For comparison the reactions were carried out under both conventional and ultrasonic conditions. In general, improvement in rates and yields were observed when the reactions were carried out under sonication compared with classical conditions.A series of 3'-aminospiro[indoline-3,1'-pyrazolo[1,2- The challenge of achieving the ideal synthesis has been pursued aggressively since scientists began to construct molecules. Of course, there are many other factors that affect the aspects of synthesis, such as cost, starting material availability, safety, environmental concerns, and overall ease of the process, including work up and purification . TraditiH-pyrazolophthalazine-1,2-dicarboxylate derivatives. Perumal and Shanthi phthalazine]-2,5',10'-trione under ultrasound irradiation , malononitrile (2), and phthalhydrazide (3) was selected as the model reaction for the synthesis of spirophthalazine] derivatives. Initially, in order to search for a better solvent, the ultrasonic-assisted model reaction was examined using different solvents such as ethanol, methanol, water, acetonitrile and THF, respectively. The results are summarized in To optimize the reaction conditions, the reaction of isatin . No reaction was observed in the absence of base. The common base such as C4b in ethanol was then explored. The results showed that when the amount of piperidine was increased, an increase in the yields of 4b was clearly observed. When the concentration of piperidine (mol %) increased from 10% to 30%, the yield of product 4b increased rapidly from 73% to 91%. The best concentration of piperidine was 20%. The higher concentration of the catalyst did not remarkably increase the yield . Experiments performed at constant transmitted power but variable frequencies (25 and 40 kHz) show the same trend , malononitrile or ethyl cyanoacetate , phthalhydrazide  and piperidine (0.20 mmol) in ethanol (15 mL). The mixture was stirred at reflux. After the completion of the reaction (monitored by TLC), the solid that precipitated after cooling was collected by filtration. When necessary, the compounds were recrystallized from hot ethanol and DMF to afford pure products.A 100 mL flask was charged with the isatin , malononitrile or ethyl cyanoacetate , phthalhydrazide  and piperidine (0.20 mmol) in ethanol (15 mL). The mixture was sonicated in the water bath of an ultrasonic cleaner under atmospheric conditions at room temperature. After the completion of the reaction (monitored by TLC), the resulting precipitate was filtered and washed with ethanol to afford the pure product as solid in good to excellent yields.A 100 mL flask was charged with the isatin . Yellow solid; m.p. 260–261 °C; IR : 3338 (N-H), 3259 (N-H), 3192 (N-H), 2196 (CN), 1744 (C=O), 1658 (C=O), 1566 (C=C), 1497 (ArC=C); 1H-NMR : δ (ppm) 2.20 , 6.79 , 7.09 , 7.30 , 7.98–8.33 , 10.81 ; HRMS calculated for C20H13N5O3Na [M+Na]: 394.0911, found: 394.0912.3'-Amino-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carbonitrile (4b). Yellow solid; m.p. 263–265 °C (lit. : 380.0754, found: 380.0753.°C . Yellow solid; m.p. 276–277 °C; IR : 3406 (N-H), 3309 (N-H), 2970 (C-H Aliph), 2195 (CN), 1745 (C=O), 1655 (C=O), 1566 (C=C), 1475 (ArC=C); 1H-NMR : δ (ppm) 6.88 , 7.48 , 7.81–8.39 , 11.08 ; HRMS calculated for C19H1079BrN5O3Na [M+Na]: 457.9859, found: 457.9861.3'-Amino-5-chloro-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carbonitrile (4d). Yellow solid; m.p. 254–255 °C; IR : 3354 (N-H), 3250 (N-H), 3193 (N-H), 2206 (CN), 1762 (C=O), 1657 (C=O), 1565 (C=C), 1476 (ArC=C); 1H-NMR : δ (ppm) 6.88 , 7.31 , 7.86 , 7.96–8.35 , 11.03 ; HRMS calculated for C19H1135ClN5O3 [M+H]: 392.0544, found: 392.0542.3'-Amino-5-fluoro-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carbonitrile (4e). Yellow solid; m.p. 258–259 °C; IR : 3352 (N-H), 3246 (N-H), 3192 (N-H), 2208 (CN), 1759 (C=O), 1656 (C=O), 1568 (C=C), 1485 (ArC=C); 1H-NMR : δ (ppm) 6.93 , 7.15 , 7.50 , 8.01–8.37 , 10.94 ; HRMS calculated for C19H10FN5O3Na [M+Na]: 398.0660, found: 398.0660.3'-Amino-1-methyl-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carbonitrile (4f). White solid; m.p. 287–289 °C (lit. : 371.1016, found: 371.1018.°C . Light yellow solid; m.p. 278–280 °C; IR : 3439 (N-H), 3333 (N-H), 2982 (C-H Aliph), 1743 (C=O), 1663 (C=O), 1530 (C=C); 1H-NMR : δ (ppm) 0.90 , 2.17 , 3.87 , 6.72 , 7.02 , 7.14 , 7.99–8.32 , 10.62 ; HRMS calculated for C22H18N4O5Na [M+Na]: 441.1169, found: 441.1167.Ethyl 3'-amino-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carboxylate (4h). Light yellow solid; m.p. 284–285 °C (lit. : 427.1013, found: 427.1012.°C . Grey solid; m.p. 279–281 °C; IR : 3438 (N-H), 3331 (N-H), 2984 (C-H Aliph), 1747 (C=O), 1703 (C=O), 1658 (C=O), 1530 (C=C), 1478 (ArC=C); 1H-NMR : δ (ppm) 0.92 , 3.89 , 6.80 , 7.40 , 7.63 , 8.01–8.32 , 10.91 ; HRMS calculated for C21H1579BrN4O5Na [M+Na]: 505.0118, found: 505.0115.Ethyl 3'-amino-5-chloro-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carboxylate (4j). Green solid; m.p. 284–286 °C; IR : 3442 (N-H), 3332 (N-H), 2983 (C-H Aliph), 1750 (C=O), 1705 (C=O), 1660 (C=O), 1530 (C=C), 1480 (ArC=C); 1H-NMR : δ (ppm) 0.92 , 3.89 , 6.84 , 7.27 , 7.52 , 7.99–8.32 , 10.90 ; HRMS calculated for C21H1535ClN4O5Na [M+Na]: 461.0623, found: 461.0609.Ethyl 3'-amino-5-fluoro-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carboxylate (4k). White solid; m.p. 294–295 °C; IR : 3442 (N-H), 3338 (N-H), 3071 (C-H Ar), 2983 (C-H Aliph), 1749 (C=O), 1706 (C=O), 1656 (C=O), 1530 (C=C), 1488 (ArC=C); 1H-NMR : δ (ppm) 0.91 , 3.88 , 6.82 , 7.02–7.07 , 7.33 , 7.99–8.32 , 10.79 ; HRMS calculated for C21H15FN4O5Na [M+Na]: 445.0919, found: 445.0916.Ethyl 3'-amino-1-methyl-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carboxylate (4l). Light yellow solid; m.p. 288–289 °C; IR : 3439 (N-H), 3318 (N-H), 3064 (C-H Ar), 2967 (C-H Aliph), 1729 (C=O), 1705 (C=O), 1668 (C=O), 1616 (ArC=C), 1532 (C=C); 1H-NMR : δ (ppm) 0.83 , 3.22 , 3.81 , 6.98 , 7.05 , 7.31–7.39 , 8.00–8.32 ; HRMS calculated for C22H18N4O5Na [M+Na]: 441.1169, found: 441.1171.Ethyl 3'-amino-4-chloro-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carboxylate (4m). White solid; m.p. 292–293 °C; IR : 3412 (N-H), 3341 (N-H), 2985 (C-H Aliph), 1753 (C=O), 1704 (C=O), 1665 (C=O), 1616 (ArC=C), 1513 (C=C); 1H-NMR : δ (ppm) 0.93 , 3.91 , 6.86 , 6.92 , 7.28 , 8.03–8.35 , 11.02 ; HRMS calculated for C21H1535ClN4O5Na [M+Na]: 461.0623, found: 461.0625.Ethyl 3'-amino-6-bromo-2,5',10'-trioxo-5',10'-dihydrospirophthalazine]-2'-carboxylate (4n). Light yellow solid; m.p. 286–287 °C; IR : 3447 (N-H), 3341 (N-H), 3258 (N-H), 2985 (C-H Aliph), 1751 (C=O), 1705 (C=O), 1659 (C=O), 1527 (C=C); 1H-NMR : δ (ppm) 0.92 , 3.89 , 6.89 , 7.09 , 7.31 , 7.99–8.31 , 10.90 ; HRMS calculated for C21H1579BrN4O5Na [M+Na]: 505.0118, found: 505.0118.4d were obtained by slow evaporation from ethanol and DMF. X-ray diffractions were recorded on a Siemens P4 diffractometer. The ORTEP view of the compound with atomic numbering is shown in Yellow crystals of compound b]phthalazine]-2,5',10'-trione derivatives by an one-pot and three-component reaction in ethanol. In comparison with conventional and reported methods, the main advantage of ultrasound application is the significant decrease in the reaction time and all the proposed reactions allowed the preparation of products in good yield without any further complicated refinement.In summary, we have successfully combined the advantages of ultrasound technology with multicomponent reactions to facilitate the rapid construction of 3'-aminospiro[indoline-3,1'-pyrazolo[1,2-


In particular, the IC50 for A549 cells was 2.24 µM, compared with an IC50 of 9.20 µM for doxorubicin, the positive control. Four synthetic routes of the key intermediate 3 of 1a were explored and 1a was prepared via route D on the gram scale for further research. Two analogs of 1a were synthesized and their preliminary structure-activity relationships were studied. Flow cytometric analysis revealed that compound 1a could significantly induce apoptosis in A549 cells in vitro at low micromolar concentrations. These results suggest that the target compound 1a and its analogs with the pyrazolopyrimidine scaffold might potentially constitute a novel class of anticancer agents, which requires further studies.In a cell-based screen of novel anticancer agents, the hit compound  Cancer is a major health problem worldwide. Improvements in treatment and prevention have led to a decrease in cancer deaths, but the number of new diagnoses continues to rise . Chemoth1a pyrimidine derivatives has been reported pyrimidine derivatives such as 1a is being reported for the first time by our group pyrimidine scaffold, was attempted in order to generate brominated product 3b(Route C in Scheme 3). Under mild bromination conditions (Br2/HAc at 60 °C) pyrimidine scaffold might potentially constitute a novel class of anticancer agents, which require further studies.As expected, the biological activity of 1a, flow cytometric analysis was used to identify and measure the apoptotic cells (sub-G1 cells) and the cell cycle after propidium iodide (PI) staining, as described previously +.5-Amino-1-phenyl-1H-pyrazole-4-carboxamide (4). To a solution of 6  in ethanol (10.0 mL) was added dropwise a solution of 33.3% sodium hydroxide . The mixture was then heated under reflux for 5 h. After cooling to room temperature, the reaction mixture was concentrated under vacuum and 6.00 mol/L HCl solution were added until pH 4. After cooling in ice water for 4 h, the precipitated solid was filtered, washed with water and dried to give 1.21 g of white product (90% yield).1H-NMR (DMSO-6d): δ 8.32 , 7.50–7.90 , 7.32–7.40 , 6.85 , 6.37 .ESI-MS: m/z 201.16 [M-H]+.Ethyl 4-oxo-1-phenyl-4,5-dihydro-1H-pyrazolopyrimidine-6-carboxylate (10). A mixture of 4  and diethyl oxalate (8.2 mL) was refluxed for 5 h and the reaction mixture was allowed to cool to room temperature overnight. The precipitate was collected, washed with petroleum ether and dried to give 1.75 g of a white solid. Yield: 50%. 1H-NMR (DMSO-6d): δ 12.87 , 8.44 , 8.03 , 7.60 , 7.45 , 4.39 , 1.35 . ESI-MS: m/z 283.20 [M-H]+.6-(Hydroxymethyl)-1-phenyl-1H-pyrazolopyrimidin-4(5-H)-one (11). To a solution of 10  in THF/H2O (170 mL/55 mL) was added calcium chloride anhydrous  and sodium borohydride , the resulting mixture was stirred at room temperature overnight. The reaction mixture was poured in ethyl acetate (150 mL) and the organic phase separated was washed twice with brine (50 mL) and then dried over sodium sulfate. The residue after evaporating in vacuo was recrystallized from ethanol to afford 1.74 g of a solid. Yield: 89%. 1H-NMR (DMSO-6d): δ 12.04 , 8.30 , 8.06–8.08 , 7.54–7.59 , 7.37–7.42 , 5.74 , 4.45 . ESI-MS: m/z 241.15 [M-H]+.6-(Bromomethyl)-1-phenyl-1H-pyrazolopyrimidin-4(5H)-one (3b). 11  and carbon tetrabromide  were dissolved in dichloromethane (5.0 mL), a solution of triphenyl phosohine  in dichloromethane (1.5 mL) was dropped in at 0 °C. After stirring at room temperature for 3 h, the reaction mixture were concentrated in vacuo and the residue is chromatographed on silica gel using ethyl acetate/petroleum ether (1/4 by volume) as eluent to afford 63.0 mg of a white solid. Yield: 50%. 1H-NMR (DMSO-6d): δ 12.76 , 8.35 , 8.02–8.05 , 7.55–7.62 , 7.40–7.44 , 4.47 . ESI-MS: m/z 305.29 [M-H]+.2-methylthio)quinazolin-4(3H)-one (1a). To a solution of ethyl 2-mercaptoquinazolin-4(3H)-one (2)  in ethanol (2.5 mL) was added 2 mol/L solution of NaOH (0.2 mL), the temperature was raised to 50 °C. And (3b)  was added. About 3 h later, the reaction mixture was cooled and poured into a solution of 2 mol/L NaHSO4 (2.0 mL). The precipitated solid was collected and crystallized to give 57.6 mg of a green solid. Yield: 70%. 1H-NMR (DMSO-6d): δ 12.80 , 12.73 , 8.36 , 8.05–8.11 , 7.82–7.86 , 7.38–7.59 , 4.63 . ESI-MS: m/z 401.00 [M-H]+.6-methyl)-1-phenyl-1H-pyrazolopyrimidin-4(5H)-one (1d). To a solution of 2-mercaptopyrimidin-4(3H)-one  in ethanol (3.0 mL) was added 2 mol/L solution of NaOH (0.3 mL), the temperature was raised to 60 °C and then 3b  was added. About 10 h later, the reaction mixture was cooled and NaHSO4 (0.06 g) was added followed by agitation. The precipitated solid was collected and chromatographed on silica gel with ethyl acetate/petroleum ether (1/4 by volume) as eluent to afford 93.8 mg of a green solid. Yield: 53%. 1H-NMR (DMSO-6d): δ 11.00–14.12 , δ 8.30 , 8.05 , 7.75 , 7.53 , 7.39 , 5.94 , 4.32 . ESI-MS: m/z 353.07 [M-H]+.2-methylthio)quinazolin-4(3H)-one (1e). To a solution of 2-mercaptopyrimidin-4(3H)-one  in ethanol (6.0 mL) was added a 2 mol/L solution of NaOH (1.0 mL), the temperature was raised to 50 °C and 2-chloroquinazolin-4(3H)-one  was added to the resulting mixture. After stirring overnight, the reaction mixture was cooled and NaHSO4 (0.12 g) was added. The precipitated solid was collected and and chromatographed on silica gel with ethyl acetate/petroleum ether (1/4 by volume) as eluent to afford 299.5 mg of a white solid. Yield: 83%. 1H-NMR (DMSO-6d): δ 12.70 , 8.10 , 8.03 , 7.77 , 7.62 , 7.50 , 7.40 . ESI-MS: m/z 337.17 [M-H]+.3a, which is the key intermediate of a promising target compound 1a found in a cell-based screen of anticancer agents, were examined. Based on these findings, 1a was prepared on the gram scale for further research. Compound 1a exhibited high anticancer activity in vitro, especially for the lung cancer cell line A549 (the IC50 for A549 cells was 2.24 µM). Flow cytometric analysis of compound 1a revealed that it could significantly induce apoptosis in A549 cells in vitro at low micromolar concentrations. Moreover, two analogs of 1a were synthesized and their in vitro anticancer activities were tested. Further SAR and mechanism studies are currently in progress.In conclusion, four synthetic routes to


The toxicity of the synthesized compounds was evaluated and the anti-inflammatory study of the triazole compound 9 established an appreciable anti-inflammatory activity that is comparable with that of other nonsteroidal anti-inflammatory agents.New 1,3,4-thiadiazole,  N-acylated amino acids are known for their hepatoprotective + species at m/z = 319.07 and Na adducts of two oxazolone molecules were also detected. The accurate mass measurements allowed the evaluation of the isotopic profile for the peaks situated at 297.09, 298.09 and 299.09.The structure of oxazolone 2-oxazolin-5-one 2 with hydrazine hydrate (98% solution) in anhydrous dioxane under reflux for 3 hours, N-(p-nitrobenzoyl)-D,L-phenylalanine hydrazide (3) was obtained  as well as elemental analyses data. The IR spectra showed two characteristic bands for NH groups at 2,857 cm-1 and 3,269 cm-1, respectively, and the absorption band for CO amide group appeared at 1,661 cm-1. In the 1H-NMR spectrum the NH groups appeared as two signals at 9.0 ppm and 10.40 ppm, while the proton signals of the NH2 group were identified at 5.37 ppm.The structure of hydrazide 4, 5 were obtained through reaction of hydrazide 3 with p-bromophenyl- and p-tolylisothiocyanates in methanol media under reflux . Beside the Na adduct there were observed (M+H)+ and (M+K)+ adducts at m/z = 542 and 580, respectively. Two cluster molecules charged with Na were also observed at m/z = 1105 (541×2 + 23). For the MS spectra of thiosemicarbazide 5 the base peak was observed at m/z = 500.13 and it is associated with the presence of the Na adduct of this thiosemicarbazide (477+23). The peaks accompanying the base peak are (M+H)+ at m/z = 478 and (M+K)+ at 516. An additional peak at m/z = 977 representing the Na adduct of two thiosemicarbazide molecules (977=477×2+23) was also observed. The formation of thiosemicarbazides 4, 5 in acid and alkaline media, respectively, new 1,3,4-thiadiazole and 1,2,4-triazole compounds 6, 7 and 8, 9 were obtained  analyses. In the IR spectra of the 1,3,4-thiadiazoles 6, 7 the >C=N group appears as an absorption band at 1,665-1,672 cm-1, and the C-S bond was identified at 640-652 cm-1. In the 1H-NMR spectra of 1,3,4-thiadiazoles 6, 7 the proton signal for the amine group appears as singlet at 6.00-6.05 ppm and that for the amide group appears at 9.08-9.10 ppm. The CH3 group is present as a singlet at 2.34 ppm. In the mass spectrum of thiadiazole 6 the base peak was observed at m/z = 524, corresponding to the [M-H]+ ions of the product. The base peak for thiadiazole 7 is situated at m/z = 460.13 corresponding to the [M-H]+ ions of the product.The structure of the final compounds was confirmed by elemental and spectral . The base peak observed in the case of triazole 9 is situated at m/z = 460.13 corresponding to the [M-H]+ ion of the product. In the IR spectra of 1,2,4-triazoles 7 and 1,2,4-thiazole 9, both having a p-tolyl moiety in the structure.The synthesized compounds were investigated for their toxicity . It was 9) comparative with standard nonsteroidal anti-inflammatory drugs  was also investigated  only indometacine has an appreciable anti-inflammatory activity (60.30% inhibition of oedema) while at higher doses (1/5 DL50) all tested compounds reduced the nystatin-induced oedema (45.33-61.20% inhibition of oedema). The activity of the 1,2,4-triazole (9) is similar to the phenylbutazone’s activity (49.03% inhibition of oedema). This effect could be explained through both the lysosomal stabilization and the inhibition of prostaglandin biosynthesis -ethyl-5-(p-bromophenylamino)-1,3,4-thiadiazole (6): light yellow solid, 223 mg (71.50% yield), mp 151-153°C; IR (cm-1) v: 3,263, 3,066 (NH), 1,672 (C=N), 1,620 (CO), 1,520 (asymmetric NO2 vibrations), 1,341 (symmetric NO2 vibrations), 812 (aromatic CH), 750 (C-Br), 640 (C-S); 1H-NMR  δ: 2.60 – 2.65 , 4.76 , 6.00 , 6.56 , 7.63 – 7.78 , 8.30 , 8.40 , 8.80 , 9.10 ; MS  m/z: 524 (base peak), 1047 (1.5%); Anal. calcd. for C23H18N5O3BrS (%): C, 52.67; H, 3.43; N, 13.35; Br, 15.26; S, 6.10; found: C, 53.02; H, 3.89; N, 13.84; Br, 15.67; S, 6.58.2-[1-(p-Nitrobenzoylamino)-2-phenyl]-ethyl-5-(p-tolylamino)-1,3,4-thiadiazole (7): yellow solid, 195 mg (71.27% yield), mp 180-182°C; IR (cm-1) v: 3,259, 3,192 (NH), 1,665 (C=N), 1,620 (CO), 1,552 (asymmetric NO2 vibrations), 1,384 (symmetric NO2 vibrations), 815 (aromatic CH), 652 (C-S); 1H-NMR  δ: 2.34 , 2.60 – 2.63 , 4.76 , 6.05 , 6.58 , 7.63-7.80 , 8.33 , 8.45 , 8.75 , 9.08 ; MS  m/z: 460.13 (base peak), 482.02 (1%), 919.14 (2%), 941.12 (1%); Anal. calcd. for C24H21N5O3S (%): C, 62.74; H, 4.57; N, 15.25; S, 6.97; found: C, 63.22; H, 4.91; N, 15.70; S, 7.47.6, 7 (0.0014 mol), a solution of NaOH 2N (10 mL) was added. The reaction mixture was heated under reflux at 80-90 °C for four hours and then a solution of HCl 1N was added until it reached pH 4.5 when a solid product was formed. The rough product was separated and dried under vacuum at 55-60ºC and then it was recrystallized from ethanol. To corresponding thiosemicarbazide 4-(p-bromophenyl)-5-[1-(p-nitrobenzoylamino)-2-phenyl-ethyl]-3-thio-1,2,4-triazole (8): yellow-orange solid, 46 mg (64.45% yield), mp 147-149°C; IR (cm-1) v: 3,062 (NH), 1,664 (CO), 1,595 (C=N), 1,546 (asymmetric NO2 vibrations), 1,300 (C=S), 1,332 (symmetric NO2  vibrations), 925, 873 (aromatic CH), 752 (C-Br); 1H-NMR  δ: 2.62 – 2.66 , 4.86 , 7.25 – 7.30 , 7.80 , 8.39 , 8.50 , 8.75 , 9.05 , 12.80 ; MS  m/z: 524.03 (base peak), 541.94 (33%), 563.92 (25%), 1069.01 (9%); Anal. calcd. for C23H18N5O3BrS (%): C, 52.67; H, 3.43; N, 13.35; Br, 15.26; S, 6.10; found: C, 53.08; H, 3.85; N, 13.85; Br, 15.26; S, 6.49.5-[1-(p-nitrobenzoylamino)-2-phenyl-ethyl]-3-thio-4-(p-tolyl)-1,2,4-triazole (9): brown solid, 43 mg (69% yield), mp 172-174°C; IR (cm-1) v: 3,437 (NH), 1,670 (CO), 1,566 (C=N), 1,520 (asymmetric NO2 vibrations), 1,320 (C=S), 1,338 (symmetric NO2  vibrations), 962, 785 (aromatic CH); 1H-NMR  δ: 2,32 , 2.57 – 3.35 , 7.14 – 7.19 , 7.78 – 7.82 , 8.45 , 8.75 , 9.08 , 12.80 ; MS  m/z: 460.13 (base peak), 482.04 (18%), 941.13 (11%). Anal. calcd. for C24H21N5O3S (%): C, 62.74; H, 4.57; N, 15.25; S, 6.97; found: C, 63.16; H, 5.06; N, 15.62; S, 7.41.2-9 as a suspension in Tween 80 to groups of six male mice, each weighing 20-22 g, according to the classical laboratory methodology [50 was established using the Spearman-Karber method [The acute toxicity was estimated by intraperitoneal administration of the compounds hodology . The anir method .et al. method [9 and the standard anti-inflammatory drugs  were administered through tube-feeding, in 1/10 and 1/5 of DL50 dose as a suspension in CMC 0.5%, 20 ml/kg body. The control groups received only vehicle. The final volume of each paw was measured four hours later, and the difference between the initial and the final paw volume was established. This difference, expressed in mL/100 g body, represents the inflammatory oedema; the mean paw volume in the drug-treated and control groups respectively was also established. The percentage oedema was calculated, using student’s – t test for statistic interpretation. The nystatin-induced paw oedema in rats was realized according to the Niemegeers . method . A group. method , the ani6-7) and 1,2,4-triazole (8-9) compounds having a D,L-phenylalanine moiety were synthesized by intramolecular cyclization of 1,4-disubstituted thiosemicarbazide in acid and alkaline medium, respectively. The corresponding thiosemicarbazides  were obtained by addition of N-(p-nitrobenzoyl)-D,L-phenylalanine hydrazide to the aromatic isothiocyanates. The DL50 values for all synthesized compounds were established, all compounds having a low toxicity. The potential anti-inflammatory effects of the thiazole 9 by using the nystatin-induced paw oedema in rats was also studied. The triazole 9, at a dose of 160 mg/kg body (1/5 DL50), reduced the inflammatory oedema considerably, this action being comparable with that of the tested standard anti-inflammatory drugs.New 1,3,4-thiadiazole (


Porcelia macrocarpa R.E. Fries (Annonaceae) displayed in vitro antitrypanosomal activity against trypomastigote forms of T. cruzi by the colorimetric MTT assay (IC50 of 65.44 μg/mL). Using chromatographic fractionation over SiO2, this extract afforded a fraction composed by one active compound (IC50 of 10.70 µg/mL), which was chemically characterized as 12,14-octadecadiynoic acid (macrocarpic acid). Additionally, two new inactive acetylene compounds  were also isolated from the hexane extract. The complete characterization of the isolated compounds was performed by analysis of NMR and MS data as well as preparation of derivatives.Chagas’ disease is caused by a parasitic protozoan and affects the poorest population in the world, causing high mortality and morbidity. As a result of the toxicity and long duration of current treatments, the discovery of novel and more efficacious drugs is crucial. In this work, the hexane extract from seeds of  Trypanosoma cruzi, is recognized by World Health Organization as a neglected disease, affecting 16 million of people in America + molecule at m/z 890.8, and the product ion scan  confirmed the identity of α,α'-dimacrocarpoyl-β-oleylglycerol (2) by the neutral losses of 276 Da  and 282 Da , as proposed in m/z 890.7231, 873.6971 and 597.4875 corresponding, respectively, to [M+NH4]+, [M+H]+ and [M-(C18H28O2)]+.The LRESIMS (positive polarity) results showed the + molecule at m/z 632.5234. The ectively ,21,22,23P. macrocarpa was evaluated in vitro against trypomastigote forms of T. cruzi. As can be seen in 50 = 65.44 μg/mL) and was subjected to chromatographic separation procedures over SiO2 to afford the active fraction III (IC50 = 5.32 μg/mL) with no toxicity against NCTC mammalian cells (CC50 > 100 μg/mL). After several separation procedures, compound 1 was isolated as main derivative of this fraction that killed 100% of the trypomastigote forms of T. cruzi at the highest tested concentration, resulting in an IC50 value of 10.70 µg/mL. However, the antitrypanosomal activity of pure compound 1 was lower than that determined for fraction III, suggesting the possibility of some synergistic action of this compound with other minor metabolites or the presence of strongly active compounds not isolated during the purification procedures. Despite the toxicity to NCTC cells (CC50 = 44.27 μg/mL or 160.40 μM—SI = 4.1), compound 1 was approximately ten times more effective than the standard drug (benznidazole), which resulted in an IC50 of 139.00 μg/mL or 534.2 μM (SI = 0.9). The selectivity index and antitrypomastigote activity for benznidazole is in accordance to our previous studies using this T. cruzi strain 4]+m/z 890.7231 . 1H-NMR (CDCl3), δ/ppm: 2.27 , 1.25 , 2.17 , 1.27 , 0.81 , 4.07 , 4.02 , 2.31 , 1.20 , 1.95 , 5.27 , 1.20 , 0.80 . 13C- NMR (CDCl3), δ/ppm: 173.3 (C-1), 33.7 (C-2), 31.2 - 24.8 (C-3 to C-10), 19.1 (C-11), 77.3 (C-12), 65.3 (C-13), 65.2 (C-14), 77.3 (C-15), 19.2 (C-16), 22.6 (C-17), 14.0 (C-18), 68.8 (C-α/α'), 64.9 (C-β), 173.7 (C-1'), 34.2 (C-2'), 24.7-29.9 (C-3' to C-7'), 27.2 (C-8'), 129.6 (C-9'), 129.9 (C-10'), 29.2 - 29.8 (C-11' to C-17'), 14.1 (C-18').Amorphous solid; HRESIMS [M+NH4]+m/z 632.5234 . 1H-NMR (CDCl3), δ/ppm: 2.27 , 1.27 , 2.16 , 1.27 , 0.76 , 4.02 , 4.17 , 5.15 , 2.25 , 1,20 , 1.95 , 5.22 , 1.20 , 0.80 . 13C-NMR (CDCl3), δ/ppm: 173.1 (C-1), 172.5 (C-1''), 33.9 (C-2 and C-2''), 31.7–24.7 , 19.1 (C-11 and C-11''), 77.6 (C-12 and C-12''), 65.1 (C-13 and C-13''), 65.2 (C-14 and C-14''), 77.1 (C-15 and C-15''), 19.2 (C-16 and C-16''), 22.5 (C-17 and C-17''), 13.9 (C-18 and C-18''), 68.7 (C-α and C-α'), 62.0 (C-β), 173.7 (C-1'), 34.2 (C-2'), 24.7–29.9 (C-3' to C-7'), 27.2 (C-8'), 129.9 (C-9'), 129.9 (C-10'), 29.7–31.9 (C-11' to C-17'), 14.1 (C-18').Amorphous solid; HRESIMS −m/z 283; 1H-NMR (CDCl3), δ/ppm: 2.41 , 1.60 , 1.21 , 0.87 .Compound 2O (2.3 mL) and EtOH (8.3 mL) was added a solution of Diazald (7.2 g) dissolved in Et2O (80 mL). This mixture was heated and the product distillated to afford ether solution of diazomethane (0.75 g). Immediately, an excess of diazomethane was added to 1 (5.0 mg). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. Purification over SiO2 column chromatography (hexane/EtOAc 99:1) afforded methyl 12,14-octadecadyinoate  (4.6 mg). Amorphous solid. LRESIMS [M+H]+m/z 291; 1H-NMR (CDCl3), δ/ppm: 3.71 , 2.25 , 1.27 , 2.06 , 1.26 , 0.80 .To a solution of KOH (1.7 g) in H1 (12.0 mg) was stirred for 1 h with aqueous solution of KMnO4 (1%). The reaction products were acidified with concentrated HCl and extracted with Et2O to afford free fatty acids (9.0 mg). This material was esterified with CH2N2 and subjected to CG-LREIMS analysis.A sample of 2 or 3 (7.0 mg) were added to a 1 M of NaOMe/MeOH and stirred for 1 h at room temperature. Product reaction was extracted using Bligh & Dyer method [Samples of r method . The obtT. cruzi trypomastigotes (Y strain) were maintained in LLC-MK2 (ATCC CCL 7) cells using RPMI-1640 medium supplemented with 2% FBS at 37 °C and 5% CO2-humidified incubator [ncubator .1–3 were dissolved in DMSO and diluted in RPMI-1640 medium to determine the 50% inhibitory concentration (IC50) [6/well in 96-well microplates. Tested compounds were incubated to the highest concentration of 300 μg/mL for 24 h at 37 °C in a 5% CO2 humidified incubator; using benznidazole as standard drug. The viability of the trypomastigotes was verified by the MTT assay as previously described [Crude extracts; fractions and compounds n (IC50) . Free trescribed ,36.2 [NCTC (ATCC clone 929) cells were maintained in RPMI-1640 (without phenol red and supplemented with 10% FBS) at 37 °C in a humidified atmosphere containing 5% CO2 .50) was determined in NCTC clone 929 cells. NCTC cells were seeded at 6 × 104 cells/well in 96-well microplates at 37 °C in a 5% CO2. The mammalian cells were incubated with tested crude extracts, fractions and compounds 1–3 to the highest concentration of 100 μg/mL for 48 h at 37 °C. The viability of the cells was determined by MTT assay at 570 nm [50 NCTC cells/IC50 trypomastigotes. Compounds with SI > 1.0 were considered selective [The 50% cytotoxic concentration , and the 95% confidence intervals are included in parentheses.The data obtained represent the mean and standard deviation of duplicate samples from three independent assays. The ICPorcelia macrocarpa lead to the isolation of three new acetylene derivatives: macrocarpic acid (1), α,α'-dimacrocarpoyl-β-oleylglycerol (2) and α-macro-carpoyl-α'-oleylglycerol (3) which were fully characterized by NMR and MS analysis. These compounds were evaluated for their antitrypanosomal activity and 1 exhibited activity against trypomastigotes of T. cruzi (IC50 = 10.70 μg/mL or 38.77 μM), ten times more effective than the standard drug (benznidazole). Otherwise, compounds 2 and 3 were inactive. Studies with the clinically more relevant intracellular amastigote form of T. cruzi will have to show whether macrocarpic acid can be considered a promising prototype for the development of new treatments of Chagas’ disease.Fractionation of the hexane extract of seeds of


Their structures were elucidated by chemical and spectroscopic methods . Compounds 1−6 showed triglyceride (TG) accumulation inhibitory effects in free fatty acid-induced HepG2 cells.Five new syringyl acylated flavonol glycosides, named leonurusoides A ( Leonurus japonicus Houtt is from the Lamiaceae family. As a Traditional Chinese Medicine (TCM), it has been commonly used to treat disorders of mammary gland 25D –13.0° ]. Its molecular formula was determined to be C36H38O19 by positive-ion HRESI-TOF-MS . The IR spectrum exhibited absorption bands typical of OH (3356 cm−1), ester carboxyl group (1699 cm–1), α,β-unsaturated ketone (1654 cm–1), aromatic ring , and an O-glycosidic linkage (1056 cm–1). The UV data of 1 showed the characteristic absorption maxima of a kampferol aglycon at 351 nm (4.00) and 268 nm (4.24). Acid hydrolysis of 1 with 1 M HCl afforded D-galactose and L-rhamnose, whose absolute configurations were determined by HPLC analysis using an optical rotation detector , together with an α-L-rhamnopyranosyl and a β-D-galactopyranosyl moieties  in the structure. On the other hand, the appearance of a methoxyl signal at δ 3.81  and a sharp aromatic singlet signal at δ 7.19  in the 1H-NMR spectrum, together with the signals at δ 55.9 , 107.0 , 119.2 (C-1''''), 140.9 (C-4''''), 147.4 , 164.7 (C-7'''') in the 13C-NMR one, indicated the presence of a syringyl group. Furthermore, the assignment of glycoside protons was determined by proton-proton correlations observed from 1H-1H COSY and HSQC experiments. Finally, the linkages of α-L-rhamnopyranosyl, β-D-galactopyranosyl, and syringyl moieties were clarified on the basis of HMBC experiment, which showed long-range correlations between δH 5.30 (H-1'') and δC 133.2 (C-3); δH 4.57 (H-1''') and δC 65.3 (C-6''), and δH 4.92  and δC 164.7 (C-7''''). On the basis of above mentioned, the structure of leonurusoide A was determined as kampferol-3-O-(2'''-syringyl)-α-L-rhamnopyranosyl(1→6)-β-D-galactopyranoside (1).detector ,12. The DMSO-d6, , which wLeonurusoide B (2) was obtained as a yellow powder and exhibited a negative rotation [[α]25D –16.6° ]. The IR spectrum of 2 indicated the presences of carboxyl (1699 cm–1), γ-pyrone (1653 and 1569 cm–1) functions, and a glycoside structure . HRESI-TOF-MS analysis suggested that 2 had the same molecular formula as 1 . In the HMBC spectrum, the long-range correlations between δH 5.32 (H-1'') and δC 133.2 (C-3); δH 4.53  and δC 66.8 (C-6''), and δH 4.95  and δC 164.7 (C-7'''') (2 were demonstrated by the acid hydrolysis like those of 1 25D: −27.2°  for 3; and −11.0°  for 4], too. The same molecular formula, C36H38O19, of them were determined from positive-ion HRESI-TOF-MS +, calcd for C36H38O19Na 797.1900]. Under acid hydrolysis with 1 M HCl . And both of them owned a syringyl moiety . Finally, the linkage position of α-L-rhamnopyranosyl, β-D-galacto-pyranosyl (in 3)/β-D-glucopyranosyl (in 4), and syringyl moieties were clarified on the basis of HMBC experiment (O-(3'''-syringyl)-α-L-rhamnopyranosyl(1→6)-β-D-galactopyranoside (3) and kampferol-3-O-(3'''-syringyl)-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside (4), respectively. 1 M HCl , 3 gave DMSO-d6,  spectra,periment . ConsequLeonurusoide E (5) was obtained as a yellow powder with negative rotation [[α]25D –18.2° ]. The molecular formula, C36H38O20, of 5 was determined by positive-ion HRESI-TOF-MS . The 1H and 13C-NMR , 7.20 ], together with a hyperoside moiety {}.DMSO-d6, , and var spectra  of 5 revδH 4.93 ] and ester carboxyl carbon [δC 164.7 (C-7"'')]. Finally, acid hydrolysis of 5 with 1 M HCl afforded L-rhamnose and D-galactose determined by HPLC analysis  to afford nine fractions (Fr. 1−9). Fraction 2 (15.0 g) was separated by ODS CC [MeOH-H2O ], and 12 fractions (Fr. 2-1−2-12) were obtained. Fraction 2-9 (0.97 g) was separated by preparative HPLC (PHPLC) [CH3CN-H2O ] to afford eight fractions (Fr. 2-9-1−2-9-8). Fractions 2-9-7 (85.3 mg) and 2-9-8 (90.1 mg) were further purified by PHPLC [MeOH-H2O ] to give leonurusoides E  and C , respectively. Fraction 2-10 (1.0 g) was separated by PHPLC [CH3CN-H2O ] to yield 10 fractions [Fr. 2-10.1−2-10.10]. Fraction 2-10-4 (440.1 mg) was purified by PHPLC [MeOH-H2O ] to give leonurusoide E . Fraction 2-10-10 (61.8 mg) was further separated by PHPLC [MeOH-H2O  to afford leonurusoide D . Fraction 2-11 (1.13 g) was separated by PHPLC [CH3CN-H2O ] to give six fractions (Fr. 2-11-1−2-11-6). Fractions 2-11-1 (381.8 mg) and 2-11-3 (56.2 mg) were purified by PHPLC [MeOH-H2O ] to give 2'''-syringylrutin  and leonurusoide A . Fraction 2-11-4 (188.9 mg) was further subjected to PHPLC [MeOH-H2O ] to afford leonurusoide B .The aerial parts of Leonurusoide A (1). Yellow powder. [α]25D –13.0° ; IR νmax (KBr) cm–1: 3356, 2922, 2840, 1699, 1654, 1608, 1516, 1457, 1361, 1281, 1210, 1180, 1116, 1056, 841, 762; UV λmax (MeOH) nm (log ε): 351 (4.00), 268 (4.24). 1H-NMR (DMSO-d6) and 13C-NMR (DMSO-d6) spectroscopic data, see m/z 775.2076 [M+H]+ .Leonurusoide B (2). Yellow powder. [α]25D –16.6° ; IR νmax (KBr) cm–1: 3218, 2948, 2841, 1699, 1653, 1608, 1569, 1517, 1458, 1361, 1281, 1210, 1180, 1113, 1055, 841, 762; UV λmax (MeOH) nm (log ε): 349 (3.88), 267 (4.13). 1H-NMR (DMSO-d6) and 13C-NMR (DMSO-d6) spectroscopic data, see m/z 775.2070 [M+H]+ .Leonurusoide C (3). Yellow powder. [α]D25 –27.2° ; IR νmax (KBr) cm–1: 3231, 2972, 2860, 1699, 1653, 1608, 1588, 1516, 1457, 1362, 1281, 1210, 1181, 1116, 1056, 765, 680; UV λmax (MeOH) nm (log ε): 348 (4.16), 268 (4.41). 1H-NMR (DMSO-d6) and 13C-NMR (DMSO-d6) spectroscopic data, see m/z 797.1904 [M+Na]+ .Leonurusoide D (4). Yellow powder. [α]25D –11.0° ; IR νmax (KBr) cm–1: 3322, 2921, 2843, 1699, 1653, 1589, 1518, 1458, 1357, 1290, 1213, 1181, 1118, 1055, 772, 677; UV λmax (MeOH) nm (log ε): 346 (3.86), 267 (4.13). 1H-NMR (DMSO-d6) and 13C-NMR (DMSO-d6) spectroscopic data, see m/z 797.1901 [M+Na]+ .Leonurusoide E (5). Yellow powder. [α]25D –18.2° ; IR νmax (KBr) cm–1: 3323, 2960, 1697, 1652, 1608, 1516, 1457, 1424, 1340, 1302, 1206, 1117, 1070, 815, 764, 675; UV λmax (MeOH) nm (log ε): 361 (4.24), 265 (4.43). 1H-NMR (DMSO-d6) and 13C-NMR (DMSO-d6) spectroscopic data, see m/z 813.1833 [M+Na]+ .1–5, each 1.5 mg) in 1 M HCl (1 mL) was heated under reflux for 3 h, respectively. The reaction mixture was neutralized with Amberlite IRA-400 (OH– form) and removed by filtration. The aqueous layer was subjected to the HPLC analysis under the following condition, respectively: HPLC column, Kaseisorb LC NH2-60-5 ; detection, optical rotation [Chiralyser ]; mobile phase, CH3CN-H2O ; flow rate 1.0 mL/min. Identification of L-rhamnose (i) and D-galactose (iii) from 1, 3 and 5; L-rhamnose (i) and D-glusose (ii) from 2 and 4 presented in the aqueous was carried out by comparison of its retention time and optical rotation with that of authentic samples, Rt: (i) 7.5 min , (ii) 13.9 min , and (iii) 14.5 min .A solution of leonurusoides A–E . After 48 h incubation, the amount of intracellular triglycerides was determined with a Triglycerides kit  after cell lysis.The hepatic cell line HepG2  were maintained in high glucose Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin under a humidified atmosphere of 5% COp < 0.05 was considered to indicate statistical significance.Values are expressed as mean ± S.D. All the grouped data were statistically performed with SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA) and Tukey’s Studentized range test was used for post hoc evaluations. 1−5, together with the known compound, 2'''-syringylrutin (6) were obtained from the aerial parts of L. japonicas. Although various acylated flavonol glycosides distribute widely in the plant kingdom [1−6 are quite rare. Compound 6 and quercetin 3-(3'''-syringylrobinobioside), with a syringyl acylated at positions 2 and 3 of rhamnose, respectively, are the first examples of natural flavonol glycosides from the same genus, Leonurus [1−6 showed TG accumulation inhibitory effects in free fatty acid-induced HepG2 cells.In summary, five new syringyl acylated flavonoid glycosides  kingdom , syringagylrutin  were obtLeonurus , and are


O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl(1→4)-α-l-rhamnopyranosyl(1→2)-[α-l-rhamno-pyranosyl(1→3)]-β-d-fucopyranosyl gypsogenin 3-O-β-d-glucopyranosyl (1→2)-β-d-glucopyranosiduronic acid (C1) and 28-O-β-d-xylopyranosyl(1→4)-α-l-rhamnopyranosyl (1→2)-[α-l-rhamnopyranosyl(1→3)]-β-d-fucopyranosyl gypsogenin 3-O-β-d-gluco-pyranosyl(1→2)-β-d-glucopyranosiduronic acid (C2) were isolated together with two known compounds from the seeds of Momordica charantia L. Their structures were elucidated by the combination of mass spectrometry (MS), one and two-dimensional NMR experiments and chemical reactions. Two new bidesmoside triterpenoid saponins which were identifed as 28- Momordica charantia L. (Cucurbitaceae) have been used to treat diabetes. Over 100 compounds have been isolated from the fruits, seeds, leaves, canes and roots of this genus, mainly cucurbitane- and oleanene-type triterpenes, and recently studies have discovered that many new cucurbitane triterpenoids from the fruits and the roots of M. charantia L. -β-d-fucopyranosyl gypsogenin 3-O-β-d-glucopyranosyl (1→2)-β-d-glucopyranosiduronic acid (C1) and 28-O-β-d-xylopyranosyl(1→4)-α-l-rhamnopyranosyl(1→2)-[α-l-rhamnopyranosyl(1→3)]-β-d-fucopyranosyl gypsogenin 3-O-β-d-gluco-pyranosyl(1→2)-β-d-glucopyranosiduronic acid (C2) and two known compounds identified as 3-O-β-d-glucopyranosyl-24β-ethyl-5α-chole-sta-7,trans-22E,5(7)trien-3β-ol (C3) and momordicoside S(C4)   on the bn-BuOH-soluable portions of the 95% EtOH extract of seeds of M. charantia L. was subjected to silica gel, RP-18, and Sephadex LH-20 column chromatographies and semipreparative HPLC to yield two new oleanane-type triterpenoids C1 and C2 and two known compounds identified as 3-O-β-d-glucopyranosyl-24β-ethyl-5α-cholesta-7-trans-22E,5(7)trien-3β-ol (C3) and momordicoside S (C4).The C1 was an amorphous white powder, which gave a positive result in the Liebermann- Burchard test. Acid hydrolysis of compound C1 with 2 mol/L HCl–1,4-dioxane  furnished d-xylose, l-rhamnose, d-glucose, and d-fucose in the ratio of 2:2:1:1 by HPLC analysis of the corresponding thiazolidine derivatives following conversion to the 1-[(S)-N-acetyl-(R)-methylbenzylamino]-1-deoxyalditol acetate derivatives − and m/z 1,533 [M+Na]+ respectively, HR-ESI-MS  analysis revealed the molecular formula of C1 to be C69H112O33Na . The five fragment ions at m/z 1,377 [M–132–H], 1,329 [M–162–H], 1,171 [M–162–176–H], 807 [M–146 × 3–132 × 2–H] and 469 [M–146 × 3–132 × 2–162–176–H], indicated the sequential losses of seven sugar moieties (five hexoses and two pentoses).Compound ivatives . In the 1H (pyridine-d5) spectra of C1 revealed the presence of nine methyl proton signals at δH 0.76 (Me-25), 0.89 (Me-29), 0.92 (Me-26), 0.96 (Me-30), 1.18 (Me-27), 1.36 (Me-24), 9.89 (H-29) and included signals due to a gyosogenin skeleton , two β-d-xylopyranosyl moieties , and a gyosogenin moiety . The identities of the monosaccharides and the oligosaccharide sequence were determined by a combination of DEPT and two-dimensional NMR experiments . The sequence of the glycan part was deduced from the following HMBC correlations: the anomeric proton signals at δH 4.76 and δC 83.5 (H-1 of the β-d-glucopyranosiduronic acid attached to the C-3 of the aglycone), δH 5.38 and δC 82.6 (H-1 of the β-d-glucopyranosyl moiety attached to the C-2 of the β-d-gluco-pyranosiduronic acid), and on the other hand, the pentasaccharide part at C-28 was established by the following HMBC information between the following protons and carbons: Fuc-1-H and 28-C, RhmI-1-H and Fuc-2-C, RhmII-1-H and Fuc-3-C, XylI-1-H and RhmI-4-C, XylII-1-H and XylI-3-C (C1 was determined as 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl(1→4)-α-l-rhamnopyranosyl(1→2)-[α-l-rhamnopyranosyl-(1→3)]-β-d-fucopyranosylgypsogenin 3-O-β-d-glucopyranosyl (1→2)-β-d-glucopyranosiduronic acid.The  13C-NMR  spectra XylI-3-C . On the C2 was obtained as a white, amorphous powder, In the (−)- and (+)-ESI-MS of C2, quasimolecular ion peaks were observed at m/z 1,377 [M-H]− and, m/z 1,401 [M+Na]+, respectively, and HR-ESI-MS analysis revealed the molecular formula of C2 to be C65H102O31 . Eight fragment ions at 1,247 [M–132+H], 1,217 [M–162+H], 1,085 [M–162–132+H], 939 [M–132–162–146+H], 909 [M–162–132–176+H], 793 [M–162–132–146–146+H], 647 [M–132–162–146–146–146+H] and 436 [M–132–162–146–146–146–176+H], indicated the sequential losses of one pentose and five hexoses. Acid hydrolysis of compound C2 with 2 mol/L HCl–1,4-dioxane  furnished d-xylose, l-rhamnose, d-glucose, d-fucose in the ratio of 1:2:1:1 which were identified by HPLC analysis of the thiazolidine derivatives following conversion to the corresponding 1-[(S)-N-acetyl-(R)-methylbenzylamino]-1-deoxyalditol acetate derivatives -β-d-fucopyranosyl gypsogenin 3-O-β-d-gluco-pyranosyl(1→2)-β-d-glucopyranosiduronic acid.Comparison of the pound C2  with thaRhmI-4-C . On the 4-100x micromelting apparatus  and are uncorrected. Optical rotations were measured with a Perkin-Elmer 241 MC polarimeter . IR spectra were obtained of Nicolet 5700 IR spectrometer . NMR spectra were recorded on an Inova 500  spectrometer . ESI-MS was performed with Agilent 1100 LC/MSD . For column chromatography, silica gel , ODS , and Sephadex LH-20  were used. The analytical HPLC was performed on an Agilent 1200 LC equipped with a DAD and the preparative HPLC was performed on a Shimadzu LC-20A  equipped with a YMC-Pack ODS column .Melting points were determined of an XTMomordica charantia L. were purchased from Anguo of Hebei Province in 2011, and identified by Lin Ma at Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College. A voucher specimen has been deposited at our lab at the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College.The seeds of M. charantia L. (15.0 kg) were defatted three times by petroleun ether (90 L each time), and then extracted three times by heating to reflux with 95% ethanol (90 L each time), and the combined solution was concentrated under reduced pressure to yield an extract (1.6 kg). The alcohol extract was partitioned successively with CHCl3, EtOAc and n-BuOH. The n-BuOH-soluble portions (175 g) was subjected to normal phase silica gel column chromatography with gradient elution  to give eleven fractions (Fr1-11). Compound C1 (8.0 mg) was purified from Fr9 (7.6 g) by normal-phase silica gel and Sephadex LH-20 chromatography. Compound C2 (5.0 mg) was purified from Fr10 (20.6 g) by repeated column chromatography as well as compound C1. Compound C3 (210.0 mg) was purified from Fr10 (20.6 g) by repeated column chromatography over silica gel with eluent as CHCl3–MeOH–H2O 4:1:0.1. Compound C4 (102.0 mg) was purified from Fr11 (12.5 g) by repeated column chromatography over silica gel with eluent as CHCl3–MeOH–H2O 6:4:0.5, and further purified by Sephadex LH-20 with eluent as MeOH and preparative HPLC . The seeds of d-Glucose, d-galactose, l-rhamnose, d-fucose, d-xylose and l-arabinose aqueous solution  PMP CH3OH solution  and aqueous NaOH solution  were heated at 70 °C for 30 min, cooled to room temperature for 10 min, HCl aqueous solution added  and extracted with CHCl3 . The aqueous fractions were identified by HPLC analysis ; flow phase A: CH3CN-20 mmol/L NH4OAc aqueous solution (15:85), B: CH3CN-20 mmol/L NH4OAc aqueous solution (40:60), flow rate: 1.2 mL/min; gradient elution, 0→20 min, volume fraction of B from 0 to 60%; detection wavelengths: 245 nm; sample volume: 20 μL).d-xylose, l-rhamnose, d-glucose, and d-fucose in a ratio of 2:2:1:1, and compound C2 furnished d-xylose, l-rhamnose, d-glucose and d-fucose in the ratio of 1:2:1:1, which were identified by HPLC analysis of the thiazolidine derivatives following conversion to the 1-[(S)-N-acetyl-(R)-methylbenzylamino]-1-deoxyalditol acetate derivatives -β-d-fucopyranosyl gypsogenin 3-O-β-d-glucopyranosyl (1→2)-β-d-glucopyranosiduronic acid (C1) and 28-O-β-d-xylopyranosyl(1→4)-α-l-rhamnopyranosyl(1→2)-[α-l-rhamnopyranosyl(1→3)]-β-d-fucopyranosyl gypsogenin 3-O-β-d-glucopyranosyl(1→2)-β-d-glucopyranosiduronic acid (C2) were isolated from the seeds of M. charantia L. together with two known ones identified as 3-O-β-d-glucopyranosyl-24β-ethyl-5α-cholesta-7-trans-22E,5(7)trien-3β-ol (C3) and momordicoside S (C4). This finding represents an addition to the ongoing research on the pharmacological activity of these compounds, which may be helpful to understand the use of this plant in traditional medicine and should continue to clarify its actual health benefits.Two new bidesmoside triterpenoid glycosides identified as 28-


Their structures including absolute configurations were determined by spectroscopic data and electronic circular dichroism (ECD) spectra. Compounds 4 and 5 showed cytotoxicity against human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cell lines.Two new naphthalenones, corynenones A and B ( Corynespora cassiicola XS-200900I7, collected from the Xisha Islands coral reef in the South China Sea − .Corynenone B (2): brown powder; c 0.40, MeOH); UV (MeOH) λmax (log ε) 220 (4.31), 290 (4.24); ECD  213 (+5.67), 284 (−2.75), 314 (+1.34); IR (KBr) νmax 3607, 2364, 1697, 1536, 1447 cm−1; 1H- and 13C-NMR data, m/z 239.0564 [M − H]− .Corynesidone E (3): white powder; UV (MeOH) λmax (log ε) 230 (4.56), 274 (4.01); IR (KBr) νmax 3431, 2950, 1634, 1414, 1266, 1117 cm−1; 1H-NMR  δ 6.86 , 6.52 , 6.52 , 3.95 , 2.39 , 2.30 . 13C-NMR  δ 163.5 (C-11), 156.4 (C-4a), 155.3 (C-8), 151.5 (C-3), 145.8 (C-9a), 143.8 (C-5a), 143.0 (C-2), 132.0 (C-6), 127.5 (C-1), 115.0 (C-11a), 114.3 (C-7), 105.7 (C-9), 101.7 (C-4), 56.7 (C-13), 16.1 (C-14), 13.3 (C-12). ESIMS: 303.2 [M + H]+, 325.2 [M + Na]+, 627.3 [2M + Na]+. HRESIMS m/z 303.0868 [M + H]+ .Balanus amphitrite Darwin based on the literature procedures [The antifouling activity against the larval settlement of the barnacle was determined using cyprids of ocedures . SeaNineocedures , and theocedures . Adriamy1 and 2), and one depsidone, corynesidone E (3), together with three known compounds (4–6) were isolated from the sponge-derived fungus Corynespora cassiicola XS-20090I7. Their structures were determined by spectroscopic data and ECD spectra. Compounds 4 and 5 showed cytotoxicity against HL-60 and HeLa cell lines.In this study, three new compounds including two naphthalenones, corynenones A and B (


Selaginella tamariscina (S. tamariscina) (Beauv.) Spring (Selaginellaceae) has been used in oriental medicine for the treatment of dysmenorrhea, chronic hepatitis, hyperglycemia, amenorrhea, hematuria, prolapse of the anus and metrorrhagia. In the present study, we isolated two strong anti-inflammatory compounds, the biflavonoids hinokiflavone (H) and 7′-O-methyl hinokiflavone (mH), from S. tamariscina and examined their anti-inflammatory activities in lipopolysaccharide (LPS)-mediated murine macrophages (RAW 264.7) and colon epithelial cells (HT-29). H and mH suppressed the production of the inflammatory mediators nitric oxide (NO), interleukin (IL)-6, IL-8, and tumor-necrosis factor (TNF)-α, which are most highly activated in inflammatory bowel disease (IBD). In addition, Western blot analysis revealed that H and mH suppressed the LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and the activation of nuclear factor-κB (NF-κB) and extracellular regulated kinases (ERK) 1/2. These results suggest that H and mH are compounds having potent anti-inflammatory effects that could be used to treat such diseases as IBD. Inflammation is part of our innate immunity, and is an important host defense response to injury, tissue ischemia, and autoimmune responses or pathogens, and consists of a complicated set of events regulated by various chemical mediators released by both resident and infiltrating cells ,2. InflaNuclear factor-kappa B (NF-κB) is a proinflammatory transcription factor and is primarily regulated by inflammatory mediators which have important roles in the pathogenesis of inflammatory diseases, such as IBDs, and are produced in response to lipopolysaccharide (LPS). Mitogen-activated protein kinases (MAPKs) such as extracellular regulated kinase (ERK), p38, and c-Jun-N-terminal kinase (JNK) in signaling plays an important role in NF-κB activation, and the downregulation of mucosal NF-κB signaling may prevent IBD ,10,11,12Selaginella tamariscina (S. tamariscina) (Beauv.) Spring (Selaginellaceae) has been used in oriental medicine for the treatment of dysmenorrhea, chronic hepatitis, hyperglycemia, amenorrhea, hematuria, prolapse of the anus, and metrorrhagia. Pharmacological studies on S. tamariscina have reported its anti-inflammatory, antibacterial, anti-hypertensive and anti-hyperglycemic activities +; 1H-NMR : δH 8.02 , 8.00 , 7.58 , 7.15 , 6.85 , 6.81 , 6.72 , 6.39 , 6.40 , 6.19 ; 13C-NMR : δC 183.1 (C-4), 182.7 (C-4′′), 165.1 (C-2′′), 164.8 (C-2), 164.7 (C-7′′), 162.8 (C-7), 162.4 (C-4′), 162.0 (C-5′′), 161.5 (C-5), 160.5 (C-4′′′), 158.3 (C-9′′), 155.5 (C-9), 132.4 (C-2′′′), 129.2 , 128.8 (C-6′′′), 122.4 (C-1′), 122.0 (C-3′′′), 121.0(C-1′′′), 117.1 (C-5′′), 116.7 , 104.9 (C-8), 104.7 (C-10), 104.0 (C-3), 103.6 (C-3′′), 99.8 (C-6′′), 99.6 (C-6), 95.0 (C-8′′).Amentoflavone (O-dimethyl-amentoflavone (2). Yellow powder; C32H22O10; ESIMS (negative mode): m/z 565.12 [M − H]−; 1H-NMR : δH 8.02 , 8.00 , 7.68 , 7.15 , 6.94 , 6.92 , 6.82 , 6.68, 6.44 , 6.16 , 3.84 , 3.77 ; 13C-NMR : δC 182.5 (C-4), 181.8 (C-4′′), 169.1 (C-7′), 164.5 (C-7′′), 163.7 , 162.4 (C-8), 161.5 , 157.5 (C-4′′′), 153.7 (C-9′′), 135.7 (C-2′), 128.2 , 125.9 (C-6′), 122.9 (C-1′), 119.6 (C-1′′′), 117.4 (C-3′), 116.3 (C-5′), 114.6 , 105.1 (C-10), 103.7 (C-10′′), 103.3 , 95.6 (C-8′′), 94.2 , 91.3 (C-8), 57.0 (OCH3-7′′), 56.1 (OCH3-7).7,7′′-3). White powder; C30H18O10; ESIMS (negative mode): m/z 537.08 [M-H]-; 1H-NMR : δH 8.02 , 7.97 , 7.04 , 6.94 , 6.86 , 6.85 , 6.71 , 6.49 , 6.20 ; 13C-NMR : δC 182.0 (C-4′′), 181.8 (C-4), 164.3 (C-7), 164.1 (C-2′′), 163.2 (C-2), 161.4 (C-5), 161.3 (C-4′′′), 160.7 (C-4′), 157.5 (C-9), 157.3 (C-9′′), 153.8 (C-7′′), 153.1 (C-5′′), 128.6 , 128.3 , 124.7 (C-6′′), 124.2 (C-1′), 121.1 (C-1′′′), 116.0 , 115.3 , 103.9 , 103.8 (C-3), 102.5 (C-3′′), 98.9 (C-6), 94.6 (C-8′′), 94.0 (C-8).Hinokiflavone (O-Methyl hinokiflavone (4). Yellow powder; C31H20O10; ESIMS (negative mode): m/z 552.08 [M − H]−; 1H-NMR : δH 8.00 , 7.09 , 6.99 , 6.89 , 6.92  6.85 , 6.48 , 6.20 , 3.89 ; 13C-NMR : δC 182.7 (C-4′′), 182.3 (C-4), 164.9 (C-7), 164.8 (C-2′′), 163.6 (C-2), 162 (C-5), 162 (C-4′′′), 161.1 (C-4′), 159 (C-9), 157.9 (C-9′′), 154.6 (C-5′′), 152.9 (C-7′′), 129.2 (C-2′′′), 129.2 (C-6′′′), 128.9 , 125.3 (C-1′), 124.9 (C-1′′′), 116.5 , 115.7 , 105.7 (C-10), 104.5 (C-10′′), 104.3 (C-3), 103.4 (C-3′′), 99.4 (C-6), 94.5(C-8), 92.6 (C-8′′), 57.3 (OCH3-7′′).7”-O-dimethyl-amentoflavone were previously reported [S. tamariscina has not yet been examined. In the present study, we focused on the anti-inflammatory molecular activities of biflavonoids isolated form S. tamariscina in LPS-induced murine macrophage and colon epithelial cells leading to the protection of IBD.Among the four compounds with an inhibitory action on NO production, the anti-inflammatory effects of amentoflavone and 7,7′′-reported ,26,27. TS. tamariscina was assessed by quantitating the cell viabilities in the presence of these flavonoids. The cells were pretreated with H and mH, stimulated with LPS for 24 h, and then analyzed for cytotoxicity using CCK-8. These flavonoids exhibited no toxic effects on the cells over the concentration range from 1 to 10 μM in comparison to control cells that received no treatment in LPS-induced RAW 264.7 cells  was deposited at the laboratory of Pharmacognosy, College of Pharmacy, Sunchon National University, Suncheon-si, Jeollanam-do, Korea.The S. tamariscina was extracted three times with 80% methanol for 3 h using sonication. The extracted solution was filtered and dried to give the methanol extract (364.6 g). The extract was suspended in H2O and partitioned in a regular sequence with n-hexane, CHCl3, EtOAc, and n-butanol to obtain 64.4 g, 37.0 g, 31.7 g, and 50 g of residue, respectively. Among these fractions, the n-hexane, CHCl3, and EtOAc fractions showed a significant inhibitory effect on NO production in RAW 264.7 cells. The EtOAc fraction was used for further isolation work. The EtOAc fraction was used for VLC using a gradient solvent system (CHCl3 → CH3OH) to obtain 10 subfractions (E1~E10). E4 was subjected to MPLC  to afford 61 subfractions (E4-1~E4-61). Compound 1 (17.8 mg) was isolated from E4-8. E3 was separated by VLC  to yield 12 subfractions (E3-1~E3-12). E3-2 was subjected to MPLC  to obtain 61 subfractions (E3-2-1~E3-2-61). Compound 2 (9.1 mg) was obtained by recrystallization from E3-2-40. E6 was separated by MPLC  to yield 17 subfractions (E6-1~E6-17). E6-12 and E6-16 were subjected to MPLC  to obtain 61 subfractions referred to as E6-12-1~E6-12-61 and E6-16-1~E6-16-61, respectively. Compounds 3 (9.5 mg) and 4 (9.0 mg) were obtained from E6-12-2 and E6-16-14, respectively.The dried aerial part (3 kg) of 2. The cells were cultured in serum-free DMEM medium with various concentrations of H and mH for 1 h, then stimulated with LPS for the indicated times. The cell viability was evaluated using a CCK-8 assay kit . All experiments were carried out using 1 μg/mL of LPS for the induction of inflammation.The murine macrophage, RAW 264.7, human colonic epithelial cells, and HT-29 cells were obtained from the Korean Cell lines bank. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, and Penicillin and Streptomycin Solution , and cultured at 37 °C in a humidified atmosphere with 5% CO2 serial dilution standard curve and the nitrite production was measured. Relative NO production (%) was calculated as 100 × (nitrite concentration of LPS + sample-treated nitrite concentration of control)/(nitrite concentration of LPS-treated − nitrite concentration of control).The cells were treated with BCS for 1 h, then induced by stimulating them with LPS for 24 h. The culture media was mixed with an equal amount of Griess reagent, reacted at RT for 15 min, and measured at 550 nm using a microplate reader . Serum-free culture medium was used as the blank in all experiments. The amount of nitrite in the samples was obtained by means of the NaNOThe protein expression of COX-2, iNOS, MAPK, NF-κB was measured by Western blot analysis. The cells were pretreated with various concentrations of BCS and stimulated with LPS for the indicated times, and the whole cell lysates were extracted with a protein extraction kit . Equal amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membrane was blocked with 5% skim milk in plain buffer (20 mM Tris (pH 7.4), and 136 mM NaCl) at RT for 1 h, then incubated with primary antibodies overnight at 4 °C. Then, the membrane was incubated with 500-times diluted specific secondary HRP-conjugated antibodies at RT for 1 h, and the immunoreactive bands were exposed to enhanced chemiluminescence Western blot analysis detection reagents , then were analyzed using a Bio imaging-system .TM, San Diego, CA, USA).The levels of proinflammatory cytokines, such as IL-1β, IL-6, TNF-α, and IL-8, in the culture media produced RAW 264.7 cells and HT-29 cells, which were measured by enzyme linked-immunosorbent assay (ELISA) kits  of at least three independent experiments. One-way ANOVA was used for comparisons of multiple group means followed by the Student′s


Penicillum citreonigrum XT20-134 (MCCC 3A00956) is a fungus with cytotoxic activity, derived from deep-sea sediment. Five new compounds, adeninylpyrenocine (1), 2-hydroxyl-3-pyrenocine-thio propanoic acid (2), ozazino-cyclo- (3), 5,5-dichloro-1--1,4-dihydroxypentan-2-one (4), and 2,3,4-trihydroxybutyl cinnamate (5), together with 19 known compounds (6–24), were isolated from an ethyl acetate (EtOAc) extract of its fermentation. The structures of the new compounds were comprehensively characterized by high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS), 1D and 2D nuclear magnetic resonance (NMR). All isolates were evaluated for their cytotoxic activities. The heteroatom-containing new compounds 2 and 4 showed potent cytotoxicity to the human hepatoma tumor cell Bel7402 with IC50 values of 7.63 ± 1.46, 13.14 ± 1.41 μM and the human fibrosarcoma tumor cell HT1080 with IC50 values of 10.22 ± 1.32, 16.53 ± 1.67 μM, respectively. Considering the extreme environment of high salinity, darkness, high pressure, and high/low temperature , the disPenicillum citreonigrum XT20-134 (MCCC 3A00956), a fungal strain that originated from the deep-sea sediment in the southeast Indian Ocean, was found to possess cytotoxic activity. From its ethyl acetate (EtOAc) extract, five new compounds, adeninylpyrenocine (1), 2-hydroxyl-3-pyrenocine-thio propanoic acid (2), ozazino-cyclo- (3), 5,5-dichloro-1--1,4-dihydroxypentan-2-one (4), and 2,3,4-trihydroxybutyl cinnamate (5) were isolated together with 19 known compounds (6–24)  . The str1) was isolated as a white amorphous powder. The infrared (IR) spectrum of 1 indicated the presence of conjugated ketone and conjugated lactone carbonyl signals at 1695 and 1642 cm−1, respectively. Its molecular formula of C16H17N5O4 was established by HR-ESI-MS at 344.1357 [M + H]+ . The unsaturation degree of 11 indicated the presence of heteroaromatic rings. With the aid of heteronuclear single quantum coherence (HSQC) spectra, three singlet signals in the low field of 1H NMR at Hδ 8.21 , 8.11 , and 5.62  were attributed to olefinic protons. Their corresponding carbon signals were found in 13C NMR at Cδ 140.3, 152.6, and 87.8, respectively. The signal at Hδ 7.16  should be assigned to the primary amino group due to the absence of carbon correlation in the HSQC spectrum. In the sp3 region of the 1H NMR, two singlet signals, belonging to a methoxyl at Hδ 3.74  and a methyl at Hδ 1.85 , could be found. In addition, a CH2–CH–CH3 fragment could be established, based on the ABXC3 spin system at Hδ , Hδ 5.06 , and the doublet methyl signal and 1.53 . Considering the five nitrogen atoms in the molecular formula and the carbon signals at Cδ 156.4, 152.6, 149.5, 140.3, and 119.5, an adenine moiety is present in 1. With the help of distortionless enhancement by polarization transfer (DEPT-135) along 13C NMR, the four quaternary carbons at Cδ 156.4, 152.6, 149.5, 140.3, and the methine at Cδ 87.8 were attributed to an α-pyrone structure unit. In the 1H–1H homonuclear chemical shift correlation spectroscopy (COSY) spectrum, only the correlations in the CH2CH–CH3 were presented. In the 1H detected heteronuclear multiple bond correlation (HMBC) spectrum, key correlations were found to reveal the total structure. The correlations between H-9 (Hδ 5.06) and C-4’ (Cδ 149.5) and C-8’ (Cδ 140.3) indicated that the adenine moiety was connected to C-9 at N-9’. The conjugated ketone signal at Cδ 198.8 (C-7) in 13C NMR, is considered to link to C-8, due to the HMBC correlation between H-8 and C-7. The other two methyl groups were also assigned, as shown in 9R-1 and 9S-1 were further calculated and compared with the experimental ones to determine the absolute configurations. As shown in 9S-1 and the absolute configuration of 1 was determined as 9S.Adeninylpyrenocine  as a light yellow powder. In the IR spectrum of 2, unconjugated carbonyl and α-pyrone ketone signals emerged at 1714 and 1626 cm−1, respectively. Its molecular formula of C14H18O7S, which gave six degrees of unsaturation, was established by the positive HR-ESI-MS ion peaks at m/z 331.0845 [M + H]+, 353.0670 [M + Na]+ and negative ion peaks at m/z 329.0706 [M − H]−, respectively. The presence of a sulfur atom was supported by the isotope quasi-molecular ion peaks at m/z 333.0808 [M(34S) + H]+, 355.0627 [M(34S) + Na]+ and 331.0662 [M(34S) – H]−, respectively. In the 1H NMR spectrum of 2, an α-pyrone olefinic proton, a methyl and a methoxyl signal were found at Hδ 5.69 , 1.23  and 3.87 , respectively. In the 1H–1H COSY spectrum, the correlations between Hδ 5.69 (H-10), Hδ 3.30 (H-9) and Hδ 2.99 & 2.90 (H-8) revealed the presence of a CH2CH–CH3 structure unit. In the 13C NMR, signals belong to the pyrenocine moiety were similar to those in 1, except for the C-9 signal at Cδ 36.3, indicating that they differed in the substituent at C-9  and Hδ ; and three additional carbon signals were at Cδ 174.5 (C-1’), 71.1 (C-2’) and 34.7 (C-3’). With the help of 1H–1H COSY and HMBC spectra, they were attributed to a –CH2–CH(OH)–COOH structure fragment. The HMBC correlations between H-3’  and C-9 (Cδ 36.3), and between H-9 (Hδ 3.30) and C-3’ (Cδ 34.7) revealed that the two structure units connected at C-9 and C-3’. The 1H-NMR and 13C-NMR signals at positions C-1’, 2’, 3’ and C-8, 9, whose peak intensities were halved and appeared in pairs, indicated that compound 2 is a pair of epimers  (3) was isolated as a white powder. The HR-ESI-MS cationized ion peaks revealed the presence of three nitrogen atoms. In the IR spectrum of 3, amide carbonyl signal was found at 1669 cm−1. The ultravoilet (UV) maximum absorption wavelengths at λmax (log ε) 240 (1.92) nm and 299 (1.55) nm, belong to the amide carbonyl and aromatic rings, respectively. In the 1H NMR spectrum of 3, a set of mono-substituted benzene ring signals could be found at Hδ 6.98 , 6.86  and 6.75 , respectively. Another ortho-substituted benzene ring was elucidated by the signals at Hδ 6.90 , 6.56 , 7.00 , and 6.56 . In addition, two ABX spin system signals were present at Hδ 4.19 , , and at Hδ 4.17 , [1.91  and 1.03  H-β], and were attributed to two –CH–CH2– structure units. All these fragments were confirmed by the 13C NMR and DEPT spectra. In addition, two amide carbonyls at Cδ 166.1 and 161.5, together with a quaternary carbon at Cδ 74.7 and a methylidyne Cδ 99.3, indicated that 3 is a compound comprising two amino acid units, one of which is phenylalanine and the other one is a tryptophane-like structure unit. Similar structures have been isolated from P. citroviride , 4.07 , and 5.96 . The fragments were confirmed by the 1H–1H COSY correlations of 4. The 13C NMR of 4 showed an unconjuated ketone carbonyl at Cδ 207.8. The correlation between Hδ 4.85  and Cδ 79.7 (C-1), found in the HSQC spectrum, revealed an oxygen-linked methylidyne. The HMBC correlations between Hδ 4.85 (H-1) and Cδ 141.7 (C-1’), 207.8 (C-2), and between Hδ 2.66 and 2.52 (H-3) and Cδ 207.8 (C-2), allowed the elucidation of the structure of 4 -1,4-dihydroxypentan-2-one (ure of 4 .5) as a white powder. The molecular formula of C13H16O5 was revealed by the HR-ESI-MS cationized ion peaks at m/z 275.0882 [M + Na]+  in positive mode, and m/z 251.0922 [M − H]−  in negative mode. A conjugated ester carbonyl IR signal was present at 1700 cm−1. A mono-substituted benzene ring was deduced by the 1H NMR signal at Hδ 7.72  and Hδ 7.74 . A pair of trans-alkene proton signals were exhibited at Hδ 7.69  and Hδ 6.64 . With the help of 1H–1H COSY spectrum, two ABX spin systems at Hδ , 3.66 , and at Hδ , 3.44 , were connected to a structure fragment as O–CH2–CH(O)–CH(O)–CH2–O (Hδ 4.33 & 4.11), H-8 (Hδ 6.64) to C-9 (Cδ 166.9), and from H-3, 5 (Hδ 7.44), H-8 (Hδ 6.64) to C-1 (Cδ 134.5), allowed the elucidation of 2,3,4-trihydroxybutyl cinnamate. The chlorogenic acid contained in the potato medium may be the original precursor of this compound.We isolated 2,3,4-trihydroxybutyl cinnamate (O)–CH2–O . In the 6–24 were elucidated by the comparison of their MS and NMR data with those reported in the literature, and they were identified as: 4-methyl-5,6-dihydropyren-2-one (6) +  and 366.1172 [M + Na]+  in positive mode, and m/z 342.1220 [M − H]−  in negative mode. 1H NMR  and 13C NMR  spectra data are listed in Adeninylpyrenocine (2): light yellow powder; +  and 353.0670 [M + Na]+  in positive mode, and m/z 329.0706 [M − H]−  in negative mode. 1H NMR  and 13C NMR  spectra data are listed in 2-Hydroxyl-3-pyrenocine-thio propanoic acid  (3): white powder; +  and 388.1265 [M + Na]+  in positive mode, and m/z 364.1303 [M − H]−  in negative mode. 1H NMR  and 13C NMR  spectra data are listed in Ozazino-4): white powder; +  and 345.0261 [M + Na]+  in positive mode, and m/z 321.0304 [M − H]−  in negative mode. 1H NMR  Hδ: 6.37 , 6.27 , 5.96 , 4.85 , 4.07 , 3.57 , ; 13C NMR  Cδ: 207.8 (C-2), 160.9 , 141.7 (C-1′), 105.3 , 100.0 (C-4′), 79.7 (C-1), 77.7 (C-5), 71.1 (C-4), 55.6 , 40.9 (C-3). The spectra are provided in 5,5-Dichloro-1--1,4-dihydroxypentan-2-one (5): white powder; +  in positive mode, and m/z 251.0922 [M − H]−  in negative mode. 1H NMR  Hδ: 7.69 , 7.44 , 7.44 , 7.72 , 6.64 , 3.44 , 3.66 , , ; 13C NMR  Cδ: 166.9 (C-9), 144.8 (C-7), 134.5 (C-1), 130.9 (C-4), 129.4 , 128.8 , 118.8 (C-8), 72.8 (C-3′), 69.9 (C-2′), 67.0 (C-1′), 63.5 (C-4′). The spectra are provided in 2,3,4-Trihydroxybutyl cinnamate . The inhibition rate = (A control − A treated)/A control × 100. The IC50 was the concentration at which it caused 50% inhibition of cell proliferation .Cytotoxicity assay was carried out according to the instructions of CCK-8 kit . Briefly


R,23R,24R)-5α,8α-epidioxy-22,23-methylene-24-methyl-cholest-6,9(11)-dien-3β-ol (1), and two known sterols, -5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2) and 24-methylenecholestane-1α,3β,5α, 6β,11α-pentol (3), were isolated from the soft coral Sinularia gaweli. The structure of sterol 1 was established by spectroscopic methods and by comparison of the spectral data with those of known analogues. The cytotoxicity of sterols 1–3 towards various tumor cells is reported.A new sterol, (22 Sinularia have been well-recognized as marine organisms containing various natural products that show interesting bioactivities + molecule at m/z 463.3192 in HRESIMS . The 13C-NMR and DEPT spectra of 1 showed this compound to have 29 carbons , 78.4 (C-8) and 66.3 (CH-3) in the 13C-NMR spectrum and an oxymethine proton at δH 4.02  in the 1H-NMR spectrum was determined. This sterol was further recognized as a 5α,8α-epidioxysterol by the presence of the characteristic signals for H-6  and H-7  in the 1H-NMR spectrum +; HRESIMS: m/z 463.3192 .-5α,8α-Epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2):  +20  ); IR (neat) υmax 3438, 1638 cm−1; 1H  and 13C  NMR data were found to be in full agreement with those reported previously [m/z 465 [M+Na]+; HRESIMS: m/z 465.3347 .22R,23R,2R-5α,8α-E22R,23R,2R-5α,8α-Eeviously ; ESIMS m24-Methylenecholestane-1α,3β,5α,6β,11α-pentol (3):  −3  ); IR (neat) υmax 3380, 1216 cm−1; 1H  and 13C  NMR data were found to be in full agreement with those reported previously [m/z 487 [M+Na]+; HRESIMS: m/z 487.3402 .3) -2,5-diphenyltetrazolium bromide] colorimetric method. Cytotoxicity assays were carried out according to previously described procedures [The cytotoxicity of sterols ocedures ,26.Sinularia spp. octocorals distributed in the waters off Taiwan [Sinularia gaweli have led to the isolation of a new epidioxysterol, -5α,8α-epidioxy-22,23-methylene-24-methyl- cholest-6,9(11)-dien-3β-ol (1), along with two known sterols, -5α,8α-epidioxy-22,23-methylene-24-methylcholest-6-en-3β-ol (2) and 24-methylenecholestane-1α,3β,5α,6β,11α-pentol (3). Sterol 3 was found to exhibit significant cytotoxicity against HL-60 tumor cells, and this result suggested that sterol 3 is worthy of further biomedical investigation. The soft coral S.gaweli has begun to be transplanted to culturing tanks with a flow-through sea water system located in the National Museum of Marine Biology and Aquarium, Taiwan for the extraction of additional natural products in order to establish a stable supply of bioactive material.Steroid metabolites are major constituents of the extracts of f Taiwan ,11,12,13


Cnidium officinale were investigated. Four compounds, namely falcarindiol (1), 6-hydroxy-7-methoxy-dihydroligustilide (2), ligustilidiol (3), and senkyunolide H (4) were isolated from the extract of the rhizome of Cnidium officinale and their structures were elucidated by analysis of their spectroscopic data and by comparison with previously reported data. These compounds showed anti-inflammatory activities, measured as inhibition of nitric oxide (NO) release in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells, with IC50 values of 4.31 ± 5.22, 152.95 ± 4.23, 72.78 ± 5.13, and 173.42 ± 3.22 μM, respectively. They also inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression induced by LPS. Among these compounds, falcarindiol (1) was found to have anti-proliferative effect against MCF-7 human breast cancer cells by induction of a G0/G1 cell cycle block of the cells, with an IC50 value of 35.67 μM. Typical apoptotic effects were observed by phase contrast microscopy and were also exhibited in fluorescence microscopy with Hoechst 33342 staining. In addition, falcarindiol induced apoptosis through strongly increased mRNA expression of Bax and p53, and slightly reduced Bcl-2 mRNA levels in a dose dependent manner. This study suggested that C. officinale extract and its components would be valuable candidates in therapeutic applications for anti-inflammatory and anti-cancer agents.The anti-inflammatory and anticancer activities of a methanol extract of the rhizome of  Cnidium officinale Makino, called “Chunkung” in Korea, has been used in Asia as a natural drug for a long time. Chunkung is a perennial plant of the family Umbelliferae and is an important medicines used to fight diverse diseases  , H-12; δ 1.42 , H-11; δ 2.27 , H-10; and δ 5.24 , H-9], a methoxyl group , a hydroxylmethine and a methoxylmethine protons , as well as two methylene groups . By comparison with the literatures  and glycol group in the cyclohexene ring and their vicinal hydroxymethine protons appeared at δ 3.90  and 3.39  in senkyunolide H, also clearly confirmed by comparison with the reference data − m/z 259.23, C17H24O2, 1H-NMR  δ: 0.88 , 1.28 , 1.37 , 2.10 , 4.94 , 5.20 , 5.25 , 5.46 , 5.51 , 5.60 , 5.91 ; 13C-NMR  δ: 14.4 (C-17), 22.9 (C-16), 27.9 (C-11), 29.4 , 29.5 (C-12), 32.0 (C-15), 58.7 (C-8), 63.6 (C-3), 68.9 (C-6), 70.5 (C-5), 78.5 (C-4), 80.1 (C-7), 117.6 (C-1), 127.8 (C-9), 134.8 (C-10), 136.0 (C-2).(3Z)-3-Butylidene-4,5,6,7-tetrahydro-6-hydroxy-7-methoxy-1(3H)-isobenzofuranone (2). ESI MS [M+H]+ m/z 261.13, C13H18O4, 1H-NMR  δ: 0.87 , 1.42 , 1.89 , 2.27 , 2.43 , 3.45 , 3.87 , 4.09 , 5.24 ; 13C-NMR  δ: 14.0 (C-12), 17.4 (C-4), 22.4 (C-11), 24.3 (C-5), 28.2 (C-10), 58.7 (–OCH3), 67.2 (C-7), 73.4 (C-6), 114.2 (C-9), 124.2 (C-8), 148.7 (C-3), 154.8 (C-2), 170.1 (C-1).3-Butylidene-4,5,6,7-tetrahydro-6,7-dihydroxy-1(3H)-isobenzofuranone (3). ESI MS [M+Na]+ m/z 247.11, C12H16O4, 1H-NMR  δ: 0.94 , 1.49 , 1.99 , 2.35 , 2.56 , 3.94 , 4.48 , 5.29 ; 13C-NMR  δ: 13.9 (C-12), 19.2 (C-4), 22.4 (C-11), 26.5 (C-5), 28.2 (C-10), 67.4 (C-7), 71.6 (C-6), 114.4 (C-9), 125.9 (C-8), 148.2 (C-3), 153.4 (C-2), 169.4 (C-1).(3Z)-3-Butylidene-6,7-dihydroxy-4,5,6,7-tetrahydro-2-benzofuran-1-one (4). ESI MS [M+Na]+ m/z 247.11, C12H16O4, 1H-NMR  δ: 0.88 , 1.42 , 1.90 , 2.27 , 2.44 , 3.39 , 3.90 , 5.24 ; 13C-NMR  δ: 14.0 (C-12), 18.9 (C-4), 22.5 (C-11), 26.2 (C-5), 28.3 (C-10), 66.8 (C-7), 71.5 (C-6), 114.4 (C-9), 125.8 (C-8), 148.3 (C-3), 154.0 (C-2), 169.7 (C-1).2 at 37 °C. Cells were treated with compounds isolated from C. officinale dissolved in dimethyl sulfoxide (DMSO). The final DMSO concentration in all cultures was set to 0.1%.A murine macrophage cell lines (RAW 264.7), and MCF-7 human breast cancer cell lines were purchased from the Korean Cell Line Bank (KCLB). Cell lines were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, in a humidified incubator containing 5% COet al. . The mixture was incubated at room temperature for 10 min, and the absorbance at 540 nm was measured using a microplate reader. Standards were prepared using a serially diluted stock solution of sodium nitrite. Measuring nitric oxide accumulation each experiment was performed in triplicates and repeated at least three times.To investigate the anti-inflammatory effects of isolated compounds, NO production was examined with LPS-stimulated RAW 264.7 cells by Griess reaction . For NO 5 cells/well) were plated into a 6 well plate and exposed to various concentrations  of falcarindiol for 24 h. After treatment, cells were harvest by centrifugation for 6 min at 1000 rpm. Following centrifugation the cells were fixed with 100% ethanol and stored at −20 °C. On the day of analysis, the cells were treated with 1 ml DNA staining solution (20 μg/mL propidium iodide (PI), 200 μg/mL RNase and 0.1% Triton X-100). Cells incubated in the dark at 37 °C for 30 min and analyzed with a flow cytometry . For each measurement, at least 10,000 cells were counted. We used MCF-7 human breast cancer cell lines for cell cycle analysis . MCF-7 c5 cells/well into a 6 well plate. After 24 h, the cells were treated with various concentration (5~50 μM) of falcarindiol. Morphological changes of cells were examined with inverted microscope (400×) at 24 h after treatment. The cells were fixed in 100% EtOH and incubated in 10 μg/mL Hoechst 33342 solution for 30 min in the dark. The cells were then examined using a fluorescence microscope .MCF-7 cells were seeded at a density of 5 × 10Total RNAs (300 ng) of the cells treated with each compound were converted into cDNA using SuperScript II reverse transcriptase and random hexamer primers. RT-generated cDNAs encoding iNOS, COX-2 for anti-inflammatory, Bcl-2, Bax, p53 for anti-cancer, and β-actin were amplified by PCR using selective primers . PCR amp1), 6-hydroxy-7-methoxy-dihydroligustilide (2), ligustilidiol (3) and senkyunolide H (4) were isolated from a methanol extract of the dried rhizome of C. officinale. The three phthalides and a polyacetylene reduced the NO production for LPS-stimulated RAW 264.7 macrophage cells. Besides, falcarindiol showed a high cytotoxicity on MCF-7 cells with the lowest IC50 value (35.67 μM). It was found that the four compounds inhibited COX-2 and iNOS expressions in LPS-stimulated macrophage cells. This inhibitory effect probably acts at the transcriptional level, as evidenced by dose-dependent reductions in their mRNA levels. Although, inhibition of these mRNA expression was presented in LPS-stimulated RAW 264.7 cells by the four compounds, it has not cytotoxicity on RAW 264.7 cells, as assessed by MTT assay and the expression of the house-keeping gene, β-actin. It had been also demonstrated that falcarindiol indicated the cytotoxicity of breast cancer cell lines and induced apoptotic cell death with a dose dependant manner.In this study, four compounds identified as falcarindiol . Also, falcarindiol induced apoptosis through strongly increased expression of Bax and p53 mRNA, and slightly reduced expression of Bcl-2 mRNA levels in a dose dependent manner. This study suggested that C. officinale extract and its components would be valuable candidates in the therapeutic applications as anti-inflammatory or anti-cancer agents.To determine whether the cytotoxicity of falcarindiol on MCF-7 cells involved the cell cycle arrest, we examined cell cycle phase distribution of the treated cells by flow cytometry. Data showed that falcarindiol induced a G


Steroidal sapogenins have shown antiproliferative effects against several tumor cell lines; and their effects on human cancer cells are currently under study. Changes in the functionality on the steroidal structure make it possible to modify the biological activity of compounds. Herein, we report the synthesis and in vitro antitumor activity of two steroidal oxime compounds on cervical cancer cells. These derivatives were synthesized from the steroidal sapogenin diosgenin in good yields. The in vitro assays show that the steroidal oximes show significant antiproliferative activity compared to the one observed for diosgenin. Cell proliferation, cell death, and the cytotoxic effects were determined in both cervical cancer cells and human lymphocytes. The cancer cells showed apoptotic morphology and an increased presence of active caspase-3, providing the notion of a death pathway in the cell. Significantly, the steroidal oximes did not exert a cytotoxic effect on lymphocytes. After a period of 1 h organic extraction was performed , the organic phase was washed with distilled water (2 × 15 mL), dried with Na2SO4 and concentrated under reduced pressure. Compound 4 was obtained  as a white solid, recrystallized from CH2Cl2. m.p. 180–181 °C (CH2Cl2). [α]D = −24° (c = 0.3). υmax 3309, 1732, 1666, 1636, 949. 1H-NMR (CDCl3) δ: 5.34 , 4.58  4.38 , 3.48 ; 3.38 , 2.62 , 2.31, 2.17 , 2.02 , 1.90 , 1.60 , 1.00 , 0.94 , 0.82 , 0.67 . 13C-NMR (CDCl3) −δ: 11.9 (CH3-COO-3), 14.1 (C-21), 16.1 (C-18), 17.0 (C-27), 19.3 (C-19), 20.8 (C-11), 21. 5 (C-232), 27.7 (C-24), 30.4 (C-8), 31.2 (C-9), 31.7 (C-1), 32.1 (C-2), 32.4 (C-10), 36.7 (C-7), 36.9 (C-15), 37.7 (C-4), 38.1 (C-13), 39.7 (C-20), 40.4 (C-24), 45.6 (C-23), 49.8 (C-25), 56.3 (C-14), 61.1 (C-17), 66.1 (C-26), 73.8 (C-3), 81.0 (C-16), 109.7 (C-22), 122.1 (C-6), 139.4 (C-5), 159.3 (C-231), 170.3 (CH3COO-3). HRMS-EI: [M + H]+ calcd. for C31H47O5N, 513.3454; found: 513.3445.Hydroxylamine chlorhydrate  was added to a solution of 1  in CH2Cl2 (40 mL) and acetone (132 mL). The temperature of the reaction mixture should not exceed 10 °C. At the end of the reaction some drops of isopropanol were added and the stirring continued, at room temperature, for 3 h, then filtered through florisil. The crude product was neutralized with saturated aqueous solution of NaHCO3, and the organic phase was washed with brine (2 × 50 mL) and distilled water (2 × 50 mL). The organic phase was dried with Na2SO4, concentrated under reduced pressure, and finally purified on a silica gel column, with hexane/EtOAc (8:2) as eluent, to afford 9  as a white solid. m.p. 193–194 °C. [α]D = −106.2° (c = 0.0014). υmax 2949, 1685. 1H-NMR (CDCl3) δ: 6.18 , 4.43 , 3.48 , 3.36 , 2.71 , 1.19 , 0.99 , 0.84 , 0.80 . 13C-NMR (CDCl3) δ: 36.6 (C-1), 33.9 (C-2), 199.4 (C-3), 125.5 (C-4), 160.6 (C-5), 201.8 (C-6), 32.1 (C-7), 35.1 (C-8), 53.7 (C-9), 38.6 (C-10), 20.8 (C-11), 39.6 (C-12), 40.3 (C-13), 55.6 (C-14), 31.6 (C-15), 80.6 (C-16), 61.9 (C-17), 17.3 (C-18), 16.3 (C-19), 41.6 (C-20), 14.5 (C-21), 109.2 (C-22), 31.3 (C-23), 28.7 (C-24), 30.2 (C-25), 66.8 (C-26), 17.1 (C-27).Jones Reagent (10 mL) was added dropwise at −10 °C to a solution of 9  in ethanol (30 mL) and pyridine (1 mL), NH2OH·HCl  was added. The reaction mixture was stirred at reflux for 1 h. The organic phase was separated, washed with distilled water (2 × 15 mL), dried with Na2SO4 and concentrated under reduced pressure. The reaction crude was purified on silica gel column, with hexane/EtOAc (6:4) as eluent, to afford 5  as white crystals, Mp 150–152 °C, [α]D = +37.8° (c = 0.0067), υmax 3313, 1681. 1H-NMR (CDCl3) δ: 6.58 , 4.43 , 3.48 , 3.37 , 3.07 , 3.44 , 1.06 , 0.98 , 0.82 , 0.79 . 13C-NMR (CDCl3) δ: 39.3 (C-1), 18.7 (C-2), 156.4 (C-3), 119.3 (C-4), 147.3 (C-5), 156.6 (C-6), 46.0 (C-7), 32.7 (C-8), 49.8 (C-9), 38.8 (C-10), 21.1 (C-11), 33.4 (C-12), 40.3 (C-13), 61.8 (C-14), 31.4 (C-15), 80.6 (C-16), 56.3 (C-17), 16.2 (C-18), 19.0 (C-19), 41.6 (C-20), 14.4 (C-21),109.3 (C-22), 31.3 (C-23), 28.7 (C-24), 30.2 (C-25), 66.9 (C-26), 17.1 (C-27). HRMS-EI: [M + H]+ calcd. for C27H40O4N2, 456.2988; found: 456.2982.To a solution of 2 at 37 °C. All cell-based assays were performed using cells in the exponential growth phase.HeLa and CaSki cell lines were purchased from the American Type Culture Collection  and cultured in RPMI-1640 medium  containing 5% newborn calf serum  with red phenol supplemented by benzylpenicillin. Cultures were maintained in a humidified atmosphere with 5% CO50) was determined after 24 h by crystal violet staining [Assays were performed by seeding 7500 cells/well in 96-well tissue culture plates at a volume of 100 μL of RPMI-1640 medium supplemented with 5% NCS per well. Cells were allowed to grow for 24 h in the culture medium prior to exposure to the compounds. Cisplatin was used as positive control. Additionally, 1% of vehicle  was added to the control cells. Antiproliferative activity  following the manufacturer’s instructions. LDH oxidizes lactate to pyruvate, which then reacts with the INT to produce formazan. The increased amount of formazan generated in the culture supernatant directly correlates to the increase in the number of lysed cells. Formazan dye is water-soluble and can be detected with a spectrophotometer at 500 nm [Cytotoxic activity was determined by means of LDH Cytotoxicity Assay Kit . ELPs were re-suspended in RPMI-1640 medium at a concentration of 1 × 106 cells/mL. CFSE (Sigma-Aldrich) was added to the cell suspension at a final concentration of 2 mM and then incubated for 15 min at room temperature in the dark. Labeling was completed by adding the same volume of NCS during 5 min at room temperature to quench the free CFSE. Labeled cells were washed five times with sterile PBS containing 10% NCS, counted, and then re-suspended in RPMI-1640 medium at 1 × 106 cells/mL [5 cells/well in 96-well, flat-bottomed cell culture plates, and five replicate samples for each treated amount were prepared. Cells were incubated in a 5% CO2 incubator at 37 °C for 72 h. Cultured cells were harvested, washed twice with PBS, fixed with 1% formaldehyde, then analyzed by flow cytometry, acquiring a minimum of 20,000 events from each sample. Data analysis was performed using FACSDiva software .Heparinized blood samples were obtained from healthy human volunteers. PBMCs were isolated using standard Hypaque  density gradient centrifugation. The PBMCs were then washed twice with RPMI 1640 (GIBCO BRL) medium containing 10% NCS, penicillin (100 U/mL), and streptomycin (100 μg/mL). The lymphocyte population was further enriched (ELP) by eliminating adherent cells , and diluted in PBS (1:500) for 18 h. Next, the cells were washed and incubated with the secondary goat anti-rabbit antibody with FITC diluted to 1:200 in PBS for 2 h. The samples were analyzed by flow cytometry, acquiring a minimum of 10,000 events from each sample. Data analysis was performed using FACSDiva 4.0 software.HeLa and CaSki cells were seeded at 10p-value < 0.05 (Student’s t-test) was considered significant.Means and standard deviations (SD) were calculated using Excel . Statistical analysis of differences was carried out by ANOVA using SPSS 10.0 for Windows. A 4 and 5 generate interesting results in the antiproliferative activity in vitro compared to the activity observed with diosgenin (1). The antiproliferative activity of 4 was 2.3–2.8 times that of 1 due to the oxime group present in the spiroketalic side chain of 4; the oxime groups present on the steroidal A/B rings of 5 decreased the antiproliferative activity 1.2–1.7 times, suggesting that the oxime group improves the activity of spirostans if it is present in the side chain. A relevant aspect of compounds 4 and 5 is that they produce low-cytotoxicity effects in two cellular types: cancer cells and normal cells (lymphocytes), since apoptosis, more than necrosis, was the pathway for cancer cell elimination with evidenced apoptotic morphology and an increased presence of active caspase-3. The fact that compounds 4 and 5 did not affect proliferation of normal lymphocytes confers a certain advantage to these compounds. It has been shown that the double bond between positions C4 and C5 on the steroidal ring A, confers a negative cytotoxic effect on cancer cells for 3-hydroxyimino-substituted compounds [4 and 5, related to the conformational difference on the rings A/B (a double bond shift) and it is possible that compound 4 may be recognized more easily by the active site in the cell by binding to protein- associated signaling molecules [Two new steroidal sapogenins were synthesized, with the incorporation of the functional group oxime to measure their antiproliferative activity, in the search for compounds with an increased broad biological activity profile. The assays showed that the steroidal oximes ompounds . This suolecules . From th


Elsholtzia rugulosa Hemsl. is known in China as a local herbal tea, medicinal herb and honey plant. Chemical examination of E. rugulosa led to the isolation of two new flavonoid glycosides, apigenin 4'-O-α-D-glucopyranoside (1) and 5,7,3',4'-tetrahydroxy-5'-C-prenylflavone-7-O-β-D-glucopyranoside (2), together with nine known flavonoids. Their structures were elucidated on the basis of spectroscopic evidence. Elsholtzia rugulosa Hemsl. (Lamicaeae), which is distributed in the Yunnan, Sichuan and Guizhou provinces of China, is known as a local herbal tea, medicinal herb and honey plant -) and 13C-NMR spectrum. Comparison of the NMR data with those of apigenin . The J value (3.7 Hz) of the anomeric proton indicated the α-configuration of the glucose moiety -) in combination with the presence of 26 carbon signals in its 13C-NMR spectrum, and the further 2D-NMR spectral data allow to elucidate the structure of compound 2 as shown in Compound 3- proton on a flavone C-ring, and two aromatic proton signals at δ 6.61  and 6.48  due to H-6, 8 on A-ring protons, respectively, two broad singlet signals at high field in the aromatic region  on B-ring were observed in 1H-NMR spectrum, which suggested the occurrence in the molecule of a flavone skeleton with a tetra-substituted B-ring. In addition, one glucopyranosyl unit  was evident in the 1H- and 13 C-NMR of 2. On enzymatic hydrolysis, compound 2 liberated D-glucose and the J value (8.1 Hz) of the anomeric proton concluded the β-configuration of the D-glucose moiety. The HMBC correlations of glucosyl H-1'' [δ 5.08 ] in 2 with the C(7) (δ 166.7) confirmed that the location of glucopyranosyl groups were at C-7 in 2. These NMR features were resembled to those of luteolin 7-O-β-D-glucoside (4) - .Compound2: Yellow amorphous powder. UV-Visible λmax (nm) MeOH: 256, 267, 346; IR : 3,450, 2,920, 1,650, 1,573, 1,515, 990-600; 1H-NMR : 7.40 , 6.95 , 6.78 , 6.61 , 6.48 , 5.16 , 5.08 , 3.78-4.40 , 3.62 , 1.68 , 1.62  ppm; 13C-NMR : 183.7 , 166.7 , 162.1 , 158.9 , 151.1 , 147.3 , 130.1 , 123.7 , 127.1 , 122.1 , 117.6 , 114.6 , 107.1 , 104.5 , 101.9 , 101.0 , 96.1 , 78.5 , 77.9 , 74.7 , 71.2 , 62.9 , 28.4 , 25.6 , 17.8 ; HR-FAB-MS (neg.): 515.1913 [M-H]- .1 (3 mg) and maltase (1 mg) was incubated at 37 °C for 80 h. The solution was extracted with CHCl3 and aglycone produced was identified as apigenin by comparison with compound 6 on silica gel TLC using CHCl3-MeOH-H2O (8:2:0.2), fR = 0.68. The aqueous layer was concentrated to a residue, which was dissolved by water and examined for identification of the component sugar, and D-glucose was identified by direct comparison on silica gel TLC with an authentic sample, using CHCl3-MeOH-H2O (7:3:0.5). fR = 0.23.An aqueous solution of 2 (2 mg) in H2O (1 mL) were treated with crude cellulase (7 mg) at 37 °C for 60 h. The reaction mixture was diluted with H2O (2 mL), and extracted with CHCl3 (3 mL × 2). The aqueous layer was concentrated to a residue, which was dissolved by water and examined for identification of the component sugar, and D-glucose was identified by direct comparison on silica gel TLC with an authentic sample, using CHCl3-MeOH-H2O (7:3:0.5). fR = 0.23.A solution of E. rugulosa led to the isolation of two new flavonoid glycosides, apigenin 4'-O-α-D-glucopyranoside (1) and 5,7,3',4'-tetrahydroxy-5'-C-prenylflavone-7-O-β-D-glucopyranoside (2), together with nine known compounds (3-11). Among them, compounds 7-10 were isolated for the first time from E. rugulosa.A detailed phytochemical investigation on