Ornithogalum saundersiae, an ornamental perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides, some of which were structurally unique and showed potent cytotoxic activity against cultured tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides (1–12) and seven known compounds (13–19). The structures of the new compounds (1–12) were identified via NMR-based structural characterization methods, and through a sequence of chemical transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of the isolated compounds (1–19) and the derivatives (1a and 6a) against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10–12, 16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent cytotoxicity with an IC50 value of 0.16 µM against HL-60 cells and induced apoptotic cell death via a mitochondrion-independent pathway.Previous phytochemical studies of the bulbs of  Ornithogalum saundersiae (Baker), of the Asparagaceae (formerly Liliaceae) family, is an ornamental perennial plant native to South Africa. Our systematic phytochemical examinations of a methanolic extract of Ornithogalum saundersiae bulbs resulted in the isolation of 29 new cholestane glycosides including 16,23-epoxy-5β-cholestane glycoside , , and the molecular formula C41H68O14 based on the high-resolution electrospray ionization mass spectrometry  and 13C-NMR spectra. The 1H-NMR spectrum of 1 showed signals for five steroidal methyl groups at δH 1.31 , 1.23 , 1.05 , 1.03 , 0.99 ; an olefinic proton at δH 5.44 ; and two anomeric protons at δH 5.09 ) and 5.05 ). Furthermore, the presence of an acetyl group in 1 was revealed via IR (1729 cm−1), 1H-NMR , and 13C-NMR (δC 170.4 C=O and 21.0 Me) spectroscopy (1 with 3% NaOMe in MeOH afforded a deacyl derivative (1a), and subsequent acid hydrolysis of 1a with 1 M HCl in dioxane/H2O (1:1) yielded (22S)-cholest-5-ene-3β,11α,16β,22-tetrol -11α,22-dihydroxycholest-5-en-16β-yl 2-O-acetyl-α-L-rhamnopyranoside.Compound 2 (C43H70O15) was shown to be closely related to 1 by the 1H- and 13C-NMR spectra, differing from 1 only by the presence of two acetyl groups moieties ; δC 170.2 (C=O) and 20.5 (Me), and δC 170.0 (C=O) and 20.6 (Me)) -3β-[(β-d-glucopyranosyl)oxy]-11α,22-dihydroxycholest-5-en-16β-yl 2,3-di-O-acetyl-α-l-rhamnopyranoside.Compound 3 (C49H74O16) and 4 (C51H78O18) were suggested to be essentially analogous to 2 based on the 1H- and 13C-NMR spectra; however, the signals for one acetyl group attached to the rhamnosyl group were not present. Instead, the signals assignable to a p-methoxybenzoyl group and a 3,4,5-trimethoxybenzoyl group were detectable in the 1H- and 13C-NMR spectra of 3 and 4, respectively. Upon alkaline methanolysis of 3 and 4 with 3% NaOMe in MeOH, methyl p-methoxybenzoate and 1a were obtained from 3, while methyl 3,4,5-trimethoxybenzoate and 1a were obtained from 4. HMBC analysis of 3 revealed long-range correlations between H-2 of Rha at δH 5.78  and the carbonyl carbon of the acetyl group at δC 169.9, and between H-3 of Rha at δH 6.05  and the carbonyl carbon of the p-methoxybenzoyl group at δC 165.8. Similarly, correlation peaks were observed between H-2 of Rha at δH 5.81  and the carbonyl carbon of the acetyl group at δC 170.0, and between H-3 of Rha at δH 6.08  and the carbonyl carbon of the 3,4,5-trimethoxybenzoyl group at δC 165.9 in that of 4. Thus, the structures of 3 and 4 were elucidated as (22S)-3β-[(β-d-glucopyranosyl)oxy]-11α,22-dihydroxycholest-5-en-16β-yl 2-O-acetyl-3-O-p-methoxybenzoyl-α-L-rhamnopyranoside and (22S)-3β-[(β-d-glucopyranosyl)oxy]-11α,22-dihydroxycholest-5-en-16β-yl 2-O-acetyl-3-O--α-l-rhamnopyranoside, respectively.Compound 5 (C35H56O9) was obtained as an amorphous powder. The 1H-NMR spectrum of 5 displayed signals arising from two tertiary methyl groups at δH 1.41 and 1.00 ; a secondary methyl group at δH 1.26 ; two methyl groups on a double bond at δH 1.78 (Me-27) and 1.73 (Me-26); two olefinic protons at δH 5.82  and 5.65 ; an anomeric proton at δH 5.03 ; and the methyl group of an acetyl moiety at δH 2.03 (s). These spectral features of 5 showed similarity to those of 18, and alkaline degradation of 5 with 3% NaOMe in MeOH yielded 18. A long-range correlation was observed between the H-2 of Rha at δH 5.65  and the carbonyl carbon of the acetyl group at δC 170.6 in the HMBC spectrum of 5, providing evidence that the acetyl group was located at C-2 of the α-l-rhamnopyranosyl moiety. The structure of 5 was defined as (22S)-3β,11α,22-trihydroxycholesta-5,24-dien-16β-yl 2-O-acetyl-α-l-rhamnopyranoside.Compound 6 (C43H68O15), for which 1H-NMR analysis showed two anomeric proton signals at δH 5.07  and 5.03 , was assumed to be a bisdesmosidic cholestane glycoside closely related to 2. The two compounds differed only in the signals of the methyl groups assignable to Me-26 and Me-27. The two three-proton doublets for Me-26 and Me-27 observed in the 1H-NMR spectrum of 2 were displaced by two three-proton singlets at δH 1.77 and 1.73 in that of 6, suggesting that 6 was the C-24/C-25 dehydro derivative of 2. This was confirmed by alkaline methanolysis of 6, followed by enzymatic hydrolysis using β-d-glucosidase, to yield 18 and d-glucose. The linkage position of the glucosyl moiety of 6 was ascertained by an HMBC correlation between H-1 of Glc and C-3 of the aglycone at δC 78.1. The structure of 6 was elucidated as (22S)-3β-[(β-d-glucopyranosyl)oxy]-11α,22-dihydroxycholesta-5,24-dien-16β-yl 2,3-di-O-acetyl-α-l-rhamnopyranoside.Compound 7 (C49H72O16) and 8 (C51H76O18) were suggested to be the corresponding C-24/C-25 dehydro derivatives of 3 and 4, respectively, based on their spectral properties  and the carbonyl carbon of the acetyl group at δC 170.0, and between H-3 of Rha δH 6.08  and the carbonyl carbon of the p-methoxybenzoyl group at δC 165.9. Similarly in 8, correlation peaks were observed between H-2 of Rha at δH 5.81  and the carbonyl carbon of the acetyl group at δC 170.1, and between H-3 of Rha at δH 6.11  and the carbonyl carbon of the 3,4,5-trimethoxybenzoyl group at δC 166.0. The structures of 7 and 8 were assigned as (22S)-3β-[(β-d-glucopyranosyl)oxy]-11α,22-dihydroxycholesta-5,24-dien-16β-yl 2-O-acetyl-3-O-p-methoxybenzoyl-α-l-rhamnopyranoside and (22S)-3β-[(β-d-glucopyranosyl)oxy]-11α,22-dihydroxycholesta-5,24-dien-16β-yl 2-O-acetyl-3-O--α-l-rhamnopyranoside, respectively.Compound operties . This as9 was obtained as an amorphous solid and shown to have the molecular formula C49H78O19 based on HRESIMS  and 13C-NMR spectral data. The deduced molecular formula of 9 was higher than that of 6 by C6H10O4, and the 1H-NMR spectrum showed signals for three anomeric protons at δH 6.39 and 5.07 , and 5.07 , and the methyl groups of two 6-deoxy sugars at δH 1.75 . Acid hydrolysis of 9 yielded l-rhamnose and d-glucose. When the 13C-NMR spectrum of 9 was compared with that of 6 (l-rhamnopyranosyl unit (Rha’) (δC 102.1 (CH), 72.6 (CH), 72.9 (CH), 74.2 (CH), 69.5 (CH), and 18.7 (Me)) were observed. The resonances attributable to C-2 of the inner Glc unit and its neighboring carbons varied, while all the other signals remained almost unaffected. In the HMBC spectrum of 9, long-range correlations were observed between H-1 of Rha at δH 6.39 (br s) and C-2 of Glc at δC 77.9, and between H-1 of Glc at δH 5.07 (J = 7.3 Hz) and C-3 of the aglycone at δC 78.2. Thus, the structure of 9 was determined as (22S)-3β-[(α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl)oxy]-11α,22-dihydroxycholesta-5,24-dien-16β-yl 2,3-di-O-acetyl-α-l-rhamnopyranoside.Compound hat of 6 , a set o10 (C43H70O14) was suggested to be a bisdesmosidic cholestane glycoside closely related to 2, both possessing acetyl groups at C-2 and C-3 of the α-l-rhamnopyranosyl moiety attached to the aglycone’s C-16 hydroxy group. However, the molecular formula of 10 contained one oxygen atom less than 2. When the 13C-NMR spectrum of 10 was compared with that of 2, the hydroxymethine carbon signal observed at δC 67.8 (C-11) in the spectrum of2 was displaced by a methylene carbon signal at δC 21.1 in that of 10, indicating that 10 was the C-11 dehydroxy derivative of 2. Alkaline methanolysis of 10 yielded the structure (22S)-3β-[(β-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-16β-yl α-l-rhamnopyranoside (10a), which we have previously isolated from Galtonia candicans [10 was assigned as (22S)-3β-[(β-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-16β-yl 2,3-di-O-acetyl-α-l-rhamnopyranoside.Compound 11 (C33H54O8) was a cholestane rhamnoside with similar spectral properties to those of 18. However, the molecular formula of 11 lacked one oxygen atom in comparison to 18, and the 13C signals assignable to C-11 differed between the two compounds. The hydroxymethine carbone signal observed at δC 68.1 (C-11) in the 13C-NMR spectrum of 18 was displaced by a methylene carbon signal at δC 21.1 in that of 11, allowing 11’s structure to be determined as (22S)-3β,22-dihydroxycholesta-5,24-dien-16β-yl α-l-rhamnopyranoside.Compound 12 had the molecular formula C43H68O14, possessing two fewer hydrogen atoms than 10. The two three-proton doublets for Me-26 and Me-27 observed in the 1H-NMR spectrum of 10 were displaced by two three-proton singlets at δH 1.79 and 1.75 in that of 12, suggesting that 12 was the C-24/C-25 dehydro derivative of 10. Catalytic hydrogenation of 12 over Pd-C in an H2 atmosphere yielded 10. Thus, the structure of 12 was formulated as (22S)-3β-[(β-d-glucopyranosyl)oxy]-22-hydroxycholesta-5,24-dien-16β-yl 2,3-di-O-acetyl-α-l-rhamnopyranoside.Compound 1–19) and the deacetyl derivatives (1a and 6a) against HL-60 cells was evaluated using a modified 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay  showed considerable cytotoxicity to HL-60 cells, with an IC50 value of 0.16 µM. On the contrary, acylation of the C-2 hydroxy group of the rhamnosyl moiety with acetic acid, and the C-3 hydroxy group with an acetic acid or a benzoic acid derivative, enhanced the cytotoxicity. Although 10 and 12 had a glucosyl moiety at the C-3 hydroxy group of the aglycone, and 16 and 17 had a hydroxy group at C-11 of the aglycone, they simultaneously possessed acyl groups at C-2 and C-3 of the rhamnosyl moiety and exhibited more potent cytotoxic activity against HL-60 cells than the positive controls. Compounds 10–12, 16, and 17 also showed significant cytotoxic activity against A549 cells (The cytotoxic activity of the isolated compounds (T) assay . Compoun49 cells .11) showed potent cytotoxicity against HL-60 cells. Therefore, the apoptosis-inducing properties of 11 in HL-60 cells were evaluated. After HL-60 cells were exposed to 11 at a concentration of 10 µM for 24 h, the cells were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed via fluorescence microscopy. The cells exhibited nuclear chromatin condensation and nuclear disassembly as shown in 11, cell cycle distribution, DNA fragmentation, and caspase-3 activity were evaluated in 11-treated HL-60 cells. When HL-60 cells were cultured with 11 at 10 µM for 20 h, the sub-G1 and G2/M phase populations, of which the vehicle control were 3.3 ± 0.20% and 22.4 ± 0.10%, increased to 12.3 ± 0.70% and 32.2 ± 2.44%, respectively  or a DPX-600 (600 MHz for 1H-NMR) spectrometer using standard Bruker pulse programs at 300 K . Chemical shifts were given as δ with reference to tetramethylsilane as an internal standard. Please see 18-OPN . Thin-layer chromatography (TLC) was conducted on precoated silica gel 60 F254 or RP18 F254S plates , and the spots were detected by spraying the plates with 10% H2SO4 aqueous solution, followed by heating. HPLC was performed with a system consisting of a CCPM pump , an RI-8021  or a Shodex OR-2  detector, and a Rheodyne injection port . A TSK gel ODS-100Z column  was used for preparative HPLC. The following materials and biochemical-grade reagents were used for the cell culture assays: a Spectra Classic microplate reader ; a 96-well flat-bottom plate ; HL-60 cells (JCRB 0085) and A549 cells (JCRB 0076) ; fetal bovine serum (FBS) ; 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution, RPMI-1640 medium, minimum essential medium (MEM), Triton X, cisplatin, etoposide and MTT ; penicillin G sodium salt and streptomycin sulfate ; ribonuclease A (RNase), paraformaldehyde and phosphate-buffered saline (PBS) ; and propidium iodide (PI) .Optical rotations were measured using a JASCO P-1030  automatic digital polarimeter. Ultraviolet and IR spectra were recorded on a JASCO V-630 UV-Vis spectrophotometer and on a JASCO FT-IR 410 spectrophotometer, respectively. NMR spectroscopic data were obtained on a DRX-500  in 2013. A voucher specimen has been deposited at the herbarium of this university (KS-2013-006).The fresh bulbs of O. saundersiae (9.7 kg) were extracted with MeOH (3 × 18 L). After evaporating the solvent in vacuo, the MeOH extract (510 g) was applied to a Diaion HP-20 column (80 mm i.d. × 470 mm) and successively separated by MeOH/H2O (1:4), EtOH, and EtOAc (each 3 L). The EtOH-eluate (75.0 g) was subjected to silica gel (100 mm i.d. × 490 mm) and eluted with a stepwise gradient mixture of CHCl3/MeOH , and finally with MeOH alone, to yield 10 subfractions (A–J). Fraction C (11.7 g) was separated by an ODS silica gel column (75 mm i.d. × 350 mm) and eluted sequentially with MeCN/H2O (2:3) into 10 subfractions (C.1–C.10). Fraction C.5 was passed through an ODS silica gel column (50 mm i.d. × 300 mm) using MeCN/H2O (2:3) to afford fractions C.5.1–C.5.5. Fraction C.5.3 (485 mg) was further divided by silica gel CC (35 mm i.d. × 390 mm) and eluted with CHCl3/MeOH (9:1) into fractions C.5.3.1–C.5.3.15. Fraction C.5.3.12 (142 mg) was purified by preparative HPLC using MeOH/H2O (3:2) to yield 14 (20.8 mg). Fraction C.5.5 (1.03 g) was separated by a silica gel column (50 mm i.d. × 300 mm) and eluted with CHCl3/MeOH (9:1) and MeOH into fractions C.5.5.1–C.5.5.9. Fraction C.5.5.2 (276 mg) was subjected to ODS silica gel CC (43 mm i.d. × 320 mm) and eluted with MeOH/H2O (7:3) to yield 19 (127 mg). Fraction C.5.5.3 (567 mg) was further separated by ODS silica gel CC (60 mm i.d. × 250 mm) and eluted with MeOH/H2O (7:3) and MeOH into fractions C.5.5.3.1–C.5.5.3.18. Fraction C.5.5.3.8 (133 mg) was purified by ODS silica gel CC (43 mm i.d. × 275 mm) using MeCN/H2O (9:11) to yield 15 (64.2 mg). Fraction C.5.5.3.13 (19.1 mg) was subjected to silica gel CC (28 mm i.d. × 225 mm) and eluted with CHCl3/MeOH/H2O (19:1:0.1) to yield 11 (12.7 mg). Fraction C.5.5.4 (84.2 mg) was chromatographed on ODS silica gel (60 mm i.d. × 265 mm) and eluted with MeOH/H2O (7:3) to yield 12 (8.6 mg). Fraction C.5.5.5 (145 mg) was subjected to CC (43 mm i.d. × 335 mm) on ODS silica gel, eluted with MeCN/H2O (2:3) and MeOH/H2O (7:3), to afford 4 (11.5 mg). Fraction C.8 (4.17 g) was subjected to CC (65 mm i.d. × 490 mm) on silica gel, eluted with CHCl3/MeOH (6:1), followed by preparative HPLC using MeOH/H2O (7:3) to afford 16 (15.3 mg) and 17 (2.6 mg). Fraction D (6.61 g) was separated by ODS silica gel CC (90 mm i.d. × 100 mm) using MeCN/H2O (1:2) to nine subfractions (D.1–D.9). Fraction D.2 (584 mg) was subjected to an ODS silica gel column (50 mm i.d. × 300 mm), using MeCN/H2O (1:3) as the eluent, and preparative HPLC using MeOH/H2O (3:2) to afford 18 (23.9 mg). Fraction D.3 (670 mg) was subjected to an ODS silica gel column, using MeCN/H2O  and MeOH/H2O (3:2) as the eluents, and a silica gel column with CHCl3–MeOH (7:1) to afford 13 (55.3 mg). Fraction D.5 (242 mg) was separated using ODS silica gel CC and eluted with MeOH/H2O (3:2) into fractions D.5.1–D.5.8. Fraction D.5.3 (100 mg) was subjected to a silica gel column and eluted with CHCl3/MeOH/H2O  to yield 5 (38.4 mg). Fraction D.6 (317 mg) was separated using ODS silica gel CC and eluted with MeOH/H2O (3:2) into fractions D.6.1–D.6.7. Fraction D.6.2 (68.6 mg) was added to an ODS silica gel column (40 mm i.d. × 320 mm), with MeCN/H2O (3:7) as the eluent, and a silica gel column with CHCl3/MeOH/H2O (8:1:0.1) to yield 6 (23.7 mg). Fraction D.9 (4.15 g) was separated using ODS silica gel CC (40 mm i.d. × 300 mm) and eluted with MeOH/H2O (7:3) into fractions D.9.1–D.9.19. Fraction D.9.4 (14.3 mg) and D.9.7 (108 mg) were subjected to preparative HPLC using MeCN/H2O (2:3) to yield 2 (4.1 mg) and 8 (15.6 mg), respectively. Fraction D.9.8 (161 mg) was subjected to ODS silica gel CC (34 mm i.d. × 410 mm) and eluted with MeCN/H2O (11:9) to yield fractions D.9.8.1–D.9.8.10. Fraction D.9.8.2 (77.4 mg) was further divided by CC (38 mm i.d. × 360 mm) on ODS silica gel and eluted with MeOH/H2O (7:3) into fractions D.9.8.2.1–D.9.8.2.8. Purification of fraction D.9.8.2.3 (20.1 mg) by preparative HPLC using MeCN/H2O (2:3) led to the isolation of 7 (2.52 mg). Compound 3 (15.2 mg) was obtained from fraction D.9.8.2.5 (18.3 mg) by subjecting it to ODS silica gel CC (35 mm i.d. × 340 mm) using MeCN/H2O (2:3). Fraction D.9.12 (150 mg) was subjected to an ODS silica gel column (38 mm i.d. × 340 mm) with MeCN/H2O (2:3) and MeOH/H2O (7:3) as the eluents, and a silica gel column with CHCl3/MeOH (6:1) to afford 10 (6.5 mg). Fraction F (7.9 g) was subjected to ODS silica gel CC (70 mm i.d. × 320 mm) and eluted with MeOH/H2O (3:2) to yield 10 subfractions (F.1–F.10). Fraction F.4 (375 mg) was subjected to preparative HPLC using MeCN/H2O (1:2) to yield 9 (4.8 mg), and 1 with minor impurities, which was purified by ODS silica gel CC (20 mm i.d. × 235 mm) using MeOH/H2O (1:1) to furnish pure 1 (10.9 mg).The fresh bulbs of 1: Amorphous solid; c = 0.05, MeOH); IR (film) νmax: 3393 (OH), 2927 (CH), 1729 (C=O) cm−1; HRESIMS m/z: 807.4518 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.44 , 4.41 , 4.26 , 4.06 , 4.04 , 1.31 , 1.23 , 1.05 , 1.03 , 0.99 .Compound 2: Amorphous solid; c = 0.05, MeOH); IR (film) νmax: 3362 (OH), 2931 (CH), 1746 (C=O) cm−1; HRESIMS m/z: 849.4618 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.40 , 4.40 , 4.21 , 4.00 , 3.98 , 1.24 , 1.18 , 1.04 , 0.98 , 0.94 .Compound 3: Amorphous solid; c = 0.10, MeOH); UV λmax (MeOH) nm (log ε): 257 (4.12), 206 (4.14); IR (film) νmax: 3389 (OH), 2932 (CH), 1748 (C=O), 1606, 1512, 1463 (aromatic ring) cm−1; HRESIMS m/z: 941.4871 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.42 , 4.46 , 4.25 , 4.05 , 4.02 , 1.28 , 1.23 , 0.99 , 0.99 , 0.95 .Compound 4: Amorphous solid; c = 0.10, MeOH); UV λmax (MeOH) nm (log ε): 262 (3.91), 257 (3.91), 215 (4.25); IR (film) νmax: 3432 (OH), 2926 (CH), 1732 and 1716 (C=O), 1616, 1508, 1460 (aromatic ring) cm−1; HRESIMS m/z: 1001.5087 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.44 , 4.48 , 4.26 , 4.07 , 4.00 , 1.29 , 1.25 , 1.01 , 1.00 , 0.96 .Compound 5: Amorphous solid; c = 0.10, MeOH); IR (film) νmax: 3361 (OH), 2925 (CH), 1719 (C=O) cm−1; HRESIMS m/z: 643.3820 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.82 , 5.65 , 4.42 , 4.28 , 4.22 , 3.91 , 1.78 , 1.73 , 1.41 , 1.26 , 1.00 .Compound 6: Amorphous solid; c = 0.098, MeOH); IR (film) νmax: 3389 (OH), 1747 (C=O), 1449 (C=C) cm−1; HRESIMS m/z: 847.4457 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.77 , 5.41 , 4.41 , 4.23 , 4.12 , 4.01 , 1.77 , 1.73 , 1.26 , 1.22 , 0.94 .Compound 7: Amorphous solid; c = 0.042, MeOH); UV λmax (MeOH) nm (log ε): 256 (4.17), 205 (4.56); IR (film) νmax: 3389 (OH), 2922 (CH), 1746 and 1724 (C=O), 1606, 1512, 1462 (aromatic ring) cm−1; HRESIMS m/z: 939.4721 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.78 , 5.43 , 4.46 , 4.24 , 4.18 , 4.02 , 1.71 , 1.70 , 1.27 , 1.26 , 0.98 .Compound 8: Amorphous solid; c = 0.10, MeOH); UV λmax (MeOH) nm (log ε): 267 (3.92), 214 (4.38); IR (film) νmax: 3389 (OH), 2936 (CH), 1724 (C=O), 1590, 1504, 1455 (aromatic ring) cm−1; HRESIMS m/z: 999.4930 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.79 , 5.43 , 4.48 , 4.24 , 4.18 , 4.03 , 1.72 , 1.70 , 1.28 , 1.27 , 0.98 .Compound 9: Amorphous solid; c = 0.05, MeOH); IR (film) νmax: 3412 (OH), 2925 (CH), 1730 (C=O) cm−1; HRESIMS m/z: 993.5050 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.79 , 5.41 , 4.44 , 4.25 , 4.14 , 3.90 , 1.79 , 1.75 , 1.45 , 1.24 , 0.96 . Compound 10: Amorphous solid; c = 0.08, MeOH); IR (film) νmax: 3391 (OH), 2929 (CH), 1748 (C=O) cm−1; HRESIMS m/z: 833.4661 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.33 , 4.40 , 4.02 , 3.95 , 1.23 , 1.09 , 1.03 , 0.94 , 0.90 .Compound 11: Amorphous solid; c = 0.10, MeOH); IR (film) νmax: 3376 (OH), 2932 (CH), 1448 (C=C) cm−1; HRESIMS m/z: 585.3765 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.56 , 5.40 , 4.41 , 4.13 , 3.85 , 1.70 , 1.67 , 1.27 , 1.08 , 0.96 .Compound 12: Amorphous solid; c = 0.10, MeOH); IR (film) νmax: 3359 (OH), 2930 (CH), 1747 (C=O) cm−1; HRESIMS m/z: 831.4511 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.79 , 5.62 , 4.40 , 4.15 , 3.94 , 1.79 , 1.75 , 1.24 , 0.91 , 0.87 .Compound 12: Compound 12 (2.0 mg) was dissolved in MeOH (2 mL) and reduced with 10% Pd–C (15 mg) under a H2 atmosphere for 4 h at room temperature. The reaction mixture, after removal of the catalyst by filtration, was subsequently passed through a Sep-Pak C18 cartridge  and then chromatographed on ODS silica gel, using MeOH/H2O (7:3) as an eluent, to yield 10 (1.33 mg).Catalyti hydrogenation of 1–8 and 10: Compounds 1–2, 4–5 and 10 (each 1.0 mg), 3 (5.0 mg), 6 (10.0 mg), 7 (2.52 mg), and 8 (6.32 mg), were separately treated with 3% NaOMe in MeOH (2.0 mL) at room temperature for 1 h. The reaction mixture was neutralized by passing it through an Amberlite IR120  column and then chromatographed on silica gel, using CHCl3/MeOH/H2O  as an eluent, to yield 18 (0.54 mg) from 5, 1a (0.8 mg) from 1, 1a (0.72 mg) from 2, 1a (2.24 mg) and methyl p-methoxybenzoate (1.17 mg) from 3, 1a (0.65 mg) and methyl 3,4,5-trimethoxybenzoate (0.15 mg) from 4, 6a (6.15 mg) from 6, 6a (1.73 mg) and methyl p-methoxybenzoate (0.28 mg) from 7, 6a (3.88 mg) and methyl 3,4,5-trimethoxybenzoate (0.90 mg) from 8, and 10a (0.71 mg) from 10. Alkaline methanolysis of 1a: Amorphous solid; c = 0.05, MeOH); IR (film) νmax: 3390 (OH), 2921 (CH) cm−1; HRESIMS m/z: 765.4369 [M + Na]+ . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N)δH: 5.43 , 4.43 , 4.29 , 4.40 , 3.99 , 1.30 , 1.21 , 1.01 , 0.83 , 0.82 . Compound 1a: Compound 1a (3.2 mg) was dissolved in 1 M HCl  and heated at 95 °C for 1 h under an Ar atmosphere. After cooling, the reaction mixture was neutralized via passage through an Amberlite IRA-96 column (Organo) and then chromatographed on silica gel, using CHCl3/MeOH (19:1) as an eluent, to yield (22S)-cholest-5-ene-3β,11α,16β,22-tetrol (0.5 mg) + . For 1H-NMR spectral data of the sugar and acyl moieties, see 13C-NMR spectral data, see 1H-NMR spectral data of the aglycone moiety (C5D5N) δH: 5.53 , 5.44 , 4.42 , 4.26 , 4.12 , 4.04 , 1.69 , 1.67 , 1.31 , 1.25 , 1.01 .Compound 6a: Compound 6a (3.0 mg) was treated with β-d-glucosidase (10 mg) in AcOH/AcONa buffer  at room temperature for 47 h. The crude hydrolysate was chromatographed on silica gel and eluted with CHCl3/MeOH/H2O (9:1:0.1), followed by MeOH, to yield 18 (1.7 mg) and a sugar fraction (0.6 mg). The glucose present in the sugar fraction was identified under the same conditions as in the case of 2a. tR (min): 14.0 .Enzymatic hydrolysis of 9: A solution of 9 (1.0 mg) in 1 M HCl  was heated at 95 °C for 90 min under an Ar atmosphere. After cooling, the reaction mixture was neutralized via passage through an Amberlite IRA-96 (Organo) column. The reaction mixture was chromatographed on silica gel, eluted with CHCl3–MeOH (4:1), to yield a sugar fraction, but genuine aglycones could not be obtained. HPLC analysis of the sugar fraction showed the presence of l-rhamnose and d-glucose; tR (min): 8.21 , 15.1 .Acid hydrolysis of v/v) heat-inactivated fetal bovine serum supplemented with l-glutamine, 100 unit/mL penicillin G sodium salt, and 100 μg/mL streptomycin sulfate. Cells were stored in a humidified incubator at 37 °C with 5% CO2. Cell growth was measured with an MTT reduction assay, as previously described [4 cells/mL, A549 cells: 1 × 104 cells/mL) were continuously treated with each compound for 72 h, and cell viability was measured with the MTT reduction assay. A dose–response curve was plotted for 3, 7–12, 15–17, and 19, all of which resulted in less than 50% cell growth at a concentration of 10 µM, and the exact concentration at which 50% inhibition (IC50) of cell growth occurred was calculated. Each assay was performed in triplicate.HL-60 cells or A549 cells were maintained in RPMI 1640 medium or MEM, respectively, containing 10% (escribed . The cel5 cells/well) were plated on coverslips in 96-well plates. After 24 h, HL-60 cells were treated with either 10 µM of 11 or 15 µM of etoposide for 24 h. The cells were fixed with 1% glutaraldehyde for 30 min at room temperature before staining with DAPI (0.5 µg/mL in PBS) at room temperature. They were then observed immediately under a CKX41 fluorescence microscope .The cells (5 × 105 cells/well) were treated with 10 µM of 11, or 1 µM of colchicine as a control, for 20 h. The cells were collected, centrifuged at 1000 rpm for 5 min, then washed with PBS, and centrifuged again at 3000 rpm for 5 min. The cells were fixed with 70% EtOH at −20 °C, and then treated with FxCycleTMPI/RNase Staining Solution . Analysis of the cell cycle distribution was performed using a FACSCanto II flow cytometer .The cells (5 × 1011 (10 µM), or etoposide (15 µM), as a control. DNA was extracted with the Wizard Genomic DNA Purification Kit . In brief, cells were centrifuged for 5 min at 1200 rpm. The cell pellet was re-suspended in nuclei lysis solution (600 µL). Then, 4 mg/mL RNaseA solution (3 µL) was added to the cell lysate, and the solution was incubated at 37 °C for 30 min. Protein precipitation solution (200 µL) was added to the RNaseA-treated cell lysate, after which the mixture was incubated for 5 min on ice, and then centrifuged at 15,000 rpm for 5 min. The supernatant was transferred to a clean 1.5 mL microcentrifuge tube containing isopropyl alcohol (600 µL), and was mixed by inversion. After centrifugation at 15,000 rpm for 5 min, DNA was visible as a small white pellet, which was washed with ethanol/water . Finally, the pellet was re-suspended in DNA rehydration solution (25 µL) and incubated at 65 °C for 1 h. The sample (8 µL) was subjected to 2% agarose gel electrophoresis in 40 mM Tris/acetate buffer (pH 7.4) at 50 V for 1 h. A DNA molecular weight marker  and DNA from HL-60 cells that underwent etoposide (15 µM)-induced apoptosis, were used for calibration. The DNA fragmentation pattern was examined in photographs taken under ultraviolet illumination.The cells were incubated at 37 °C for 20 h with 11 or 15 µM of the control reagent etoposide for 24 h, and the cells were then centrifuged at 400× g for 5 min and collected. Cell pellets were suspended in ice-cold cell lysis buffer (160 µL), and incubated on ice for 10 min. This cell pellet suspension was centrifuged at 10,000× g for 5 min, and the supernatant was collected. The cell lysate  was mixed with reaction buffer (2 × 50 µL) containing the substrate for caspase-3 (DEVD-pNA (p-nitroanilide)). After incubation for 6 h at 37 °C, the absorbance of the liberated chromophore pNA was measured at 405 nm in a microplate reader. The activity of caspase-3 was evaluated in triplicate.The activity of caspase-3 was measured using the Appocyto Caspase-3 Colorimetric Assay Kit . HL-60 cells were treated with either 10 µM of ΔΨm) was investigated using the MitoCapture™ Apoptosis Detection Kit , according to the manufacturer’s protocol. HL-60 cells (5 × 105 cells/mL) were treated with either 10 µM of 11 or 33 µM of cisplatin for 6 h, and then were centrifuged at 500 × g for 5 min and collected. Cell pellets were resuspended in 250 µL of MitoCapture™ solution, incubated for 15 min at 37 °C, and then centrifuged again for 5 min. Pellets were resuspended in 200 µL of incubation buffer and observed via fluorescence microscopy.The mitochondrial membrane potential , according to the manufacturer’s protocol. HL-60 cells (2.7 × 107 cells/mL) were treated with either 10 µM of 11 or 33 µM of cisplatin as a control for 6 h, and the cells were centrifuged and collected. Cells were homogenized and isolated as cytosolic and mitochondrial fractions by employing the appropriate reagents. The cytosolic and mitochondrial fractions (10 µg) were then subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). A standard western blot procedure was performed using monoclonal mouse anti-cytochrome c antibody, according to the manufacturer’s protocol.The release of cytochrome p < 0.01 was considered statistically significant.Data are expressed as means ± standard error of the mean (S.E.M.). Significant differences were calculated using Dunnett’s test using Excel-Toukei 2010 software . O. saundersiae, with 12 novel compounds. Compounds 10–12, 16, and 17 exhibited significant cytotoxic effects, with IC50 values ranging from 0.05–0.16 µM against HL-60 cells, and from 0.27–2.41 µM against A549 cells. Compound 11 showed potent cytotoxicity against HL-60 cells by inducing apoptosis. The evaluation of mitochondrial membrane potential and cytochrome c release into the cytoplasm revealed no differences between HL-60 cells treated with the control or compound 11, suggesting that 11 induces apoptosis through a mitochondrion-independent apoptotic pathway.In conclusion, 19 cholestane rhamnosides were isolated from the bulbs of


C2-symmetrical “salen” ligand (+)-9 bearing two [5]ferrocenophane substituents has been prepared in five steps starting from readily available diacetylferrocene, p-hydroxybenzaldehyde and -N,N’-diphenylethylenediamine. Reaction of (+)-9 with Mn(OAc)3, Co(OAc)2 ZnEt2 or UO2(OAc)2 gave the corresponding metal-complexes which were characterised by spectroscopic methods. The  N,N’-bisethylendiamine  compounds are very popular ligands because of their easy formation and rich coordination chemistry with a large variety of metal ions, that has allowed their use as catalysts in different asymmetric reactions ferrocenophane 4 as a yellow solid, practically insoluble in all the common organic solvents, even after its O-deprotection. Deoxygenation of 4 by treatment with excess of borane in CH2Cl2 and removal of the benzyl group with hydrogen over Pd/C gave the alkylderivative 6 in 85% overall yield, this compound being soluble and more suitable for the following step of formylation on the aryl ring. The same reaction sequence was applied to prepare the isomeric [5]ferrocenophane 7 starting from 1 and 3 -9 by condensation of 8 with (+)--diphenylethylendiamine [-dpen] -dpen] .9 was confirmed by its 1H-NMR spectrum and diagnostic resonances at δ 8.41 and 13.16 for the iminic and phenolic protons, respectively, together with a singlet at δ 4.76 for the protons on the diimine bridge were observed. The methylenic protons on the ferrocenophane bridge gave rise to four separate multiplets in the range 1.8-2.4 ppm whereas the methinic signal was overlapped by the cycplopentadienyl resonances. Complete assignment of the carbon resonances by 2D-NMR spectra and molecular peaks at m/z 924.2 in the ESI-MS spectrum also supported the structure of ligand (+)-9.The symmetrical structure of (+)-9 with Mn(OAc)3 or Co(OAc)2 in EtOH gave the corresponding complexes (−)-10 and (+)-11 in high yield; due to their paramagnetic nature they were characterised by their ESI-MS and circular dichroism spectra (vide infra). The treatment of (+)-12, prepared by reacting the free ligand (+)-9 with Et2Zn in toluene, was mainly established by NMR analysis. The 1H-NMR spectrum of (–)-12 in CDCl3 was fully unresolved whereas in (CD3)2SO all the resonances could be assigned and the symmetric nature of the complex was evidenced; the main features of the spectrum are the absence of the phenolic signal and a slight upfield shift for both the resonances of azomethine and N-methinic protons compared with those of the free ligand. Complexes 10-12 -es 10-12  are quitSalen-uranyl(VI) complexes have been prepared as models for the pseudo-octahedral salen Mn=O species responsible for the selective oxygen transfer in the asymmetric epoxidation of olefins  and rece9 with (UO2)(OAc)2 gave the uranyl complex (+)-13 as a brown solid, whose structure was fully assigned from its 1H- and 13C-NMR spectra registered in (CD3)2SO. The lack of phenolic resonance in the protonic spectrum of 13 and the marked deshielding of the azomethinic and aminic protons  were taken as an evidence of the nitrogen and oxygen coordination with the metal, which acts as an electron withdrawing group. In the 13C-NMR spectrum, the diagnostic resonances for the C-OH and C=N carbons were also shifted downfield upon complexation showing large Δδ values .The reaction of (+)-1H-NMR spectrum of 13 registered in CDCl3 showed broad resonances for all the protons, but two broad singlets in 1:1 ratio at 298 K were distinguishable for the azomethinic group and assigned to two diastereomeric conformers which slowly interconverted on the NMR time scale. By warming, the above resonances tended to collapse and at 338K a 80:20 ratio of the two conformers was measured. In the case of uranyl complex of 14, with the same aminic moiety of (+)-13 and taken as a reference, two conformers were also observed in CDCl3, but not in (CD3)2SO, with a coalescence temperature of 300K. The slower interconversion rate of (+)-13 compared with the (UO2)-14 complex could be ascribed to the presence of bulky ferrocenophane substituents.The 1H-NMR but no evidence of such coordination was obtained for (+)-13. The additional coordination of a molecule of ethanol to the uranium atom has been reported for some salen-uranyl complexes on the basis of elemental analyses data  and it cCircular dichroism (CD) spectroscopy has been applied as a useful tool in the assignment of absolute configuration as well as conformational analysis of chiral molecules and biological polymers ,37,38. T9 and the corresponding metal complexes were registered in CH3CN, except for (–)-12 that was unsoluble in this solvent, and no substantial differences were observed in CH3Cl. The electronic and CD spectra of the free ligand (+)-9 the azomethine π-π* transition band was observed at 338 nm and the benzene π-π* transitions gave rise to overlapping bands below 250 nm. Although two bands assigned to metal-ligand and intraligand charge transfer transitions could be expected for the ferrocene chromophore in the range 300-600 nm ; Anal. Calcd. For C21H22FeO: C, 72.85; H, 6.40. Found: C, 73.58; H, 6.47.To a solution of 6, the isomeric derivative 7 was obtained in 78% overall yield (referred to 1). 1H-NMR (CDCl3) δ: 2.00 , 2.28 , 2.54 , 4.08 , 4.10 , 4.22 , 4.26 , 4.61 , 4.88 , 6.78 , 6.95 , 7.10 , 7.31 ; 13C-NMR (CDCl3) δ: 24.08 (CH2), 31.71 (CH2), 32.00 (CH), 66.50 (CpH), 67.74 (CpH), 68.19 (CpH), 69.50 (CpH), 90.15 (Cp), 115.22 (Ar-H), 121.03 (Ar-H), 126.60 (Ar-H), 128.49 (Ar-H), 134.30 (Ar), 152.98 (Ar-OH); mp 134-135 °C, ESI-MS: m/z 346.6 [M+]; Anal. Calcd. For C21H22FeO: C, 72.85; H, 6.40. Found: C, 73.34; H, 6.46.Starting from salicylaldehyde and applying the same reaction sequence described for compound 6  was dissolved in CHCl3 (20 mL) and 13M KOH (2 mL) was added. The mixture was then refluxed for 24 h under vigorous stirring and the reaction monitored by TLC analysis (n-hexane:EtOAc 85:15). The reaction was then quenched at about 25% conversion of substrate, when 8 was detected as the sole product, by addition of H2O and CH3COOH to acidic pH. After extraction the organic layer was dried over MgSO4 and taken to dryness. Column chromatography on Si gel (n-hexane:EtOAc 85:15) gave pure 8  and the unreacted 6 , which was then recycled. mp 139-140 °C; 1H-NMR (CDCl3) δ: 1.96 , 2.06 , 2.22 , 2.48 , 4.04 , 4.09 , 4.21 , 6.99 , 7.49 , 7.53 , 9.95 , 10.90 ; 13C-NMR (CDCl3) δ: 23.82 (CH2), 32.45 (CH2), 39.36 (CH), 66.21 (CpH), 67.11 (CpH), 68.11 (CpH), 69.06 (CpH), 89.99 (Cp), 117.55 (Ar-H), 120.50 (Ar), 131.97 (Ar-H), 136.31 (Ar-H), 139.73 (Ar), 160.25 (Ar-OH) 196.61 (CHO); ESI-MS: m/z 374.3 [M+]; Anal. Calcd. For C22H22FeO2: C, 70.60; H, 5.93. Found: C, 70.98; H, 6.01.Compound 8  in absolute ethanol (100 mL) -1,2-diphenylethylenediamine  was added and the reaction mixture maintained under stirring at room temperature for 2 days. When the TLC analysis indicated the disappearance of the aldehyde, the solvent was removed in vacuo and the residue applied to a silica gel column (n-hexane:EtOAc 9:1) to give pure (+)-9  as a pale orange solid, mp 139-140 °C; [α]D = +20.2 ; 1H-NMR (CDCl3) δ: 1.85 , 1.96 , 2.16 , 2.41 , 3.98 , 4.00 , 4.07 , 4.20 , 4.76 , 6.95 , 7.11 , 7.24 , 8.41 , 13.15 ; 13C-NMR (CDCl3) δ: 23.88 (CH2), 32.50 (CH2), 32.56 (CH2), 39.35 (CH), 66.20 (CpH), 67.16 (CpH), 68.00 (CpH), 69.20 (CpH), 69.25 (CpH), 80.39 (CH-N), 89.20 (Cp), 116.77 (Ar-H), 118.40 (Ar), 127.58 (Ar-H), 127.94 (Ar-H), 128.36 (Ar-H), 130.64 (Ar-H), 132.00 (Ar-H), 138.45 (Ar), 139.48 (Ar), 159.08 (Ar-OH), 166.46 (C=N); ESI-MS: m/z 924.2 [M+], 462.4 [M++]; Anal. Calcd. For C58H56Fe2N2O2: C, 75.33; H, 6.10; N, 3.03. Found: C, 75.91; H, 6.16; N, 3.08.To a solution of aldehyde 9  in absolute EtOH (10 mL), the appropriate M(OAc)n·xH2O , Co(II) or [UO2]2+) was added and the mixture left to react at rt under stirring until TLC analysis showed the complete conversion of substrate. The complex was separated from the solution by filtration and air-dried (80–90% yield)To a solution of (+)-Mn(III)-complex (–)-10: [α]D = –642.1 ; ESI-MS: m/z 1023.3 [M + EtOH]+; CD: λ (Δε) 625 (+1.08), 370 (+2.08), 306 (–3.82), 268 (+0.38), 243 (–7.16); Anal. Calcd. for C60H57Fe2MnN2O4: C, 69.51; H, 5.54; N, 2.70. Found: C, 69.48; H, 5.50; N, 2.67.Co(II)-complex (+)-11: [α]D = +610.2 ; ESI-MS: m/z 981.2 [M] +, 490.8 [M] ++; CD: λ (Δε) 562 (+4.90), 470 (+1.83), 416 (–0.77), 372 (+0.42), 300 (–3.78), 257 (+2.07), 232 ( –8.31); Anal. Calcd. For C58H54CoFe2N2O2 : C, 70.69; H, 5.54; N, 2.85. Found: C, 70.66; H, 5.49; N, 2.82.2 complexUO (+)-13: [α]D = +142.7 ; 1H-NMR (6d-DMSO) δ: 1.91 , 2.03 , 2.12 , 2.42 , 4.07 , 4.10 , 4.19 , 6.22 , 6.97 , 7.20 , 7.49 , 7.62 , 7.66 , 9.44 ; 13C-NMR (6d-DMSO) δ: 23.94 (CH2), 32.98 (CH2), 38.81 (CH), 66.35 (CpH), 67.34 (CpH), 68.17 (CpH), 69.22 (CpH), 80.61 (CH-N), 89.00 (Cp), 120.70 (Ar-H), 123.22 (Ar), 127.60 (Ar-H), 127.79 (Ar-H), 128.68 (Ar-H), 133.84 (Ar-H), 135.08 (Ar-H), 136.06 (Ar), 142.2 (Ar), 168.21 (Ar-O), 171.99 (C=N); ESI-MS: m/z 1192.3 [M]+; CD: λ (Δε) 575 (+0.68), 398 (+1.16), 331 (–2.52), 287 (+8.60), 250 (+12.45), 228 (–11.48); Anal. Calcd. For C58H54Fe2N2O4U: C, 58.40; H, 4.56; N, 2.35. Found: C, 58.38; H, 4.49; N, 2.31.2Zn in toluene  was added to a solution of (+)-9  in toluene (5 mL) and the mixture stirred at rt overnight. The solvent was removed under vacuum, the residue suspended in CHCl3 and the solid removed by filtration. The final solution was taken to dryness to give pure complex in quantitative yield: [α]D = – 21.3 ; 1H-NMR (6d-DMSO) δ: 1.77 , 1.92 , 2.05 , 2.37 , 3.86 , 4.00 , 4.03 , 4.06 , 4.14 , 5.05 , 6.64 , 7.00 , 7.17 , 7.26 , 7.36 , 7.44 , 8.36 ; 13C-NMR (6d-DMSO) δ: 23.87 (CH2), 32.56 (CH2), 39.23 (CH), 66.20 (CpH), 67.22 (CpH), 68.02 (CpH), 69.33 (CpH), 73.13 (CH-N), 89.40 (Cp), 118.9 (Ar), 123.09 (Ar-H), 127.85 (Ar-H), 127.92 (Ar-H), 128.87 (Ar-H), 131.4 (Ar), 133.44 (Ar-H), 133.56 (Ar-H), 142.37 (Ar), 170.32 (Ar-O), 170.65 (C=N); ESI-MS: m/z 987.3 [M]+, CD: λ (Δε) 416 (+0.63), 389 (–2.74), 353 (3.58), 289 (–3.25); Anal. Calcd. For C58H54Fe2N2O2Zn : C, 70.50; H, 5.51; N, 2.83. Found: C, 70.46; H, 5.48; N, 2.80.A 2M solution of Et9 bearing two [5]ferrocenophane para-substituents on the salicylideneamine rings has been synthesized starting from readily available diacetylferrocene, p-hydroxybenzaldehyde and -dpen and its complexes with four different metals have been also prepared and characterised. The evaluation of the use of these complexes as ligands in asymmetric synthesis is currently under investigation.A novel salen ligand (+)-


The envisaged structures of the all the synthesized ligands have been confirmed on the basis of their spectral analysis FT-IR, mass spectroscopy, 1H- and 13C-NMR. In vitro screening of their antibacterial and antifungal potential against Escherichia coli bacterium and Fusarium oxysporum f.sp albedinis (F.o.a) fungus, respectively, revealed that all the ligands showed no significant antibacterial activity, whereas most of them displayed good antifungal activity. Homology modeling and docking analysis were performed to explain the antifungal effect of the most and least active compound against two F.o.a fungus proteins.Twelve new Schiff base derivatives have been prepared by the condensation reaction of different amino substituted compounds (aniline, pyridin-2-amine,  Nowadays, much attention is paid to the study of Schiff base compounds, not only for their broad range of biological activities—such as antimalarial, antiviral, antitubercular, anticancer, anti-inflammatory, antifungal, and antibacterial properties ,3,4,5,6—Fusarium oxysporum f.sp albedinis (F.o.a) represents the leading dangerous agent among all pathologies of date palms cultivation, particularly in North Africa + = 183.N-((E)-pyridin-3-ylmethylidene)aniline (2): White powder. Yield 92%; m.p. 176–181 °C; IR : 3080 ; 2965; 1609 ; 1592 (C=N); 1568 (C=C); 1H-NMR : 7.4 , 7.45 , 8.8 , 2.4 ; 13C-NMR : 15.7 (C15- methylbenzene), 122.2 (C14- methylbenzene), 123.9 (C5-pyridine), 127.1 (C13-methylbenzene), 127.2 (C12-methylbenzene), 130.4 , 130.6 (C10-methylbenzene), 137.2 (C4-pyridine), 147 (C9- methylbenzene), 151.5 (C2-pyridine), 152 (C6-pyridine), 155.92 (C7-imine). Mass spectrum (EIMS) [M]+ = 167.2-methyl-N-((E)-pyridin-3-ylmethylidene)aniline (3): Crystal. Yield 85%; m.p. 145–150 °C; IR : 3080 ; 1609 ; 1592 (C=N); 1568 (C=C); 1360 ; 1H-NMR : 6.8 , 7.48 , 8.8 ; 13C-NMR : 122.4 (C11-nitrobenzene), 123.2 (C14-nitrobenzene), 123.9 (C12-nitrobenzene), 128.2 (C3-pyridine), 130.4 (C13-nitrobenzene), 136.2 (C4-pyridine), 137.2 (C10-nitrobenzene), 141.9 (C9-nitrobenzene), 144.1 (C2-pyridine), 151.5 (C6-pyridine), 154.69 (C7-imine). Mass spectrum (EIMS) [M]+ = 228.2-nitro-N-((E)-pyridin-3-ylmethylidene)pyridin-2-amine (4): White powder. Yield 96%; m.p. 119–125 °C; IR : 3084 ; 1610 ; 1597 (C=N); 1570 (C=C); 1H-NMR : 7.8 , 8.50 , 8.8 ; 13C-NMR : 116.2 (C3-pyridine), 122.4 (C5-pyridine), 123.9 (C13-pyridine), 130.4 (C9-pyridine), 137.2 (C14-pyridine), 137.3 (C4-pyridine), 150.4 (C6-pyridine), 151.5 (C10-pyridine), 152 (C12-pyridine), 156.5 (C2-pyridine), 160 (C7-imine). Mass spectrum (EIMS) [M]+ = 184.N-((E)-(4-nitrophenyl)methylidene)aniline (5): Yellow powder. Yield 85%; m.p. 145–150 °C; IR : 3080 ; 1609 ; 1592 (C=N); 1568 (C=C); 1360 ; 1H-NMR : 8.2 , 8.10 ; 13C-NMR : 121.1 , 121.2 , 122.4 , 123.2 , 128.2 , 130.1 , 130.2 , 136.2 , 139.9 , 141.9 , 148.3 , 150.35 , 150.7. Mass spectrum (EIMS) [M]+ = 272.2-nitro-N-((E)-(3-nitrophenyl)methylidene)aniline (6): Yellow powder. Yield 96%; m.p. 181–183 °C; IR : 3080 ; 1609 ; 1592 (C=N); 1568 (C=C); 1360 ; 1H-NMR : 8.2 , 8.75 ; 13C-NMR : 122.4 , 123.2 , 123.4 , 124.1 , 128.2 , 129.8 , 134.7 , 135.3 , 136.2 , 141.8 , 148.3 , 148.42 , 148.5. Mass spectrum (EIMS) [M]+ = 272.2-nitro-N-((E)-(2-nitrophenyl)methylidene)aniline (7): Yellow powder. Yield 83%; m.p. 181–183 °C; IR : 3080 ; 1609 ; 1592 (C=N); 1568 (νC=C); 1360 ; 1H-NMR : 8.2 , 8.05 ; 13C-NMR : 122.4 , 123.2 , 123.4 , 124.1 , 128.2 , 129.8 , 134.7 , 135.3 , 136.2 , 141.8 , 148.3 , 145 , 148.5. Mass spectrum (EIMS) [M]+ = 272.2-nitro-N-((E)-(4-nitrophenyl)methylidene)pyridin-2-amine (8): Crystal. Yield 85%; m.p. 150–155 °C; IR : 3084 ; 1601 ; 1590 (C=N); 1562 (νC=C); 1364 ; 1H-NMR : 8.2 , 8.10 , 7.8 ; 13C-NMR : 116.2 , 121.2 , 121.3 , 130.1 , 137.3 , 139.9 , 148.42 , 150.4 ,150.7 , 160.7 . Mass spectrum (EIMS) [M]+ = 228.N-((E)-(3-nitrophenyl)methylidene)pyridin-2-amine (9): Crystal. Yield 79%; m.p. 142–145 °C; IR : 3085 ; 1606 ; 1597 (C=N); 1565 (C=C); 1364 ; 1H-NMR : 8.5 , 8.15 , 8.00 ; 13C-NMR : 116.2 , 121.2 , 121.3 , 130.1 , 137.3 , 139.9 , 148.42 , 150.4 ,150.7 , 160.7 . Mass spectrum (EIMS) [M]+ = 228.N-((E)-(2-nitrophenyl)methylidene)pyridin-2-amine (10): Yellow powder. Yield 95%; m.p. 208–212 °C; IR : 3085 ; 1606 ; 1597 (C=N); 1565 (C=C); 1364 ; 1H-NMR : 8.2 , 8.6 , 8.05 ; 13C-NMR : 116.2 , 121.2 , 121.3 , 130.1 , 137.3 , 139.9 , 148.42 , 150.4 ,150.7 , 160.7 . Mass spectrum (EIMS) [M]+ = 228.Z)-(2-nitrophenyl)methylidene)amino)propan-1-ol (11): Yellow powder. Yield 85%; m.p. 187–190 °C; IR : 3500 (O-H); 3078 ; 2870 (C-H2); 1597 (C=N); 1565 (C=C); 1364 ; 1070 (C-O); 1H-NMR : 7.8 , 8.7 , 3.55(N-2CH-CH2-CH2-O), 1.84 (N-CH2-CH2-CH2-O), 3.53(N-CH2-CH2-CH2-O), 2.1 ; 13C-NMR : 34.2 , 57.9 , 60.5, 122.4, 123.2 , 128.2 , 136.2 , 141.9 , 148.3 , 157.20 . Mass spectrum (EIMS) [M]+ = 209.3-(((E)-pyridin-2-ylmethylidene)amino)phenol (12): White powder. Yield 80%; m.p. 185–188 °C; IR : 3550 (O-H); 3068 ; 1587 (C=N); 1568 (C=C); 1072 (C-O); 1H-NMR : 7.8 , 7.8 , 8.13 , 13C-NMR : 117.3 , 123.8 , 123.9 , 126.2 , 136.1 , 141.6 , 149.2 , 137.83 , 153.6 , 157 . Mass spectrum (EIMS) [M]+ = 199.4- fungus, as previously described [F.o.a fungus were placed into the middle of each Petri plate. Thereafter, all the plates were incubated at 28 °C for 7 days. The growth of the microorganism was followed by a measurement of mycelium diameter. The inhibition percentage of a molecule is equal to the ratio of the mycelium diameter of the culture in the presence of a dose of the tested compound over the mycelium diameter of the reference culture multiplied by 100. The minimum inhibition concentration (MIC) is the least dose of the compound which caused inhibition of the microorganism growth.The in vitro antifungal activities of all the new compounds were determined by the agar diffusion technique against escribed ,24. For F.o.a in the absence and in the presence of the tested compound, respectively. Due to lack of a known antibiotic that can treat this infection directly, (DMSO/EtOH + distilled water) mixture was used as negative control without any standard reference drug.Escherichia coli bacterium strain in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) recommendations [E.coli strain was subcultured on Muller–Hinton agar into Petri plates. Sterile WHATMAN paper discs of 6 mm diameter were put in the middle of each plate and then soaked up by DMSO/EtOH solution of the tested compounds at different concentrations. The incubation was carried out at 37 °C for 24 h. The zones of inhibition around each disc were noted.The prepared compounds were screened for their in vitro antibacterial potential against ndations  using thThe structures of all Schiff base derivatives were drawn using ACD/ChemSketch software (version 12.01(free version), FSO, Oujda, Morocco) . GeometrBoth the Fusarium oxysporum Guanine nucleotide-binding protein beta subunit (Fgb1) and the Fusarium oxysporum isolate FO 3-phytase-like (Fophy) proteins were used in the present work. The target primary amino acid sequence of Fgb1 was retrieved from the UniProtKB database (accession number Q96VA6), while the DNA sequence of Fophy was received from the NCBI GenBank database with accession number KC708486, and then translated to the equivalent amino acid sequence with the aid of Discovery Studio client v16 software (DS) (version 16 (academic version), Oujda, Morocco) . To findMolecular docking studies of the most and the least active antifungal compounds against the homology models of the Fgb1 and Fophy proteins were performed using the SwissDock server  . The CHA1H- and 13C-NMR. The preliminary antibacterial and antifungal screening against E. coli bacterium and Fusarium oxysporum f.sp albedinis (F.o.a) fungus, respectively, proved that most of these compounds exhibited good to excellent antifungal activity, but very weak antibacterial effect. Furthermore, theoretical investigations by homology modeling and docking studies were undertaken in order to illustrate and understand the possible binding mode of the most and least active compounds towards the Fgb1 and Fophy proteins. The results obtained were found to be in good agreement with the experimental findings in both cases. However, more appropriate in vitro experiments on the inhibition of Fgb1 or Fophy with the novel Schiff base compounds should be carried out, and their interactions should be confirmed by others, performing methods such as free-energy calculations and molecular dynamic simulations.A series of twelve Schiff base derivatives were synthesized and characterized by FT-IR, mass spectroscopy,


Ircinia echinata led to the isolation of six new 9α-hydroxy-5α,6α-epoxysterols: 5α,6α-epoxycholesta-7,22(E)-dien-3β,9α-diol (1), 5α,6α-epoxycholesta-7,24(28)-dien-3β,9α-diol (2), (24R)-5α,6α-epoxy-24-ethyl-cholesta-7-en-3β,9α-diol (3), 5α,6α-epoxycholesta-7-en-3β,9α-diol (4), (24S)-5α,6α-epoxyergosta-7,22-dien-3β,9α-diol (5), and (24R)-5α,6α-epoxy-24-methyl-cholesta-7-en-3β,9α-diol (6) along with the known 5α-6α-epoxysterols: 5α,6α-epoxystigmasta-7-en-3β-ol (7), 5α,6α-epoxystigmasta-7,22-dien-3β-ol (8), and 5α,6α-epoxyergosta-7-en-3β-ol (9). Their structures and their configurations were established on the basis of high resolution mass spectra and extensive 1D and 2D NMR spectroscopic data and by comparison with the literature. Their cytotoxic activity, evaluated against three human cancer cell lines, MCF-7, Hep-G2 and LU-1, revealed that only compounds 3 and 4 exhibited significant antiproliferative activity and compound 3 showed a selective inhibition towards the MCF-7 human breast cancer cells.Chemical investigation of the methanol extract of the Vietnamese marine sponge  Ircinia are known as a rich source of varied bioactive natural products, including fatty acids + , corresponding for compound 2 to a molecular formula of C28H44O3 with seven degrees of unsaturation. The 1H and 13C NMR data showed that compound 2 shares the same Δ7 sterol nucleus as 1, with the presence of an epoxy group at C-5/C-6 and a hydroxyl group at C-9  and 34.9 (C-25) located the double bond at position C-24/C-28. Careful analysis of 2D NMR spectra revealed the structure of compound 2 as 5α,6α-epoxycholesta-7,24(28)-dien-3β,9α-diol , corresponding for compound 3 to a molecular formula of C29H48O3 with six degrees of unsaturation. Examination of the NMR data indicated that compound 3 also shared the same Δ7-5,6-epoxyhydroxy sterol nucleus as compounds 1 and 2, but possesses a different side chain  was determined to be located at C-24 from the COSY correlations between the methyl at δH 0.88 (CH3-29) and the methylene at δH 1.35–1.18 (CH2-28), which in turn correlated with the methine at δH 0.96 (CH-24). Although only one diastereomer was isolated, the difference between C-26 and C-27 chemical shifts, which is 0.70 ppm, suggests the 24 R configuration for compound 3 (instead of 0.55 ppm in the case of S-configuration) + , corresponding for compound 4 to a molecular formula of C27H44O3 with six degrees of unsaturation. The 1H NMR signals of 4 were close to those of 1, except for the presence of two methylenes instead of the C-22/C-23 double bond. Analysis of 2D NMR spectra, in particular of HMBC correlations , corresponding for compound 5 to a molecular formula of C28H44O3 with seven degrees of unsaturation. A comparison of the 1H and 13C NMR data of compound 5 with that of sterol 1 revealed the presence of an additional methyl group at δH 0.94  and its corresponding resonance at δC 18.6, suggesting the replacement of a methylene at C-24 in compound 1 by a methine linked to a methyl group for compound 5. The location of the additional methyl group in C-24 was confirmed by HMBC correlations from the methyl at δH 0.94 (CH3-28), with the carbons at δC 133.4 (C-23) and 34.5 (C-25). The configuration at C-24 of compound 5 was determined by comparison of the 13C NMR chemical shifts with those of the related structures 25D −15.0 . The HRESIMS results showed [M-2H2O + H]+ found at m/z 379.2953 , and [M-3H2O + H]+ found at m/z 361.2913 . For 1H and 13C NMR, see Compound 2: white microcrystals; m.p. 181–182 °C; [α]25D −11.0 . The HRESIMS results showed [M-2H2O + H]+ found at m/z 393.3070 , and [M-3H2O + H]+ found at m/z 375.3035 . For 1H and 13C NMR, see Compound 3: white microcrystals; m.p. 172–173 °C; [α]25D −17.0 . The HRESIMS results showed [M-H2O + H]+ found at m/z 427.3597 , and [M-2H2O + H]+ found at m/z 409.3499 , and [M-3H2O + H]+ found at m/z 391.3371 . For 1H and 13C NMR, see Compound 4: white microcrystals; m.p. 158–159 °C; [α]25D −46.0 . The HRESIMS results showed [M-H2O + H]+ found at m/z 399.3259 , [M-2H2O + H]+ found at m/z 381.3161 , and [M-3H2O + H]+ found at m/z 363.3053 . For 1H and 13C NMR, see Compound 5: white microcrystals; m.p. 190–192 °C; [α]25D −32.0 . The HRESIMS results showed [M-H2O + H]+ found at m/z 411.3284 , [M-2H2O + H]+ found at m/z 393.3128 , and [M-3H2O + H]+ found at m/z 375.3044 . For 1H and 13C NMR, see Compound 6: white microcrystals; m.p. 207–208 °C; [α]25D −15.0 . The HRESIMS showed [M-H2O + H]+ found at m/z 413.3414 , [M-2H2O + H]+ found at m/z 395.3306 , and [M-3H2O + H]+ found at m/z 377.3202 . For 1H and 13C NMR, see Compound −1), streptomycin (100 μg.mL−1), and gentamicin (10 μg.mL−1). Stock solutions of the compounds were prepared in DMSO/H2O (1/9), and the cytotoxicity assays were carried out against cancer cells (3 × 103 cells.mL−1). After 72 h of incubation at 37 °C in air/CO2 (95:5), with or without the test compounds, cell growth was estimated by colorimetric measurement of stained living cells using neutral red. Optical density was determined at 540 nm with a Titertek Multiscan photometer. The IC50 value was defined as the concentration of the sample necessary to inhibit cell growth to 50% of the control. Ellipticine was used as a reference compound.Cytotoxicity assays were carried out in triplicate in a 96-well microtiter plates against HepG-2, Lu-1, and MCF-7, using a modification of the published method . Cells w


O-β-D-glucopyranosyl-(1→2)-β-D-glucoside, and two alkamides, N-cis- and N-trans-p-coumaroyl-3-O-methyldopamine, were isolated from stems of Aristolochia gigantea, together with the known compounds allantoin, E-nerolidol, β-sitosterol, (+)-kobusin, (+)-eudesmin, trans-N-feruloyltyramine, trans-N-coumaroyltyramine, trans-N-feruloyl-3-O-methyldopamine, aristolactam Ia-N-β-D-glucoside, aristolactam Ia 8-β-D-glucoside, aristolactam IIIa, and magnoflorine. Their structures were determined by spectroscopic analyses.A new aristolactam, aristolactam 9- Aristolochia.The Aristolochiaceae family consists of 450 to 600 species, among which more than 200 have been at least partially studied . Most ofAristolochia herbs have been used in weight-loss regimens. The clinical application of aristolochic acid (AA) has been limited due to its severe nephrotoxic activity. Recent studies have revealed that AA-I can cause direct damage to renal tubular cells, and its carcinigenicity is associated with the formation of promutagenic AA-DNA adducts −. The IR spectrum of compound 10 showed characteristic absorption bands of a lactam group at 1,654 cm−1 and hydroxyl groups at 3,442 and 1,088 cm−1. The DEPT and 13C-NMR spectra of 10 (C 167.4), methylenedioxy (δC 103.0), and methoxyl (δC 56.0) groups. A molecular formula of Cra of 10  showed s1H-NMR spectrum showed signals characteristic of CONH at δ 10.18 and only four aromatic hydrogens at δ 8.26 , 7.56 , 7.23 , and 7.65 (s). In addition, signals for carbons and hydrogens for a diglycosyl were observed. These data suggested that compound 10 was an aristolactam. 1H-1H COSY and 1D-TOCSY experiments allowed us to determine that the glycosyl units were β-glucosyl-β-glucosyl (1→2). Furthermore, the negative ESI-MS/MS of the ions at m/z 632.1 gave rise to ions at m/z 308.1 and 469.9 that suggested C9-O and O-C1'' fragmentations, respectively. The substituent positions on the aristolactam structure were assigned with the help of gHMBC experiments (C 132.6) and H-1' (δH 5.08); C-3(δC 148.2) and CH2O2 (δH 6.46); C-8 (δC 157.2) and OCH3 (δH 3.94) and H-6 (δH 7.56), as well as between C-2′ (δC 81.0) and H-1″ (δH 4.65). The eriments . These e1H- and 13C-NMR, IR, and UV spectroscopic data of 10 were very similar to those reported in the literature for triangularine -β-D-glucoside.The rine 16;  25], th, th1H- a1H- and 13C-NMR spectra of 14 + 15  deduced from the HRMS spectra was also consistent with the lack of an OCH3 substituent. Based on the integration of the signals corresponding to the olefinic hydrogens  it was possible to determine that the isolated mixture was in a 1:2 cis/trans proportion. Although cis and trans isomers can isomerize under UV light, both alkamide isomers may be natural compounds  gave 1 (16.5 mg) and 11 (17.5 mg). Fraction 21 (1.10 g) was subjected to RP CC  to give 17 subfractions (ca. 125 mL each). Subfractions 10 and 11 (98.4 mg) after HPLC  gave 7 (4.0 mg), 8 (9.6 mg), 9 (1.8 mg), and 10 (10.1 mg). The stem crude ethanol extract (10.0 g) was fractionated over Sephadex LH-20  to give 17 fractions. Fraction 6 (166 mg) was subjected to two HPLC runs  to give 4 (2.0 mg), 12 (2.0 mg), 13 (1.5 mg), and 14+15 (1.0 mg)].The rhizomes (433.6 g) and aerial stems (379.4 g) were ground and exhaustively extracted successively at room temperature with hexane, acetone, and ethanol. The residues were extracted with ethanol in a Soxhlet apparatus and the extracts were individually concentrated. A portion of the crude ethanol extract of rhizomes (8.10 g) was washed with CHAllantoin (1). Yellow needles. mp 233–234 °C [lit. 232–235 °C]  . IR, 1H-reported .E-Nerolidol (2). Yellow oil.  −17 °  [lit. −12.5 ° ]  . 1H-NMR β-Sitosterol (3). Colorless crystals.  −15.2 °  [lit. −26.1 ° ] [13C- NMR data were consistent with those previously reported [reported .Kobusin (4). Yellow solid.  +51.4 °  [lit. +58.0 ° ]  . 1H- NMREudesmin (5). Yellow oil.  +17.5 °  [lit. +61 ° ]  [1H-NMR data were consistent with those previously reported [c 0.07)] . 1H-NMR reported .Aristolactam Ia 8-β-D-glucoside (8). Amorphous solid. −8.2 °  [lit. −10.5 ° ] [1H-NMR data were consistent with those previously reported [ c 0.2)] . 1H-NMR reported .Aristolactam IIIa (9). Amorphous solid. 1H-NMR (DMSO-6d) δ 7.62 , 7.97 , 7.10 , 7.79 , 7.05 , 6.48 , 10.65 .Aristolactam 9-O-β-D-glucopyranosyl-(1→2)-β-D-glucoside (10). Amorphous solid. −3.9 ° . 1H-NMR (CDCl3) and 13C-NMR (CDCl3) spectra see m/z (rel. int.): 632. 1614 [M − H]− (100) ; ESI-MS/MS (probe) 4,500 V from ions at m/z 632.1 (100), m/z (rel. int.): 308.1 [M − glc-glc]− (92).Magnoflorine (11). Amorphous solid. +164.4 °  [lit. +150.0 ° ]  . 1H-NMR trans-N-Coumaroyltyramine (12). 1H-NMR (DMSO-6d) δ 6.38 , 7.30 , 7.37 , 6.78 , 3.31 , 2.64 , 7.00 , 6.67 , 7.94 .trans-N-Feruloyl-3-O-methyldopamine13). (1H-NMR (DMSO-6d) δ 6.43 , 7.30 , 7.10 , 6.78 , 6.97 , 3.32 , 2.65 , 6.77 , 6.68 , 6.60 , 3.79 , 3.74 , 7.94 .N-cis- and N-trans-p-Coumaroyl-3-O-methyldopamine (14 + 15). Colorless oil. 1H-NMR (CDCl3) spectra see 13C-NMR (DMSO-6d) δ 137.8 (C-3), 127.0 , 116.0 , 113.2 (C-2′′′), 120.9 (C-6′′′), 147.4 (C-3′′′), 145.1 (C-4′′′), 130.6 (C-1′′′), 126.3 (C-1′), 159.0 (C-4′), 56.0 (OCH3). ESI-HR-TOF-MS (probe) 4,000V, m/z (rel. int.): 312.1233 [M − H]− (100) .A. gigantea showed a diverse chemical composition. As previously observed, the characteristic chemical constituents of the leaves of this species arebisbenzylisoquinolinic and 8-benzylberberinic alkaloids, whereas stems contain lignans at high concentrations, alkamides, and aristolactams. Among the compounds that were isolated from stems, two alkamides and an aristolactam are described here for the first time.Extracts from different parts of


A cholesterol-succinobucol prodrug was also prepared in order to enhance the absorption of succinobucol through the intestinal membrane into the organism and to target the drug into the place of lipid metabolism—The enterohepatic circulation system. Their low toxicity towards mice fibroblasts at maximal concentrations, their antioxidant activity, comparable or even higher than that of ascorbic acid as determined by direct quenching of the DPPH radical, and their potential for significantly altering total and LDL cholesterol levels, suggest that these conjugates merit further studies in the treatment of cardiovascular or other related diseases. A brief discussion of succinobucol’s ability to quench the radicals, supported with a computational model of the electrostatic potential mapped on the electron density surface of the drug, is also presented. Synthesis, detailed structural characterization , and studies of toxicity, antioxidant activity and bioavailability of unique potent anti-atherosclerotic succinobucol-steroid conjugates are reported. The conjugates consist of, on one side, the therapeutically important drug succinobucol (4-{2,6-di- Coronary artery diseases still remain one of the major causes of morbidity and mortality in the developed world. The pathogenesis of atherosclerosis lies in abnormalities in the lipoprotein metabolism leading to pathological interactions with blood vessel walls and a release of inflammatory components, which further aggravate the disease conditions. The accumulation of lipids within arteries remains the initial impulse for the pathogenesis of atherosclerosis; however, both oxidative stress and inflammation are considered to play crucial roles in this process ,2,3,4.tert-butyl-4-[(1-{3-tert-butyl-4-hydroxy-5-(propan-2-yl)phenyl]sulfanyl}ethyl)sulfanyl]phenoxy}-4-oxo-butanoic acid] (succinobucol) a derivative of probucol ]bis).Many large-scale clinical trials have documented a decrease in cardiovascular morbidity and mortality by lowering the LDL-cholesterol level by several therapeutic (hypolipidemics) and/or life style modification  strategies in patients with multiple risk factors. Among the hypolipidemics our attention is directed to fatty acid esters of plant stanols/sterols and  represents the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%. For reasons of clarity we speak in terms of 1/EC50 or the antiradical power (ARP): the larger the ARP, the more efficient the antioxidant is D20: Optical rotation was measured on polarimeter Autopol IV by Rudolph Research Analytical (USA). Sodium D doubleline was used, wavelength 589 nm. Measurements were made in chloroform and corrected to 20 °C. UV: Spectra were recorded on spectrometer Specord 210 from Analytic Jena (Germany) in the range 200–700 nm, equipped by software WinAspect. X-ray crystallography: Crystallographic data were collected at 123(2) K on a Nonius KappaCCD diffractometer with graphite monochromated Mo-Kα radiation. COLLECT data collection software was utilized and the data were processed with DENZO-SMN  (1)  was added to the cloudy solution and the mixture was stirred for 45 min under a nitrogen atmosphere at ambient temperature, upon which time it turned orange. Succinic acid anhydride was added to the mixture which turned brown, later dark blue +, 656 (48) [M+K]+; HRMS-FAB m/z [M+Na]+ and [M+K]+ calcd. for C35H52O5S2: 639.3154 and 655.2893, found: 639.6405 and 655.6061; Anal. calcd for C35H52O5S2: C 68.14, H 8.50, S 10.40, found: C 68.13, H 8.53, S 10.38.A nitrogen-purged flask was charged with dry tetrahydrofuran (THF) (30 mL) and potassium ark blue . The realsewhere . For ato2 with stanol/sterols were prepared by Steglich esterification D20=+11.2 (c=1.4 μM in CHCl3); 1H-NMR: δ = 0.65 , 0.82 , 0.92–0.80 , 1.34 , 1.44 , 1.47 , 2.68 , 2.98 , 4.73 , 5.36 , 7.45 , 7.62 ppm ; 13C-NMR: δ = 11.97 (C-19’), 12.06 (C-18’), 12.22 (C-29’), 18.73 (C-21’), 19.03 (C-27’), 19.81 (C-26’), 21.20 (C-11’), 22.62 (C-2’), 23.05 (C-28’), 24.21 (C-15’), 26.07 (C-23’), 27.41 (C-4’), 28.26 (C-16’), 28.59 (C-6’), 29.05 (C-3), 29.14 (C-25), 30.27 , 30.56 , 30.72 (C-2), 30.72 (C-1’), 31.47 , 31.98 (C-7’), 33.94 (C-22’), 34.32 , 35.47 , 35.47 (C-8’), 36.16 (C-10’), 36.73 (C-20’), 39.97 (C-12’), 42.58 (C-13’), 44.63 (C-24’), 45.83 (C-5), 54.22 (C-9’), 56.16 (C-17’), 56.41 (C-14’), 59.45 (C-11), 74.38 (C-3’), 122.03 (C-14), 129.43 (C-8), 134.14 , 134.71 , 135.98 , 142.66 , 148.62 (C-5), 155.01 (C-17), 171.47 (C-4), 172.12 (C-1) ppm; ATR IR: ν = 3495, 2929, 2855, 2119, 1757, 1717, 1447, 1425, 1362, 1332, 1256, 1174, 1146, 1128, 1100 cm−1; UV/Vis (acetonitrile): λmax (ε) = 202 (11.69 cm2·mg−1); MS (ESI-TOF) m/z (%): 431 (100), 445 (48), 667 (8), 1038 (10) [M+Na]+; HRMS-FAB m/z [M+Na]+ calcd for C64H102O5S2: 1037.7067, found: 1037.6096; Anal. calcd for C64H102O5S2: C 75.69, H 10.12, S 6.31, found: C 75.68, H 10.13, S 6.32. β-Sitosterol conjugate4: From β-sitosterol ; the product 4 was obtained as a white crystalline solid . Analyses: Rf = 0.31 (light petroleum ether/diethyl ether 2:1); mp: 165–167 °C; [α]D20 = −17.3 (c = 1.6 μM in CHCl3); 1H-NMR: δ = 0.68 , 0.88 , 0.87 , 0.92 , 1.02 , 1.34 , 1.35 , 1.45 , 1.47 , 2.33 , 2.70 , 3.00 , 4.65 , 5.36 , 5.37 , 7.45 , 7.63 ppm ; 13C-NMR: δ = 11.85 (C-18’), 18.72 (C-21’), 19.30 (C-19’), 21.03 (C-11’), 22.54 (C-27’), 22.79 (C-26’), 23.82 (C-23’), 24.27 (C-15’), 27.71 (C-2’), 27.99 (C-25’), 28.21 (C-16’), 29.11 (C-3), 30.29 , 30.59 , 30.73 (C-2), 31.48 , 31.87 (C-7’), 31.90 (C-8’), 34.31 , 35.47 , 35.77 (C-4’), 36.19 (C-22’), 36.59 (C-10’), 36.97 (C-20’), 38.06 (C-1’), 39.52 (C-24’), 39.74 (C-12’), 42.32 (C-13’), 50.06 (C-9’), 56.16 (C-17’), 56.70 (C-14’), 59.47 (C-11), 74.55 (C-3’), 122.12 (C-14), 122.69 (C-6’), 129.51 (C-8), 134.10 , 134.65 , 136.02 , 139.58 (C-5’), 142.67 , 148.62 (C-5), 154.99 (C-17), 171.29 (C-4), 172.07 (C-1) ppm; ATR IR: ν = 3592, 2950, 2866, 2119, 1756, 1723, 1466, 1425, 1362, 1325, 1254, 1174, 1146, 1129, 1100 cm−1; UV/Vis (acetonitrile): λmax (ε) = 206 (12.96 cm2·mg−1); MS (ESI-TOF) m/z (%): 431 (100), 445 (32), 1008 (37) [M+Na]+; HRMS-FAB m/z [M+Na]+ and [M+K]+ calcd for C62H96O5S2: 1008.6597 and 1023.6336, found: 1007.5692 and 1023.5323; Anal. calcd for C62H96O5S2: C 75.56, H 9.82, S 6.51, found: C 75.54, H 9.80, S 6.52.Stigmasterol conjugate5: From stigmasterol ; the product 5 was obtained as a white crystalline solid . Analyses: Rf = 0.65 (light petroleum ether/diethyl ether 6:1); mp: 129–132 °C; [α]D20 = −18.0 (c = 1.7 μM in CHCl3); 1H-NMR: δ = 0.70 , 0.80 , 0.81 , 0.85 , 1.02 , 1.03 , 1.34 , 1.34 , 1.44 , 1.47 , 2.33 , 2.70 , 3.00 , 4.65 , 5.02 , 5.16 , 5.36 , 5.37 , 7.45 , 7.62 ppm ; 13C-NMR: δ = 12.05 (C-18’), 12.22 (C-29’), 18.99 (C-26’), 19.31 (C-19’), 21.03 (C-11’), 21.06 (C-27’), 21.21 (C-21’), 24.36 (C-15’), 25.39 (C-28’), 27.72 (C-2’), 28.88 (C-16’), 29.13 (C-3), 30.30 , 30.60 , 30.75 (C-2), 31.49 , 31.88 (C-7’), 31.88 (C-8’), 31.89 (C-25’), 34.32 , 35.48 , 36.62 (C-10’), 36.98 (C-1’), 38.08 (C-4’), 39.65 (C-12’), 40.45 (C-20’), 42.23 (C-13’), 50.09 (C-9’), 51.24 (C-24’), 55.99 (C-17’), 56.81 (C-14’), 59.48 (C-11), 74.56 (C-3’), 122.13 (C-14), 122.70 (C-6’), 129.33 (C-23’), 129.52 (C-8), 134.12 , 134.67 , 136.03 , 138.28 (C-22’), 139.60 (C-5’), 142.69 , 148.64 (C-5), 155.01 (C-17), 171.32 (C-4), 172.08 (C-1) ppm; ATR IR: ν = 3627, 2955, 2868, 2119, 1761, 1737, 1420, 1361, 1175, 1149, 1100 cm−1; UV/Vis (acetonitrile): λmax (ε) = 202 (8.44 cm2·mg−1); MS (ESI-TOF) m/z (%): 431 (100), 1034 (10) [M+Na]+; HRMS-FAB m/z [M+Na]+ and [M+K]+ calcd for C64H98O5S2: 1033.6753 and 1049.6493, found: 1033.7561 and 1049.7605; Anal. calcd for C64H98O5S2: C 75.99, H 9.76, S 6.34, found: C 75.96, H 9.73, S 6.36.Cholesterol conjugate6: From cholesterol ; the product 6 was obtained as a white crystalline solid . Analyses: Rf = 0.66 (light petroleum ether/diethyl ether 6:1); mp: 166–168 °C; [α]D20 = −12.8 (c = 1.5 μM in CHCl3); 1H-NMR: δ = 0.68 , 0.94–0.80 , 1.02 , 1.34 , 1.44 , 1.47 , 2.33 , 2.70 , 3.00 , 4.65 , 5.36 , 5.37 , 7.45 , 7.62 ppm ; 13C-NMR: δ = 11.83 (C-18’), 11.96 (C-29’), 18.75 (C-21’), 19.01 (C-27’), 19.29 (C-19’), 19.80 (C-26’), 20.99 (C-11’), 23.04 (C-28’), 24.28 (C-15’), 26.04 (C-23’), 27.67 (C-2’), 28.22 (C-16’), 29.11 (C-3), 29.69 (C-25’), 30.26 , 30.54 , 30.72 (C-2), 31.45 , 31.82 (C-7’), 31.89 (C-8’), 33.91 (C-22’), 34.30 , 35.45 , 36.13 (C-4’), 36.56 (C-20’), 36.96 (C-10’), 38.04 (C-1’), 39.69 (C-12’), 42.29 (C-13’), 45.81 (C-24’), 49.99 (C-9’), 56.00 (C-17’), 56.66 (C-14’), 59.43 (C-11), 74.53 (C-3’), 121.99 (C-6’), 122.70 (C-14), 129.40 (C-8), 134.12 , 134.69 , 135.95 , 139.55 (C-5’), 142.64 , 148.60 (C-5), 154.99 (C-17), 171.33 (C-4), 172.11 (C-1) ppm; ATR IR: ν = 3500, 2955, 2870, 1757, 1720, 1467, 1326, 1255, 1173, 1146, 1128, 1100 cm−1; UV/Vis (acetonitrile): λmax (ε) = 203 (12.32 cm2·mg−1); MS (ESI-TOF) m/z (%): 431 (100), 445 (32), 1036 (20) [M+Na]+; HRMS-FAB m/z [M+Na]+ calcd for C64H100O5S2: 1035.6910, found: 1035.7633; Anal. calcd for C64H100O5S2: C 75.84, H 9.94, S 6.33, found: C 75.84, H 9.98, S 6.32. All conjugates were recrystallized from an acetonitrile/diethyl ether mixture and their single crystals were studied in detail by X-ray crystallography [3 bottle with 10 mL of cultivation medium . The cells were kept in an incubator saturated by water vapor at 37 °C and under an atmosphere of 5% CO2; the medium was changed every 48–72 h. When a mono-layer was reached, the cells were washed with sterilized PBS (5 mL), released by incubation in a 0.25% solution of trypsin with EDTA . 5 mL of the cultivation medium was added and centrifuged . The pellet was re-suspended in the cultivation medium (20 mL) and the cells were transferred into a 75 cm3 cultivating bottle and further cultivated. When the mono-layer was reached, the cells were washed by sterilized PBS (10 mL) and released by incubation in the 0.25% solution of trypsin with EDTA , and were re-suspended in the cultivation medium (10 mL). The suspension was centrifuged . The pellet was re-suspended in the cultivation medium (10 mL) and the cells were finally used for experiments. The cells were used in 5 to 10 passages.Mouse fibroblasts the Balb/c 3T3 cell line (No. 86110401) were purchased from ECACC . Cryo-preserved cells were taken out of a deep-freezing box and left to warm up for 1 min at the ambient temperature. Subsequently, they were transferred to a 25 cm1, 2, and conjugates 3–6 were prepared in ethanol in a concentration range of 7.81–2,000 µmol L−1.Stock solutions were prepared in ethanol to a concentration of 2% (v/v) of ethanol in the medium. The solutions of the tested compounds were stirred in vortex (2 min) and further treated in an ultrasound bath (5 min) to fully dissolve. Control cells were incubated in the medium with a corresponding volume of ethanol only. The solutions of 4 cell mL−1 in a 0.2 mL well−1. After a 24 h incubation of the culture , the cultivation medium was exchanged for a medium without serum containing the tested compounds and this was further incubated for 24 h under the same conditions. After this period, the cell damage was observed by detection of mitochondrial dehydrogenase activity (MTT) test [Cell concentrations were determined by the trypan blue coloring method. The cells were dissolved in the cultivation medium and seeded in a 96-well plate in a concentration of 3.10TT) test , whereby50 values were calculated from the measured values of the absorbance. Tests were carried out in triplicate. The solutions of 1, 2 and conjugates 3–6 were prepared in a concentration range of 0.16–40 µmol L−1 of medium. The cells were washed by aseptic PBS after incubation with the tested compounds and, subsequently, 100 µL of fresh medium without serum and 10 µL of MTT solution  were applied. After 3 h of incubation , the medium with MTT was evacuated and 200 µL DMSO with 1% ammonia was added into wells and the plate was shaken. The absorbance was measured at 540 nm.The IC−1) were prepared in methanol  and checked for possible degradation by TLC. Ascorbic acid was used as a positive control of antioxidant capacity. The reaction mixture contained 2.25 mL of a DPPH solution  and 0.75 mL of the tested compound solution or methanol alone in the control sample. The samples were kept in a closed dark box to protect them from the light and were taken out for the measurements only. The decrease in the absorbance at 522 nm  was measured with a spectrophotometer until a constant difference between the absorbance of the sample and the control sample (24 hours). The assay was carried out in triplicate.The scavenging activity was evaluated using the DPPH radical, a stable violet radical with a maximum absorbance at 522 nm that can be reduced to a yellow hydrazine derivative. The decrease in the absorbance corresponds to the scavenging activity of the test compound. Stock solutions of the tested compounds , plant sterols (β-sitosterol and stigmasterol) and an animal sterol (cholesterol), were prepared in good yields by Steglich esterification and fully characterized at their molecular and submolecular level. 3–6.The conjugates, together with parental probucol and succinobucol, were tested for their toxicity on mouse fibroblasts Balb/c 3T3 by the MTT test, where the compounds showed low toxicity at their maximal concentrations. Esterification of succinobucol’s free carboxylic group lowers its toxicity, as observed in the comparison with the conjugates The tested compounds showed similar or even higher antioxidant activity compared to a standard ascorbic acid in the scavenging of the DPPH radical. This was ascribed to the participation of sulfur atoms in the radical scavenging rather than to an activity of phenolic moieties.The prepared conjugates, as esters which can resist gastric acidic hydrolysis, are expected to be cleaved by pancreatic non-selective lipases into succinobucol and stanol/sterol in the upper part of the small intestine. The released succinobucol may be incorporated into mixed bile acid salt micelles, transported into the enterohepatic circulation, and further distributed within the body incorporated in lipoproteins. The released free plant stanols or sterols then either block the absorption of cholesterol from intestine or reduce its serum level after absorption by influencing its distribution and metabolism. The absorptive pathway of succinobucol may be multiplied in the case of its cholesterol conjugate.The development of succinobucol-steroid conjugates, which, on one side, target anti-inflammatory and antioxidant processes, and on the other side, decrease the LDL-cholesterol level, increase the absorption and targeting of the drug into the enterohepatic circulation. This may add to the armamentarium of agents used in the treatment of atherosclerotic diseases and type 2 diabetes. Nevertheless, further biological studies are needed to discover their full medicinal potential.


Tinospora species. A previous study pointed to the necessary presence of quaternary nitrogens for strong acetylcholinesterase (AChE) inhibitory activity in such alkaloids. Repeated column chromatography of the vine of Tinospora crispa extract led to the isolation of one new protoberberine alkaloid, 4,13-dihydroxy-2,8,9-trimethoxydibenzoquinolizinium (1), along with six known alkaloids—dihydrodiscretamine (2), columbamine (3), magnoflorine (4), N-formylannonaine (5), N-formylnornuciferine (6), and N-trans-feruloyltyramine (7). The seven compounds were isolated and structurally elucidated by spectroscopic analysis. Two known alkaloids, namely, dihydrodiscretamine and columbamine are reported for the first time for this plant. The compounds were tested for AChE inhibitory activity using Ellman’s method. In the AChE inhibition assay, only columbamine (3) showed strong activity with IC50 48.1 µM. The structure–activity relationships derived from these results suggest that the quaternary nitrogen in the skeleton has some effect, but that a high degree of methoxylation is more important for acetylcholinesterase inhibition.Quaternary alkaloids are the major alkaloids isolated from  Tinospora species + based on the HREIMS. In the mass spectrum of 1, the m/z peak 310.0366 has virtual maximum intensity (100%), which indicates a high stability of the ion [C17H17NO5]+ with respect to electron impact. Similar fragmentation processes involving direct elimination of CH2 from the molecular ion with m/z 352.0075 to m/z 310.0366 characterized the presence of three methoxy groups in the sample. The ion at m/z 337.0475 was determined as the neutral loss of the radical group radical CH3∙ from C-9. The ion at m/z 308.0654 can be produced by loss of the carbonyl group formed in C-9 of the D ring via molecular rearrangement. The fragmentation patterns of these compounds are similar to those of protoberberine compounds quinolizinium compounds  (DTNB), bovine serum albumin fraction V (0.1%), physostigmine were obtained from Sigma-Aldrich . Tris-hydrochloric acid buffer (50 mM) at pH 8.0 (Sigma-Aldrich) was used to prepare enzyme solutions . Industrial grade solvents were used for extraction processes, and analytical grade solvents (Merck) were used for chromatography and recrystallization.Ultraviolet (UV) spectra were recorded on a Genesys 10S UV-VIS  visible spectrophotometer in methanol solution. Infrared (IR) spectra were recorded on a Perkin Elmer 100 FT-IR  spectrophotometer as KBr pellets or thin films. NMR spectra were recorded on a Bruker Avance 500 Spectrometer  Tinospora crispa vines were collected in Bentong, Pahang, identified by the botanist, Berhaman Ahmad , and a voucher specimen FRI54832 deposited at the Forest Research Institute Malaysia (KEP).Tinospora crispa vines (5.0 kg) were first de-fatted by percolation with hexane andfiltration (fraction A). The plant material was dried, then extracted with MeOH-H2O (4:1) by sonication for three cycles of 30 min each and then filtered to give a brown extract (fraction B) which was concentrated under low pressure. The concentrated extract was acidified with H2SO4 to pH 2 and then partitioned with CHCl3. The CHCl3 phase was collected and concentrated, leaving a brownish gummy residue (fraction C) of non-alkaloidal components. The aqueous phase was basified with NH4OH to pH 10 and extracted with MeOH-CHCl3 (3:1) to yield a dark brown gummy residue (fraction D) most likely containing alkaloids. Fraction D (25.0 g) was chromatographed over a column of silica gel slurry packed in CHCl3. Elution was initiated with CHCl3 and progressed through the solvent series of 10%, 15%, 30%, 50%, and 70% MeOH in CHCl3, 100% MeOH, and 10% to 15% of NH4OH in MeOH to afford nine subfractions (F1–F9). Subfraction 5 was subjected to silica gel column chromatography eluting with MeOH-CHCl3 (6:4) to yield compounds 1 (4.5 mg), 2 (5.5 mg) and 3 (10 mg). Subfraction 8 was eluted with MeOH-CHCl3 gradually. After reaching 50%, a few drops of NH4OH were added to yield compound 4 (8 mg). Subfraction 2 was eluted with EtOAc-Hex (7:3) to yield compounds 5 (3.5 mg) and 6 (45 mg). Subfraction 3 was eluted with CHCl3-MeOH (3:1) to yield compound 7 (40 mg).Powdered, dried 1: Rf 0.40 (CHCl3-MeOH-Hex 7:3:1). IR (KBr): 3449, 1584, 1637, 1091 cm−1. UV/Vis λmax (MeOH) nm (log ε): 213, 291. 1H-NMR : 3.81 , 4.07 , 4.21 , 6.52 , 6.56 , 6.86 , 7.28 , 7.35 , 7.87 , 7.96 . 13C-NMR : 44.2 (8-OCH3), 54.9 (9-OCH3), 55.0 (2-OCH3), 103.8 (C-3), 107.6 (C-1), 111.8 (C-12), 114.7 (C-10), 118.5 (C-11), 21.3 (C-5), 121.5 (C-8a), 127.1 (C-12a), 127.2 (C-4a), 130.31 (C-6), 132.0 (C-14a), 136.9 (C-14), 147.2 (C-9), 147.5 (C-13), 150.4 (C-8), 162.3 (C-2), 163.8 (C-4). HRESIMS: m/z (%) 352.0075 [M+] (2), 339.0653 (25), 311.0237 (77), 310.0366 (100), 296.0022 (13), 282.0120 (16), 151.0560 (14). Yield .Compound 2: Rf 0.42 (CHCl3-MeOH 6:4). IR (KBr): 3371, 1584, 1361, 1090 cm−1. UV/Vis λmax (MeOH) nm (log ε): 293, 372. 1H-NMR : 3.23 , 3.95 , 4.07 , 4.85 , 7.05 , 7.49 , 7.65 , 7.77 , 8.44 , 9.39 . 13C-NMR  26.4 (C-5), 55.2 (2-OCH3), 55.9 (C-6), 60.0 (9-OCH3), 110.5 (C-4), 111.2 (C-1), 119.6 (C-13), 119.9 (C-14a), 123.0 (C-8a), 123.2 (C-12), 126.4 (C-4a), 132.2 (C-12a), 133.9 (C-11), 135.4 (C-14), 141.6 (C-8), 142.1 (C-9), 146.6 (C-3), 150.5 (C-2), 151.02 (C-10). ESIMS: m/z (%) 324 [M+] (10), 311 (8), 226 (16), 175(13), 148 (26), 136 (46), 114 (100). Yield .Compound 3: Rf 0.30 (CHCl3-MeOH 7:3). IR (KBr): 3459, 1639, 1092 cm-1. UV/Vis λmax (MeOH) nm (log ε): 290, 348. 1H-NMR : 3.29 , 3.99 , 4.13 , 4.23 , 4.85 , 7.05 , 7.59 , 8.03 , 8.14 , 8.67 , 9.77 . 13C-NMR : 26.4 (C5), 55.3 (3-OCH3), 56.3 (C-6), 56.4 (10-OCH3), 61.2 (9-OCH3), 110.6 (C-4), 111.7 (C-1), 119.2 (C-14a), 119.7 (C-13), 121.8 (C-8a), 123.1 (C-12), 126.6 (C-11), 127.4 (C-4a), 133.8 (C-12a), 138.5 (C-14), 144.2 (C-10), 144.8 (C-8), 146.6 (C-2), 150.5 (C-3), 151.1 (C-3). ESIMS: m/z (%) 338 [M+] (10), 279 (75), 165(80), 149 (100), 57(95). Yield .Compound 4: Mp: 250–251 °C (lit  (2), 312 (14), 298 (58), 284 (84), 270 (100), 256 (79), 240 (37). Yield .Compound  °C (lit  248 °C).5: Mp: 216–217 °C (lit.  (52), 251(43), 236(27), 235 (100). Yield .Compound °C (lit.  218–219 6: Mp: 220–221 °C (lit.  (90), 250(100), 237(11). Yield .Compound °C (lit.  222–224 7: Mp 89–90 °C (lit.  (21), 193 (26), 192(41), 177(100), 145 (12), 120 (15). Yield .Compound °C (lit.  91 °C). 2∙6H2O). In the 96-well plates, 25 μL of 15 mM acetylthiocholine iodide (ATChI) in water, 125 μL of 3 mM DTNB in buffer C, 50 μL of buffer B, and 25 μL of isolated compounds dissolved in MeOH at concentrations ranging from 62.5 to 1000 μg/mL were added. Then 25 μL of 0.22 UmL−1 of AChE was added and the absorbance measured at 405 nm was read every 10 min twice. To avoid false positive results, a parallel test omitting the DTNB was done. and none of the test substances were found to interact with thiocholine formed during the assay. Physostigmine served as the positive control. All assays were performed in duplicate in a 96-well microplate reader (Tecan Infinite 200 Pro). The percentage inhibition was calculated using the formula (1):50 values were calculated by plotting percentage inhibition against the extract concentration (The three compounds were tested for AChE inhibiting activity using a modified Ellman’s colorimetric method . Three bntration .T. crispa, as well as the genus Tinospora, due to their strong presence within this genus. The results of the present investigation further point to the potential of this genus as a source of new AChE inhibitors.The study on AChE inhibitory activity of isolated compounds showed that it was influenced by the alkaloid skeleton but especially by the ring substituents. Three different types of alkaloids were isolated, namely, aporphine, oxoaporphine and quaternary protoberberines. The AChE inhibitory results showed that the most active compound was the least polar one, columbamine. One of the outcomes of this investigation is that it suggests that the alkaloids are suitable chemical markers for


Cimicifuga simplex Wormsk roots. Their structures were established by chemical tests and detailed spectroscopic analysis as 25-O-acetyl-7,8-didehydrocimigenol-3-O-β-D-galactopyranoside (1), 7,8-didehydrocimigenol-3-O-β-D-galactopyranoside (2) and 7,8-didehydro-24S-O-acetylhydroshengmanol-3-O-β-D-galactopyranoside (3), respectively.Three new cycloartenol triterpene saponins, named shengmaxinsides A-C, have been isolated from the ethyl acetate soluble fraction of an ethanol extract of  C.simplex (Shengma in Chinese) is a deciduous perennial herb widely distributed in China. Traditionally, the root of C.simplex has been used in oriental countries as an anti-inflammatory and anti-viral agent + and [M+Na]+ signals at m/z 631.3884  and 671.3802 , respectively, indicating nine degrees of unsaturation.Compound 13C-NMR spectrum , 691.4102  and 713.3837 , respectively, indicating nine degrees of unsauration.Compound 1H-NMR spectrum , six tertiary methyl groups at δH 1.02, 1.18, 1.29, 1.37, 1.49, and 1.74, a secondary methyl at δH 0.95 , an acetyl methyl at δH 2.01, one anomeric proton at δH 4.84 , four oxygenated methine protons  and a series of overlapped signals suggesting a cycloartane-type triterpene glycoside. The 13C-NMR spectrum . Three further signals at δH 4.32 , 4.85  and 4.21  in the region of the aglycon moiety suggested the presence of three additional oxygen-bearing carbons. This hypothesis was confirmed by the HMBC spectrum, which showed cross-peaks between the proton signal at δH 4.32  with C-14, C-16, C-13 and C-17, between the proton signal at δH 4.85  with C-23 and C-25, and between the proton signal at δH 4.21  with C-22 and C-24, thus unambiguously identifying the oxygen-bearing carbons as C-15, C-23, and C-24. In the HMBC spectrum, significant correlations between δH 4.84(H-1') and 88.4(C-3) suggested that the galactopyranosyl was located at the C-3 position. Moreover, the long-range correlations between H-24 (δH 4.85) with an acetyl carbon (δC 170.7) indicated that the acetyl unit locating at C-24. Other key long-range correlations were found for H-1/C-3, H-19/C-19, 23-H/C-22 and C-24, H-24/C-25  analysis revealed the presence of NMR data , and the-24/C-25 .R when C-16 chemical shift in the 13C-NMR spectrum should be 102.9~103.7, while for S it appears to be 106.1~106.8 25D: +0.02 (MeOH); IR (KBr): 3431.13, 3423.41, 2956.67, 2937.38, 2871.81, 1730.03, 1367.44, 1240.14, 1151.42, 1070.42, 1058.85, 1043.42, 975.91 cm−1; HRESIMS from the [M-H2O+H]+ and [M+Na]+ signals at m/z 673.3964  and 713.3894 ; 1H-NMR and 13C-NMR data are shown in Shengmaxinside B (2). Colorless needles; [α]25D: +0.03 (MeOH); IR (KBr): 3431.13, 3421.48, 2960.53, 2931.6, 2871.81, 2358.78, 2341.42, 2331.78, 1155.28, 1056.92, 987.49, 977.84 cm−1; HRESIMS from the [M-H2O+H]+ and [M+Na]+ signals at m/z 631.3887  and 671.3804 ; 1H-NMR and 13C-NMR data are shown in Shengmaxinside C (3). White amorphous powder; [α]25D: +0.02 (MeOH); IR (KBr): 3411.84, 2952.81, 2935.46, 1718.46, 1379.01, 1244.00, 1163.00, 1151.42, 1062.7, 1031.85, 981.7 cm-1; HRESIMS from the [M-2H2O+H]+, [M-H2O+H]+ and [M-H2O+Na]+ signals at m/z 673.3975 , 691.4102  and 713.3837 ; 1H-NMR and 13C-NMR data are shown in 4 (40 mg), followed by acidification with dilute CH3COOH, and then co-distilled with pure CH3OH to remove excess boric acid. The reduced sugars were acetylated with 1:1 pyridine-Ac2O in a boiling water bath for 2 h to give the corresponding alditol acetates, which were analyzed by GLC on a HP 6890 N gas chromatograph (Agilent) equipped with a flame ionization detector FID) using N2 as carrier gas. The instrument was fitted with a HP-5 capillary column (30 m×0.32mm×0.25 μm). The injector temperature was set at 250 °C and the column temperature program was as follows: the initial temperature of 120 °C was increased by 3°/min to the final temperature of 210 °C, then was held 4 min. The detector temperature was set at 300 °C. The standard monosaccharides were subjected to the same reaction and GC analysis under the same conditions Acid hydrolysis was performed by a previously described method . For thiC. simplex, three new cycloartenol triterpene saponins with galactopyranosyl moieties, shengmaxinsides A-C, were isolated. Their structures were established on the basis of spectroscopic analysis and chemical evidence. Their biological activities will be further researched in our laboratory. It has been reported that 9,19-cyclolanostane triterpene glycosides exhibit varied biological activities, including antiosteoporosis, antitumor, anti-complement, antioxidant and anti-inflammatory effects ,24,25. A


The structures of novel compounds were confirmed by 1H-, 19F- and 13C-NMR and MS. Compounds 8 and 10 containing unsaturated fluorophenylalkyl side chains showed better inhibitory effect than their saturated fluorophenylalkylated pyrimidine counterparts 7 and 9. A conformational study based on NOE enhancements showed the importance of the double bond and substitution in the side chain for the conformational preferences in relation to inhibitory activity. Among all tested compounds, C-5 furyl (12) and phenyl (13 and 15) substituted pyrimidine derivatives showed significant cytostatic activities against all tested tumor cell lines.The synthetic route for introduction of fluorophenylalkyl (compounds  The application of fluorine-containing compounds in the pharmaceutical and agrochemical fields has a very short history ,2. The s6, 9, 14 and 15), fluorophenylalkenyl  and 5-furyl-6-methyl (12) pyrimidine derivatives.Recently, we have reported that fluorinated propyl or propenyl C-6 acyclic pyrimidine derivatives containing the 2-hydroxy-3,3,3-trifluoro-1-propenyl side chain exhibited a pronounced effect against breast carcinoma (MCF-7), while the compound with a 2-fluoromethyl-2-acetoxypropyl chain exhibited moderate effect against cervical carcinoma (HeLa) . We have1), 2,4-dimethoxy-5,6-dimethylpyrimidine (2) and 5-bromo-2,4-dimethoxy-6-methylpyrimidine (3) were prepared according to the procedure described in the literature -2,4-dimethoxy-pyrimidine (5), 6-[2-(4’-fluorophenyl)-2-hydroxypropyl]-2,4-dimethoxy-5-methyl-pyrimidine (7), 2,4-dimethoxy-6-methyl-5-phenylpyrimidine (11), 5-furyl-2,4-dimethoxy-6-methylpyrimidine (12) and 6-[2-(4’-fluorophenyl)-2-hydroxypropyl]-5-phenyl-2,4-dimethoxypyrimidine (15) were synthesized in accord with original procedures given in the literature +; Anal. calcd. for C14H13F2N2O2: C, 64.61; H, 5.03; N, 10.76. Found: C, 64.48; H, 5.04; N, 10.78.9: 13C-NMR (DMSO-d6) δ: 165.34 (C4), 161.42 , 150.84 (C2), 147.54 (C6), 144.63 , 127.42 , 114.84 , 107.22 (C5), 74.14 (C2'), 43.97 (C1'), 29.46 (C2’-CH3), 10.64 (C5-CH3); MS m/z 277 [M-H]; Anal. calcd. for C14H15FN2O3: C, 60.42; H, 5.43; N, 10.07. Found: C, 60.30; H, 5.45; N, 10.09.7  was dissolved in acetyl chloride (7 mL) and the reaction mixture was refluxed for 5 h. Water (2 mL) was then added and the reaction mixture was stirred overnight at room temperature. The solvent was evaporated at the reduced pressure and the remaining yellow oil was chromatographed using dichloromethane as eluent, which yielded compounds 9 and 10. 9: Yield = 60 mg (44.3%); 10: Yield = 20 mg (9.8%); mp = 66–68 °C.Compound 10: 13C-NMR (DMSO-d6) δ: 169.52 (C4), 162.01 , 162.0 (C6), 162.0 (C2), 143.03 (C2'), 139.22 , 128.20 , 122.11 , 115.24 , 108.42 (C5), 54.10 (C2-OCH3), 53.92 (C4-OCH3), 17.85 (C2'-CH3), 9.89 (C5-CH3); MS m/z 289 [MH]+; Anal. calcd. for C16H17FN2O2: C, 66.65; H, 5.94; N, 9.72. Found: C, 66.92; H, 5.93; N, 9.74.13 was performed in the same manner as described for 4. Reagents used were: compound 11 , LDA  and ethyl 4-fluorobenzoate  in anhydrous THF (6 mL). After column chromatography using cyclohexane-ethyl acetate = 6:1 as eluent compound 13 was isolated as crude oil. Yield = 44.3 mg (16.6%). 13C-NMR (DMSO-d6) δ: 167.99 (C4), 163.55 , 163.03 (C2'), 162.35 (C6), 160.52 (C2), 134.00 , 132.88 (Cϕ1'), 131.08 , 130.79 (Cϕ2'), 129.12 (Cϕ3'), 128.00 (Cϕ4'), 127.87 , 122.33 (C1'), 114.08 (C5), 54.79 (C2-OCH3), 54.55 (C4-OCH3); MS m/z 353 [MH]+; Anal. calcd. for C20H17FN2O3: C, 68.17; H, 4.86; N, 7.95. Found: C, 68.37; H, 4.84; N, 7.97.The synthesis of 4, the synthesis of 14 was performed using compound 11 , LDA  and 2-fluorophenylacetone  in anhydrous THF (6 mL) as reagents. After column chromatography using petroleum ether-ethyl acetate = 6:1 as eluent compound 14 was isolated. Yield = 40.3 mg (25.7%). 13C-NMR (DMSO-d6) δ: 169.19 (C4), 161.95 , 166.56 (C6), 163.02 (C2), 133.91 , 133.54 (Cϕ1’), 131.25 (Cϕ2’), 130.90 , 129.58 (Cϕ3’), 125.16 (Cϕ4’), 116.24 , 123.77 , 115.00 , 116.25 (C5), 72.91 (C2'), 44.28 (C1'), 54.89 (C2-OCH3), 54.51 (C4-OCH3). 40.85 , 27.14 (C2'-CH3); MS m/z 383 [MH]+; Anal. calcd. for C22H23FN2O3: C, 69.09; H, 6.06; N, 7.33. Found: C, 69.29; H, 6.04; N, 7.31.According to the procedure described for L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 °C.The colon carcinoma (HCT 116 and SW 620), breast carcinoma (MCF-7) and lung carcinoma (H 460) cells were cultured as monolayers and maintained in Dulbecco's modified Eagle's medium (DMEM), while MOLT-4 cells (acute lymphoblastic leukemia) were cultured in suspension in RPMI medium, both supplemented with 10% fetal bovine serum (FBS), 2 mM -8 to 10-4 mol/L) and incubated for further 72 hours. Working dilutions were freshly prepared on the day of testing. The solvent (DMSO) was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in working concentrations . After 72 hours of incubation, the cell growth rate was evaluated by performing the MTT assay [The growth inhibition activity was assessed as described previously, according to the slightly modified procedure of the National Cancer Institute, Developmental Therapeutics Program ,10. Brie50, which is the concentration necessary for 50% of inhibition. The IC50 values for each compound are calculated from dose-response curves using linear regression analysis by fitting the test concentrations that give PG (percentage of growth) values above and below the reference value (i.e. 50%). Each result is a mean value from three separate experiments.Each test point was performed in quadruplicate in three individual experiments. The results are expressed as IC5 cells were seeded per well in a 6-well plate. After overnight incubation, tested compounds were added. After desired length of time, the attached cells were trypsinized, combined with floating cells, washed with phosphate buffered saline (PBS) and fixed with 70% ethanol. Immediately before the analysis, cells were washed with PBS and incubated with 0.1 μg/μL RNAse A at 37 °C for 15 minutes. Subsequently, cells were stained with 50 µg/ml of propidium iodide (PI) and analyzed by Becton Dickinson FACScalibur flow cytometer. For each analysis, 20,000 events were measured. Measurements were performed in duplicate for two independent experiments. The percentage of the cells in each cell cycle phase was based on the obtained DNA histograms and determined using the ModFit LTTM software (Varity House). Statistical analysis was performed in Microsoft Excel by using the ANOVA at p < 0.05.2 × 106, 9, 14 and 15), fluoropenylakenyl  and 5-furyl-6-methyl (12) pyrimidine derivatives were synthesized and evaluated for their cytostatic activities. Among all tested compounds, C-5 furyl (12) and phenyl (13 and 15) substituted pyrimidine derivatives showed the most significant cytostatic activities against all tumor cell lines (IC50 are in low micromolar range). In the series of C-6 fluorophenylalkyl 5-methylpyrimidines, compounds 8 and 10 containing unsaturated side chains showed better inhibitory effects  than their saturated pyrimidine derivatives 7 and 9. Furthermore, among C-6 fluorophenylalkyl 5-phenyl-pyrimidines, compounds 13 and 15 with a p-fluorophenyl moiety in the side chain exhibited the most pronounced antiproliferative activity . Besides, 12 and 15 induced strong changes in the cell cycle of tumor cells (accumulation of cells in G1 phase and drastic decrease in the number of cells in S phase), which eventually caused death of tumor cells. Thus, these compounds emerged as the most interesting leading compounds that could be used for further structural optimization. C-6 fluorophenylalkyl (