Geranium collinum Steph is known in Tajik traditional medicine for its hepatoprotective, antioxidant, and anti-inflammatory therapeutic effects. The present study was conducted to evaluate of potential antidiabetic, antioxidant activities, total polyphenolic and flavonoid content from the different extracts  and individual compounds isolated of the root parts of G. collinum. The 50% aqueous-ethanolic extract possesses potent antidiabetic activity, with IC50 values of 0.10 μg/mL and 0.09 μg/mL for the enzymes protein-tyrosine phosphatase (1B PTP-1B) and α-glucosidase, respectively. Phytochemical investigations of the 50% aqueous-ethanolic extract of G. collinum, led to the isolation of ten pure compounds identified as 3,3′,4,4′-tetra-O-methylellagic acid (1), 3,3′-di-O-methylellagic acid (2), quercetin (3), caffeic acid (4), (+)-catechin (5), (–)-epicatechin (6), (–)-epigallocatechin (7), gallic acid (8), β-sitosterol-3-O-β-d-glucopyranoside (9), and corilagin (10). Their structures were determined based on 1D and 2D NMR and mass spectrometric analyses. Three isolated compounds exhibited strong inhibitory activity against PTP-1B, with IC50 values below 0.9 μg/mL, more effective than the positive control (1.46 μg/mL). Molecular docking analysis suggests polyphenolic compounds such as corilagin, catechin and caffeic acid inhibit PTP-1B and β-sitosterol-3-O-β-d-gluco-pyranoside inhibits α-glucosidase. The experimental results suggest that the biological activity of G. collinum is related to its polyphenol contents. The results are also in agreement with computational investigations. Furthermore, the potent antidiabetic activity of the 50% aqueous-ethanolic extract from G. collinum shows promise for its future application in medicine. To the best of our knowledge, we hereby report, for the first time, the antidiabetic activity of G. collinum.The root of  In recent years herbal medicines have started to gain importance as a source of hypoglycemic agents. It is estimated that more than one thousand plant species are being used as folk medicine for diabetes . The bioProtein tyrosine phosphatase (PTP-1B), is a negative regulator of insulin signaling; it inhibits insulin-stimulated tyrosine phosphorylation of insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and is a therapeutic target for type 2 diabetes . AcarbosGeranium collinum Stephan ex Willd. is a perennial plant that belongs to the family Geraniaceae. In tropical and subtropical areas of the globe, there are 810 species in the family, which belong to eight genera. In Tajikistan there are more than 20 species, which belong to three genera, namely Pelargonium L’Hér., Geranium L. and Erodium L’Hér. G. collinum is one of the most common plants in Central Asia [G. collinum grows at an altitude of 800–2700 m above sea level, in the belts of black wood, juniper, mixed grass steppes and subalpine meadows, mainly along rivers and streams. The literature shows that the plant contains phenols , saponins, tannins, carbohydrates , vitamin C and flavonoids  [ral Asia . In TajiG. collinum has been used for the treatment of rheumatism, gout, dysentery, external and internal bleeding, as well as in the treatment of skin wounds, eczema, scabies, tenosynovitis and pruritus [G. collinum by studying their effects on inhibition of α-glycosidase enzymes and protein-tyrosine phosphatase (PTP-1B). Additionally, antioxidant activity along with total polyphenolic compounds and total flavonoids contents in the root extracts of the G. collinum plant were also determined.pruritus . This plpruritus . Studiespruritus . The pre2O, 30%, 50%, and 100% ethanol were 19 g/100 g, 21 g/100 g, 28 g/100 g and 22 g/100 g, respectively.The method of extracting plant material under reflux was selected due to its routine application, requiring easily available apparatus and glassware, and high extractive yield. Different combinations of ethanol with water  and pure water were evaluated in order to determine the corresponding extraction efficiencies and to identify the optimal solvent and time of extraction. Extraction was conducted for various extraction time durations using the optimal extraction conditions. The extract recovery yield appeared to increase as the extraction time increased up to 3 h, but further extension of the extraction time did not result in any further increase of extract recovery yield. No big difference in the extractive yield was observed between 2 h and 3 h and in the case of 70% ethanol 2 h extraction delivered more yield than 3 h. Therefore, in order to save energy and time, the optimal extraction time was set to be 2 h. All of the extracts obtained with 2 h extraction times were used for quantification of total polyphenols, flavonoids and their pharmacological activities. 1H- and 13C-NMR, including HMQC, HMBC and DEPT) and by comparison with published literature data. They were thus identified as 3,3′,4,4′-tetra-O-methylellagic acid [O-methylellagic acid [O-β-d-glucopyranoside [The structures of all isolated compounds were elucidated by analyzing their spectral data -epigic acid , gallic gic acid , β-sitosranoside  and coriranoside  . The highest amounts of total polyphenolic compounds were calculated as 349.84 ± 0.21 and 180.14 ± 0.11 mg GAE/g of the dried extract in 50% ethanol and 70% ethanol extracts, respectively . Using this equation, quantity of total flavonoids (x) with high contents in 50% and 70% ethanol extracts were determined as 96.07 ± 0.08 and 75.31 ± 0.07 mg quercetin equivalents (QE)/g of the dried extract  of the 50% ethanol, 70% ethanol, absolute ethanol and water extracts were: 11.21 ± 0.49, 12.69 ± 0.6, 11.23 ± 0.7 and 15.17 ± 0.84, respectively. The antioxidant activity of several phenolic compounds such as catechin, gallic acid, caffeic acid and chlorogenic acid have been previously evaluated . The majG. collinum were obtained after a period of extraction of 2 h. They were assayed for their inhibitory activities against PTP-1B and α-glycosidase, delivering strong activity, with IC50 values ranging from 0.1 µg/mL to 1.98 µg/mL . Results of α-glucosidase inhibitory activities of root extracts are presented on 1–10 from G. collinum were evaluated. Catechin, epicatechin and corilagin delivered the strongest activities, with IC50 values of 0.62, 0.23 and 0.87, respectively. That was more than the known PTP-1B inhibitor (1.46 μg/mL). Catechin, epicatechin, corilagin and quercetin have shown potent antidiabetic activities in the α-glucosidase inhibition assay. IC50 values of the all the samples are presented in Flavonoids and phenolic glycosides of plant extracts have shown promising effect as inhibitors of PTP-1B and α-glucosidase. They can be used in pharmaceutical formulations against type 2 diabetes mellitus ,20,21. T98 µg/mL . Among tThe experimental results suggest promise for the future application of plant extracts for the development of phytopharmaceuticals and food formulations against diabetes. Nevertheless, toxicological studies should be carried out to ascertain the boundary between health beneficial effects and the risk of possible toxicity.dock) and normalized docking scores (DSnorm) of the G. collinum ligands with the protein targets are summarized in In order to provide some insight as to which compound(s) may be responsible for the antidiabetic activity, a molecular docking study was carried out on two different protein structures of human PTP-1B and two different protein structures of human α-glucosidase. Molegro re-rank energies (EO-β-d-glucopyranoside), on the other hand, showed docking preference for α-glucosidase.Polyphenolic ligands such as epigallocatechin or caffeic acid showed preferential docking with HsPTP1B, although the docking energies were not as exothermic as the co-crystallized ligands for these proteins. Daucosterol (β-sitosterol-3-d-glucose moiety of daucosterol overlays the 6-amino-4-(hydroxymethyl)-4-cyclo-hexene-1,2,3-triol group of the co-crystallized α-acarbose, occupying a cavity on the protein surrounded by hydrogen-bonding amino acids His1584, Asp1526, and Asp1279  spectrometer. Melting points were determined using a BUCHI Melting Point B-540 apparatus . Column chromatographic separation was performed on Sephadex LH-20 gel  and silica gel . The spots on the TLC plate were identified by spraying with 5% H2SO4 solution in EtOH and heating the plate at about 105 °C. UPLC analysis was performed on a Waters Acquity UPLC™ system  equipped with binary solvent delivery pump, an auto sampler, and a photodiode array detector (PAD). The instrument was controlled by the Waters Empower 2 software. The chromatographic separation was performed using a Waters Acquity BEH Shield C18 column , operated at 35 °C. Absolute ethanol and acetone were purchased from Tianjinshi Baishi Chemicals Company . DMSO, DPPH, pNPP, NaOH, NaCl, vitamin C, quercetin (98%), gallic acid (≥97%), aluminum chloride, sodium acetate and Folin-Ciocalteau reagent (2 N) were purchased from Sigma-Aldrich GmbH . All the chemicals and reagents were of analytical grade and double-distilled water was used throughout the experiment. NMR spectra recorded on Varian MR-400  was weighed accurately and extracted with each of the aforesaid solvents (500 mL) for 1, 2 or 3 h. The extracts were reduced to dryness under reduced pressure. The dried residue of 50% aqueous ethanol extract (8 g) was mixed thoroughly with 32 g of dry silica gel (200–300 mesh). The mixture was dried in the oven at 45 °C and adsorbed on a 320 g silica gel column (1500 cm × 3.5 cm). Elution was performed in a gradient mode starting with petroleum ether−ethyl acetate (7:1). The polarity of the eluent was gradually increased using petroleum ether–ethyl acetate (4:1–0:1) and ethyl acetate−methanol (9:1–2:1), respectively. The fractions were analyzed by silica gel TLC using the mobiles phases . Spots were visualized by heating at 110 °C silica gel plates sprayed with 5% H2SO4 in EtOH. Similar fractions were combined after developing their thin layer chromatography (TLC) profiles, giving six fractions. Fraction 2 was applied onto a silica gel (100–200 mesh) column (750 cm × 3 cm) and eluted with chloroform–methanol (8:1) to yield compounds 1, 2, 3. Compound 4 was obtained from combined fraction 3 on a Sephadex LH-20 (1200 cm × 1.5 cm) column by eluting with chloroform–methanol (4:1). Fraction 4 was purified with methanol–chloroform (9:1) on a Sephadex LH-20 (1200 cm × 1.5 cm) column to give compounds 5 and 6. Pure compounds 7 and 8 were purified from fraction 5 with methanol–acetone (1:1) using Sephadex LH-20 (1200 cm × 1.5 cm) column chromatography. Compound 9 was isolated from fraction 6 using methanol–water (4:1) as eluent via Sephadex LH-20 (1200 cm × 1.5 cm) column chromatography. Compound 10 was isolated from fraction 5 via UPLC with PDA detector at the Rt = 18.26 min. The mobile phase consisted of 0.2% formic acid-water (A) and acetonitrile (B) with a gradient elution of 95% A (0–3 min), 95–90% A (3–20 min), 90–89% A (20–20.5 min), 89% A (20.5–29 min). Chromatograms were recorded at 254 nm and a flow rate of 250 µL·min−1 was used injecting 500 µL of the sample.Extraction efficiency was determined based on the total percent value of the amount extracted using different mixtures of aqueous ethanol  and pure water employing the reflux extraction procedure. Each time root part of 3,3′,4,4′-Tetra-O-methylellagic acid (1). Colorless needle-like crystals (from pyridine), m.p. 223–225 °C, 1H-NMR : δ 7.65 , 4.10 , 4.12 ; 13C-NMR : δ 111.12 , 140.92 , 140.15 , 153.74 , 107.41 , 113.29 , 158.23 , 60.97 (2 × OCH3), 56.69 (2 × OCH3).3,3′-Di-O-methylellagic acid (2). Yellow powder (MeOH), m.p. 210–212 °C, 1H-NMR : δ 7.52 , 4.04 , 10.73 ; 13C-NMR : δ 111.59 , 141.34 , 140.36 , 152.32 , 111.59 , 112.25 , 158.60 , 61.09 (2 × OCH3).Caffeic acid (3). Pale yellow crystals (MeOH), m.p. 223–225 °C, 1H-NMR : δH 7.36 , 6.97 , 6.91 , 6.70 , 6.12 , 13C-NMR : δc 167.95 (C-9), 148.18 (C-4), 145.57 (C-3), 144.63 (C-7), 125.70 (C-1), 121.19 (C-6), 115.75 (C-5), 115.12 (C-8), 114.65 (C-2).Quercetin (4). Yellow needles (MeOH), m.p. 314–315 °C, 1H-NMR : δ 6.17 , 6.40 , 6.86 , 7.51 , 7.65 , 12.47 , 10.77 , 9.59 , 9.36 , 9.30 ; 13C-NMR : δ 146.79 (C-2), 135.72 (C-3), 175.84 (C-4), 160.72 (C-5), 98.18 (C-6), 163.89 (C-7), 93.36 (C-8), 156.13 (C-9), 103.01 (C-10), 121.95 (C-1′), 115.61 (C-2′), 145.06 (C-3′), 147.71 (C-4′), 115.05 (C-5′), 119.97 (C-6′). (+)-Catechin (5). Colorless crystals (MeOH), m.p. 228–229 °C, 1H-NMR : δ 4.57 , 4.00 , 2.91 , 2.53 , 6.03 , 5.88 , 6.90 , 6.80 , 6.76 , 3.90 , 8.17 , 7.98 , 7.82 , 7.88 ; 13C-NMR : δ 82.72 (C-2), 68.34 (C-3), 28.82 (C-4), 157.20 (C-5), 96.13 (C-6), 157.73 (C-7), 95.46 (C-8), 156.92 (C-9), 100.66 (C-10), 132.22 (C-1′), 115.24 (C-2′), 145.62 (C-3′), 145.69 (C-4′), 115.71 (C-5′), 120.07 (C-6′). (–)-Epicatechin (6). Pale red powder (MeOH), m.p. 224–226 °C, 1H-NMR : δ 5.40 , 4.75 , 3.44 , 3.57 , 6.71 , 6.69 , 7.30 , 7.37 , 7.95 , 11.26 , 11.47 ; 13C-NMR : δ 80.59 (C-2), 67.43 (C-3), 30.18 (C-4), 159.20 (C-5), 97.14 (C-6), 159.12 (C-7), 96.28 (C-8), 158.12 (C-9), 100.68 (C-10), 132.64 (C-1′), 116.85 (C-2′), 147.43 (C-3′), 147.33 (C-4′), 116.60 (C-5′), 119.89 (C-6′).(–)-Epigallocatechin  (7). Colorless crystals (MeOH), m.p. 236–238 °C, 1H-NMR : δ 4.52 , 3.98 , 2.87 , 2.53 , 6.02 , 5.88 , 6.46 , 3.92 , 8.35–7.50 ; 13C-NMR : δ 82.74 (C-2), 68.30 (C-3), 28.43 (C-4), 157.19 (C-5), 96.08 (C-6), 157.68 (C-7), 95.42 (C-8), 156.85 (C-9), 100.57 (C-10), 131.53 (C-1′), 107.19 (C-2′), 146.25 (C-3′), 133.29 (C-4′), 146.25 (C-5′), 107.19 (C-6′).Gallic acid (8). Colorless needles (MeOH), m.p. 245–246 °C, 1H-NMR : δ 7.07 ; 13C-NMR : δ 170.38 (COOH), 146.3, 110.30 , 139.53 (C-4), 121.88 (C-1). d-glucopyranosideβ-Sitosterol-3-O-β- (9). Colorless powder (MeOH/H2O), m.p. 210–211 °C. Compound 9 was identified by comparison with an authentic standard of β-sitosterol-3-O-β-d-glucopyranoside by TLC and comparision with published literature data.d-glucose)Corilagin . Pale yellow powder (MeOH). The 1H- and 13C-NMR spectra of 10 showed characteristic signals for a hydrolyzable tannin with galloyl, 1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl (HHDP) and glucose units. 1H-NMR : 7.03 , 6.59 , 6.50 , 6.25 , 4.62 , 4.36 , 4.29–4.22 , 3.96 , 3.88 ; 13C-NMR : 165.3 , 145.9 , 139.8 , 119.3 , 109.4 , 167.4 and 167.0 (C=O HHDP), 145.1 and 145.0 (C-5′ and C-5′′), 144.6 and 144.3 (C-3′ and C-3′′), 135.8 and 135.7 (C-4′ and C-4′′), 124.1 and 123.4 (C-1′ and C-1′′), 116.1 and 115.8 (C-6′ and C-6′′), 107.2 and 106.3 (C-2′ and C-2′′), 92.5 (C-1′′′ anomeric carbon), 77.8 (C-3′′′), 76.6 (C-5′′′), 71.9 (C-2′′′), 62.4 (C-4′′′), 64.2 (C-6′′′).G. collinum was reconstituted in 20 mL of 70% methanol and this extract was used for the determination of total polyphenolic compounds and total flavonoids contents. Total polyphenolic compounds were determined by Folin-Ciocalteau [Each dried root extract (1 g each) of ocalteau ,26 methoocalteau . The enzyme activity was measured using the PTP-1B -2-ethylbenzofuran-6-sulfonic acid-(4-(thiazol-2-ylsulfamyl)-phenyl)-amide) enzyme inhibition assay according to the literature ,28,29,30norm) based on the molecular weight: DSnorm = 7.2 × Edock/MW⅓ [Protein-ligand docking studies were carried out based on the structures of two human protein tyrosine phosphatase 1B crystal structures  scavenging assay procedure as published in the literature ,41. The G. collinum growing in Tajikistan can provide a natural source of antidibetical drugs and antioxidants. Our investigations suggest the development of herbal formulations based on the roots parts of G. collinum growing in Tajikistan, however, further toxicological and dose determination studies are required to realize an effective phytopharmaceutical. The 50% ethanol extract delivered 10 pure biologically active compounds. Based on molecular docking, we conclude that polyphenolic constituents are likely responsible for the inhibition of PTP-1B while the sterol glucoside (daucosterol) is a likely inhibitor of human α-glucosidase. Our investigation clearly showed that other fractions also have potent activities and deserve further investigation in the future. In the present investigation, phenolic compounds and steroid glycoside were identified for the first time from roots of G. collinum. The alcoholic extracts of G. collinum exhibited strong antidiabetic activity.The underground part of


H-1,3-thiazine-2-thiones constitute a so far rather unexplored class of compounds, with the latest report dating back more than two decades. Thiazine-2-thiones contain an endocyclic dithiocarbamate group, which is often found in pesticides, in substrates for radical chemistry and in synthetic intermediates towards thioureas and amidines. We now report the multicomponent reaction (MCR) of in situ-generated 1-azadienes with carbon disulfide. With this reaction, a one-step protocol towards the potentially interesting 3,6-dihydro-2H-1,3-thiazine-2-thiones was established and a small library was synthesized.Non-fused 3,6-dihydro-2 An example of the use of MRSs in diversity generating MCRs is the coupling of in situ generated 1-azadienes with a range of iso(thio)cyanates .,22.21,22via stabilized intermediate 12.The subsequent reaction with carbon disulfide may proceed by either of two possible routes . The fir10 were prepared in situ as described by us earlier (6Me6) as a quantitative internal standard, crude 1H-NMR measurements showed complete product formation after sixteen hours (68%) and only little degradation of the product, even after three days (66%). The structure of 4,6-diphenyl-3,6-dihydro-2H-1,3-thiazine-2-thione (1a) was confirmed by X-ray crystallography .The 1-azadienes  earlier  2]. Car. Car10 wlography . CCDC 86H-1,3-thiazine-2-thiones was synthesized to study the scope of the reaction. Aliphatic and aromatic, as well as sterically demanding inputs, were used  and 1g (entry 8) could not be isolated in pure form by flash chromatography. In case of 1g (entry 8), the product could not be separated from the remaining aldehyde starting material, while in case of 1f (entry 7) the product was contaminated with unknown side products.Because of the presence of excess of starting materials and side products, attempts were made to purify the crude reaction products by recrystallization. Methanol proved to be a suitable solvent for crystallization of products 1d (entry 5) was not formed, most likely due to steric hindrance, and an equimolar mixture of 2,6-dimethylbenzonitrile and the mesitylene carboxaldehyde was recovered after column chromatography. This suggests that first stage of the reaction, i.e., the nucleophilic attack of the deprotonated phosphonate on the nitrile, did not take place and apparently is particularly sensitive to steric hindrance. The second stage of the reaction, i.e., the Horner-Wadsworth-Emmons-type reaction of the aldehyde, seems less influenced by steric bulk, as indicated by the fact that 1e (entry 6) is formed, albeit in lower yield (30%). Steric hindrance does not seem to play a significant role in case of mono-ortho-substitution , as products 1h, 1j, and 1l are obtained in similar yields as 1a. Furthermore, electronic factors do not seem to play a significant role, as both electron-donating and electron-withdrawing groups are allowed on both the nitrile and the aldehyde input with comparable yields in all cases (entries 8–13).Aliphatic nitrile or aldehyde inputs give reduced yields in this reaction entries 3 and 4), which is in agreement with earlier observations ,3. Compo and 4, w1 are stable in the solid state, it should be noted that the products slowly decompose over time in solution. This is one of the reasons for the difficulties in purifying some of the products, e.g., 1f and 1g. The degradation is more pronounced in crude mixtures in the presence of air, than in purified products kept under inert atmosphere. However, more studies are necessary to pinpoint the nature of this degradation.Although all products 1H- and 13C- nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 (500.23 MHz and 125.78 MHz respectively) or a Bruker Avance 400 (400.13 MHz and 100.61 MHz respectively) instrument with chemical shifts (δ) reported in ppm downfield from tetramethylsilane. Electrospray Ionization (ESI) mass spectrometry was carried out using a Bruker micrOTOF-Q instrument in positive ion mode . Column chromatography was performed with flash silica gel (40–63 μm) and a mixture of ethyl acetate and cyclohexane. Compounds in Thin Layer Chromatography (TLC) were visualized by UV detection. THF was dried and distilled from sodium potassium alloy and benzophenone prior to use. Diethyl methylphosphonate was prepared as described earlier  284.0538  [MH+−CS2] 208.1108 ; Melting point: 125–128 °C (decomp.).4-Isopropyl-6-phenyl-3,6-dihydro-2H-1,3-thiazine-2-thione (1b)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , isobutyronitrile , benzaldehyde  and carbon disulfide followed by flash column chromatography (EtOAc/c-hex = 1:15) afforded 1b as a yellow solid . 1H-NMR  δ (ppm) 8.62 , 7.39–7.24 , 5.15 , 4.88 , 2.46 , 1.22 , 1.21 ; 13C-NMR  δ (ppm) 193.3 (C), 143.6 (C), 140.5 (C), 129.1 (2 × CH), 128.3 (CH), 127.8 (2 × CH), 99.6 (CH), 46.3 (CH), 32.4 (CH), 20.8 (CH3), 20.7 (CH3); HRMS [MH+] 250.0712  [MH+−CS2] 174.1275 ; Melting point: 94–95 °C.6-Isopropyl-4-phenyl-3,6-dihydro-2H-1,3-thiazine-2-thione (1c)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , benzonitrile , isobutyraldehyde  and carbon disulfide followed by flash column chromatography (EtOAc/c-hex = 1:15) afforded 1c as a light yellow solid . 1H-NMR  δ (ppm) 8.91 , 7.43 , 5.45 , 3.68 , 2.03 , 1.07 , 1.06 ; 13C-NMR  δ (ppm) 194.9 (C), 138.4 (C), 134.9 (C), 129.8 (CH), 129.2 (2 × CH), 125.9 (2 × CH), 102.6 (CH), 49.7 (CH), 34.7 (CH), 18.9 (CH3), 18.6 (CH3); HRMS [MH+] 250.0710  [MH+−CS2] 174.1273 ; Melting point: 93–95 °C.6-Mesityl-4-phenyl-3,6-dihydro-2H-1,3-thiazine-2-thione (1e)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , benzonitrile , mesitylene carboxaldehyde  and carbon disulfide followed by flash column chromatography (EtOAc/c-hex = 1:15) afforded 1e as a yellow foam . 1H-NMR  δ (ppm) 9.02 , 7.48–7.42 , 6.89 , 5.88 , 5.54 , 2.45 , 2.28 ; 13C-NMR  δ (ppm) 196.6 (C), 138.3 (3 × C), 137.5 (C), 134.6 (C), 129.9 (C), 129.8 (2 × CH), 129.3 (3 × CH), 125.8 (2 × CH), 105.6 (CH), 42.6 (CH), 20.9 (3 × CH3); HRMS [MH+] 326.1020  [MH+−CS2] 250.1585 .6-(2-(Trifluoromethyl)phenyl)-4-(4-(trifluoromethyl)phenyl)-3,6-dihydro-2H-1,3-thiazine-2-thione (1f)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , p-trifluoromethylbenzonitrile , o-trifluoromethylbenzaldehyde  and carbon disulfide followed by flash column chromatography (EtOAc/c-hex = 1:15) afforded an inseparable mixture of 1f and impurities (367 mg) 1H-NMR  δ (ppm) 9.12 , 8.10–7.20 , 5.60 , 5.47 ; HRMS [MH+] 420.0255  [MH+−CS2] 344.0847 .6-(2-methoxyphenyl)-4-(4-(trifluoromethyl)phenyl)-3,6-dihydro-2H-1,3-thiazine-2-thione (1g)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , n-butyllithium , p-trifluoromethylbenzonitrile , p-methoxybenzaldehyde  and carbon disulfide followed by flash column chromatography (EtOAc/c-hex = 1:15) afforded an inseparable mixture of 1g and impurities (599 mg). 1H-NMR  δ (ppm) 9.10 , 7.71 , 7.61 , 7.28 , 6.90 , 5.67 , 5.47 , 3.81 ; HRMS [MH+] 382.0515  [MH+−CS2] 306.1085 .4-(4-Methoxyphenyl)-6-(2-(trifluoromethyl)phenyl)-3,6-dihydro-2H-1,3-thiazine-2-thione (1h)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , p-methoxybenzonitrile , o-trifluoromethylbenzaldehyde  and carbon disulfide and subsequent recrystallization from MeOH afforded 1h as a light yellow solid . 1H-NMR  δ (ppm) 9.09 , 7.71 , 7.67 , 7.60 , 7.46-7.38 , 6.95 , 5.44 , 5.41 , 3.84 ; 13C-NMR  δ (ppm) 192.4 (C), 161.0 (C), 139.5 (C), 138.2 (C), 133.0 (CH), 130.8 (CH), 127.4 , 127.4 (2 × CH), 126.0 , 125.1 (CH) 123.5  114.6 (2 × CH), 101.1 (CH), 55.5 (CH3), 42.2 (CH); HRMS [MH+] 382.0523  [MH+−CS2] 306.1084 ; Melting point: 147–148 °C (decomp.).6-(2-Methoxyphenyl)-4-(4-methoxyphenyl)-3,6-dihydro-2H-1,3-thiazine-2-thione (1i)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , p-methoxybenzonitrile , p-methoxybenzaldehyde  and carbon disulfide followed by flash column chromatography (EtOAc/c-hex = 1:15) afforded 1i as an orange foam . 1H-NMR  δ (ppm) 9.04 , 7.40 , 7.28 , 6.95 , 6.89 , 5.50 , 5.04 , 3.84 , 3.80 ; 13C-NMR  δ (ppm) 193.7 (C), 160.9 (C), 159.7 (C), 138.3 (C), 131.7 (C), 129.2 (2 × CH), 127.4 (2 × CH), 127.0 (C), 114.6 (2 × CH), 114.5 (2 × CH), 102.5, (CH), 55.5 (CH3), 55.4 (CH3), 46.4 (CH); HRMS [MH+] 344.0741  [MH+−CS2] 268.1310 .6-(2-Methoxyphenyl)-4-(4-methoxyphenyl)-3,6-dihydro-2H-1,3-thiazine-2-thione (1j)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , p-methoxybenzonitrile , o-methoxybenzaldehyde  and carbon disulfide and subsequent recrystallization from MeOH afforded 1j as a light orange solid . 1H-NMR  δ (ppm) 9.00 , 7.43 , 6.98–6.93 , 6.90 , 5.50 , 5.36 , 3.87 , 3.85 ; 13C-NMR  δ (ppm) 194.3 (C), 160.8 (C), 156.4 (C), 138.8 (C), 129.4 (CH), 128.2 (CH), 127.4 (2 × CH), 127.2 (C), 120.9 (CH), 114.6 (2 × CH), 110.8 (CH), 100.8 (CH), 55.6 (CH3), 55.5 (CH3), 40.0 (CH); HRMS [MH+] 344.0755  [MH+−CS2] 268.1318 ; Melting point: 139–143 °C (decomp.).4,6-Bis(4-bromophenyl)-3,6-dihydro-2H-1,3-thiazine-2-thione (1k)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , p-bromobenzonitrile , p-bromobenzaldehyde  and carbon disulfide followed by flash column chromatography (EtOAc/c-hex = 1:15) afforded 1k as a red foam . 1H-NMR  δ (ppm) 9.02 , 7.58 , 7.50 , 7.34 , 7.22 , 5.57 , 5.01 ; 13C-NMR  δ (ppm) 193.0 (C), 138.6 (C), 138.1 (C), 133.3 (C), 132.5 (2 × CH), 132.4 (2 × CH), 129.5 (2 × CH), 127.6 (2 × CH), 124.4 (C), 122.7 (C), 103.2 (CH), 46.1 (CH); HRMS [MNa+] 461.8557  [MH+−CS2] 363.9304 .4,6-Bis(4-bromophenyl)-3,6-dihydro-2H-1,3-thiazine-2-thione (1l)According to General procedure I, the reaction between diethyl methylphosphonate, n-butyllithium , o-bromobenzonitrile , m-bromobenzaldehyde  and carbon disulfide followed by flash column chromatography (EtOAc/c-hex = 1:15) afforded 1l as a light pink foam . 1H-NMR  δ (ppm) 8.93 , 7.66 , 7.57 , 7.44 , 7.38 , 7.36-7.28  7.25 , 5.42 , 4.97 ; 13C-NMR  δ (ppm) 191.9 (C), 142.2 (C), 138.3 (C), 135.3 (C), 133.6 (CH), 131.6 (CH), 131.5 (CH), 131.3 (CH), 131.0 (CH), 130.7 (CH), 128.0 (CH), 126.6 (CH), 123.1 (C), 122.5 (C), 105.1 (CH), 46.1 (CH); HRMS [MNa+] 461.8569  [MH+−CS2] 363.9309 .in situ generated 1-azadienes with carbon disulfide to furnish unfused 3,6-dihydro-2H-1,3-thiazine-2-thiones in reasonable to good yields. In this reaction, electron-donating as well as electron-withdrawing substituents are tolerated on both the nitrile and aldehyde input. Moreover, aliphatic nitriles and aldehydes can be used in this reaction, although these sometimes lead to lower yields. In conclusion, rapid and easy access to a rather underexplored class of compounds was established with a wide range of potential inputs.We report the novel MCR of


E)-N-cinnamoyl-2-methoxypiperidine (1) and (R)-1-(2-oxopyrrolidin-3-yl)-5,6-dihydropyridin-2(1H)-one (2), four new amide glucosides, retrofractosides A–D (3–6), and two new phenylpropanoid glucosides, retrofractosides E (7) and F (8), together with 24 known compounds (9–32) were isolated from the fruits of Piper retrofractum. The chemical structures of these new compounds were elucidated based on extensive spectroscopic analysis. All of these isolates (1–32) were evaluated for inhibitory activity against mouse platelet aggregation induced by the peptide AYPGKF-NH2. (E)-N-(Tetrahydro-2H-pyran-2-yl)cinnamamide (9) showed a weak inhibitory effect, with an inhibition ratio of 52.0% at a concentration of 150 μM.Two new amides (The online version of this article (10.1007/s13659-019-0208-z) contains supplementary material, which is available to authorized users. Piper retrofractum Vahl (Piperaceae) is primarily distributed in Southeast Asia and cultivated in Indonesia and Thailand , one E-double bond [δH 7.48  and 7.05 ], one trisubstituted phenyl ring [δH 7.58 (s) and 7.26 ], and one carbonyl group (δC 167.8). By comparing the NMR data with those of (E)-3-phenyl-1-(piperidin-1-yl)prop-2-en-1-one (17) and (E)-3--1-(piperidin-1-yl)prop-2-en-1-one , one trisubstituted phenyl ring , and one carbonyl group (δC 169.7). However, the double bond in 4 possessed a Z-configuration [δH 6.65  and 6.03 ] rather than an E-configuration. Based on the COSY and HMBC correlations -N-caffeoylpiperidine moiety in 4 was confirmed. The HMBC correlations  was determined to be (Z)-N- caffeoylpiperidine.Retrofractoside B (5) was determined to be C26H37NO12, based on the 13C NMR data . The 1H and 13C NMR data of 5  and 4.43 ], a p-disubstituted phenyl ring [δH 7.31  and 7.07 ], a Z-double bond [δH 6.64  and 5.97 ], and a carbonyl group [δC 169.8]. By comparing its NMR data with those of 3 and 4, the amide moiety of 5 was deduced to be (Z)-N-p-coumaroylpiperidine, which was supported by the COSY and HMBC correlations  was determined to be (Z)-N-p-coumaroylpiperidine 4′-O-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranoside.The molecular formula of retrofractoside C (6) was found to have the molecular formula, C27H39NO13, based on 13C NMR data  and 4.44 ], one 1,2,4-trisubstituted phenyl ring , one Z-double bond [δH 6.64  and 6.00 ], a carbonyl group [δC 169.8], and a methoxy group . Based on the HMBC correlations from OMe to C-3′ -N-feruloylpiperidine from the COSY and HMBC correlations -Glc-O-C-4′, as that in 5, based on the HMBC correlations from H-1′′′ to C-4′′, from H-1′′ to C-4′, and from H-4′′ to C-6′′ and C-1′′′ as well as the ROESY correlations of H-1′′/H-5′′ and the COSY correlations H-5′′/H-6′′  was determined to be (Z)-N-feruloylpiperidine 4′-O-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranoside.Retrofractoside D (13C NMR (Table 7) and F (8) were determined to have the same molecular formula, C27H40O16. The 1H and 13C NMR data , 4.51 , and 4.39  in 7; δH 4.40 , 4.38 , and 4.31  in 8] and an (E)-cinnamyl alcohol moiety  in these two compounds, which could derive from rozin (29) , tride (18) , piperci18) +, 513 [2M + Na]+; HREIMS m/z 245.1423 [M]+ .White amorphous powder; mentclass2pt{minimc = 0.23, MeOH); UV (MeOH) λmax (logε ) 246 (2.32), 232 (2.27), 196 (3.05) nm; 1H NMR  δH 6.76 , 6.23 , 5.87 , 3.38 , 2.51 , 2.51 , 2.44 , 2.36 , 2.01 ; 13C NMR  δC 180.7 (C-2′), 166.6 (C-2), 143.6 (C-4), 125.2 (C-3), 65.0 (C-3′), 39.7 (C-6), 30.5 (C-5′), 25.7 (C-4′), 25.3 (C-5); ESIMS (positive) m/z 203 [M + Na]+, 383 [2 M + Na]+; HRESIMS (positive) m/z 203.0793 [M + Na]+ .White amorphous powder; c = 0.10, MeOH); UV (MeOH) λmax (logε ) 290 (4.33), 248 (3.93), 213 (4.28), 207 (4.28), 196 (4.33) nm; 1H NMR and 13C NMR data see Table m/z 594 [M + Na]+; HRESIMS (positive) m/z 594.2160 [M + Na]+ .White amorphous powder; entclass1pt{minimac = 0.12, MeOH); UV (MeOH) λmax (logε) 264 (3.95), 242 (3.82), 197 (4.30) nm; 1H NMR and 13C NMR data see Table m/z 594 [M + Na]+; HRESIMS (positive) m/z 594.2161 [M + Na]+ .White amorphous powder; entclass1pt{minimac = 0.04, MeOH); UV (MeOH) λmax (logε ) 265 (3.83), 203 (4.06) nm; 1H NMR and 13C NMR data see Table m/z 578 [M + Na]+; HRESIMS (positive) m/z 578.2206 [M + Na]+ .White amorphous powder; gε ) 265 .83, 203 c = 0.10, MeOH); UV (MeOH) λmax (logε ) 293 (3.96), 266 (4.16), 205 (4.46) nm; 1H NMR and 13C NMR data see Table m/z 608 [M + Na]+; HRESIMS (positive) m/z 608.2317 [M + Na]+ .White amorphous powder; c = 0.10, MeOH); UV (MeOH) λmax (logε ) 250 (3.51), 231 (3.42), 197 (4.03) nm; 1H NMR and 13C NMR data see Table m/z 643 [M + Na]+; HRESIMS (positive) m/z 643.2218 [M + Na]+ .Brown syrup; ), 231 3.2, 197 ; UV (MeOH) λmax (logε ) 249 (4.29), 224 (3.90), 200 (4.51) nm; 1H NMR and 13C NMR data see Table m/z 643 [M + Na]+; HRESIMS (positive) m/z 643.2217 [M + Na]+ .Brown syrup; c = 0.07, MeOH); UV (MeOH) λmax (logε ) 322 (1.71), 276 (2.36), 207 (2.76) nm; 1H NMR  δH 7.66 , 7.50 , 7.37 , 7.36 , 6.36 , 5.98 , 5.26 , 4.01 , 3.66 , 1.91 , 1.88 , 1.65 , 1.55 , 1.45 ; 13C NMR  δC 165.2 (C-9), 142.4 (C-7), 134.8 (C-1), 130.0 (C-4), 129.0 , 128.0 , 120.4 (C-8), 78.1 (C-2′), 67.6 (C-6′), 31.9 (C-3′), 25.2 (C-5′), 23.0 (C-4′); ESIMS (positive) m/z 254 [M + Na]+, 485 [2M + Na]+; HRESIMS (positive) m/z 254.1152 [M + Na]+ .White amorphous powder; 2Cl2-MeOH-H2O, 500:10:0, 300:10:0, 200:10:1, 100:10:1) to afford d-glucopyranose (1.2 mg), which was identified based on its 1H NMR spectrum and optical rotation value: c 0.12, H2O)  × 100%, where X is the maximum aggregation rate of vehicle-treated gel-filtered platelets and Y is the maximum aggregation rate of sample-treated gel-filtered platelets.In vitro platelet aggregation was measured according to the turbidimetric method, using a four-channel aggregometer  , 44. GelPiper retrofractum in this study led to the identification of 32 compounds, including two new amides, four new amide glucosides, and two new phenylpropanoid glucosides. In vitro platelet aggregation assays of all the isolates were conducted, and the results showed that (E)-N-(tetrahydro-2H-pyran-2-yl)cinnamamide (9) possessed weak inhibitory activity against mouse platelet aggregation induced by AYPGKF-NH2, which is a gold agonist of protease-activated receptor 4.The phytochemical investigation of the fruits of Authors declare that there are no conflicts of interest associated with this work.Supplementary file1 (PDF 6444 kb)Supplementary material associated with this article . Below is the link to the electronic supplementary material.


These compounds were obtained by cultivating fungus on wort agar medium with the addition of potassium bromide. Structures of the isolated metabolites were established based on spectroscopic methods. The effects of some isolated glycosides and aglycons 15–18 on urease activity and regulation of Reactive Oxygen Species (ROS) and Nitric Oxide (NO) production in macrophages stimulated with lipopolysaccharide (LPC) were evaluated.Ten new diterpene glycosides virescenosides Z Dichotomomyces cejpii −. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formula of 2 as C26H38O9. 1H and 13C NMR spectra of 2  and one secondary alcohol function at δC 80.0. The remaining functionality, corresponding to the carbon signals at δ 202.9 (C), 168.7 (C) and 130.3 (C), suggested the presence of the tetrasubstituted enone chromophore. The structure of the aglycon part of 2 was found by extensive NMR spectroscopy to be the same as that of virescenoside P [In HRESIMS virescenoside Ztra of 2  indicate11 (3) showed the quasimolecular ion at m/z 509.2408 [M–H]−. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formula of 3 as C26H38O10. The structure of the aglycon moiety of 3 was found by extensive NMR spectroscopy  (The HRESIMS of virescenosides Zd NOESY)  to be th13C and 1H NMR spectra of the sugar moieties of virescenoside Z10 (2) and Z11 (3) showed a close similarity of all proton and carbon chemical shifts with those of virescenosides Z7 and Z8 + and m/z 533.2718 [M + Na]+, respectively. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formula of 4 and 5 as C27H42O8 and C27H42O9, respectively. A close inspection of the 1H and 13C NMR data of 4 (12 (4) was structurally identical to virescenosides B -isopimara-7,15-dien-3β-ol, and the structure of virescenoside Z13 (5) was established as 19-O-[-uronat]-isopimara-7,15-dien-2α,3β-diol.The NMR spectra of glycosides moieties . Initial14 (6), Z15 (7) and Z16 (8) suggested the presence of one sugar . The 1H and 13C NMR spectra of the sugar moieties of 6, 7 and 8 showed a close similarity of all proton and carbon chemical shifts and proton multiplicities. These data and HMBC correlations from anomeric protons to C-5′ methine groups and from H-5′  to C-6′  and from H3-7′  to C-6′ suggested the presence of the methyl ester of a β-altruronopyranoside unit in 6, 7 and 8. The 7.0-, 7.3-, 8.0-Hz splitting between H-4 and H-5 indicated that both were axial and conformation of sugar parts in 6, 7 and 8 is 4C1.The NMR data  of vires14 (6) showed the quasimolecular ion at m/z 547.2508 [M + Na]+. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formula of 6 as C27H40O10. The structure of the aglycon moiety of 6 was found by extensive NMR spectroscopy  (The HRESIMS of virescenosides Zd NOESY)  to be th15 (7) was determined as C27H40O10 based on the analysis of HRESIMS  and NMR data. The 1H and 13C NMR data (7 closely resembled those obtained for virescenoside Z10 (2) with the exception of the C-1‒C-4 carbon and proton signals of ring A. The HMBC correlations from H-5 (δH 1.75) to C-3 (δC 84.4), H-3 (δH 3.00) and from H2-1  to C-2 (δC 69.2) and downfield chemical shifts of C-2 placed an additional hydroxy group at C-2 of ring A. The relative stereochemistry of protons on C-2 and C-3 was defined based on the 1H-1H coupling constant (J=9.8) and assigned as axial. Previously, a similar aglycon has been described for virescenoside M [The molecular formula of virescenoside ZNMR data  observed16 (8) showed the quasimolecular at m/z 515.2617 [M + Na]+. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formula of 8 as C27H40O8 /H-19a  and H-1′/C-19  in the NOESY and HMBC spectra, respectively. From all these data, virescenoside Z14 (6) was structurally identified as 19-O-[-uronat]-7-oxo-isopimara-8(14),15-dien-2α,3β-diol, virescenoside Z15 (7) as 19-O-[-uronat]-7-oxo-isopimara-8(9),15-dien-2α,3β-diol and virescenoside Z16 (8) as 19-O-[-uronat]-3-oxo-isopimara-7,15-dien.The attachment of a carbohydrate chains at C-19 of aglycon moieties of 17 (9) showed the quasimolecular ion at m/z 575.3194 [M + Na]+. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formula of 9 as C30H48O9. The 1H and 13C NMR data observed for aglycon and sugar (C-1′‒C-6′) parts of 9 (13 (5). The correlations observed in the COSY and HSQC spectra of 9 indicated the presence of the isolated spin system: ‒CH2‒CH2‒CH2‒CH3 (C-7′‒C-10′). These data and HMBC correlations from H3-10′ (δH 0.96) to C-8′ (δC 32.3), C-9′ (δC 20.7) and from Ha-7′ (δH 4.15) to C-6′ (δC 172.9), C-8′ and C-9′ suggested the presence of the butyl ester of a β-altruronopyranoside unit of 1C4 form in 9. On the basis of all the data above, the structure of virescenosides Z17 (9) was established as 19-O-[-uronat]-isopimara-7,15-dien-2α,3β-diol.The HRESIMS of virescenoside Zrts of 9  matched 18 (10) showed the quasimolecular at m/z 517.2773 [M + Na]+. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formula of 10 as C27H42O8. The 1H and 13C NMR data observed for the aglycon part of 10 . The 1H and 13C NMR spectra of the sugar part of 10 indicated the presence of the methoxycarbonyl group . A comparison of the 13C NMR spectrum with the data published for α- and β-D-mannopyranoses as well as a good coincidence of carbon signals C-1′‒C-4′ with those of virescenoside Q together with magnitudes of 1H-1H spin coupling constants in 1H NMR spectrum of 10 elucidated the presence of β-D-mannouronopyranoside unit of 4C1 form in 10 -isopimara-7,15-dien-3β-ol.The HRESIMS of virescenoside Zrt of 10  matched rm in 10 ,23. A lo12 (4) and Z13 (5) which were characterized by 1H and 13C NMR spectra. Furthermore, we observed compounds 4-8 and 10 in subfraction II by HPLC-MS method  and G (12), lactone of virescenoside G (13) c 0.15, MeOH); 1H and 13C NMR data, see m/z 553.2618 [M + Na]+ .Virescenoside Z10 (2): amorphous solids; [α]c 0.09, MeOH); UV (MeOH) λmax (log ε) 248 (3.91) nm; 1H and 13C NMR data, see m/z 493.2446 [M–H]− .Virescenoside Z11 (3): amorphous solids; [α]c 0.10, MeOH); UV (MeOH) λmax (log ε) 248 (3.64) nm; 1H and 13C NMR data, see m/z 509.2403 [M–H]− .Virescenoside Z12 (4): amorphous solids; [α]c 0.10, MeOH); 1H and 13C NMR data, see m/z 517.2770 [M + Na]+ .Virescenoside Z13 (5): amorphous solids; [α]c 0.13, MeOH); 1H and 13C NMR data, see m/z 533.2718 [M + Na]+ .Virescenoside Z14 (6): amorphous solids; [α]c 0.10, MeOH); UV (MeOH) λmax (log ε) 249 (3.81) nm; 1H and 13C NMR data, see m/z 547.2508 [M + Na]+ .Virescenoside Z15 (7): amorphous solids; [α]c 0.15, MeOH); UV (MeOH) λmax (log ε) 248 (3.99) nm; 1H and 13C NMR data, see m/z 547.2515 [M + Na]+ .Virescenoside Z16 (8): amorphous solids; [α]c 0.05, MeOH); 1H and 13C NMR data, see m/z 515.2617 [M + Na]+ .Virescenoside Z17 (9): amorphous solids; [α]c 0.10, MeOH); 1H and 13C NMR data, see m/z 575.3194 [M + Na]+ , m/z 551.3229 [M‒H]− calcd. for C29H47O9, 551.3226, Δ ‒0.6 ppm).Virescenoside Z18 (10): amorphous solids; [α]c 0.12, MeOH); 1H and 13C NMR data, see m/z 517.2773 [M + Na]+ .Virescenoside Z11): amorphous solids; 1H NMR  δ: 5.80 , 5.38 , 4.92 , 4.86 , 4.85 , 4.27 ,4.24 , 4.12 , 3.93 , 3.78 , 3.76 , 3.73 , 2.98 , 2.11 , 2.04 , 1.99 , 1.92 , 1.73 , 1.60 , 1.50 , 1.39 , 1.39 , 1.34 , 1.14 , 1.11 , 0.92 , 0.86 . 13C NMR  δ: 174.0 (C-6′), 151.9 (C-15), 136.9 (C-8), 123.2 (C-7), 110.4 (C-16), 103.5 (C-1′), 85.8 (C-3), 76.6 (C-5′), 74.3 (C-19), 70.9 (C-2′), 70.4 (C-3′), 70.2 (C-4′), 69.7 (C-2), 54.1 (C-9), 53.4 (C-5), 48.0 (C-1), 47.6 (C-14), 44.4 (C-4), 38.4 (C-13), 37.9 (C-10), 37.9 (C-12), 25.1 (C-6), 24.2 (C-18), 22.6 (C-17), 22.1 (C-11), 17.7 (C-20); + .Virescenoside F (12): amorphous solids; 1H NMR  δ: 5.80 , 5.37 , 4.92 , 4.86 , 4.84 , 4.30 ,4.24 , 4.10 , 3.93 , 3.84 , 3.78 , 3.24 , 2.07 , 2.01 , 1.97 , 1.91 , 1.90 , 1.74 ,1.68 ,1.66 , 1.57 , 1.47 , 1.37 , 1.38 , 1.26 , 1.23 , 1.12 , 0.86 , 0.85 . 13C NMR  δ: 173.7 (C-6′), 152.0 (C-15), 137.1 (C-8), 123.2 (C-7), 110.4 (C-16), 103.9 (C-1′), 81.7 (C-3), 76.7 (C-5′), 73.9 (C-19), 70.6 (C-4′), 70.4 (C-3′), 70.1 (C-2′), 54.1 (C-9), 53.7 (C-5), 47.7 (C-14), 43.7 (C-4), 40.0 (C-1), 38.4 (C-13), 37.9 (C-12), 36.8 (C-10), 29.4 (C-2), 25.0 (C-6), 23.6 (C-18), 22.6 (C-17), 22.1 (C-11), 17.0 (C-20); + .Virescenoside G (13): amorphous solids; 1H NMR  δ: 5.80 , 5.66 , 5.56 , 5.37 , 5.29 , 4.93 , 4.85 , 4.68 , 4.41 , 4.39 , 4.14 , 4.12 , 3.55 , 3.49 , 3.48 , 2.22 , 1.93 , 1.91 , 1.88 , 1.87 , 1.71 , 1.64 , 1.55 , 1.46 , 1.44 , 1.31 , 1.25 , 1.22 , 1.12 , 0.89 , 0.82 . 13C NMR  δ: 174.0 (C-6′), 149.9 (C-15), 135.5 (C-8), 121.1 (C-7), 109.8 (C-16), 93.8 (C-1′), 84.0 (C-3′), 80.1 (C-3), 71.4 (C-2′), 70.1 (C-4′), 68.5 (C-5′), 68.4 (C-19), 50.8 (C-9), 49.9 (C-5), 45.4 (C-14), 36.5 (C-13), 36.1 (C-4), 35.9 (C-1), 35.5 (C-12), 34.7 (C-10), 25.5 (C-18), 21.7 (C-6), 21.3 (C-17), 21.2 (C-2), 19.7 (C-11), 15.7 (C-20); m/z 485.2508 [M + Na]+ .Lactone of virescenoside G (14): amorphous solids; 1H NMR  δ: 5.80 , 5.37 , 4.93 , 4.85 , 4.14 , 3.79 , 3.50 , 3.09 , 2.11 , 1.98 , 1.97 , 1.91 , 1.92 , 1.73 , 1.61 , 1.50 , 1.39 , 1.35 , 1.21 , 1.12 , 0.93 , 0.86 . 13C NMR  δ: 151.9 (C-15), 137.1 (C-8), 123.1 (C-7), 110.4 (C-16), 86.5 (C-3), 69.7 (C-2), 66.6 (C-19), 54.0 (C-9), 53.0 (C-5), 47.7 (C-1), 47.6 (C-14), 44.4 (C-4), 38.4 (C-13), 37.9 (C-10), 37.8 (C-12), 24.8 (C-6), 24.5 (C-18), 22.6 (C-17), 22.1 (C-11), 17.9 (C-20), m/z 343.2241 [M + Na]+ .Aglycon of virescenoside A  and 5 µL of test compounds dissolved in water  was preincubated at 37 °C for 60 min in 96-well plates. Then 55 µL of phosphate buffer solution with 100 µM urea was added to each well and incubated at 37 °C for 10 min. The urease inhibitory activity was estimated by determining of ammonia production using indophenol method [The reaction mixture consisting of 25 µL enzyme solution (urease from l method . BrieflyThe suspension of macrophages on 96-well plates (2 × 104 cells/well) were washedwith the PBS and treated with 180 µL/well of the tested compounds (10 μM) for 1 h and 20 µL/well LPS from E. coli serotype 055:B5 , which were both dissolved in PBS and cultured at 37 °C in a CO2-incubator for one hour. For the ROS levels measurement, 200 μL of 2,7-dichlorodihydrofluorescein diacetate  fresh solution was added to each well, and the plates were incubated for 30 min at 37 °C. The intensity of DCF-DA fluorescence was measured at λex 485 n/λem 518 nm using the plate reader PHERAstar FS  .The suspension of macrophages on 96-well plates (2 × 104 cells/well) were washed withthe PBS and treated with 180 µL/well of the tested compounds (10 μM) for 1 h and 20 µL/well LPS from E. coli serotype 055:B5 , which were both dissolved in PBS and cultured at 37 °C in a CO2-incubator for one hour. For the RNS levels measurement, 200 μL Diaminofluorescein-FM diacetate  fresh solution was added to each well, and the plates were incubated for 40 min at 37 °C, then replaced with fresh PBS, and then incubated for an additional 30 min to allow complete de-esterification of the intracellular diacetates. The intensity of DAF FM-DA fluorescence was measured at λex 485 n/λem 520 nm using the plate reader PHERAstar FS .Mice BALB/c were sacrificed by cervical dislocation. Peritoneal macrophages were isolated using standard procedures. For this purpose, 3 mL of PBS (pH 7.4) was injected into the peritoneal cavity and the body intensively palpated for 1–2 min. Then the peritoneal fluid was aspirated with a syringe. Mouse peritoneal macrophage suspension was applied to a 96-well plate left at 37 °C in an incubator for 2 h to facilitate attachment of peritoneal macrophages to the plate. Then a cell monolayer was triply flushed with PBS (pH 7.4) for deleting attendant lymphocytes, fibroblasts and erythrocytes and cells were used for further analysis.All animal experiments were conducted in compliance with all rules and international recommendations of the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes. All procedures were approved by the Animal Ethics Committee at the G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences , according to the Laboratory Animal Welfare guidelines.Average value, standard error, standard deviation and p-values in all experiments were calculated and plotted on the chart using SigmaPlot 3.02 . Statistical difference was evaluated by t-test, and results were considered as statistically significant at p < 0.05.9‒Z18 (1‒10) were isolated from a marine strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Virescenoside Z9 (1) is an altroside of a new 7-oxo-isopimara-15-en-2α,3β,6α,8β-tetraol aglycon. Virescenosides Z12-Z16 (4‒8) were determined as the monosides having unique methyl esters of altruronic acid as their sugar moieties. Carbohydrate chain of virescenoside Z18 (10) was structurally identified as the methyl ester of mannuronic acid. The effects of some isolated glycosides and aglycons 15‒18 on urease activity and regulation of ROS and NO production in macrophages stimulated with lipopolysaccharide (LPC) were evaluated.Ten new diterpene glycosides, virescenosides Z


N-oxide derivatives as potential trypanocidal agents. Additionally, molecular docking and enzymatic assays on trypanothione reductase (TR) were performed to provide a basis for their potential mechanism of action. Seven compounds showed better trypanocidal activity on epimastigotes than the reference drugs, and only four displayed activity on trypomastigotes; T-085 was the lead compound with an IC50 = 59.9 and 73.02 µM on NINOA and INC-5 strain, respectively. An in silico analysis proposed compound T-085 as a potential TR inhibitor with better affinity than the natural substrate. Enzymatic analysis revealed that T-085 inhibits parasite TR non-competitively. Compound T-085 carries a carbonyl, a CF3, and an isopropyl carboxylate group at 2-, 3- and 7-position, respectively. These results suggest the chemical structure of this compound as a good starting point for the design and synthesis of novel trypanocidal derivatives with higher TR inhibitory potency and lower toxicity.Chagas disease or American trypanosomiasis is a worldwide public health problem. In this work, we evaluated 26 new propyl and isopropyl quinoxaline-7-carboxylate 1,4-di- Trypanosoma cruzi [American trypanosomiasis or Chagas disease is a neglected tropical disease caused by the parasitic protozoa ma cruzi . The infma cruzi ,3. The dma cruzi . Chagas ma cruzi .Chagas disease can be treated with nifurtimox and benznidazole ,7. Both Mycobacterium tuberculosis [Entamoeba histolytica [Trichomonas spp. [Plasmodium falciparum [Leishmania spp. [T. cruzi. Cerecetto et al. [N-oxide derivatives with anti-T. cruzi activity against the epimastigote life cycle form. They proposed that the N-oxide group acts as a pharmacophore. Subsequently, the trypanocidal activity of new quinoxaline N-N´-oxide derivatives was reported by Aguirre et al. [T. cruzi, generating oxygen reactive species harmful to the parasite. Another study by Ancizu et al. [N-oxide derivatives (CAQDO) which were further evaluated in vitro; two molecules obtained IC50 values similar to the reference drug nifurtimox. The study by Benitez, et al. [N-oxide ring increased biological activity. The most recent study by Torres et al. [N-oxides against T. cruzi; three compounds , showed good activity against bloodstream trypomastigotes NINOA and INC-5 of T. cruzi with about 50% lysis at 5 µg/mL . In the same study, molecular docking analysis suggested that these compounds could be trypanothione reductase (TR) inhibitors [T. cruzi. Additionally, molecular docking analysis and enzymatic assays were performed on TR to confirm the mechanism of action of our derivatives.Several researchers have been demonstrating the trypanocidal effect of quinoxaline derivatives. Quinoxalines are heterocyclic compounds formed by a benzene ring and a pyrazine ring. This chemical structure offers many possibilities for structural modification. It has been shown that the oxidation of both nitrogen atoms increases activity against rculosis , Entamoetolytica , Trichomnas spp. , Plasmodlciparum , Leishmania spp.  and parto et al.  reportede et al. , who sugu et al.  reported, et al.  found ths et al.  confirmehibitors . The con50, CC50 and SI values obtained for propyl and isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives against T. cruzi INC-5 epimastigotes are showed. Seven derivatives showed an IC50 less or equal to 10 µM and were more active than the reference drugs, nifurtimox and benznidazole. T-085 was the most active compound with an IC50 value < 2.5 μM, while T-069, T-070, T-071, T-116, and T-124 showed IC50 values ranging from 2.83 to 12.12 μM. Additionally, T-067 was more active than benznidazole, but not nifurtimox.In T-089 and T-124, which possess a trifluoromethyl group at 3-position. The incorporation of this group was key in the trypanocidal activity; for example, T-090 has a methyl group at 3-position, but when it is substituted for trifluoromethyl in T-124, biological activity increases 36-fold.A structure-activity relationship (SAR) analysis showed that the propyl derivatives had low biological activity, except compounds T-067, T-069, T-070, T-071, T-085 and T-116) had better biological activity than compounds that have a methyl group . Again, the effect of the trifluoromethyl group is evident; for example, in compounds T-065 and T-098, there was a change from the methyl to trifluoromethyl group to obtain compounds T-116 and T-071, respectively, enhancing considerably (>25-fold) their trypanocidal activity. These results are in agreement with a previous report by Benitez et al. [N-oxide derivatives with trypanocidal activity. The trifluoromethyl group is stable and is related to the lipophilicity of the molecules [On the other hand, SAR analysis of isopropyl derivatives confirmed that compounds with a trifluoromethyl group at 3-position ; this same biological behavior occurs between T-069 and T-089 . In our previous work, the effect on trypanocidal activity between the methyl and ethyl ester group at 7-position on quinoxaline 1,4-di-nclusive ; howeverT-126 and T-130 had the lowest cytotoxic effect; however, they were inactive on epimastigotes with both compounds having a methyl group in R3. On the other hand, T-071 was the most toxic compound with a benzyl group in R2. When analyzing the cytotoxic effect of quinoxaline derivatives on mammalian cells, the compounds 50 of mammalian cells and the parasite IC50 [T-070 and T-085 showed the highest SI, becoming lead compounds from this study against epimastigotes of T. cruzi; both compounds have an isopropyl carboxylate group, a trifluoromethyl group, and a short aliphatic chain at R7-, R3- and R2-position, respectively. In general, all compounds showed lower selectivity than the reference drugs; therefore, we propose that rational structural modifications can be made to these molecules to decrease toxicity.The selectivity index (SI), which reflects the impact of the compound on the parasite, was determined by the ratio of the CCite IC50 ,26. CompT-069, T-071, T-085 and T-089. The compound T-085 showed a better trypanocidal activity than the reference drugs against trypomastigotes  to identify molecules with the best activity against this stage of the parasite and to select those that induce at least 50% lysis. Compounds with a better lysis percentage on both strains were stigotes . HoweverN-oxide derivatives than bloodstream trypomastigotes; only half of the compounds that showed activity against epimastigotes were active against trypomastigotes.Epimastigotes were more sensitive under the conditions tested to quinoxaline 1,4 di-T-067, T-070, T-116 and T-124 showed good activity on epimastigotes, but significantly reduced their activity on bloodstream trypomastigotes. T-067, T-116 and T-124 have a carboxylate group at R2- and compound T-070 has a carbonyl group at R2-, followed by a primary carbon. Interestingly, the four compounds that were active against bloodstream trypomastigotes have a carbonyl group followed by a tertiary carbon instead. The previous SAR analysis highlights the importance of maintaining a carbonyl group in R2 to conserve activity in both stages of the parasite.Compounds T. cruzi, the epimastigote form is found in the vector and the bloodstream trypomastigote form in the mammal [In the life cycle of e mammal , each bie mammal . N-oxide derivatives docked in the TR protein are shown in T-003 or M2, T-021 or M6 and T-044 or M8) [p-value = 0.0003). This statistical analysis indicates that compounds with better binding energy are influenced by a summation of atom-pair interaction  caused by their high MW. In addition, a Spearman correlation analysis  was not significant, showing that the predicted affinity for those compounds is due to specificity and not to the size of the molecule [T-085 showed notable in vitro trypanocidal effects in epimastigotes and trypomastigotes. It has been reported that the essential residues involved in the catalysis of the TR protein are Cys53 and Cys58 and the active-site base His461 [T-085 with a focus on those essential amino acids  . A totalmolecule . On the e His461 . Thus, wno acids . T-085 iN-oxide derivatives could bind and inhibit TR. This molecular mechanism of action might explain the observed antiparasitic activity against T. cruzi. On this basis, three methyl  and one isopropyl (T-085) quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives  against both enzymes and the results are graphically depicted in T-085 has a similar IC50 value of 50 µM against TcTR and hGR. The collected results suggest that T-085 is a good starting point for future medicinal chemistry studies to identify novel derivatives with higher TR inhibitory potency, an improved selectivity profile over the host GR and lower toxicity.To assess selectivity over the human homologue glutathione reductase (GR), N-oxide derivatives were synthetized as described by Gomez-Caro et al. [Propyl and isopropyl quinoxaline-7-carboxylate 1,4-di-o et al. . The como et al. . NifurtiT-064: Isopropyl methyl-3-methyl-quinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 7.9% yield from isopropyl benzofuroxane-5-carboxylate and methyl acetoacetate. IR (KBr): 2986 (C-H), 1744 and 1712 (C=O), 1330 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.38 2CH), 2.43 , 4.03 , 5.21–5.25 2-CH-), 8.34 , 8.51 , 8.88 . Calculated analysis for C15H16N2O6: C, 56.25; H, 5.04; N, 8.75. Found: C, 56.10; H, 4.75; N, 8.35T-065: Isopropyl ethyl-3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 3.5% yield from isopropyl benzofuroxane-5-carboxylate and ethyl acetoacetate. IR (KBr): 2984 (C-H), 1740 and 1710 (C=O), 1332 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.39 2CH and CH2CH3), 2.44 , 4.51 , 5.22–5.25 2-CH-), 8.34 , 8.51 , 8.85 . Calculated analysis for C16H18N2O6: C, 57.48; H, 5.43; N, 8.38. Found: C, 57.10; H, 5.05; N, 7.90.T-066: Isopropyl tert-butyl-3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 20% yield from isopropyl benzofuroxane-5-carboxylate and tert-butyl acetoacetate. IR (KBr): 2978 (C-H), 1736 and 1711 (C=O), 1331 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.38 2CH), 1.61 3), 2.46 , 5.22–5.25 2CH), 8.37 , 8.54 , 8.86 . Calculated analysis for C18H22N2O6: C, 59.66; H, 6.12; N, 7.73. Found: C, 59.42; H, 5.82; N, 7.56.T-067: Isopropyl ethyl-3-(2-ethoxy-2-oxoethyl) quinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 5.4% yield from isopropyl benzofuroxane-5-carboxylate and diethyl 3-oxoglutarate. IR (KBr): 2988 (C-H), 1740 and 1719 (C=O), 1328 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.15 , 1.30 , 1.36 2CH-), 3.77 , 4.10 , 4.48 , 5.19–5.22 2CH-), 8.40 , 8.53 , 8.85 . Calculated analysis for C19H22N2O8: C, 56.15; H, 5.46; N, 6.89. Found: C, 56.20; H, 5.20; N, 6.40.T-069: Isopropyl 2-(thiophene-2-carbonyl)-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 2.5% yield from isopropyl benzofuroxane-5-carboxylate and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione. IR (KBr): 2968 (C-H), 1721 and 1661 (C=O), 1332 (N-oxide), 1285 and 1153 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 1.40 2CH), 5.24–5.29 2-CH-), 7.31 , 8.24 , 8.30 , 8.51 , 8.54 , 8.94 . Calculated analysis for C18H13F3N2O5S: C, 50.71; H, 3.07; N, 6.57. Found: C, 50.30; H, 2.85; N, 6.21.T-070: Isopropyl 2-acetyl-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 8.0% yield from isopropyl benzofuroxane-5-carboxylate and 1,1,1-trifluoro-2,4-pentanedione. IR (KBr): 2988 (C-H), 1726 (C=O), 1337 (N-oxide), 1285 and 1153 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 1.41 2CH), 2.62 , 5.22–5.28 2-CH-), 8.5 , 8.6 , 8.9 . Calculated analysis for C15H13F3N2O5: C, 50.29; H, 3.66; N, 7.82. Found: C, 49.88; H, 3.30; N, 7.56.T-071: Isopropyl 2-benzoyl-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 7.5% yield from isopropyl benzofuroxane-5-carboxylate and 4,4,4-trifluoro-1-phenyl-1,3-butanedione. IR (KBr): 2997 (C-H), 1720 and 1683 (C=O), 1328 (N-oxide), 1292.49 and 1162 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 1.42 2CH), 5.24–5.29 2-CH-), 7.6 , 7.77 , 8.16 , 8.51–8.53 , 8.95 . Calculated analysis for C20H15F3N2O5: C, 57.15; H, 3.60; N, 6.66. Found: C, 56.90; H, 3.18; N, 6.21.T-072: Isopropyl 2-(naphthyl-2-carbonyl)-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 4.5% yield from isopropyl benzofuroxane-5-carboxylate and 4,4,4-trifluoromethyl-1-(2-naphthyl)-1,3-butanedione. IR (KBr): 2981 (C-H), 1724 and 1673 (C=O), 1336 (N-óxido), 1282 and 1172 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 1.42 2CH), 5.25-5.29 2-CH-), 7.66 , 7.75 , 8.0 , 8.1 , 8.14 , 8.5 , 8.87 , 9.0 . Calculated analysis for C24H17F3N2O5: C, 61.28; H, 3.64; N, 5.96. Found: C, 61.01; H, 3.35; N, 5.62.T-073: Isopropyl 2-(furyl-2-carbonyl)-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 17.30% yield from isopropyl benzofuroxane-5-carboxylate and 4,4,4-trifluoro-1-(2-furyl)-1,3-butanedione. IR (KBr): 2993 (C-H), 1714 and 1664 (C=O), 1332 (N-oxide), 1255 and 1168 (Ar-CHF2) cm−1. 1H-NMR  δ ppm: 1.40 2CH), 5.23–5.29 2-CH-), 6.84 , 7.9 , 8.26 , 8.5 , 8.6 , 8.94 . Calculated analysis for C18H13F3N2O6: C, 52.69; H, 3.19; N, 6.83. Found: C, 52.36; H, 2.74; N, 6.41.T-085: Isopropyl 2-isobutyryl-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 16.0% yield from isopropyl benzofuroxane-5-carboxylate and 1,1,1-trifluoromethyl-5-methyl-2,4-hexanedione. IR (KBr): 2983 (C-H), 1715 and 1678 (C=O), 1325 (N-oxide), 1283 and 1178 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 1.30 2), 1.33 2CH), 1.4 2), 5.25 2-CH-), 8.5 , 8.6 , 8.88 . Calculated analysis for C17H17F3N2O5: C, 52.85; H, 4.44; N, 7.25. Found: C, 52.40; H, 4.12; N, 7.04.T-088: n-Propil methyl-3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 11.85% yield from n-propil benzofuroxane-5-carboxylate and methyl acetoacetate. IR (KBr): 2964.93 (ArC-H), 1745.58 (C=O), 1331 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.0 2O), 1.74–1.84 2O), 2.44 , 4.03 , 4.32–4.36 2O), 8.35–8.40 , 8.51–8.57 , 8.85–8.90 . Calculated analysis for C15H16N2O6: C, 56.25; H, 5.04; N, 8.75. Found: C, 56.1; H, 4.9; N, 8.4.T-089: n-Propil 2-(thiophene-2-carbonyl)-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 6.51% yield from n-propil benzofuroxane-5-carboxylate and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione. IR (KBr): 2965 (ArC-H), 1722 and 1662 (C=O), 1331 (N-oxide), 1284 and 1165 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 1.0 2O), 1.77–1.86 2O), 4.37–4.40 2O), 7.30–7.34 , 8.25 , 8.31 , 8.5–8.7 , 8.96 . Calculated analysis for C18H13F3N2O5S: C, 50.71; H, 3.07; N, 6.57. Found: C, 50.35; H, 2.85; N, 6.26.T-090: n-Propil ethyl-3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 17.85% yield from n-propil benzofuroxane-5-carboxylate and ethyl acetoacetate. IR (KBr): 2965 (ArC-H), 1742.53 and 1721.70 (C=O), 1329 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.01 2O), 1.36 , 1.76–1.82 2O), 2.45 , 4.32–4.36 2O), 4.49–4.54 , 8.35–8.40 , 8.5 , 8.9 . Calculated analysis for C16H18N2O6: C, 57.48; H, 5.43; N, 8.38. Found: C, 57.20; H, 5.18; N, 8.15.T-091: n-Propil tert-butyl-3-methylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 7.58% yield from n-propil benzofuroxane-5-carboxylate and tert-butyl acetoacetate. IR (KBr): 2966 (ArC-H), 1740 and 1721 (C=O), 1325 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.01 2O), 1.61 3), 1.8 2O), 2.46 , 4.33–4.36 2O), 8.4 , 8.6 , 8.92 . Calculated analysis for C18H22N2O6: C, 59.66; H, 6.12; N, 7.73. Found: C, 59.30; H, 5.80; N, 7.45.T-097: Isopropyl 2-phenylamide-3-methylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 18.5% yield from isopropyl benzofuroxane-5-carboxylate and 3-oxo-N-phenylbutanamide. IR (KBr): 2984 (C-H), 1720 and 1680 (C=O), 1332 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.39 2CH), 2.90 , 5.23 2-CH-), 7.20 , 7.4 , 7.8 , 8.17–8.30 , 8.73 , 10.85 . Calculated analysis for C20H19N3O5: C, 62.99; H, 5.02; N, 11.02. Found: C. 62.56; H, 4.87; N, 10.70.T-098: Isopropyl 2-benzoyl-3-methylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 45% yield from isopropyl benzofuroxane-5-carboxylate and 1-phenyl-1,3-butanedione. IR (KBr): 2983 (C-H), 1715 and 1678 (C=O), 1326 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.40 2CH), 2.30 , 5.3 2-CH-), 7.6 , 7.8 , 8.1 , 8.4 , 8.5 , 8.97 . Calculated analysis for C20H18N2O5: C, 65.57; H, 4.95; N, 7.65. Found: C, 65.23; H, 4.47; N, 7.32.T-107: Isopropyl 2-(tert-butyl-2-carbonyl)-3-tert-butylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 5% yield from isopropyl benzofuroxane-5-carboxylate and 2,2,6,6-tetramethyl-3,5-heptanedione. IR (KBr): 2978 (C-H), 1736 and 1711 (C=O), 1331 (N-oxide) cm−1. 1H NMR  δ ppm: 1.34 3), 1.35 3), 1.38 2CH), 5.13–519 2-CH-), 7.96 , 8.17 , 8.67 . Calculated analysis for C21H28N2O5: C, 64.93; H, 7.27; N, 7.21. Found: C, 64.73; H, 7.03; N, 6.95.T-108: Isopropyl 2-amide-3-methyl-quinoxaline-7-carboxilate 1,4-di-N-óxide. This compound was obtained in 4.0% yield from isopropyl benzofuroxane-5-carboxylate and methyl acetoacetamide. IR (KBr): 2982 (C-H), 1738 and 1714 (C=O), 1328 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.38 2CH), 2.48 , 5.20-2.27 2-CH-), 8.20 , 8.38 , 8.58 , 8.90 . Calculated analysis for C14H16N3O5: C, 55.08; H, 4.95; N, 13.76. Found: C, 54.76; H, 4.36; N, 13.35T-116: Isopropyl ethyl-3-trifluoromethylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 6.5% yield from isopropyl benzofuroxane-5-carboxylate and ethyl 4,4,4-trifluoroacetoacetate. IR (KBr): 2981 (C-H), 1736 and 1708 (C=O), 1331 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.39 2CH and CH2CH3), 4.5 , 5.21–5.25 2-CH-), 8.34 , 8.51 , 8.85 . Calculated analysis for C17H17F3N2O5: C, 52.85; H, 4.44; N, 7.25. Found: C, 52.51; H, 4.05; N, 6.90.T-117: Isopropyl tert-butyl-3-heptafluoropropylquinoxaline-2,7-dicarboxylate 1,4-di-N-oxide. This compound was obtained in 3.0% yield from isopropyl benzofuroxane-5-carboxylate and 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedione. IR (KBr): 2980 (C-H), 1733 and 1714 (C=O), 1332 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.38 2CH), 1.42 3), 5.21–5.24 2CH), 8.38 , 8.52 , 8.87 . Calculated analysis for C20H19F7N2O6: C, 48.01; H, 3.83; N, 5.60. Found: C, 47.85; H, 3.56; N, 5.34.T-118: Isopropyl 2-pentafluorocarbonyl-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di N-oxide. This compound was obtained in 4.0% yield from isopropyl benzofuroxane-5-carboxylate and 1,1,1,5,5,6,6,6-octafluoro-2,4-hexanedione. IR (KBr): 2986 (C-H), 1728 (C=O), 1332 (N-oxide), 1284 and 1152 (CF3) cm−1. 1H-NMR  δ ppm: 1.38 2CH), 5.22–5.26 2-CH-), 8.51 , 8.62 , 8.9 . Calculated analysis for C16H10F8N2O5: C, 41.57; H, 2.18; N, 6.06. Found: C, 41.16; H, 2.05; N, 5.84.T-124: n-Propyl 2-propionyl-3-trifluoromethylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 7.52% yield from n-propyl benzofuroxane-5-carboxylate and 1,1,1-trifluoro-2,4-hexanedione. IR (KBr): 2981 (Ar C-H), 1724 (C=O), 1329 (N-oxide), 1256 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 1.0 2O), 1.35 , 1.8 2O), 4.3–4.35 2O), 4.5 , 8.40–8.45 , 8.55 , 8.9 .Calculated analysis for C16H15F3N2O5: C, 51.62; H, 4.06; N, 7.52. Found: C, 51.4; H, 3.85; N, 7.20.T-125: n-Propyl 2-phenylamide-3-methylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 12.36% yield from n-propyl benzofuroxane-5-carboxylate and 3-oxo-N-phenylbutanamide. IR (KBr): 3245 (N-H), 2972 (Ar C-H), 1720 (C=O), 1328 (N-oxide) cm−1. 1H-NMR  δ ppm: 1.02 2O), 1.8 2O), 2.51 , 4.36 2O), 7.20 , 7.42 , 7.67 ) 8.4 , 8.6 , 9.0 , 11.02 . Calculated analysis for C20H19N3O5: C, 62.99; H, 5.02; N, 11.02. Found: C, 62.60; H, 4.85; N, 10.70.T-126: n-Propyl 2-acetyl-3-methylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 8.95% yield from n-propyl benzofuroxane-5-carboxylate and 2,4-pentanedione. IR (KBr): 2984 (Ar C-H), 1726 (C=O), 1330 (N-oxide), 1258 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 1.0 2O), 1.8 2O), 2.55 , 3.97 , 4.36 2O), 8.40 , 8.5 , 8.92 . Calculated analysis for C15H16N2O5: C, 59.21; H, 5.30; N, 9.21. Found: C, 58.95; H, 5.15; N, 8.85.T-130: n-Propyl 2-amide-3-methylquinoxaline-7-carboxylate 1,4-di-N-oxide. This compound was obtained in 5.68% yield from n-propyl benzofuroxane-5-carboxylate and acetoacetamide. IR (KBr): 2976 (Ar C-H), 1728 (C=O), 1334 (N-oxide), 1254 (Ar-CF3) cm−1. 1H-NMR  δ ppm: 0.99 2O), 1.8 2O), 2.47 , 4.4 2O), 8.27 , 8.39 , 8.6 , 8.96 . Calculated analysis for C14H15N3O5: C, 55.08; H, 4.95; N, 13.76. Found: C, 54.75; H, 4.62; N, 13.35.T. cruzi isolated in Mexico were used. T. cruzi NINOA strain (MHOM/MX/1994/NINOA) was obtained from a patient with acute Chagas disease [T. cruzi INC-5 (MHOM/MX/1994/INC5) was isolated from a patient in the chronic phase of the disease [T. cruzi to obtain bloodstream trypomastigote samples. Animal experiments were performed according to Norma Oficial Mexicana (NOM-062-Z00-1999) published on 22 August, 2009.In this study, two strains of  disease  and T. c disease . EpimastT. cruzi epimastigotes using 3--2,5-diphenyltetrazolium bromide (MTT) were performed. Briefly, the parasites were harvested during the exponential growth phase for a period of five days. In a 96-well microplate 1x106 epimastigotes were deposited in brain-heart infusion (BHI) medium supplemented with 10% FBS and 100 U/mL antibiotic-antifungal mixture (Gibco). Five concentrations serial twofold from 50 μg/mL to 3.12 μg/mL for each of the quinoxaline 1,4-di-N-oxide derivatives and the reference drugs (Nfx and Bnz) were evaluated. Each well reached a final volume of 100 µL. Each condition was assessed in triplicate. The microplate was incubated for 24 h at 27 °C in darkness. Afterwards, 10 μL of a 5 mg/mL MTT solution was added and the plate was incubated for 20 h at 27 °C. Then 200 μL of 10% dodecyl sulfate (SDS)/0.1% HCl solution was added to dissolve formazan salts; the plate was incubated an additional 4 h. Finally, the plate was read at 570 nm in a spectrophotometer . The amount of viable cells was proportional to the amount of formazan produced. The inhibitory concentration of 50% of the population (IC50) was determined by the Probit statistical tool. Later the results were converted to micromolar units. Compounds with an IC50 less or equal to 10 µM were selected. As a positive control, parasites were incubated only in culture medium with a 1% DMSO concentration, corresponding to the highest sample dilution of DMSO [Viability assays of  of DMSO ,35,36.T. cruzi bloodstream trypomastigotes of the INC-5 and NINOA strains. The course of infection continued for 4–6 weeks. At the peak of parasitemia blood was obtained by cardiac puncture using sodium heparin as an anticoagulant. The blood was adjusted to 1 × 106 bloodstream trypomastigotes/mL. CD1 female mice 6–8 weeks old were infected with N-oxide derivatives or reference drug dilutions were seeded, with each well reaching a final volume of 200 µL. Each assay was performed in triplicate. Initially, all compounds were tested at 50 μg/mL. Afterwards, the compounds with a percentage of lysis >50 were tested at five concentrations (100 to 10 μg/mL) to obtain a lysis concentration of 50% of the population (LC50). As a negative control of lysis, wells with untreated blood trypomastigotes were used, and as a positive control, wells with the reference drugs were used. The microplates were incubated at 4 °C for 24 h. Bloodstream trypomastigotes were quantified by the method of Brener-Pizzi, 5 μL of blood were placed on a slide and covered with a coverslip 13 × 13 mm. The mobile protozoa were counted in 15 fields at 40× magnification using an optical microscope. The percentage lysis was determined by comparing the remaining trypomastigotes against the negative control. LC50 was determined with the Probit statistical tool. The results were later converted to micromolar units [In each well of the 96-well plate, 195 μL of infected blood and 5 μL of quinoxaline 1,4-di-ar units ,37,38.2 and humidity. Trypsinized J774.A1 macrophages were washed and viability was assessed by the trypan blue dye  exclusion assay. In a 96-well microplate, 50,000 macrophages were seeded per well and a dose-response assay was performed. Compounds were evaluated using six-fold serial dilutions ranging from 100 µg/mL to 0.3 µg/mL of final concentration. Each compound was tested in triplicate. After 20 h incubation, Alamar Blue was added (10% v/v) and cells were incubated for an additional 4 h period. Results were obtained as previously described and CC50 was calculated. As a negative control of cytotoxicity, cells were incubated only in the presence of 0.1% DMSO [The murine macrophage cell line J774.A1 was maintained in culture flasks with RPMI 1640 medium supplemented with 10% FBS, 1% MEM-NEAA medium (Gibco) and 100 U/mL antibiotic-antifungal mixture (Gibco). Cells were incubated at 37 °C with 5% CO.1% DMSO ,40.50/IC50 [The selectivity index (SI) was calculated to evaluate the selectivity of the molecules for the parasite. SI is defined as the ratio of CC50/IC50 .In silico molecular docking studies were performed with Vina software [N-oxide derivatives on the active site of the TR protein from T. cruzi. The TR crystal structure with PDBID: 1BZL [www.rcsb.org). This protein exists co-crystalized with its natural substrate trypanothione disulfide, which is located on the active site formed by the interface of the two dimers that comprise the whole protein. The crystal structure was prepared as a receptor by removing the waters and substrates and adding the Gasteiger charges using AutoDock software [N-oxide derivatives including trypanothionine. The compounds were drawn and energy minimized with MarvinSketch software (www.chemaxon.com). The hydrogens were added with babel software and the Gasteiger charges were added during the transformation to pdbqt format with the “prepare-ligand.py” script from AutoDock tools. For each docked ligand the predicted binding energy reported in Vina scores  [t-test were performed with R software [software  in orderID: 1BZL  was downsoftware . Then thh other)  was usedsoftware . 2) and recombinant T. cruzi TR were prepared following published procedures [2 and NADPH consumption was followed at 340 nm.Trypanothione disulfide  contain2PO4, 26.5 mM K2HPO4, 200 mM KCl, 1 mM EDTA, pH 6.9). The assays contained 100 µM NADPH and 5–10 mU GR and varying concentrations of the inhibitor. The reaction was started by adding 100 µM GSSG and the absorption decrease was followed at 340 nm.GR activity was measured at 25 °C in a total volume of 1 mL GR assay buffer  for Excel .Five compound concentrations were used ranging from 100–5 μM. All measurements were performed twice in three independent series. Enzyme activity was plotted versus increasing inhibitor concentrations. IC2 . The inhibitory constant Ki was calculated from the direct plot using nonlinear least-squares data fitting in Excel spreadsheet [The type of inhibition and the inhibitor constants were derived from Lineweaver-Burk plot. The activity of TR was measured in the absence and presence of two to three constant concentrations of inhibitor varying the concentration of TSeadsheet ,49.N-oxide derivatives against epimastigotes and trypomastigotes of T. cruzi were made. Four compounds were active against both forms of the parasite; one of them, T-085, showed a better trypanocidal activity than the reference drugs, exhibited a moderate selectivity against the epimastigote and bloodstream tripomastigotes life cycle of the parasite and a high affinity in docking and enzymatic analysis for TR. The latter demonstrated that T-085 acts as a noncompetitive inhibitor. Structurally, the presence of an isopropyl carboxylate group in R7, a trifluoromethyl group in R3, and a carbonyl group in R2 of quinoxaline 1,4-di-N-oxide derivatives, favors its anti-trypanocidal activity as a TR inhibitor.In this study, in vitro evaluations of propyl and isopropyl 7 quinoxaline carboxylate 1,4-di-


More recently its presence in the milk of different farm animals has been reported. Pterosin B (Ptb) and bromopterosin (BrPt) represent the most convenient analogues in the detection of ptaquiloside by mass spectrometry. Pterosin sesquiterpenes are also involved in many patented biomedical protocols. In this work we introduce a new and convenient approach to the synthesis in three steps and more than 80% yield of d A mixture of the alcohol 3 , iodomethane , anhydrous K2CO3 , and DMF (20 mL) was stirred at room temperature for 12 h. After the reaction was completed the reaction mixture was diluted with water (50 mL) and extracted with chloroform (3 ° 50 mL). The combined organic phases were washed with water, brine (100 mL each), and then dried over Na2SO4. After filtration of the mixture and removal of the solvent by rotary evaporation, the residue was purified by column chromatography ; to give methyl 2-ethyl ether-d4 (4a). Yield: 1.66 g ; colourless oil; IR (film): ν = 2880 (s), 1100 (s) cm−1; 1H-NMR (CDCl3): δ = 6.92–7.01 , 3.25 , 2.35 ; 13C-NMR (CDCl3): δ = 136.9, 128.2, 126.2, 120.1, 60.6, 55.4, 20.0, 15.8; GC-MS : m/z = 168 [M+] (25), 154 (20), 121 (100), 107 (15), 92 (20).4)-1,3-dimethylbenzene2-. To a solution of the alcohol 3  in glacial acetic acid (4 mL) was added HBr , and the mixture was heated at 100 °C in a sealed tube for 12 h. After cooling to room temperature, the reaction mixture was poured onto a cold solution of saturated NaHCO3 (50.00 mL), and the mixture was extracted with chloroform (3 × 30 mL). The combined organic phases were washed with brine, and then dried over Na2SO4. After filtration of the mixture and removal of the solvent by rotary evaporation, the residue was purified by column chromatography ; to give 1-bromo-2-ethyl-d4 (4b). Yield: 1.99 g ; colourless oil; IR (film): ν = 2880 (s), 1440 (s), 1298 (s) cm–1; 1H-NMR (CDCl3): δ = 6.92–7.01 , 2.35 ; 13C-NMR (CDCl3): δ = 136.9, 128.2, 126.2, 120.1, 29.6, 20.0, 15.8; GC-MS : m/z = 218 [M++2] (14), 216 [M+] (15), 137 (80), 121 (100), 93 (30).4)-2R,5,7-trimethyl-2,3-dihydro-1H-inden-1-one6-. Methacrylic anhydride  was added to a suspension of methyl 2-ethyl ether-d4  in PPA (30 g). Because of the high viscosity of the PPA the reaction mixture was stirred by mechanical stirrer for 48 h at room temperature, and after diluted with ice water (50 mL), and extracted with chloroform (3 × 30 mL). The combined organic phases were washed with brine, and then dried over Na2SO4. After filtration of the mixture and removal of the solvent by rotary evaporation, the residue was purified by column chromatography ; to give d4-pterosin methyl ether 5. Yield: 1.1 g ; colourless oil; [α]25D −31 (c 5 mg/mL); IR (film): ν = 1740 (m), 1700 (s), 1675 (w), 1590 (s) cm−1; 1H-NMR (CDCl3): δ = 7.0 , 3.51 , 3.28 , 2.59–2.77 , 2.35 , 2.24 , 1.26 ; 13C-NMR (CDCl3): δ = 210.1, 152.2, 141.2, 137.3, 134.1, 131.3, 122.8, 60.9, 55.1, 42.0, 33.5, 31.7, 20.1, 16.3, 13.5; GC-MS : m/z = 236 [M+] (30), 221 (50), 189 (100).4)-2R,5,7-trimethyl-2,3-dihydro-1H-inden-1-one6-. To a solution of d4-pterosin methyl ether 5 (1.08 g 4.60 mmol) in chloroform (10 mL), was added BBr3  at −20 °C, the solution was stirred at the same temperature for 2 h. The reaction mixture was added to a saturated solution of NaHCO3 (100 mL), and extracted with chloroform (3 × 50 mL). The combined organic phases were washed with brine, and then dried over Na2SO4. After filtration of the mixture and removal of the solvent by rotary evaporation, the residue was purified by column chromatography ; to give d4-pterosin (2a). Yield: 0.96 g ; mp. 90–92 °C; IR (KBr): ν = 3410 (s), 1736 (s), 1668 (s), 1590 (s), 1040 (s) cm−1; 1H-NMR (CDCl3): δ = 7.0 , 3.65 , 3.53 , 2.56–2.76 , 2.35 , 2.23 , 1.27 ; 13C-NMR (CDCl3): δ = 210.1, 152.2, 141.2, 137.3, 134.1, 131.3, 122.8, 60.9, 42.0, 33.5, 31.7, 20.1, 16.3, 13.5; GC-MS : m/z = 222 [M+] (40), 207 (70), 189 (100).4)-2R,5,7-trimethyl-2,3-dihydro-1H-inden-1-one6-. Methacrylic anhydride  was added to a suspension of methyl 2-ethyl ether-d4  in PPA (30 g). The reaction stirred by mechanical stirrer for 48 h at room temperature, and after diluted with ice water (50 mL), and extracted with chloroform (3 × 30 mL). The combined organic phases were washed with brine, and then dried over Na2SO4. After filtration of the mixture and removal of the solvent by rotary evaporation, the residue was purified by column chromatography ; to give d4-bromopterosin 2b. Yield: 1.19 g ; yellow crystals; mp. (108–109 °C); [α]25D −21 (c 5 mg/mL); IR (KBr): ν = 2880 (w), 1736 (s), 1668 (s), 1590 (s), 1440 (w), 1040 (s) cm−1; 1H-NMR (CDCl3): δ = 7.0 , 3.51 , 2.60-2.76 , 2.37 , 2.26 , 1.29 ; 13C-NMR (CDCl3): δ = 210.1, 152.2, 141.2, 137.3, 134.1, 131.3, 122.8, 42.0, 33.9, 33.6, 20.1, 15.3, 15.1, 13.6; GC-MS : m/z = 286 [M++2] (50), 284 [M+] (50), 205 (100), 189 (50).4-pterosin B in four steps with a total yield of 78%, and d4-bromopterosin in three steps with a total yield of 80%. The synthesized compounds can be used as internal standards for the assay of pterosin sesquiterpenes and analogues in food and biological fluids and could provide the basis for the formation of labelled analogues of other members of this peculiar family of natural compounds.In conclusion, we have developed a new facile and convenient total synthesis of d