Natural and synthetic coumarins are often considered privileged scaffolds for obtaining pharmacological agents with hypoglycemic activity. Chemical modification of coumarins often leads to antidiabetic agents with greater efficacy. In the present work, twenty monoterpene-substituted 7-hydroxycoumarins were synthesized. A new approach using the Mitsunobu reaction was shown to be effective for the synthesis of target compounds. All of the synthesized compounds were evaluated in an oral glucose tolerance test, and two of them containing geranyl and (-)-myrtenyl substituents showed in vivo hypoglycemic action. A possible mechanism of action of these compounds may include inhibition of DPP IV, which was proved in an in vitro test. H-chromene-2-one) exhibit a variety of biological activities and are often considered privileged scaffolds for obtaining more active pharmacological agents on their basis hept-2-en-2-yl)methoxy)-4-methyl-2H-chromen-2-one (6c). Yield 30%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-methyl-2H-chromen-2-one (7a). Yield 37%.(E)-7--2,3-dihydrocyclopenta[c]chromen-4(1H)-one (7b). Yield 40%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one (7c). Yield 55%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one (7d). Yield 29%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)ethoxy)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one (8a). Yield 53%.(E)-3--7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (8b). Yield 38%.3--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (8c). Yield 38%.3--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (8d). Yield 32%.3--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)ethoxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (9a). Yield 56%.(E)-7--4-phenyl-2H-chromen-2-one (9b). Yield 40%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-phenyl-2H-chromen-2-one (9c). Yield 46%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-phenyl-2H-chromen-2-one (9d). Yield 12%.7--6,6-dimethylbicyclo [3.1.1]hept-2-en-2-yl)ethoxy)-4-phenyl-2H-chromen-2-one (10a).(E)-7--4-(4-fluorophenyl)-2H-chromen-2-one (Yield 35%10b). Yield 53%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-(4-fluorophenyl)-2H-chromen-2-one (10c). Yield 35%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-(4-fluorophenyl)-2H-chromen-2-one (10d). Yield 37%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)ethoxy)-4-(4-fluorophenyl)-2H-chromen-2-one  . The resulting solution was stirred for 2 h while cooling in an ice bath and another 48 h at room temperature. The solvent was evaporated, and the residue was chromatographed on silica gel (eluent-hexane/ethyl acetate 20:1) to result in compounds 6–10.A total of 0.42 mmol of corresponding compound 6a). Yield 64%.(E)-7--4-methyl-2H-chromen-2-one (6b). Yield 70%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-methyl-2H-chromen-2-one (6c). Yield 60%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-methyl-2H-chromen-2-one (6d). White solid.Yield 50%. m.p. 112.9–114.6 °C. [α]25D-13 .7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)ethoxy)-4-methyl-2H-chromen-2-one  (ppm) δ: 0.80 , 1.15 , 1.25 , 2.04–2.10 , 2.14–2.32 , 2.33–2.40 , 2.37 , 2.41–2.50 , 4.00 , 5.34 , 6.10 , 6.76 , 6.81 , 7.44 . 13C-NMR  (ppm) δ: 154.8 (C-1), 161.6 (C-2), 112.3 (C-3), 152.2 (C-4), 113.0 (C-5), 125.0 (C-6), 111.4(C-7), 160.9 (C-8), 100.9 (C-9), 18.2 (C-10), 66.5 (C-11), 35.7 (C-12), 143.5 (C-13), 118.7 (C-14), 30.92(C-15), 40.2 (C-16), 37.6 (C-17), 45.4 (C-18), 31.2 (C-19), 20.8 (C-20), 25.8 (C-21). HRMS for C21H23O3+ calcd 324.1720, found 323.1643 [M]+.7a). Yield 71%.(E)-7--2,3-dihydrocyclopenta[c]chromen-4(1H)-one (7c). Yield 56%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one (8a). Yield 69%.(E)-3--7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (8c). Yield 54%.3--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (9c). Yield 58%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-phenyl-2H-chromen-2-one (9d). Yield 60%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)ethoxy)-4-phenyl-2H-chromen-2-one (10b). Yield 70%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)methoxy)-4-(4-fluorophenyl)-2H-chromen-2-one (10d). Yield 47%.7--6,6-Dimethylbicyclo [3.1.1]hept-2-en-2-yl)ethoxy)-4-(4-fluorophenyl)-2H-chromen-2-one . Oral glucose loading (2.5 g/kg) was performed in all the groups of mice. Prior to dissolution in water, all compounds were mixed with two drops of Tween 80. Vildagliptin tablets  were dissolved in water. The tested compounds were given by oral gavage 30 min prior to the glucose load. Blood glucose was quantified with a ONE TOUCH Select blood glucose meter  before dosing (time 0) and at 30, 60, 90 and 120 min after the glucose load. The area under the glycemic curve was calculated using Tai’s model [The test was performed on mice after a 12 h fast (’s model .6–10. Sitagliptin (as part of a kit) was used as positive control inhibitor. Substances were diluted in dimethylsulfoxide ) and tested at a final concentration of 100 µM (in 10% DMSO). For two compounds , inhibitory activity was also determined at concentrations of 0.1, 1, 10 and 1000 µM to estimate IC50. The assay was based on liberation of AMC (7-amino-4-methyl-coumarin) from DPP-IV substrate, Gly-Pro-AMC. Changes in fluorescence due to cleavage of the molecule by DPP-IV were measured with an excitation and emission wavelength at 350 and 450 nm using a ClarioStar . Percentage inhibition of DPP-IV activity was calculated relative to baseline activity  using the formula: %inhibition = (baseline activity − activity with inhibitor) / baseline activity * 100. IC50 (for compounds 7b and 9a) was calculated using a four-parameter logistic regression model.A fluorometric assay kit (DPP (IV) Inhibitor Screening Assay Kit, Cayman chemical) was used to determine the DPP-IV inhibitory activity of compounds p < 0.05 were considered statistically significant.Statistical analysis was performed by the Mann–Whitney U test. Data are shown as mean ± SEM. Data with Twenty 7-O-monoterpenylcoumarins differing in the structure of the terpenyl substituent and the substituent in positions 3 and 4 of the coumarin backbone were synthesized using the previously described and new approaches to the synthesis. It was shown that synthesis by the Mitsunobu reaction leads to a marked increase in the yields of the target compounds. The hypoglycemic effect of 7-O-terpenylcoumarins in the oral glucose tolerance test (OGTT) in animals was studied for the first time and their inhibitory effect on DPP IV was evaluated. The hypoglycemic effect was shown to depend both on the structure of the terpenyl substituent and on modifications of the piranone ring of the coumarin backbone. Two compounds containing the geranyl and (−)-myrtene substituents showed a significant decrease in the glucose level in the OGTT when administered at doses of 10 and 15 mg/kg, respectively. In vitro test data on DPP IV inhibition correlate with glucose tolerance test data, which may indicate the participation of DPP IV in the mechanism of hypoglycemic action of terpenylcoumarins.


Edelithusgen. nov. is described based on the discovery and description of two new species from Xishuangbanna, Yunnan Province, China: E.puersp. nov. and E.shenmiguosp. nov. Both species are described in detail and illustrated. Types are deposited in theInstitute of Zoology, Chinese Academy of Sciences (IZCAS) in Beijing, China. Phrurolithidae Banks, 1892 includes 20 genera and 313 species from America, Australia, and Eurasia  in Beijing, China.Specimens were examined using a SZ6100 stereomicroscope. Both male and female copulatory organs were dissected and examined in 80% ethanol using an Olympus CX43 compound microscope with a KUY NICE CCD camera. The epigynes were cleared with pancreatin solution. Specimens, including dissected male palps and epigynes, were preserved in 75% ethanol after examination. For SEM photographs, the specimens were kept under natural dry conditions, coated with gold with a small ion-sputtering apparatus ETD-2000, and photographed with a Zeiss EVO LS15 scanning electron microscope. Types are deposited in the Institute of Zoology, Chinese Academy of Sciences (The measurements were taken using a stereomicroscope (Axio Vision SE64 rel. 4.8.3) and are given in millimetres. The body lengths of all specimens exclude the chelicerae and spinnerets. Terminology of the male and female genitalia follows Leg measurements are given as total length . Promarginal and retromarginal teeth on the chelicerae are given as the fproximal, median and distal and counted from the base of the fang to the distal groove. Leg spines are documented by dividing each leg segment into four aspects: dorsal (d), prolateral (p), and retrolateral (r), and indicating the ventral (v) spines as single (1) or paired (2), e.g., femur I d2, pv1111; tibia d1, I v2222. The abbreviations used in the figures are as follows:EyesALE anterior lateral eye;AME anterior median eye;MOA median ocular area;PLE posterior lateral eye;PME posterior median eye.LegsCS chemsensory seta;CTC claw tuft clasper;LO lyriform organ;MPB metatarsal preening brush;MTS metatarsal dorsal stopper;Sc scale;SS slit sensillum;TS tenent setae.CheliceraePES promarginal escort seta;PRS promarginal rake seta;RES retromarginal escort seta;SS slit sensillum;WS whisker seta.Male palpdTA distal tegular apophysis;DTA dorsal tibial apophysis;Em embolus;FA femoral apophysis;rTA retrolateral tegular apophysis;RTA retrolateral tibial apophysis;SD sperm duct;sTA subdistal tegular apophysis;VTA ventral tibial apophysis.EpigyneBu bursa;CD copulatory duct;CO copulatory opening;CT connecting tube;FD fertilization duct;GA glandular appendage;MS median septum;Spe spermathecae.Taxon classificationAnimaliaAraneaePhrurolithidaeLiu & Ligen. nov.7CE30421-DCC1-5A2F-93C3-2F8ACA55333Fhttps://zoobank.org/59555B23-B0D4-4DD9-B5C9-3BCF0E6C98EEEdelithusshenmiguo Liu & Li sp. nov. by designation herein.Labialithus Kamura, 2021   and three prolateral spines  , the male scutum covering nearly 1/2 of abdomen  and by the palpal tibia with a dorsal apophysis . It can be separated from Otacilia (see Otacilia)       Figs , 6D, 11Dus) Figs , 8A, 11Fia) Figs , 6A, 11Aia) Figs , 8C, 11Gia) Figs , 10 and ia) Figs , 10. Malus) Figs , 9E, 10Ium) Figs , 12. Furoth Fig. , while pedentatus”  and the latter half of Phrurolithus. The gender is masculine.The name is a combination of the first three letters of “AME clearly smaller than other eyes, PME with indistinct black pigment around eye cups, smaller than ALE and PLE, nearly separated by their diameter. Chelicera .Taxon classificationAnimaliaAraneaePhrurolithidaeLiu & Lisp. nov.FA7670D1-A2AE-5DB0-A1D4-03511C9F1D19https://zoobank.org/73417E12-1129-408E-9A01-046FC40865FFHolotype ♂ (Phu-147), 21°54.607'N, 101°17.005'E, elevation ca 633 m, XTBG, Menglun Township, Mengla County, Xishuangbanna, Yunnan Province, China, 4–11.IV.2007, G. Zheng leg. Paratypes 1 ♂, 2 ♀, the same data as holotype; 1 ♀, 4–11.IV.2007, other data as holotype (JSIII-2-18); 1 ♀, 10–20.VI.2007, other data as holotype (JSIII-1-20); 1 ♀, 1–15.VIII.2007, other data as holotype (JSIII-3-23); 3 ♂, 16–31.III.2007, other data as holotype (JSIII-5-16); 1 ♀, 10–20.VI.2007, other data as holotype (JSIII-2-20); 1 ♀, 16–31.V.2007, other data as holotype (JSIII-1-20); 2 ♂, 1–15.IV.2007, other data as holotype (JSIII-5-17); 5 ♂, 1 ♀, 1–15.IV.2007, other data as holotype (JSIII-2-17); 2 ♂, 1–15.IV.2007, other data as holotype (JSIII-4-17); 3 ♂, 2 juveniles, 1–15.IV.2007, other data as holotype (JSIII-3-17); 1 ♀, 19–26.V.2007, other data as holotype (JSIII-2-17); 1 ♀, 16–31.VI.2007, other data as holotype (JSIII-5-22); 3 ♀, 16–31.IV.2007, other data as holotype (JSIII-5-18); 2 ♀, 4–11.V.2007, other data as holotype (JSIII-3-18); 1 ♀, 4–11.V.2007, other data as holotype (JSIII-1-19); 1 ♀, 19–26.V.2007, other data as holotype (JSIII-2-17); 3 ♀, 16–31.IV.2007, other data as holotype (JSIII-3-22); 1 ♀, 4–11.V.2007, other data as holotype (JSIII-1-18); 1 ♀, 19–26.IV.2007, other data as holotype (JSIII-3-17); 1 ♀, 1–15.V.2007, other data as holotype (JSIII-5-19); 1 ♀, 10–20.VI.2007, other data as holotype (JSIII-3-20); 1 ♂, 16–31.IV.2007, other data as holotype (JSIII-4-18); 1 ♀, 16–31.V.2007, other data as holotype (JSIII-3-20); 1 ♀, 19–26.IV.2007, other data as holotype (JSIII-4-17); 2 ♀, 19–26.V.2007, other data as holotype (JSIII-2-19); 6 ♂, 1 ♀, 16–31.IV.2007, other data as holotype (JSIII-1-18); 1 ♀, 19–26.V.2007, other data as holotype (JSIII-4-19); 6 ♂, 1 ♀, 16–31.III.2007, other data as holotype (JSIII-1-16); 3 ♂, 16–31.III.2007, other data as holotype (JSIII-1-16); 2 ♂, 16–31.III.2007, other data as holotype (JSIII-3-16); 1 ♀, 1–15.V.2007, other data as holotype (JSIII-2-19); 1 ♀, 19–25.XI.2007, other data as holotype (JSIII-3-03); 4 ♂, 2 ♀, 1–15.IV.2007, other data as holotype (JSIII-1-17); 2 ♂, 1–15.III.2007, other data as holotype (JSIII-3-15); 1 ♂, 16–31.IV.2007, other data as holotype (JSIII-3-18); 1 ♂, 1 ♀, 16–31.IV.2007, other data as holotype (JSIII-2-18); 2 ♀, 16–31.VI.2007, 21°55.428'N, 101°16.441'E, elevation ca 598 m, other data as holotype (CZI-3-22); 1 ♀, 16–31.VI.2007, other data as holotype (CZI-5-22); 1 ♀, 16–31.VI.2007, other data as holotype (CZI-2-22); 4 ♂, 16–31.VI.2007, 21°54.984'N, 101°16.982'E, elevation ca 656 m, other data as holotype (JSIII-5-18); 1 ♀, 4–11.V.2007, other data as previous (JSII-3-18); 1 ♀, 10–20.VI.2007, other data as previous (JSII-2-20); 1 ♀, 16–31.VI.2007, other data as previous (JSIII-4-18); 2 ♂, 1–15.III.2007, other data as previous (JSII-5-15); 1 ♀, 1–15.V.2007, other data as previous (JSII-2-19); 3 ♀, 4–11.IV.2007, other data as previous (JSII-2-16); 5 ♂, 19–26.III.2007, other data as previous (JSII-4-15); 7 ♂, 1–15.IV.2007, other data as previous (JSII-2-17); 2 ♀, 1–15.V.2007, other data as previous (JSII-5-19); 4 ♂, 16–31.III.2007, other data as previous (JSII-4-16); 2 ♂, 1–15.III.2007, other data as previous (JSII-1-15); 2 ♀, 19–26.V.2007, other data as previous (JSII-4-19); 2 ♂, 16–31.III.2007, other data as previous (JSII-5-16); 2 ♂, 1–15.IV.2007, other data as previous (JSII-4-17); 6 ♂, 2 ♀, 16–31.IV.2007, other data as previous (JSII-3-18); 3 ♂, 1–15.IV.2007, other data as previous (JSII-1-17); 2 ♀, 4–11.V.2007, other data as previous (JSII-2-18); 1 ♀, 16–31.IV.2007, other data as previous (JSII-5-22); 3 ♀, 4–11.V.2007, other data as previous (JSII-4-18); 1 ♀, 19–26.V.2007, other data as previous (JSII-1-19); 2 ♀, 1–15.V.2007, other data as previous (JSII-1-19); 2 ♀, 16–31.IV.2007, other data as previous (JSII-4-22); 6 ♂, 16–31.III.2007, other data as previous (JSII-1-16); 2 ♂, 1 ♀, 16–31.IV.2007, other data as previous (JSII-2-18); 4 ♂, 16–31.III.2007, other data as previous (JSII-3-16); 6 ♂, 1–15.IV.2007, other data as previous (JSII-3-17); 3 ♀, 19–26.IV.2007, other data as previous (JSII-4-17); 2 ♀, 4–16.IV.2007, other data as previous (JSII-4-16); 1 ♀, 10–20.VI.2007, other data as previous (JSII-4-20); 1 ♀, 16–31.V.2007, other data as previous (JSII-3-20); 3 ♂, 1 ♀, 1–15.IV.2007, other data as previous (JSII-5-17); 1 ♀, 16–31.V.2007, other data as previous (JSII-5-20); 1 ♀, 19–26.IV.2007, other data as previous (JSII-1-17); 1 ♀, 19–26.IV.2007, other data as previous (JSII-2-17); 5 ♂, 16–31.III.2007, other data as previous (JSII-2-16); 3 ♀, 1–15.VI.2007, other data as previous (JSII-5-21); 1 ♀, 1–15.VII.2007, other data as previous (JSII-5-23); 2 ♀, 1–15.VI.2007, other data as previous (JSII-3-21); 1 ♀, 1–15.VI.2007, other data as previous (JSII-2-21); 2 ♀, 1–15.VII.2007, other data as previous (JSII-2-23); 1 ♂, 16–31.III.2007, 21°54.718'N, 101°16.940'E, elevation ca 645 m, other data as holotype (JSI-4-16); 1 ♀, 19–26.IV.2007, other data as previous (JSI-3-17); 2 ♂, 1–15.III.2007, other data as previous (JSI-3-15); 1 ♂, 16–31.IV.2007, other data as previous (JSI-5-18); 4 ♂, 1–15.IV.2007, other data as previous (JSI-4-17); 4 ♀, 16–31. VII.2007, other data as previous (JSI-2-24); 2 ♂, 10–20.VI.2007, other data as previous (JSI-3-20); 2 ♀, 1–15.V.2007, other data as previous (JSI-2-19); 1 ♀, 1–15.IV.2007, other data as previous (JSI-4-21); 1 ♀, 10–20.IV.2007, other data as previous (JSI-1-20); 2 ♀, 1–15.VI.2007, other data as previous (JSI-2-21); 2 ♀, 1–15.VII.2007, other data as previous (JSI-2-23); 5 ♂, 16–31.III.2007, other data as previous (JSI-1-16); 1 ♂, 1–15.IV.2007, other data as previous (JSI-3-17); 2 ♀, 16–31.V.2007, other data as previous (JSI-5-20); 1 ♀, 16–24.X.2007, other data as previous (JSI-2-06); 3 ♂, 1 ♀, 16–31.V.2007, other data as previous (JSI-1-20); 1 ♀, 16–31.VII.2007, other data as previous (JSI-3-24); 1 ♀, 4–11.V.2007, other data as previous (JSI-2-18); 3 ♂, 1–15.IV.2007, other data as previous (JSI-5-17); 1 ♀, 16–31.VII.2007, other data as previous (JSI-5-24); 2 ♂, 2 ♀, 19–26.IV.2007, other data as previous (JSI-4-17); 1 ♀, 4–11.V.2007, other data as previous (JSI-3-18); 2 ♂, 1–15.III.2007, other data as previous (JSI-2-15); 1 ♂, 2 ♀, 16–31.V.2007, other data as previous (JSI-4-20); 1 ♀, 1–15.V.2007, other data as previous (JSI-5-19); 2 ♀, 4–11.IV.2007, other data as previous (JSI-1-16); 1 ♀, 19–26.IV.2007, other data as previous (JSI-2-17); 1 ♀, 19–26.V.2007, other data as previous (JSI-3-19); 1 ♂, 1 ♀, 16–31.V.2007, other data as previous (JSI-2-20); 1 ♀, 10–20.VI.2007, other data as previous (JSI-4-20); 1 ♀, 19–26.V.2007, other data as previous (JSI-2-19); 1 ♂, 1 ♀, 1–15.V.2007, other data as previous (JSI-1-19); 1 ♀, 4–11.IV.2007, other data as previous (JSI-2-16); 1 ♀, 10–20.VI.2007, other data as previous (JSI-2-20); 1 ♀, 16–31.IV.2007, other data as previous (JSI-4-18); 6 ♂, 1 ♀, 16–31.IV.2007, other data as previous (JSI-3-18); 5 ♂, 4 ♀, 16–31.IV.2007, other data as previous (JSI-2-18); 8 ♂, 16–31.III.2007, other data as previous (JSI-2-16); 4 ♂, 1–15.IV.2007, other data as previous (JSI-1-17); 5 ♂, 1 ♀, 16–31.III.2007, other data as previous (JSI-3-16); 10 ♂, 2 ♀, 1–15.IV.2007, other data as previous (JSI-2-17); 1 ♂, 16–31.V.2007, other data as previous (JSI-3-30); 1 ♀, 19–26.V.2007, other data as previous (JSI-4-19); 2 ♂, 2 ♀, 16–31.IV.2007, other data as previous (JSI-1-18).The specific name refers to a famous tea from Xishuangbanna, Pu’er tea, which is planted on the mountainsides of Xishuangbanna and has a long history in China; noun in apposition.E.shenmiguo sp. nov. . Habitus as in Fig. AME 0.04, ALE 0.06, PME 0.05, PLE 0.06; AME–AME 0.03, AME–ALE 0.01, PME–PME 0.04, PME–PLE 0.04, AME–PME 0.05, AME–PLE 0.09, ALE–ALE 0.13, PLE–PLE 0.21, ALE–PLE 0.03; PME separated by slightly less than their diameters. MOA 0.14 long, frontal width 0.11, posterior width 0.13. Chelicerae  lack prolateral spine on femora I, but a few specimens (ca 1/10) with one prolateral spine which locate at the distal part of femora I.Known only from the type locality in Yunnan Province, China.Taxon classificationAnimaliaAraneaePhrurolithidaeLiu & Lisp. nov.98D60652-0B6F-52BC-9A7B-159B69C2249Fhttps://zoobank.org/C4A6AD1-0DEE-4CCF-82A6-F14ABA2A6B66Holotype ♂ , 21°57.669'N, 101°11.893'E, elevation ca 790 m, XTBG, Menglun Township, Mengla County, Xishuangbanna, Yunnan Province, China, 5–12.I.2007, G. Zheng leg. Paratype 2 ♂, 1 ♀, the same data as holotype (GBII-2-17); 11 ♂, 1 ♀, 16–31.III.2007, other data as holotype (GBII-1-16); 3 ♀, 4–11.IV.2007, other data as holotype (GBII-4-16); 5 ♂, 4 ♀, 16–31.III.2007, other data as holotype (GBII-2-16); 25 ♂, 3 ♀, 16–31.III.2007, other data as holotype (GBII-4-16); 2 ♂, 16–31.III.2007, other data as holotype (GBII-4-12); 2 ♂, 5–12.II.2007, other data as holotype (GBII-3-10); 1 ♀, 16–31.VII.2007, other data as holotype (GBII-4-24); 1 ♂, 2 ♀, 16–31.IV.2007, other data as holotype (GBII-4-18); 2 ♀, 1–15.V.2007, other data as holotype (GBII-1-19); 4 ♀, 10–20.VI.2007, other data as holotype (GBII-1-20); 3 ♀, 1–15.VII.2007, other data as holotype (GBII-3-23); 2 ♀, 19–26.IV.2007, other data as holotype (GBII-4-17); 3 ♂, 1 ♀, 5–12.I.2007, other data as holotype (GBII-2-10); 6 ♀, 4–11.V.2007, other data as holotype (GBII-3-18); 9 ♂, 3 ♀, 1–15.III.2007, other data as holotype (GBII-2-15); 1 ♂, 5–12.II.2007, other data as holotype (GBII-4-12); 2 ♂, 2 ♀, 19–26.III.2007, other data as holotype (GBII-3-15); 2 ♂, 5–12.I.2007, other data as holotype (GBII-2-12); 1 ♂, 1–15.I.2007, other data as holotype (GBII-2-11); 1 ♀, 10–20.VII.2007, other data as holotype (GBII-4-21); 3 ♀, 19–26.IV.2007, other data as holotype (GBII-2-17); 2 ♀, 1–15.I.2007, other data as holotype (GBII-1-23); 1 ♀, 10–20.VII.2007, other data as holotype (GBII-1-21); 3 ♂, 1 ♀, 1–15.III.2007, other data as holotype (GBII-5-15); 7 ♀, 19–26.V.2007, other data as holotype (GBII-1-19); 1 ♂, 19–26.V.2007, other data as holotype (GBII-3-19); 2 ♂, 1–15.II.2007, other data as holotype (GBII-4-13); 9 ♂, 1 ♀, 16–31.III.2007, other data as holotype (unspecified); 1 ♂, 16–31.III.2007, other data as holotype (GBII-2-20); 2 ♀, 5–12.III.2007, other data as holotype (GBII-4-14); 1 ♀, 1–15.V.2007, other data as holotype (GBII-4-19); 1 ♀, 1–15.IV.2007, other data as holotype (GBII-2-21); 1 ♀, 1–15.IV.2007, other data as holotype (GBII-1-21); 1 ♀, 5–12.XII.2007, other data as holotype (GBII-1-08); 2 ♀, 1–15.IV.2007, other data as holotype (GBII-4-21); 4 ♂, 1–15.III.2007, other data as holotype (GBII-2-13); 3 ♂, 1–15.III.2007, other data as holotype (GBII-3-15); 10 ♂, 2 ♀, 16–31.IV.2007, other data as holotype (GBII-1-18); 9 ♀, 4–11.V.2007, other data as holotype (GBII-1-18); 2 ♂, 1–15.IV.2007, other data as holotype (GBII-1-17); 2 ♀, 10–20.VI.2007, other data as holotype (GBII-4-20); 1 ♀, 10–14.VIII.2006, other data as holotype (GBII-4-01); 2 ♀, 19–26.IV.2007, other data as holotype (GBII-2-17); 7 ♂, 1 ♀, 16–31.III.2007, other data as holotype (GBII-5-16); 1 ♂, 1 ♀, 19–25.II.2007, other data as holotype (GBII-4-13); 4 ♂, 1 ♀, 1–15.IV.2007, other data as holotype (GBII-5-17); 7 ♂, 16–31.II.2007, other data as holotype (GBII-2-14); 2 ♂, 1 ♀, 1–15.IV.2007, other data as holotype (GBII-4-17); 1 ♂, 16–31.II.2007, other data as holotype (GBII-3-14); 1 ♀, 4–11.V.2007, other data as holotype (GBII-2-18); 1 ♀, 19–26.IV.2007, other data as holotype (GBII-3-17); 1 ♀, 4–11.IV.2007, other data as holotype (GBII-1-16); 5 ♀, 19–26.V.2007, other data as holotype (GBII-4-19); 5 ♀, 4–11.V.2007, other data as holotype (GBII-4-18); 1 ♀, 2–12.III.2007, other data as holotype (GBII-3-14); 1 ♀, 5–12.III.2007, other data as holotype (GBII-2-14); 2 ♂, 1 ♀, 19–25.I.2007, other data as holotype (GBII-2-11); 7 ♂, 16–31.III.2007, other data as holotype (GBII-3-16); 5 ♀, 19–26.V.2007, other data as holotype (GBII-2-19); 2 ♀, 10–20.VI.2007, other data as holotype (GBII-2-20); 1 ♀, 10–20.VI.2007, other data as holotype (GBII-3-20); 2 ♀, 16–31.IV.2007, other data as holotype (GBII-5-18); 4 ♀, 4–11.IV.2007, other data as holotype (GBII-2-16); 1 ♀, 16–31.IV.2007, other data as holotype (GBII-3-22); 1 ♂, 21°54.813'N, 101°12.634'E, elevation ca 876 m, 1–15.IV.2007, other data as holotype (GBII-4-17); 5 ♂, 1–15.IV.2007, other data as previous (GBIII-3-17); 1 ♂, 1–15.IV.2007, other data as previous (GBIII-5-17); 5 ♂, 1–15.IV.2007, other data as previous (GBIII-2-17); 1 ♀, 1–15.VII.2007, other data as previous (GBIII-2-23); 1 ♀, 19–26.IV.2007, other data as previous (GBIII-4-17); 4 ♂, 16–31.IV.2007, other data as previous (GBIII-3-18); 2 ♀, 4–11.V.2007, other data as previous (GBIII-3-18); 2 ♀, 19–26.IV.2007, other data as previous (GBIII-2-19); 1 ♂, 4–11.IV.2007, other data as previous (GBIII-1-16); 2 ♂, 1–15.III.2007, other data as previous (GBIII-1-15); 1 ♀, 16–31.V.2007, other data as previous (GBIII-3-20); 1 ♂, 16–31.IV.2007, other data as previous (GBIII-4-18); 2 ♀, 10–20.IV.2007, other data as previous (GBIII-4-20); 3 ♀, 19–26.V.2007, other data as previous (GBIII-3-19); 1 ♂, 1 ♀, 4–11.IV.2007, other data as previous (GBIII-4-16); 2 ♀, 16–26.V.2007, other data as previous (GBIII-4-19); 2 ♀, 4–11.V.2007, other data as previous (GBIII-4-18); 2 ♂, 1 ♀, 16–31.III.2007, other data as previous (GBIII-4-16); 4 ♂, 16–31.III.2007, other data as previous (GBIII-5-16); 1 ♀, 10–20.VII.2007, other data as previous (GBIII-4-18); 5 ♀, 21°57.445'N, 101°12.997'E, elevation ca 744 m, 4–11.V.2007, other data as holotype (GBIII-1-18); 1 ♀, 16–31.V.2007, other data as previous (GBIII-4-20); 3 ♀, 19–26.IV.2007, other data as previous (GBIII-3-17); 3 ♀, 19–26.III.2007, other data as previous (GBIII-4-15); 1 ♂, 6 ♀, 4–11.V.2007, other data as previous (GBIII-3-18); 4 ♀, 4–11.V.2007, other data as previous (GBIII-2-18); 2 ♀, 4–11.IV.2007, other data as previous (GBIII-3-16); 1 ♂, 5–12.II.2007, other data as previous (GBIII-4-12); 5 ♀, 4–11.V.2007, other data as previous (GBIII-4-18); 1 ♀, 16–31.V.2007, other data as previous (GBIII-1-20); 8 ♂, 1 ♀, 16–31.III.2007, other data as previous (GBIII-1-16); 2 ♀, 10–20.VI.2007, other data as previous (GBIII-1-20); 3 ♀, 16–31.VI.2007, other data as previous (GBIII-2-22); 1 ♀, 1–15.V.2007, other data as previous (GBIII-2-19); 6 ♀, 19–26.IV.2007, other data as previous (GBIII-4-17); 2 ♀, 1–15.V.2007, other data as previous (GBIII-4-19); 1 ♂, 1–15.III.2007, other data as previous (GBIII-5-15); 2 ♀, 19–26.IV.2007, other data as previous (GBIII-1-17); 8 ♀, 19–26.V.2007, other data as previous (GBIII-4-19); 1 ♂, 19–26.III.2007, other data as previous (GBIII-3-15); 7 ♂, 1 ♀, 16–31.III.2007, other data as previous (GBIII-3-16); 4 ♂, 1–15.IV.2007, other data as previous (GBIII-1-17); 1 ♂, 5–12.I.2007, other data as previous (GBIII-4-10); 1 ♀, 5–12.VI.2006, other data as previous (GBIII-1-06); 2 ♂, 19–26.III.2007, other data as previous (GBIII-2-15); 1 ♂, 16–31.III.2007, other data as previous (GBIII-3-05); 3 ♀, 4–11.IV.2007, other data as previous (GBIII-4-16); 1 ♀, 16–31.II.2007, other data as previous (GBIII-4-14); 6 ♂, 1–15.IV.2007, other data as previous (GBIII-2-17); 3 ♀, 19–26.V.2007, other data as previous (GBIII-1-19); 1 ♀, 16–31.V.2007, other data as previous (GBIII-3-20); 1 ♀, 1–15.IV.2007, other data as previous (GBIII-5-21); 1 ♀, 5–12.III.2007, other data as previous (GBIII-3-14); 1 ♂, 19–26.III.2007, other data as previous (GBIII-1-15); 1 ♀, 10–20.VI.2007, other data as previous (GBIII-3-20); 2 ♀, 10–20.VI.2007, other data as previous (GBIII-4-20); 13 ♂, 16–31.III.2007, other data as previous (GBIII-4-16); 7 ♂, 3 ♀, 1–15.IV.2007, other data as previous (GBIII-4-17); 7 ♂, 3 ♀, 16–31.III.2007, other data as previous (GBIII-2-16); 2 ♀, 4–11.IV.2007, other data as previous (GBIII-2-16); 1 ♀, 10–20.VI.2007, other data as previous (GBIII-3-21); 1 ♂, 5–12.III.2007, other data as previous (GBIII-2-14); 1 ♀, 16–24.IX.2006, other data as previous (GBIII-3-04); 2 ♂, 1–15.IV.2007, other data as previous (GBIII-5-17); 1 ♂, 1–15.IV.2007, other data as previous (GBIII-3-17); 3 ♂, 8 ♀, 5–12.III.2007, other data as previous (GBIII-4-14); 11 ♂, 10–31.III.2007, other data as previous (GBIII-5-16); 2 ♂, 5–12.III.2007, other data as previous (GBIII-1-14); 5 ♀, 16–29.VI.2007, other data as previous (GBIII-3-19); 2 ♀, 5–12.XI.2007, other data as previous (GBIII-2-06); 1 ♀, 16–31.IV.2006, other data as previous (GBIII-5-18); 2 ♀, 16–31.VI.2006, other data as previous (GBIII-4-22); 1 ♂, 4 ♀, 21°55.035'N, 101°16.500'E, elevation ca 558 m, 16–31.V.2007, other data as holotype (GZI-4-20); 1 ♀ (GBIII-4-12).Synsepalumdulcificum (Schumach. & Thonn.) Daniell, 1852, shenmiguo, which was introduced to XTBG from Ghana; noun in apposition.The specific name refers to the Chinese name of E.puer sp. nov. . Habitus as in Fig. AME 0.04, ALE 0.06, PME 0.05, PLE 0.06, AME–AME 0.03, AME–ALE 0.01, PME–PME 0.05, PME–PLE 0.03, AME–PME 0.04, AME–PLE 0.09, ALE–ALE 0.12, PLE–PLE 0.22, ALE–PLE 0.03. MOA 0.13 long, frontal width 0.10, posterior width 0.14. Chelicerae (Fig. rae Fig.  with thrrae Fig. , 7B sligrae Fig. , 7B widerae Fig. , longer rae Fig. : I 3.29 rae Fig. : femora rae Fig.  nearly cColouration (Fig. AME, ALE and PLE with dark layer of black pigment around the eye cup, but PME absent. Chelicerae, endites, and labium yellow. Sternum yellow, mottled around margin. Legs yellow, without dark stripes. Abdomen yellow-brown, mottled, with three light yellow chevrons posteriorly and many yellow spots on surface; venter yellow.ion Fig. . CarapacPalp (Figs alp Figs , 10. FemFemale. Habitus as in Fig. AME 0.04, ALE 0.06, PME 0.04, PLE 0.06, AME–AME 0.01, AME–ALE 0.01, PME–PME 0.05, PME–PLE 0.04, AME–PME 0.04, AME–PLE 0.08, ALE–ALE 0.12, PLE–PLE 0.20, ALE–PLE 0.02. MOA 0.13 long, frontal width 0.10, posterior width 0.13. Leg measurements (Fig. nts Fig. : I 2.99 nts Fig. : femora Colouration (Fig. ion Fig. . LighterEpigyne (Fig. yne Fig. . EpigynaE.puer sp. nov.Prolateral spine on femora I same detail as in Known only from the type locality in Yunnan Province, China.


H)-ones. The probability of cytoprotective action was assessed by measuring cell viability using two tests . It was found that some derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1H)-ones under the conditions of our experiment had a pronounced cytoprotective activity, providing better cell survival in vitro, including the MTT test and conditions of blood hyperviscosity. To correlate the obtained results in vitro, molecular docking of the synthesized derivatives was also carried out. The standard drug omeprazole  was used as a standard. It was shown that all synthesized derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1H)-ones had higher affinity for the selected protein than the standard gastro-cytoprotector omeprazole. The studied derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1H)-ones also fully satisfy Lipinski’s rule of five (RO5), which increases their chances for possible use as orally active drugs with good absorption ability and moderate lipophilicity. Thus, the results obtained make it possible to evaluate derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1H)-ones as having a relatively high cytoprotective potential.Currently, studies are being conducted on the possible role of the cytoprotective effect of biologically active substances in conditions of cerebral hypoxia or cardiomyopathies. At the same time, oxidative stress is considered one of the important mechanisms of cellular cytotoxicity and a target for the action of cytoprotectors. The aim of this study is to search for derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1 H)-ones based on the intramolecular cyclization of N-amides. Almost all of the obtained 3-aminopyridin-2(1H)-ones possess high antiradical activity ..H)-ones.H)-one derivatives showed antiradical activity against DPPH and ABTS radicals. In addition, these synthesized compounds were tested in vivo for anxiolytic activity using a light–dark box test and antidepressant activity by Porsolt’s “behavioral despair” test. Several derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1H)-one with higher potential neurotropic activity than that of the comparator drugs (mexidol and amitriptyline) were found  was carried out. As a standard of antiradical activity, vitamin E (Trolox) is present in the form of Trolox equivalent of antioxidant capacity (TEAC) .Evaluating the antiradical effect of the presented samples in relation to the ABTS•+ radical, it can be seen that the studied compounds have a moderate potential for antiradical activity, approaching in terms of the severity of the quantitative assessment the indicators inherent in vitamin E (Trolox).The results presented in In accordance with the aim of the study, the next step after establishing the antiradical effect of the investigated 3-(arylmethylamino)pyridone compounds was to study their probable cytoprotective effect. The results of measuring the level of viability of transplanted MCF-7 (Michigan Cancer Foundation-7) cells during incubation with the studied 3-(arylmethylamino)pyridine compounds are presented in As For the compound with the most pronounced ability to protect cells under conditions of cultivation of the MCF-7 monolayer during the 24-h period, namely compound 3d, additional studies were performed to confirm the level of viability of MCF-7 cells in the test with neutral red. This dye is known to selectively stain cells with fully functionally preserved membranes. The results of the study of changes in the viability of MCF-7 cells under in vitro conditions under the influence of the studied samples in the test with neutral red are presented in 3d compound with the neutral red dye, which is able to selectively stain the cell only with a functionally and structurally preserved cell membrane, indicate that the cell viability after contact with the 3d compound was significantly increased.As can be seen, the results of the interaction of cells pre-incubated with the In experiments on the study of hemorheological activity of samples , it was Currently, modern cytoprotectors are successfully used in the treatment of cerebral hypoxia of various etiologies, as well as in acute and chronic cardiovascular pathology ,15,16. IIn this regard, to evaluate a possible biological target and confirm the previously obtained results of the cytoprotective activity of compounds 3a–h, we used the method of molecular docking. The oxidoreductase enzyme receptor (PDB identifier: 4KEW) , which iThree-dimensional (3D) structures were obtained from the RCSB Protein Data Bank , while t3a.The docking results showed  that for3a–h, the number of intermolecular hydrogen bonds, the binding energies of the stable ligand–receptor complex (4KWE), and the number of nearest amino acid residues were determined Molecular weight less than or equal to 500;(2)LogP (octanol-water partition coefficient) must be less than or equal to 5;(3)Hydrogen bond donors must be less than or equal to 5;(4)There should not be more than 10 hydrogen bond acceptors .To model the bioavailability of new physiologically active substances for their possible use as an orally active drug, one of the ways to identify compounds with good absorption capacity and moderate lipophilicity is to screen compounds using Lipinski’s rule of five (RO5). The rule states that good absorption or penetration of a drug is possible if the chemical structure of the drug does not violate more than one of the following criteria/rules:3a–h with potential cytoprotective activity as the likelihood of their use in medical practice.The Molinspiration online tool  was usedThe results obtained are shown by us in The results of the computer screening indicate that none of the studied compounds 3a–h violated any of the RO5 rules, indicating their possible use as drugs with antiradical and cytoprotective activity.The results of the MTT test and the neutral red test can be considered evidence of the presence of cytoprotective activity in 3-(arylmethylamino)pyridone compounds in the compound codenamed 3d. As was determined in the same series of experiments, the investigated compounds of 3-(arylmethylamino)pyridone compounds, including 3d, have a pronounced potential for antiradical activity against the standard DPPH and ABTS•+ tests. The data obtained allow us to suggest a potential relationship between the presence of antiradical activity and manifestations of a cytoprotective effect. The results obtained under the conditions of our experiment support the hypothesis of the possible protection of cellular and intracellular membranes of eukaryotic cells from the damaging effects of free radicals by substances that bind and neutralize free radicals . In partAn absolute limitation of this study is the lack of comparison of antiradical and cytoprotective effects in vivo using adequate models. This is what is expected to be done in the future.Thus, for 3-(arylaminomethyl)pyridone compounds, an antiradical effect on free radicals DPPH and ABTS•+ has been proven, and the results of in vitro studies on cell culture have been obtained, suggesting the possibility of a cytoprotective effect. Both results obtained under the conditions of this experiment deserve, in our opinion, detailed consideration in the forthcoming extended experiments.1H and 13C NMR spectra were recorded on a Bruker DRX400  and Bruker AVANCE 500  instruments using DMSO-d6 , DMSO-d6 + CF3COOH (compound 2d), or CDCl3 (remaining compounds) with TMS as internal standard. Elemental analysis was performed on a Carlo Erba 1106 CHN instrument. Melting points were determined using a Koffler hot bench. Monitoring of the reaction course and the purity of the products was carried out by TLC on Sorbfilplates and visualized using iodine vapor or UV light.IR spectra were recorded on an Infralum FT-801 spectrometer for KBr pellets. H)-one 1 was synthesized according to procedure [3-Amino-6-methyl-4-phenylpyridine-21H-one 1 w3a–h based on 3-amino-6-methyl-4-phenylpyridine-2(1H)-one 1. 3-(Arylmethylamino)pyridones 3a–g were obtained in good yields by reacting pyridine-2-one 1 with a series of aromatic aldehydes followed by reduction of imines 2a–g with sodium borohydride in 2-propanol at 25–35 °C (In order to create a library of 3-(arylaminomethyl)pyridone derivatives and then evaluate them for possible antiradical and cytoprotective activity, we performed the synthesis of compounds 25–35 °C , their p2a–h. The mixture of 3-amino-pyridin-2(1H)-one 1 , aromatic aldehyde (1.2 mmol), and a catalytic amount of formic acid in 5 mL of 2-propanol was refluxed for 1–3 h. After cooling the reaction mixture, precipitated imines 2a–h were filtered off and washed with hexane.Typical procedure for the synthesis of imines 3a–h. To a suspension of imines 2a–h (1 mmol) in 2-propanol (15 mL) were added water (3 mL) and sodium borohydride  with stirring at a temperature of 25–35 °C; the reaction mixture was stirred for 10–15 h. Then the reaction mixture was poured into a beaker with ice-cold water (150 mL). The aqueous layer was extracted with chloroform (3 × 25 mL), the organic layer was dried over Na2SO4, the solvent was removed by distillation, and the residue was triturated with hexane. The crude product was recrystallized from a 1:2 mixture of 2-propanol and hexane.Typical procedure for the synthesis of compounds E)-3-(Benzylideneamino)-6-methyl-4-phenylpyridin-2(1H)-one (2a). Yield: 236 mg (82%), yellow crystals, m.p.: 214–215 °C (2-propanol - chloroform). IR : 3450, 3352, 1636. 1H NMR  δ ppm 2.41 , 6.25 , 7.35–7.40 , 7.48–7.53 , 7.75 , 9.35 , 13.18 . 13C NMR  δ ppm 18.7, 108.7, 121.6, 127.5, 128.0, 128.4, 128.6, 130.0, 130.6, 131.0, 131.7, 137.7, 140.8, 146.3, 161.2, 162.6. Anal. Calcd. for C19H16N2O: C, 79.14; H, 5.59; N, 9.72. Found: C, 79.52; H, 5.87; N, 9.91.(E)-3-((2-Hydroxybenzylidene)amino)-6-methyl-4-phenylpyridin-2(1H)-one (2b). Yield: 289 mg (95%), yellow crystals, m.p.: 251–252 °C (2-propanol). IR : 3454, 2826, 1626, 1466, 1282. 1H NMR  δ ppm 2.24 ; 6.13 ; 6.71 ; 6.86 ; 7.26 ; 7.36–7.44 ; 9.86 ; 12.16 ; 12.59 . 13C NMR  δ ppm 18.3, 107.1, 116.3, 118.8, 119.7, 127.9, 128.27, 128.31, 128.5, 132.3, 132.4, 137.9, 142.8, 147.2, 159.1, 159.9, 164.3. Anal. Calcd. for C19H16N2O2: C, 75.31; H, 5.66; N, 9.57. Found: C, 74.98; H, 5.30; N, 9.20.(E)-3-((4-Methoxybenzylidene)amino)-6-methyl-4-phenylpyridin-2(1H)-one (2c). Yield: 261 mg (82%), yellow crystals, m.p.: 217–219 °C (2-propanol- chloroform). IR : 2901, 1625, 1574, 1248. 1H NMR  δ ppm 2.40 ; 3.83 ; 6.23 ; 6.90 ; 7.38 ; 7.51 ; 7.70 ; 9.40 ; 13.05 . 13C NMR  δ ppm 18.7 (CH3), 55.3 (OCH3), 108.1, 113.9 , 127.5 , 128.1 , 128.9, 130.0 , 132.2, 138.1, 140.2, 145.4, 159.9, 161.3, 161.8, 162.0. Anal. Calcd. for C20H18N2O2: C, 75.45; H, 5.70; N, 8.80. Found: C, 75.04; H, 6.06; N, 8.38.(E)-6-Methyl-3-((3-nitrobenzylidene)amino)-4-phenylpyridin-2(1H)-one (2d). Yield: 260 mg (78%), orange crystals, m.p.: 290–291 °C. IR : 2893, 1655, 1624, 1534. 1H NMR  δ ppm 2.16 ; 5.98 ; 7.38 ; 7.70 ; 8.17 ; 8.36 ; 8.55 ; 9.99 ; 13.18 . 13C NMR  δ ppm 19.5 (CH3), 108.2 (C-5), 116.9, 125.1, 129.0, 129.5 (2C Ph), 129.7, 130.4 (2C Ph), 130.9, 131.9, 136.1, 136.3, 138.8, 146.4, 148.2, 149.9, 192.3. Anal. Calcd. for C19H15N3O3: C, 68.46; H, 4.54; N, 12.61. Found: C, 68.88; H, 4.93; N, 12.18.(E)-3-amino)-6-methyl-4-phenylpyridin-2(1H)-one (2e). Yield: 294 mg (84%), yellow crystals, m.p.: 220–224 °C. IR (KBr cm−1): 2831, 1605, 1583, 1271. 1H NMR  δ ppm 2.21 ; 3.79 ; 3.84 ; 6.13 ; 6.53 ; 6.60 ; 7.33–7.38 ; 7.42–7.43 ; 7.64 ; 9.54 ; 11.81 . 13C NMR  δ ppm 18.3 (CH3), 55.4 (OCH3), 55.6 (OCH3), 98.0 , 106.2 (C-5), 106.5 (C-5 Ar), 118.4 (C-6 Ar), 127.4 , 127.5, 127.7, 129.7 , 132.1, 138.2, 140.2, 143.2, 155.5, 159.2, 160.4, 163.1. Anal. Calcd. for C21H20N2O3: C, 72.40; H, 5.79; N, 8.04. Found: C, 72.05; H, 6.21; N, 8.39.(E)-3-amino)-6-methyl-4-phenylpyridin-2(1H)-one (2f). Yield: 296 mg (85%), yellow crystals, m.p.: 241–244 °C. IR (KBr cm−1): 2838, 1635, 1621, 1262, 1230. 1H NMR  δ ppm 2.23 ; 3.68 ; 3.78 ; 6.19 ; 6.79 ; 7.22 ; 7.28 ; 7.34 ; 7.39 ; 7.48 ; 9.41 ; 11.91 . 13C NMR  δ ppm 18.3 (CH3), 55.1 (OCH3), 55.6 (OCH3), 106.6 (C-3 Ar), 109.0 (C-5), 111.3 (C-5 Ar) 123.0 (C-6 Ar), 127.4 (2C Ph), 127.8, 129.9 (2C Ph), 130.6, 130.7, 138.1 140.1, 144.4, 148.9, 151.2, 159.3, 160.0. Anal. Calcd. for C21H20N2O3: C, 72.40; H, 5.79; N, 8.04. Found: C, 72.08; H, 6.18; N, 8.41.(E)-3-((5-Bromo-2-hydroxybenzylidene)amino)-6-methyl-4-phenylpyridin-2(1H)-one (2g). Yield: 362 mg (94%), yellow crystals, m.p.: 294–297 °C. IR : 2922, 1625, 1621, 1162, 820. 1H NMR  δ ppm 2.27 ; 6.14 ; 6.69 ; 7.36–7.44 ; 7.63 9.83 ; 12.03 ; 12.50 . Anal. Calcd. for C19H15BrN2O2: C, 59.55; H, 3.95; N, 7.31. Found: C, 59.98; H, 4.38; N, 7.70.(E)-3-((4-Dimethylamino)benzylidene)amino)-6-methyl-4-phenylpyridin-2(1H)-one (2h). The mixture of 3-aminopyridin-2(1H)-one 1 , 654 mg 4-(dimethylamino)benzaldehyde (43 mmol) and catalytic amount of formic acid in 30 mL 2-propanol was refluxed for 8 h. After cooling the reaction mixture, precipitated imine 2h was filtered off and washed with hexane. Yield: 310 mg (93%), yellow crystals, m.p.: 258–261 °C (2-propanol). IR (KBr): ν, cm−1: 3748, 2894, 1633, 1614. 1H NMR  δ ppm 2.36 ; 3.01 2); 6.20 ; 6.66 ; 7.33–7.36 ; 7.51 ; 7.63 ; 9.20 ; 12.55 . 13C NMR  δ ppm 18.8 (CH3); 40.2 (N(CH3)2) 108.5 (C-5); 111.4 ; 125.7; 127.5 ; 127.7 (C-4 Ph); 130.1 ; 130.2 ; 133.3 (C-1’ Ar); 138.2; 139.1; 143.9; 152.2; 161.3 (C-2); 162.9 (N=CH). Anal. Calcd. for C21H21N3O: C, 76.11; H, 6.39; N, 12.68. Found: C, 75.74; H, 6.81; N, 12.28.(H)-one (3a). Yield: 235 mg (81%), yellow crystals, m.p.: 167–169 °C (2-propanol-hexane). IR : 3334, 3274, 2925, 1831, 1628. 1H NMR  δ ppm 2.27 ; 3.73 ; 5.19 ; 5.87 ; 6.88 ; 7.0 ; 7.3 ; 12.38 ; 13C NMR  δ ppm 18.2; 49.9; 110.6; 126.5; 127.3; 127.6; 128.0; 128.2; 128.3; 132.2; 132.6; 132.9; 139.0; 139.9; 160.2. Anal. Calcd. for C19H18N2O: C, 78.59; H, 6.25; N, 9.65; Found: C, 74.91; H, 6.05; N, 9.86.3-(Benzylamino)-6-methyl-4-phenylpyridin-2(1H)-one (3b). Yield: 217 mg (71%), yellow crystals, m.p.: 187–188 °C (2-propanol-hexane). IR : 3367, 2920, 1632, 1542. 1H NMR  δ ppm 2.47 ; 3.62 ; 4.24 ; 6.10 ; 6.74 ; 6.91 , 6.99 , 7.18 , 7.37–7.43 , 7.47 , 10.05 ; 13.38 . 13C NMR  δ ppm 18.6, 48.8, 109.5, 116.7, 118.5, 124.4, 127.8, 128.5, 129.2, 129.3, 131.8, 137.2, 137.4, 140.9 157.5, 162.7. Anal. Calcd. for C19H18N2O2: C, 74.49; H, 5.92; N, 9.14; Found: C, 74.81; H, 5.74; N, 9.26.3-((2-Hydroxybenzyl)amino)-6-methyl-4-phenylpyridin-2(1H)-one (3c). Yield: 176 mg (55%), brown crystals, m.p.: 154–157 °C (2-propanol-hexane). IR : 3303, 2838, 1884, 1630, 1509. 1H NMR  δ ppm 2.27 ; 3,74  3.75 ; 5.00 ; 5.91 ; 6.74 ; 6.97 ; 7.32 ; 7.38–7.41 ; 7.45–7.47 ; 12.77 . 13C NMR  δ ppm 18.3 (CH3), 49.8 (N-CH2), 55.2 (OCH3), 109.4 (C-5), 113.5 , 127.5, 128.3 (4C Ph), 128.7 , 132.1, 132.3, 133.0, 139.3, 158.4, 161.6. Anal. Calcd. for C20H20N2O2: C, 74.98; H, 6.29; N, 8.74; Found: C, 74.54; H, 6.66; N, 8.34.3-((4-Methoxybenzyl)amino)-6-methyl-4-phenylpyridin-2(1H)-one (3d). Yield: 255 mg (76%), orange crystals, m.p.: 160–163 °C (2-propanol). IR : 3359, 3303, 2923, 1844, 1642. 1H NMR  δ ppm 2.28 ; 3.96 ; 5.19 ; 5.89 ; 7.32–7.36 ; 7.37–7.38  7.80 ; 8.01  12.74 . 13C NMR  δ ppm 18.3 (CH3), 49.2 (N-CH2), 109.4 (C-5), 121.9 (C-4 Ar), 122.4 (C-2 Ar), 127.9, 128.2 (2C Ph), 128.3 (2C Ph), 129.0, 131.9, 132.3, 132.9, 133.4, 142.4, 148.0, 161.5. Anal. Calcd. for C19H17N3O3: C, 68.05; H, 5.11; N, 12.53; Found: C, 68.48; H, 5.52; N, 12.93.6-Methyl-3-((3-nitrobenzyl)amino)-4-phenylpyridin-2(1H)-one (3e). Yield: 179 mg (51%), yellow crystals, m.p.: 132–133 °C (2-propanol-hexane). IR : 3341, 2931, 1629, 1518. 1H NMR  δ ppm 2.26 ; 3.75 ; 3,78 ; 3,82 ; 5.04 ; 5.90 ; 6.56 ; 6.57 ; 6.70 ; 7.32 ; 7.39 ; 7.43 ; 12.74 . 13C NMR  δ ppm 18.3 (CH3), 50.0 (N-CH2), 55.6 (OCH3), 55.8 (OCH3), 109.4 (C-5), 110.7 (C-2 Ar), 110.7 (C-5 Ar), 119.5 (C-6 Ar), 127.5, 128.3 (2C Ph), 128.3 (2C Ph), 131.6, 132.1, 132.8, 133.0, 139.2, 147.7, 148.6, 161.6. Anal. Calcd. for C21H22N2O3: C, 71.98; H, 6.33; N, 7.99; Found: C, 72.39; H, 6.72; N, 8.36.3-amino)-6-methyl-4-phenylpyridin-2(1H)-one (3f). 3-amino)-6-methyl-4-phenylpyridin-2(1−1): 3304, 2938, 1865, 1644. 1H NMR  δ ppm 2.25 ; 3,64 ; 3,75  3.84 ; 5.07 ; 5.88 ; 6.30 ; 6.32 ; 6.81 ; 7.31 ; 7.36–7.43 ; 12.54 . 13C NMR  δ ppm 18.3 (CH3), 45.0 (N-CH2), 55.0 (4-OCH3), 55.2 (2-OCH3), 98.2 (C-3 Ar), 103.3 (C-5), 109.2 (C-5 Ar), 120.8, 127.3, 128.3 (4C Ph), 129.8, 131.7, 131.9, 133.3, 139.4, 154.5, 159.9, 161.6. Anal. Calcd. for C21H22N2O3: C, 71.98; H, 6.33; N, 7.99; Found: C, 71.54; H, 6.75; N, 8.42.Yield: 214 mg (61%), yellow crystals, m.p.: 181–184 °C (2-propanol-hexane). IR -one (3g). Yield: 285 mg (74%), yellow crystals, m.p.: 203–206 °C (2-propanol-hexane). IR : 3370, 3279, 1842, 1643. 1H NMR  δ ppm 2.09 ; 3.66 ; 5.10 ; 5.81 ; 6.62 ; 6.83 ; 7.11–7.14 ; 7.34 ; 7.39–7.42 ; 9.8 ; 11.6 . 13C NMR  δ ppm 17.8 (CH3), 44.5 (N-CH2), 107.5 (C-5 Ar), 109.5 (C-5), 116.9 (C-4 Ar), 127.5 (C-3 Ar), 127.9 (2C Ph), 128.5 (2C Ph), 128.9, 129.1, 130.3 (C-6 Ar), 130.9, 132.0, 132.6, 139.0, 154.7, 160.1. Anal. Calcd. for C19H17BrN2O2: C, 59.23; H, 4.45; N, 7.27; Found: C, 59.63; H, 4.84; N, 7.70.3-((5-Bromo-2-hydroxybenzyl)amino)-6-methyl-4-phenylpyridin-2(1H)-one (3h). 3-((4-Dimethylaminobenzyl)amino)-6-methyl-4-phenylpyridin-2 in 2-propanol (50 mL) was added water (3 mL) and sodium borohydride  with stirring at a temperature of 25–35 °C; the reaction mixture was stirred for 10 h. Then the reaction mixture was poured into a beaker with ice-cold water (150 mL). The aqueous layer was extracted with chloroform (3×25 mL), the organic layer was dried over Na2SO4, the solvent was removed by distillation, and the residue was triturated with hexane. The crude product was recrystallized from a 1:2 mixture of 2-propanol and hexane. Yield: 250 mg (75%), yellow crystals, m.p.: 165–168 °C (2-propanol-hexane). IR : 2922, 1643, 1518. 1H NMR  δ ppm 2.27 ; 2.89 2); 3.70 ; 4.97 ; 5.93 ; 6.60 ; 6.95 ; 7.32 ; 7.41 ; 7.49 ; 12.83 . 13C NMR  δ ppm 18.3 (CH3); 40.7 (N(CH3)2); 50.0 (CH2-Ar); 109.4 (C-5); 112.5 ; 127.4 (C-4 Ph); 128.2; 128.2 ; 128.3 ; 128.5 ; 131.0; 131.9; 133.4; 139.5; 149.6 (C-4’ Ar); 161.6 (C-2). Anal. Calcd. for C21H23N3O: C, 75.65; H, 6.95; N, 12.60; Found: C, 75.22; H, 5.65; N, 9.94.To a suspension of imine 3h, 3e, and 3d was routinely studied in relation to the radicals of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) [The antiradical action of the compounds (ABTS•+) .3h, 3e, and 3d), the standard DPPH test purchased from Sigma Aldrich, Steinheim, Germany was used in accordance with the manufacturer’s instructions. To select substances with pronounced antiradical activity, 2 mL of a 100 μM ethanol solution of DPPH were mixed with 20 μL of the test samples dissolved in methanol at a concentration of 5 mM. As a result, the final concentration of the test substance in the reaction mixture was 50 µM. Ten minutes after adding the test compound solution to the DPPH radical solution, the decrease in absorbance at 515 nm was measured. For substances capable of reducing optical density by more than 50%, a test was performed for interaction with the DPPH radical at final concentrations of the studied substances of 50, 25, 20, 15, 10, 5, and 2.5 µM. After that, the concentration of the test substance capable of reducing the optical density by 50% was determined—IC50 (DPPH). As a reference drug, a substance with known antiradical properties, ascorbic acid, was used.To assess the antiradical activity of the studied samples (3h and 3e) were dissolved in DMSO to a concentration of 10 mM, then twice 10-fold dissolved in 1×Assay Buffer to a final concentration of 0.1 mm. All test substances were tested at a concentration of 0.1 mM.The antiradical activity of the presented samples was studied against the radical cation 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) using the Antioxidant Assay Kit (Sigma Aldrich). The principle of the method is the formation of a ferryl myoglobin radical from metmyoglobin and hydrogen peroxide, which oxidizes ABTS with the formation of a cation radical: ABTS•+. ABTS•+ is a stable radical that can exist in aqueous solutions for quite a long time, however, the introduction of various antiradical agents into the solution leads to their interaction with ABTS•+ and rapid consumption (“quenching”) of the latter. The consumption of ABTS•+ is accompanied by characteristic spectral changes, which make it possible to record the reaction rate. The ability to dose the initial concentration of radicals in the system and control the rate of their ”quenching”" led to the widespread use of ABTS•+ to standardize the antiradical activity of various compounds . We comp3h, 3e, and 3d) were evaluated in the MTT test on the breast cancer cell line MCF-7. Of the test sample, 1 mg was dissolved in 1 mL of DMSO. 10 μL of the dissolved substance was added to 100 μL of the nutrient medium with MCF-7 cells. Cells in nutrient medium without the addition of test compounds served as controls. After cells were incubated for 24 h with the test objects, viability was determined in the MTT test using the In Vitro Toxicology Assay Kit, MTT based (Sigma Aldrich) according to the manufacturer’s instructions. The cytoprotective properties of the presented samples  was used on the MCF-7 cell line according to the manufacturer’s manual to access compounds with pronounced cytoprotective activity.Hyperviscosity syndrome (HBIS) was reproduced in vitro by blood incubation at 43.0 °C for 60 min. Blood viscosity was measured on a Brookfield DV2T rotational viscometer at various spindle speeds (from 2 to 60 rpm).−5 g/mL of blood. Blood samples to which DMSO solvent was added in an equivolume amount served as controls. As a reference drug, a substance with known hemorheological properties, pentoxifylline, was used at a concentration of 10−5 g/mL of blood [Studies of the hemorheological activity of 6 samples were carried out on 12 Wistar female rats, 12 weeks old, weighing 220–240 g. After blood sampling, the initial blood viscosity was determined in laboratory animals, and then blood samples were incubated with the test substances at a temperature of 43.0 °C for 60 min and then measured the parameters under study. The blood was incubated with the test objects dissolved in DMSO; the final concentration of the compounds was 10of blood ,35. Blooof blood . The iniAll research work with laboratory animals was performed in accordance with generally accepted ethical standards for the treatment of animals, based on standard operating procedures that comply with the rules adopted by the European Convention for the Protection of Vertebrate Animals used for Research and other Scientific Purposes . The study protocol of the project “Search for means of pharmacological correction of increased blood viscosity syndrome associated with endocrine pathology” was approved on 7 August 2020 (Protocol No. 3) by the Local Ethics Commission of the Republican State Enterprise “National Center for Biotechnology” .Statistical processing of the results was carried out using the Excel program. The results obtained are presented as mean ± standard error of the mean.H)-ones, it was found that they have a fairly high cytoprotective potential. It was shown that some derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1H)-ones under the conditions of our experiment have a pronounced cytoprotective activity, providing better cell survival in vitro, including in hyperviscosity conditions.Ss a result of the bioscreening performed on two tests by measuring cell viability , as well as in the test under conditions of blood hyperviscosity of synthesized derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1H)-ones have a higher affinity for the selected protein than the standard gastro-cytoprotector omeprazole.Correlation of the results obtained in vitro was performed using molecular docking on the example of the selected target protein of the oxidoreductase enzyme receptor (PDB identifier: 4KEW); the oxidoreductase enzyme receptor showed that all synthesized derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1H)-ones also fully satisfy Lipinski’s rule of five (RO5), which increases their chances for possible use as orally active drugs with good absorption capacity and moderate lipophilicity.The investigated derivatives of 3-(arylmethylamino)-6-methyl-4-phenylpyridin-2(1


Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense STIB900. Cytotoxicity of all compounds towards L6 cells was evaluated and the respective selectivity indices (SI) were calculated. We found that benzyl naphthoquinone 5c was the most active and selective molecule against T. brucei rhodesiense . Furthermore, the antiprotozoal assays revealed no specific effects. In addition, some key physicochemical parameters of the synthesised compounds were calculated.Quinones and quinols are secondary metabolites of higher plants that are associated with many biological activities. The oxidative dearomatization of phenols induced by hypervalent iodine(III) reagents has proven to be a very useful synthetic approach for the preparation of these compounds, which are also widely used in organic synthesis and medicinal chemistry. Starting from several substituted phenols and naphthols, a series of cyclohexadienone and naphthoquinone derivatives were synthesized using different hypervalent iodine(III) reagents and evaluated for their in vitro antiprotozoal activity. Antiprotozoal activity was assessed against  Quinone derivatives are reported to exhibit a broad spectrum of biological activities such as anti-inflammatory + = 200.0485; found: 200.0499.Methyl 2-acetate (4c). Compound 4c was obtained after stirring for 10 min and purified by flash chromatography using CH2Cl2/MeOH (25:1). Yellowish oil; yield 63%; Rf = 0.43 (CH2Cl2:MeOH = 25:1); 1H NMR , 6.98 , 6.30 , 4.16 , 3.78 , 2.78 , 2.73  ppm; 13C NMR : δ = 178.0 (C-4), 171.0 (C-8), 149.1 (C-6), 144.6 (C-2), 132.8 (C-3), 127.2 (C-5), 69.3 (C-1), 52.5 (C-9), 43.0 (C-7) ppm; HRMS (ESI) calcd. for C9H10ClO4 [M + H]+ = 217.0268; found: 217.0262.Methyl 2-acetate (4d). Compound 4d was obtained after stirring for 10 min and purified by flash chromatography using toluene/EtOAc (5:1). Yellow oil; yield 62%; Rf = 0.44 (toluene:EtOAc = 5:1); 1H NMR : δ = 7.43 , 6.98 , 6.31 , 4.08 , 3.78 , 2.77 , 2.72  ppm; 13C NMR : δ = 177.7 (C-4), 171.0 (C-8), 148.9 , 126.8 (C-5), 124.8 (C-3), 69.9 (C-1), 52.5 (C-9), 42.7 (C-7) ppm; HRMS (EI) calcd. for C9H9BrO4 [M]+ = 259.9684; found: 259.9693.Methyl 2-acetate (4e). Compound 4e was obtained after stirring for 10 min and purified by flash chromatography using cyclohexane/EtOAc (2:1). Yellowish oil; yield 68%; Rf = 0.28 (cyclohexane:EtOAc = 2:1). The spectroscopic data were found to be identical to the ones described in Ref + = 249.9800; found: 249.9813.d in Ref . AlthougMethyl 2-acetate (4f). Compound 4f was obtained after stirring for 10 min and purified by flash chromatography using toluene/EtOAc (5:1). Yellowish oil; yield 62%; Rf = 0.38 (toluene:EtOAc = 5:1). The spectroscopic data were found to be identical to the ones described in Ref + = 337.8789; found: 337.8789.d in Ref . AlthougMethyl 3-propanoate (4g). Compound 4g was obtained after stirring for 20 min and purified by flash chromatography using CHCl3/CH3CN (5:2). Amber solid; yield 67%; Rf = 0.31 (CHCl3:CH3CN = 5:2); m.p.: 49-50 °C; 1H NMR : δ = 6.83 , 6.20 , 3.67 , 2.60 , 2.36 , 2.12  ppm; 13C NMR : δ = 185.1 (C4), 173.4 (C-9), 150.2 , 128.6 , 69.2 (C-1), 52.0 (C-10), 34.5 (C-7), 28.6 (C-8) ppm; HRMS (ESI) calcd. for C10H13O4 [M + H]+ = 197.0814; found: 197.0808.1-Oxaspiro [4.5]deca-6,9-diene-2,8-dione (4h). Compound 4h was obtained after stirring for 20 min and purified by flash chromatography using CHCl3/CH3CN (5:2). Amber solid; yield 8%; Rf = 0.54 (CHCl3:CH3CN = 5:2); m.p.: 105–106 °C : δ = 6.86 , 6.29 , 2.79 , 2.38  ppm; 13C NMR : δ = 184.1 (C-4), 175.1 (C-9), 145.5 , 129.2 , 78.4 (C-1), 32.3 (C-7), 28.0 (C-8) ppm. °C (lit  m.p. 104 °C (lit . AlthougMethyl (2E)-3-prop-2-enoate (4i). Compound 4i was obtained after stirring for 10 min and purified by flash chromatography using CHCl3/CH3CN (7:1). Amber oil; yield 15%; Rf = 0.28 (CHCl3:CH3CN = 7:1). The spectroscopic data were found to be identical to the ones described in Ref + = 248.0837; found: 248.0837. °C . Compound 5d was obtained after stirring for 90 min and purified by flash chromatography using cyclohexane/EtOAc (3:1). Amber solid; yield: 87%; Rf = 0.46 (cyclohexane:EtOAc = 3:1); m.p.: 119–120 °C : δ = 8.14 , 7.73 , 7.43 , 7.02 , 6.99  ppm; 13C NMR : δ = 183.9 , 183.6 , 166.1 , 138.9 (C-3), 138.6 , 134.5 , 129.8 , 128.5 , 121.2 , 113.2  ppm. °C . Compound 5e was obtained after stirring for 150 min at room temperature and purified by flash chromatography using cyclohexane/EtOAc (3:1). Amber solid; yield: 82%; Rf = 0.50 (cyclohexane:EtOAc = 3:1); mp: 128–130 °C. With the exception of the melting point, the compound was described as oil in the reference, and all spectroscopic data are identical to those given in Ref − = 194.0179; found: 194.0183.n in Ref . AlthougP. falciparum using the drug-sensitive strain NF54. Parasite proliferation was assessed by incorporation of [3H]-hypoxanthine using a modified version of [Antimalarial activity was determined in vitro against the erythrocytic stages of rsion of ; for detT. b. rhodesiense STIB900 as described in Refs. [Trypanocidal activity was determined in vitro against axenically grown bloodstream-forms of in Refs. ,77 and dCytotoxicity was determined in vitro against rat L6 myoblasts as described ; detailsμ-oxo-dimer 1 can be used instead of PIDA and PIFA for oxidative dearomatizations in aqueous media., This reagent provided the highest yields, especially in the conversion of phenols into substituted 4-hydroxy-cyclohexa-2,5-dienones. However, the laborious preparation prevents a general use of this oxidant.The 5c . In contrast, assays on antiprotozoal activity revealed high activity but no specific effects. The results presented in this paper demonstrate the potential of quinols and quinones for the development of new anti-infectives and identify PIDA as the most probable reagent for the preparation of these valuable compoundsMany of the synthesised compounds showed promising biological activities  as well


Trans-resveratrol is a natural polyphenol showing numerous biological properties, especially anti-tumoral and antioxidant activity. Among numerous resveratrol derivatives, aza-stilbenes, which bear an imine bound, show interesting biological activities. In the present study, we synthesized a series of imine analogs of trans-resveratrol (seven aza-stilbenes) following an easy and low-cost procedure of green chemistry. The toxicity of synthesized aza-stilbenes, which is currently unknown, was evaluated on murine neuronal N2a cells, comparatively to trans-resveratrol, by considering: cell density evaluated by staining with sulforhodamine 101; esterase activity, which is a criteria of cell viability, by staining with fluorescein diacetate; and transmembrane mitochondrial potential, which is known to decrease during cell death, by staining with DiOC6(3) using flow cytometry. In addition, the antioxidant activity was quantified with the KRL (Kit Radicaux Libres) assay, the DPPH  assay and the FRAP (ferric reducing antioxidant power) assay. The PAOT  score was also used. The aza-stilbenes provide different cytotoxic and antioxidant activities, which are either higher or lower than those of trans-resveratrol. Based on their cytotoxic and antioxidant characteristics, all synthesized aza-stilbenes are distinguished from trans-resveratrol. Among tAZA-ST  was purchased from Aldrich and 4-bromobenzaldehyde [1122-91-4] is purchased from TCI Europe .All reagents and solvents are purchased from commercial suppliers and used without further purifications. 4-aminophenol [123-30-8], 3-hydroxybenzaldehyde [100-83-4], salicylaldehyde [90-02-8] and 3,5-dihydroxybenzaldehyde [26153-38-8] are purchased from Alfa Aesar ; 4-hydroxybenzaldehyde [123-08-0] and −1. 1H (500 MHz) and 13C (126 MHz) NMR spectra were recorded on Brucker 500 MHz spectrometer. The chemical shifts are given in ppm relative to DMSO d6 . Coupling constants J are given in Hz. Multiplicities are given as follows: singlet (s), doublet (d), triplet (t), quadruplet (q) and multiplet (m). “CPh” designates protons and carbons of the aromatic ring bound to the carbon atom of imine, and “NPh” designates protons and carbons of the aromatic ring bound to the nitrogen atom of imine ”. High-resolution mass spectra (HRMS) were obtained on a Thermo LTQ-Orbitrap XL with ESI source. FTIR spectra were obtained on a Brucker Alfa spectrometer  in the range of 400–4000 cmof imine .General procedure for the synthesis of (hydroxyphenyliminomethyl)phenols1a–1g.2)  was stirred with an equimolar amount of an aromatic aldehyde 3a–g in 20 mL of distillated water, during 4 to 5 h at room temperature (20 °C). The solid product obtained 1a–1g was filtered, washed with water, air dried and recrystallized from ethanol, acetone, ethyl acetate or acetonitrile.4-aminophenol (1H and 13C NMR spectra and the HMBC spectra of aza-stilbenes 1a to 1g are shown in The 4-{[(4-hydroxyphenyl)imino]methyl}phenol (1a): recrystallized from acetone; yield 63%; m.p. 212–214 °C. IR: 3485.4 (ν/O-H), 1639.3 (ν/C = N), 1239.5 (ν/C-O). 1H NMR (DMSO-d6) δ: 10.00 , 9.38 , 8.43 , 7.72 , 7.11 , 6.85 , 6.77 . 13C NMR (DMSO-d6) δ: 160.1 (CPh C-1), 156.9 (C = N), 155.7 (NPh C-1), 143.2 (NPh C-4), 130.1 , 127.9 (CPh C-4), 122.1 , 115.6 , 115.5 . HRMS (ESI+) m/z: 214.0861 [M + H]+ calc. for C13H12NO2+, 214.0863, found 214.0861.3-{[(4-Hydroxyphenyl)imino]methyl}phenol (1b): no recrystallization; yield 87%; m.p. 193–195 °C. IR: 3296.5 (ν/O-H), 1622.2 (ν/C = N), 1214.7 (ν/C-O). 1H NMR (DMSO-d6) δ: 9.61 , 9.47 , 8.50 , 7.32 , 7.28 , 7.27 , 7.17 , 6.90–6.86 , 6.79 . 13C NMR (DMSO-d6) δ: 157.6 (CPh C-1), 157.2 (C = N), 156.2 (NPh C-1), 142.6 (NPh C-4), 137.8 (CPh C-3), 129.7 (CPh C-6), 122.5 , 119.9 (CPh C-2), 118.1 (CPh C-5), 115.7 , 113.9 (CPh C-4). HRMS (ESI+) m/z: 214.0862 [M + H]+ calc. for C13H12NO2+, 214.0863, found 214.0862.2-{[(4-Hydroxyphenyl)imino]methyl}phenol (1c): no recrystallization; yield 11%; m.p. 138–139 °C. IR: 3392.1 (ν/O-H), 3280.5 (ν/O-H), 1613.8 (ν/C = N), 1209.1 (ν/C-O). 1H NMR (DMSO-d6) δ: 13.41 , 9.67 , 8.90 , 7.59 , 7.37 , 7.32 , 6.96 , 6.93 , 6.84 . 13C NMR (DMSO-d6) δ: 160.7 (C = N), 160.6 (CPh C-1), 157.4 (NPh C-1), 139.7 (NPh C-4), 133.0 (CPh C-5), 132.7 (CPh C-6), 123.1 , 119.9 (CPh C-2), 119.4 (CPh C-3), 116.9 (CPh C-4), 116.4 . HRMS (ESI+) m/z: 214.0861 [M + H]+ calc. for C13H12NO2+, 214.0863, found 214.0 861.5-{[(4-Hydroxyphenyl)imino]methyl}benzene-1,3-diol (1d): washed with hot ethyl acetate; yield 60%; m.p. decomposed at 238 °C. IR: 3481.2 (ν/O-H), 3273.2 (ν/O-H), 1624.0 (ν/C = N), 1213.8 (ν/C-O). 1H NMR (DMSO-d6) δ: 9.46 , 9.44 , 8.39 , 7.16 , 6.78 , 6.76 , 6.32 . 13C NMR (DMSO-d6) δ: 158.6 (C = N), 157.4 (CPh C-1), 156.1 (NPh C-1), 142.6 (NPh C-4), 138.3 , 122.5 , 115.7 , 106.4 , 105.2 (CPh C-4). HRMS (ESI+) m/z: 230.0811 [M + H]+ calc. for C13H12NO3+, 230.0812, found 230.0811.4-{[(4-Hydroxyphenyl)imino]methyl}anisole (1e): recrystallized from ethanol; yield 40%; m.p. 188–190 °C. IR: 1602.8 (ν/C = N), 1221.3 (ν/C-O). 1H NMR (DMSO-d6) δ: 9.42 , 8.51 , 7.83 , 7.15 , 7.04 , 6.78 , 3.82 . 13C NMR (DMSO-d6) δ: 161.5 (CPh C-1), 156.6 (C = N), 155.9 (NPh C-1), 143.0 (NPh C-4), 130.0 , 129.4 (CPh C-4), 122.3 , 115.7 , 114.2 , 55.4 (CH3). HRMS (ESI+) m/z: 228.1018 [M + H]+ calc. for C14H14NO2+, 228.1019, found 228.1018.4-{[(4-Hydroxyphenyl)imino]methyl}bromobenzene (1f): recrystallized from acetonitrile; yield 33%; m.p. 205–206 °C. IR: 1618.7 (ν/C = N), 1223.9 (ν/C-O). 1H NMR (DMSO-d6) δ: 9.54 , 8.61 , 7.84 , 7.70 , 7.22 , 6.80 . 13C NMR (DMSO-d6) δ: 156.5 (CPh C-1), 155.9 (C = N), 142.2 (NPh C-1), 135.7 (NPh C-4), 131.8 , 130.0 , 124.3 (CPh C-4), 122.7 , 115.7 . HRMS (ESI+) m/z: 276.0018 [M + H]+ calc. for C13H11NOBr+, 276.0019, found 276.0018.2-{[(4-Hydroxyphenyl)imino]methyl}bromobenzene (1g): recrystallized from acetonitrile; yield 35%; m.p. 152–153 °C. IR: 1613.7 (ν/C = N), 1236.2 (ν/C-O). 1H NMR  δ: 9.62 , 8.79 , 8.11 , 7.74 , 7.50 , 7.44 , 7.23 , 6.83 . 13C NMR  δ: 156.9 (CPh C-1), 155.0 (C = N), 142.2 (NPh C-1), 134.3 (CPh C-2), 133.2 (CPh C-3), 132.6 (CPh C-4), 128.5 (CPh C-6), 128.1 (CPh C-5), 125.0 (NPh C-4), 122.7 , 115.9 . HRMS (ESI+) m/z: 276.0018 [M + H]+ calc. for C13H11NOBr+, 276.0019, found 276.0018.v/v) of heat-inactivated fetal bovine serum (FBS)   and 1% (v/v) of penicillin (100 U/mL)/streptomycin (100 mg/mL) (Pan Biotech). The cells were incubated at 37 °C in a humidified atmosphere  and passaged twice a week. The cells were seeded at 60,000 cells per well containing 1 mL of DMEM supplemented with 10% (v/v) heat-inactivated FBS and 1% antibiotics  in 24-well plates . The stock solutions of resveratrol  and aza-stilbenes (AZA-ST 1a to 1g) were prepared as follows: resveratrol  was prepared at 50 mM in absolute ethanol ; AZA-ST 1a to 1g were prepared at 50 mM in dimethyl sulfoxide . The aspects of resveratrol and aza-stilbenes in solution are shown in v/v).The mouse neuro-2a (N2a) neuroblastoma cell line , Manassas, VA, USA) was maintained in Dulbecco’s modified Eagle medium  containing 10% (Sulforhodamine 101 (SR101)  assay was used to assess the cytotoxic effect of resveratrol and AZA-ST on N2a cells at different concentrations ranging from 1.5 to 100 µM. SR101 is an anionic dye that electrostatically binds to cellular proteins . SR101 pCell viability was measured with the fluorescein diacetate (FDA)  assay, which considers esterase ,41. The 6(3)) . This fluorochrome accumulates in the mitochondria proportionally to the ΔΨm value [6(3) at 40 nM . The cells were immediately analyzed on a BD Accuri™ C6 flow cytometer . The loss of ∆Ψm is indicated by a decrease in the intensity of the green fluorescence collected through a band pass filter of 520 ± 10 nm. For each sample, 10,000 cells were acquired, and the data were analyzed with FlowJo  software. All assays were performed in triplicate.The variation of the mitochondrial transmembrane potential (∆Ψm) was measured using 3,3′-dihexyloxacarbocyanine iodide  test was used to assess the oils’ overall antioxidant activity by their ability to protect erythrocytes against a controlled free radical attack at varying concentrations ,21. DiluThe DPPH• radical is a stable soluble molecule characterized by its deep-violet color, with an absorption maximum at 515 nm. Antioxidants (AH) or other radical species (R•) are able to react with this stable radical (DPPH•) by providing an electron or hydrogen atom, thus reducing it to 2,2-diphenyl-1-hydrazine (DPPH-H) or a substituted analogous hydrazine (DPPH-R) characterized by colorless or pale-yellow color that could be easily monitored with a spectrophotometer. In the present study, the free radical scavenging activity was determined by the DPPH assay as previously described . DPPH• w3+ (ferric ions) to Fe2+ (ferrous ions), and the assay was carried using previously described methods [3+-TPTZ) is reduced to ferrous (Fe2+-TPTZ) with the formation of an intense blue color with an absorption maximum at 593 nm. Briefly, 2.3 mL of FRAP reagent  was mixed with 0.7 mL of the product (resveratrol or aza-stilbene 1a–1g) at different concentrations. The mixture was then incubated at 37 °C for 30 min in the dark. The absorbance was measured at 593 nm against a blank containing all the reagents, except the sample, using a spectrophotometer . The increase in absorbance of the reaction mixture indicates an increase in reduction capacity. Results of the samples were expressed as µMol Fe2+ reduced per mM of resveratrol and aza-stilbenes from a standard calibration curve of FeSO4∙7H2O.Ferric reducing antioxidant power (FRAP) is based on the reduction of Fe methods ,24. At l® Technology . This method was based on an electrochemical reaction with a molecule in a free radical state (mediator M•), following Equation 1 (Equation (1)) [The total antioxidant power of resveratrol and aza-stibenes was also determined by PAOT Liquidion (1)) .OxidizeAnalysis was started with electrochemical potential measurement (EP control t0) of the reaction medium (1 mL), and then 20 µL of sample was added. The potential product (EP product t4) was registered after 4 min of interaction between the sample that contains antioxidants and reduced mediator M•The variation ratio of the oxidized and reduced forms of the mediator M• during reaction gives an estimation of antioxidant activity in the sample by using Equation 2 (Equation (2))The results were expressed as PAOT score per liter (PAOT Score/L) of the analyzed sample.p-value less than 0.05 was considered statistically significant.The experimental results were statistically analyzed with GraphPad Prism 8.0 software . Data were expressed as the mean ± standard deviation (SD) and compared with a Student’s t-test. A


The structures of 1 and 2 were elucidated on the basis of extensive spectroscopic data and application of the CD technique. The potential biological activities of secoatractylohexone A were predicted by network pharmacology in silico, the result of which indicated that secoatractylohexone A may be used to treat type II diabetes.Secoatractylohexone A ( Atractylodes, belonging to the Asteraceae family, consists of about seven species distributed widely in Eastern Asia and among them, five species are found in China. Atractylodes lancea (Thunb.) DC., which is a perennial herb known as “Cangzhu”, has been reputed in Traditional Chinese Medicine for “strengthening spleen, removing cold, and improving eyesight” + and [2M+Na]+ peaks at m/z 453.2067  and 883.4214, corresponding to a molecular formula of C21H34O9, with five degrees of unsaturation. TLC analysis displayed a yellow spot with the spraying of 1% vanillin-H2SO4 reagent, followed by heating at 105 °C after 1–2 min, and a positive reaction of the Molish reagent.Compound 1  with C-1 (δ 40.3), C-3 (δ 170.3), and C-10 (δ 35.6); H-1  with C-2 (δ 36.0), C-4 (δ 211.2), C-5 (δ 59.8), C-9 (δ 27.0), C-10 (δ 35.6), and C-14 (δ 73.7); H-2ax  with C-1 (δ 40.3), C-3 (δ 170.3), C-5 (δ 59.8), and C-10 (δ 35.6); H-5  with C-1 (δ 40.3), C-2 (δ 26.0), C-4 (δ 211.2), C-6 (δ 28.3), C-7 (δ 46.2), and C-10 (δ 35.6); H-10  with C-8 (δ 26.9) and C-14 (δ 73.7). However, the correlation between C-3 (δ 170.3) and 15-CH3 , H-5  was not detected. Based on the information from the HBMC spectrum, it can be speculated that there was no direct bond between C-3 and C-4; and C-3, C-14 participated in the formation of a six-membered δ-lactone ring. Furthermore, the position of the glucosyl group was located at C-11 by the observed cross peak between the glucosyl H-1′  and C-11 (δ 80.3) in the HMBC spectrum. Therefore, compound 1 can be defined as a novel secoguaiane-type glucoside with a six membered δ-lactone.Several correlations were observed in HMBC spectrum such as H-14ax , resulting in a molecular structure as shown in The relative stereochemistry of 1 was assigned by a comparison of the measured and simulated electronic circular dichroism (ECD) spectra . As shown in S,5R,7R,10R)-1 reproduced the sign and intensity of the experimentally determined positive cotton effect (CE) at 216 and 287 nm, and the negative CE at 219 nm. Therefore, the absolute configuration of 1 was assigned to . The trivial name secoatractylohexone A was given to compound 1, and its description is -secoatractylo-δ-lactone 11-O-β-d-glucopyranoside. Compound 1 has an unexampled secoguaiane skeleton featuring a six-membered lactone ring. Searching the literature, it was found that no compound similar to the skeleton has been reported.The absolute configuration of compound S,5R,7R,10R)-seco-atractylolactone and -seco-atractylolactone 11-O-β-d-glucopyranoside +, [2M+Na]+, [M-C6H12O6+H]+ peak at m/z 437.2124 , 851.4292, 235.1697, respectively, in its ESI-HRMS spectrum, with five degrees of unsaturation. The TLC analysis displayed a cyan spot with the spraying of the 1% vanillin-H2SO4 reagent followed by heating at 105 °C after 1–2 min, and a positive reaction of the Molish reagent.Compound 1H and 13C NMR spectroscopic data in combination with analysis of the DEPT and HSQC spectrum indicated the presence of a carbonyl carbon, two quaternary carbons, ten -CH-, five sp3 methylenes, and three methyl carbons. The 13C NMR spectrum showed a carbonyl at δ 219.1, two alkene carbons δ 129.9 and 143.9, and three CH3 at δ 12.8, 23.9, and 24.0, respectively. Careful analysis of the NMR data indicated that it was a dehydrated atractyloside derivative. Acid hydrolysis and gas chromatographic analysis of the persilanizated derivative of the sugar indicated that the sugar was d-glucopyranose.The 1H NMR spectrum and 1H-1H COSY correlation spectra for 2 displayed signals for three methyl groups at δ 1.10 , δ 1.39 , and δ 1.40 , and five sp3 methylene proton signals (-CH2-CH2-) at δ 2.75 , 3.03 ; δ 1.26 , 2.61 ; δ 2.05 , 2.91 ; δ 4.28 , and δ 4.31 , 4.46 , respectively, and ten sp2 methines . The methines  and a olβ. The calc. ECD pattern for -2 showed good accordance with the experimental ECD of 2  to give seven fractions (frs. A–G). Fraction A (1.3 g) was chromatographed over silica gel CC to give four fractions (frs.A1–A4), fraction A4 (0.15 g) was passed through silica gel CC repeatedly (EtOAc:MeOH = 10:1) to give 6 (4.0 mg). Fraction B (3.0 g) was passed through a RP-C18 column  to give six fractions (frs. B1–B6), fraction B3 (0.9 g) was subjected to Sephadex LH-20  to give three fractions (frs. B3a–B3c) and fraction B4 (0.8 g) to give two fractions (frs. B4a–B4b), fraction B3a (35 mg) was subjected to semi preparative RP-HPLC  to 1 (5 mg), and fraction B4b (105 mg) was subjected to repeated semi preparative RP-HPLC (MeOH:H2O = 33:67) to give 2 (6 mg) and 5 (14 mg). Fraction C (8.0 g) was applied to a silica gel column eluted in a step gradient manner with CHCl3–MeOH (from 10:1 to 4:1) to afford fractions C1–C7. Fraction C3 (4.0 g) was subjected to Sephadex LH-20  to give four fractions (C3a-C3d), C3b (1.3 g) was passed through a RP-18 column  to give three fractions (C3b-1-C3b-3), and C3b-1 and C3b-3 were applied to a silica column to give 4 (7.5 mg) and 7 (90 mg), respectively. Fraction F (4.9 g) was applied to a silica gel column again to afford four fractions (frs. F1–F4), and fraction F4 (1.2 g) was passed through a RP-C18 column (MeOH: H2O = 5:95) to give 3 (140 mg).The fresh rhizome of A. lancea (42 kg) was extracted three times with 95% alcohol (60 L per time) at room temperature, the ethanol extract was concentrated at 50 °C under vacuum conditions, and then the residue was suspended in distilled water (10 L) to obtain an aqueous solution and successively extracted three times with ethyl acetate (10 L per time). The aqueous layer was passed through non-polar macroporous resin D-101 and eluted with water, 50% EtOH, and 90% EtOH. The 50% alcohol eluate (64 g) was chromatographed over a silica gel  column + , 883.4214 [2M+Na]+; 1H and 13C NMR shown in c 0.01, MeOH) λmax 206 (+), 219 (−), 287 (+) nm.-11,14-Dihydroxy-9-guaien-3-one 11-O-β-d-glucopyranoside (2): White amorphous powder; + , 851.4292 [2M+Na]+, 235.1697 [M-C6H12O6+H]+; 1H and 13C NMR shown in c 0.01, MeOH) λmax 198 (+), 346 (+) nm. (13): 13C-NMR  δ 44.0 (C-1), 37.9 (C-2), 79.6 (C-3), 75.3 (C-4), 52.5 (C-5), 31.9 (C-6), 45.9 (C-7), 24.4 (C-8), 31.2 (C-9), 81.2 (C-10), 80.6 (C-11), 24.0 (C-12), 24.9 (C-13), 68.6 (C-14), 16.0 (C-15), 98.6 (C-1’), 75.8(C-2’), 78.8 (C-3’), 71.8 (C-4’), 78.2 (C-5’), 62.8 (C-6’) [Atractyloside B -10,11,14-Trihydroxyguai-3-one 11-O-β-d-glucopyranoside (4): 13C-NMR  δ 46.7 (C-1), 39.3 (C-2), 219.4 (C-3), 52.2 (C-4), 44.1 (C-5), 36.7 (C-6), 50.8 (C-7), 23.4 (C-8), 37.0 (C-9), 74.5 (C-10), 80.3 (C-11), 23.9 (C-12), 24.4 (C-13), 69.6 (C-14), 12.9 (C-15), 98.6 (C-1’), 75.3 (C-2’), 78.9 (C-3’), 71.9 (C-4’), 78.1 (C-5’), 63.0 (C-6’) [-11,14-Dihydroxyguai-3-one 11-O-β-d-glucopyranoside (5): 13C-NMR  δ 43.6 (C-1), 45.6 (C-2), 218.8 (C-3), 52.3 (C-4), 48.9 (C-5), 35.7 (C-6), 50.1 (C-7), 25.6 (C-8), 28.8 (C-9), 48.0 (C-10), 80.4 (C-11), 24.1 (C-12), 24.2 (C-13), 66.0 (C-14), 12.6 (C-15), 98.6 (C-1’), 75.4 (C-2’), 78.9 (C-3’), 72.0 (C-4’), 78.1 (C-5’), 63.1 (C-6’) [-Seco-atractylolactone (6): 13C-NMR  δ 41.1 (C-1), 37.3 (C-2), 175.9 (C-3), 209.4 (C-4), 55.5 (C-5), 32.0 (C-6), 53.9 (C-7), 25.2 (C-8), 32.6 (C-9), 91.4 (C-10), 71.4 (C-11), 26.0 (C-12), 28.5 (C-13), 68.7 (C-14), 28.6 (C-15) [-Seco-atractylolactone 11-O-β-d-glucopyranoside (7): 13C-NMR  δ 41.1 (C-1), 37.4 (C-2), 175.8 (C-3), 209.9 (C-4), 55.5 (C-5), 31.8 (C-6), 52.2 (C-7), 25.2 (C-8), 32.8 (C-9), 91.4 (C-10), 79.6 (C-11), 22.7 (C-12), 25.5 (C-13), 68.7 (C-14), 28.8 (C-15), 98.9 (C-1’), 75.4 (C-2’), 79.0 (C-3’), 72.1 (C-4’), 78.3 (C-5’), 63.0 (C-6’) [ at 80 °C for 3 h in a water bath. Dioxane was removed and the solution was extracted with EtOAc (1 mL × 3). The EtOAc portion was washed with water and removed the EtOAc. The aqueous solution of the acid hydrolysis of each glycoside was neutralized by passing through an Amberlite MB-3 resin column eluted with water, then concentrated and dried. The dried sugar mixture was dissolved in pyridine (0.5 mL), and then treated with hexamethyl-disilazane (0.2 mL) and trimethylchlorosilane (0.1 mL) at room temperature for 6 h. After centrifugation, the above fraction was analyzed by GC analysis with authentic monosaccharides.Each glycoside (2 mg) was heated in 1 mL of 1 M HCl -1 and -2 were conducted by the Spartan 14 program [−1 energy window were initially optimized at the density functional theory (DFT)/Becke-3-Lee-Yang-Parr (B3LYP)/6-31G+(d) level. The frequency calculation was then conducted for the previously optimized conformers to obtain the corresponding relative Gibbs free energies (∆G). Boltzmann weighting factors (iP%) of each conformer were calculated on the basis of ∆G to eliminate the conformers that possess an inappreciable contribution. Subsequently, the conformers selected for ECD calculation were re-optimized by DFT calculations at the CAM-B3LYP/TZVP level. Then, the 20 lowest electronic transitions were calculated using the time-dependent density functional theory (TD-DFT) method at the CAM-B3LYP/TZVP level, and with methanol as the solvent employing the PCM model. All quantum chemical calculations were conducted using the Gaussian 09 program package [1 and 2, respectively.The conformational search for  with the target database limited to “Human Protein Targets Only” by the reverse docking algorithm. The screened targets were renamed to official gene names by using UniProt  with human normalization. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) ver. 6.8  and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways  were used to search for information about the functional annotation of candidate genes for extracting potential pathways that showed high accordance with these proteins. The Search Tool for the Retrieval of Interacting Genes (STRING) database  was utilized to hunt for the protein–protein interaction between candidate targets, with the organism defined to “Homo sapiens”, a confidence score > 0.9, and interaction number < 50. The protein–protein interaction networks were constructed using Cytoscape 3.7.2. Topological parameters of the network  were implemented using the network analyzer plug-in in Cytoscape to judge the importance of nodes in the given network.The candidate targets of compound Atratylodes lancea revealed the isolation and purification of two new (1 and 2) and five (3–7) known compounds. Compound 1 was clarified to be -secoatractylo-δ-lactone 11-O-β-d-glucopyranoside, named secoatractylohexone A, which has an unexampled secoguaiane skeleton featuring a six-membered lactone ring. Compound 2 has been clarified as -11,14-dihydroxyl-9-guaien-3-one 11-O-β-d-glucopyranoside, which has an uncommon scaffold. Furthermore, network pharmacology predicted the treatment of diabetes with secoatractylohexone A, along with the potential targets and mechanism. According to all of these shreds of evidence, it is believed that the potency of secoatractylohexone A is attributed to its dissimilar orientation. Therefore, further studies are required to confirm the potency of secoatractylohexone A.The crude extract of


Trypanosoma cruzi and affects 6–7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl  glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα[Galα]Galβ. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.Chagas disease (CD) is caused by the parasite  Trypanosoma cruzi. Historically limited to Latin America, CD has spread beyond its geographical boundaries due to global migratory flows and currently affects 6–7 million people worldwide Galβ covalently linked via 4-(N-succinimidomethyl)cyclohexane-1-carbonyl linkers. The oligosaccharide contains a 2,6-branched Gal unit present in some of the tGPI-MUC eriments . The cap strains ,45 were SHG11) with two terminal nonreducing α-Gal moieties and its conjugation with commercially available maleimide-derivatized BSA is presented. Structural analysis of tGPI-MUC O-glycans of T. cruzi  Galβ (TU TcII)  and preltGPI-MUC ,46.1 using the stereoselective introduction of α-Gal moieties with Kiso’s α-Gal donor 2 equipped with a 4,6-O-di-tert-butyl silylene group Galβ units/BSA, was determined by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS)  tri-TOF-MS) B. Subtra-TOF-MS) B.T. cruzi Colombiana (TcI tGPI-MUC) and Y (TcII tGPI-MUC) strains, as described in Materials and Methods. We used tGPI-MUC preparations from these two genotypes because they are responsible for most T. cruzi infections in North, Central, and South America and Spain Galβ or a similar glycotope, one could hypothesize that NGP11b (with 29 GU/BSA) has a higher density of that glycan than TcI and TcII tGPI-MUC. Glycan density has been a critical factor for modulating the binding of anti-glycan antibodies or lectins in different types of ELISA- or glycan array-based immunoassays, as previously demonstrated  were measured from one-dimensional spectra. Mass spectrometry (MS) of the carbohydrate derivatives was performed on a high-resolution JEOL Accu Time-of-Flight (TOF) mass spectrometer using an electrospray ionization (ESI) source. Protein derivatives were measured by MALDI-TOF-MS . The thiol-ene reaction was performed in a Rayonet RPR200 photochemical reactor  equipped with 16 UV lamps, at 350 nm.All chemicals were purchased as reagent grade and used without further purification from Thermo Fisher Scientific, Sigma-Aldrich, or Acros Organic, USA. The ACS grade solvents used for reactions were obtained from Thermo Fisher Scientific, and if necessary, further dried following standard procedures. Molecular sieves (MS 3 and 4 Å) were purchased from Alfa Aesar or Fisher Scientific, respectively, and activated under high vacuum and heat prior to use. Reactions were performed under an argon (Ar) atmosphere, under strictly anhydrous conditions, and monitored by thin-layer chromatography (TLC) on silica gel 60 F254 plates from EMD Millipore or Dynamic Adsorbents, Inc. Spots were detected under UV light and/or by charring with 2% sulfuric acid in ethanol. Compound purifications were performed by column chromatography on silica gel (40–60 μm) from Fisher Chemical, and the ratio between silica and crude product ranged from 50:1 to 200:1 --3,4-O-isopropylidene-β-D-galactoside (3): To a solution of the known galactoside acceptor 1 +: 1303.5305, found: 1303.5257; see Allyl 2,3-di-ceptor 1  --3,4-O-isopropylidene-β-D-galactoside (4): The fully protected trisaccharide 3  was dissolved in 12 mL of anhydrous THF in a plastic conical tube and cooled to 0 °C. Then, 120 μL of HF-pyr (70%) was added and stirred for 30 min at 0 °C, and then 1 h at rt under Ar. The reaction mixture was cooled again to 0°C and quenched with saturated NaHCO3 solution. Finally, the mixture was extracted with EtOAc, washed with water and brine, dried over MgSO4, concentrated, and purified by flash column chromatography on silica gel (EtOAc/hexanes = 4:1) to furnish the desilylated trisaccharide 4  as a white powder. fR 0.26 (EtOAc/hexanes = 4:1). 2Cl2). 1H NMR  δ 8.03–7.93 , 7.55–7.43 , 7.41–7.29 , 5.77–5.60 , 5.48 , 5.36 , 4.96 , 4.88 , 4.45 , 4.31 , 4.17 , 4.15–4.04 , 4.01–3.89 , 3.89–3.83 , 3.74 , 3.64–3.50 , 3.40–3.26 , 2.86–2.76 , 1.45 , 1.06 , 0.91–0.79  ppm. 13C NMR  δ 166.1 (C=O), 166.0, (2 × C=O), 165.8 (C=O), 133.5 , 133.4 , 133.3 , 129.9 , 129.8 , 129.7 , 129.6 , 129.5 , 129.4 , 128.6 , 128.5 , 117.8 (CH2), 110.3 (C), 101.3 , 96.8 , 96.7 , 78.1 (CH), 77.4 (CH), 73.5 (CH), 71.4 (CH), 71.0 (CH), 70.9 (CH), 70.4 (CH), 70.2 (CH2), 69.8 (CH), 69.3 (CH), 69.0 (2 × CH), 68.6 (CH), 66.6 (CH2), 63.8 (CH2), 63.2 (CH2), 28.1 (CH3), 26.1 (CH3) ppm. HR ESI-TOF-MS m/z calcd for C52H56NaO20 [M+Na]+: 1023.3263, found: 1023.3252, see Allyl 2,3-di-O-benzoyl-α-D-galactosyl-(1→2)--β-D-galactoside (5): To a solution of trisaccharide 4  in DCM (8 mL), H2O (1.0 mL) and TFA (1.0 mL) were consecutively added, and the mixture was vigorously stirred at rt for 30 min. After disappearance of starting material based on TLC, the resulting solution was twice co-evaporated with EtOH (10 mL). The residue was then further dried under vacuum and purified by flash column chromatography on silica gel (DCM/MeOH = 9:1) to afford the partially deprotected 5  as a colorless syrup. Rf 0.33 (DCM/MeOH = 9:1). 2Cl2). 1H NMR  δ 8.02-7.86 , 7.55–7.46 , 7.45–7.29 , 5.72–5.61 , 5.55 , 5.31 , 4.94 , 4.86–4.79 , 4.61 , 4.37–4.27 , 4.10 , 4.02–3.91 , 3.85–3.69 , 3.52  ppm. 13C NMR  δ 166.1 (C=O), 166.0 (2 × C=O), 165.8 (C=O), 133.7 , 133.2 , 133.1 , 133.0 , 129.7 , 129.6 , 129.5 , 129.4 , 129.3 , 129.2 , 128.2 , 128.1 , 116.1 (CH2), 102.6 , 96.2 , 96.1 , 75.6 (CH), 72.8 (CH), 72.2 (CH), 71.3 (2 × CH), 71.0 (CH), 70.32 (CH), 69.7 (CH2), 69.5 (CH), 69.2 (CH), 69.0 (CH), 67.7 (CH), 67.5 (CH), 66.4 (CH2), 61.1 (CH2), 61.0 (CH2) ppm. HR ESI-TOF-MS m/z calcd for C49H52NaO20 [M + Na]+: 983.2950, found: 983.2975; see Allyl 2,3-di-O-benzoyl-α-D-galactosyl-(1→2)--β-D-galactoside (6): To a solution of allyl trisaccharide 5  and AIBN  in anhydrous THF (3 mL) under Ar, thioacetic acid  was added, and the mixture was stirred under water cooling (~ 25 °C) for 6 h in a Rayonet UV reactor equipped with 350 nm lamps. The solution was then co-evaporated with toluene and concentrated to near dryness. The crude product was purified by flash column chromatography on silica gel (CHCl3/MeOH = 14:1) to afford the acyl-protected trisaccharide 6  as a white solid. Rf 0.33 (CHCl3/MeOH = 7:1). 1H NMR  δ 8.02–7.85 , 7.61–7.28 , 5.72–5.59 , 5.49 , 5.31 , 4.66–4.57 , 4.37–4.28 , 4.23 , 4.16–3.89 , 3.89-3.63 , 3.59–3.48 , 2.95–2.83 , 2.71–2.55 , 2.23  ppm. 13C NMR  δ 195.9 (C=O), 166.1 (C=O), 166.0 (2 × C=O), 165.8 (C=O), 133.2 , 133.1 , 133.0 , 129.7 , 129.6 , 129.5 , 129.3 , 129.2 , 128.3 , 128.2 , 128.1 , 103.5 , 96.1 , 96.0 , 75.6 (CH), 72.8 (CH), 72.1 (CH), 71.3 (2 × CH), 71.0 (CH), 70.2 (CH), 69.6 (CH), 69.3 (CH), 69.1 (CH), 67.7 (CH), 67.7 (CH2), 67.6 (CH), 66.3 (CH2), 61.0 (2 × CH2), 29.3 (CH2), 25.3 (CH2) ppm. HR ESI-TOF-MS m/z calcd for C51H56NaO21S [M+Na]+: 1059.2932, found: 1059.2925; see 3-(Acetylthio)propyl 2,3-di-S)2 was dissolved in 20 mL of anhydrous 0.25 M NaOMe, and stirred for 2 h under Ar at rt. The solution was then neutralized with Amberlyst-15, filtered through celite, concentrated, and finally dissolved in water and lyophilized. Initially, the unprotected mercaptopropyl trisaccharide SHG11 was produced, which oxidizes by handling on air within hours to the disulfide S)2 as an off-white solid. Rf 0.25 [(iPrOH/H2O = 5:1 w/3 drops AcOH (27 μL added to 12 mL of eluent)]. 2O). 1H NMR  δ 5.34 , 4.93 , 4.50 , 4.14–4.29 , 3.53–4.06 , 3.31 , 2.71–2.88 , 1.95–2.11 , 1.87  ppm. 13C NMR  δ 103.3 (C-1), 98.1 (C-1), 97.9 (C-1), 73.0 (CH), 71.5 (CH), 70.9 (CH), 70.6 (CH), 69.5 (CH), 69.3 (CH), 69.2 (2 × CH), 69.1 (CH), 68.7 (CH2), 68.3 (CH), 68.2 (CH), 66.3 (CH2), 61.2 (CH2), 60.9 (CH2), 34.3 (CH2), 28.4 (CH2), 23.3 (CH) ppm. HR ESI-TOF-MS m/z calcd for C42H74NaO32S2 [M+Na]+: 1177.3502, found: 1177.3310; see 3-Thiopropyl α-D-galactosyl-(1→2)-[α-D-galactosyl-(1→6)]-β-D-galactoside  solution (4 mg DTNB in 1 mL reaction buffer), and reacted for 15 min at rt. The absorbance at 412 nm was measured in a UV-vis spectrophotometer. The thiol concentration was determined using the molar extinction coefficient of 2-nitro-5-thiobenzoic acid (TNB), ε = 14,150 M−1 cm−1, and the amount of sugar conjugated  was calculated.The kit for the conjugation of the thiol-containing glycan ublished ,46. Brieg, rt. The mixture was washed with 1 mL of ultrapure water three times following the same procedure. The tube with the filtrate was then removed, and 500 µL of ultrapure water was added to the NGP11b solution remaining in the filter. Since a small amount of aggregation can occur, the solution/suspension was transferred onto a 2-mL Zeba spin desalting column , provided in the kit, that had been previously washed with 1 mL of ultrapure pure water 4 times by centrifugation at 1000× g for 2 min, rt. This procedure removed all salts and aggregated protein. The filtrate was lyophilized, and the solid NGP can be stored at −50 °C for at least 6 months. In our hands, this combination of filtration and size-exclusion chromatography avoids or minimizes aggregation of the NGP. To determine the NGP11b quantity, a solution of 1–2 mg of it in 1–3 mL of ultrapure water was prepared, and the concentration was determined with a Pierce BCA Protein Assay Reagent kit using a spectrophotometer at 562 nm.The conjugation mixture was diluted with ultrapure water to a volume of 1 mL and desalted using an Amicon Ultra 3K centrifugal filter and was centrifuged for 20 min at 4000× 7) and NGP11b, 1 µL of 7 (125 µg/mL) was combined in a 1.5 mL microcentrifuge tube with 1 µL NGP11b (670 µg/mL) and 2 µL of matrix . Two microliters of the sample-matrix mixture were spotted onto a 48-well steel MALDI plate and allowed to crystallize at room temperature for approximately 20 min. The mass spectra were acquired using a Shimadzu MALDI-8020 mass spectrometer set to linear mode with dithering at a scan range of 10,000 to 100,000 m/z. Data acquisition included a laser power of 110, laser repetition rate (Hz) of 50, accumulated shots 5, blast shots 2, profiles at 200, pulse extraction set to 66431, and a blanking mass of 15,000. Spectra were processed by Threshold Apex set at constant threshold, Gaussian smoothing, smoothing filter width of 200, and peak width of 2. MS-grade BSA standard  was used for calibration and internal references set at [BSA + H]+ = 66,431 and [BSA + 2H]2+ = 33,216, with a 5 ppm mass tolerance.To determine the mass of the maleimide-derivatized BSA  and Y  strains Galβ glycotope may be an immunodominant structure in infective forms of T. cruzi, or a mimic thereof. These results, together with the previous observation that CCD patient sera and NHS showed only a small difference in Ab reactivity to an NGP with α-Gal monosaccharide moieties Galβ-(CHmoieties , supportT. cruzi-positive and -negative sera into two distinct groups. These results showed that NGP11b was of superior diagnostic value when compared to the purified TcI or TcII tGPI-MUC and, therefore, has the potential to replace tGPI-MUC as a BMK for CCD in serological assays. By means of the reversed glycomics approach, we also demonstrated for the first time that a synthetic glycotope likely present on T. cruzi tGPI-MUC can potentially be used for the early assessment of parasiticidal drug efficacy in CCD patients. We demonstrated that the seroreactivity to the double α-Gal glycotope of NGP11b was significantly reduced 24 months following BZN chemotherapy in all six CCD patients. Nevertheless, further validation through a prospective, well-designed phase II clinical trial will determine the true value of NGP11b as a prognostic tool.Using a large cohort of 85 individual sera from CCD patients and healthy donors, we demonstrated by CL-ELISA with NGP11b that we can place T. cruzi-positive and -negative sera and on extending chemotherapy studies to longer periods of follow-up with various NGPs for a more accurate assessment of treatment outcomes.In summary, our results corroborate the antigenicity and specificity of the α-Gal epitopes in NGP11b. Due to significant differential antibody reactivities between sera of CCD patients and healthy individuals, NGP11b seems suitable as a diagnostic CCD BMK, and also shows potential for monitoring the follow-up serological status of CCD patients after chemotherapy. Current efforts are focused on the discovery and immunological evaluation of glycan-based BMKs that produce even greater antibody-differential reactivities between