E-2-chlorovinyl) selenides and bis(E-2-bromovinyl) selenides in quantitative yields by reactions of selenium dichloride and dibromide with alkynes were developed. The reactions proceeded at room temperature as anti-addition giving products exclusively with (E)-stereochemistry. The glutathione peroxidase-like activity of the obtained products was estimated and compounds with high activity were found. The influence of substituents in the products on their glutathione peroxidase-like activity was discussed.Highly efficient stereoselective syntheses of novel bis( The term “click chemistry” was coined by K. Barry Sharpless, in 1998 and was first fully described by Sharpless, Kolb, and Finn in 2001 ,4,5,6.Organylselenenyl halides are widely used in modern organic synthesis ,3,4,5,6.1) was obtained in 80% yield from elemental selenium and acetylene in an aqueous solution of potassium hydroxide using tin dichloride as a reducing agent at 105–115 °C for 15 h  under acetylene pressure (14 atm) in an autoclave  waZ)-allyl alcohols, unsaturated aldehydes, and enyne derivatives, were obtained based on vinyl selenides [Vinyl selenides are versatile intermediates and synthons for organic synthesis. A series of valuable products, including functionalized alkenes, ketones, - and (E)-enyne derivatives in good yields with retention of stereochemical configuration is very important [The synthesis of resveratrol and its derivatives was realized in several stages from vinyl selenides . Resveramportant . Vinyl smportant , hepatopmportant , and antmportant  activityE-2-chlorovinyl) selenide (2) and bis(E-2-bromovinyl) selenide (3) by electrophilic addition of selenium dihalides to acetylene [Previously, we developed efficient synthesis of bis-3,3′-selanediylbis(2-propenamides), based on the reaction of sodium selenide with 3-trimethylsilyl-2-propynamides [Z,Z)-3,3′-selanediylbis(2-propenamides) [Recently we developed the efficient regio- and stereoselective synthesis of the novel class of divinyl selenides, (ynamides . Like ebnamides) . 1, bis(E-2-chlorovinyl) selenide 2, and bis(E-2-bromovinyl) selenide 3 was planned, and their activity compared with that of the obtained bis selenides. In addition, synthesis of bis selenoxides by oxidation of corresponding bis selenides was scheduled. The bis selenoxides were supposed to be intermediates in the process of oxidation of dithiothreitol by tert-butyl hydroperoxide on the assessment of glutathione peroxidase-like activity of the obtained bis selenides. The goal of the present work was to develop efficient stereoselective syntheses of bis(2-chloroethyl) selenides and bis(2-bromoethyl) selenides by electrophilic addition of selenium dihalides to dialkylacetylenes  and the assessment of glutathione peroxidase-like activity of the obtained bis selenides. Evaluation of the glutathione peroxidase-like activity of unsubstituted divinyl selenide E-2-chlorovinyl) selenides (4–7) in quantitative yields was developed by electrophilic addition of selenium dichloride to dialkylacetylenes . The reaction proceeded in methylene chloride or chloroform at room temperature in a stereoselective fashion as anti-addition producing products exclusively with (E)-stereochemistry  selenides 8–11 (quantitative yields), which did not require additional purification. The reaction proceeded in a stereoselective mode as anti-addition affording products only with (E)-configuration  selenides 4–11. Selenide tert-butyl hydroperoxide . The control experiment was conducted under the same reaction conditions but in the absence of the catalyst. The glutathione peroxidase-like activity of the obtained products was estimated using the model reaction of dithiothreitol oxidation by peroxide  in the p1 showed the best activity among the tested selenides  = C(Bu)SeC(Bu) = C(Bu)X, the activity decreased and was lower than the activity of 4-octyne derivatives 6 and 10, XC(Pr) = C(Pr)SeC(Pr) = C(Pr)X X . We assuIt is worth noting that this is the first example of glutathione peroxidase-like activity assessment of divinyl selenide and bis selenides, which do not contain additional heteroatoms. tert-butyl hydroperoxide on the assessment of glutathione peroxidase-like activity of the corresponding selenides. Bis(2-chlorovinyl) and bis(2-bromovinyl) selenoxides were supposed to be intermediates in the catalytic process of oxidation of dithiothreitol by 12–15 (16–19 (tert-butyl hydroperoxide were developed. The application of sodium metaperiodate for the oxidation of the selenides made it possible to obtain cleaner products in comparison with the use of tert-butyl hydroperoxide. The efficient syntheses of novel families of bis(2-chlorovinyl) selenoxides 12–15  and bisselenides and bis(E-2-bromovinyl)selenides in quantitative yields by electrophilic addition reactions of selenium dichloride and selenium dibromide to dialkylacetylenes were developed. The glutathione peroxidase-like activity of the obtained products was estimated and compounds with high activity were found. Thus, stereoselective syntheses of novel bis(1H (400.1 MHz) and 13C (100.6 MHz) NMR spectra . Elemental analysis was performed on a Thermo Scientific Flash 2000 Elemental Analyzer . The organic solvents were dried and distilled according to standard procedures.The ctra see  were recDivinyl selenide (1). Hydrazine hydrate (7 mL) and a cold solution of KOH  in water (40 mL) were added to selenium powder  and the mixture was stirred overnight. The next day, the resulting mixture was heated (70–80 °C) in a 1 L rotating autoclave under the pressure of acetylene (10–12 atm) for 5 h. The lower organic phase was separated from the reaction mixture by a separatory funnel, dried over Na2SO4 and distilled at reduced pressure (85–95 mm Hg) giving divinyl selenide [selenide ,28 (12.1Bis(E-2-chloro-1-methyl-1-propenyl) selenide (4). A solution of selenium dichloride (1 mmol) in methylene chloride (2 mL) was added dropwise to a solution of 2-butyne  in methylene chloride (18 mL). The mixture was stirred for 1 h at room temperature. The solvent was removed by a rotary evaporator and the residue was dried in vacuum giving compound 4 (258 mg) as a light yellow oil in quantitative yield. 1H-NMR (400 MHz): 2.49 , 2.54 . 13C-NMR (100 MHz): 23.7 (CH3), 26.3 (CH3), 121.8 , 130.7 (CCl). IR (KBr): λ = 2950, 2916, 2848, 1621 (C=C), 1435 cm−1.8H12Cl2Se (258.05): C 37.24, H 4.69, Cl 27.48, Se 30.60%. Found: C 37.51, H 4.78, Cl 27.23, Se 30.38%.Anal. calcd. for CBis(E-2-chloro-1-ethyl-1-butenyl) selenide (5) was obtained under the same conditions as compound 4 from selenium dichloride and 3-hexyne as a light yellow oil in quantitative yield. 1H-NMR (400 MHz): 1.16 , 1.23 , 2,48 , 2.84 . 13C-NMR (100 MHz): 12.7 (CH3), 13.3 (CH3), 30.0 (CH2), 33.1 (CH2), 128.6 , 137.4 (CCl). IR (KBr): λ = 2971, 2932, 2873, 1606 (C=C), 1458 cm−1.12H20Cl2Se (314.15): C 45.88, H 6.42, Cl 22.57, Se 25.13%. Found: C 46.13, H 6.56, Cl 22.45, Se 24.80%.Anal. calcd for CBis(E-2-chloro-1-propyl-1-pentenyl) selenide (6) was obtained under the same conditions as compound 4 but during 2 h from selenium dichloride and 4-octyne as a light yellow oil in quantitative yield. 1H-NMR (400 MHz): 1.00 , 1.02 , 1.58–1.73  2.38–2.45 , 2.76–2.81 . 13C-NMR (100 MHz): 13.2 (CH3), 13.7 (CH3), 21.3 (CH2), 21.4 (CH2), 37.8 (CH2), 40.8 (CH2), 128.3 , 135.9 (CCl). IR (KBr): λ = 2961, 2931, 2871, 1606 (C=C), 1461 cm−1.16H28Cl2Se (370.26): C 51.90, H 7.62, Cl 19.15, Se 21.33%. Found: C 51.79, H 7.71, Cl 18.86, Se 21.50%.Anal. calcd. for CBis(E-2-chloro-1-butyl-1-hexenyl) selenide (7) was obtained under the same conditions as compound 4 but for 2 h from selenium dichloride and 5-decyne as a light yellow oil in quantitative yield. 1H-NMR (400 MHz): 0.91 , 0.94 , 1.28–1.40 , 1.44–1.59  2.34–2,40 , 2.70–2.76 . 13C-NMR (100 MHz): 14.2 (CH3), 22.1 (CH2), 22.5 (CH2), 30.1 (CH2), 30.4 (CH2), 35.8 (CH2), 39.0 (CH2), 128.1 , 135.9 (CCl). IR (KBr): λ = 2957, 2928, 2860, 1607 (C=C), 1463 cm−1.20H36Cl2Se (426.36): C 56.34, H 8.51, Cl 16.63, Se 18.52%. Found: C 56.06, H 8.75, Cl 16.42, Se 18.86%.Anal. calcd. for CBis(E-2-bromo-1-methyl-1-propenyl) selenide (8). A solution of selenium dibromide (1 mmol) in methylene chloride (2 mL) was added dropwise to a solution of 2-butyne  in methylene chloride (18 mL). The mixture was stirred for 1 h at room temperature. The solvent was removed by a rotary evaporator and the residue was dried in vacuum giving compound 8 (347 mg) as a light yellow oil in quantitative yield. 1H-NMR (400 MHz): 2.19 , 2.54 . 13C-NMR (100 MHz): 27.1 (CH3), 29.2 (CH3), 121.9 (CBr), 123.7 . IR (KBr): λ = 2949, 2914, 2847, 1621 (C=C), 1433 cm−1.8H12Br2Se (346.95): C 27.69, H 3.49, Br 46.06, Se 22.76%. Found: C 27.91, H 3.30, Br 45.83, Se 23.02%.Anal. calcd. for CBis(E-2-bromo-1-ethyl-1-butenyl) selenide (9) was obtained under the same conditions as compound 8 but for 2 h from selenium dibromide and 3-hexyne as a light yellow oil in quantitative yield. 1H-NMR (400 MHz): 1.05 , 1.10 , 2,39 , 2.83 . 13C-NMR (100 MHz): 11.8 (CH3), 13.2 (CH3), 32.4 (CH2), 34.9 (CH2), 129.3 CBr), 129.7 . IR (KBr): λ = 2970, 2932, 2873, 1607 (C=C), 1456 cm−1.16H28Br2SeO (403.06): C 35.76, H 5.00, Br 39.65, Se 19.59%. Found: C 36.03, H 5.17, Br 39.38, Se 19.34%.Anal. calcd. for CBis(E-2-bromo-1-propyl-1-pentenyl) selenide (10) was obtained under the same conditions as compound 8 but for 2 h from selenium dibromide and 4-octyne as a light yellow oil in quantitative yield. 1H-NMR (400 MHz): 0.83–0.91 , 1.37–1.68  2.46–2.58 . 13C-NMR (100 MHz): 13.6 (CH3), 14.2 (CH3), 21.6 (CH2), 22.4 (CH2), 41.3 (CH2), 43.5 (CH2), 128.8 (CSe JC-Se 108 Hz), 130.2 (CCl). IR (KBr): λ = 2960, 2930, 2871, 1604 (C=C), 1460 cm−1.16H28Br2SeO (459.16): C 41.85, H 6.15, Br 34.80, Se 17.20%. Found: C 42.03, H 6.27, Br 35.05, Se 16.99%.Anal. calcd. for CBis(E-2-bromo-1-butyl-1-hexenyl) selenide (11) was obtained under the same conditions as compound 8 but for 3 h from selenium dibromide and 5-decyne as a light yellow oil in quantitative yield. 1H-NMR (400 MHz): 0.92 , 0.95 , 1.30–1.39 , 1.48–1.56  2.35–2,42 , 2.80–2.87 . 13C-NMR (100 MHz): 14.1 (CH3), 14.1 (CH3), 21.9 (CH2), 22.4 (CH2), 30.0 (CH2), 31.0 (CH2), 38.9 (CH2), 41.3 (CH2), 128.5 (CBr), 129.7 . IR (KBr): λ = 2956, 2926, 2859, 1606 (C=C), 1462 cm−1.20H36Br2Se (515.27): C 46.62, H 7.04, Br 31.01, Se 15.32%. Found: C 46.34, H 7.00, Br 30.89, Se 15.45%.Anal. calcd. for CBis(E-2-chloro-1-methyl-1-propenyl) selenoxide (12). Sodium metaperiodate  was added to a solution of selenide 4  in absolute methanol (15 mL). The mixture was stirred overnight (16 h) at room temperature. The mixture was filtered. The solvent was removed by a rotary evaporator from the filtrate and the residue was dried in vacuum giving compound 12 (266 mg) as a light yellow oil in 97% yield. 1H-NMR (400 MHz): 1.99 , 2.00 , 2.31 , 2.32 . 13C-NMR (100 MHz): 12.7 (CH3) 24.4 (CH3), 135.1 , 135.6 (CCl). IR (KBr): λ = 2958, 2919, 2852, 1640 (C=C), 1441, 918, 840 (Se=O) cm−1.8H12Cl2SeO (274.05): C 35.06, H 4.41, Cl 25.87, Se 28.81%. Found: C 35.34, H 4.57, Cl 25.58, Se 28.61%.Anal. calcd. for CBis(E-2-chloro-1-ethyl-1-butenyl) selenoxide (13) was obtained under the same conditions as compound 12 by oxidation of selenide 5 as a light yellow oil in 95% yield. 1H-NMR (400 MHz): 1.09 , 1.19 , 251–2.75 . 13C-NMR (100 MHz): 12.4 (CH3), 13.2 (CH3), 21.1 (CH2), 30.1 (CH2), 140.1 , 142.2 (CCl). IR (KBr): λ = 2975, 2937, 2877, 1623 (C=C), 1458, 913, 842 (Se=O) cm−1.12H20Cl2SeO (330.15): C 43.66, H 6.11, Cl 21.48, Se 23.92%. Found: C 43.44, H 5.98, Cl 21.22, Se 24.18%.Anal. calcd. for CBis(E-2-chloro-1-propyl-1-pentenyl) selenoxide (14) was obtained under the same conditions as compound 12 by oxidation of selenide 6 as a light yellow oil in 96% yield.1H-NMR (400 MHz): 0.81–0.87 , 1.33–1.65  234–2.56 . 13C-NMR (100 MHz): 13.0 (CH3), 14.0 (CH3), 20.9 (CH2), 22.0 (CH2), 29.7 (CH2), 39.0 (CH2), 139.8 , 140.8 (CCl). IR (KBr): λ = 2963, 2932, 2873, 1621 (C=C), 1461, 911, 841 (Se=O) cm−1.16H28Cl2SeO (386.26): C 49.75, H 7.31, Cl 18.36, Se 20.44%. Found: C 50.03, H 7.45, Cl 18.50, Se 20.26%.Anal. calcd. for CBis(E-2-chloro-1-butyl-1-hexenyl) selenoxide (15) was obtained under the same conditions as compound 12 by oxidation of selenide 7 as a light yellow oil in 99% yield. 1H-NMR (400 MHz): 0.76–0.82 , 1.19−1.58  2.34–2,52 . 13C-NMR (100 MHz): 13.5 (CH3), 13.6 (CH3), 21.8 (CH2), 22.7 (CH2), 27.5 (CH2), 29.7 (CH2), 30.6 (CH2), 37.0 (CH2), 139.8 , 140.6 (CCl). IR (KBr): λ = 2959, 2931, 2872, 1620 (C=C), 1464, 909, 837 (Se=O) cm−1.20H36Cl2SeO (442.36): C 54.30, H 8.20, Cl 16.03, Se 17.85%. Found: C 54.07, H 8.31, Cl 15.92, Se 18.09%.Anal. calcd. for CBis(E-2-bromo-1-methyl-1-propenyl) selenoxide (16) was obtained under the same conditions as compound 12 by oxidation of selenide 8 as a light yellow oil in 95% yield. 1H-NMR (400 MHz): 2.02 , 2.03 , 2.51 , 2.51 . 13C-NMR (100 MHz): 15.77 (CH3), 27.0 (CH3), 126.4 (CBr), 137.4 . IR (KBr): λ = 2957, 2918, 2852, 1639 (C=C), 1440, 916, 840 (Se=O) cm−1.8H12Br2SeO (362.95): C 26.47, H 3.33, Br 44.03, Se 21.76%. Found: C 26.71, H 3.48, Br 43.88, Se 21.96%.Anal. calcd. for CBis(E-2-bromo-1-ethyl-1-butenyl) selenoxide (17) was obtained under the same conditions as compound 12 by oxidation of selenide 9 as a light yellow oil in 96% yield. 1H-NMR (400 MHz): 1.09 , 1.19 , 2.53–2.72 . 13C-NMR (100 MHz): 12.9 (CH3), 13.2 (CH3), 23.5 (CH2), 37.8 (CH2), 134.7 (CBr), 141.7 . IR (KBr): λ = 2974, 2935, 2876, 1621 (C=C), 1457, 915, 843 (Se=O) cm−1.12H20Br2SeO (419.05): C 34.39, H 4.81, Br 38.14, Se 18.84%. Found: C 34.15, H 4.84, Br 37.88, Se 19.12%.Anal. calcd. for CBis(E-2-bromo-1-propyl-1-pentenyl) selenoxide (18) was obtained under the same conditions as compound 12 by oxidation of selenide 10 as a light yellow oil in 97% yield.1H-NMR (400 MHz): 0.94–1.00 , 1.45–1.79  2.46–2.55 . 13C-NMR (100 MHz): 12.9 (CH3), 14.0 (CH3), 21.8 (CH2), 21.9 (CH2), 32.1 (CH2), 40.9 (CH2), 133.3 (CBr), 141.8 . IR (KBr): λ = 2962, 2931, 2872, 1620 (C=C), 1460, 912, 840 (Se=O) cm−1.16H28Br2SeO (475.16): C 40.44, H 5.94, Br 33.63, Se 16.62%. Found: C 40.21, H 6.13, Br 33.47, Se 16.88%.Anal. calcd. for CBis(E-2-chloro-1-butyl-1-hexenyl) selenoxide (19) was obtained under the same conditions as compound 12 by oxidation of selenide 11 as a light yellow oil in 98% yield. 1H-NMR (400 MHz): 0.81–0.90 , 1.22–1.55  2.34–2.71 . 13C-NMR (100 MHz): 13.6 (CH3), 13.8 (CH3), 21.9 (CH2), 22.8 (CH2), 30.3 (CH2), 30.6 (CH2), 30.7 (CH2), 39.3 (CH2), 134.1 (CBr), 141.0 . IR (KBr): λ = 2959, 2930, 2872, 1618 (C=C), 1463, 909, 837 (Se=O) cm−1.20H36Br2SeO (531.27): C 45.22, H 6.83, Br 30.08, Se 14.86%. Found: C 44.87, H 6.93, Br 29.84, Se 15.12%.Anal. calcd. for CE-2-chlorovinyl) and bis(E-2-bromovinyl) selenides in quantitative yields by electrophilic addition reactions of selenium dichloride and selenium dibromide to dialkylacetylenes were developed. The reactions proceeded as anti-addition producing products exclusively with (E)-stereochemistry. The reactions can be regarded as selenium dihalides click chemistry due to the quantitative yields and 100% stereoselectivity.The stereoselective syntheses of novel bis selenides, in general, exceeds the activity of bis(2-chlorovinyl) selenides. This trend was explained in terms of electron density on the selenium atom in divinyl selenides. The chlorine atom was superior to the bromine atom in electronegativity, and the glutathione peroxidase-like activity of bis(2-bromovinyl) selenides exceeded the activity of chloro-containing selenides. Unsubstituted divinyl selenide does not have electronegative heteroatoms, and it showed the best activity among the tested selenides.7 and 11, the activity decreased and was lower than the activity of 4-octyne derivatives 6 and 10. The steric factor was assumed to manifest itself in the latter case, and the selenium atom in the 5-decyne derivatives became sterically less accessible for redox processes.Another observed trend was the increase in the glutathione peroxidase-like activity with the increasing length of the carbon skeleton in the tested molecules from XCH = CHSeCH = CHX to XC(Pr) = C(Pr)SeC(Pr) = C(Pr)X . However, in the case of 5-decyne derivatives tert-butyl hydroperoxide. The synthesis of novel families of bis(2-chlorovinyl) selenoxides and bis(2-bromovinyl) selenoxides in 95–99% yields by oxidation of corresponding bis selenides with sodium metaperiodate was developed. The selenoxides were supposed to be intermediates in the catalytic process of oxidation of dithiothreitol by


The scope of the process was systematically assessed.Methoximes are important as a class of intermediates and products, among fine chemicals and specialties. The development of a new, facile and efficient method for their synthesis is reported. The methoximes were properly accessed from the corresponding aromatic aldehydes and ketones in good to excellent yields, under mild conditions, employing the inexpensive and environmentally friendly MnCl The synthesis of methoximes is a highly relevant transformation with both theoretical and practical implications in the chemistry of carbonyl compounds . Methoxi1  were also able to conveniently assist the methoximation of a model ketone, with the chloride salt exhibiting the best performance xime (1) −1. 1H NMR: δ = 8.45 , 7.78 , 7.33 , 6.94  and 6.88 , 3.96 , 3.83 . 13C NMR: δ = 157.5 (C-2′), 144.8 (C-1), 131.1 (C-4′), 126.4 (C-6′), 120.8 (C-1′ and C-5′), 111.0 (C-3′), 61.9 (N-OMe) and 55.6 (OMe).Colourless oil; yield: 81%. IR : 3075, 3003, 2961, 2938, 2899, 2837, 2816, 1607, 1601, 1487, 1464, 1456, 1439, 1341, 1302, 1285, 1252, 1161, 1111, 1055, 1028, 920, 853 and 754 cmE)-2-Bromobenzaldehyde O-methyl oxime (2) [(29]xime (2) ,291H NMR: δ = 8.45 , 7.87 , 7.55 , 7.30 , 7.21 , 4.00 . 13C NMR: δ = 147.9 (C-1), 133.1 (C-3′), 131.5 (C-1′), 131.0 (C-4′), 127.5 (C-6′), 127.5 (C-5′), 123.8 (C-2′), 62.3 (N-OMe).Yellow oil; yield: 98%. E)-2-Chlorobenzaldehyde O-methyloxime (3) [(]xime (3) −1. 1H NMR: δ = 8.48 , 7.88 , 7.38–7.21  and 3.99 . 13C NMR: δ = 145.6 (C-1), 135.1 (C-2′), 133.8 (C-5′), 130.7 (C-1′), 129.8 (C-4′), 127.1 (C-6′), 126.9 (C-3′) and 62.2 (N-OMe).Yellow oil; yield: 97%. IR : 2936, 1601, 1472, 1433, 1342, 1209, 1063, 1045, 924, 851, 754, 704, 619 and 602 cmE)-2-(Trifluoromethyl)benzaldehyde O-methyloxime (4) [(]xime (4) −1. 1H NMR: δ = 8.43 , 8.06 , 7.67 , 7.54 , 7.46  and 4.02 . 13C NMR: δ = 145.2 (C-1), 131.9 (C-5′), 130.4 (C-1′), 129.4 (C-4′), 128.4 (C-2′), 128.2 , 127.2 (C-6′), 125.8  and 62.3 (N-OMe). EI-MS : 203 , 152 (77), 145 (100), 125 (26) and 75 (51). HRMS (ESI-TOF): m/z [M + H]+ calcd. for C9H9F3NO: 204.0611; found: 204.0608.Colourless oil; yield: 68%. IR : 2968, 2941, 2903, 2822, 1487, 1454, 1360, 1315, 1283, 1171, 1125, 1067, 1049, 1034, 962, 932, 854, 768, 750, 662 and 629 cmE)-3,4-Dihydroxybenzaldehyde O-methyloxime (5) [(]xime (5) 1H NMR: δ = 7.95 , 7.19 , 6.96 , 6.86 , 5.60  and 3.94 . 13C NMR: δ = 148.5 (C-1), 145.7 (C-4′*), 143.6 (C-3′*), 125.2 (C-1′), 121.7 (H-6′), 115.4 (H-5′), 112.9 (C-2′) and 61.8 (N-OMe).Yellow oil; yield: 94%. E)-3,4-Dimethoxybenzaldehyde O-methyloxime (6) [(]xime (6) 1H NMR: δ = 8.00 , 7.25 , 7.01 , 6.84 , 3.96 , 3.93  and 3.90 . 13C NMR: δ = 150.7 (C-3′*), 149.3 (C-4′*), 148.4 (C-1), 125.1 (C-1′), 121.6 (C-6′), 110.7 (C-5′), 108.0 (C-2′), 61.9 (N-OMe) and 55.9 (3′-OMe and 4′-OMe).White solid; yield: 94%; mp 56–57°C. E)-2,4,6-Trimethoxybenzaldehyde O-methyloxime (7), 6.13 , 3.97 , 3.84  and 3.83 . 13C NMR: δ = 162.4 (C-2′ and C-6′), 160.1 (C-4′*), 144.4 (C-1), 102.4 (C-1), 90.7 (C-3′ and C-5′), 61.5 (N-OMe), 56.0 (2′-OMe and 6′-OMe) and 55.3 (4′-OMe′). HRMS (ESI-TOF): m/z [M + H]+ calcd. for C11H16NO4: 226.1074; found: 226.1075.White solid; yield: 97%; mp 82–84°C. IR : 3566, 3545, 3481, 3420, 3416, 2999, 2941, 2901, 2843, 2820, 2365, 2344, 1614, 1599, 1570, 1474, 1458, 1339, 1227, 1202, 1155, 1126, 1067, 1053, 1030 and 810 cmE)-2-Bromo-5-methoxybenzaldehyde O-methyloxime (8), 7.42 , 7.39 , 6.81 , 4.00  and 3.83 . 13C NMR: δ = 158.9 (C-5′), 148.0 (C-1), 133.7 (C-3′), 132.1 (C-1′), 118.5 (C-4′), 114.6 (C-2′), 111.2 (C-6′), 62.3 (N-OMe) and 55.6 (OMe). HRMS (ESI-TOF): m/z [M + H]+ calcd. for C9H11BrNO2: 243.9968; found: 243.9970.Yellow oil; yield: 87%. IR : 3003, 2963, 2936, 2899, 2833, 2818, 1591, 1564, 1464, 1418, 1404, 1348, 1292, 1273, 1233, 1171, 1061, 1047, 1016 and 910 cmE)-2,5-Dimethoxybenzaldehyde O-methyloxime (9) [(]xime (9) −1. 1H NMR: δ = 8.42 , 7.33 , 6.90 , 6.82 , 3.97 , 3.80  and 3.79 . 13C NMR: δ = 153.7 (C-2′*), 152.1 (C-5′*), 144.6 (C-1), 121.3 (C-1′), 117.6 (C-4′), 112.8 (H-3′), 110.1 (C-6′), 61.9 (N-OMe), 56.3 (2-OMe*) and 55.8 (5-OMe*).Yellow oil; yield: 83%. IR : 3001, 2940, 2901, 2835, 1506, 1495, 1456, 1339, 1221, 1053 and 908 cmE)-1-(4-Methoxyphenyl)ethan-1-one O-methyloxime (10) [(]ime (10) −1. 1H NMR: δ = 7.60 , 6.89 , 3.98 , 3.82  and 2.20 . 13C NMR: δ = 160.4 (C-4), 154.2 (C-1), 129.2 (C-1′), 127.4 (C-2′and C-6′), 113.8 (C-3′and C-5′), 61.8 (N-OMe), 55.3 (OMe) and 12.5 (C-2).White solid; yield: 84%; mp 54–55°C. IR : 3414, 1616, 1522, 1258, 1049, 891 and 827 cmE)-1-(p-Tolyl)ethan-1-one O-methyloxime (11) [(]ime (11) 1H NMR: δ = 7.54 , 7.17 , 3.98 , 2.36  and 2.21 . 13C NMR: δ = 154.6 (C-1), 139.0 (C-4′), 133.8 (C-1′), 129.1 (C-3′ and C-5′), 125.9 (C-2′ and C-6′), 61.8 (N-OMe), 21.2 (4′-Me) and 12.6 (C-2′).Colourless oil; yield: 77%. E)-1-(4-Bromophenyl)ethan-1-one O-methyloxime (12) [(33]ime (12) ,33E/Z = 87:13); yield: 93%. IR : 2936, 1609, 1587, 1485, 1395, 1317, 1084, 1049, 1009, 895 and 824 cm−1. 1H NMR: δ = 7.55–7.46 , 3.99  and 2.19 . 13C NMR: δ = 153.5 (C-1), 135.5 (C-1′), 131.5 (C-3′ and C-5′), 127.6 (C-2′ and C-6′), 123.3 (C-4′), 62.0 (N-OMe) and 12.4 (C-2).Colourless oil (isomeric mixture E)-1-(4-Fluorophenyl)ethan-1-one O-methyloxime (13) [(34]ime (13) ,34E/Z = 94:6); yield: 61%. IR : 2959, 2926, 2853, 2818, 1730, 1603, 1512, 1369, 1315, 1233, 1159, 1084, 1051, 1015, 895, 835, 575, 444, 436, 430, 411 and 403 cm−1. 1H NMR: δ = 7.67–7.59 , 7.09–7.00 , 3.99  and 2.21 . 13C NMR: δ = 163.3 , 153.6 (C-1), 132.8 (C-1′), 127.9 (C-2′*), 127.8 (C-6′*), 115.5 (C-3′*), 115.2 (C-4′*), 61.9 (N-OMe) and 12.6 (C-2).Yellowish oil (mixture E)- and (Z)-1-ethan-1-one O-methyloxime (14) [(]ime (14) E/Z = 85:15); yield: 86%; mp 39–41°C. IR : 3005, 2938, 2899, 2820, 2374, 2345, 1751, 1734, 1653, 1541, 1472, 1375, 1315, 1140, 1051 and 1028 cm−1.White solid , 7.49 , 7.42 , 4.00  and 2.18 . 13C NMR: δ = 152.3 (C-1), 136.5 (C-1′), 133.0 (C-3′*), 132.7 (C-4′*), 130.3 (C-5′), 127.8 (C-2′), 125.1 (C-6′), 62.2 (N-OMe) and 12.2 (C-2).Z-isomer: 1H NMR: δ = 7.61 , 7.43 , 7.33 , 3.86  and 2.17 . 13C NMR: δ = 151.1 (C-1), 136.5 (C-1′), 133.9 (C-3′*), 132.4 (C-4′*), 130.1 (C-2′*), 130.1 (C-5′*), 127.4 (C-6′), 61.9 (N-OMe) and 21.3 (C-2).E)-1-ethan-1-one O-methyloxime (15) [(]ime (15) −1. 1H NMR: δ = 7.30 , 7.14 , 6.85 , 3.99 , 3.93 , 3.90  and 2.21 . 13C NMR: δ = 154.2 (N-OMe), 150.0 (C-4′), 148.9 (C-3′), 129.4 (C-1′), 119.1 (C-6′), 110.6 (C-5′), 108.6 (C-2′), 61.8 (N-OMe), 55.9 (3′-OMe and 4′-OMe) and 12.5 (C-2).White solid; yield: 96%; mp 58–59°C. IR : 2961, 1576, 1516, 1464, 1416, 1339, 1277, 1252, 1231, 1175, 1150, 1045, 1020, 918, 866 and 812 cmE)-1-(2-Nitrophenyl)ethan-1-one O-methyloxime (16) [(]ime (16) E/Z = 77:23); yield: 89%. IR : 2938, 2820, 1611, 1537, 1346, 1047, 893 and 787 cm−1. 1H NMR: δ = 8.00 , 7.64 , 7.53 , 7.46 , 3.96  and 2.17 . 13C NMR: δ = 154.2 (C-1), 148.0 (C-2′), 133.6 (C-1′), 133.3 (C-6′), 130.6 (C-4′), 129.5 (C-5′), 124.6 (C-3′), 62.1 (N-OMe) and 16.0 (C-2).Yellow oil (isomeric mixture E)-1-(2-Hydroxy-4-methoxyphenyl)ethan-1-one O-methyloxime (17) [(]ime (17) 1H NMR: δ = 11.49 , 7.29 , 6.49 , 6.45 , 3.97 , 3.80  and 2.26 . 13C NMR: δ = 161.7 (C-4′*), 159.6 (C-2′*), 158.1 (C-1), 128.6 (C-6′), 111.7 (C-1′), 106.2 (C-5′), 101.6 (C-3′), 62.2 (N-OMe), 55.3 (OMe), 11.4 (C-2).White solid; yield: 97%; mp 52–54°C. E)-1-(2-Hydroxyphenyl)ethan-1-one O-methyloxime (18) [(]ime (18) −1. 1H NMR: δ = 11.27 , 7.41 , 7.25 , 6.97 , 6.89 , 4.00 , 2.31 . 13C NMR: δ = 158.3 (C-2′), 157.9 (C-1), 130.7 (C-4′), 127.5 (C-6′), 119.0 (C-5′), 118.3 (C-1′), 117.3 (C-3′), 62.4 (N-OMe), 11.4 (C-2).Yellow oil; yield: 92%. IR : 3003, 2965, 2938, 2899, 2820, 2376, 2349, 2318, 2311, 1620, 1607, 1506, 1456, 1300, 1250, 1053 and 920 cmE)-N-Methoxypropanimidoyl]benzene-1,3-diol (19) [4-[(1]iol (19) −1. 1H NMR: δ = 11.73 , 7.27 , 6.46 , 6.41 , 5.67 , 3.96 , 2.77  and 1.17 . 13C NMR: δ = 163.2 (C-1), 160.0 (C-1′), 157.9 (C-3′), 128.8 (C-5′), 110.7 (C-6′), 107.1 (C-4′), 103.9 (C-2′), 62.3 (N-OMe), 18.8 (Me) and 11.4 (C-3). EI-MS : 195 , 180 , 164 (33), 135 (100) and 108 (57).Yellowish oil; yield: 86%. IR : 3379, 2978, 2938, 1703, 1634, 1614, 1520, 1454, 1250, 1047, 970, 891, 851 and 743 cmE)-1-(4-Methoxyphenyl) propan-1-one O-methyloxime (20) [(]ime (20) 1H NMR: δ = 7.58 , 6.89 , 3.96 , 3.82 , 2.72  and 1.12 . 13C NMR: δ = 160.3 (C-1), 159.4 (C-4′), 128.1 (C-1′), 127.6 (C-2′ and C-6′), 113.8 (C-3′ and C-5′), 61.7 (N-OMe), 55.3 (OMe), 20.0 (C-2) and 11.2 (C-3).Colourless oil; yield: 88%. S,E)-4-(prop-1-en-2-yl)cyclohex-1-ene-1-carbaldehyde O-methyl oxime (21) [(]ime (21) E). [α]D16 = –134.4 . IR : 2936, 2899, 1643, 1454, 1435, 1373, 1179, 1059, 1045, 947, 897 and 667 cm−1. 1H NMR: δ = 7.65 , 5.98 , 4.75 , 4.72 , 3.86 , 2.47 , 2.37–2.15 , 2.15–2.01 , 1.93–1.83 , 1.75  and 1.48 . 13C NMR: δ = 151.6 (C-1), 149.2 (C-2″), 134.9 (C-2′), 132.6 (C-1′), 109.0 (C-1″), 61.6 (N-OMe), 40.9 C-4′), 31.3 (C-3′), 26.8 C-6′), 23.9 (C-5′) and 20.7 (C-3″). EI-MS : 179 , 164 , 138 (4), 110 (50), 80 (100) and 68 (44).Colourless oil; yield: 80% -2-Methyl-5-(prop-1-en-2-yl)cyclohexan-1-one O-methyl oxime (22) [(2]yl oxime  [22]E). [α]D16 = − 86.0 . IR : 2963, 2930, 2855, 1643, 1447, 1378, 1261, 1051, 905, 880 and 849 cm−1. 1H NMR: δ = 4.73 , 3.82 , 3.32 , 2.24–2.11 , 2.05 , 1.99–1.90 , 1.90–1.79 , 1.73 , 1.58 , 1.41 , 1.24 , 1.10 . 13C NMR: δ = 161.7 (C-1), 148.7 (C-2′), 109.2 (C-1′), 61.1 (N-OMe), 44.9 (C-5), 37.2 (C-2), 35.4 (C-4), 30.9 (C-3), 29.9 (C-6), 20.8 (C-3′) and 16.4 (Me-2). EI-MS : 181 , 166 , 149 (2), 125 (8), 109 (13), 97 (50) and 71 (100).Colourless oil; yield: 81% , was examined.As shown in 2, it was found that the counterion seems to be relevant, since MnCl2.4H2O turned the transformation most efficiently, resulting in full depletion of the starting ketone in only 30 min (Entry 5 versus Entry 3). Therefore, the latter was employed for further studies.Although it may be argued that the addition of NaOAc to the reaction may afford Mn(OAc)2.4H2O is also a GRAS substance [2.4H2O was also supported on montmorillonite K-10, and used in the catalytic synthesis of benzaldehyde derivatives, by the H2O2-mediated oxidation of the corresponding alcohols [MnClubstance , suitablubstance , esterifubstance ,46, Michubstance , glucoseubstance , condensubstance , carbonyubstance  and the ubstance . The salubstance ,53 or C–ubstance ,55, and ubstance –59. MnClalcohols .Once the base, as well as the promoter and its level were defined, the performances of MeOH (Entry 6), and 2-PrOH (Entry 7) were compared against the use of EtOH (Entry 5) as reaction medium. It was verified that in the latter case the transformation concluded in just 30 min, whereas the transformation was slower in the other media, not reaching completeness even after 75 min. This observation enabled us to conclude that EtOH should be the solvent of choice. Fortunately, this is the less expensive and less toxic one among the tested solvents.2.4H2O catalysis at 27°C (Entry 8), 40°C (Entry 9) and 50°C (Entry 5) revealed that the rate of the transformation was markedly slowed as the system approached room temperature, and confirmed that 50°C was a suitable reaction condition, still being mild when compared with methods requiring solvent reflux .Finally, comparing the methoximations run in EtOH under MnClOnce the reaction conditions for optimum efficiency were established, and it was demonstrated that the transformation is efficient under low catalyst load and without the need for addition of external ligands, we began the exploration of the scope of the method, by submitting to methoxime formation an array of aldehydes and ketones bearing different steric and electronic characteristics . In ordeortho-substituted aldehydes (Entries 1–4) or those carrying additional functionalities at other positions (Entries 7–9) were reasonably similar to those arising from aldehydes lacking ortho-substituents (Entries 5 and 6).In general, it was observed that the transformation was efficient, proceeding in over 75% yield, except for the fluorinated compounds (Entries 4 and 13). The aldehydes (Entries 1–9) were more reactive than the ketones, and the reactions were generally completed in 5 min. Furthermore, the process seemed to be insensitive to steric effects, since the yields of the methoximes of ortho-substituent (Entries 10–15) afforded the corresponding products in 15–30 min, while some of their congeners with an ortho-functionalization (Entries 16–19) reacted at a pace which depended on the nature of the substituent.On the other hand, the results evidenced that the ketones lacking an ortho-nitro group (Entry 16) caused a reduced reaction rate, and the same outcome was observed with the presence of an ortho-OH substituent (Entries 17–19). This effect may be a result of the known hydrogen bonding capability of the OH group with the nearby carbonyl [An carbonyl ,62. Inteortho-OH group.Finally, the effect of steric hindrance on the side chain was examined (Entries 19 and 20). It was detected that the reaction afforded highly satisfactory yields of the expected products (86–88%); however, it took longer (Entry 20 versus Entry 10), especially in the case of Entry 19, where the ketone displays an E/Z (anti/syn) mixtures of methoximes (with one of them clearly prevailing) were easily visible in the NMR spectra of the compounds. Unfortunately, however, these mixtures were not chromatographically separable on a silica gel column.Interestingly, in most of the cases, signs of In addition, we confirmed that the reaction has a wide scope, observing that the conditions are also useful for efficiently accessing methoximes derived from non-aromatic substrates, as exemplified with the derivatives of perillaldehyde (Entry 21) and dihydrocarvone (Entry 22). Further, thanks to the very mild reaction conditions, no signs of the potentially competing Michael addition were observed. All these reaction features suggest that, contrary to previous beliefs , the syn3.7H2O as catalyst and the proposed methodology employing MnCl2.4H2O as promoter  was carried out on a set of eight compounds, including aromatic aldehydes and ketones, as well as a couple of alicyclic derivatives. The analysis revealed that MnCl2.4H2O displays a similar and often even better performance than the cerium(III) salt, and that it can conveniently act as its alternative or substitute under analogous reaction conditions. Furthermore, advantageously, the manganese(II) salt is a GRAS compound, being easily available and less costly than CeCl3.7H2O.With all the data in our hands, a comparison between our previous results with CeCl2 to the oxygen of the carbonyl motif of model compound A triggers the transformation, yielding the activated intermediate (i) and releasing chloride anion.At the present stage, the intimate details of the reaction mechanism are not clear. However, 4 and MnCl2.Plausibly, the extent of the coordination depends on the nature of the counteranion in the Mn(II) Lewis acid catalyst, which would explain the differences in reactivity between MnSOi) can suffer a nucleophilic attack by the nitrogen atom of methoxylamine, which would result in the tetrahedral intermediate (ii), O- and N-coordinated to the manganese centre, able to undergo an intramolecular proton transfer to afford the protonated hemiaminal-type intermediate (iii), analogous to that reported during the titanium(IV)-mediated preparation of oxime ethers [B).In turn, this intermediate (e ethers . The latA) to the last intermediate (iii) are reversible, unlike the step from intermediate (iii) toward (B), which under similar reaction conditions is also the rate-limiting step of the sequence [B) was formed, it is less likely that the latter could be hydrolyzed to the starting material (A). The validity of this conjecture stems from the fact that oxime ethers are relatively stable to hydrolysis [It is very likely that all the steps from -catalysed reaction for aromatic aldehydes and ketones based on the easily available MnCl2.4H2O, the catalyst is also a competent, highly convenient and more sustainable substitute of the previously reported CeCl3.7H2O. These characteristics suggest that the proposed approach is a promising protocol with potentially high synthetic value, that avoids the use of high temperature, costly or undesirable solvents, excess reagents and/or more expensive or even toxic additives. The currently disclosed findings may also be conceived as the starting point for the development of other Mn-based catalysts for this reaction.The reaction evidences a unique and still unreported catalytic application of this scarcely employed manganese salt. This derivatization of carbonyl compounds can be efficiently performed with a minimalistic setup, under mild conditions and in short time, being a suitable alternative to methoximation processes which so far have been carried out in generally harsher reaction scenarios and/or longer times, usually employing less convenient reagents. These features advance the state of the art of the methoximation reaction; additionally, it should be considered that due to the marked abundance of manganese on Earth and the eco-friendliness of MnClClick here for additional data file.


Pleosporales sp. SF-7343 revealed four known secondary fungal metabolites: alternate C (1), altenusin (2), alternariol (3), and altenuene (4). The compound structures were identified primarily by NMR and MS analyses. Atopic dermatitis, an inflammatory disease, is driven by the abnormal activation of T helper (Th) 2 cells and barrier dysfunction. We attempted to identify the anti-inflammatory components of SF-7343. Initial screening showed that compounds 1 and 3 inhibited the secretion of interleukin-8 and -6 in tumor necrosis factor-α/interferon-γ-treated HaCaT cells, and these compounds also showed inhibitory effects on CCL5 and CCL22. Compounds 1 and 3 also downregulated the protein expression levels of intercellular adhesion molecule-1 and upregulated the expression of filaggrin and involcurin. The mechanism study results showed that compounds 1 and 3 inhibited nuclear translocation of nuclear factor-kappa B p65 and the phosphorylation of STAT1 and STAT3. Compound 1, but not compound 3, significantly promoted the expression of heme oxygenase (HO)-1. The effects of compound 1 were partly reversed by co-treatment with a HO-1 inhibitor, tin protoporphyrin IX. Taken together, this study demonstrates the potential value of Antarctic fungal strain SF-7343 isolates as a bioresource for bioactive compounds to prevent skin inflammation.Chemical investigation of the Antarctic fungi  Atopic dermatitis (AD) is a chronic inflammatory skin disease, and the causes of this disease are complex and multifactorial. The prevalence of AD is quite high, at 20% in children and 10% in adults . Some inKeratinocytes are the most abundant cells in the epidermis and represent the first line of the host defense system. These cells form the physical skin barrier by sensing pathogens via innate immune receptors, initiating anti-microbial responses, and producing various cytokines, chemokines, and anti-microbial peptides . In AD-dPleosporales sp. SF-7343, which led to the isolation of four compounds such as alternate C (compound 1), alternariol (compound 2), alternariol (compound 3), and altenuene (compound 4). In a previous study, alternariol are only reported to suppress LPS-induced inflammation in THP-1-derived macrophages + . 1H NMR : 10.22 , 8.73 , 8.70 , 6.55 , 6.42 , 6.40 , 6.13 , 3.72 , 3.41 , 1.87 ; 13C NMR : 168.4 (C-1), 160.8 (C-5), 157.4 (C-3), 144.2 (C-10), 143.0 (C-7), 142.2 (C-11), 130.9 (C-8), 125.2 (C-13), 116.9 (C-12), 116.1 (C-9), 113.0 (C-2), 107.3 (C-6), 99.7 (C-4), 55.2 (OCH3-5), 51.4 (OCH3-1), 18.9 (C-14).Brown amorphous powder; HR-ESI-MS m/z: 291.0878 [M + H]+ . 1H NMR : 8.67 , 8.63 , 6.54, , 6.43 , 6.42 , 6.10 , 3.76 , 1.86 ; 13C NMR : 171.6 (C-1), 162.0 (C-5), 161.5 (C-3), 145.0 (C-7), 144.0 (C-10), 142.2 (C-11), 132.4 (C-8), 125.0 (C-13), 116.7 (C-12), 116.0 (C-9), 108.9 (C-6), 108.8 (C-2), 99.6 (C-4), 55.3 (OCH3-5), 18.9 (C-14).Yellowish powder; HR-ESI-MS m/z: 259.0606 [M + H]+ ; 281.0428 [M + Na]+ . 1H NMR : 7.22 , 6.70 , 6.62 , 6.35 , 2.68 ; 13C NMR : 165.6 (C-5), 164.7 (C-1), 164.1 (C-3), 158.4 (C-11), 152.6 (C-9), 138.3 (C-13), 138.1 (C-7), 117.5 (C-12), 109.0 (C-8), 104.4 (C-6), 101.6 (C-10), 100.9 (C-4), 97.2 (C-2), 25.2 (C-14).White powder; HR-ESI-MS α]25D −25.5 . HR-ESI-MS m/z: 293.1028 [M + H]+ ; 315.0852 [M + Na]+ . 1H NMR : 11.28 , 6.75 , 6.51 , 6.30 , 5.31 , 5.13 , 3.95 , 3.86 , 3.70 , 2.26 , 1.95 , 1.47 .Brown amorphous powder; [HaCaT cells were donated by Hyeonsook Cheong, Chosun University , and the cells were cultured in DMEM with 10% FBS , and tin protoporphyrin . Recombinant human TNF-α and IFN-γ, and ELISA kits for IL-8, IL-6, and RANTES were obtained from BioLegend . Antibodies against ICAM-1, FLG, IVL, actin, PCNA, HRP-conjugated anti-mouse, and anti-rabbit IgG were purchased from Santa Cruz Biotechnology . Antibodies against p-IκBα, IκBα, p65, p-stat1, p-stat3, HO-1, and Nrf2 were purchased from Cell Signaling Technology . Other chemical reagents were purchased from Sigma-Aldrich Chemical Company .1 (20–80 μM), compound 2 (5–40 μM), compound 3 (12.5–20 μM) and compound 4 (10–80 μM). After incubation for 24 h, the cell culture medium was removed from each well and replaced with 500 μL of fresh medium in each well. The cells were incubated with 0.5 mg/mL of 3--2,5-diphenyltetrazolium bromide (MTT) for 1 h, and the formed formazan was dissolved in dimethyl sulfoxide . The absorbance of the dissolved formazan was measured at a wavelength of 540 nm by using an ELISA microplate reader .To determine cell viability, cells were maintained at a density of 2 × 104 cells in 48-well plates and then treated with compound 6 cells/mL) were seeded in a 12-well plate. For ELISA analysis, the cells were pre-treated with compounds for 3 h and then stimulated with TNF-α/IFN-γ (each for 5 ng/mL), with co-treatment for 24 h. After that, the cell culture supernatant was collected and IL-6, IL-8, RANTES, and MDC levels were evaluated using an ELISA kit, according to the manufacturer’s instructions. For experiments of compound 1 with Snpp, firstly, HaCaT cells were treated with Snpp (40 μM) for 2 h, and then the compound 1 was added into the culture media for another 3 h, Finally, we stimulated TNF-α/IFN-γ with HaCaT cell for 24 h. After that, the cell culture supernatant was collected and IL-6, IL-8, and MDC were detected using an ELISA kit .HaCaT cells  for 24 h. For analysis of p-IκBα, IκBα, p65, p-stat1, and p-stat3, the cells were induced with TNF-α/IFN-γ for 15 min.For total protein, the cells were scraped, harvested, and lysed with RIPA buffer . Nuclear and cytoplasmic protein fractions were extracted using a Nuclear Extraction Kit , according to the manufacturer’s instructions. All proteins were stored at −80 °C until use. HaCaT cells were pre-treated with indicated concentrations of compound Proteins were separated by electrophoresis on an SDS-PAGE gel and transferred to nitrocellulose membranes. The membrane was blocked with 5% skim milk (dissolved in TBST) for 45–60 min at room temperature. They were then incubated overnight at 4 °C with primary antibodies (1:1000 dilution). After washing thrice with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 1 h at room temperature. After washing with TBST, specific proteins were detected using ECL solution. The membranes were analyzed using the ImageJ software .1 with Snpp, firstly, HaCaT cells were treated with Snpp (40 μM) for 2 h, and then we added the compound 1 into the culture media for another 3 h, Finally, we stimulated HaCaT cells with TNF-α/IFN-γ for 15 min. After that, the cell was collected, nuclear protein fractions were extracted using a Nuclear Extraction Kit.For experiments of compound NF-κB p65-DNA binding was detected using an NF-κB p65 Transcription Factor Assay Kit . The nuclear fraction was used to quantify the relative nuclear NF-κB p65-DNA binding activity. This was evaluated in accordance with the manufacturer’s instructions.#p < 0.05, ##p < 0.01, ###p < 0.001 vs. control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group. △p < 0.05, △△p < 0.01, △△△p < 0.001 vs. compound 1+TNF-α/IFN-γ-treated groupThe result values for each group were represented as the mean ± standard deviation (SD), and the GraphPad Software  was used to carry out one-way analysis of variance (ANOVA), followed by Duncan’s multiple comparison tests to test the significance. Statistical significance was set at 1) and alternariol (3) had inhibitory effects on IL-6, IL-8, MDC, and RANTES secretion; the expression of ICAM-1 was also decreased. Furthermore, alternate C (1) and alternariol (3) also upregulated the expression of FLG and IVL. Further mechanistic studies showed that alternate C (1) and alternariol (3) inhibited the nuclear translocation of NF-κB p65 and STAT1 and STAT3 activation. Alternate C (1) significantly induced the expression of HO-1, which exerts anti-inflammatory effects by inhibiting the secretion of IL-6, IL-8, MDC, and RANTES. These results demonstrate that the compounds obtained from the fungal strain SF-7343 metabolites could be further developed as preventive agents on the regulation of skin inflammation in human keratinocytes. These data may be used as a basis for the development of preventive materials strategies for atopic dermatitis.In this study, the effects of 4 compounds isolated from fungal strain SF-7343 metabolites on skin inflammation were examined. Alternate C (


Their structures were elucidated on the basis of comprehensive spectroscopic analysis. The absolute configurations of 1 and 4 were assigned on the basis of experimental and calculated electronic circular dichroism (ECD) data, and those of enantiomers 2 and 3 were determined by a modified Mosher’s method and basic hydrolysis. To the best of our knowledge, phomochromenones D–F (1–4) possessing a 3-substituted-chroman-4-one skeleton are rarely found in natural sources. Diaporchromone A (5) showed moderate to weak immunosuppressive activity against T and/or B lymphocyte cells with IC50 of 34 μM and 117 μM.Four new chromones, phomochromenones D–G ( Phomopsis. Chemical investigation of this fungal genus has resulted in the discovery of over 70 potentially bioactive secondary metabolites, such as subintestinal vessel plexus (SIV) accelerator phomopsis-H76 A chromene nucleus displayed significant immunosuppressive activity compared to compounds  with a chromone nucleus and compound 8 with a 10H-chromenopyridine nucleus. The additional 11-OH group in 5 is essential for its stimulated splenic lymphocyte inhibitory compared to compound 4.Our primary application of immunosuppressive activity screening indicated that a crude extract of ectively , whereas1H, 13C and 2D NMR spectra were recorded on a Bruker AV 400 NMR spectrometer using TMS as an internal standard. High-resolution ESI-MS were performed on an LTQ Orbitrap XL instrument  using peak matching. TLC and column chromatography (CC) were carried out over silica gel , or a Sephadex-LH-20 , respectively. UPLC analysis  was recorded using a Waters system equipped in ESI mode on an Acquity UPLC H-Class connected to an SQ Detector 2 mass spectrometer using a BEH RP C18 column . Semi-preparative HPLC was performed using a Waters equipped with a 2998 PDA detector  and a RP C18 column .Specific rotations were obtained on a WYA-2S digital Abbe refractometer . UV spectra were determined using a Shimadzu UV-2401 PC spectrophotometer , while CD spectra were measured on a JASCO J-715 spectra polarimeter . Phomopsis asparagi was isolated with PDA medium from the fresh root of the mangrove plant Rhizophora mangle, collected in October 2015 in Dong Zhai Gang-Mangrove Garden on Hainan Island, China. The fungus (strain no.DHS-8) was identified using a molecular biological protocol by DNA amplification and sequencing of the ITS region (GenBank Accession no.MT126606) 20D − 13 ; UV (MeOH) λmax 219, 245, 295 nm; 1H and 13C NMR data, see m/z 307.1179 [M+H]+ .Phomochromenone D (2): yellow gum (MeOH); [α]20D +17 ; UV (MeOH) λmax 220, 245, 294 nm; 1H NMR data, see m/z 321.0989 [M−H]− .Phomochromenone E ((3): yellow gum (MeOH); [α]20D − 19 ; UV (MeOH) λmax 220, 245, 294 nm; 1H NMR data, see m/z 321.0989 [M−H]− .Phomochromenone F 2/3 by Mosher’s Method. The mixture of compounds 2 and 3 (1.5 mg) was treated with (R)- or (S)-MPA (1.0 mg) with DCC (1.0 mg) and DMAP (0.3 mg) in anhydrous CH2Cl2 (0.5 mL). After being stirred at room temperature for 4 h at 0 °C, the solvent from the reaction mixture was removed in vacuo to furnish a residue, which was then subjected to semi-preparative RP-HPLC eluting with CH3CN-H2O  to obtain R-MTPA-2/S-MTPA-3 (tR 18.8 min) and R-MPA-3/S-MPA-2 (tR 19.2 min).Preparation of MPA Esters of R)-MPA ester of 2: yellow gum (MeOH); 1H NMR δH 7.63–7.41 , 7.25–7.18 , 7.22 , 5.26 , 4.98 , 4.68 , 3.94 , 3.92 , 3.55 , 3.20 , 2.77 , 2.62 , 1.92 , 1.09 ; HR-ESI-MS m/z 619.2164 [M + H]+ . (S)-MPA ester of 2: yellow gum (MeOH); 7.72–7.40 , 7.16 , 7.05–6.82 , 5.30 , 4.64 , 4.28 , 3.96 , 3.85 , 3.55 , 3.27 , 2.88 , 2.61 , 1.75 , 1.32 ; HR-ESI-MS m/z 619.2172 [M+H]+ .(R)-MPA ester of 3: yellow gum (MeOH); 1H NMR δH 7.65–7.42 , 7.16 , 7.05–6.82 , 5.30 , 4.64 , 4.28 , 3.96 , 3.85 , 3.55 , 3.27 , 2.88 , 2.62 , 1.75 , 1.32 ; HR-ESI-MS m/z 619.2184 [M+H]+ . (S)-MPA ester of 3: yellow gum (MeOH); 1H NMR δH 7.63–7.41 , 7.25–7.19 , 7.22 , 5.26 , 4.98 , 4.68 , 3.94 , 3.92 , 3.55 , 3.20 , 2.77 , 2.60 , 1.92 , 1.09 ; HR-ESI-MS m/z 619.2157 [M+H]+ .(2) and F(3). (R)- and (S)-MPA ester of 2 were combined (1.2 mg) and dissolved in 10% NaOH (1.0 mL) and stirred for 1 h at room temperature (rt). The reaction mixture was extracted with EtOAc repeatedly and the organic layer was evaporated in vacuo to give optically pure (+)-2 (0.7 mg). Similarly, (−)-3 (0.8 mg) was prepared from the alkaline hydrolysis of (R)- and (S)-MPA ester of 3  in the same manner.Preparation of phomochromenones E(4): white amorphous powder (MeOH); [α]20D − 15 ; UV (MeOH) λmax 205, 241, 303 nm; 1H and 13C NMR data, see m/z 431.1314 [M+Na]+ .Phomochromenone G (5): yellow gum (CHCl3); [α]20D − 40 ; UV (MeOH) λmax 219, 294 nm; 1H NMR δH 6.89, 6.87, 4.72, 3.98, 3.90, 2.86, 2.76, 1.63; 13C-NMR  δC 173.7 (C-4), 169.8 (C-13), 163.2 (C-7), 158.4 (C-2), 158.0 (C-8a), 134.7 (C-5), 115.6 (C-3), 114.4 (C-4a), 113.0 (C-6), 101.7 (C-8), 95.4 (C-11), 57.5 (C-9), 56.2 (C-15), 53.2 (C-14), 37.1 (C-10), 29.4 (C-12). ESI-MS m/z 321.09 [M+H]+.Diaporchromone A (6): yellow gum (CHCl3); UV (MeOH) λmax 215, 239, 275, 315 nm; 1H NMR δH 6.54, 6.46, 4.71, 4.57, 4.38 , 3.94, 3.84, 2.66, 2.88, 2.71, 2.18; 13C NMR  δC 210.0 (C-3′), 190.2 (C-4), 169.7 (C-9), 165.5 (C-8a), 164.0 (C-7), 136.3 (C-5), 111.7 (C-4a), 109.8 (C-6), 102.0 (C-8), 68.2 (C-2), 64.0 (C-1′), 56.1 (C-11), 53.1 (C-10), 50.6 (C-3), 47.7 (C-2′), 30.9 (C-4′). ESI-MS m/z 323.09 [M+H]+.Diaporchromanone C (7): yellow gum (CHCl3); UV (MeOH) λmax 215, 239, 275, 315 nm; 1H NMR δH 6.56, 6.46, 4.60, 4.51, 4.47 , 3.93, 3.85, 2.96, 2.80, 2.74, 2.18; 13C NMR  δC 208.8 (C-3′), 190.9 (C-4), 169.7 (C-9), 165.6 (C-8a), 164.1 (C-7), 136.3 (C-5), 112.3 (C-4a), 109.8 (C-6), 102.2 (C-8), 68.6 (C-2), 66.4 (C-1′), 56.1 (C-11), 53.1 (C-10), 49.6 (C-3), 46.8 (C-2′), 31.0 (C-4′). ESI-MS m/z 323.09 [M+H]+.Diaporchromanone D (8): yellow gum (MeOH); UV (MeOH) λmax 206, 236, 308 nm; 1H NMR δH 9.34, 7.30, 6.98, 4.06, 4.02, 2.70; 13C-NMR  δC 176.3 (C-10), 171.7(C-12), 165.4 (C-3), 162.9 (C-4a), 153.9 (C-7), 150.1 (C-1), 146.6 (C-5a), 139.1 (C-6), 124.6 (C-9), 116.7 (C-10a), 115.4 (C-9a), 112.7 (C-4), 109.6 (C-8), 57.3 (C-14), 53.4 (C-13), 24.4 (C-11). ESI-MS m/z 316.12 [M+H]+.Phomochromenone C . The lowest energy conformers within 2 kcal/mol were subjected to further DFT calculations. The geometries of the conformers were optimized at the B3LYP/6-31+G level in the gas phase using the Gaussian 09 program . The theoretical calculation of ECD was conducted with IEFPCM solvent model for CH3OH using TDDFT at the B3LYP/6-31+G level for all conformers of compounds .Female BALB/c mice  were purchased from the Department of Laboratory Animal Science . The mice were sacrificed by cervical dislocation, and their spleens were collected in complete RPMI 1640 medium, which was minced with surgical scissors in a germ-free condition. The suspension was then filtered through a sterile sieve mesh to obtain the single cell suspension. After centrifugation (1000 rpm at 4 °C for 5 min), the resulting cells were treated with erythrocytes lysis buffer, followed by washing twice with cold phosphate-buffered saline (PBS). Then, the cells were adjusted to the concentration of 5 × 101–8 were measured using the tetrazolium salt-based CCK-8 assay according to a previously described protocol with some modifications [6 cells/mL onto 96-well plates containing 100 μL of RPMI1640 complete medium (triplicate wells). Then, the cells were treated with 100 μL of various concentrations  of isolated compounds or cyclosporine (CsA) at 37 °C with a 5% CO2 incubator for 44 h. At the end of the culture, the cell culture plate was removed and observed under an inverted microscope followed by 20 μL of CCK-8 reagent (5 mg/mL). The cells were further incubated for 4 h at 37 °C with 5% CO2. The optical density was measured at 450 nm on a microplate reader.Cell viability of compounds ications . Spleen 1–8 were evaluated for immunosuppressive activity against the proliferation of concanavalin A (ConA)-induced T and lipopolysaccharide (LPS)-induced B murine splenic lymphocyte in vitro using a CCK-8 method according to previously reported methods [Compounds  methods . Cyclosp


Compounds 9, 11, and 12 showed inhibitory activity against human pathogenic fungi. Compounds 11, 12 (flavanones) and 13 (isoflavone) were the most active against clinical isolated Staphylococcus aureus MRSA, showing that structural requirements as prenylation at position C-6 or C-8 and OH at positions C-5, 7, and 4′ are key to the antibacterial activity. The combination of 11 or 12 with commercial antibiotics synergistically enhanced the antibacterial activity of vancomycin, ciprofloxacin, and methicillin in a factor of 10 to 100 times against drug-resistant bacteria. Compound 11 combined with ciprofloxacin was able to decrease the levels of ROS generated by ciprofloxacin. According to docking results of S enantiomer of 11 with ATP-binding cassette transporter showed the most favorable binding energy; however, more studies are needed to support this result.Prenylated flavonoids are an important class of naturally occurring flavonoids with important biological activity, but their low abundance in nature limits their application in medicines. Here, we showed the hemisynthesis and the determination of various biological activities of seven prenylated flavonoids, named  IR (KBr): νmax cm−1: 3431, 2960, 2927, 2858, 1731, 1647, 1615, 1579, 1507, 1384, 1363, 1275. 1H-NMR (DMSO-d6) δ ppm: 12.75 ; 10.6–11.0 ; 8.03 ; 7.59–7.57 ; 6.95 ; 6.30 ; 5.18 2); 3.44 2); 1.74 ; 1.61 . 13C-NMR (DMSO-d6) δ ppm: 17.8 (-CH = CCH3CH3); 21.3 (-CH2CH = CCH3CH3); 25.4 (CH2CH = CCH3CH3); 98.5 (ArC-6); 103.9 (ArC-4a); 105.0 (ArC-3); 106.3 (ArC-8); 122.4 (CH = C(CH3)2); 126.3 ; 129.2 ; 131.1 (CH = C(CH3)2); 132.0 (ArC-4′); 154.6 (ArC-8a); 159.1 (ArC-5); 161.9 (ArC-7); 163.1 (ArC-2); 182.2 (4-C = O).2-phenyl-8-(3-methyl-2-buten-1-yl)-5,7-dihydroxy-48 was obtained as a yellow powder  mp: 221–222 °C : νmax cm−1: 3400, 2969, 2918, 2651, 1639, 1580, 1553, 1483, 1449; 1H-NMR (DMSO-d6) δ ppm: 13.08 ; 8.05 ; 7.60–7.56 ; 6.95 ; 6.57 ; 5.17 2); 3.22 2); 1.72 ; 1.61 . 13C-NMR (DMSO-d6) δ ppm: 17.7 (-CH = CCH3CH3); 21.0 (-CH2CH = CCH3CH3); 25.5 (CH2CH = CCH3CH3); 93.4 (ArC-8); 103.7 (ArC-4a); 105.0 (ArC-3); 111.2 (ArC-6); 122.1 (CH = C(CH3)2); 126.4 ; 129.1 ; 130.7 (CH = C(CH3)2); 130.8 (ArC-1′); 131.9 (ArC-4′); 155.2 (ArC-8a); 158.3 (ArC-5); 162.2 (ArC-7); 162.9 (ArC-2); 181.9 (4-C = O).Compound °C (lit.  213–214 11) and 5,7-dihydroxy-8-(3-methyl-2-buten-1-yl)-2-(4-hydroxyphenyl)chroman-4-one (12) were obtained as racemic mixtures because these were synthesized from (±) naringenin (4) (3.7 mmol) commercially available, 3-methyl-2-buten-1-ol (6) (7.3 mmol), and ZnCl2 (7.3 mmol) as described above. The crude mixture was purified using ethyl acetate-hexane in a gradient system (0 → 70% of ethyl acetate) as the mobile phase to afford 11 as a yellow powder ; mp: 206–207 °C : νmax cm−1: 3420, 2960, 2926, 2857, 1728, 1633, 1586, 1519, 1457, 1384, 1296. 1H-NMR (DMSO-d6) δ ppm: 12.40 ; 10.74 ; 9.55  7.29 ; 6.78 ; 5.94 ; 5.39 ; 5.11 2); 3.22 ; 3.10 2); 2.66 ; 1.68 ; 1.60 . 13C-NMR (DMSO-d6) δ ppm: 17.6 (-CH = CCH3CH3); 20.6 (-CH2CH = CCH3CH3); 25.4 (-CH2CH = CCH3CH3); 42.0 (CH2-3); 78.3 (ArC-2); 94.3 (ArC-8); 101.5 (ArC-4a); 107.5 (ArC-6); 115.1 ; 122.6 (CH = C(CH3)2); 128.3 ; 128.98 (ArC-1′); 130.2 (CH = C(CH3)2); 157.7 (ArC-4′); 160.5 ; 164.3 (ArC-5); 196.4 (4-C = O).5,7-dihydroxy-6-(3-methyl-2-buten-1-yl)-2-(4-hydroxyphenyl)chroman-4-one  mp: 193 °C : νmax cm−1: 3169, 2966, 2912, 1635, 1519, 1439, 1383, 1347. 1H-NMR (DMSO-d6) δ ppm: 12.09 ; 10.75 ; 9.56  7.30 ; 6.78 ; 5.95 ; 5.40 ; 5.07 2); 3.19 ; 3.06 2); 2.70 ; 1.57 ; 1.52 . 13C-NMR (DMSO-d6) δ ppm: 17.6 (-CH = CCH3CH3); 21.2 (-CH2CH = CCH3CH3); 25.5 (-CH2CH = CCH3CH3); 41.9 (CH2-3); 78.2 (ArC-2); 95.3 (ArC-6); 101.7 (ArC-4a); 106.9 (ArC-8); 115.1 ; 122.6 (CH = C(CH3)2); 128.0 ; 129.2 (ArC-1′); 130.1 (CH = C(CH3)2); 157.5 (ArC-4′); 159.7 (ArC-8a); 161.1 (ArC-5); 164.3 (ArC-7); 196.6 (4-C = O).Compound  powder 4 mg, 36% H-chromen-4-one  was obtained from quercetin (2) (3.3 mmol), 3-methyl-2-buten-1-ol (6) (13.2 mmol), and ZnCl2 (13.2 mmol) as described above. The crude mixture was purified using ethyl acetate-hexane in a gradient system (0 → 80% of ethyl acetate) as the mobile phase to afford the title compound as a pale green powder ; mp: 205–206 °C : νmax cm−1: 3599, 3390, 2969, 2914, 1655, 1635, 1601, 1566, 1518, 1462, 1363, 1313, 1283, 1247, 1167. 1H-NMR (DMSO-d6) δ ppm: 12.52 ; 10.75 ; 9.31 ; 9.03 ; 8.93 ; 6.83 ; 6.66 ; 6.29 ; 6.18 ; 5.10 2); 3.12 2); 1.56 ; 1.47 . 13C-NMR (DMSO-d6) δ ppm: 17.5 (CH = CCH3CH3); 25.4 (CH2CH = CCH3CH3); 31.1 (CH2CH = CCH3CH3); 93.3 (ArC-8); 98.2 (ArC-6); 103.5 (ArC-4a); 116.5 (ArC-6′); 117.1 (ArC-2′); 120.3 (ArC-5′); 123.5 (CH = C(CH3)2); 131.0 (CH = C(CH3)2); 132.2 (ArC-3′); 136.3 (ArC-2); 142.9 (ArC-1′); 147.1 (ArC-4′); 150.0 (ArC-3); 156.7 (ArC-8a); 160.9 (ArC-5); 163.8 (ArC-7); 176.2 (4-C = O).2-phenyl)-3,5,7-trihydroxy-4°C (lit.  176–178 H-chromen-4-one (10) was obtained from fisetin (3) (1.75 mmol), 3-methyl-2-buten-1-ol (6) (7 mmol), and ZnCl2 (7 mmol) as described above. The crude mixture was purified using ethyl acetate-hexane in a gradient system (0 → 80% of ethyl acetate) as the mobile phase to afford the title compound as a dark green powder ; mp: 229–230 °C; HRMS m/z, observed: 355.1176; C18H19O6 [M + H]+ requires: 355.1182. IR (KBr): νmax cm−1: 3350, 2971, 2926, 2855, 1698, 1612, 1596, 1508, 1458, 1417, 1272. 1H-NMR (DMSO- d6) δ ppm: 10.68 ; 9.25 ; 9.01 ; 8.62 ; 7.93 ; 6.89 ; 6.83 ; 6.78 ; 6.66 ; 5.11 2); 3.12 2); 1.54 ; 1.44 . 13C-NMR (DMSO- d6) δ ppm: 17.5 (CH = CCH3CH3); 25.4 (CH2CH = CCH3CH3); 31.2 (CH2CH = CCH3CH3); 101.8 (ArC-8);114,6 (ArC-6); 114.8 (ArC-4a); 116.4 (ArC-6′); 117.1 (ArC-2′); 120.9 (ArC-5′); 123.6 (CH = C(CH3)2); 126.5 (ArC-5); 130.8 (CH = C(CH3)2); 132.0 (ArC-3′); 137.8 (ArC-1′); 142.8 (ArC-2); 146.8 (ArC-4′); 148.1 (ArC-3); 156.7 (ArC-8a); 162.1 (ArC-7); 172.1 (4-C = O).2-phenyl)-3,7-dihydroxy-4H-chromen-4-one  was obtained from genistein (5) (1.9 mmol), 3-methyl-2-buten-1-ol (6) (7.4 mmol), and ZnCl2 (7.4 mmol) as described above. The crude mixture was purified using ethyl acetate-hexane in a gradient system (0 → 80% of ethyl acetate) as the mobile phase to afford the title compound as a white powder ; mp: 140 °C : νmax cm−1: 3350, 3174, 2921, 1705, 1651, 1613, 1570, 1513, 1428, 1364, 1301, 1257, 1198. 1H-NMR (DMSO-d6): δ ppm: 12.98 ; 9.58 ; 8.38 ; 7.37 ; 6.80 ; 6.30 ; 5.14 2); 3,34 2); 1.74 ; 1.62 . 13C-NMR (DMSO- d6) δ ppm: 17.7 (-CH = CCH3CH3); 21.0 (-CH2CH = CCH3CH3); 25.4 (CH2CH = CCH3CH3); 98.5 (ArC-6); 104.4 (ArC-4a); 105.8 (ArC-8); 115.0 ; 121.3 (ArC-1′); 121.9 (ArC-3); 122.1 (CH = C(CH3)2); 130.1 ; 131.0 (CH = C(CH3)2); 154.0 (ArC-2); 154.8 (ArC-8a); 157.3 (ArC-4′); 159.5 (ArC-5); 161.7 (ArC-7); 180.5 (4-C = O).3-(4-hydroxyphenyl)-8-(3-methyl-2-buten-1-yl)-5,7-dihydroxy-4°C (lit.  133–135 S. aureus (622-4 and 97-7) and one clinical isolate of E. coli 33.1 were kindly donated by Dr. Marcela Wilkens from Universidad de Santiago de Chile. S. aureus (NCTC8325-4) and E. coli (ATCC25922) were used as the control strains. The antimicrobial activities of the isolated compounds against E. coli (ATCC25922), multi-resistant E. coli (33.1), S. aureus (NCTC8325-4), and methicillin-resistant S. aureus (MRSA) 97-7 strains were assessed using the Clinical and Laboratory Standards Institute (CLSI) microdilution method , and MH with streptomycin [Broth microdilution methods were used to evaluate the effects of the compounds on the growth of ompounds ,62,63. Aptomycin  was usedThe first experiment (first kinetic assay) in which the compound was exposed to the bacterial cultures in MH was carried out over a period of 6 h. Subsequently, to determine the minimal bactericidal concentration (MBC), a second experiment (second kinetic assay) was conducted; this experiment involved taking inoculum from the first kinetic assay and adding it to MH in a new 96-well microplate containing culture medium, which was then cultured for 7 h. The aim of this second culture (second kinetic assay) was to determine whether the compounds have bactericidal or bacteriostatic properties .p < 0.05 was performed to identify significant differences among the treatment and control groups and between the two treatments.The Mann–Whitney U test (*) with 2 10 µM at pH 7.5) and mixed with 10 µM H2DCFDA dye in the reaction mixture and incubated for 15 min in the assay plate. Subsequently, 280 µL of buffer was added, and 10 µL of inhibitor with a final concentration of 10 µM was placed per well in the reaction mixture and incubated for 30 min. The reaction was started by the addition of a suitable concentration of arachidonic acid (0.5 µM). The fluorescence was read in a multimode detector Synergy™ HT Multi-Mode Microplate Reader (Biotek) at 480 nm excitation/520 nm emission after 1 h of incubation at room temperature. The inhibition percentage was obtained for analysis of oxidation of H2DCFDA dye to the highly fluorescent 2′,7′-dichloro-fluorescein (DCF) product.The commercially available enzyme by Cayman Chemicals Inc., Ann Arbor, MI, USA, was diluted (1:500) in the assay buffer  values for each compound was determined graphically.The radical scavenging activity of the prenylated compounds and starting materials towards the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was measured as described , adapted2 humidified atmosphere. Cell viability was measured using CyQuant® Direct Cell Proliferation Assay Kit (Life Technologies) following the manufacturer’s instruction. Briefly, 5.000 cells/well were seeded onto a flat-bottomed 96-well plate in a 200 μL final volume. Six hours after seeding, the culture medium was replaced with the medium containing the tested compounds at concentrations ranging from 0 to 100 μM dissolved in DMSO  for 72 h. The concentrations used to calculate the IC50 values were 100, 30, 10, 3, 1, 0.3, 0.1, 0.01, and 0 μM. Untreated cells (medium containing 0.1% DMSO) were used as controls. At the end of the incubation, 100 μL of culture medium was removed from each experimental well and replaced by 2× detection reagent. Cells were incubated for 1 h, and fluorescence emission was measured at 535 nm with excitation at 480 nm in a microplate reader . At least four independent experiments were performed for each concentration. The results from each experiment were transformed to a percentage of controls, and the IC50 values were graphically obtained from the dose–response curves. The IC50 value was obtained adjusting the dose–response curve to sigmoidal curves (variable slope), generated using GraphPad Prisma 6.0 software [The cell lines used in this work included MDA-MB-231 human breast adenocarcinoma cells, B16-F10 mouse metastatic melanoma cells, and MEF primary mouse embryonic fibroblasts. Cells were maintained in a DMEM high glucose medium  supplemented with 10% (MDA-MB-231 and B16-F10) or 15% (MEF) heat-inactivated fetal bovine serum (HyClone Laboratories), 100 IU/mL penicillin, and 100 μg/mL streptomycin and maintained at 37 °C in a 5% COsoftware .11), 8-prenylated-(±)-naringenin (12) and the 8-prenylated genistein (13) are active molecules against Staphylococcus aureus-resistant bacteria with a strong synergistically effect in combination with commercial antibiotics vancomycin, ciprofloxacin, and methicillin, enhancing their effects in a factor of 10–100 times against drug-resistant bacteria. Compound 12 is the most active against the dermatophytes T. rubrum, T. mentagrophytes, and M. gypseum fungus. Prenylation substitution was shown to be fundamental for these biological activities, specifically at positions 6 and 8 of the (±)-naringenin ; however, others structural requirements are important as well, such as hydroxylation at positions 5, 7 of ring A, and 4′ of ring B. According to the docking results of 11 with ATP-binding cassette transporter, the S isomer showed better interaction energy than the R isomer. It is relevant to continue this study with enantiomerically pure isomers of prenylated flavanones 11 and 12.Our results demonstrated that the 6-prenylated-(±)-naringenin (


Here, we examined JA-regulated root growth, apical hook curvature, flowering time, and defense against the insect Spodoptera exigua in the intermediate jaz mutants jaz1/2/5/6/10/11/12, jaz1/2/3/4/5/6/7/9, and jaz3/4/7/8/9/10/11/12. This study shows that these jaz mutants differentially affect JA responses, suggesting the complexity of JA pathway in these multiple jaz mutants.The phytohormones jasmonates (JAs) regulate diverse aspects of plant growth and defense responses. The JA-ZIM domain (JAZ) family of repressors are targeted by the JA receptor Coronatine Insensitive 1 for ubiquitination and subsequent degradation via the 26S proteasome. We previously investigated the functions of JAZs in JA responses by analyzing  On the 10/11/12 . jaz1/2/f coi1-1 .Figure 2jaz1/2/3/4/5/6/7/9/10/11/12 is more resistant than wild-type in defense against the pest Spodoptera exigua (13The S. exigua larvae fed with jaz1/2/3/4/5/6/7/9 and jaz3/4/7/8/9/10/11/12 gained less weight than those with wild-type, but more weight than those with jaz1/2/3/4/5/6/7/9/10/11/12 (S. exigua larvae reared with jaz1/2/5/6/10/11/12 gained more weight than those with wild-type (jaz1/2/5/6/10/11/12 is more susceptible to S. exigua than wild-type.a exigua .13The S.10/11/12 . On the ild-type , suggestjaz mutants exhibited different effects on JA responses, including root growth, apical hook curvature, flowering time, and defense against insects, and indicate the redundancy of JAZ members and complexity of JA pathway in high-order jaz mutants.In conclusion, these results showed that the three high-order


Paris is an excellent source of steroidal saponins that exhibit various bioactivities. Paris mairei is a unique species and has been widely used as folk medicine in Southwest China for a long time. With the help of chemical methods and modern spectra analysis, five new steroidal saponins, pamaiosides A–E (1–5), along with five known steroidal saponins 6–10, were isolated from the rhizomes of Paris mairei. The cytotoxicity of all the new saponins was evaluated against human pancreatic adenocarcinoma PANC-1 and BxPC3 cell lines.The genus  Paris (Liliaceae) includes 33 species around the world, and 27 species and more than 15 varieties have been discovered in China -a-l-arabinopyranoside.According to the ccharide . The seqectively ,19. The 56 (C-5) ; the α a2, named Pamaiosides B, a white amorphous solid, was positive to Liebermann Burchard and Molisch chemical reactions. The pseudomolecular ion peak was measured in the HR-ESI-MS spectrum at m/z 1043.4677 [M + Na]+ , corresponding to the molecular formula C48H76O23. Compared to 1, one angular methyl at δC 15.07 (C-21) and two methylenes at δC 32.59 (C-23) and 30.04 (C-24) were absent, and the chemical shifts were all markedly up-field at δC 62.95 , δC 71.20 , and δC 74.01 , respectively, which indicates that a hydroxyl group substituted at the primary carbon atom (δH 2.78 (H-20) and δC 62.95 (C-21) and between δHa 3.55, δHb 3.69 (H-21) and δC 46.04 (C-20)/δC 112.72 (C-22) authenticated the hydroxyl substituted at C-21, and it was further confirmed by the correlations for δHa 3.55, δHb 3.69 (H-21) to δH 2.78 (H-20) in the 1H-1H COSY spectrum (δH 1.91 (H-25) to δH 3.76 (H-24) and δH 3.76 (H-24) to δH 3.52 (H-23) in the 1H-1H COSY spectrum and the signal of δH 0.90 (H-27) to δC 74.01 (C-24) derived from the HMBC spectrum illustrated that the hydroxyl displaced at C-23 and C-24. In the NOESY spectra, the configurations of C-1, C-3, C-23, and C-24 were successively evidenced as β, β, α, and β orientations derived from correlations for H-1 (δH 3.40) to H-9 (δH 1.25), H-3 (δH 3.38) to H-9 (δH 1.25), H-20 (δH 2.78) to H-23 (δH 3.52), and H-24 (δH 3.76) to H-27 (δH 0.90), respectively. Using the same method as for 1, C-20, C-22, and C-25 were determined as R configuration. By summarizing the data and comparing it to the literature + , corresponding to the molecular formula C46H66O22. Compared to 2, one angular methyl δC 17.30 (C-18) was missing and two quaternary carbons, δC 179.23 (C-13) and 139.51 (C-14), and one ketone δC 207.09 (C-15) signal were detected /δHa 2.36, δHb 2.60 (H-12)/δH 2.34 (H-17)/δH 4.38 (H-16) and δC 179.23 (C-13), between δH 2.26 (H-8)/δHa 1.48, δHb 2.87 (H-7), and δC 139.51 (C-14) allowed to deduce that one double bond was located at C-13/C-14. Moreover, the location of δC 207.09 (C-15) was affirmed by correlation of δH 2.34 (H-17) to δC 207.09 (C-15) -spirost-5,13-diene-1β,3β,21,23α,24β-pentol + , corresponding to the molecular formula C46H66O22. Compared with 3, only two distinctions, the position of one acetyl group and the sugar linkages, were detected , δHa 4.19 , and δHb 4.33 . It was further acknowledged by the cross-peaks between δH 2.08 /δHa 4.19, δHb 4.33 (H-21), and δC 172.90 (CH3CO-) in the HMBC spectrum (δH 5.18) to Rha C-3 (δC 77.91), Rha H-1 (δH 5.28) to Ara C-4 (δC 75.62), and Ara H-1 (δH 4.30) to C-1 (δC 85.33) in the HMBC spectra clarified the linkages. Thus, compound 4 was characterized as 15-oxo-18-nor--spirost-5,13-diene-21-O-acetyl-1β,3β,21,23α,24β-pentol-1-O-β-d-apiofuranosyl-(1→3)-2-acetyl-α-l-rhamnopyranosyl-(1→4)-a-l-arabinopyranoside.Compound detected . The acespectrum . In addi5, named Pamaiosides E, a white amorphous solid, was positive to Liebermann Burchard and Molisch chemical reactions. The pseudomolecular ion peak was measured in the HR-ESI-MS spectrum at m/z 935.3892 [M + Na]+ , corresponding to the molecular formula C44H64O20. Compared to 3, the proton signals at H-21 were replaced by one angular methyl, δH 1.16 , in an aglycone moiety. Moreover, one keto-methyl at 21.27  and one carbonyl at δC 173.78 signals were observed in the 13C-NMR spectra (δH 2.16 (C3HCO-)/δH 5.31 (H-24) and δC 173.78 (CH3CO) conjectured that one acetyl was substituted at C-24, and the up-field chemical shifts at H-24 (Δppm + 1.98) proved the hypothesis -spirost-5,13-diene-24-acetyl-1β,3β,23α,24β-tetrol.Compound  spectra . By analpothesis . Accordi5 exhibited L-Ara, L-Rha, and D-Xyl residues in a ratio of 1:1:1. The configuration of each monosaccharide was deduced by the same approach employed in compound 1, which was α-L-Ara, α-L-Rha, and β-D-Xyl, respectively. The sequence was derived from the correlations from Xyl H-1 (δH 4.44) to Ara C-4 (δC 85.51), Rha H-1 (δH 5.35) to Ara C-2 (δC 74.40), and Ara H-1 (δH 4.31) to C-1 (δC 85.57). Thus, compound 5 was identified as 15-oxo-18-nor--spirost-5,13-diene-24-acetyl-1β,3β,23α,24β-tetrol-1-O-β-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-a-l-arabinopyranoside.Acid hydrolysis and GC analysis of 6–10, were defined as 25(R)-spirost-5-en-1β,3β,21,23α,24β-pentol-1-O-β-d-apiofuranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→4)]-a-l-arabinopyranoside (6) -a-l-arabinopyranoside (7) -a-l-arabinopyranoside (8) -a-l-arabinopyranoside (9) -a-l-arabinopyranoside (10) 22D − 95.0 ; IR (KBr) νmax (cm−1): 3420, 2930, 1080, 990, and 840; positive ESI-MS m/z 995.13 [M + Na]+, negative ESI-MS m/z 971.28 [M − H]−; positive HR-ESI-MS m/z 995.4824 [M + Na]+ ; 1H-NMR  and 13C-NMR  data, see Pamaiosides A (2): white amorphous solid, [α]22D − 98.2 ; IR (KBr) maxν (cm−1): 3422, 2932, 1078, 988, and 840; positive ESI-MS m/z 1043.49 [M + Na]+; positive HR-ESI-MS m/z 1043.4677 [M + Na]+ ; 1H-NMR  and 13C-NMR  data, see Pamaiosides B (3): White amorphous solid, [α]22D − 105.2  νmax (cm−1): 3420, 2930, 1668, 1078, 989, and 842; positive ESI-MS m/z 993.48 [M + Na]+; negative ESI-MS m/z 969.28 [M − H]−; positive HR-ESI-MS m/z 993.3932 [M + Na]+ ; 1H-NMR  and 13C-NMR  data, see Pamaiosides C (4): White amorphous solid, [α]22D − 110.0 ; IR (KBr) νmax (cm−1): 3432, 2922, 1664, 1080, 990, 837; Positive ESI-MS m/z 993.18 [M + Na]+; Negative ESI-MS m/z 969.36 [M − H]−; Positive HR-ESI-MS m/z 993.3969 [M + Na]+ ; 1H-NMR  and 13C-NMR  data, see Pamaiosides D (5): White amorphous solid, [α]22D − 109.0 ; IR (KBr) νmax (cm−1): 3430, 2925, 1080, 990, and 840; positive ESI-MS m/z 935.39 [M + Na]+; positive HR-ESI-MS m/z 935.3892 [M + Na]+ ; 1H-NMR  and 13C-NMR  data, see Pamaiosides E (1–5 could be found in All the NMR (1D and 2D) and MS spectra of compounds 1–5 (each 2 mg) were mixed with 2 mol/L CF3COOH (2 mL) and heated in a sealed tube at 110 °C for 8 h. Distilled water (20 mL) was added when the reaction was over and extracted with EtOAc (20 mL) three times. The aqueous layer was concentrated in vacuo by repeated mixing with methanol until the solvent was completely evaporated. The residue was dissolved in a 1 mL pyridine solution of 2 mg/L of L-cysteine methyl ester hydrochloride. After warming at 60 °C for 1 h, the solvent was evaporated under N2 protection. The reaction products were dissolved in the mixed solution of 0.2 mL N-(trimethylsilyl)imidazole and 2 mL anhydrous pyridine, and the mixture was warmed at 60 °C for another 1 h. Then, the solvent was evaporated under N2 protection. The residue was suspended in cyclohexane and water, the cyclohexane layer was the trimethylsilyl ether derivatives of monosaccharide. The mixture was filtered through a 0.45 µm membrane to remove the precipitate and analyzed by GC. Separations were carried out on an HP-5 capillary column . Highly pure N2 was used as a carrier gas (1.0 mL/min flow rate), and the FID detector operated at 250 °C (column temperature 250 °C). The carbohydrates were determined by comparing the retention times with standard trimethylsilyl ether derivatives prepared from authentic sugars using the same procedure performed for the sample. Retention times for authentic sugars after being derivatized were 11.23 min (D-Api), 12.20 min (L-Ara), 13.34 min (D-Xyl), and 14.48 min (L-Rha), respectively.The assay was performed according to the procedure of Qiang F., et al.  with sli2. In the exponential phase of the growth, cells were plated onto 96-well plates at a concentration of 8000 cells/well for 24 h. Compounds 1–5 were prepared to various concentrations  and incubated in 96-well plates (each concentration in six-fold wells) for 72 h. Gemcitabine  was offered as the positive control. Cell viability was determined according to reported assay methods using the commercial CCK8 kit  [50).The human pancreatic adenocarcinoma PANC-1 and BxPC3 cell lines were purchased from the Cell Bank of the Chinese Academy of Science  and cultured in DMEM  supplemented with 10% FBS  and 1% Penicillin–Streptomycin  at 37 °C with 5% CO, China) . The optParis mairei, including five new spirostane saponins. None of the new compounds exhibited cytotoxicity against PANC-1 and BxPC3 pancreatic cell lines, implying that the polyglucosides at 1-hydroxy in spirostane saponins may significantly decreased the activities of antitumor.This study afforded 10 compounds from the rhizomes of


Toddalia asiatica (L.) Lam. (Rutaceae) has shown a broad spectrum of biological properties, such as anti-inflammatory, antioxidant, antimicrobial, anti-HIV, and anticancer properties. The present study is concerned with the separation of the main components with broad partition coefficients  from T. asiatica, using linear gradient high-speed counter-current chromatography (LGCCC) combined with an off-line two-dimensional (2D) mode. Similar to the binary gradient HPLC, the LGCCC mode is operated by the adjustment of the proportion between the mobile phase of 5:5:1:9 (v/v) (pump A) and 5:5:4.5:5.5 (v/v) (pump B) in an n-hexane/ethyl acetate/methanol/water solvent system. The off-line 2D-CCC mode was used in this study for the secondary separation of two similar KD value compounds with n-hexane/ethyl acetate/methanol/water . Notably, six coumarins, namely, tomentin (1), toddalolactone (2), 5,7,8-trimethoxycoumarin (3), mexoticin (4), isopimpinellin (5), and toddanone (6), were efficiently separated. The structures of the pure compounds were elucidated by spectral techniques and compared with the literature. Toddalia asiatica (L.) Lam. (Rutaceae) is used in traditional medicine and is reported to treat wounds, pain, swelling, fever, malaria, diarrhea, cancer, coughs, and stomach infections. In addition, this plant exhibits anti-inflammatory, antioxidant, antibacterial, and antifungal properties +. 1H-NMR  δH: 8.83 , 8.00 , 6.67 , 6.15 , 3.92 , 3.89 . 13C-NMR  δC: 160.6 (C-2), 152.3 (C-7), 149.2 (C-9), 143.9 (C-5), 139.5 (C-4), 127.6 (C-6), 110.9 (C-3), 103.4 (C-10), 93.2 (C-8), 56.9 (-OCH3), 56.8 (-OCH3).Tomentin 1 ,  m/z 309.1328 [M + H]+. 1H-NMR  δH: 8.01 , 6.85 , 6.28 , 3.87 , 3.85 , 3.55 , 3.17 , 2.66 , 2.52 , 1.81 , 1.13 . 13C-NMR  δC: 162.0 (C-2), 160.4 (C-7), 156.0 (C-5), 154.3 (C-9), 139.8 (C-4), 119.2 (C-6), 111.8 (C-3), 106.8 (C-10), 95.4 (C-8), 75.8 (C-2′), 72.1 (C-3′), 63.1 (7-OMe), 56.4 (5-OMe), 25.9 (C-5′), 25.6 (C-4′), 25.4 (C-1′).Toddalolactone 3 ,  m/z 237.0727 [M + H]+. 1H-NMR  δH: 8.02 , 6.69 , 6.19 , 3.95 , 3.93 , 3.74 . 13C-NMR  δC: 160.9 (C-2), 156.6 (C-5), 152.6 (C-7), 148.3 (C-4), 139.5 (C-8b), 129.7 (C-8), 111.2 (C-3), 103.4 (C-8a), 93.2 (C-6), 61.2 (-OCH3), 57.0 (-OCH3), 56.9 (-OCH3).5,7,8-Trimethoxycoumarin 4 ,  m/z 309.1285 [M + H]+. 1H-NMR  δH: 8.00 , 6.87 , 6.27 , 3.87 , 3.85 , 3.72 , 2.72 , 2.67 , 1.15 , 1.13 . 13C-NMR  δC: 162.3 (C-2), 160.6 (C-7), 156.4 (C-5), 154.6 (C-9), 140.1 (C-4), 119.4 (C-10), 112.2 (C-3), 107.0 (C-8), 95.8 (C-6), 77.5 (C-2′), 73.3 (C-3′), 63.5 , 56.8 , 25.9 (C-1′), 22.1 (C-5′), 20.6 (C-4′).Mexoticin 5 ,  m/z 247.0600 [M + H]+. 1H-NMR  δH: 8.19 , 8.10 , 7.40 , 6.35 , 4.18 , 4.04 . 13C-NMR  δC: 159.5 (C-2), 149.4 (C-7), 146.3 (C-2′), 144.3 (C-5), 143.0 (C-8a), 139.7 (C-4), 127.1 (C-8), 114.4 (C-6), 112.5 (C-3), 106.8 (C-4a), 105.6 (C-3′), 61.2 (OCH3), 60.8 (OCH3).Isopimpinellin 6 ,  m/z 291.1184 [M + H]+. 1H-NMR  δH: 8.01 , 6.90 , 6.30 , 3.82 , 3.80 , 3.73 , 2.79 , 1.09 . 13C-NMR  δC: 211.4 (C-12), 161.5 (C-7), 160.5 (C-2), 156.2 (C-5), 155.3 (C-9), 139.7 (C-4), 115.0 (C-6), 112.6 (C-3), 106.9 (C-10), 96.0 (C-8), 63.6 , 56.9 , 40.3 (C-13), 35.6 (C-11), 18.6 (C-14), 18.6 (C-15).Toddanone HPLC-grade acetonitrile was obtained from the Tedia Company, Inc. . The solvents used for extraction and separation were of analytical grade . The deionized water was disposed by an osmosis Milli-Q system.T. asiatica were obtained from a local medicine store in Zhaoqing  and identified by Dr. Xiao Wang  with a voucher specimen (2018060911) in the Shandong Analysis and Test Center.The roots of 18 column  was used for the sample detection process .TBE-300C HSCCC equipment was used with three multilayer columns of a total of 300 mL (the diameter of the PTFE tube was 2.6 mm) and a 20 mL sample loop . An LC3050N integrated system was used to contact the HSCCC equipment, composed with a P3050N gradient pump and a UV3050N monitor . A DC-0506 low-constant-temperature bath was used to control the temperature at 30 °C. An Agilent 1120 with a Zobax SB CT. asiatica was prepared in our laboratory as previously described [T. asiatica  were extracted twice under reflux with 5 L of 95% ethanol (2 h each time). The extracts were filtrated, combined, and concentrated under vacuum by rotary evaporation at 50 °C. When the ethanol was removed, the pH was adjusted to 2 with 2% diluted hydrochloric acid. The solution was extracted three times with isometric petroleum ether, the pH of the degreased aqueous solution was slowly increased to 8.5 using aqueous ammonia, and the sample was extracted three times with isometric ethyl acetate. The upper ethyl acetate layer was combined and concentrated by rotary evaporation at 50 °C. Finally, the crude extract (6.3 g) was obtained after vacuum concentration and stored in a refrigerator at 5 °C.The crude sample of escribed . FirstlyKD values of the compounds were determined by HPLC. Firstly, different proportions of the solvent systems were prepared in a 5 mL tube and violently shaken. Two milliliters of each phase of the equilibrated two-phase solvent system was added to the crude extract (∼3 mg). After being shaken vigorously for 1 min and full separation achieved, 1 mL of each layer was removed and dried under nitrogen. The remnant was re-dissolved in 1 mL of methanol and analyzed by HPLC. The KD value was defined as the peak area of a compound in the stationary phase divided by the peak area of a compound in the mobile phase with the following equation:AU and AL are the peak areas of target compounds in the stationary and mobile phases, respectively.The n-hexane/ethyl acetate/methanol/water  and  were placed into the individed separating funnel. After being shaken vigorously, the solutions were kept still for several minutes and were separated into two phases for the experiment. The upper organic phase of n-hexane/ethyl acetate/methanol/water  was used as the stationary phase. The lower aqueous phase of n-hexane/ethyl acetate/methanol/water  was used as mobile A and  as mobile B. For the off-line 2D isocratic HSCCC elution, the solvent system of n-hexane/ethyl acetate/methanol/water  was placed into the separating funnel. After being shaken vigorously, the solutions were kept still for several minutes and were separated into two phases for the experiment. The upper organic phase was used as the stationary phase, while the lower aqueous phase was used as the mobile phase. All the stationary and mobile phases were ultrasonic degassed prior to use.For the LGCCC elution, the solvent systems of v/v). For the off-line dimensional HSCCC separation, 45 mg of fraction A in the linear gradient eluton was set in 10 mL of isometric solution .For the linear gradient HSCCC separation, 200 mg of the crude sample was dissolved in 10 mL of solution  was set at a flowrate of 2.0 mL/min in head-to-tail mode and monitored with a portable recorder at 320 nm. All the fractions were collected in 10 mL test tubes according to UV absorption. The retention of the stationary phase was defined as the stationary phase relative to the total column capacity after separation.For the LGCCC separation, the stationary phase was first introduced into the HSCCC column at a flowrate of 20.0 mL/min. The HSCCC equipment was rotated clockwise at 800 rpm at 30 °C, while mobile phase A was pumped into the CCC column at a flowrate of 5.0 mL/min in head-to-tail mode. After the establishment of a hydrodynamic equilibrium, the sample solution was injected through the injection valve. The prescribed mobile phase gradient combinations of A and B  were app18 column . The HPLC mobile phase comprised of water (A) and acetonitrile (B) with a linear gradient elution of 0–30 min, 22–55% B; 30–31 min, 55–95% B; and 34–35 min, 95–22% B. The flowrate was 1.0 mL/min, the selected wavelength was 320 nm, and the sample injection volume was 10 µL.HPLC analyses of crude extract and CCC fractions were performed on an Agilent 1120 apparatus with a Zobax SB C1H and 13C NMR spectra. The ESI-MS experiment was performed on an Agilent 6520 Q-TOF . NMR spectra were recorded on a Bruker AV-400 spectrometer  with DMSO. Solvent and chemical shift (δ) values are expressed, respectively, in parts per million (ppm) and coupling constant (J) in Hz.The separated compounds were identified by ESI-MS as well as T. asiatica. Similar to a binary gradient in HPLC, the solvent system was varied by adjusting the proportions of the two solvent systems (lower phase of n-hexane/ethyl acetate/methanol/water 5:5:1:9 and 5:5:4.5:5.5). Furthermore, a mixture was separated by off-line 2D-CCC. Six main coumarin moieties, namely, tomentin (1), toddalolactone (2), 5,7,8-trimethoxycoumarin (3), mexoticin (4), isopimpinellin (5), and toddanone (6), were efficiently separated, with purities above 98%. The established LG and 2D-CCC provided an efficient method for separating coumarins from T. asiatica. It may also be applied to separate compounds with broad KD values from other complex natural products.The current study demonstrates an alternate and more efficient separation method using two-dimensional HSCCC linear gradient and isocratic elution modes for the separation of the main components from


A certain influence of the azasteroid substituents concerning absorption and fluorescent properties was observed. Compounds anchored with a bulky pivaloyl group or without a C=O carbonyl group have shown increased fluorescence intensity.We report here the synthesis and optical spectral properties of several new azasteroid derivatives. The formation of these compounds was explained based on the most probable mechanism. The luminescent heterocycles were synthesized by 1,3-dipolar cycloaddition reactions between benzo[f]quinoline and methylpropiolate or dimethyl acetylenedicarboxylate (DMAD). A selective and efficient way for [3+2]-dipolar cycloaddition of benzo[f]quinolinium ylides under ultrasound (US) irradiation (20 kHz processing frequency) is presented. We report substantially higher yields under US irradiation, whereas the solvent amounts required are at least three-fold less compared to classical heating. The azasteroid derivatives are blue emitters with λ Azasteroids are an important class of heterocycles that has received increasing interest in recent years , due to 2 hybridization of all the atoms in the fused ring and is preserved upon substitution with different groups.Azasteroid derivatives are in fact benzo-azaindolizines, a class with an 18 π-electron N-fused heterocycle, containing a bridgehead nitrogen atom shared by an electron-excessive pyrrole and an azine electron-deficient six-membered ring. This structural arrangement being a ‘pure’ blue-emitting moiety. ,15,16,17In recent years, ultrasound-assisted reactions have proved to be a new versatile tool in synthetic organic chemistry ,19, offeOur group has presented in previous work several studies on the synthesis of azine derivatives and their biological activities ,32,33,345a–c and 7a–c, involves two steps. In the first step, an N-alkylation of the benzo[f]quinoline with bromoketones 3a–c take place and benzo[f]quinolinium bromides are obtained as previously reported quinolinium ylides 4a–c. In the case of the benzo[f]quinolinium ylide 4a, we have studied the time conversion of the 6a intermediate (isolated after 24 h of reflux) to the final cycloadduct 7a , with a nominal power of 200 W, and an operating frequency of 20 kHz. The used reactor allowed us to control the pulse sequence, as well as the amplitude  and the irradiation time. All these parameters are expected to influence the reaction. The cycloaddition reactions were performed using 80% of the instrument nominal power and were completed after 2 h of irradiation.3c with DMAD, under US irradiation, we isolated less decarbonylated cycloadduct 8c which makes this reaction more selective to the fully aromatized cycloadduct 7c.The data from −5 mol/L) prepared in cyclohexane and trichloromethane, respectively. The dilution of each solution was adjusted, thus the absorbances maxima were measured in the 340–440 nm range, and reported on a 10 mm cuvette, to fit in a 0.5–0.9 unit range.Optical properties of the synthesized azasteroids were investigated on diluted solutions , while for the sample 5c the maximum of the absorption is situated on 386 nm in trichloromethane and 384 nm in cyclohexane. Sample 5c presents the same absorption bands as 5a and 5b with a small bathochromic shift.In the case of the samples 7a–c and 8c, the absorption bands have similar intensities and profiles. In the case of the samples 7a and 7b, the absorption bands are not so well separated in trichloromethane but well separated in cyclohexane, while for the samples 7c and 8c these bands are separated both in trichloromethane and in cyclohexane. The maximum of the absorption of the sample 7a presents a small bathochromic shift in comparison with the sample 7b–c and 8c. The maxima of the absorption are 381 nm for 7a, 375 nm for 7b, 370 nm for 7c, and 369 nm for 8c in trichloromethane, and 378 nm for 7a, 376 nm for 7b, 373 nm for 7c, and 374 nm for 8c in cyclohexane.In the case of the samples max = 7 nm (5a compared with 7a), of Δmax = 7 nm (5b compared with 7b) and of Δmax = 15 nm (5c compared with 7c), could be observed in trichloromethane (max = 2 nm (5a compared with 7a), of Δmax = 2 nm (5b compared with 7b), and of Δmax = 3 nm (5c compared with 7c), could be observed in cyclohexane  the fluorescence intensity is maximum.When the substituent from the 3rd position of the azasteroid skeleton is the bulky pivaloyl group, an increase in the fluorescence intensity can be observed. A possible explanation can be the fact that the bulky group determines the deviation of the ketone group out of the molecule’s plane which breaks the conjugation between C=O carbonyl and the rest of the molecule, thus, the quenching of the fluorescence by this carbonyl group is reduced. When this carbonyl group from the 3rd position is missing  and in cyclohexane (right column), the absorption and emission spectra of the other compounds  are presented in The optical absorption and emission maxima of the azasteroid derivatives in cyclohexane and trichloromethane are summarized in max of fluorescence around 430–450 nm). Compounds 7c (in cyclohexane) and 8c (both in trichloromethane and cyclohexane) have higher intensity in the emission spectra. Different behavior (in terms of the maximum of the emission) of the samples 5a,b comparing with 5c, and of the samples 7a,b comparing with 7c shown in 5a,b and 7a,b related to 5c and 7c, respectively. In the case of the compounds 5a,b and 7a,b, the substituent from the C=O keto group is methyl and ethyl, respectively, the difference between them is negligible, while in the case of the compounds 5c and 7c the substituent from the C=O keto group is tert-butyl, a bulkier one.1H and 125 MHz for 13C. The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet. Chemical shifts were reported in delta (δ) units, part per million (ppm), and coupling constants (J) in Hz. Infrared (IR) data were recorded in powder on the diamond crystal ATR mode  on an FT-IR Vertex-70  spectrophotometer. Ultrasound-assisted reactions were carried out using a Bandelin Ultrasound reactor , with a nominal power of 200 W and a frequency of 20 kHz. The booster horn SH 213 G was fixed tightly to the ultrasonic converter. The titanium flat probe tip TT13  was fixed tightly to the booster horn. The titanium probe tip was immersed in the used solvent. UV-Vis spectra were recorded on an  1800 PC spectrophotometer in cyclohexane and trichloromethane (spectroscopic grade) solution. The fluorescence measurements were made using an  F900 photoluminescence spectrometer, in the same solvents as for the UV-Vis spectra, with the excitation wavelength set to the absorption band maximum. For all spectral determinations, the solutions were kept in 10 mm path length quartz cells. The fluorescence quantum yield was determined at room temperature with an .All the reagents and solvents were purchased from commercial sources and used without further purification except bromoacetone which was synthesized by the reaction of acetone with bromine in acetic acid as the catalyst. Melting points were recorded on an Electrothermal MEL-TEMP  apparatus in open capillary tubes and are uncorrected. Analytical thin-layer chromatography was performed with commercial silica gel plates 60 F254  and visualized with UV light. The NMR spectra were recorded on a  Avance III 500 MHz spectrometer operating at 500 MHz for 3a–c  and methyl propiolate  or dimethyl acetylenedicarboxylate  was suspended in 30 mL 1,2-butylene oxide. The stirring and refluxing were continued for 48 h. After the reaction was finished (TLC), the obtained solution was cooled down at room temperature and evaporated under reduced pressure to give the crude product. The purification of the crude product was done by column chromatography on silica gel (eluted with 99.5/0.5 CH2Cl2/CH3OH).A mixture of benzo[f]quinolinium salt 3a–c  and methyl propiolate  or dimethyl acetylenedicarboxylate  in 10 mL 1,2-butylene oxide was placed into the reaction vessel and exposed to irradiation .  and 0.635 g, 80% (under ultrasounds)) as a straw-yellow crystals, m.p. 215–216 °C); Rf (99.5/0.5 CH2Cl2/CH3OH) 0.44; IR (cm−1): 3034, 3013 (C-H arom.), 2963, 2927 , 1704 , 1639 , 1610, 1547, 1497, 1442, 1415, 1406, 1353 (aromatic and heteroaromatic ring), 1243, 1178, 1157, 1098, 1064 ; 1H NMR : δ 8.56 , 8.47 , 8.10 , 7.96 , 7.71 , 7.63 , 3.97 , 2.75 ; 13C NMR : δ 186.8 (CO keto group from 3rd position), 164.6 (CO keto ester from 1st position), 140.5 (C-12a), 132.5 (C-4a), 131.0 (C-6a), 129.7 (C-10a), 129.1 (C-6), 128.9 (C-7), 128.6 (C-5), 128.6 (C-3), 127.7 (C-9), 126.8 (C-8), 124.2 (C-11), 123.0 (C-10), 121.2 (C-10b), 120.4 (C-2), 117.5 (C-12), 106.7 (C-1), 51.6 (CH3 of methoxycarbonyl group from 1st position), 28.4 (CH3 of acetyl group from 3rd position). Anal. calc. for C20H15NO3 (317.34): C 75.70, H 4.76, N 4.41; found: C 75.65, H 4.70, N 4.35.Methyl 3-propionylbenzo[f]pyrroloquinoline-1-carboxylate (5b).  and 0.671 g, 81% (under ultrasounds)) as a straw-yellow crystals, m.p. 186–187 °C); Rf (99.5/0.5 CH2Cl2/CH3OH) 0.58; IR (cm−1): 3034, 3001 (C-H arom.), 2978, 2940 , 1705 , 1645 , 1610, 1547, 1496, 1442, 1414, 1359 (aromatic and heteroaromatic ring), 1238, 1180, 1156, 1128, 1088, 1073 ; 1H NMR : δ 8.56 , 8.54 , 8.46 , 8.08 , 8.01 , 7.96 , 7.70 , 7.62 , 3.97 , 3.10 , 1.38 ; 13C NMR : δ 190.9 (CO keto group from 3rd position), 164.6 (CO keto ester from 1st position), 140.3 (C-12a), 132.5 (C-4a), 131.0 (C-6a), 129.7 (C-10a), 129.1 (C-6), 128.8 (C-7), 128.6 (C-3), 127.7 (C-8), 127.6 (C-2), 126.8 (C-9), 123.9 (C-11), 123.0 (C-10), 121.2 (C-10b), 120.3 (C-5), 117.6 (C-12), 106.7 (C-1), 51.5 (CH3 of methoxycarbonyl group from 1st position), 33.7 (CH2 of propionyl group from 3rd position), 9.9 (CH3 of propionyl group from 3rd position). Anal. calc. for C21H17NO3 (331.37): C 76.12, H 5.17, N 4.23; found: C 76.07, H 5.13, N 4.17.Methyl 3-pivaloylbenzo[f]pyrroloquinoline-1-carboxylate (5c).  and 0.701 g, 78% (under ultrasounds)) as a straw-yellow crystals, m.p. 192–193 °C); Rf (99.5/0.5 CH2Cl2/CH3OH) 0.67; IR (cm−1): 3057 (C-H arom.), 2982, 2950, 2930 , 1700 , 1648 , 1611, 1545, 1507, 1493, 1444, 1415, 1367 (aromatic and heteroaromatic ring), 1235, 1152, 1138, 1119, 1084 ; 1H NMR : δ 8.50 , 8.40 , 7.92 , 7.90 , 7.65 , 7.57 , 7.54 , 3.96 , 1.58 ; 13C NMR : δ 199.7 (CO keto group from 3rd position), 164.8 (CO keto ester from 1st position), 138.9 (C-12a), 131.9 (C-4a), 130.6 (C-6a), 129.9 (C-10a), 129.3 (C-5), 128.8 (C-7), 127.7 (C-9), 126.9 (C-3), 126.6 (C-8), 124.1 (C-2), 122.9 (C-10), 122.8 (C-11), 120.8 (C-10b), 119.2 (C-6), 117.6 (C-12), 105.9 (C-1), 51.4 (CH3 of methoxycarbonyl group from 1st position), 44.4 (C of tert-butyl group from 3rd position) 29.1 (3xCH3 of tert-butyl group from 3rd position). Anal. calc. for C23H21NO3 (359.43): C 76.86, H 5.89, N 3.90; found: C 76.80, H 5.86, N 3.85.Dimethyl 3-acetylbenzo[f]pyrroloquinoline-1,2-dicarboxylate (7a).  and 0.666 g, 71% (under ultrasounds)) as a straw-yellow crystals, m.p. 197–198 °C); Rf (99.5/0.5 CH2Cl2/CH3OH) 0.26; IR (cm−1): 3067, 3003 (C-H arom.), 2952 , 1739, 1695 , 1662 , 1613, 1547, 1491, 1468, 1444, 1405 (aromatic and heteroaromatic ring), 1247, 1219, 1197, 1166, 1154, 1096 ; 1H NMR : δ 8.55 , 8.51 , 8.40 , 7.95 , 7.73 , 7.64 , 4.05 , 3.95 , 2.60 ; 13C NMR : δ 189.0 (CO keto group from 3rd position), 167.0 (CO keto ester from 2nd position), 163.6 (CO keto ester from 1st position), 137.8 (C-12a), 131.7 (C-4a), 130.9 (C-6a), 130.5 (C-10a), 129.7 (C-5), 129.7 (C-2), 128.9 (C-7), 128.1 (C-9), 127.1 (C-8), 126.9 (C-3), 123.9 (C-11), 123.0 (C-10), 121.5 (C-10b), 119.3 (C-6), 117.9 (C-12), 104.9 (C-1), 53.4 (CH3 of methoxycarbonyl group from 1st position), 52.0 (CH3 of methoxycarbonyl group from 2nd position), 29.5 (CH3 of acetyl group from 3rd position). Anal. calc. for C22H17NO5 (375.38): C 70.39, H 4.56, N 3.73; found: C 70.33, H 4.52, N 3.69.Dimethyl 3-propionylbenzo[f]pyrroloquinoline-1,2-dicarboxylate (7b).  and 0.672 g, 69% (under ultrasounds)) as a straw-yellow crystals, m.p. 206–207 °C); Rf (99.5/0.5 CH2Cl2/CH3OH) 0.30; IR (cm−1): 3032, 3000 (C-H arom.), 2981, 2951 , 1731, 1697 , 1668 , 1612, 1546, 1494, 1467, 1444, 1406, 1364 (aromatic and heteroaromatic ring), 1266, 1223, 1182, 1163, 1145, 1095, 1089 ; 1H NMR : δ 8.54 , 8.46 , 8.38 , 7.95 , 7.72 , 7.63 , 7.56 , 4.03 , 3.94 , 2.85 , 1.31 ; 13C NMR : δ 194.3 (CO keto group from 3rd position), 166.7 (CO keto ester from 2nd position), 163.7 (CO keto ester from 1st position), 137.2 (C-12a), 131.5 (C-4a), 130.8 (C-6a), 129.8 (C-5), 129.7 (C-10a), 128.9 (C-7), 128.8 (C-2), 128.1 (C-9), 127.1 (C-8), 127.0 (C-3), 123.2 (C-11), 123.0 (C-10), 121.4 (C-10b), 118.7 (C-6), 118.1 (C-12), 104.7 (C-1), 53.3 (CH3 of methoxycarbonyl group from 1st position), 51.9 (CH3 of methoxycarbonyl group from 2nd position), 35.6 (CH2 of propionyl group from 3rd position), 9.1 (CH3 of propionyl group from 3rd position). Anal. calc. for C23H19NO5 (389.41): C 70.94, H 4.92, N 3.60; found: C 70.87, H 4.88, N 3.55.Dimethyl 3-pivaloylbenzo[f]pyrroloquinoline-1,2-dicarboxylate (7c).  and 0.678 g, 65% (under ultrasounds)) as a straw-yellow crystals, m.p. 184–185 °C); Rf (99.5/0.5 CH2Cl2/CH3OH) 0.35; IR (cm−1): 3067, 3023 (C-H arom.), 2981, 2971, 2953 , 1729, 1694 , 1682 , 1612, 1559, 1546, 1505, 1464, 1418 (aromatic and heteroaromatic ring), 1258, 1208, 1177, 1161, 1119, 1091, 1059 ; 1H NMR : δ 8.56 , 8.35 , 7.95 , 7.73 , 7.64 , 3.95 , 3.93 , 1.15 ; 13C NMR : δ 207.9 (CO keto group from 3rd position), 165.6 (CO keto ester from 2nd position), 164.0 (CO keto ester from 1st position), 134.4 (C-12a), 131.8 (C-4a), 130.8 (C-6a), 130.1 (C-5), 129.8 (C-10a), 128.9 (C-7), 128.3 (C-9), 127.0 (C-8), 126.4 (C-2), 123.0 (C-10), 122.9 (C-3), 120.9 (C-11), 120.7 (C-10b), 118.7 (C-12), 116.9 (C-6), 104.6 (C-1), 52.8 (CH3 of methoxycarbonyl group from 1st position), 51.9 (CH3 of methoxycarbonyl group from 2nd position), 47.2 (C of tert-butyl group from 3rd position), 27.4 (3xCH3 of tert-butyl group from 3rd position). Anal. calc. for C25H23NO5 (417.16): C 71.93, H 5.55, N 3.36; found: C 71.88, H 5.50, N 3.32.Dimethyl benzo[f]pyrroloquinoline-1,2-dicarboxylate (8c).  and 0.042 g, 5% (under ultrasounds)) as a straw-yellow crystals, m.p. 198–199 °C); Rf (99.5/0.5 CH2Cl2/CH3OH) 0.28; IR (cm−1): 3145, 3091, 3066 (C-H arom.), 2982, 2963, 2942 , 1710, 1675 , 1610, 1563, 1545, 1507, 1463, 1441, 1406 1369 (aromatic and heteroaromatic ring), 1270, 1255, 1231, 1217, 1197, 1164 1095, 1060 ; 1H NMR : δ 8.41 , 8.21 , 8.11 , 8.06 , 7.88 , 7.66 , 7.56 , 3.97 , 3.96 ; 13C NMR : δ 165.0 (CO keto ester from 2nd position), 164.5 (CO keto ester from 1st position), 134.9 (C-12a), 130.8 (C-6a), 130.5 (C-6), 130.1 (C-4a), 129.8 (C-10a), 129.0 (C-7), 128.0 (C-9), 126.5 (C-8), 122.7 (C-10), 120.7 (C-2), 119.5 (C-11), 119.3 (C-10b), 118.6 (C-12), 116.1 (C-3), 113.9 (C-5), 105.4 (C-1), 52.3 (CH3 of methoxycarbonyl group from 1st position), 51.6 (CH3 of methoxycarbonyl group from 2nd position). Anal. calc. for C20H15NO4 (333.34): C 72.06, H 4.54, N 4.20; found: C 72.00, H 4.49, N 4.16.5a–c were obtained, while in the case of reactions with DMAD fully aromatized compounds 7a–b and a mixture of fully aromatized compound 7c and a fully aromatized and decarbonylated derivative 8c were obtained. A feasible reaction mechanism for the azasteroid derivatives formation is described in this study. Under ultrasound irradiation, we isolated fully aromatized cycloadducts with increased yield, and in the case of the reaction of bromide 3c with DMAD, we isolated less decarbonylated cycloadduct 8c which make this reaction more selective to the fully aromatized cycloadduct 7c.In conclusion, we report herein an efficient and straightforward pathway for obtaining a new class of blue fluorescent azasteroid derivatives, under conventional  heating as well as under unconventional (ultrasound) irradiation. Blue fluorescent azasteroid derivatives have been obtained using a cycloaddition reaction of benzo[f]quinolinium ylides with symmetrically and unsymmetrically activated alkynes. Under conventional heating, in the case of reactions with methyl propiolate only the fully aromatized compounds The absorption and emission maxima of the obtained azasteroid derivatives were studied, some of these compounds being blue emitters. The absorption and emission spectra are dependent on their structure, compounds with a similar structure having spectra with small differences, whereas the compounds with a bulky pivaloyl group or without a C=O keto group in the 3rd position present more intense fluorescence emission.We also note that under ultrasound irradiation, the reactions occur with increased selectivity regarding the decarbonylated compound and offers several advantages in terms of yield, easier workup of the reaction, a substantial decrease in consumed solvents, a substantial reduction in reaction time (from days to hours), thus, consequent diminution in energy consumption. Taking into consideration these advantages, the proposed method should be considered environmentally friendly.