaldehyde 8 with ethyl malonate 10 in anhydrous ethanol in the presence of piperidine. The ammonolysis of compound 11 with aromatic amines 1 in anhydrous ethanol under reflux led to desired compound 12.The synthesis of coumarin sulfonamides derivatives 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.The in vitro cytotoxicity of the synthesized compounds  was eval7a–7b and 9a–9c, 7-diethylaminocoumarin sulfonamides 9a–9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a–7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a–9c had better cytotoxicity compared to amides derivatives 12a–12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a–9c and 12a–12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency.In addition, it was observed that among coumarin sulfonamides 9c on the migration of MDA-MB-231 cells with a wound-healing assay. The results showed that the scratch gaps in compound 9c (10 and 20 μM) treated MDA-MB-231 cells were wider than the control with increased treating time  the number of invaded cells at the concentration of 20 μM  had stronger fluorescence intensity compared with the control group, which indicated that compound 9c promoted apoptosis of MDA-MB-231 cells. Meanwhile, as shown in 9c, the percentages of apoptotic MDA-MB-231 cells increased from 3.85% in control cells to 7.72%, 20.11%, 40.71%, 53.50%, and 57.46%, respectively. The results implied that compound 9c could induce the apoptosis of MDA-MB-231 cells in a concentration-dependent manner.To further evaluate whether compound poptosis a. The ce9c. Intracellular ROS can oxidize 2′,7′-dichlorofluorescin (DCFH) into the fluorescent 2′,7′-dichlorofluorescein (DCF) whose intensity is proportional to the amount of intracellular ROS. After these cells were subjected to various doses of compound 9c , the fluorescence intensity increased obviously  fluorescence probe to further evaluate the changes in intracellular ROS level after treating MDA-MB-231 cells with compound bviously , reveali9c. The protein expression levels of caspase-3 and Bcl-2 were detected since caspase-3 played a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins, and Bcl-2 is the well-known anti-apoptotic marker. After exposure of MDA-MB-231 cells to compound 9c, the expression level of caspase-3 was dramatically increased, while no obvious effect on the expression of Bcl-2 was observed  and are uncorrected. Infrared spectra were determined with a TENSORII spectrometer . NMR spectra were measured using 300 MHz Bruker AV III spectrometers  and 400 MHz JNM-ECZ400S/L1 spectrometers  with tetramethylsilane (TMS) as an internal standard. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad singlet. Coupling constant (J) is reported in Hertz (Hz). HR-MS spectra were obtained on Finnigan LCQ and Micromass Auto Spec Ultima-Tof spectrometer . Analytic thin-layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates  using petroleum ether-ethyl acetate as solvent system. All the chemicals were of analytic grade. The detailed preparation procedures of intermediates, byproducts, and target compounds are described below.2  was added dropwise to a solution of aromatic amines 1 (4.35 mmol) in Et3N (10 mL). The reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, a solution of 10% HCl was added slowly to the mixture to adjust the pH to 7. The mixture was then extracted with DCM (12 mL × 3). The combined organic layer was dried over anhydrous Na2SO4 and evaporated in a vacuum to give a crude residue which was purified by column chromatography (cyclohexane: ethyl acetate = 15:1–1:1) to yield compounds 3a–3c. The 1H-NMR spectra of compounds 3a–3c are in the Methyl 2-chlorosulfonylacetate 3a)Methyl 2-(N-phenylsulfamoyl)acetate  δ 7.42–7.30 , 7.29–7.21 , 7.05 , 3.96 , 3.82 . HR-MS (ESI): calcd for C9H12NO4S [M + H]+ 230.0487, found 230.0481.ate (3a)  Yellow s3b)Methyl 2-(N-(4-bromophenyl)sulfamoyl)acetate  δ 7.52–7.46 , 7.25–7.18 , 3.94 , 3.82 . HR-MS (ESI): calcd for C9H11BrNO4S [M + H]+ 307.9592, found 307.9603.ate (3b) . Yellow 3cMethyl 2-(N-(4-methoxyphenyl) sulfamoyl) acetate  + 260.0593, found 260.0602.ate (3c) . Yellow 4  and NaHCO3  were dissolved in anhydrous DMF (2 mL), followed by the addition of the bromide 5 (1.45 mmol). The reaction mixture was stirred at 110 °C. After completion of the reaction, the reaction mixture was diluted with DCM (20 mL) and washed with water (10 mL) and saturated brine (10 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated in a vacuum to give a crude residue which was purified by column chromatography (cyclohexane: ethyl acetate = 200:1) to yield compounds 6a–6b. The 1H-NMR spectra of compounds 6a–6b are in the 2,4-dihydroxybenzaldehyde 6a)4-Ethoxy-2-hydroxybenzaldehyde  δ 11.48 , 9.70 , 7.41 , 6.52 , 6.40 , 4.08 , 1.43 . HR-MS (ESI): calcd for C9H11O3 [M + H]+ 167.0708, found 167.0704.yde (6a) . Light y6b)2-hydroxy-4-propoxybenzaldehyde  δ 11.48 , 9.70 , 7.41 , 6.55–6.51 , 6.41 , 3.97 , 1.84 , 1.04 . HR-MS (ESI): calcd for C10H13O3 [M + H]+ 181.0865, found 181.0862.yde (6b)  Light ye2 atmosphere, compound 3a  and compound 6 (0.466 mmol) were dissolved in anhydrous ethanol (2 mL), and piperidine  was added dropwise. The resulting mixture was stirred at 87 °C. After completion of the reaction, the solvent was removed under reduced pressure and water (10 mL) was added to the residue and then extracted with DCM (10 mL × 3). The combined organic layers were dried over anhydrous Na2SO4 and evaporated in a vacuum to give a crude residue which was purified by column chromatography (cyclohexane: ethyl acetate = 100:1–5:1) to yield compounds 7a–7b. The NMR spectra of compounds 7a–7b are in the Under a N7a)7-Ethoxy-2-oxo-N-phenyl-2H-chromene-3-sulfonamide  νmax (cm−1) 3288, 1722, 1595, 1494; 1H-NMR  δ 8.35 , 7.45 , 7.30 , 7.13 , 6.88 , 6.80 , 4.27 , 1.46 . 13C-NMR  δ 164.50 (-COO-), 156.85 (-CH=C), 155.11 (-CH=C), 149.06 (-CH=C), 137.61 (-CH=C), 132.19 (-CH=C), 129.28 (-CH=C), 124.30 (-CH=C), 120.75 (-CH=C), 120.20 (-CH=C), 114.06 (-CH=C), 110.55 (-CH=C), 100.94 (-CH=C), 64.60 (-O2CHCH3), 14.28 (-OCH23CH). HR-MS (ESI): calcd for C17H16NO5S [M + H]+ 346.0749, found 346.0755.7b).7-propoxy-2-oxo-N-phenyl-2H-chromene-3-sulfonamide  νmax (cm−1) 3314, 1717, 1615, 1546; 1H-NMR  δ 8.34 , 7.45 , 7.24 , 7.13 , 6.90 , 6.81 , 4.00 , 1.85 , 1.05 . 13C-NMR  δ 164.62 (-COO-), 156.81 (-CH=C), 155.09 (-CH=C), 149.03 (-CH=C), 137.17 (-CH=C), 132.14 (-CH=C), 129.13 (-CH=C), 124.05 (-CH=C), 120.75 (-CH=C), 119.92 (-CH=C), 113.98 (-CH=C), 110.52 (-CH=C), 100.91 (-CH=C), 70.19 (-O2CHCH2CH3), 21.70 (-OCH22CHCH3), 10.21 (-OCH2CH23CH). HR-MS (ESI): calcd for C18H18NO5S [M + H]+ 360.0906, found 360.0897.2 atmosphere, compound 3 (1 mmol) and 4-(diethylamino) salicylaldehyde 8  were dissolved in anhydrous ethanol and piperidine  was then added dropwise. The resulting mixture was heated under reflux with stirring for 30 min. The solvent was removed under reduced pressure and water (10 mL) was added to the residue, and then extracted with DCM (10 mL × 3). The combined organic layers were dried over anhydrous Na2SO4 and evaporated in a vacuum to give a crude residue which was purified by column chromatography (cyclohexane: ethyl acetate = 10:1) to yield compounds 9a–9c. The NMR spectra of compounds 9a–9c are in the Under a N9a).7-(Diethylamino)-2-oxo-N-phenyl-2H-chromene-3-sulfonamide  νmax (cm−1) 3273, 2974, 1708, 1614; 1H-NMR  δ 10.19 , 8.55 , 7.64 , 7.29–7.08 , 6.98 , 6.76 , 6.51 , 3.44 , 1.09 . 13C-NMR  δ 157.73 (-COO-), 155.75 (-CH=C), 152.99 (-CH=C), 148.47 (-CH=C), 137.62 (-CH=C), 132.05 (-CH=C), 129.04 (-CH=C), 123.68 (-CH=C), 119.59 (-CH=C), 114.85 (-CH=C), 110.13 (-CH=C), 105.96 (-CH=C), 96.01 (-CH=C), 44.41 (-N2CHCH3), 12.23 (-NCH23CH). HR-MS (ESI): calcd for C19H21N2O4S [M + H]+ 373.1222, found 373.1229.9b).N-(4-bromophenyl)-7-(diethylamino)-2-oxo-2H-chromene-3-sulfonamide  νmax (cm−1) 3267, 2976, 1711, 1613; 1H-NMR  δ 8.17 , 7.38–7.33 , 7.30 , 7.14–7.07 , 6.61 , 6.44 , 3.44 , 1.22 . 13C-NMR  δ 157.73 (-COO-), 155.75 (-CH=C), 152.99 (-CH=C), 148.47 (-CH=C), 137.62 (-CH=C), 132.05 (-CH=C), 129.04 (-CH=C), 123.68 (-CH=C), 119.59 (-CH=C), 114.85 (-CH=C), 110.13 (-CH=C), 105.96 (-CH=C), 96.01 (-CH=C), 44.41(-N2CHCH3), 12.23 (-NCH23CH). HR-MS (ESI): calcd for C19H20BrN2O4S [M + H]+ 451.0327; found: 451.0334.9c)7-(Diethylamino)-N-(4-methoxyphenyl)-2-oxo-2H-chromene-3-sulfonamide  νmax (cm−1) 3271, 2928, 1796, 1615; 1H-NMR  δ 8.12 , 7.27 , 7.16 , 6.76 , 6.60 , 6.46 , 3.72 , 3.42 , 1.22 .13C-NMR  δ 157.70 (-COO-), 156.37 (-CH=C), 155.92 (-CH=C), 152.90 (-CH=C), 148.20 (-CH=C), 131.95 (-CH=C), 129.97 (-CH=C), 123.14 (-CH=C), 115.03 (-CH=C), 114.22 (-CH=C), 110.0 (-CH=C)9, 105.99 (-CH=C), 96.03 (-CH=C), 55.09 (-OCH3), 44.40 (-N2CHCH3), 12.25 (-NCH23CH). HR-MS (ESI): calcd for C20H23N2O5S [M + H]+: 403.1328; found: 403.1333.2 atmosphere, 4-(diethylamino) salicylaldehyde 8  and diethyl malonate 10  were dissolved in anhydrous ethanol (10 mL) and piperidine  was added dropwise. The reaction mixture was stirred at room temperature for 3 h. 10% HCl was then added into the reaction mixture to adjust the pH to 8. The mixture was then extracted with ethyl acetate (10 mL × 3). The combined organic layer was dried over anhydrous Na2SO4 and evaporated in a vacuum to give a crude residue which was purified by column chromatography (petroleum ether: ethyl acetate = 8:1) to yield compound 11. The NMR spectra of compound 11 are in the Under a N11)Ethyl 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylate  δ 8.40 , 7.33 , 6.58 , 6.43 , 4.34 , 3.42 , 1.37 , 1.21 . 13C-NMR  δ 164.37 (-COO-), 158.54 (-COO-), 152.96 (-CH=C), 149.33 (-CH=C), 131.14 (-CH=C), 109.62 (-CH=C), 108.97 (-CH=C), 107.76 (-CH=C), 96.78 (-CH=C), 61.25 (-O2CHCH3), 45.19 (-N2CHCH3), 14.49 (-OCH23CH), 12.52 (-NCH23CH). HR-MS (ESI): calcd for C16H20NO4 [M + H]+ 290.1392, found 290.1386.ate (11) . Yellow 11  and aromatic amines 1 (1.2 mmol) were dissolved in ethanol (10 mL). The reaction mixture was heated under reflux. After completion of the reaction, the solvent was evaporated in a vacuum followed by the addition of water (10 mL) and 10% HCl to adjust the pH to 8. The mixture was then extracted with ethyl acetate (20 mL × 3). The combined organic layer was dried over anhydrous Na2SO4 and evaporated in a vacuum to give a crude residue which was purified by column chromatography (petroleum ether: ethyl acetate = 5:1–1:1) to yield compounds 12a–12c. The NMR spectra of compounds 12a–12c are in the Compound 12a).7-(diethylamino)-2-oxo-N-phenyl-2H-chromene-3-carboxamide  νmax (cm−1) 3125, 1699, 1605. 1H-NMR  δ 10.87 , 8.80 , 7.74 , 7.47 , 7.33–7.38 , 7.12 , 6.67 , 6.53 , 3.47 , 1.25 . 13C-NMR  δ 163.20 (-COO-), 161.21 (-CONH-), 157.86 (-CH=C), 152.89 (-CH=C), 148.59 (-CH=C), 138.43 (-CH=C), 131.42 (-CH=C), 129.04 (-CH=C), 124.20 (-CH=C), 120.46 (-CH=C), 110.27 (-CH=C), 108.66 (-CH=C), 96.68 (-CH=C), 45.26 (-N2CHCH3), 12.55 (-NCH23CH). HR-MS (ESI): calcd for C20H21N2O3 [M + H]+: 337.1552; found: 337.1561.12b)N-(4-bromophenyl)-7-(diethylamino)-2-oxo-2H-chromene-3-carboxamide  νmax (cm−1) 3431, 1685, 1617. 1H-NMR  δ 8.38 , 7.48 , 7.15 , 7.10 , 6.25 , 6.18 , 3.40 , 1.21 . 13C-NMR  δ 162.90 (-COO-), 159.82 (-CONH-), 156.78 (-CH=C), 150.96 (-CH=C), 147.07 (-CH=C), 132.87 (-CH=C), 131.22 (-CH=C), 121.47 (-CH=C), 107.93 (-CH=C), 102.89 (-CH=C), 96.66 (-CH=C), 43.61 (-N2CHCH3), 11.68 (-NCH23CH). HR-MS (ESI): calcd for C20H20BrN2O3 [M + H]+: 415.0657; found: 415.0646.12c).7-(diethylamino)-N-(4-methoxyphenyl)-2-oxo-2H-chromene-3-carboxamide  νmax (cm−1) 3260, 1687,1588. 1H-NMR  δ 8.36 , 7.20 , 7.14 , 6.92 , 6.24 , 3.82 , 3.40 , 1.20 . 13C-NMR  δ 163.14 (-COO-), 157.48 (-CONH-), 156.78 (-CH=C), 150.87 (-CH=C), 132.70 (-CH=C), 123.34 (-CH=C), 122.45 (-CH=C), 120.53 (-CH=C), 113.52 (-CH=C), 107.84 (-CH=C), 102.85 (-CH=C), 96.90 (-CH=C), 54.52 (-OCH3), 43.62 (-N2CHCH3), 11.70 (-NCH23CH). HR-MS (ESI): calcd for C21H23N2O4 [M + H]+: 367.1658; found: 367.1666.® HTB-26™), KB (ATCC® CCL-17™) and HCT-116 (ATCC® CCL-247™) cells were purchased from the American Type Culture Collection . Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) media with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin at 37 °C in a 5% CO2 incubator.MDA-MB-231 . After a 72-h incubation, 3--2,5-diphenyltetrazolium bromide (MTT) were added, and the cells were incubated for another 4 h prior to the addition of DMSO. Absorbance was recorded at a wavelength of 540 nm with a reference length of 655 nm, and the results shown were collected from three independent experiments. The IC50 was determined by using GraphPad Prism software version 7.0 and nonlinear regression (curve fit).4 × 105 MDA-MB-231 cells per well were cultured in a 6-well plate and allowed to reach 80% confluency for 12 h. The monolayer was then scratched using a 200 µL pipet tip to produce a wound. The culture medium was removed, and each plate was washed with phosphate buffer saline (PBS) and treated with different concentrations of compound 9c  for 24 and 48 h, respectively. Images were captured at the identified time point  and processed with the ImageJ software.5 × 104 MDA-MB-231 cells per well were seeded in the upper part of the insert filled with 200 µL of DMEM without FBS, and for the lower part, the chamber was filled with 600 µL DMEM containing 10% FBS. The cells were allowed to grow for 48 h followed by treatment with different concentrations of compound 9c . The invaded cells were fixed with paraformaldehyde for 2 h and stained with crystal violet solution (0.2%) for 1 h, and non-invading cells in the top chambers of the transwell plates were scraped away by cotton swabs. Every insert was imaged at 10× magnification and analyzed using ImageJ software.For the assay, 24-well transwell chambers coated with 45 µL matrigel were used. In brief, 6 × 105 MDA-MB-231 cells per well were seeded in a 6-well plate for 12 h and were treated with different concentrations of compound 9c  for 48 h. After treatment, the cells were harvested, washed twice with ice-cold PBS and resuspended in 500 µL of 1× Annexin V binding buffer and then mixed with 5 µL of Annexin V-FITC and 5 µL of PI solution at room temperature in the dark for 10 min. Then, one part of the cells was immediately analyzed with a flow cytometer. The other part was washed twice with PBS, and the cells were photo-graphed by fluorescence microscope.5 × 105 MDA-MB-231 cells were plated and treated with different concentrations  of compound 9c for 48 h. ROS assay kit (Beyotime) was used to measure intracellular ROS level. DCFH-DA diluted with serum-free medium with 1:1000 was added into the cells at 37 °C for 20 min. The cells were collected after washing three times with serum-free medium. Images were acquired using an inverted fluorescence microscope, and the fluorescence intensity was detected at an excitation wavelength of 488 nm and an emission wavelength of 525 nm with microplate reader.In 6-well plates, 2 × 10For Western blotting analyses, the collected cells were rinsed in PBS and lysed in sodium dodecyl sulfate (SDS) lysis buffer. Equal amounts of protein were separated on 10% SDS-polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked with 5% nonfat dry milk in tris-buffered saline and tween 20 (TBST) for 1 h. The membranes were then incubated with primary antibodies  overnight at 4 °C. After being washed three times with TBST for 10 min, the membranes were incubated with appropriate secondary antibodies in TBST for 1 h. After several washes of TBST, the blots were developed by enhanced chemiluminescence (ECL) solution.p-value less than 0.05 are considered as significant and statistical significance is represented as **** (p < 0.0001), *** (p < 0.001), ** (p < 0.01), * (p < 0.05), ns (not significant).GraphPad Prism software version 7.0 was used to determine the differences between groups. Values with a 9c was found to be the most potent derivative, which displayed favorable antiproliferative activities toward MDA-MB-231 cells and inhibited migration and invasion and induced apoptosis in MDA-MB-231 cells. Furthermore, it was verified that compound 9c promoted MDA-MB-231 cell apoptosis by increasing intracellular ROS levels and upregulating the expression of caspase-3. This work presents information that is helpful for the design and synthesis of new coumarin derivatives as potential antitumor drug candidates.A novel series of coumarin sulfonamides and amides derivatives were designed and synthesized. Compound


E)-5-phenyl-1-(pyridin-2-yl)pent-2-en-4-yn-1-one underwent spontaneous [2 + 2] cycloaddition reaction while (E)-5-(4-methylphenyl)-1-(pyridin-2-yl)pent-2-en-4-yn-1-one remained photoinert.Upon recrystallization from ethylene glycol in daylight, ( E)-5-phenyl-1-(pyridin-2-yl)pent-2-en-4-yn-1-one at room temperature from ethylene glycol in daylight afforded [3,4-bis(phenylethynyl)cyclobutane-1,2-diyl)bis, while (E)-5-(4-methylphenyl)-1-(pyridin-2-yl)pent-2-en-4-yn-1-one, C17H13NO (2), remained photoinert. This is the first experimental evidence that pentenynones can be photoreactive when fixed in nearly coplanar parallel positions. During the photoreaction, the bond lengths and angles along the pentenyne chain changed significantly, while the disposition of the pyridyl ring towards the keto group was almost unchanged. The cyclobutane ring adopts an rctt conformation.Recrystallization of ( Only the rctt isomer of a 1,2,3,4-tetrasubstituted cyclobutane was obtained of four theoretically possible (based on XRD data).The asymmetric unit of ketone es Fig. 1. Their c2 and 3 is probably affected by intramolecular C—H⋯N contacts  = 3.206 (3) Å, and in the head-to-tail chains r(C⋯O) and r(C⋯N) = 3.379 (2) and 3.465 (3) Å, respectively. The corresponding C—H⋯O and C—H⋯N angles are, respectively, 139, 143 and 136°. Note, that only one of two independent molecules in 2 forms head-to-tail chains via a pair of intermolecular C—H⋯O and C—N⋯N bonds. None of the previously reported pyridine-2-yl-containing chalcones nor 3 forms such associates. Instead, the nitrogen atoms interact with the hydrogen atoms of the alkyl and aryl groups. For example, in the crystal of 3, the hydrogen atoms of a pyridine-2-yl ring take part in C—H⋯N interactions [Fig. 3r(C⋯N) = 3.445 (3)–3.665 (2) Å]. Oxygen atoms take part in C—H⋯O bonding with hydrogen atoms of the phenyl and pyridin-2-yl rings. In addition, in 2 and 3, numerous hydrophobic interactions can be found.As the independent molecules of ketone fs Fig. 3. In the 1, and 5-(4-methylphenyl)-1-(pyridin-2-yl)pent-2-en-4-yn-1-one, 2, were synthesized according to the previously described method pent-2-en-4-yn-1-one, 1: yellowish needles, yield 61%, m.p. 348–351 K (from a mixture of water and ethanol). 1H NMR , δ, ppm: 8.48 s , 8.09–8.16 m , 7.84–7.79 m , 7.20–7.52 m . 13C NMR , δ, ppm: 188.5 (C1), 152.6, 149.0, 132.2, 131.9, 129.5, 128.6, 128.2, 128.0, 127.1, 122.1, 99.6 (C5), 88.9 (C4). Found, %: C 82.44; H 5.41. C16H11NO. Calculated, %: C 82.38; H 4.75.For 2: yellowish needles, yield 34%, m.p. 373–374 K , 1649 (C=O). Found, %: C 82.44; H 5.33. C17H13NO. Calculated, %: C 82.57; H 5.30.For 2 were collected at the K4.4 ‘Belok’ beamline of the Kurchatov Synchrotron Radiation Source  at a wavelength of 0.80248 Å using a Rayonix CCD 165 detector. Image integration was performed using iMosflm software  = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020000055/ff2164sup1.cifCrystal structure: contains datablock(s) global, 2, 3. DOI: 10.1107/S2056989020000055/ff21643sup2.hklStructure factors: contains datablock(s) 3. DOI: Click here for additional data file.10.1107/S2056989020000055/ff21643sup4.cmlSupporting information file. DOI: 10.1107/S2056989020000055/ff21642sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989020000055/ff21642sup5.cmlSupporting information file. DOI: 1975319, 1975318CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Salvia reuterana Boiss. is an aromatic perennial plant traditionally used for its anxiolytic and sedative properties. In the present study, various fractions and essential oil of S. reuterana aerial parts were investigated to find its free radical scavenging principles. Hydroalcoholic fraction with IC50 value of 112.6 ±3.2 μg mL-1 in DPPH assay demonstrated the highest free radical scavenging activity and was selected to further phytochemical investigation. RP-18 and Sephadex LH-20 column chromatography of the hydroalcoholic fraction resulted in the isolation and structural elucidation of four phenolic derivatives, including apigenin-7-O-β-D-glucopyranoside (1), luteolin-7-O-β-D-glucopyranoside (2), rosmarinic acid (3), and luteolin (4). Isolated compounds showed potent free radical scavenging activities (5.1-34.2 μg mL-1), compared with BHT (21.30 ± 1.9 μg mL-1). Twenty four compounds were also identified in GC-MS analysis of the plant essential oil, of which benzyl benzoate (26.64%), n-hexyl benzoate (22.99%) and n-hexyl isovalerate (6.04%) were the main compounds. The results of the present study introduced S. reuterana as a valuable source of natural phenolic antioxidants which can be utilized in prevention of oxidative stress related diseases. Moreover, interesting composition of S. reuterana essential oil, dominated by non-terpenes compounds (76.17%) especially aromatic derivatives, make it an appropriate candidate for more detailed studies. Nowadays, the role of free radicals, particularly reactive oxygen species (ROS), have been well recognized in development of many pathological disorders such as cardiovascular diseases, diabetes, cancers, inflammatory, and neurodegenerative diseases  . So, in Salvia L., commonly known as Sage, is one of the largest genera in the Lamiaceae family, mainly distributed in temperate and subtropical regions of the world  and ferric reducing power , among the other tested Salvia species  in comparison with its leave extract , in DPPH assay  soluble  main fractions.The air dried and grinded plant aerial parts (400 g) were macerated with 80% methanol in water (5 × 2.5 L). The total hydroalcoholic extract (82 g) was then dissolved in 500 mL of methanol-water mixture (8:2) and extracted by enough volumes of Essential oil extractionHydrodistillation method using Clevenger apparatus was used to essential extraction from 100 g of dried and comminuted plant material for 3 hours. The pale yellowish oil was dried over anhydrous sodium sulphate and kept at 4 °C until analysis.DPPH free radical scavenging assay et al. (-3 mg mL-1) were made from samples, individually (each 2 mL). Two milliliter of freshly prepared DPPH (Sigma) solution (80 µg mL-1) was then added to each test tube. After 30 min, absorptions of the solutions were recorded at 517 nm using an Optizen 2120 UV PLUS spectrophotometer. Butylated hydroxy toluene (BHT) (Sigma), a commercial synthetic antioxidant, was also used as positive control. For each sample concentration causing a 50% reduction in absorption of DPPH solution (40 µg mL-1) was calculated as IC50. The experiment was repeated three times and results were expressed as Mean ± SD.Antioxidant activity of the total extract, main fractions and essential oil of plant aerial parts were evaluated by DPPH  free radical scavenging assay method described by Sarker et al. . BrieflyIsolation and purification of compounds1 (18 mg) and 2 (43 mg) were isolated from the fraction M3 (320 mg) on a Sephadex LH-20 column (Fluka) eluted by methanol:water (8:2) as solvent system. Colum chromatography of the fraction M4 (147 mg) on a Sephadex LH-20 column with methanol resulted in the isolation of compound 3 (28 mg). Compound 4 (18 mg) was isolated from the fraction M5 (95 mg) through the reversed-phase column chromatography . It was more purified on a Sephadex LH-20 column using methanol as eluent. All column chromatographies were monitored using thin layer chromatography  and the fractions giving same spots under 254 and 366 UV wavelengths were combined.Hydroalcoholic fraction with the highest free radical scavenging activity  was subjGC and GC-MS analysis -1), temperature program: 50 °C for 5 min, 50-280 °C at 10 °C/min, injector temperature: 280 °C, injection volume: 1 μL. A homologous series of n-alkanes was also injected in conditions equal to the oil sample in order to calculate retention indices (RI). The compounds were identified by computer matching with the Wiley7n.L and NIST05a.L libraries, as well as by comparison of RIs and mass fragmentation patterns with those published in literature for standard compounds  connected to an Agilent 5977A mass selective detector (70 eV) under the following conditions; carrier gas: helium , luteolin-7-O-β-D-glucopyranoside (2), rosmarinic acid (3) and luteolin (4) (1-4) were characterized using 1H-NMR and 13C-NMR spectral analysis  and confirmed in accordance with bibliographic data  . The strhic data .Spectroscopic data of isolated compoundsApigenin-7-O-β-D-glucopyranoside (Cosmosiin) (1); 1H-NMR : δ 7.94 , 6.94 , 6.90 , 6.84 , 6.43 , 5.46 , 3.1-3.9 . 13C-NMR : δ 181.93 (C-4), 164.25 (C-2), 162.94 (C-7), 161.84 (C-4′), 161.3 (C-5), 156.94 (C-9), 128.62 (C-2′), 128.50 (C-6′), 120.91 (C-1′), 116.16 (C-3′), 115.87 (C-5′), 105.32 (C-10), 103.25 (C-3), 102.85(C-1″), 99.91 (C-6), 94.60(C-8), 77.18 (C-5″), 76.43 (C-3″), 73.09 (C-2″), 69.56 (C-4″), 60.60 (C-6″) (Luteolin-7-O-β-D-glucopyranoside (Cynaroside) (2); 1H-NMR : δ 7.45 , 7.43 , 6.94 , 6.82 , 6.74 , 6.46 , 5.08 , 3.2-3.6 . 13C-NMR : δ 182.13 (C-4), 164.82 (C-2), 163.17 (C-7), 161.47 (C-5), 157.25 (C-9), 150.61 (C-4′), 146.15 (C-3′), 121.35 (C-1′), 119.51 (C-6′), 116.34 (C-5′), 113.60 (C-2′), 105.69 (C-10), 103.27 (C-3), 100.24(C-1″), 100.14 (C-6), 95.00 (C-8), 77.34 (C-5″), 76.58 (C-3″), 73.37 (C-2″), 69.88 (C-4″), 60.93 (C-6″) .Rosmarinic acid  (3); 1H-NMR : δ 7.36 , 7.06 , 6.89 , 6.74 , 6.68 , 6.60 , 6.48 , 6.17 , 4.86 , 3.02 , 2.75 . 13C-NMR : δ 172.97 (C-9′), 166.34 (C-9), 149.04 (C-4), 146.17 (C-7), 145.06 (C-3), 144.45(C-3′), 143.69 (C-4′), 129.81 (C-1′), 125.25 (C-1), 120.81 (C-6), 119.59 (C-6′), 116.78 (C-2′), 116.05 (C-5), 115.49 (C-5′), 115.04 (C-2), 114.54(C-8), 75.97 (C-8′), 37.17 (C-7′) (7 (C-7′) .Luteolin  (4); 1H-NMR : δ 7.42 , 7.39  6.89 , 6.66 , 6.44 , 6.18 .13C-NMR : δ 181.61 (C-4), 164.12 (C-2), 163.86 (C-7), 161.45 (C-5), 157.26 (C-9), 149.68 (C-4′), 145.71 (C-3′), 121.46 (C-1′), 118.93 (C-6′), 116.26 (C-5′), 113.32 (C-2′), 103.65 (C-10), 102.82 (C-3), 98.79 (C-6), 93.80 (C-8) (80 (C-8) .1-4) were also assessed by DPPH test. As shown in 4), rosmarinic acid (3) and luteolin-7-O-β-D glucopyranoside (2) were found to have a potent free radical scavenging activity with IC50 values of 5.1 ± 0.6, 9.6 ± 1.2, 17.3 ± 2.1 µg mL-1, higher than positive control, BHT (IC50: 21.3 ± 1.9 µg mL-1). Thus, these compounds can be assumed as the major free radical scavengers present in S. reuterana aerial parts. Free radical scavenging activities of the isolated compounds  from the aerial parts of this medicinal species. These compounds, however, have been isolated from various other Salvia species  has been reported for its anxiolytic (4) and its 7-O-glucopyranoside derivative (2) have shown anti-inflammatory , which has also been reported as a chemotaxonomic marker of the subfamily Nepetoideae  produces antidepressant-like effect in the forced swimming test in mice  and n-hexyl isovalerate (6.04%) . However, the low yield of essential oil extraction (yield: 0.2% (v/w)) attenuates the importance of the plant essential oil in antioxidant properties of S. reuterana.GC-MS analysis of the essential oil resulted in the identification of twenty four compounds, representing the 98.48% of the total oil. The essential oil was rich in non-terpene compounds (76.17%), mainly benzyl benzoate (26.64%),  (6.04%) . EssentiS. reuterana aerial parts collected from different regions of Iran , β-elemene (4.5-13.9%), germacrene D (2.6-7.2%), spathulenol (1.0-8.0%) and n-hexyl acetate (1.2-6.8%) were identified as major compounds (n-hexyl benzoate (22.99%), another main compound identified in the present study was not detected by Fattahi et al. in their examined essential oils of different S. reuterana populations (n-hexyl benzoate has been characterized at high amounts (17.0%) in essential oil of S. reuterana flowers, collected from Kashan region, center of Iran  can be considered as a valuable source of natural phenolic antioxidants. Literature review on biological activities of the isolated compounds also provides some molecular explanations for anxiolytic and antidiabetic properties, previously reported from S. reuterana. Moreover, interesting chemical composition of S. reuterana essential oils, which is dominated by non-terpenes compounds (76.17%), especially aromatic derivatives, make it an appropriate candidate for further studies.The results of present study verify that


Gymnema sylvestre, a medicinal plant, has been used in Indian ayurvedic traditional medicine for the treatment of diabetes. Phytochemical investigation of Gymnema sylvestre led to the isolation of five new pregnane glycosides, gymsylosides A–E (1–5) and four known oleanane saponins, 3β-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (6), gymnemoside-W1 (7), 3β-O-β-D-xylopyranosyl-(1→6)-β-D- glucopyranosyl-(1→6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-glucopyranosyl ester (8), and alternoside XIX (9). Their structures were identified based on spectroscopic evidence and comparison with those reported in the literature. All compounds were evaluated for their α-glucosidase and α-amylase inhibitory activities. Compounds 2–4 showed significant α-amylase inhibitory activity, with IC50 values ranging from 113.0 to 176.2 µM. Gymnema sylvestre (Retz.) R.Br. ex Sm. (Apocynaceae) is a perennial woody climber native to tropical and subtropical regions, such as India, Africa, and southeast Asia. In folk medicine, G. sylvestre have been used to treat snake bites, arthritis, digestive, and enhancing laxative , together with three secondary methyl groups , confirmed the presence of three sugar units. The 13C NMR and DEPT spectra indicated that 1 contained one carbonyl, seven non-protonated carbons (three oxygenated), nineteen methines (sixteen oxygenateds), nine methylenes, and eleven methyl carbons (three methoxys). The 1H and 13C-NMR spectroscopic data suggested that 1 was a pregnane glycoside  suggested that the configuration of H-12 was axial  and Hα-1 (δH 1.09)/ Hα-4 (δH 2.33), and between H-12 (δH 4.68) and H-9 (δH 1.49)/Hα-15 (δH 1.88) suggested the configurations of the oxygenated groups at C-3 and C-12, the hydroxy groups at C-8 and C-14 to be β. The HMBC correlations between Tig H-5 (δH 1.85) and Tig C-1 (δC 169.1)/Tig C-2 (δC 130.0)/Tig C-3 (δC 139.5) and between Tig H-4 (δH 1.80) and Tig C-2/ Tig C-3 and NOESY correlations between Tig H-4 (δH 1.80) and Tig H-5 (δH 1.85) suggested the presence of (E)-tigloyl moiety. In addition, the position of this moiety at C-12 was confirmed by HMBC correlation from H-12 (δH 4.68) to Tig C-1 (δC 169.1). Acid hydrolysis of 1 gave three monosaccharides, which were identified as D-cymarose +, corresponding to the molecular formula of C54H88O20. The 1H and 13C-NMR spectra of 3 showed the presence of one pregnane aglycone, four sugar units, and one tigloyl unit (3 were compared to gymsyloside A (1) and found the addition of one sugar unit in the sugar linkages. Acid hydrolysis of 3 gave three monosaccharides, which were identified as D-cymarose, D-oleandrose, and D-thevetose. The tetrasaccharide was determined to be β-D-thevetopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, by the analysis of HMBC and COSY correlations. The location of sugar linkages at C-3 was confirmed by the HMBC correlation between Cym I H-1 (δH 4.84) and C-3 (δC 79.3). Consequently, the structure of 3 was determined to be (20S)-12β-tigloyloxy-3β,8β,14β,17β, 20-pentahydroxypregn-5-ene 3-O-β-D-thevetopyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, a new compound named gymsyloside C.The HRESIMS of oyl unit . The NMR4 was determined as C54H88O20 by the HRESIMS. The 1H and 13C-NMR data  +. The 1H and 13C NMR data of 5 were similar to 2, except for an additional sugar unit at Thv C-4 (δH 4.41) and Thv C-4 (δC 82.9), Thv H-1 (δH 4.43) and Ole C-4 (δC 84.1), Ole H-1 (δH 4.57) and Cym C-4 (δC 83.8), and between Cym H-1 (δH 4.84) and aglycone C-3 (δC 79.3) confirmed the sequence of sugar linkages, previously reproted from G. sylvestre + ; 1H  and 13C-NMR  data, see White amorphous powder; c 0.1, MeOH); C47H76O17, HRESIMS m/z: 935.4996 [M + Na]+ ; 1H  and 13C-NMR  data, see White amorphous powder; c 0.1, MeOH); C54H88O20, HRESIMS m/z: 1079.5769 [M + Na]+ ; 1H  and 13C-NMR  data, see White amorphous powder; c 0.1, MeOH); C54H88O20, HRESIMS m/z: 1079.5778 [M + Na]+ ; 1H  and 13C-NMR  data, see White amorphous powder; c 0.1, MeOH); C53H86O22, HRESIMS m/z: 1097.5530 [M + Na]+ ; 1H  and 13C NMR  data, see White, amorphous powder; 1–5, 3.0 mg) was separately dissolved in 1.0 N HCl  and heated to 80 °C in a water bath for 3 h. The acidic solution was dried under N2 overnight. After extraction with CHCl3, the aqueous layer was dried using N2 to give aqueous residue (A). The aqueous residue (A) was separated by silica gel CC eluting with CH2Cl2–MeOH  and then further fractionated by RP-18 CC using a solvent gradient of MeOH–H2O , to give the monosaccharides (50% yield). The specific rotations of these sugars were determined. The specific rotations (2O for 24 h and compared to the literature (lit):D-cymarose: found +50.1 , lit +51.8 [c 0.4, H2O), lit +11.7 [c 0.4, H2O); lit +42.3 [O-methyl-D-allose: found +10.9 ; lit +10.0 [c 0.4, H2O); lit +48.0 [1 and 3: D-cymarose, D-oleandrose, and D-thevetose; compounds 2 and 4: D-cymarose, D-oleandrose, and 6-deoxy-3-O-methyl-D-allose; compound 5: D-cymarose, D-oleandrose, D-thevetose, and D-glucose.Each compound  in 1.0 mL of 5% KOH/MeOH was heated at 40 °C four 4 h and then neutralized with HCl 0.1 M. After that, the solution was partitioned with CHCl3 to give CHCl3 layer. CHCl3 layer was separated on HPLC system: J’sphere H-80 column (150 × 20 mm), solvent condition of 55% acetonitrile, to give sarcostin (54% yield). In a similar way, sarcostin was found as aglycone of compounds 2–5.A solution of compound The α-glucosidase  enzyme inhibition assay was performed according to the previously described method . The samThe α-amylase  enzyme inhibitory activity was measured using the reported method . SubstraG. sylvestre. Besides oleanane saponins-hightly species-specific of G. sylvestre, new pregnane glycosides in this report will provide secondary metabolisms as specific compounds in Gymnema genus. Compound 4 showed the weak α-glucosidase inhibitory activity. Compounds 2, 3, and 4 showed significant α-amylase inhibitory activity. This is the first report of α-amylase and α-glycosidase inhibitory activities of compounds from G. sylvestre.In summary, five new pregnane glycosides and four known oleanane saponins were isolated and identified from


Aspergillus tubingensis G131, a non-toxigenic strain. The radical scavenging activity of these NγPs was evaluated using ABTS assay. The Trolox equivalent antioxidant capacity on the seven isolated NγPs ranged from 2.4 to 14.6 μmol L−1. The toxicity and ability of the NγPs to prevent H2O2-mediated cell death were evaluated using normal/not cancerous cells (CHO cells). This cell-based assay showed that NγPs: (1) Are not toxic or weakly toxic towards cells and (2) are able to protect cells from oxidant injuries with an IC50 on H2O2-mediated cell death ranging from 2.25 to 1800 μmol mL−1. Our data show that A. tubingensis G131 strain is able to produce various NγPs possessing strong antioxidant activities and low toxicities, making this strain a good candidate for antioxidant applications in food and cosmetic industries.Seven naphtho-gamma-pyrones (NγPs), including asperpyrone E, aurasperone A, dianhydroaurasperone C, fonsecin, fonsecinone A, fonsecin B, and ustilaginoidin A, were isolated from  Aspergillus spp. [Naphtho-gamma-pyrones (NγPs) are polyketide pigments that have been found to occur in higher plants belonging to the genera Cassia. They are also widely produced by fungi and, notably, lus spp. ,2. Theirlus spp. . The NγPlus spp. . This falus spp. ,6,7. Antlus spp. . Nowadaylus spp. . Few stulus spp. ,11,12,13Aspergillus tubingensis G131 and evaluated their AOA using the radical 2,2′-azino-bis assay, commonly used in the agrifood sector and H2O2-mediated cell death assay.Among the published activities, only fonsecin shows a strong AOA compared to ascorbic acid, fonsecinone A having a moderate AOA and aurasperone A, a moderate or no significant AOA compared to pure references ascorbic acid or BHT. In the present study, we isolated various NγPs  and coupling constants (J) are reported in hertz (Hz). The one- (1D) and two-dimensional (2D) spectra were processed using Topspin. The NMR spectra were measured as solutions in chloroform-d or methanol-d following the solubility of the compounds in 5 mm outer diameter tubes. The structural identifications of the compounds were achieved by comparing their spectroscopic data  with the values found in the literature.Nuclear magnetic resonance (NMR) spectra were recorded with an Avance III Bruker NMR spectrometer operating at 600 MHz for −1 and an injection volume of 40 μL. Elution was performed using acidified water (H2O with 2% acetic acid) (solvent A) and acetonitrile (ACN) (solvent B) with a linear gradient over 55 min at a flow rate of 1 mL min−1, starting from 30 to 100% of solvent B during 45 min, followed by 100% solvent B for 5 min. The UV absorbance of the eluent was monitored at 280 nm.Dry samples were dissolved in ethanol and filtered. A C18 RP, Zorbax Eclipse XDB column 150 × 5 mm, 4.6 μm particle size  fitted with a C18 guard column was used with a flow rate of 1 mL min−1 and an injection volume of 100 μL, following a linear gradient starting from H2O/ACN  to 100% ACN during 60 min. The UV absorbance of the eluent was monitored at 280 nm.Separation of the compounds was performed using HPLC apparatus . The column was a C18   with a flow rate of 3 mL minA. tubingensis G131 was provided by École Nationale Supérieure Agronomique de Toulouse (France); this fungus is non-mycotoxigenic as the synthetic genes required for the production ochratoxin A and fumonisin are absent from its genome [v/v) Tween 80  and scraping with a magnetic stirrer to recover the conidia. The quantity of conidia was counted using a Malassez cell prior to inoculation of the solid medium.s genome . It was 7 conidia g−1  at an initial humidity of 66% and incubated at 25 °C during 7 days.The SSF was performed in Raimbault columns on 50 g  of moist solid medium composed by 50% of vine shoots, 25% of wheat bran, and 25% of potato flakes. The medium was inoculated with 2.101–7. Compounds 1 (16 mg) and 3–7 were isolated as yellowish powder , whereas compound 2 (11 mg) was presented as red needles.One kilogram of moist fermented solid matrix was extracted using 2 L of ethanol at 25 °C during 2 h. The obtained extract was filtered with Whatman No. 1 filter paper and concentrated under reduced pressure using a rotary evaporator at 40 °C. The extract (8.32 g) was suspended in 250 mL of distilled water before being partitioned with hexane (84.7 mg), dichloromethane (334.9 mg), ethyl acetate (143.52 mg), and butanol (107.5 mg). The fractions that exhibited the most powerful antioxidant activity, thus the methylene chloride and the ethyl acetate extracts, were subsequently purified by semi-preparative HPLC. Twenty fractions were obtained (F1–F20), and subsequent semi-preparative HPLC on F6, F9, F16, F17, F19, and F20 allowed to isolate compounds The spectroscopic data of the known NγPs were in accordance with literature.Fonsecin (1). 1H-NMR  δ: 6.43 , 6.38 , 6.31 , 3.89 , 3.01 , 2.76 , 1.65 .13C-NMR  δ: 198.8 (C-4), 165.6 (C-5), 163.0 (C-6), 162.1 (C-8), 155.0 (C-10a), 144.9 (C-9a), 107,2 (C-5a), 104,0 (C-4a), 102.8 (C-9), 102.8 (C-10), 100.9 (C-2), 97.5 (C-7), 56.2 (6-CH3O), 48.2 (C-3), 28.1 (2-CH3).Ustilaginoidin A (2). 1H-NMR  δ: 6.90 , 6.41 , 5.98 , 3.94 , 2.36 .13C-NMR  δ: 185.4 , 169.4 , 164.8 , 161.8 , 160.8 , 153.9 , 142.3 , 108.2 , 107.4 , 104.3 , 101.4 , 101.9 , 98.2 , 56.4 , 20.7 .Fonsecin B (3). 1H-NMR  δ: 6.46 , 6.42 , 6.23 , 3.88 , 3.82 , 2.93 , 2.80 , 1.63 .13C-NMR  δ: 197.1 (C-4), 164.2 (C-5), 162.4 (C-8), 161.3 (C-6), 153.8 (C-10a), 143.4 (C-9a), 107.2 (C-5a), 103.3 (C-4a), 102.7 (C-10), 99.9 (C-2), 98.6 (C-9), 96.7 (C-7), 56.0 (6-CH3O), 55.4 (8-CH3O), 47.5 (C-3), 27.8 (2-CH3).Dianhydroaurasperone C (4). 1H-NMR  δ: 7.14 , 6.99 , 6.48 , 6.36 , 6.32 , 6.25 , 4.02 , 3.63 , 3.61 , 2.56 , 2.47 .13C-NMR  δ: 167.1 (C-2), 166.8 (C-2′), 162.5 (C-8), 159.9 (C-10), 157.1 (C-10′), 156.5 (C-5′), 156.2 (C-8′), 116.1 (C-9′), 110.9 (C-3′), 110.7 (C-3), 109.5 (C-4′a), 108.4 (C-4a), 108.4 (C-10′a), 106.2 (C-7′), 105.8 (C10a), 97.6 (C-9), 96.4 (C-7), 61.8 (10′-CH3O), 56.3 (10-CH3O), 55.5 (8-CH3O), 20.8 (2′-CH3), 20.8 (2-CH3).Asperpyrone E (5). 1H-NMR  δ: 7.14 , 6.99 , 6.48, , 6.36 , 6.32 , 6.25, , 4.02 , 3.63 , 3.61 , 2.56 , 2.47 .13C-NMR  δ: 167.1 (C-2), 166.8 (C-2′), 162.5 (C-8), 159.9 (C-10), 157.1 (C-10′), 156.5 (C-5′), 156.2 (C-8′), 116.1 (C9′), 110.9 (C-3′), 110.7 (C-3), 109.5 (C-4′a), 108.4 (C10′a), 108.4 (C-4a), 106.2 (C-7′), 105.9 (C-6′), 105.8 (C10a), 97.6 (C-9), 96.4 (C-7), 61.8 (10′-CH3O), 56.3 (10-CH3O), 55.5 (8-CH3O), 20.8 (2-CH3), 20.8 (2′-CH3).Fonsecinone A (6). 1H-NMR  δ: 7.23 , 7.12 , 6.43, , 6.53 , 6.25 , 6.10 , 3.99 , 3.81 , 3.59 , 3.48 , 2.53 , 2.16 .13C-NMR  δ: 182.3 (C-4′), 170.0 (C-2′), 169.6 (C-2), 163.1 (C-8′), 162.1 (C-6′), 161.2 (C-8), 157.6 (C-10), 157.2 (C-10b), 118.3 (C-9), 110.8 (C-3), 110.0 (C-4a), 108.9 (C10a), 108.7 (C5′a), 107.5 (C-3′), 106.9 (C-10′), 106.8 (C-6), 105.0 (C4′a), 102.8 (C-7), 98.0 (C-7′), 97.2 (C-9′), 61.7 (10-CH3O), 56.3 (8-CH3O), 56.3 (6′-CH3O), 55.4 (8′-CH3O), 20.1 (2-CH3), 20.1 (2′-CH3).Aurasperone A (7). 1H-NMR  δ: 7.37 , 7.26 , 6.51 , 6.25 , 6.15 , 6.09 , 3.98 , 3.81 , 3.61 , 3.48 , 2.45 , 2.16 .13C-NMR  δ: 185.8 (C-4), 170.4 (C-2), 169.9 (2′), 162.9 (8′), 161.9 (6′), 161.4 (8), 158.9 (6), 154.3 (10a), 142.3 (9a), 118.5 (7), 111.7 (5a), 108.8 (5′a), 107.7 (C-3), 107.4 (3′), 106.7 (10′), 105.2 (C4a), 104.5 (4′a), 102.8 (9), 102.8 (10), 98.0 (7′), 97.2 (9′), 62.3 (6-CH3O), 56.3 (8-CH3O), 56.3 (6′-CH3O), 55.4 (8′-CH3O), 20.3 (2-CH3), 20.1 (2′-CH3).•+ as it reacts with the NγPs. The decolorization was monitored in a 96-well microplate using a microplate reader Infinite 200 . The reaction between the sample and the ABTS•+ solution was performed for 5 min at 25 °C and the reading was made at 734 nm. The scavenging activity is expressed in Trolox equivalent antioxidant capacity (TEAC). A calibration curve of 3,4-dihydro-6-112 hydroxy-2,5,7,8-tétramethyl-2H-1-benzopyran-2-carboxylic acid (Trolox) ranging from 15.625 to 1000 μmol L−1 was made. To compare the various TEAC, the concentrations of the sample were set to 10 μmol L−1.0 is the initial absorbance and Af the absorbance after 5 min.A colorimetric assay was adapted from Re et al. . It cons2 flasks in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% l-glutamine, and 1% antibiotics , and maintained in a 5% CO2 incubator at 37 °C. For innocuity testing, CHO cells grown on flasks were trypsinized, diluted in culture media, and seeded onto 96-well plates  at 20,000 cells per well [1–7 or ascorbic acid for 24 h at 37 °C before evaluation of the cell viability using a resazurin-based in vitro toxicity assay kit (Sigma-Aldrich), as previously described [Innocuity was evaluated on normal/non-cancerous cells, i.e., Chinese hamster ovary cells . Cells were routinely grown on 25 cmper well . After sescribed ,18.Briefly, resazurin stock solution was diluted 1:100 in sterile PBS containing calcium and magnesium . Plates were aspirated and 100 μL of the diluted solution was added per well. After 1 h incubation at 37 °C, fluorescence intensity was measured using a microplate reader  with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The fluorescence values were normalized by the controls (untreated cells) and expressed as percent viability.2O2-mediated cell death, confluent cells on 96-well plates were exposed for 30 min to increasing doses of compounds 1–7 or ascorbic acid before treatment with 300 μmol L−1 of hydrogen peroxide for 24 h at 37 °C prior to evaluation of the cell viability using resazurin-based assay, as explained above. Results were expressed as percentage of inhibition of the hydrogen peroxide effect on the cells.For protective effect against HAll experiments were performed in triplicate. The results were fitted using R software version 3.5.1.1), ustilaginoidin A (2), fonsecin B (3), dianhydroaurasperone C (4), asperpyrone E (5), fonsecinone A (6), and aurasperone A (7) [1 and 3 are monomeric NγPs and compounds 4–7 are dimeric NγPs. The NγPs contain a fully conjugated system, giving rise to very characteristic UV/VIS spectra, with an absorption maximum at 280 nm.Most of the studies evaluating production of NγPs involve a microorganism growing on synthetic media ,19,20,21ne A (7) ,25,26,27•+ was tested for the seven NγPs isolated at a concentration of 10 μmol L−1. Compounds 1–7 exhibited various AOA activity, expressed in TEAC, i.e., 13.3, 14.6, 5.1, 4.6, 3.3, 2.4, to 2.4 μmol L−1 respectively  and ustilaginoidin A (2).ectively . In compA linear relation between the degree of substitution with hydroxyl groups per NγP unit and the TEAC was observed for the isolated NγPs . The degThe proposed radical scavenging mechanism is based on a resonance stabilization of the unpaired electron through its redistribution on the aromatic cores of the NγPs .2O2-mediated cell death were investigated using an in vitro model, i.e., CHO cells. With regards to the innocuity of the compounds, only compounds 1 and 2—with the highest radical scavenging potential—reduced the cells’ viability at higher doses with 56.5 and 35% reduction in cell viability for 3.4 and 1.9 mmol L−1 of compounds .The innocuity and the protective effect of the various compounds against Hompounds . Compoun1–7 or ascorbic acid. At the end of the incubation period, cell viability was evaluated using Alamar blue assay. Error bars represent the variability associated with the measurements of triplicate samples.CHO cells were exposed for 24 h to increasing concentrations of compounds 1–7 against H2O2-mediated cell death, and as expected, incubation of CHO cells for 24 h with 300 μmol L−1 of H2O2 decreases cell viability by 75.5 ± 0.7%. Pre-exposure of CHO cells to increasing concentrations of compounds 1–7 or ascorbic acid resulted in variable degrees of protection against H2O2 , MIA Pa Ca-2 (pancreatic cancer), MCF-7 (breast cancer), and SF-268  [−1 . In acconeration .2O2-mediated cell death were investigated using an in vitro model, i.e., CHO cells. Indeed, since the Free Radical Theory of Aging, attention has been focused on the role of reactive oxygen species (ROS) in the initiation and progression of the aging process [2+ to produce OH• radicals. These radicals are the most noxious and interactive of the radical species and are known to initiate rounds of peroxidative damage to membrane lipids [2O2 in the cytosol induces an oxidative stress that eventually results in cell death [2O2-mediated cell death in normal/non-cancerous cells such as CHO cells is a good way to assess the efficiency of antioxidant potential of this compound in biological conditions [The evaluation of the radical scavenging potential of a given substance on cellular models might be a good requisite for eventual applications in cosmetics ,40. It i process . ROS are process . Among te lipids . Hence, ll death . Protectnditions .Finally, although some studies have been carried out on this strain to understand its metabolism in order to optimize NγP production, further analyses are required in the case of an industrial application ,47.A. tubingensis G131 have different antioxidant effect using ABTS and cell-based assays. Amongst all of the antioxidant NγPs, fonsecin and ustilaginoidin A exhibit interesting results, particularly against ROS-mediated cell death. The hypothesis behind this increased radical scavenging potential is that the higher the number of hydroxyl substitutes on the NγP skeleton is, the higher this potential will be. Such properties could be applied in the food and cosmetic industries.NγPs are fungal secondary metabolites exhibiting interesting radical scavenging properties. This effect has been documented in vitro for some members of this family. In this study, we demonstrated that seven NγPs isolated from