S,3R)-methyl 7-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-3-(hydroxymethyl)-2,3-dihydrobenzofuran-5-carboxylate (1) and -5--4-isopropyldihydrofuran-2-one (2), tentatively named norcurlignan and limlactone, respectively, were isolated from Liriope muscari, together with the known compound (−)-pinoresinol (3). The structures of these compounds were elucidated and characterized on the basis of 1D NMR, 2D NMR, CD and MS data. The in vitro antioxidant activities of compounds 1–3 were assessed by the DPPH and ABTS scavenging methods. Two new compounds, (2 Liriope muscari (Decne.) Bailey (Liliaceae) is called duantingshanmaidong in China. Due to the lack of Radix Ophiopogonis (maidong in Chinese) resources and the similar pharmacological activities of these two herbal medicines, L. muscari is used locally in Fujian province as a substitute for maidong − ion peak at m/z 345.0962  in the HR-ESI-MS. 1H-NMR  showed eight downfield proton signals, including two phenolic hydroxyl group signals , an AB-pattern for two aromatic protons at δ 7.40  and δ 7.33 , and an ABX-pattern for three aromatic protons at δ 6.93 , δ 6.78 , and δ 6.75 . Ten highfield proton signals, including two singlets , a doublet  coupled to a multiplet  and a double doublet  were also observed. The 13C-NMR data -4-isopropyldihydrofuran-2-one -methyl 7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydrobenzofuran-5-carboxylate. This novel natural product is tentatively named norcurlignan after its homologue curlignan.The absolute stereochemistry at C-2 and C-3 of compound urlignan , both sh2 was obtained as colorless feathery crystals; c 0.0075, CH2Cl2), UV (CH3OH) λmax (nm) (lgε): 289.5 (3.2). Its molecular formula was assigned as C15H20O3, suggesting six degrees of unsaturation, on the basis of the [M−H]− ion peak at m/z 247.1331  in the HR-ESI-MS. 1H-NMR ; a multiplet ; a methylene signal ; two singlets  and a group of isopropyl proton signals . The 13C-NMR data -4-isopropyldihydrofuran-2-one.In the HMBC spectrum , the obs2 was confirmed by the analysis of the 1H-1H COSY spectrum, which further revealed the presence of a CH (H-5)-CH (H-4)-CH2 (H-3), CH3-CH (H-6)-CH3 fragment (The assigned structure for compound fragment .J = 9.0 Hz), H-4 and H-5 was determined to be trans-oriented − . The 1H- and 13C-NMR spectral data are listed in Limlactone (2): Colorless feathery crystals (CHCl3). c 0.0075, CH2Cl2). UV (CH3OH) λmax (nm) (lgε): 289.5 (3.2). HR-ESI-MS: m/z 247.1331 [M−H]− . The 1H- and 13C-NMR spectral data are listed in (−)-Pinoresinol (3): Colorless oil. c 0.012, CH2Cl2). UV (CH3OH) λmax (nm) (lgε): 231, 281 . C20H22O6 . 1H-NMR  δ: 6.92, 6.90 , 6.84 , 4.76 , 4.27 , 3.90 , 3.92 , 3.13 . 13C-NMR  δ: 146.7 , 145.3 , 132.9 , 119.0 , 114.3 , 108.6 , 85.9 , 71.7 , 56.0 (2×OCH3), 54.2 . The 1H- and 13C-NMR spectral data are consistent with the published data [hed data ,18.1) and limlactone (2), together with the known compound (−)-pinoresinol (3) were isolated from L. muscari. Their antioxidant activities were evaluated using DPPH and ABTS assays. In both methods, compound 3 showed potential activity . Compound 1 and compound 2 showed potential activity in the ABTS assay, with IC50 values of 46.4 µM and 23.1 µM, respectively.Two new compounds, norcurlignan (


Their structures were elucidated on the basis of spectroscopic analyses using 1D and 2D-NMR spectra and electron ionization mass spectrometry (EIMS). The effects of compounds 1–10 on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages were evaluated. Compounds 4, 6, and 10 exhibited similar NO inhibitory activities to NG-monomethyl-l-arginine acetate (l-NMMA). These compounds did not exhibit cytotoxicity at an effective concentration. The results of present study suggest that compounds 4, 6, and 10 have potential as anti-inflammatory disease agents.Three novel lupane-, bauerane-, and euphane-type triterpenoids ( Taraxacum officinale (commonly known as dandelion) has a number of culinary and medicinal uses, despite being generally regarded as a weed. Dandelion is the general name of approximately two thousand species of plants that belong to the genus Taraxacum (Compositae). Young leaves and inflorescences are used as ingredients in salads and stir-fries. Dandelion roots have long been largely used on the continent, and the plant is cultivated largely in India as a remedy for liver complaints. Dandelion roots contain bitter principles that have a tonic effect on the liver and digestive system  to afford the novel lupane, bauerane, euphane-type triterpenes, as well as seven known triterpenes.The methanol extract of the roots of 1 was obtained as colorless needles, the molecular ion peak at m/z 514.4022 in high resolution (HR) electron ionization (EIMS) of which showed the molecular formula C33H54O4 . Its IR spectrum showed the presence of an ester group at 1739 and 1246 cm−1. Detailed assignments of 1H- and 13C-NMR spectra ), two secondary methyls ), an acetyl methyl (δH 2.04 (s), δC 171.0 (s)), nine methylenes, six methines including two oxymethines , δC 83.2 (d); δH 4.47 , δC 80.9 (d)), five quarternary carbons, and two oxygenated sp3 quaternary carbons (δC 71.9 (s), 77.0 (s)). EIMS showed fragment ion peaks characteristic of the cleavage of the C-ring at m/z 249 (C16H25O2) and 189 (249 − HOAc), which revealed the presence of an acetyl group at the C-3 position . The 1H- and 13C-NMR spectra  (each s)); a secondary methyl (δH 0.87 (d)), two vinyl methyls ), an acetyl , δC 171.0 (s)); eight sp3 methylene, five sp3 methines including an oxymethine (δH 4.47 (dd)); four sp3 quaternary carbons, two trisubstituted olefins (δH 5.09 (tt), δC 124.8 (d), 131.2 (s)), (δH 5.69 (d), δC 124.8 (d), 170.8 (s)), and an αβ-unsaturated ketone (199.4 (s)). Cross peaks in the 1H-1H COSY spectrum are shown in bold lines in H 0.82 (s))/C-12, C-13, C-17; Me-19 (δH 0.88 (s))/C-1, C-9, C-10; Me-21 (δH 0.87 (d))/C-17, C-20, C-22; Me-26 and Me-27 )/C-24 (δC 124.8 (d)), C-25 (δC 131.2 (s)); Me-28 and Me-29 )/C-3 (δC 80.6 (d)), C-4, C-5; Me-30 (δH 1.05 (s))/C-8, C-13, C-14, C-15; H-7 (δH 5.69 (d))/C-5, C-6 (δC 199.4 (s)), C-8, C-9, C-14; H-24 (δH 5.09 (tt))/C-22, C-23, C-25 (δC 131.2 (s)), C-26, C-27. The relative configuration of 3 was elucidated on the basis of NOESY correlations  inhibitor—were evaluated for their inhibitory effects on NO production .Macrophages may be a potential therapeutic target for inflammatory diseases . Activatoduction . Among t1H- and 13C-NMR spectra were obtained on an Agilent VNMRS 600 spectrometer  with standard pulse sequences, operating at 600 and 150 MHz, respectively. CDCl3 was used as the solvent, and tetramethylsilane (TMS) as the internal standard. HREIMS were recorded on a JEOL-7000 mass spectrometer . Column chromatography was performed over silica gel , while medium pressure liquid chromatography (MPLC) was conducted with silica gel . HPLC was run on a JASCO PU-1586 instrument  equipped with a differential refractometer (RI 1531). Fractions obtained from column chromatography were monitored by thin layer chromatography (TLC) .Melting points were measured on a Yanagimoto micro-melting point apparatus  and were uncorrected. Optical rotations were measured using a JASCO DIP-1000 digital polarimeter . IR spectra were recorded using a Perkin-Elmer 1720X FTIR spectrophotometer . T. officinale (Compositae) were collected in Takatsuki city, Osaka, Japan in April 2014. A voucher specimen (TR-01) was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.The roots of T. officinale (5.81 kg) were extracted with MeOH at 60 °C for 10 days, and the dried extract obtained (890 g) was partitioned between EtOAc and H2O. The organic layer (290 g) was condensed in vacuo and subjected to column chromatography (silica gel (3 kg); hexane:EtOAc (5:1→1:1→0:1) and EtOAc:MeOH (1:1→0:1)), affording residues A , B , C , D , E , F , G , H , I , J , K , L , M , N , O , P , Q , R , and S . Residue C was rechromatographed over silica gel to give a triterpene fraction, which was subjected to HPLC  to afford compounds 1 (1.38 mg) and 9 (1.12 mg) + ; IR (KBr) νmax cm−1: 2956, 2925, 1739 (O-C=O), 1456, 1381, 1367, 1246; 1H- and 13C-NMR, see m/z (relative intensity (rel. int.)): 514 [M]+ (100), 483 (26), 416 (45), 325 (4), 233 (6), 191 (9), 135 (12).3β-Acetoxybauer-7-en-6-one (2): Colorless crystals; mp 110–112 °C; c 0.1, EtOH); HREIMS m/z: 482.3759 [M]+ ; UV λmax (EtOH) nm (log ε): 245.0 (3.97); IR (KBr) νmax cm−1: 2954, 2869, 1733, and 1247 (O-C=O), 1665 (O=C-C=C), 1466, 1388; 1H- and 13C-NMR, see m/z (rel. int.): 482 [M]+ (100), 422 [M − HOAc]+ (48), 407 (31), 379 (30), 300 (45), 219 (46), 206 (44), 203 (42), 191 (35), 161 (40).3β-Acetoxyeupha-7,24-dien-6-one (3): Colorless crystals; mp 140–143 °C; c 0.04, EtOH); HREIMS m/z: 482.3764 [M]+ ; UV λmax (EtOH) nm (log ε): 244.5 (3.99); IR (KBr) νmax cm−1: 2958, 1731, and 1247 (O-C=O), 1664 (O=C-C=C), 1437, 1384; 1H- and 13C-NMR, see m/z (rel. int.): 482 [M]+ (59), 467 (56), 422 [M − HOAc]+ (26), 407 (34), 369 (100), 300 (43), 217 (16), 206 (44), 189 (17), 161 (45).4 cells in 100 μL) were seeded on a 96-well microplate and incubated for 24 h. Dulbecco's modified eagle's medium (DMEM)-containing test samples  dissolved in dimethyl sulfoxide  was added. After being treated for 24 h, thiazolyl blue tetrazolium bromide (MTT) solution was added. After a 3-h incubation, 20% sodium dodecyl sulfate in 0.1 M HCl was added to dissolve the formazan produced in the cells. Absorbance in each well was read at 570 nm using a microplate reader. The optical density of vehicle control cells was assumed to be 100%.RAW264.7 cell proliferation was examined in accordance with a previously reported method . Briefly4 cells in 100 μL) were seeded on a 96-well microplate and incubated for 24 h. DMEM-containing test samples  dissolved in DMSO  and LPS  was added. After being treated for 24 h, the supernatant of the culture medium was transferred to another 96-well microplate, and 50 μL of 0.15% N-(1-naphtyl)ethylenediamine in H2O and 1.5% sulfanilamide in 7.5% phosphoric acid were then added. After being incubated for 30 min, absorbance in each well was read at 570 nm using a microplate reader. The optical density of vehicle control cells was assumed to be 100%An inhibitory assay of NO production was examined in accordance with a previously reported method  with min1), as well as a novel bauerane-type triterpenoid (2) and a novel euphane-type triterpenoid (3) were isolated from the roots of T. officinale (Compositae), together with seven known triterpenoids (4–10). Their structures were elucidated by spectroscopic analyses. In the NO inhibitory assay, Compounds 4, 6, and 10 exhibited similar NO inhibitory activities  to l-NMMA (IC50 23.9 μM). These compounds did not exhibit cytotoxicity at an effective concentration . These results suggest that compounds 4, 6, and 10 have potential as anti-inflammatory disease agents.A novel lupane-type triterpenoid (


Among these compounds, eight dammarane-type saponins, 2, 5, 6, 7, 11, 12, 13, and 15, exhibited the greatest antiproliferative effects against two human tumor cell lines (A549 and HepG2).Six new dammarane-type saponins, gypenosides CP1-6 ( Gynostemma pentaphyllum (Thunb.) Makino (family Cucurbitaceae) is an ethnomedicine frequently used in Asian countries as a functional food and tea 26D +11.5 , has the molecular formula C55H92O24, as established by NMR and HRESIMS , indicating ten degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl, carbonyl, and olefinic groups at 3358, 1736, and 1638 cm−1, respectively. In the 1H-NMR spectrum ] and an olefinic proton [δH 5.13 ]. The 13C-NMR (C 16.3 (C-18), 17.4 (C-30), 17.7 (C-29), 17.8 (C-19), 18.0 (C-27), 22.4 (C-21), 25.9 (C-26), 28.6 (C-28)], 4 oxygenated carbons , and a pair of olefinic carbons . The 1H-1H COSY and HMBC correlations fully established the planar structure of 1 /H-2 (δH 3.72)/H-3 (δH 2.95) and H-9 (δH 1.48)/H2-11 /H-12 (δH 3.74)/H-13 (δH 1.73)/H-17 (δH 2.28) /H2-16 /H2-15  as well as the HMBC long-range correlation of H-3 with carbon signals at δC 41.8 (C-4) and 57.2 (C-5). The molecular formula and 1D and 2D-NMR spectroscopic data of 1 suggested a dammarane-type saponin, a typical constituent of Gynostemma species, with the same aglycone as that of 2α,3β,12β,20(S)-tetrahydroxydammar-24-ene [C 20.9 (CH3) and 172.8 (C=O)] in the 13C NMR spectrum. Four anomeric signals were observed at δH 4.30 /δC 105.5, δH 4.42 /δC 104.7, δH 4.56 /δC 98.1, and δH 4.71 /δC 105.0. HMBC cross peaks of H-1′ to C-3, H-1″ to C-2′, H-1‴ to C-20, and H-1″″ to C-6‴ determined the positions of the four sugars. Acid hydrolysis of 1 yielded d-glucose (Glc) and d-xylose (Xyl) in a ratio of 3:1 based on HPLC analysis of the component monosaccharides compared with the standard sugars -20-O-[β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside].Gypenoside CP1  was isolated as a white powder. Its molecular formula was determined to be C52H86O20 from HRESIMS and 13C-NMR spectroscopic analysis. Comparison of the 1H and 13C-NMR spectroscopic data of 1 and 2  but lacks the xylose and acetyl group found in 1. The location of the but-2-enoyl group at Glc-C-6″ was confirmed by the correlations observed in the HMBC between δH 4.18, 4.37 (H-6″) and δC 168.1  /δC 104.6, δH 4.71 /δC 105.2, δH 4.60 /δC 98.3], which were confirmed to be from d-glucose via acid hydrolysis. Long-range correlations were also observed between δH 4.42 (H-1′) and δC 96.5 (C-3), δH 4.71 (H-1″) and δC 82.7 (C-2′), and δH 4.60 (H-1‴) and δC 84.9 (C-20) indicating that the three sugars were attached to C-3, C-2′, and C-20, respectively. Thus, the structure of gypenoside CP2 (2) was elucidated as 2α,3β,12β,20S-tetra-hydroxydammar-24-ene-3-O-{[6-O-(E)-but-2-enoyl]-β-d-glucopyranosyl-(1→2)-β-d-glucoyranosyl}-20-O-β-d-glucopyranoside.Gypenoside CP2  . The NMR3) was isolated as a white powder. Its molecular formula was determined as C57H94O24 from HRESIMS and 13C-NMR spectroscopic analysis. The 1H- and 13C-NMR spectra -but-2-enoyl]-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl} moiety and a 20-O-[β-d-xylopyranosyl-(1→6)-β-d-glucopyranosyl] moiety, which were virtually superimposable onto those of 2; however, carbon signals  consistent with an additional sugar moiety were also present. Four anomeric signals were found at δH 4.29 /δC 105.6, δH 4.41 /δC 104.6, δH 4.56 /δC 98.1, and δH 4.70 /δC 105.2. Acid hydrolysis of 3 yielded d-glucose and d-xylose (3:1). The signals for CH2-6‴  in 3 were shifted downfield compared to those in 2 , indicative of the attachment of a d-xylose at CH2-6‴ in 3 . Moreover, long-range correlations (HMBC) between δH 4.41 (H-1′) and δC 96.5 (C-3), δH 4.70 (H-1″) and δC 82.7 (C-2′), δH 4.56 (H-1‴) and δC 84.9 (C-20), and δH 4.29 (H-1″″) and δC 70.1 (C-6‴) indicated the following sugar locations, d-glucose at C-3, C-2′, and C-20, respectively and d-xylose at C-6‴. Accordingly, compound 3 (gypenoside CP3) was determined as 2α,3β,12β,20S-tetrahydroxydammar-24-ene-3-O-{[6-O-(E)-but-2-enoyl]-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl}-20-O-[β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside].Gypenoside CP3 ( spectra  of 3 sho4) was isolated as a white powder. The HRESIMS and 13C-NMR spectroscopic data of 4 suggested its molecular formula to be C57H94O23. Analysis of the 1H- and 13C-NMR spectra  by a methylene  at aglycone in 4. Detailed checking the NMR data together with the analysis of acid hydrolysis, the glycone moiety of 4 were composed 3 units of of d-glucose and one d-xylose. Thus, gypenoside CP4 was determined as 3β,12β,20S -trihydroxydammar-24-ene-3-O-{[6-O-(E)-but-2-enoyl]-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl}-20-O-[β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside].Gypenoside CP4 ( spectra  gave 57 5) showed a quasimolecular ion at m/z 1129.5928 [M + Na]+ , corresponding to the molecular formula C58H90O20. Like previous isolates, compound 5 has a 2α,3β,12β,20 (S)-tetrahydroxydammar-24-ene skeleton, due to the similarity of the 1H- and 13C-NMR spectroscopic data  and δH 173.4 (C-10a) in the HMBC spectrum of 5 (2 was replaced by a (E)-4-phenylbut-3-enoyl unit in 5. Accordingly, the structure of 5 (gypenoside CP5) was confirmed as 2α,3β,12β,20S-tetrahydroxydammar-24-ene-3-O-{[6-O-(E)-4-phenyl-but-3-enoyl]-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl}-20-O-β-d-glucopyranoside.The HRESIMS of gypenoside CP5 , which was 132 amu more than the molecular ion of 5, presumably corresponding to a xylose group. The 1D and 2D NMR spectroscopic data of 6 showed similar signals as those of 5 except for an additional unit in 6 characterized by a xylose signals . Acidic hydrolysis of 6 also furnished d-glucose and d-xylose. Based on the above corroborations, the structure of 6 (gypenoside CP6) was determined as 2α,3β,12β,20S-tetrahydroxydammar-24-ene-3-O-{[6-O-(E)-4-phenyl-but-3-enoyl]-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl}-20-O-[β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside].The positive HRESIMS of compound 1-6 were proved as novel chemical structures and named as gypenosides CP1-CP6, respectively. The remaining nineteen isolates were identified as 2α,3β,12β,20S-tetrahydroxydammar-24-ene-3-O-β-d-glucopyranosyl-20-O-[β-d-6-O-acetylglucopyranosyl-(1→2)-β-d-glucopyranoside (7) [8) [9) [10) [11) [12) [S-tetrahydroxydammar-24-ene-3-O-β-d-glucopyranosyl-20-O-β-d-glucopyranoside (13) [14) [15) [S-trihydroxydammar-24-ene-3-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-20-O-[β-d-xylo-pyranosyl-(1→6)-β-d-glucopyranoside] (16) -3,5,5-trimethyl-2-cyclohexen-1-one (25)  (16) , togetheide (17) , quercet17) 26D +11.5 ; IR (KBr) νmax 3358, 2945, 2876, 1736, 1638, 1082 cm−1; 1H-  and 13C-  NMR data, see m/z 1159.5874 [M + Na]+ .Gypenoside CP2 (2), White amorphous powder; [α]26D +15.7 ; IR (KBr) νmax 3362, 2941, 2872, 1715, 1650, 1074 cm−1; 1H-  and 13C-  NMR data, see m/z 1053.5607 [M + Na]+ .Gypenoside CP3 (3), White amorphous powder; [α]26D +12.9 ; IR (KBr) νmax 3370, 2928, 2876, 1715, 1650, 1078 cm−1; 1H-  and 13C-  NMR data, see m/z 1185.6030 [M + Na]+ . Gypenoside CP4 (4), White amorphous powder; [α]26D +6.6 ; IR (KBr) νmax 3350, 2937, 2868, 1728, 1650, 1078 cm−1; 1H-  and 13C-  NMR data, see m/z 1169.6084 [M + Na]+ .Gypenoside CP5 (5), White amorphous powder; [α]26D +7.7 ; IR (KBr) νmax 3370, 2921, 2872, 1679, 1074 cm−1; 1H-  and 13C-  NMR data, see m/z 11129.5928 [M + Na]+ .Gypenoside CP6 (6), White amorphous powder; [α]26D +12.9 ; IR (KBr) νmax 3370, 2921, 2864, 1687, 1070 cm−1; 1H-  and 13C-  NMR data, see m/z 1261.6340 [M + Na]+ .2Cl2 to afford the aglycone portion, and the aqueous layer was neutralized with Na2CO3 and filtered. To the evaporated filtrate was added 1-(trimethylsilyl)imidazole and pyridine (0.2 mL), and the mixture was stirred at 60 °C for 5 min. After the reaction mixture was dried under a stream of N2, each residue was partitioned between CHCl3 and H2O. Each CH2Cl2 fraction was subjected to gas chromatography . Under these conditions, the sugars of each reactant were identified by comparison with authentic standards (d-glucose and d-xlyose).Each isolated compound (1.0 mg) was treated with 2 N methanolic HCl (2 mL) under conditions of reflux at 90 °C for 1 h. Each mixture was extracted with CHl-glutamine and 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies) under standard culture conditions. After treatment with serial dilutions of tested compounds for 48 h, the alamar blue assay  was used to obtain the half maximal inhibitory concentration (IC50). Doxorubicin was used as a positive control. Plates were incubated at 37 °C for 6 h prior to measure the absorbance at 570 nm and at 600 nm wavelengths using a spectrophotometric plate reader . Experimental data were normalized to control values. Mitomycin c was used as a positive control .The isolates were tested for antiproliferative effects against HepG2 (human hepatocellular carcinoma), A549 (human lung adenocarcinoma) tumor cell lines and the M10  cell line in vitro using the MTT  colorimetric method based on previously published procedures . Two celG. pentaphyllum. All the new chemical structures of compounds 1–6 were elucidated completely and tentatively named gypenosides CP1–CP6. These new isolates were obtained from the titled plant for the first time, and not yet found in other natural resources or synthesized molecules before. For the derived chemical structures of 5 and 6, (E)-4-phenylbut-3-enyl unit is first time to be found in nature. This study adds to the present phytochemical and properties information on this plant species, together with the studies performed and compiled by others [In this study, we isolated and characterized six new and ten known dammarane-type triterpene saponins as well as eight known flavonoids and two known sesquiterpene glucosides from a 95% EtOH extract of dried aerial parts of


Eucalyptus perriniana as biocatalysts was investigated. The cells of E. perriniana glycosylated genistein and glycitein to give the corresponding 4'-O-β-glucosides, 7-O-β-glucosides, and 7-O-β-gentiobiosides, which were two new compounds. The β-glucosides of genistein and glycitein showed 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity and superoxide-radical scavenging activity. On the other hand, 7-O-β-glucosides of genistein and glycitein and the 7-O-β-gentiobioside of glycitein exerted inhibitory effects on IgE antibody production.The synthesis of gentiooligosaccharides of genistein and glycitein using cultured cells of  Eucalyptus perriniana to produce the corresponding 4'-O-β-glucosides, 7-O-β-glucosides, and 7-O-β-gentiobiosides. We also report their 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, superoxide-radical scavenging activity, and inhibitory activity for IgE antibody formation.The soy isoflavonoids genistein and glycitein have been recognized as natural antioxidants, and have been reported to show anti-allergic activities, such as inhibitory effects on histamine release from mast cells ,2,3,4,5.E. perriniana were incubated with genistein (1) for five days, the glycosylated products 2-4 were isolated from the cells by extraction with MeOH. On the other hand, none were detected in the medium. No additional glycosylation products were detected in the MeOH extracts of the cells despite careful HPLC analyses. On the basis of their HRFABMS, 1H- and 13C-NMR, H-H COSY, C-H COSY, and NOE-spectroscopic analyses, the products , genistein 7-O-β-glucoside , and genistein 7-O-β-gentiobioside , of which 4 is a new compound. After cultured cells of products  were det4 was established as C27H30O15 based on its HRFABMS spectrum, which included a pseudomolecular ion [M + Na]+ peak at m/z 617.1488 . HRFABMS suggested that 4 was composed of one molecule of 1 and two hexoses. Its 1H-NMR spectrum showed two anomeric proton signals at δ 4.25  and 5.10 . This suggested the presence of two β-anomers. The 13C-NMR spectrum included two anomeric carbon signals at δ 99.5 and 102.8. The sugar component of 4 was determined to be β-D-glucopyranose based on the chemical shifts of the carbon signals. The 13C resonance of C-6'' was shifted downfield to δ 68.6. Correlations were observed between the anomeric proton signal at δ 5.10 (H-1'') and the carbon signal at δ 163.0 (C-7), and between the anomeric proton signal at δ 4.25 (H-1''') and the carbon signal at δ 68.6 (C-6'') in the HMBC spectrum. These findings confirmed that the inner glucopyranosyl residue was attached to the phenolic hydroxyl group at C-7 of genistein (1), and that the pair of β-D-glucopyranosyl residues was 1,6-linked. Thus, 4 was identified as genistein 7-O-[6-O-(β-D-glucopyranosyl)]-β-D-glucopyranoside (7-O-β-gentiobioside).The molecular formula of 5) was subjected to the same biotransformation system. Glycoside products 6-8 were obtained from the MeOH extracts of the cells. The products were identified as glycitein 4'-O-β-glucoside , glycitein 7-O-β-glucoside , and glycitein 7-O-β-gentiobioside   , of whic8 included a pseudomolecular ion [M + Na]+ peak at m/z 631.1645, consistent with a molecular formula of C28H32O15 . The 1H-NMR spectrum of 8 included proton signals at δ 4.50  and 5.12 , indicating the presence of two β-anomers in the sugar moiety. The 1H- and 13C-NMR spectra of 8 indicated that it was a β-gentiobiosyl analogue of 5. Furthermore, the HMBC spectrum included correlations between the anomeric proton signal at δ 5.12 (H-1'') and the carbon signal at δ 152.5 (C-7), and between the anomeric proton signal at δ 4.50 (H-1''') and the carbon signal at δ 68.9 (C-6''). These findings confirm that the inner β-D-glucopyranosyl residue was attached to the phenolic hydroxyl group at C-7 of glycitein (5) and that the pair of β-D-glucopyranosyl residues were 1,6-linked. Thus, the product 8 was identified as glycitein 7-O-[6-O-(β-D-glucopyranosyl)]-β-D-glucopyranoside (7-O-β-gentiobioside).The HRFABMS spectrum of β-glycosides 2-4 and glycitein β-glycosides 6-8 was determined by in vitro bioassay. The antioxidant activities were expressed as IC50 values and are summarized in O-β-D-glucoside (2), genistein 7-O-β-D-glucoside (3), glycitein 4'-O-β-D-glucoside (6), and glycitein 7-O-β-D-glucoside (7) showed DPPH free-radical scavenging activity. On the other hand, the β-gentiobiosides of genistein and glycitein (4 and 8) had no antioxidant activity. The results obtained here suggested that mono-glucosides of genistein and glycitein would be useful free-radical scavenging antioxidants with aqueous-solubility.The DPPH free-radical scavenging activity of genistein β-glycosides 2-4 and glycitein β-glycosides 6-8 was expressed as IC50 values summarized in O-β-D-glucoside (2), genistein 7-O-β-D-glucoside (3), glycitein 4'-O-β-D-glucoside (6), and glycitein 7-O-β-D-glucoside (7) showed superoxide-radical scavenging activity. The present data demonstrated that gentiobiosylation of isoflavones drastically reduced the antioxidant activity in comparison with isoflavone glucosides. A recent paper reported that glycitein 7-O-maltotrioside showed no antioxidant activity, although glycitein 7-O-glucoside exerted relatively high antioxidant activity, and that maltosylation at C-7 of glycitein also reduced the antioxidant activity in comparison with glycitein 7-O-glucoside . Genistein and glycotein were purchased from Sigma-Aldrich Co. The NMR spectra were recorded in DMSO-E. perriniana was induced in our laboratory, and has been cultivated for over 20 years  to give glycosylated products.Substrate (0.08 mmol) dissolved in EtOH (300 μL) was individually administered to a 500-mL flask containing suspension cultured cells of Spectral data of new compounds are as follows.Genistein 7-O-β-gentiobioside (4): IR (νmax): 3417, 1655, 1612, 1582, 1510, 1495, 1360, 1241; HRFABMS: m/z 617.1488 [M + Na]+ ; 1H-NMR: δ 3.15-3.70 , 4.25 , 5.10 , 6.51 , 6.70 , 6.90 , 7.51 , 8.40 ; 13C-NMR: δ 60.8 (C-6'''), 68.6 (C-6''), 69.2 (C-4'''), 69.8 (C-4''), 72.7 (C-2'''), 73.3 (C-2''), 75.9 (C-5'''), 76.3 (C-5''), 76.4 (C-3'''), 76.9 (C-3''), 99.5 (C-1''), 100.2 (C-8), 102.8 (C-1'''), 101.6 (C-6), 106.0 (C-10), 115.0 , 122.1 (C-1'), 124.1 (C-3), 130.2 , 153.5 (C-2), 156.2 (C-4'), 157.5 (C-9), 160.2 (C-5), 163.0 (C-7), 178.0 (C-4). Anal. Calcd for C27H30O15: C, 52.54; H, 4.90. Found: C, 52.45; H, 4.85.Glycitein 7-O-β-genitiobioside (8): IR (νmax): 3410, 1651, 1611, 1577, 1510, 1490, 1362, 1235; HRFABMS m/z: 631.1645 [M + Na]+ ; 1H-NMR: δ 3.09-3.72 , 3.90 , 4.50 , 5.12 , 7.04 , 7.46 , 7.51 , 7.76 , 8.41 ; 13C-NMR: δ 56.1 (OCH3), 60.5 (C-6'''), 68.9 (C-6''), 69.5 (C-4'''), 69.9 (C-4''), 72.9 (C-2'''), 73.3 (C-2''), 75.7 (C-5'''), 76.7 (C-5''), 76.8 (C-3'''), 77.0 (C-3''), 99.0 (C-1''), 101.9 (C-1'''), 101.0 (C-8), 105.1 (C-5), 115.1 , 117.6 (C-10), 122.5 (C-1'), 124.5 (C-3), 130.3 , 148.8 (C-6), 151.0 (C-9), 152.5 (C-7), 153.5 (C-2), 157.0 (C-4'), 178.0 (C-4). Anal. Calcd for C28H32O15: C, 53.28; H, 5.11. Found: C, 53.25; H, 5.01.β-glycosides of genistein and glycotein were determined as follows: DPPH was dissolved in ethanol (500 μM) [Ac − tA) / Ac] × 100, where At and Ac are the respective absorbance at 517 nm of sample solutions with and without the test compounds. Antioxidant activity was expressed as the 50% inhibitory concentration (IC50).DPPH free-radical scavenging activities of (500 μM) . The sam50 value is shown as the sample concentration at which 50% of superoxide-radical was scavenged.Superoxide was generated by the xanthine-xanthine oxidase system . Reactioβ-glycosides of genistein and glycotein on IgE antibody formation was examined as follows. 7S-Globulin was used as the antigen (1 mg/rat), and Al(OH)3 and pertussis vaccine were used as the adjuvants . Sensitization was made by injection of a mixture (0.6 mL) of the antigen and the adjuvant into the paws of each rat . Paw edema was measured 24 h after injection and the treated rats were divided in groups with an equal average swelling volume. Each sample was dissolved in physiological saline containing 10% Nikkol and the solution was injected daily into the rat for 11 d starting on the day of grouping. Hydrocortisone was used as the positive control. The amount of IgE was measured by the passive cutaneous anaphylaxis method on the 15th day [The inhibitory action of 15th day . The resE. perriniana. The β-glucosides of genistein and glycitein showed DPPH free-radical scavenging activity and superoxide-radical scavenging activity. On the other hand, 7-O-β-glucosides of genistein and glycitein, and 7-O-β-gentiobioside of glycitein exerted inhibitory effects on IgE antibody production.The gentiooligosaccharides of genistein and glycitein were successfully produced by biocatalytic glycosylation with cultured cells of


H-pyrazolooxazin-4-one (3) was prepared by hydrolysis of ethyl 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate (1) to afford the corresponding carboxylic acid 2, which was reacted with acetic anhydride to give 3. The pyrazolooxazin-4-one 3 was reacted with hydroxylamine hydrochloride, urea, thiourea, thiosemicarbazide, phenylhydrazine and aromatic amines to afford the corresponding pyrazolopyrimidin-4-ones 4, 5a,b, 6, 7, 8a–e, respectively. Condensation of pyrazoloxazine derivative 3 with 99% hydrazine hydrate afforded the 5-aminopyrazolopyrimidine derivative 9. Coupling of 9 with aromatic aldehydes yielded a series of 3,6-dimethyl-5-(4-substitutedbenzylideneamino)-1-phenyl-1,5-dihydropyrazolopyrimidin-4-ones 10a–e. The new compounds were tested for their antitumor activity on the MCF-7 human breast adenocarcinoma cell line. Almost all the tested compounds revealed antitumor activity, especially 3,6-dimethyl-5-(4-nitrobenzylideneamino)-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (10e) which displayed the most potent inhibitory activity with a half maximal inhibitory concentration (IC50) of 11 µM.3,6-Dimethyl-1-phenyl-1 Cancer remains one of the most life-threatening diseases, taking nearly 7 million lives each year worldwide. It is realized that neither surgery nor radiation nor the two in combination can adequately control metastatic cancer , therefod]pyrimidine derivatives has received great attention due to their structural similarity with purines and hence several pyrazolopyrimidine derivatives exhibit promising anticancer activity pyrimidin-4-ones were reported to possess antiproliferative activity against breast carcinoma, MCF7 pyrimidin-4-ones has been synthesized and screened in vitro for antitumor activity. The series comprises the derived 5,6-disubstituted pyrazolopyrimidin-4-one pharmacophore that is structurally related to erlotinib and lapatinib  pyrimidin-4-one (4). The presence of a singlet OH band at 3,426 cm−1 and C=O at 1,680 cm−1 confirmed the formation of 4. Its 1H-NMR spectrum showed the appearance of a D2O exchangeable singlet signal at δ 11.52 ppm corresponding to the OH proton. The target compounds 5a,b and 6 were synthesized by fusion at 200 °C of pyrazoloxazine derivative 3 with urea, thiourea and thiosemicarbazide, respectively. The formation of compounds 5a,b and 6 was confirmed by their 1H-NMR spectra that indicated the appearance of exchangeable singlet signals at δ 11.15–12.45 ppm corresponding to NH and NH2. In addition, the mass spectra agreed with the calculated molecular weights of the expected products. On the other hand, reaction of pyrazoloxazine derivative 3 with phenylhydrazine afforded 3,6-dimethyl-1-phenyl-5-phenylamino-1,5-dihydropyrazolopyrimidin-4-one (7). The 1H-NMR of this compound revealed in addition to the corresponding integration for aromatic protons, the presence of NH at δ 9.09 ppm. The mass spectrum also showed a molecular ion peak at m/z 331 as the base peak.The synthesis of the target compounds is outlined in late (1)  gave 5-aacid (2) . The for3 with appropriate aromatic amines furnished 3,6-dimethyl-1-phenyl-5-(4-substitutedphenyl)-1,5-dihydropyrazolopyrimidin-4-ones 8a–e -pyrazolopyrimidine derivatives 10a‒e. The structures of 10a–e were established on the basis of their elemental analysis and spectral data. The 1H-NMR spectra of 10a–e showed singlet downfield CH=N signals at δ 8.32–10.94 ppm. It is worth mentioning that the lack of NH2 bands in the IR spectra and D2O exchangeable NH2 signals in the 1H-NMR spectra confirmed the production of the pyrazolopyrimidine derivatives 10a–e.Moreover, reaction of compound nes 8a–e . The strin vitro cytotoxic activity against human breast cell line (MCF7) using doxorubicin as the reference drug according to the method described as reported by Vichai and Kirtikara pyrimidin-4-one (10e) with an IC50 value of 11 µM, followed by 10d which showed an IC50 value of 12 µM. Compound 9 exhibited the least cytotoxic activity.The obtained data revealed that most of the newly synthesized compounds showed potent antitumor activity. Among the tested compounds, the most potent cytotoxic effect against MCF-7 cell line was obtained with the compound 5-(4-nitrobenzylideneamino)pyrazolopyrimidin-4-one derivative 8b (IC50 = 25) and 5-hydroxy-3,6-dimethyl-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (4) (IC50 = 49). Also, the introduction of a 5-amino function on the pyrazolopyrimidine core produced inactive compound 9 (IC50 = 84), while the introduction of an anilino function favors the anticancer activity as shown by compound 7 (IC50 = 14). Conversion of inactive compound 9 into a 5-substituted benzylidene amino function yielded compounds 10a–e, the most active members of this study. The influence of substituents on the benzylideneamino group on the antitumor activity was in the order NO2 > F > Cl > OCH3 > H. Moreover, 5-(4-substituted benzylideneamino) derivatives 10a–e with azomethine spacer groups were more potent than the 5-(4-substituted phenyl) derivatives 8a–e.From the antitumor screening results the against the MCF-7 cell line, some structure activity relationships can be suggested. Aromatic substitution on N5 position favors the activity; this is obvious upon comparing 5- mass spectra were recorded on a Shimadzu QP-2010 plus instrument. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was routinely employed to follow the course of reactions and to check the purity of products. All reagents and solvents were purified and dried by standard techniques. Elemental microanalyses were carried out at the Microanalytical Center, Cairo University.Melting points were determined on a Griffin apparatus and are uncorrected. IR spectra were determined on Shimadzu IR 435 spectrophotometer and values are presented in cm5-Amino-3-methyl-1-phenyl-1H-pyrazole-4-carboxylic acid (2). A mixture of ethyl 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate  and sodium hydroxide  in methanol (60 mL) was heated under reflux for 5 h. After cooling, the reaction mixture was poured into ice-cold water, then adjusting pH of the mixture to 4 using concentrated hydrochloric acid. The solid obtained was filtered, dried and crystallized from ethanol/water. Mp 156–157 °C; yield: 56%; IR (cm−1): 3389–3204 (OH & NH2); 3009 (CH aromatic); 2927 ; 1651 (C=O); 1613 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.25 ; 6.30 ; 7.36–7.54 ; 12.08 ; MS m/z: 217 ;. Anal. Calcd for C11H11N3O2 (217.23): C, 60.82; H, 5.10; N, 19.34. Found: C, 60.69; H, 5.20; N, 19.67.3,6-Dimethyl-1-phenyl-1H-pyrazolooxazin-4-one (3). A mixture of 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carboxylic acid  and acetic anhydride (5 mL) was heated under reflux for 5 h. After cooling, the formed solid was filtered, dried and crystallized from methanol. Mp 129–130 °C; yield: 49%; IR (cm−1): 3078 (CH aromatic); 2923 ; 1764 (C=O); 1599 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.46 ; 2.49 ; 7.43 ; 7.57 ; 7.90 ; MS m/z: 241 . Anal. Calcd for C13H11N3O2 (241.25): C, 64.72; H, 4.60; N, 17.42. Found: C, 65.01; H, 4.73; N, 17.19.3,6-Dimethyl-5-hydroxy-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (4). A mixture of compound 3  and hydroxylamine hydrochloride  in dry pyridine (30 mL) was heated under reflux for 8 h. The reaction mixture was concentrated to its half volume and the separated solid was filtered, washed with water, dried and crystallized from dioxane. Mp 180–181 °C; yield: 74%; IR (cm−1): 3426 (OH); 2926 ; 1680 (C=O); 1568 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.48 ; 2.54 ; 7.35 ; 7.53 ; 8.03 ; 11.52 ; 13C-NMR (DMSO-6d) δ ppm: 13.71, 21.38, 105.07, 121.58, 126.92, 129.59, 138.75, 145.98, 149.94, 155.24, 157.86. MS m/z: 256 ; Anal. Calcd for C13H12N4O2 (256.27): C, 60.93; H, 4.72; N, 21.86. Found: C, 60.81; H, 5.00; N, 21.66.H-pyrazolooxazin-4-one  and urea or thiourea (10 mmol) was fused at 200 °C for 1 h. The mixture was cooled and methanol was added to the reaction mixture. The separated solid was filtered, washed with methanol, dried and crystallized from ethanol.A mixture of 3,6-dimethyl-1-phenyl-13,6-Dimethyl-4-oxo-1-phenyl-1,4-dihydropyrazolopyrimidine-5-carboxamide (5a). Mp > 300 °C; yield: 65%; IR (cm−1): 3432–3344 (NH2); 3073 (CH aromatic); 2926 ; 1671 (broad band of 2C=O); 1592 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.35 ; 2.46 ; 7.34 ; 7.51 ; 8.02 ; 12.20 ; MS m/z: 283 ; Anal. Calcd for C14H13N5O2 (283.29): C, 59.36; H, 4.63; N, 24.72. Found: C, 59.55; H, 4.52; N, 24.94.3,6-Dimethyl-4-oxo-1-phenyl-1,4-dihydropyrazolopyrimidine-5-carbothioamide (5b). Mp > 300 °C; yield: 45%; IR (cm−1): 3425 (NH2); 2923 ; 1675 (C=O); 1596 (C=N); 1118 (C=S); 1H-NMR (DMSO-6d) δ ppm: 2.38 ; 2.48 ; 7.35 ; 7.51 ; 8.02 ; 12.45 ; Anal. Calcd for C14H13N5OS (299.36): C, 56.17; H, 4.38; N, 23.39. Found: C, 56.22; H, 4.21; N, 23.64.3,6-Dimethyl-4-oxo-1-phenyl-1,4-dihydropyrazolopyrimidine-5-carbothioic acid hydrazide (6). A mixture of 3,6-dimethyl-1-phenyl-1H-pyrazolooxazin-4-one  and thiosemicarbazide  was fused at 200 °C for 1 h. The reaction mixture was cooled and methanol was added to the reaction mixture. The separated solid was filtered, washed with methanol, dried and crystallized from acetone. Mp > 250–251 °C; yield: 63%; IR (cm−1): 3321 (broad band of NH &NH2); 3064 (CH aromatic); 2920 ; 1694 (C=O); 1590 (C=N); 1105 (C=S); 1H-NMR (DMSO-6d) δ ppm: 2.37 ; 2.49 ; 7.35 ; 7. 51 ; 8.01 ; 11.15 ; 12.19 ; MS m/z: 314 ; Anal. Calcd for C14H14N6OS (314.37): C, 53.49; H, 4.49; N, 26.73. Found: C, 53.28; H, 4.47; N, 26.89.3,6-Dimethyl-1-phenyl-5-phenylamino-1,5-dihydropyrazolopyrimidin-4-one (7). A mixture of 3,6-dimethyl-1-phenyl-1H-pyrazolooxazin-4-one  and phenylhydrazine  in ethanol (20 mL) was heated under reflux for 6 h. The separated solid was filtered while hot, dried and crystallized from butanol. Mp 265–266 °C; yield: 54%; IR (cm−1): 3250 (NH); 3115–3011 (CH aromatic); 2930 ; 1685 (C=O); 1H-NMR (DMSO-6d) δ ppm: 2.49 ; 2.53 ; 6.69 ; 6.86 ; 7.21 ; 7.39 ; 7.56 ; 8.05 ; 9.09 ; MS m/z: 331 ; Anal. Calcd for C19H17N5O (331.38): C, 68.87; H, 5.17; N, 21.13. Found: C68.68; H, 5.09; N, 21.05.H-pyrazolooxazin-4-one  and the appropriate aromatic amine (10 mmol) in dry pyridine (30 mL) was heated under reflux for 6 h. The reaction mixture was poured into ice-cold water and the mixture was neutralized with hydrochloric acid (10%). The separated product was filtered, washed with water, dried and crystallized from the appropriate solvent.A mixture of 3,6-dimethyl-1-phenyl-13,6-Dimethyl-1-phenyl-5-p-tolyl-1,5-dihydropyrazolopyrimidin-4-one (8a). Mp 170–171 °C ; yield: 50%; IR (cm−1): 2923 ; 1693 (C=O); 1568 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.16 ; 2.51 ; 2.66 ; 7.38–7.41 ; 7.56–7.62 ; 8.07–8.15 ; MS m/z: 330 ; Anal. Calcd for C20H18N4O (330.39): C, 72.71; H, 5.49; N, 16.96. Found: C, 72.77; H, 5.20; N, 16.64.3,6-Dimethyl-5-(4-hydroxyphenyl)-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (8b). Mp 250–252 °C ; yield: 76%; IR (cm−1): 3439 (OH); 2930 ; 1694 (C=O); 1566 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.17 ; 2.51 ; 6.89 ; 7.17 ; 7.38 ; 7.53 ; 8.07 ; 9.80 ; MS m/z: 332 ; Anal. Calcd for C19H16N4O2 (332.36): C, 68.66; H, 4.85; N, 16.86. Found: C, 68.30; H, 4.50; N, 16.98.5-(4-Acetylphenyl)-3,6-dimethyl-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (8c). Mp 186–187 °C ; yield: 66%; IR (cm−1): 2924 ; 1699 (broad band of 2C=O); 1591 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.17 ; 2.44 ; 2.50 ; 7.23 ; 7.26 ; 7.52–7.64 ; 8.08 ; MS m/z: 358 ; Anal. Calcd for C21H18N4O2 (358.40): C, 70.38; H, 5.06; N, 15.63. Found: C, 69.97; H, 4.98; N, 15.94.4-benzoic acid (8d). Mp 200–201 °C ; yield: 59%; IR (cm−1): 3450 (OH); 3068 (CH aromatic); 2929 ; 1713, 1678 (2C = O); 1558 (C = N); 1H NMR (DMSO-6d) δ ppm: 2.16 ; 2.51 ; 7.38 ; 7.54-7.65 ; 8.07-8.13 ; 13.15 ; MS m/z: 360 ; Anal. Calcd for C20H16N4O3 (360.38): C, 66.66; H, 4.48; N, 15.55. Found: C, 66.90; H, 4.71; N, 15.78.Ethyl 4-benzoate (8e). Mp 190–191 °C ; yield: 85%; IR (cm−1): 2925 ; 1709 (broad band of 2C=O); 1555 (C=N); 1H NMR (DMSO-6d) δ ppm: 1.36 ; 2.15 ; 2.51 ; 4.37 ; 7.39 ; 7.53–7.62 ; 8.07–8.15 ; MS m/z: 388 ; Anal. Calcd for C22H20N4O3 (388.43): C, 68.03; H, 5.19; N, 14.42. Found: C 68.21; H, 5.51; N, 14.66.5-Amino-3,6-dimethyl-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (9). A mixture of compound 3  and hydrazine hydrate  in butanol (20 mL) was heated under reflux for 6 h. After cooling, the separated solid was filtered, dried and crystallized from ethanol. Mp 129–130 °C; yield: 47%; IR (cm−1): 3322–3262 ; 3065 (CH aromatic); 2924 ; 1692 (C=O); 1H-NMR (DMSO-6d) δ ppm: 2.50 ; 2.59 ; 5.71 ; 7.34 ; 7.52 ; 8.05 ; MS m/z: 255 . Anal. Calcd for C13H13N5O (255.28): C, 61.17; H, 5.13; N, 27.43. Found: C, 61.53; H, 5.03; N, 27.23.9  and the appropriate aromatic aldehyde (12 mmol) in acetic acid (15 mL) was heated under reflux for 20 h. The reaction mixture was concentrated under vacuum then cooled. The obtained solid was filtered, washed with water, dried and crystallized from the appropriate solvent. The spectral data for compounds 10a–e are listed below.A mixture of compound (E)-5-(Benzylideneamino)-3,6-dimethyl-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (10a). Mp 250–251 °C ; yield: 70%; IR (cm−1): 3055 (CH aromatic); 2925 ; 1702 (C=O); 1555 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.50 ; 2.55 ; 7.39 ; 7.53-7.65 ; 7.98 ; 8.08 ; 8.89 ; MS m/z: 343 ; Anal. Calcd for C20H17N5O (343.39): C, 69.96; H, 4.99; N, 20.39. Found: C, 69.77; H, 5.20; N, 20.54.(E)-3,6-Dimethyl-5-(4-fluorobenzylideneamino)-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (10b). Mp 160–161 °C ; yield: 79%; IR (cm−1): 3067 (CH aromatic); 2928 ; 1709 (C=O); 1596 (C=N); 1H NMR (DMSO-6d) δ ppm: 2.10 ; 2.41 ; 7.38–7.40 ; 7.52–7.57 ; 7.99-8.01 ; 10.94 ; Anal. Calcd for C20H16FN5O (361.38): C, 66.47; H, 4.46; N, 19.38. Found: C, 66.38; H, 4.02; N, 19.61.(E)-3,6-Dimethyl-5-(4-methoxybenzylideneamino)-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (10c). Mp 255–256 °C ; yield: 55%; IR (cm−1): 3027 (CH aromatic); 1671 (C=O); 1594 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.49 ; 2.51 ; 3.84 ; 7.22 ; 7.37–7.57 ; 8.07 ; 8.85 ; MS m/z: 373 ; Anal. Calcd for C21H19N5O2 (373.42): C, 67.55; H, 5.13; N, 18.75. Found: C, 67.10; H, 5.01; N, 18.47.(E)-5-(4-Chlorobenzylideneamino)-3,6-dimethyl-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (10d). Mp > 300 °C ; yield: 60%; IR (cm−1): 3064 (CH aromatic); 2925 ; 1698 (C=O); 1596 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.09 ; 2.54 ; 7.37 ; 7.56 ; 7.66 ; 7.99 ; 8.32 ; 8.32 ; 13C-NMR (DMSO-6d) δ ppm: 13.72, 23.32, 104.40, 121.72, 127.13, 129.68, 129.83, 130.93, 131.63, 137.96, 138.66, 146.86, 150.30, 154.86, 157.74, 168.91. MS m/z: 377 , Anal. Calcd for C20H16ClN5O (377.85): C, 63.58; H, 4.27; N, 18.51. Found: C, 63.28; H, 4.04; N, 18.13.(E)-3,6-Dimethyl-5-(4-nitrobenzylideneamino)-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (10e). ; mp > 300 °C; yield: 95%; IR (cm−1): 3057 (CH aromatic); 2925 ; 1690 (C=O); 1597 (C=N); 1H-NMR (DMSO-6d) δ ppm: 2.60 ; 2.61 ; 7.39 ; 7.57 ; 8.07 ; 8.25 ; 8.42 ; 9.16 ; MS m/z: 388 . Anal. Calcd for C20H16N6O3 (388.39): C, 61.85; H, 4.15; N, 21.64. Found: C, 62.15; H, 4.44; N, 21.40.2 95% air and high humidity atmosphere in the water jacketed incubator . The cell line was regularly subcultured to be maintained in the exponential growth phase. The sterile conditions were strictly attained by working under the equipped laminar flow .Human breast cancer cell line, MCF was grown as monolayer culture in RPM 11640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cell line was incubated at 37 °C 5% COThe cytotoxicity was carried out using the sulphorhodamine-B (SRB) assay. Cells were seeded in 96 well microtiter plates at a concentration of 1,000–2,000 cells/well, 100 µL/well, After 24 h, cells will be incubated for 72 h with various concentrations of drugs . For each derivative concentration and doxorubicin, 3 wells were used. The plates were incubated for 72 h. The medium is discarded. The cells were fixed with 150 μL cold trichloroacetic acid 10% final concentration for 1 h at 4 °C.The plates were washed with distilled water using a Tecan automatic washer  and stained with 50 μL 0.4% SRB dissolved in 1% acetic acid for 30 min at room temperature in dark. The plates were washed with 1% acetic acid to remove unbound dye and air-dried (24 h).The dye was solubilized with 150 µL/well of 10 mMtris base (PH 7.4) for 5 min on a shaker at 1,600 rpm. The optical density (OD) of each well will be measured spectrophotometrically at 490 nm with an ELISA microplate reader. The mean background absorbance was automatically subtracted and mean values of each derivative and doxorubicin concentration was calculated. The experiment was repeated 3 times. The percentage of cell survival was calculated as follows:d]pyrimidin-4-ones 4, 5a,b, 6, 7, 8a–e, 10a–e were synthesized. The cytotoxic activity of the newly synthesized compounds against the human breast adenocarcinoma cell line MCF-7 was investigated. Almost all the tested compounds revealed some antitumor activity, in particular 3,6-dimethyl-5-(4-nitrobenzylideneamino)-1-phenyl-1,5-dihydropyrazolopyrimidin-4-one (10e) which exhibited the highest activity among the tested compounds with an IC50 equal to 11 µM. The antitumor screening revealed that aromatic substitution on the N5 position favors the activity. Moreover, 5-(4-substituted benzylideneamino) derivatives 10a–e with azomethine spacer groups were more potent than the 5-(4-substituted phenyl) derivatives 8a–e.In conclusion, series of new 5-substituted pyrazolo[3,4-


Acorus calamus rhizomes, its insecticidal activity against the booklouse, (Liposcelis bostrychophila) and to isolate any insecticidal constituents from the essential oil. The essential oil of A. calamus rhizomes was obtained by hydrodistillation and analyzed by GC-FID and GC-MS. A total of 32 components of the essential oil of A. calamus rhizomes was identified and the principal compounds in the essential oil were determined to be α-asarone (50.09%), (E)-methylisoeugenol (14.01%), and methyleugenol (8.59%), followed by β-asarone (3.51%), α-cedrene (3.09%) and camphor (2.42%). Based on bioactivity-guided fractionation, the three active constituents were isolated from the essential oil and identified as methyleugenol, (E)-methylisoeugenol and α-asarone. The essential oil exhibited contact toxicity against L. bostrychophila with an LD50 value of 100.21 µg/cm2 while three constituent compounds, α-asarone, methyleugenol, and (E)-methylisoeugenol had LD50 values of 125.73 µg/cm2, 103.22 µg/cm2 and 55.32 µg/cm2, respectively. Methyleugenol and (E)-methylisoeugenol possessed fumigant toxicity against L. bostrychophila adults with LC50 values of 92.21 μg/L air and 143.43 μg/L air, respectively, while the crude essential oil showed an LC50 value of 392.13 μg/L air. The results indicate that the essential oil of A. calamus rhizomes and its constituent compounds have potential for development into natural fumigants/insecticides for control of the booklice. The aim of this research was to determine the chemical composition of the essential oil of  Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae) has a worldwide distribution and has been reported infesting domestic premises, raw material stores, manufacturing factories, and also museums  and AMX500 [500 MHz (1H)] instruments using CDCl3 as the solvent with TMS as internal standard. Electron impact mass spectra (EIMS) were determined on a Micromass VG7035 mass spectrometer at 70 eV (probe).The crude essential oil of Methyleugenol  δ (ppm): 6.81 , 6.74 , 6.71 , 5.96 , 5.07 , 3.86 , 3.87 , 3.34 . 13C-NMR  δ (ppm): 148.8 (C-4), 147.3 (C-3), 137.7 (C-8), 132.6 (C-6), 120.4 (C-1), 115.6 (C-9), 111.8 (C-2), 111.2 (C-5), 55.9(-OCH3), 55.7 (-OCH3), 39.8 (C-7). EI-MS m/z (%): 178 (100), 163 (33), 151 (15), 147 (32), 135 (18), 131 (11), 107 (28), 103 (39), 91 (50), 77 (26). The spectral data matched with a previous report [genol 1, . A colors report . (E)-Methylisoeugenol  δ (ppm): 6.83 , 6.81 , 6.76 , 6.35 , 5.67 , 3.81 , 3.80 , 1.87 . 13C-NMR  δ (ppm): 148.5 (C-4), 147.7(C-3), 130.7 (C-1), 129.5 (C-7), 125.4 (C-6), 121.4 (C-5), 112.2 (C-2), 110.1 (C-8), 55.9 (-OCH3), 55.8 (-OCH3), 14.36(C-9). EI-MS m/z (%): 178 (100), 163 (44), 147 (11), 135 (18), 131 (11), 115 (138), 107 (39), 103 (28), 91 (39), 79 (17), 77 (15).The spectral data matched with a previous report [genol 2, . A colors report . α-Asarone  δ (ppm): δ: 6.95 , 6.66 , 6.48 , 6.09 , 3.90 , 3.84 , 3.81 , 1.89 . 13C-NMR  δ (ppm): 159.9 (C-4), 148.9 (C-6), 143.5 (C-1), 125.2 (C-7), 124.5 (C-8), 119.2 (C-3), 109.9 (C-2), 98.1 (C-5), 56.8 (C-10), 56.6 (C-12), 56.2 (C-11), 18.9 (C-9). EI-MS m/z (%): 208 (100), 165 (38), 137 (21), 105 (13), 91 (19), 69 (25), 41 (11). The spectral data matched with a previous report [arone 3, . A crysts report . A. calamus rhizomes and pure compounds. The essential oil and compound were diluted in acetone. The filter paper with 3.5 cm in diameter (Whatman) was treated with 150 μL of the solution. Then the filter paper after treated with solid glue  was placed in a Petri dish (3.5 cm in diameter) and 10 booklice were put on the filter paper by using a hair brush. The plastic cover with holes was put and all the Petri dishes were kept in incubators at 27–29 °C, 70%–80% r.h. for 24 h. Acetone was used as controls and pyrethrum extract was used as a positive control. Six concentrations  and five replicates of each concentration were used in all treatments and controls. Mortality of insects was observed and the observed data were corrected for control mortality using Abbott’s formula. The results from all replicates were subjected to probit analysis using the PriProbit Program V1.6.3 to determine LC50 values [Range-finding studies were run to determine the appropriate testing concentrations of the essential oil of 0 values . PyrethrA. calamus essential oil. A filter paper strip (3.5 cm × 1.5 cm) treated with 10 μL of an appropriate concentration of test essential oil/compound in acetone. The impregnated filter paper was then placed in the bottom cover of glass bottle of 250 mL. The insects, 10 adults in a small glass bottle (8 mL), were exposed for 24 h and each concentration with five replicates. Six concentrations  were used in all treatments and controls. Acetone was used as controls and dichlorvos was used as a positive control. The observed mortality data were corrected for control mortality using Abbott’s formula. The LC50 values were calculated by using Probit analysis [Range-finding studies were run to determine the appropriate testing concentrations of the pure compounds and analysis . PositivA. calamus rhizomes and its constituent compounds, methyleugenol, (E)-methylisoeugenol and α-asarone have potential for development into natural fumigants/insecticides for control of the booklice.The study indicates that the essential oil of


Sanguisorba tenuifolia was found to have a hypoglucemic effect in alloxan-induced diabetic rats. Two new triterpenoids, identified as 2-oxo-3β,19α-dihydroxyolean-12-en-28-oic acid β-D-gluco-pyranosyl ester (1) and 2α,19α-dihydroxy-3-oxo-12-ursen-28-oic acid β-D-glucopyranosyl ester (4) were isolated from this fraction, along with thirteen known triterpenoids. Their structures were elucidated by chemical and spectroscopic methods. All these compounds demonstrated inhibitory activities against α-glucosidase with IC50 values in the 0.62-3.62 mM range.The ethyl acetate soluble fraction from the roots of  S. tenuifolia is one such plant.Diabetes mellitus (DM), considered a lifestyle related diseases, is a metabolic disease with hyper-glycemia as a symptom and causes many complications . RecentlS. tenuifolia (Rosaceae) is a perennial herb, which is widely distributed in China’s Heilongjiang, Liaoning, and Jilin provinces and Inner Mongolia. The residents in Northeast China regard S. tenuifolia as a substitute for S. officinalis, and apply its roots for the treatment of diarrhea, chronic intestinal infections, duodenal ulcers, diabetes mellitus and bleeding + ion signal at m/z 666.4221  and [M + Na]+ ion signal at m/z 671.3779 . The IR spectrum showed the presence of hydroxyl groups , ester carbonyl , carbonyl  and double bond . Compound 13C-NMR spectrum and DEPT of 1 showed seven methyl, nine methylene, eleven methine, and nine quaternary carbon signals, including one ester carbonyl at Cδ 177.3, a quaternary olefinic carbonyl at Cδ 144.5, one anomeric carbon signal at Cδ 95.9, a ketone carbonyl atCδ 213.4. The 1H-NMR spectrum exhibited seven singlet methyl signals at Hδ 1.21, 0.92, 1.01, 1.15, 1.52, 1.12 and 0.95, an anomeric proton signal at Hδ 6.37 , two methine proton signals at Hδ 3.89 (s) and 3.54 , and an olefinic proton signal at Hδ 5.45 (br s), which were characteristic of the oleanolic acid skeleton. Comparison of the data 1 with those of oleanolic acid  and 2α,19α-dihydroxy-3-oxo-olean-12-en-28-oic acid β-D-glucopyranosyl ester (6) [C-2 (Cδ 216.5) and C-3 ]  urs-12,19(29)-dien-28-oic acid 28-β-D-glucopyranosyl ester (14) + , 649.3953 [M + H]+ , 666.4221 [M + NH4]+ ; 1H- and 13C-NMR (pyridine-5d) data, see α,19α-Dihydroxy-3-oxo-12-ursen-28-oic acid β-D-glucopyranosyl ester (4). White amorphous powder.  + 21.5° . IR (KBr): 3431.1, 1737.1, 1714.3, 1644.6, 1070.4, 1029.9, 991.3 cm-1. HR-ESI-MS m/z 649.3945 [M + H]+ ; 1H- and 13C-NMR (pyridine-5d) data, see 21 (5 mg) in 1 M HCl  was heated at 90 °C for 3 h under an Ar atmosphere. After the dioxane was removed, the solution was extracted with EtOAc (3 mL × 3) to remove the aglycone. The aqueous layer was neutralized by passing through an ion-exchange resin column  and concentrated to dryness under reduced pressure to give the sugar fraction. The residue was dissolved in pyridine (0.1 mL) to which 0.1 M L-cysteine methyl ester hydrochloride in pyridine (0.1 mL) was added. The mixture was heated at 60 °C for 1 h. After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with l-trimethylsilylimidazole (0.2 mL) for 2 h. The mixture was partitioned between hexane and H2O , and the hexane extracted was analyzed by GC under the following conditions: capillary column, DM-5 (0.25 mm × 30 m × 0.25 μm); detector, FID; injector temperature, 280 °C, detector temperature, 280 °C; initial temperature was maintained at 160 °C for 2 min and then raised to 195 °C at a rate of 10 °C/min; carrier gas, N2. In the acid hydrolysate of 1, D-glucose was confirmed by comparison of the retention time of their derivatives with those of D-glucose and L-glucose derivatives prepared in a similar way, which showed retention times of 28.56 and 27.72 min, respectively. The sugar from 4 (30 mg) was also identified by the same method.α-Glucosidase (EC.3.2.1.20) enzyme inhibition assay has been performed according to the literature [α-Glucosidase , various concentrations of samples (25 μL), and 67 mM phosphate buffer  were mixed at room temperature for 10 min. Reactions were initiated by the addition of 23.2 mM p-nitrophenyl-α-D-glucopyranoside (25 μL). The reaction mixtures were incubated at 37 °C for 15 min in a final volume of 250 μL, and then 1 M Na2CO3 (50 μL) was added to the incubation solution to stop the reaction. The activities of glucosidase were detected in a 96-well plate, and the absorbance was read at 405 nm by a microplate spectrophotometer . The negative control was prepared by adding phosphate buffer instead of the sample in the same way as the test. Acarbose was utilized as the positive control. The blank was prepared by adding phosphate buffer instead of α-glucosidase using the same method. The inhibition rates (%) were calculated from the following formula: terature . α-Glucoβ,19α-dihydroxyolean-12-en-28-oic acid β-D-glucopyranosyl ester (1) and 2α,19α-dihydroxy-3-oxo-12-ursen-28-oic acid β-D-glucopyranosyl ester (4) were isolated from an ethyl acetate fraction of S. tenuifolia roots, along with thirteen known triterpenoids. All these triterpenoids are reported for the first time in S. tenuifolia and demonstrated inhibitory activities against α-glucosidase with IC50 values in the 0.62-3.62 mM range. Triterpenoids of the EtOAc-soluble fraction may be the potential anti-hypoglycemic agents in this plant, as they have been shown to induce an anti-diabetic effect.Two new triterpenoids, 2-oxo-3


The derivatives 4-allylbenzene-1,2-diol (3), 4 4-[3-(acetyloxy)propyl]-1,2-phenylene diacetate (6) and 4-[3-(acetyloxy)propyl]-5-nitro-1,2-phenylene diacetate (10) showed higher cytotoxicity than the parent compound 2 in tests performed on two breast cancer cell lines (MCF-7 and MDA-MB-231). The IC50 values of 40.2 ± 6.9 μM, 5.9 ± 0.8 μM and 33.8 ± 4.9 μM, respectively, were obtained without toxicity towards dermal human fibroblast (DHF cells).Catechols were synthesized from safrole. Nine derivatives were prepared and assessed for antiproliferative effects using different human cell lines. The  In vitro, a depletion of glutathione (GSH) was observed by the addition of safrole to hepatocytes -1,2-phenylene diacetate (6) and 4-allyl-1,2-phenylene diacetate (7) from4: The compound 4  cooled to −5 °C, under an atmosphere of N2, was slowly hydroborated with a 2.0 M solution of BH3·DMS/THF (0.25 mL) at −10 °C for 15 min was added dropwise. Then the mixture was stirred at room temperature for 1 h. The resultant organoborane was oxidized by sodium perborate  and water (100 mL). The mixture was stirred at room temperature for 2 h. Then, it was extracted with portions of ethyl ether (4 × 50 mL) and the layers were separated. The organic layer was dried over MgSO4, filtered, evaporated and re-dissolved in 5 mL of CH2Cl2. It was absorbed on silica, chromatographed by CC, eluting with mixtures of petroleum ether/EtOAc of increasing polarity (18.8:1.2→17.6:2.4). Then two fractions were obtained: fraction I  of compound 5 and fraction II (147.4 mg), a mixture of polar products. This mixture was dissolved in dry CH2Cl2 (50 mL), DMAP (1.02 mg) and Ac2O  were added and the mixture was stirred at room temperature for 2 h. A cooled solution of 10% KHSO4 (approx. 50 mL) was then added to this mixture. The watery layer was discarded and the organic layer was washed to neutrality with a saturated solution of NaHCO3 and water. Then it was dried over MgSO4, filtered, evaporated and re-dissolved in 5 mL of CH2Cl2. Subsequently, it was absorbed on silica and chromatographed by CC with petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→17.8:2.2). Two products were isolated identified as the triacetylated 6  and the diacetylated 7 . Compound 6; IR (cm−1): 2925 (C-H); 1731 (C=O); 1561 (C=C); 1429 (-CH2); 1214 (C-O); 1076 (C-O); 896 (C-H). M.S. : 295 (25.7); 282 (18.4); 281 (64.2); 267 (11.5); 222 (18.7); 221 (80.6); 207 (33.6); 147 (78.6); 73 (100). 1H-NMR: 7.08 ; 7.01 ; 4.10 ; 2.68 ; 2.26 ; 2.05 ; 1.98 . 13C-NMR: 171.1 (CH3CO); 168.4 (CH3CO); 168.3 (CH3CO); 141.9 (C-2); 140.2 (C-1); 140.1 (C-4); 126.5 (C-5); 123.2 (C-3); 123.1 (C-6); 63.6 (C-3’); 31.6 (C-2’); 29.9 (C-1’); 20.9 (CH3); 20.6 (2 × CH3). Compound 7; IR (cm−1): 2943 (C-H); 1707 (C=O); 1429 (-CH2); 1371 (-CH3); 1221 (C-O); 1017 (C-O); 905 (C-H). M.S. : 236 (5.3); 194 (19.3); 153 (11.8); 152 (100); 124 (6.9); 123 (74.5); 109 (3.6); 77 (5.4). 1H-NMR: 7.06 ; 6.99 ; 2.57 ; 2.28 ; 1.63 ; 0.95 . 13C-NMR: 168.5 (CH3CO); 168.4 (CH3CO); 141.7 (C-2); 141.6 (C-1); 139.8 (C-4); 126.5 (C-5); 123.1 (C-6); 122.9 (C-3); 37.3 (C-2’); 24.2 (C-2’); 20.6 (2x CH3); 13.8 (CH3).8), 4-(2-hydroxypropyl)-5-nitrobenzene-1,2-diol (9), 4-[3-(acetyloxy)propyl]-5-nitro-1,2-phenylene diacetate (10) and 3-propyl acetate (11) from 2: The compound 2  added dropwise at −10 °C for 15 min, and later the mixture was stirred at room temperature for 1 h. The resultant organoborane was oxidized by a solution of sodium perborate  and water (100 mL) and then the mixture was stirred at room temperature for 2 h. Then, it was extracted with portions of ethyl ether (4 × 50 mL) and the layers were separated. The organic layer was dried over MgSO4, filtered, evaporated and re-dissolved in 5 mL of CH2Cl2. It was absorbed on silica, chromatographed by CC eluting with mixtures of petroleum ether/EtOAc of increasing polarity (16.0:4.0→14.0:6.0). Isolated two solids compounds. Solids were recrystallized in a mixture methanol/ether, purifying the product 8  and isomer 9 . Compound 8, dark yellow, p.f.: 98.9–99.8 °C. IR (cm−1): 3458 (O-H); 2921 (=C-H); 1593 (C=C); 1532 (NO2); 1457 (CH2); 1385 (CH3); 1331 (N=O); 880 (-C-H). 1H- NMR: 9.24 ; 8.88 ; 7.53 ; 6.85 ; 5.89 ; 4.39 ; 2.88 , 1.79 . 13C-NMR: 151.3 (C-2); 144.2 (C-1); 141.5 (C-5); 132.2 (C-4); 118.5 (C-3); 112.9 (C-6); 61.8 (C-3’); 34.4 (C-2’); 30.1 (C-1’). Compound 9, dark yellow; p.f.: 104.3–105.8 °C. IR (cm−1): 3520 (O-H); 2923 (C-H); 1599 (C=C); 1530 (NO2); 1458 (-CH2); 1376 (-CH3); 1327 (N=O); 1043 (-C-O); 885(-C-H). 1H-NMR: 9.19 ; 8.84 ; 7.53 ; 6.84 ; 4.12 ; 3.73 ; 3.36 ; 3.16 ; 1.33 . 13C-NMR: 151.1 (C-2); 145.1 (C-1); 144.2 (C-5); 125.8 (C-4); 114.6 (C-3); 108.55 (C-6); 68.4 (C-2’); 39.1 (C-1’), 24.7 (C-3’). Then, this complex mixture (0.15 g) was dissolved in dry CH2Cl2 (50 mL), DMAP (1.08 mg) and Ac2O  were added and the mixture was stirred at room temperature for 2 h. A cooled solution of 10% KHSO4 (approx. 50 mL) was then added to this mixture. The watery layer was discarded and the organic layer was washed to neutrality with a saturated solution of NaHCO3 and water. Then it was dried over MgSO4, filtered, evaporated and re-dissolved in 5 mL of CH2Cl2. Subsequently, it was absorbed on silica and chromatographed by CC with petroleum ether/EtOAc mixtures of increasing polarity (18.8:1.2→15.6:4.4). Two products were isolated and identified as nitrated triacetyl derivative 10 , and monoacetylated nitrated compound 11 . Compound 10, IR (cm−1): 2955 (C-H); 1757 (C=O); 1588 (C=C); 1531 (NO2); 1432 (-CH2); 1368 (C-NO2); 1045 (C-O). M.S. : 339 (2.8); 326 (5.7); 298 (23.3); 267 (8.2); 255 (18.3); 199 (11.3); 143 (25.3); 87 (69.6); 74 (100); 69 (14.1); 55 (22.0). 1H-NMR: 7.89 ; 7.23 ; 4.14 ; 2.98 ; 2.32 ; 2.06 ; 2.00 . 13C-NMR: 171.1 (CH3CO); 167.6 (CH3CO); 167.3 (CH3CO); 145.9 (C-5); 145.6 (C-2); 140.4 (C-1); 135.8 (C-4); 126.4 (C-3); 120.9 (C-6); 63.5 (C-3’); 29.7 (C-2’); 29.2 (C-1’); 20.9 (CH3CO); 20.6 (CH3CO); 20.5 (CH3CO). Compound 11; IR (cm−1): 3351 (O-H); 2978 (C-H); 1739 (C=O); 1633 (C=C); 1546 (NO2); 1425 (CH2); 1253 (C-O). 1H-NMR: 7.56 ; 6.85 ; 4.06 ; 2.94 ; 2.92 ; 1.99 ; 1.91 . 13C-NMR: 171.0 (CH3CO); 151.5 (C-2); 144.4 (C-1); 141.4 (C-5); 131.4 (C-4); 118.7 (C-3); 113.2 (C-6); 64.1 (C-3’); 30.3 (C-2’); 29.9 (C-1’); 20.7 (CH3CO).Synthesis of 4-(3-hydroxypropyl)-5-nitrobenzene-1,2-diol , 4- the cells were treated with different concentrations of drugs (safrole and derivatives) and incubated for 72 h under the same conditions. Stock solution of compounds was prepared in ethanol and the final concentration of this solvent was kept constant at 0.1%. Control cultures received 0.1% ethanol alone.The experimental cell cultures were obtained from American Type Culture Collection . MCF-7, MDA-MB-231 cells were grown in DMEM-F12 medium containing 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mM glutamine. Cells are seeded into 96 well microtiter plates in 100 µL at plating density of 3 × 10et al. [3 cells per well of a 96-well, flat-bottomed 200 μL microplate. Cells were incubated at 37 °C in a humidified 5% CO2/95% air mixture and treated with the compounds at different concentrations for 72 hours. At the end of drug exposure, cells were fixed with 50% trichloroacetic acid at 4 °C . After washing with destilled water, cells were stained with 0.1% sulforhodamine B , dissolved in 1% acetic acid (50 µL/well) for 30 min, and subsequently washed with 1% acetic acid to remove unbound stain. Protein-bound stain was solubilized with 100 µL of 10 mM unbuffered Tris base. The cell density was determined using a fluorescence plate reader (wavelength 540 nm). Values shown are the mean + SD three independent experiments in triplicate.The sulforhodamine B assay according to the method of Skehan et al.  was usedet al. . Briefly3 was achieved with good performance and successful hydroboration. In relation to the above method, both protected and free catechols enhanced the reaction in order to resolve the complex mixture. As a subproduct, a catechol with an n-propyl side chain was unexpectedly obtained. The safrole derivatives 6  and 10  proved to be more cytotoxic than safrole and derivative 2 against the selected cancer cells lines. These results indicate that 6  and 10 exert their cytotoxic and selective effects on the breast cancer cells lines MCF-7, MDA-MB-231 over a wide concentration range without effects in non tumoral cell line DFH. We think that the cytotoxic effect of catechol derivatives could be related to changes in intracellular free Ca2+ levels, as reported in safrole by Jan [In synthetic terms, the establishment of a new protocol for the deprotection of safrole with BFe by Jan . The aut


H,3H)-dione and 1-allyl-5-methyl-3-(oxiran-2-ylmethyl)-pyrimidine-2,4-dione, with (S)-prolinol. The new templates were obtained with good yields following a route which exploits the reactivity of epoxides in the presence of sulphated zirconia as catalyst. The key step was carried out using microwave and solvent-free conditions and proceeds with high selectivity.New azanucleosides were obtained using sulphated zirconia (ZS) as catalyst in the nucleophilic oxirane ring opening reaction of 1-allyl-3-(oxiran-2-ylmethyl)pyrimidine-2,4(1 Under aC, -2-(hydroxymethyl)pyrrolidin-1-yl)propyl)pyrimidine-2,4-dione) (8a). N-1-Allyluracil  in DMF (5 mL) were placed in a round-bottom flask. Then, potassium tert-butoxide  was added at 0 °C under a nitrogen atmosphere over 5 min. Then, the mixture was stirred for 25 min at room temperature. Finally, epibromohydrin  was added and the reaction mixture was stirred for 24 h at room temperature. After this time, the mixture was diluted with methylene chloride (5 mL) and the product was recovered by extractions with methylene chloride (5 × 15 mL) and water (5 mL) and the organic extract was dried over Na2SO4 and concentrated under vacuum to give 6a. The crude product was used directly in the next step. In a closed reaction system there were placed 6a  and (S)-prolinol  with sulphated zirconia (100 mg). The reaction mixture was irradiated in a microwave reactor (CEM Labmate®) under vigorous agitation at 60 °C for 150 min . After the reaction time methanol (10 mL) was added, the catalyst was recovered by filtration and washed with acetone (20 mL). The organic product was dried under reduced pressure. The residue was purified by flash column chromatography on silica gel  to give 8a as a colorless oil. 1H-NMR (CDCl3): δ 7.26 , 5.89 , 5.79 , 5.30 , 4.39 , 4.10 , 3.62 , 3.42 , 3.24 , 2.85 , 2.76 , 2.53 , 2.38 , 1.8 ; 13C-NMR (CDCl3): δ 164.1, 152.0, 142.7, 131.4, 119.4, 101.7, 68.1, 67.5, 66.2, 63.1, 62.8, 59.8, 59.5, 56.0, 54.9, 51.3, 45.3, 27.1, 23.5; HRMS (FAB) calc. for C15H23N3O4 309.1689, found 309.1654.1-Allyl-3-(2-hydroxy-3-((S)-2-(hydroxymethyl)pyrrolidin-1-yl)propyl)-5-methylpyrimidine-2,4-dione  (8b).N-1 Allylthymine  in DMF (5 mL) was placed in a round-bottom flask. Then, t-BuOK  was added at 0 °C under a nitrogen atmosphere over 5 min. Then, the mixture was stirred for 25 min at room temperature. Finally, epibromohydrin  was added and the reaction mixture was stirred for 24 h at room temperature. After this reaction time, the mixture was diluted with methylene chloride (5 mL) and the product was recovered by extractions with methylene chloride (5 × 15 mL) and water (5 mL) and the organic extract was dried over Na2SO4 and concentrated under vacuum to give 6a. The crude product was used directly in the next step. In a closed reaction system were placed 6b (0.454 g as crude product) and (S)-prolinol  with sulphated zirconia (100 mg). The reaction mixture was irradiated in a microwave reactor (CEM Labmate®) under vigorous agitation at 60 °C during 150 min . Next methanol (10 mL) was added, the catalyst was recovered by filtration and washed with acetone (20 mL). The organic product was dried under reduced pressure. The residue was purified by flash column chromatography on silica gel  to give 8b as a colorless oil. 1H-NMR (CDCl3): δ 7.02 , 5.79 , 5.25 , 4.29 , 4.10 , 3.61 , 3.52 , 3.27 , 2.93 , 2.61 , 2.70 , 2.53 , 1.80 , 1.85 ; 13C-NMR (CDCl3): δ 164.8, 152.0, 138.98, 131.8, 119.2, 110.2, 67.4, 66.9, 62.2, 59.9, 55.0, 51.2, 45.4, 26.9, 24.4, 12.9; HRMS (FAB) calc. for C16H25N3O4 323.1845, found 323.1839.H,3H)-dione (6a) and 1-allyl-5-methyl-3-(oxiran-2-ylmethyl) pyrimidine-2,4--dione (6b) with (S)-prolinol to give the azanucleosides 8a and 8b with good yield. The advantage of our approach is that the epoxide ring-opening was carried out under solvent-free conditions and with short reaction time using microwave irradiation. We believe that our protocol will find use in the efficient synthesis of a new kind of nucleosides analogues and facilitate the synthesis of novel bioactive molecules.We have demonstrated that sulphated zirconia is an efficient catalyst in the regioselective nucleophilic opening of the oxiranes 1-allyl-3-(oxiran-2ylmethyl) pyrimidine-2,4-(1


The structures of the new compounds were completely elucidated using a combination of 2D NMR techniques  and HR-ESI-MS analyses. The known compounds were identified by comparison of their spectroscopic and physical data with those reported in the literature.Five new iridoids, salvialosides A–E (compounds  Salvia is the largest genus in the economically and medicinally important family Labiatae  with a carbomethoxy group , an oxygenated methine , a dioxygenated methine , two aliphatic methine , a methylene group , and a methyl group . Moreover, the iridoid structure was completely verified by the following HMBC correlations  and δC 62.5 (C-6'), 71.4 (C-4'), 74.7 (C-2'), 77.9 (C-3'), 78.5 (C-5'), and 100.2 (C-1'). The HMBC correlations  and 3.93  as well as δC 57.3 (3"- and 5"-OCH3), 108.7 (C-2" and -6"), 127.4 (C-1"), 140.6 (C-4"), 154.3 (C-3" and -5"), and 166.8 (C=O) assigned to a syringic acid which made an ester linkage with 6-OH inferred at first from the deshielded H-6 (δ 5.51) and confirmed by the HMBC correlation of H-6 with syringyl C=O. The stereochemistry of H-5, H-9, and 1-O-glucosyl was supposed to adopt universally β-configuration. By careful examination of the NOESY spectrum, the β-face orientations of the 6-O-syringyl and 8-O-acetyl were proved by the presence of weak NOE correlation between the 8-CH3 (H-10) and H-6/H-1/H-7(α). In addition, the unexpected weak NOE correlations between H-9(β) and H-10(α), H-5(β) and H-6(α) were occasionally observed and it was noted that Jensen et al. +; HRESIMS m/z: 813.2424 [M+Na]+ .Colorless amorphous powder;  = −103.4 ; UV (MeOH), λmax (log ε) 220 (4.41), 277 (4.01 ) nm; IR(KBr) νmax: 3367, 1701 cm−1; 1H- and 13C-NMR see m/z (rel. int.): 609 [M+Na]+; HRESIMS m/z: 609.1791 [M+Na]+ .Colorless amorphous powder;  = −80.0 ; UV (MeOH), λmax (log ε) 220 (4.60), 277 (4.16) nm; IR(KBr) νmax: 3379, 1693 cm−1; 1H- and 13C-NMR see m/z (rel. int.): 625 [M+Na]＋; HRESIMS m/z: 625.1740 [M+Na]＋ .Colorless syrup;  = −14.5 ; UV (MeOH), λmax (log ε) 220 (4.51), 276 (4.07) nm; IR(KBr) νmax: 3329, 1697 cm−1; 1H- and 13C-NMR see m/z (rel. int.): 625 [M+Na]＋; HRESIMS m/z: 625.1742 [M+Na]＋ .Colorless amorphous powder;  = −0.80 ; UV (MeOH), λmax (log ε) 217 (3.41) nm; IR(KBr) νmax: 3383,1747, 1708 cm−1; 1H- and 13C-NMR see m/z (rel. int.): 427 [M+Na]＋; HRESIMS m/z: 427.1580 [M+Na]＋ .Colorless amorphous powder; Salvia digitaloides, including twelve iridoid glycosides, seven diterpenoids, seven triterpenoids, four caffeic acid sugar esters, and other compounds. Among, them the salvialosides A–E (compounds 1–5) were new compounds isolated for the first time as natural products.In our investigation, fifty five compounds were isolated from the roots of


The side chain of this intermediate was modified by oxidative decarboxylation to get a terminal olefin at the C22-C23 position (compound 20) and subsequent dihydroxylation of the olefin. The resulting epimeric mixture of 21a, 21b was separated and the absolute configuration at the C22 carbon for the main product 21a was elucidated by single crystal X-ray diffraction analysis of the benzoylated derivative 22. Finally, lactonization of 21a through a Baeyer-Villiger oxidation of triacetylated derivative 23, using CF3CO3H/CHCl3 as oxidant system, leads to lactones 24 and 25 in 35% and 14% yields, respectively. Deacetylation of these compounds leads to 2-deoxybrassinosteroids 18 and 19 in 86% and 81% yields. Full structural characterization of all synthesized compounds was achieved using their 1D, 2D NMR, and HRMS data.Natural brassinosteroids are widespread in the plant kingdom and it is known that they play an important role in regulating plant growth. In this study, two new brassinosteroid analogs with shorter side chains but keeping the diol function were synthesized. Thus, the synthesis of 2-deoxybrassinosteroids analogs of the 3α-hydroxy-24-nor, 22,23-dihydroxy-5α-cholestane side chain type is described. The starting material is a derivative from hyodeoxycholic acid ( Most of this work has been focused on determining the structural requirements that these compounds should possess to elicit strong biological activity /[(I+) + (I−)]  . Neverth + (I−)] . The res2CO3/MeOH, r.t.) of glycol 21a gave the brassinosteroid analog 9 in 97% yield  must be introduced in the B ring of 21a  of compound 21a  produced the new analogs of 2-deoxy-brassinosteroids 18 and 19 with 86% and 81% yields, respectively.Baeyer-Villiger oxidation of 20, 21a, 9, 22, and 23 were carried out by analysis of spectroscopic data obtained from 1H-NMR, 13C-NMR, and HRMS of pure and isolated compounds.The full structure assignment of compounds 1H-NMR of compound 20 the protons H-22, Htrans-23 and Hcis-23 appear at δH = 5.66 ppm (H-22), 4.93 ppm (Htrans-23) and 4.83 ppm (Hcis-23). In the 13C-NMR the carbons C22 and C23 appear at δC = 144.89 and 111.84 ppm, respectively  were observed at δC = 75.19, 66.02 and 63.19 ppm, , 7.58 (HAr-4’), and 7.46 (HAr-3’) ppm. Additionally, two low field shifted signals at δH = 4.50  and 4.20  ppm correspond to H-23a and H-23b. In the 13C-NMR spectrum the signal at δC = 167.01 ppm is assigned to carbonyl of aromatic ester, whereas the signals at δC = 129.86, 129.63, 128.45 and 133.22 ppm . In the 2D HMBC spectrum a heteronuclear correlation at 3JH-C between H-23a (δH = 4.50 ppm) with carbonyl of aromatic ester (δC = 167.01 ppm) was observed, confirming the presence of benzoyl ester at C23 position. The structure of compounds 18, 19, 24 and 25 were mainly elucidated by analysis of data obtained from 1H, 13C, 13C DEPT-135, 2D HSQC, 2D HMBC NMR, and HRMS measurements. The structure of derivative 3.22 ppm  are assi24, the position of the 7-oxa lactone function was established from the 1H-NMR spectrum where a signal observed at δH = 4.13–4.04 ppm , was assigned to hydrogens H-7 and correlated by 2D 1H-13C HSQC with the signal at δC = 70.31 ppm  H-7 shows 3JHC correlations with the signal at δC = 175.97 ppm ; and 2JHC correlation with signal appearing at δC = 39.42 ppm (assigned to carbon C-8); (ii) H-5α at δH = 3.03 ppm shows 3JHC correlation with signals at δC = 14.54, and 58.31 ppm, which were assigned to carbons CH3-19 and C-9, respectively  H25. Thus, in the 1H-NMR spectrum a signal at δH = 4.46 ppm was assigned to H-5α, and correlated by 2D 1H-13C HSQC with the signal at δC = 79.68 ppm (CH with impair multiplicity from DEPT-135 spectrum). Additionally, H-5α shows 2JHC correlation with signal at δC = 32.95 ppm that is assigned to carbon C-4; and 3JHC correlation with signals at δC = 11.59, 57.94 and 174.64 ppm, which are assigned to carbons CH3-19, C-9 and C-6, respectively , shows 2JHC correlation with signals appearing at δC = 38.31 and 174.64 ppm, which were assigned to carbons C-8 and C-6, respectively  appears at higher field (δH = 3.17 ppm) than in compound 19  (δH = 4.60 ppm) , while in 6-oxalactone 19 these H-atoms are displaced to high field   . Similar, m, 2H) .1H-, 13C-, 13C DEPT-135, gs 2D HSQC and gs 2D HMBC NMR spectra were recorded in CDCl3 or MeOD solutions, and are referenced to the residual peaks of CHCl3 at δ = 7.26 ppm and δ = 77.00 ppm for 1H and 13C, respectively and CD3OD at δ = 3.30 ppm and δ = 49.00 ppm for 1H and 13C, respectively, on an Avance 400 Digital NMR spectrometer  operating at 400.1 MHz for 1H and 100.6 MHz for 13C. Chemical shifts are reported in δ ppm and coupling constants (J) are given in Hz, multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet (t), quartet (q), multiplet (m). IR spectra were recorded as KBr disks in a FT-IR 6700 spectrometer  and frequencies are reported in cm−1. High-resolution mass spectra (HRMS-ESI) were recorded in a Exactive Plus mass spectrometer . The analysis for the reaction products was performed with the following relevant parameters: heater temperature, 50 °C; sheath gas flow, 5 (arbitrary unit); sweep gas flow rate, 0 (arbitrary unit) and spray voltage, 3.0 kV at negative mode. The accurate mass measurements were performed at a resolving power: 140,000 FWHM at range m/z 300–500. Optical rotations were measured on a Model AA-5 polarimeter  with a sodium lamp using a l = 0.1 dm cell and are reported as follows: 2SO4 in H2O. Chromatographic separations were carried out by conventional column on silica gel 60 (230–400 mesh) using EtOAc-hexane gradients of increasing polarity. All organic extracts were dried over anhydrous magnesium sulfate and evaporated under reduced pressure, below 40 °C.All reagents were purchased from commercial suppliers, and used without further purification. Melting points were measured on a SMP3 apparatus  and are uncorrected. 22 was mounted on a MiTeGen MicroMount  in a random orientation. Diffraction data was collected at 120 K on a D8 VENTURE diffractometer  equipped with a bidimensional CMOS Photon100 detector, using graphite monochromated Cu-Kα radiation (λ = 1.54178 Å). The diffraction frames were integrated using the APEX2 package. The structure of 22 was solved using Olex2 +; found 387.2874 [M + H]+.3α-Acetoxy-22(S), 23-dihydroxy-24-nor-5α-cholan-6-one (21a) and 3α-acetoxy-22(R), 23-dihydroxy-24-nor-5α-cholan-6-one (21b). To a solution of 20  in acetone (150 mL) was added NMO . Then the mixture was homogenized by magnetic stirring and 2.0 mL of 4% OsO4 (0.210 mmol) was added dropwise with stirring for 36 h at room temperature. The end of the reaction was verified by TLC. Then the solvent was removed (up to 25 mL approximate volume) and water (25 mL) and Na2S2O3·5H2O (25 mL saturated solution) were added. The organic layer was extracted with EtOAc (2 × 30 mL), washed with water (2 × 50 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure. The crude was re-dissolved in DCM (10 mL) and chromatographed on silica gel with PE/EtOAc mixtures of increasing polarity (19.8:0.2 → 9.8:10.2). A mixture of 21a/21b = 7.5/1.0 was obtained . Recrystallization of this mixture (MeOH/Et2O = 3/1) allows for compound 21a to be obtained as colorless solid : m.p. 250.1–253.8 °C; 3); 1H-NMR (CDCl3) δ 5.11 , 3.79 , 3.64 , 3.51 , 2.55 , 2.31 , 2.03 , 0.954 , 0.735 , 0.675 ; 13C-NMR (CDCl3) see max: 3519 (O-H), 2941 and 2885 (C-H), 1732 (C=O), 1708 (C=O), 1278 (C-O), 1050 (C-O) cm−1; HRMS-ESI (negative mode): m/z calculated for C25H40O5: 420.2876 [M]+; found 419.2811 [M − H]−.3α-Acetoxy-22(S)-hydroxy-24-nor-5α-cholan-6-oxo-23-benzoate (22). Compound 21a  was dissolved in DCM (25 mL) and pyridine (1.0 mL). Later DMAP (5.0 mg) and PhCOCl  were added with slow stirring at room temperature. The end of the reaction was verified by TLC (2 h), solvent volume was reduced to about 10 mL, and then EtOAc (20 mL) were added. The organic layer was washed with 5% KHSO4 (2 × 5 mL) and water (2 × 10 mL), dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The crude was redissolved in DCM (5 mL) and chromatographed on silica gel with PE/EtOAc mixtures of increasing polarity (19.8:0.2 → 14.2:5.8). Compound 22  was obtained as a colorless solid: m.p. 210.5–211.7 °C (MeOH/Et2O = 1/2); 3); 1H-NMR (CDCl3) δ 8.05 , 7.58 , 7.46 , 5.12 , 4.50 , 4.20 , 4.06 , 2.57 , 2.32 , 2.04 , 1.06 , 0.748 , 0.709 ; 13C-NMR (CDCl3) (see max: 3502 (O-H), 2951, 2936, 2893 and 2870 (C-H), 1730 (C=O), 1715 (C=O), 1693 (C=O), 1601 (C=C Ar), 1276 (C-O), 1070 (C-O), 716 (C-H Ar) cm−1; HRMS-ESI (positive mode): m/z calculated for C32H44O6: 524.3138 [M]+; found 525.3186 [M + H]+. Cl3) see ; IR νmax3α-22(S), 23-Trihydroxy-24-nor-5α-cholan-6-one (9). To a solution of 21a  in MeOH (30 mL) was added K2CO3 , then the suspension was stirred at room temperature for 3 h. The end of the reaction was verified by TLC. Then the solvent was removed to dryness and the residue acidified with 2% HCl (20 mL). The obtained solid was filtered and washed with 5% NaHCO3 (20 mL) and water (2 × 10 mL) and dried. Compound 9  was obtained as a colorless solid: m.p. 227.0–229.1 °C (MeOH/Et2O = 3/1); 1H-NMR (CD3OD) δ 4.04–4.03 , 3.71 , 3.60 , 3.40 , 2.74 , 2.21 , 2.11 , 2.04 , 0.943 , 0.732 , 0.716 ; 13C-NMR (CD3OD) see max: 3387 (O-H), 2940, 2906 and 2871 (C-H), 1700 (C=O), 1246 (C-O), 1050 (C-O), 754 (CH) cm−1; HRMS-ESI (positive mode): m/z calculated for C23H38O4: 378.2770 [M]+; found 379.2823 [M + H]+.3α-22(S), 23-Triacetoxy-24-nor-5α-cholan-6-one (23). Compound 21a  was dissolved in DCM (30 mL) and pyridine (3.0 mL). Later DMAP (5.0 mg) and Ac2O  were added to the solution and the reaction mixture was stirred at room temperature. The end of the reaction was verified by TLC (30 min), volume of solvent was reduced to about 5 mL and extracted with EtOAc (2 × 10 mL). The organic layer was washed with 5% KHSO4 (2 × 5 mL) and water (2 × 10 mL), dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure. The crude was redissolved in DCM (5 mL) and chromatographed on silica gel with PE/Et2O mixtures of increasing polarity (19.8:0.2 → 11.8:8.2). Compound 23  was obtained as a colorless solid: m.p. 136.1–137.6 °C (Et2O/hexane); 3); 1H-NMR (CDCl3) δ 5.10–5.09 , 4.31 , 3.99 , 2.54 , 2.30 , 2.06 , 2.04 , 2.03 , 0.973 , 0.723 , 0.651 ; 13C-NMR (CDCl3) , 1737 (C=O), 1711 (C=O), 1369 (CH3), 1242 (C-O), 1224 (C-O), 1051 (C-O), 757 (C-H) cm−1; HRMS-ESI (positive mode): m/z calculated for C29H44O7: 504.3087 [M]+; found 505.3134 [M + H]+.Cl3) see ; IR νmax3α-22(S), 23-Triacetoxy-24-nor-B-homo-7-oxa-5α-cholan-6-one (24) and 3α-22(S), 23-triacetoxy-24-nor-B-homo-6-oxa-5α-cholan-6-one (25). Preparation of oxidant: 1.0 mL of H2O2 (30%), (9.77 mmol) was slowly dripped into a solution of (CF3CO)2O  at 0 °C, diluted with CHCl3 (3 mL) and stirred for 30 min. The oxidant mixture (3.0 mL) was slowly added to the solution of compound 23  at 0 °C and slowly stirred in N2 atmosphere for 24 h. The end of reaction was verified by TLC, the mixture was filtered, then concentrated in a rotary evaporator to a volume of approximately 10 mL. Then Et2O (40 mL) was added and the organic layer was washed with saturated NaHCO3 solution (2 × 20 mL), water (2 × 15 mL), then dried over Na2SO4, and filtered. The solvent was evaporated and the crude was re-dissolved in DCM (5 mL) and chromatographed on silica gel with hexane/Et2O mixtures of increasing polarity (0.2:50.0 → 23.8:26.2). Three fractions were obtained. Fraction I (less polar): 0.363 g (35% yield), compound 24. Fraction II (medium polarity): 0.285 g, mixture of compounds 24 and 25. Fraction III (more polar): 0.146 g (14% yield) compound 25. 24 was obtained as a colorless solid: m.p. 90.3–91.8 °C (Hexane/Et2O = 2/1); 3); 1H-NMR (CDCl3) δ 5.11–5.09 , 4.32 , 4.13–4.04 , 3.99 , 3.03 , 2.08 , 2.08 , 2.05 , 0.979 , 0.897 , 0.701 ; 13C-NMR (CDCl3) , 1735 (C=O), 1438 (C-H), 1368 (CH3), 1242 (C-O), 1182 (C-O), 1052 (C-O), 754 (C-H) cm−1; HRMS-ESI (positive mode): m/z calculated for C29H44O8: 520.3036 [M]+; found 521.3085 [M + H]+.Compound Cl3) see ; IR νmax25 was obtained as a colorless solid: m.p. 169.9–170.8 °C (Hexane/Et2O = 2/1); 1H-NMR (CDCl3) δ 5.14–5.09 , 4.46 , 4.32 , 3.99 , 2.55–2.43 , 2.07 , 2.05 , 0.971 , 0.901 , 0.695 ; 13C-NMR (CDCl3) see max: 2965, 2949 and 2871 (C-H), 1743 (C=O), 1736 (C=O), 1725 (C=O), 1445 (C-H), 1368 (CH3), 1258 (C-O), 1239 (C-O), 1225 (C-O), 1044 (C-O), 1022 (C-O), 754 (C-H) cm−1; HRMS-ESI (positive mode): m/z calculated for C29H44O8: 520.3036 [M]+; found 521.3082 [M + H]+.Compound 3α-22(S), 23-Trihydroxy-24-nor-B-homo-7-oxa-5α-cholan-6-one (18). To a solution of 24  in MeOH (20 mL) was added K2CO3 , then the suspension was stirred at room temperature for 3 h. The end of the reaction was verified by TLC. Then the solvent was removed to dryness and the residue acidified with 2% HCl (10 mL). The obtained solid was filtered and washed with 5% NaHCO3 (20 mL) and water (2 × 10 mL) and dried. Compound 18  was obtained as a colorless solid: m.p. 91.8–94.5 °C (MeOH/Et2O = 3/1); 1H-NMR (CDCl3) δ 4.19–4.15 , 4.08–4.07 , 3.79 , 3.64 , 3.52 , 3.17 , 2.13 , 1.98 , 0.950 , 0.888 , 0.710 ; 13C-NMR (CDCl3/CD3OD = 2/1) see max: 3400 (O-H); 2959; 2941; 2902 and 2870 (C-H); 1709 (C=O); 1315 (C-H); 1249 (C-O); 1183 (C-O), 1065 (C-O); 1048 (C-O), 753 (C-H) cm−1; HRMS-ESI (positive mode): m/z calculated for C23H38O5: 394.2719 [M]+; found 395.2771 [M + H]+.3α-22(S), 23-Trihydroxy-24-nor-B-homo-6-oxa-5α-cholan-6-one (19). Compound 19 was obtained from 25 by the same method described above. Compound 25 , MeOH (20 mL), K2CO3 . Compound 25 , colorless solid: m.p. 227.4–229.5 °C (MeOH/Et2O =3/1); 1H-NMR (CDCl3) δ 4.60 , 4.23–4.21 , 3.79 , 3.64 , 3.50 , 2.53–2.43 , 0.944 , 0.892 , 0.701 ; 13C-NMR (CDCl3/CD3OD = 2/1) see max: 3386 (O-H); 2942; 2889; 2869 and 2851 (C-H); 1710 (C=O); 1278 (C-O); 1038 (C-O); 751 (C-H) cm−1; HRMS-ESI (negative mode): m/z calculated for C23H38O5: 394.2719 [M]+; found 393.2652 [M − H]−.9 and new compounds 18, 19, 21a, 22–25. Compound 9 was obtained from 17 in a total yield of 46%, whereas new lactones analogues 18 and 19 were obtained from glycol 21a in 29% and 11% total yields. Additionally, using 1D, 2D NMR, and HRMS we have achieved full structural determination of all compounds shown in S) by X-ray crystallography studies of the benzoylated derivative 22. This conclusion is in line with literature data reported for similar steroidal structures [A new synthetic route has been used to obtain the known brassinosteroid analog ructures . Finally