The structures of the new compounds were established on the basis of spectroscopic data, including 1D and 2D NMR and HRESIMS, and comparisons with published data.Twelve new steroidal saponins, including eleven furostanol saponins, terrestrinin J–T ( Tribulus terrestris L. is a perennial plant widely distributed around the world, especially in subtropical areas. Its dried fruit, named “Jili” in Chinese, has been used as a traditional Chinese medicine (TCM) for the treatment of edema, abdominal distention, emission, morbid leucorrhea and vitiligo -β-d-glucopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-galactopyran-oside (13) [R)-26-[(β-d-glucopyranosyl)oxy]-5α-furostane-3β,22α-diol 3-O-α-l-rhamno-pyranosyl-(1→2)-[β-d-glucopyranosyl-(1→4)]-β-d-galactopyranoside (14) [R)-26-[(β-d-glucopyranosyl)oxy-5α-furost-20(22)-en-3β-ol 3-O-β-d-xylopyranosyl-(1→3)-[β-d-xylopyranosyl-(1→2)]-β-d-glucopyranos-yl (1→4)-[α-l-rhamnopyranosyl(1→2)]-β-d-galactopyranoside (15) [S-terrestrosin I (16) [R-terrestrosin I (17) [18) [19) [The fresh whole plant of ranoside  [R)-26-[ranoside  [S-terreranoside  [S-terretrosin I  [R-terretrosin I  [R-terre1 was isolated as a white powder and its molecular formula was determined to be C61H100O31 by the HRESIMS [M − H]− ion peak at m/z 1327.6162 . The 1H-NMR spectrum of 1 showed two methyl singlets at δ 0.76  and 0.87 , two methyl doublets at δ 1.31  and 0.97 , and one olefinic proton at δ 5.09 . Additionally, it also showed six anomeric proton signals at δ 4.83 , 6.20 , 4.98 , 5.23 , 5.42 , and 4.81 , which indicated that 1 contains six sugar moieties. The 13C-NMR spectrum exhibited 61 carbons, including two olefinic carbons at δ 118.8 (C-7) and 139.0 (C-8). The 1H-1H COSY correlations for δ 5.09 (H-7)/1.67 (H-6a) and δ 2.08 (H-6b)/1.22 (H-5), as well as the HMBC correlations (2-26 (ΔδH = 0.32 ppm < 0.48 ppm) demonstrated the 25R configuration of 1 -5α-furost-7-ene-3β,22α-diol 3-O-β-d-xylo-pyranoseyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-galacto-pyranoside, which was named terrestrinin J.The large coupling constants . Comparison of the NMR data of 2 with those of 1 , 121.7 (C-6) and δ 5.28 (H-6) indicated the double bond at C-5 and C-6 in 2. Thus, the aglycone of 2 was identified as (25R)-furost-5-ene-3β,22α,26-triol. The whole structure of 2 was finally confirmed by the 1D and 2D NMR experiments which indicated the structure of 2 to be (25R)-26-[(β-d-glucopyranosyl)oxy]-furostan-5-ene-3β,22α-diol 3-O-β-d-xylo-pyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-galactopyranoside, named terrestrinin K.Compound ose of 1  indicate3 was determined to be C61H100O31 by HRESIMS . The NMR data of compound 3 were very similar to those of a known compound, -26-[(β-d-glucopyranosyl)oxy]-furost-5,22-diene-3β,20α-diol 3-O-β-d-gluco-pyranosyl-(1→4)-[a-l-rhamnopyra-nosyl-(1→2)]-β-d-glucopyranoside, reported in the literature -5α-furost-22-ene-3β,20α-diol 3-O-β-d-xylopyranosyl-(1→2)-[β-d-xylopyranos-yl-(1→3)]-β-d-glucopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-galactopyranoside, named terrestrinin L.The molecular formula of terature , which i4 was isolated as a white amorphous powder with the molecular formula C56H92O28 . Comparison of the MS and NMR data of 4 with those of 3 suggested that the structure of 4 is similar to that of 3, with one fewer sugar unit in the sugar chain linked to C-3 of the aglycone. The sugar sequence of glucose, galactose, xylose and its linkage to C-3 and C-26 of the aglycone were ascertained by correlations between δ 4.91 (H-1′) and δ 77.4 (C-3), δ 5.18 (H-1′′) and δ 79.6 (C-4′), δ 5.47 (H-1′′′) and δ 81.1 (C-2′′), δ 5.07 (H-1′′′′) and δ 85.6 (C-3′′), and δ 4.82 (H-1′′′′′) and δ 75.3 (C-26) in the HMBC spectrum. Thus, the structure of 4 was concluded to be -26-[(β-d-glucopyranosyl)oxy]-5α-furost-22-ene-3β,20α-diol 3-O-β-d-galactopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside, named terrestrinin M.Compound 5 was deduced as C56H92O28 due to the appearance of a [M − H]− ion at m/z 1211.5709 in the HRESIMS, as same as 4. According to the comparison of the 1H and 13C-NMR data of 5 with that of 4, they were deduced to share the same aglycone moiety. Further comparison of the NMR data of 5 with those of polianthoside D [5 was concluded to be -26-[(β-d-glucopyranosyl)oxy]-5α-furost-22-ene-3β,20α-diol 3-O-β-d-glucopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside and named terrestrinin N.The molecular formula of hoside D  suggeste6 showed an [M − H]− ion peak at m/z 1195.5730  in the negative HRESIMS, corresponding to a molecular formula of C56H92O27. The 13C-NMR spectrum showed two olefinic carbon signals at δ 103.7 (C-20) and 152.4 (C-22), which indicated that a double bond existed in 6. In the HMBC spectrum -26-[(β-d-glucopyranosyl)oxy]-5α-furost-20(22)-ene-3β-ol 3-O-β-d-glucopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside, named terrestrinin O.Compound trum See , the corm/z 1193.5640), compound 7 showed the same molecular formula, C56H90O27, as polygodoside G -furosta-5,20(22)-dien-3β-ol 3-O-β-d-glucopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside, and named terrestrinin P.On the basis of HRESIMS , suggesting a molecular formula of C33H48O9. The 1H-NMR spectrum of 8 , four methyl group signals at δ 0.99, 1.08, 1.75, and 1.02 , and one olefinic proton signal at δ 5.85, s, H-4). The 13C-NMR spectrum showed two double bond signals at δ 124.8 (C-4) and 168.5 (C-5), and δ 103.1 (C-20) and 153.3 (C-22), and two characteristic ketone carbons at δ 197.9 (C-3) and 211.8 (C-12). The NMR data of 8 were quite similar to those of terrestrinin A -furosta-4,20(22)-diene-3,12-dione, named terrestrinin Q.The HR ESI-MS of rum of 8  showed a9 was isolated as a white amorphous powder with a molecular formula of C61H100O32, which was determined by the negative ion HRESIMS (m/z 1343.6113 [M − H]−). Comparison of the 1H and 13C-NMR data of 9 -26-[(β-d-glucopyranosyl)oxy]-3β,22α-dihydroxy-5α-furostan-12-one 3-O-β-d-xylopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-galacto-pyranoside, and named terrestrinin R.Compound ata of 9  with tho10 displayed an [M − H]− ion at m/z 1343.6145  by HRESIMS, giving a molecular formula of C61H100O32, which was identified as a isomer of 9  of C-25 in 10 rather than an R configuration in 9 [10 was assigned as (25S)-26-[(β-d-glucopyranosyl)oxy]-3β,22α-dihydroxy-5α-furostan-12-one 3-O-β-d-xylopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-galactopyranoside, named terrestrinin S.Compound mer of 9 . A detaiion in 9 ,25. Thus11 gave a [M − H]− ion at m/z 1343.6128, indicating a molecular formula of C61H100O32. In the 13C-NMR spectrum, two carbonyl group signals at δ 205.5 (C-20) and 173.2 (C-22) were observed in 11. The key COSY and HMBC correlations  for 11. Additionally, the α-configuration of H-16 was determined from the J value of 7.8 Hz between H-16 and H-17. Based on these data, the structure of 11 was characterized as 16β-{(25R)-26-[(β-d-glucopyranosyl)oxy]-25-methylpentanoyloxy}-3β-hydroxy-5α-pregnan-20-one 3-O-β-d-xylopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-gluco-pyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-galactopyranoside, named terrestrinin T.HRESIMS of ions See  observedesides A  and 1, iguration  . The 1H-NMR spectrum displayed four steroidal methyl groups at δ 0.72 , 0.83 , 1.05 , and 1.13  , and six anomeric proton signals at δ 4.84 , 6.18 , δ 4.97 , 5.23 , 5.42 , and 4.92 , as well as six anomeric carbon signals at δ 100.2, 102.0, 105.4, 105.1, 105.8 and 106.4. The sugar units and their configurations was identified by GC analysis after acidic hydrolysis, as well as the large coupling constants. Furthermore, the NMR data of 12 were similar to those of a known spirostanol saponin, 25R-tribulosin -5α-spirost-3β-ol 3-O-β-d-xylopyranosyl-(1→2)-[β-d-xylopyranosyl-(1→3)]-β-d-glucopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-galactopyranoside, named terrestrinin U.Compound on of 12 . On the 1H at 600 MHz and 13C at 150 MHz) were taken on a UNITY INOVA 600 spectrometer  in pyridine-d5 solution, and the chemical shifts are given in ppm on the δ scale with tetramethylsilane (TMS) as an internal standard. HPLC separations were performed using a Waters 2695 series instrument equipped with an analytical Venusil XBP C18 column , a YMC  preparative column, and ELSD 2000 evaporative light-scattering detector . ODS gel , Sephadex LH-20 , and MCI gel  were used for column chromatography. TLC was carried out on silica gel GF254  plates, and spots were visualized under UV light (254 or 365 nm) or by spraying with 10% H2SO4 in 95% EtOH followed by heating.Optical rotations were obtained on a 341 digital polarimeter . IR and UV spectra were recorded on FTIR-8400S  and UV2550 spectrometer (Shimadzu Corp.), respectively. HRESIMS were recorded on Synapt Q/TOF MS . NMR spectra . A voucher specimen (No. 20130726) has been deposited at the Herbarium of the Beijing Institute of Radiation Medicine, Beijing, China.The fresh whole plant of T. terrestris was cut into small pieces and refluxed with 75% (v/v) EtOH . The combined extract was evaporated under reduced pressure and then separated by column chromatography (CC) on macroporous resin SP825 eluting with 5%, 60% and 90% EtOH in succession. The 60% EtOH eluate fraction was separated on a silica-gel column using gradient solvents of CHCl3–MeOH–H2O  to give five fractions (Fr. A-E). Fr. B were subjected to ODS CC eluted with MeCN–H2O  and semi-preparative HPLC with MeCN–H2O  to yield 16 (4.7 mg) and 17 (21.1 mg). Fr. C was subjected to column chromatography (CC) over MCI silica gel eluted with EtOH–H2O  to give five subfractions (Fr. C1 to Fr. C3). Fr. C2 was subjected to ODS CC (20% MeCN in H2O) to give three subfractions Fr. C2-1 to Fr. C2-3. Fr. C2-2 was purified by ODS semi-preparative HPLC  to afford 18 (54.5 mg). Fr. C2-3 was further purified by semi-preparative HPLC eluted with MeCN–H2O  to yield 4 (16.0 mg) and 5 (15.3 mg). Fr. C3 was separated on an MCI-gel column with Me2CO–H2O  and was purified by semi-preparative HPLC with Me2CO-H2O (29:71) to yield 19 (110.8 mg). Fr. D was fractioned by ODS chromatography (23%–30% MeCN in H2O) to give three subfractions (Fr. D1 to Fr. D3). Fr. D1 was further purified successively by ODS semi-preparative HPLC  to yield 10 (10.4 mg) and 9 (70.5 mg). Fr. D2 was purified by semi-preparative HPLC with MeCN–H2O  to yield 3 (4.8 mg). Fr. D3 was separated by an ODS column eluted with MeOH–H2O , and then was purified by semi-preparative HPLC with MeCN–H2O  to yield 11 (5.8 mg), 1 (4.4 mg), and 2 (5.0 mg). Fr. E was subjected to ODS silica-gel CC with a gradient mixture of MeCN-H2O,  as elute, and three fractions were obtained (Fr. E1 to Fr. E3). Fr. E1 was purified by semi-preparative HPLC with MeCN-H2O (32:68) to yield 12 (60.7 mg), 6 (125.4 mg), and 7 (17.0 mg), and Fr. E2 was purified by semi-preparative HPLC with MeCN-H2O (35:75) to yield 13 (9.3 mg), 8 (7.4 mg), and 15 (81.1 mg), respectively. Fr. E2 was purified successively by semi-preparative HPLC using MeCN-H2O  as mobile phase to afford 14 (6.2 mg).The fresh whole plant (20 kg) of Terrestrinin J (1): White amorphous powder, c 0.067, MeOH), HR-ESI-MS (neg.): m/z 1327.6162 [M − H]− , ESI-MS (pos.): m/z 1311.6 [M + H − H2O]+, 1179.6 [M + H − H2O − 132]+, 1017.5 [M + H − H2O − 132 − 162]+ , 885.5 [M + H − H2O − 2 × 132 − 162]+, 739.4 [M + H − H2O − 2 × 132 − 162 − 146]+, 577.4 [M + H − H2O − 2 × 132 − 2 × 162 − 146]+, 415.3 [M + H − H2O − 2 × 132 − 3 × 162 − 146]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin K (2): White amorphous powder, c 0.071, MeOH), HR-ESI-MS (neg.): m/z 1327.6230 [M − H]− , ESI-MS (pos.): m/z 1311.64 [M + H − H2O]+, 1179.6 [M + H − H2O − 132]+, 885.5 [M + H − H2O − 2 × 132 − 162]+, 739.4 [M + H − H2O − 2 × 132 − 162 − 146]+, 577.4 [M + H − H2O − 2 × 132 − 2 × 162 − 146]+, 415.3 [M + H − H2O − 2 × 132 − 3 × 162 − 146]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin L (3): White amorphous powder, c 0.059, MeOH), HR-ESI-MS (neg.): m/z 1327.6105 [M − H]− , ESI-MS (pos.): m/z 1311.6 [M + H − H2O]+, 1149.6 [M + H − H2O − 162]+, 1017.6 [M + H − H2O − 162 − 132]+, 885.5 [M + H − H2O − 162 − 2 × 132]+, 723.4 [M + H − H2O − 2 × 162 − 2 × 132]+, 577.4 [M + H − H2O − 2 × 162 − 2 × 132 − 146]+, 415.3 [M + H − H2O − 3 × 162 – 2 × 132 − 146]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin M (4): White amorphous powder, c 0.064, MeOH), HR-ESI-MS (neg.): m/zm/z 1211.5687 [M − H]− , ESI-MS (pos.): m/z 1195.57 [M + H − H2O]+, 1033.52 [M + H − H2O − 162]+, 901.48 [M + H − H2O − 162 − 132]+, 739.43 [M + H − H2O − 2 × 162 − 132]+, 577.37 [M + H − H2O − 3 × 162 − 132]+, 415.32 [M + H − H2O − 4 × 162 − 132]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin N (5): White amorphous powder, c 0.065, MeOH), HR-ESI-MS (neg.): m/z 1211.5709 [M − H]− , ESI-MS (pos.): m/z 1195.6 [M + H − H2O]+, 1033.5 [M + H − H2O − 162]+, 901.5 [M + H − H2O − 162 − 132]+, 739.4 [M + H − H2O − 2 × 162 − 132]+, 577.4 [M + H − H2O − 3 × 162 − 132]+, 415.3 [M + H − H2O − 4 × 162 − 132]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin O (6): White amorphous powder, c 0.069, MeOH), HR-ESI-MS (neg.): m/z 1195.5730 [M − H]− , ESI-MS (pos.): m/z 1035.5 [M + H − 162]+, 741.4 [M + H − 2 × 162 − 132]+, 579.4 [M + H − 2 × 162 − 2 × 132]+, 417.3 [M + H − 3 × 162 − 2 × 132]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin P (7): White amorphous powder, c 0.067, MeOH), HR-ESI-MS (neg.): m/z 1193.5640 [M − H]− , ESI-MS (pos.): m/z 1033.5 [M + H − 162]+, 739.4 [M + H − 2 × 162 − 132]+, 577.4 [M + H − 2 × 162 − 2 × 132]+, 415.3 [M + H − 3 × 162 − 2 × 132]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin Q (8): White amorphous powder, c 0.069, MeOH), HR-ESI-MS (neg.): m/z 633.3265 [M + HCOO]− , ESI-MS (pos.): m/z 427.29 [M + H − 162]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin R (9): White amorphous powder, c 0.077, MeOH), HR-ESI-MS (neg.): m/z 1343.6113 [M − H]− , ESI-MS (pos.): m/z 1327.7 [M + H − H2O]+, 1195.6 [M + H − H2O − 132]+, 901.5 [M + H − H2O − 2 × 132 − 162]+, 739.4 [M + H − H2O − 2 × 132 − 2 × 162]+, 593.4 [M + H − H2O − 2 × 132 − 2 × 162 − 146]+, 431.3 [M + H − H2O − 2 × 132 − 3 × 162 − 146]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin S (10): White amorphous powder, c 0.067, MeOH), HR-ESI-MS (neg.): m/z 1343.6145 [M − H]− , ESI-MS (pos.): m/z 1327.7 [M + H − H2O]+, 1195.6 [M + H − H2O − 132]+, 901.5 [M + H − H2O − 2 × 132 − 162]+, 739.4 [M + H − H2O − 2 × 132 − 2 × 162]+, 593.4 [M + H − H2O − 2 × 132 − 2 × 162 − 146]+, 431.3 [M + H − H2O − 2 × 132 − 3 × 162 − 146]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin T (11): White amorphous powder, c 0.067, MeOH), HR-ESI-MS (neg.): m/z 1343.6128 [M − H]− , ESI-MS (pos.): m/z 1213.60 [M + H − 132]+, 1051.6 [M + H − 132 − 162]+, 919.5 [M + H − 2 × 132 − 162]+, 757.4 [M + H − 2 × 132 − 2 × 162]+, 611.4 [M + H − 2 × 132 − 2 × 162 − 146]+, 449.3 [M + H − 2 × 132 − 3 × 162 − 146]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see Terrestrinin U (12): White amorphous powder, c 0.067, MeOH), HR-ESI-MS (neg.): m/z 1327.6173 [M − H]− , ESI-MS (pos.): m/z 1149.6 [M + H − H2O − 162]+, 903.5 [M + H − H2O − 162 − 146]+, 723.4 [M + H − H2O − 2 × 162 − 2 × 132]+, 577.4 [M + H − H2O − 2 × 162 − 2 × 132 − 146]+, 415.3 [M + H − H2O − 3 × 162 − 2 × 132 − 146]+, 13C-NMR (pyridine-d5) and 1H-NMR (pyridine-d5) data see 1–12) were individually hydrolyzed in 2N CF3COOH (5 mL) and heated at 95 °C for 5 h. After extraction with CH2Cl2 (5 mL) three times, the aqueous layer was repeatedly evaporated to dryness with EtOH until neutral and then the residue of the sugars in pyridine (1 mL) was added to l-cysteine methyl ester hydrochloride (3.0 mg), and the mixture was stirred at 60 °C for 1 h. Furthermore, HMDS-TMCS (hexamethyldisilazane–trimethylchlorosilane) (0.6 mL) was added and then kept at 60 °C for 0.5 h. Finally, the supernatant was analyzed by GC under the following conditions: 6890 gas chromatograph ; HP-5 capillary column; column temperature: 180 °C/250 °C, programmed increase, 15 °C/min; 5973 mass spectrograph detector; carrier gas: N2 (1 mL/min); injection and detector temperature: 250 °C; injection volume: 1.0 µL, split ratio: 1/50. The derivatives of L-rhamnose, D-xylose D-glucose, and D-galactose were detected, with the following retention times, tR (min): D-xylose (15.05 and 16.90), L-rhamnose (16.65 and 18.45), D-glucose (20.19 and 20.95), and D-galactose (20.80 and 22.48) compared the retention times with those of standard samples, respectively.Isolates , and HRESIMS. Among them, the aglycone of compound 1 found in this study has a rare aglycone with an unsaturation between C-7 and C-8. In addition, compounds 13, 16 and 17 were isolated as monomeric form for the first time. This work could be helpful to investigate on the bioactive compounds from T. terrestris.In conclusion, twelve new steroidal glycosides, including eleven furostanol saponins


H-1,2,4-triazol-5-yl)-1,3-thiazolidin-4-one derivatives (7c–l) was designed and synthesized. Their structures have been elucidated based on analytical and spectral data. They were evaluated for their antibacterial and antifungal activities. Compound 7h showed the highest activity against all tested strains, except P. vulgaris, with MIC 8 μg/mL and 4 μg/mL against S. aureus and C. albicans, respectively. Furthermore, Compounds 7c, 7h, and 7j demonstrated moderate anti-mycobacterium activity. The binding mode of the synthesized thiazolidinones to bacterial MurB enzyme was also studied. Good interactions between the docked compounds to the MurB active site were observed primarily with Asn83, Arg310, Arg188 and Ser82 amino acid residues.A series of 3-(2 Mycobacterium tuberculosis and Staphylococcus aureus, highlights the need for new classes of antimicrobial agents .,21.5a–b hemistry . The realdehydes . The proMycobacterium fortuitum at a concentrations ranging from 0.0625–128 μg/mL using moxifloxacin as the standard drug  of 25 mm compared to 26 mm (IZ) of moxifloxacin. On the other hand, Compounds 7c–j showed variable activities. Weak activity was observed for Compounds 7d–g with an IZ range from 9–12 mm. Good activities were observed for Compounds 7c, 7j and 7h with the IZ varying from 20–25 mm with an MIC between 32 and 64 μg/mL. Different variations on R and R1 gave compounds with a range of antimycobacterial activity. The antimycobacterial activity of the synthesized compounds against Mycobacterium fortuitum was observed when R was 4-methoxy, and R1 groups were in the order: 4-chloro > 4-nitro > 4-fluoro. Furthermore, when R was 3-chloro, R1 groups show in the order: 4-chloro > 4-methoy > 4-nitro and 4-fluoro  and the minimal inhibitory zone (MIC). As shown in 7h–l showed moderate to very good broad-spectrum antibacterial activity, while Compounds 7d–g showed no activity against all tested strains. Furthermore, it was noted that none of the tested compounds showed activity against the Gram-negative microorganism Proteus vulgaris. Compound 7h showed the highest activity, and it was effective against all tested strains, except Proteus vulgaris. This compound was the most active against Escherichia coli with an inhibition zone (IZ = 30 mm) greater than the standard drug ciprofloxacin (IZ = 27 mm) at the same tested concentration. Compound 7c showed moderate activity with ~61% inhibition zone compared to the standard drugs against Staphylococcus aureus and Escherichia coli. The MIC, as well as the zone of inhibition values for each compound are explained in 7h was 8 μg/mL against Staphylococcus aureus. Lipophilicity is an important physicochemical parameter in the development of an antibacterial agent, because it is known to be closely related to the permeation through the lipid coat of bacteria. The title compounds showed very good lipophilic characteristics with p-values of 3.4–5.2  then 7g (IZ = 18 mm), while 7k showed the least activity with IZ of 16 mm compared to the 28-mm IZ of the standard drug fluconazole  and Arg225 with the oxygen of methoxy group with (34% 2.38 Å), while it also shows cationic-arene interaction with the Arg188 residue , Ser82  and Gly81  via the NH group of the triazole ring and the carbonyl group of the thiazolidine ring, respectively, with a binding affinity of −12.81985 Kcal/mol; in addition to a cationic arene interaction with the aromatic ring with the Arg310 amino acid  and  with Arg310 and Ser82 via methoxy oxygen and the carbonyl group of the thiazolidine ring, respectively  with the Ser143 amino acid; the second is a cationic arene interaction with Arg188  and Arg310 a cationic-arene interaction with a binding energy of −12.71658 Kcal/mol  higher binding affinity, but still less than the reference ligand; (ii) the incorporation with amino acids is similar to the co-crystallized ligand, especially the hydrogen bonding interactions. These findings are considered encouraging, providing a possible mechanism of action for the tested compounds and indicating the possibility of developing novel antibacterial agents targeting an early step in peptidoglycan biosynthesis.7h are illustrated, showing a higher space of greener areas in the enzyme binding pocket, indicating a highly lipophilic active site. 7h, 7j and 7l, where the phenyl group was chosen for its steric and/or hydrophobic interaction with the protein residues. Moreover, the substitution on the phenyl ring of the parent molecule was selected as the hydrogen bond acceptor or hydrogen bond donor to form a bond or to impart hydrophobicity. It was reported that the lipophilic characteristic of the previously-tested compounds could play an important role in their binding to the MurB active site . Therefo1H-NMR and 13C-NMR spectra were run using TMS as an internal standard  on a Varian Mercury VXr-300 NMR . Mass spectra were measured on a JEOL-SX-102 instrument using electron impact ionization. Elemental analysis were performed on a Perkin-Elmer 240C analyzer  at the Analytical Center, College of Science, Cairo University, Egypt. All values were within ±0.4% of the theoretical values. The reactions were monitored by thin-layer chromatography (TLC) using TLC sheets precoated with UV fluorescent silica gel Merck 60 F254 plates and were visualized using a UV lamp and different solvents as mobile phases. All chemicals were purchased from .Melting points were measured on a Griffin melting point apparatus  and are uncorrected. The infrared spectra were recorded as KBr discs on a Nicolet IR 200 . The 7c–l, as well as intermediates, were prepared in the following steps: synthesis of 3-amino-5-(substituted phenyl)-2H-1,2,4-triazol ; Compound 5a,b was prepared according to the literature procedure +. Analysis for C17H12Cl2N4OS (Molecular weight (m. w.) 390.01); Calcd.: C, 52.18; H, 3.09; N, 14.32. Found: C, 52.44; H, 3.13; N, 14.51.3-(5-(3-Chlorophenyl)-2H-1,2,4-triazol-3-yl)-2-(4-fluorophenyl)thiazolidin-4-one (7d): Yield, 65%; m.p.: 170–172 °C. IR : 3068 (CH aromatic), 1730 (C=O), 1256 (C-S). 1H-NMR : δ = 4.21 , 4.34 , 5.68 , 7.12–7.71 , 7.91 , 88.3 , 13C-NMR : δ =33.24 (CH2-C3H3NOS), 68.15 (CH-C3H3NOS), 121.73 (CH-C6H5), 125.11 (CH-C6H5), 126.24 (CH-C6H5), 127.64 (CH-C6H5), 128.16 (CH-C6H5), 128.79 (CH-C6H5), 129.12 (CH-C6H5), 129.75 (CH-C6H5), 130.11 (C-C6H5), 131.21 (C-C6H5), 132.03 , 155.76 (C-C2HN3), 163.12 (C-C2HN3), 164.04 , 176.71 (CO-C3H3NOS), MS (m/z): 375.04 [M]+. Analysis for C17H12ClFN4OS (m. w. 374.04); Calcd.: C, 54.47; H, 3.23; N, 14.95. Found: C, 54.51; H, 3.42; N, 14.82.3-(5-(3-Chlorophenyl)-2H-1,2,4-triazol-3-yl)-2-(4-nitrophenyl)thiazolidin-4-one (7e): Yield, 66%; m.p.: 178–180 °C. IR : 3085 (CH aromatic), 1735 (C=O), 1257 (C-S). 1H-NMR : δ = 4.09 , 4.17 , 5.74 , 7.11–7.62 , 7.87 , 8.1 , 13C-NMR : δ =34.51(CH2-C3H3NOS), 68.32 (CH-C3H3NOS), 121.41 (CH-C6H5), 124.52 (CH-C6H5), 125.94 (CH-C6H5), 127.41 (CH-C6H5), 128.34 (CH-C6H5), 128.81 (CH-C6H5), 129.21 (CH-C6H5), 129.42 (CH-C6H5), 130.37 (C-C6H5), 130.13 , 141.31 (C-C6H5), 147.28 , 164.47 (C-C2HN3), 165.68 (C-C2HN3), 173.82 (CO-C3H3NOS), MS (m/z): 402.03 [M]+. Analysis for C17H12ClN5O3S (m. w. 401.03); Calcd.: C, 50.81; H, 3.01; N, 17.43. Found: C, 50.62; H, 3.42; N, 17.24.3-(5-(3-Chlorophenyl)-2H-1,2,4-triazol-3-yl)-2-(4-methoxyphenyl)thiazolidin-4-one (7f): Yield, 62%; m.p.: 207–209 °C. IR : 3068 (CH aromatic), 1732 (C=O), 1255 (C-S). 1H-NMR : δ = 3.74 , 4.24 , 4.38 , 5.84 , 7.13–7.62 , 7.92 , 8.54 , 13C-NMR : δ = 34.2 (CH2-C3H3NOS), 54.23 (C-OCH3), 68.24 (CH-C3H3NOS), 116.78 (CH-C6H5), 116.82(CH-C6H5), 126.41 (CH-C6H5), 127.41 (CH-C6H5), 128.61 (CH-C6H5), 128.96 (CH-C6H5), 129.21 (CH-C6H5), 129.47 (CH-C6H5), 131.31 (C-C6H5), 133.74 (C-C6H5), 136.25 , 157.02 , 164.22 (C-C2HN3), 164.51 (C-C2HN3), 172.89 (CO-C3H3NOS), MS (m/z): 387.06 [M]+. Analysis for C18H15ClN4O2S (m. w. 386.06); Calcd.: C, 55.88; H, 3.91; N, 14.48. Found: C, 55.93; H, 3.78; N, 14.27.3-(5-(3-Chlorophenyl)-2H-1,2,4-triazol-3-yl)-2-p-tolylthiazolidin-4-one (7g): Yield, 64%; m.p.: 225–227 °C. IR : 3070 (CH aromatic), 1745 (C=O), 1258 (C-S). 1H-NMR : δ = 2.36 , 4.16 , 4.28 , 5.57  7.08–7.79 , 7.95 , 8.14 , 13C-NMR : δ = 22.78 (C-CH3), 35.32 (CH2-C3H3NOS), 60.92 (CH-C3H3NOS), 123.77 (CH-C6H5), 124.41 (CH-C6H5), 126.42 (CH-C6H5), 126.78 (CH-C6H5), 127.1 (CH-C6H5), 127.44 (CH-C6H5), 130.29 (CH-C6H5), 130.71 (CH-C6H5), 131.05 (C-C6H5), 132.47 , 135.72 (C-C6H5), 136.47 (C-C6H5), 157.71 (C-C2HN3), 158.94 (C-C2HN3), 175.48 (CO-C3H3NOS), MS (m/z): 371.07 [M]+. Analysis for C18H15ClN4OS (m. w. 370.07); Calcd.: C, 58.30; H, 4.08; N, 15.11. Found: C, 58.45; H, 4.19; N, 14.96.2-(4-Chlorophenyl)-3-(5-(4-methoxyphenyl)-2H-1,2,4-triazol-3-yl)thiazolidin-4-one (7h): Yield, 65%; m.p.: 211–213 °C. IR : 3079 (CH aromatic), 1740 (C=O) 1256 (C-S). 1H-NMR : δ = 3.81 , 4.21 , 4.36 , 5.71 , 7.11–7.64 , 7.87 , 8.4 , 13C-NMR : δ = 36.7 (CH2-C3H3NOS), 52.43 (C-OCH3), 69.42 (CH-C3H3NOS), 121.57 (CH-C6H5), 123,89 (CH-C6H5), 127.98 (CH-C6H5), 128.71 (CH-C6H5), 128.89 (CH-C6H5), 129.24 (CH-C6H5), 129.41 (CH-C6H5), 130.87 (CH-C6H5), 131.61 (C-C6H5), 132.54 , 137.97 (C-C6H5), 155.4 , 156.62 (C-C2HN3), 156.81 (C-C2HN3), 174.5 (CO-C3H3NOS), MS (m/z): 387.06 [M]+. Analysis for C18H15ClN4O2S (m. w. 386.06); Calcd.: C, 55.88; H, 3.91; N, 14.48. Found: C, 56.04; H, 3.73; N, 14.51.2-(4-Fluorophenyl)-3-(5-(4-methoxyphenyl)-2H-1,2,4-triazol-3-yl)thiazolidin-4-one (7i): Yield, 68%; m.p.: 215–217 °C. IR : 3078 (CH aromatic), 1725 (C=O), 1259 (C-S). 1H-NMR : δ = 3.81 , 4.09 , 4.18 , 5.61 , 7.21–7.73 , 7.98 , 8.2 , 13C-NMR : δ = 32.92 (CH2-C3H3NOS), 54.13 (C-OCH3), 67.83 (CH-C3H3NOS), 121.27 (CH-C6H5), 124.52 (CH-C6H5), 126.48 (CH-C6H5), 126.61 (CH-C6H5), 128.51 (CH-C6H5), 128.89 (CH-C6H5), 129.61 (CH-C6H5), 129.42 (CH-C6H5), 130.27 (C-C6H5), 132.84 (C-C6H5), 154.62 (C-C2HN3), 155.62 (C-C2HN3), 159.84 , 163.81 , 174.18 (CO-C3H3NOS), MS (m/z): 371.09 [M]+. Analysis for C18H15FN4OS (m. w. 370.09); Calcd.: C, 58.37; H, 4.08; N, 15.13. Found: C, 58.46; H, 3.87; N, 15.47.3-(5-(4-Methoxyphenyl)-2H-1,2,4-triazol-3-yl)-2-(4-nitrophenyl)thiazolidin-4-one (7j): Yield, 68%; m.p.: 218–220 °C. IR : 3078 (CH aromatic), 1740 (C=O), 1257 (C-S). 1H-NMR : δ = 3.87 , 4.15 , 4.08 , 5.76 , 7.08–7.62 , 8.01 , 8.2 , 13C-NMR : δ = 30.92 (CH2- C3H3NOS), 55.32 (C-OCH3), 65.93 (CH-C3H3NOS), 122.47 (CH-C6H5), 124.72 (CH-C6H5), 126.18 (CH-C6H5), 126.55 (CH-C6H5), 128.81 (CH-C6H5), 128.92 (CH-C6H5), 129.12 (CH-C6H5), 129.52 (CH-C6H5), 129.94 (C-C6H5), 129.98 (C-C6H5), 140.47 , 153.52 (C-C2HN3), 154.87 (C-C2HN3), 164.61 , 176.28 (CO-C3H3NOS), MS (m/z): 398.08 [M]+. Analysis for C18H15N5O4S (m. w. 397.08); Calcd.: C, 54.40; H, 3.80; N, 17.62. Found: C, 54.64; H, 3.93; N, 17.41.2-(4-Methoxyphenyl)-3-(5-(4-methoxyphenyl)-2H-1,2,4-triazol-3-yl)thiazolid-din-4-one (7k): Yield, 68%; m.p.: 219–221 °C. IR : 3085 (CH aromatic), 1730 (C=O), 1253 (C-S). 1H-NMR : δ = 3.75 , 3.97 , 4.38 , 4.23 , 5.79 , 7.21–7.72 , 8.09 , 8.7 , 13C-NMR : δ = 32.26 (CH2-C3H3NOS), 52.44 (C-OCH3), 52.86 (C-OCH3), 67.28 (CH-C3H3NOS), 121.64 (CH-C6H5), 124.32 (CH-C6H5), 126.38 (CH-C6H5), 127.51 (CH-C6H5), 127.81 (CH-C6H5), 128.21 (CH-C6H5), 129.41 (CH-C6H5), 129.92 (CH-C6H5), 130.17 (C-C6H5), 131.56 (C-C6H5), 157.62 (C-C2HN3), 158.14 (C-C2HN3), 163.41 , 164.77 , 174.78 (CO-C3H3NOS), MS (m/z): 391.01 [M]+. Analysis for C19H18N4O3S (m. w. 382.11); Calcd.: C, 59.67; H, 4.74; N, 14.65. Found: C, 59.47; H, 4.93; N, 14.52.3-(5-(4-Methoxyphenyl)-2H-1,2,4-triazol-3-yl)-2-p-tolylthiazolidin-4-one (7l): Yield, 68%; m.p.: 216–218 °C. IR : 3070 (CH aromatic), 1728 (C=O), 1254 (C-S). 1H-NMR : δ = 2.24 , 3.76 , 4.28 , 4.21 , 5.91 , 7.21–7.72 , 8.03 , 8.4 , 13C-NMR : δ = 26.14 (C-CH3), 31.92 (CH2-C3H3NOS), 57.21 C-OCH3), 66.42 (CH-C3H3NOS), 121.57 (CH-C6H5), 124,82 (CH-C6H5), 125.88 (CH-C6H5), 126.91 (CH-C6H5), 128.11 (CH-C6H5), 128.79 (CH-C6H5), 129.61 (CH-C6H5), 129.82 (CH-C6H5), 130.07 (C-C6H5), 130.81 (C-C6H5), 134.54 (C-C6H5), 154.47 (C-C2HN3), 155.44 (C-C2HN3), 165.31 , 170.8 (CO-C3H3NOS), MS (m/z): 367.12 [M]+. Analysis for C19H18N4O2S (m. w. 366.12); Calcd.: C, 62.28; H, 4.95; N, 15.29. Found: C, 62.47; H, 5.14; N, 14.91.7c–l, were screened for their antibacterial activity against Escherichia coli ATCC 25922, Proteus vulgaris atcc 6380, Pseudomonas aeruginosa ATCC 27857, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 10400, antimycobacterial activity against Mycobacterium fortuitum ATCC 6841 and antifungal activity against Candida albicans ATCC 2091. The tested compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution (5120 μg/mL).The initial antimicrobial screening (growth inhibition zone) and determination of minimum inhibitory concentration (MIC) of the tested compounds was performed by cup plate and broth dilution methods, respectively, with different strains ,26. Ten ® Ultrospec) at 625 nm to give absorbance 0.1–0.14 (1 × 108 CFU/mL). The suspension was diluted 1:100 in Cation Adjustment Mueller–Hinton (CAMH) broth  to obtain 1 × 106 CFU/mL. The suspension was swabbed on a CAMH agar plate  and allowed to dry thoroughly. Then, four cups per agar plate were made using a cork borer. A 100 µL (512 μg) stock solution (5120 μg/mL) was transferred into the cup using a sterile pipette. The plates were kept in the refrigerator at 4°C for 30 min for diffusion. Then, the plates were incubated at 37 °C for 24 h, except for Mycobacterium fortuitum ATCC 6841, which was incubated for 48 h. After the incubation period, the diameter of the inhibition zone (including the cup diameter) was measured and recorded in mm. Ciprofloxacin, moxifloxacin and fluconazole disks BD BBL™ Sensi-Disc™  (BBL) were used as positive controls for antibacterial and antifungal activities, respectively. The assay was carried out in duplicate.Three to five pure colonies of each tested organism were taken from the overnight culture of tryptone soy agar and suspended in 5 mL of sterile normal saline. The bacterial culture was measured using the spectrophotometer (LKB6 CFU/mL) was added to all tubes, except Tube #14, to give final inocula of 5 × 105 CFU/mL, and the concentration of the tested compounds was two-fold diluted. Ciprofloxacin, moxifloxacin and fluconazole were used as positive controls as the antibacterial and antifungal, respectively. The inoculated tubes were incubated at 37 °C for 20 h, except for Mycobacterium fortuitum ATCC 6841, which was incubated for 48 h. After the incubation period, the results of the MIC were recorded manually according to the guidelines of European Committee on Antimicrobial Susceptibility testing (EUCAST), 2003.MIC values were determined for the active in the cup plate method. Briefly, 1 mL of sterilized CAMH broth was dispensed into a sterile 7-mL bijou tube . For each compound, 14 tubes were used. Tube Numbers 13 and 14 were used as the positive growth control (no tested compound) and the negative control for the medium sterility (no microorganism), respectively. A 1 mL stock solution (5120 μg/mL) was 10-fold diluted in 9 mL CAMH to obtain 512 μg/mL. One milliliter of the tested compounds was transferred into Tube #1 and mixed well to give a concentration of 256 μg/mL. Then, 1 mL was pipetted from Tube #1 and transferred to Tube #2 to make a two-fold dilution (128 μg/mL). This procedure was repeated down to Tube #12 to reach the concentration of 0.125 μg/mL. One milliliter was discarded from Tube #12. A volume of 1 mL of inocula  gradient of 0.01 kcal/mol conformational searching in torsional space were performed using the multi-conformer method. Energy minima for the above compounds were determined by a semi-empirical method Austin Model 1 (AM1) . The lowest energy conformer of each new analogue “global-minima” that was docked into the MurB enzyme-binding domain was used. The crystal structure of Staphylococcus Aureus MurB (PDB Entry Code 1HSK) [http://www.rcsb.org/pdb). During the docking process, water molecules and all of the ligands in the crystal structures were removed, except the FAD co-factor for the protein PDB Code 1HSK. All of the hydrogens were added, and the enzyme structure was subjected to a refinement protocol in which the constraints on the enzyme were gradually removed and minimized until the RMSD gradient was 0.01 kcal/mol Å. The energy minimization was carried out using the molecular mechanics force field “AMBER.” For each of the 4-thiazolidinone analogues, energy minimizations (EM) were performed using 1000 steps of steepest descent, followed by conjugate gradient minimization to an RMSD energy gradient of 0.01 Kcal/mol Å. The active site of the enzyme was defined using a radius of 10.0 Å around FAD. The energy of binding was calculated as the difference between the energy of the complex and individual energies of the enzyme and ligands [The investigated compounds were subjected to surface mapping experiments using “Molecular Operating Environment” software . The molecules were built using the Builder module of MOE. Their geometry was optimized by using the Merck Molecular Force Field (MMFF94x) force field. The molecular mechanics (MM) calculations de 1HSK)  was extr ligands ,28,29,307h followed by 7j gave the most potent activity against Staphylococcus aureus. Compounds 7h–l showed good antifungal activity against Candida albicans. The in vitro studies demonstrated that 7h, 7j and 7c showed promising inhibitory activities against Mycobacterium fortuitum. Despite that the MIC values of the tested compounds were not as strong as the reported antibiotics, however, they possessed a broad spectrum of activity against Gram-positive, Gram-negative, mycobacterium and fungal strains. Our compounds are not selective, so they may be useful as moderate to high-level disinfectants. This suggests that these compounds could be formulated as disinfectants or antiseptics if they are proven later to be nontoxic to skin/mucous membranes. The search for new antibacterial agents directed towards novel targets became highly imperative. Therefore, we performed a deeper modeling study on the molecular level to clarify the proposed binding mode of these compounds into the MurB active site and consequently provide straight forward information for further structural optimization. Our results suggested that docked ligands 7h, 7j, 7l, 7c, 7i and 7k arranged in such a conformation with a proper and stable complex with high lipophilic interaction where the p-chloro or p-methoxy phenyl ring was embedded within the hydrophobic active site residues. Our new 1,2,4-triazole-thiazolidin-4-ones hybrids presented in this work could provide a potential for further optimization towards more active derivatives against Staphylococcus aureus, as well as Candida albicans. This study has also paved the way for generating more useful thiazolidinone analogues in future studies as bacterial MurB inhibitors.We have reported here the design and synthesis of new 1,2,4-triazole-based thiazolidin-4-one hybrids along with their spectral and antimicrobial evaluation data. Compound


The anticipated worldwide increase in biodiesel production will result in an accumulation of glycerol for which there are insufficient conventional uses. The surplus of this by-product has increased rapidly during the last decade, prompting a search for new glycerol applications. We describe here the synthesis of dissymmetric chlorohydrin esters from symmetric 1,3-dichloro-2-propyl esters obtained from glycerol. We studied the influence of two solvents: 1,4-dioxane and 1-butanol and two bases: sodium carbonate and 1-butylimidazole, on the synthesis of dissymmetric chlorohydrin esters. In addition, we studied the influence of other bases (potassium and lithium carbonates) in the reaction using 1,4-dioxane as the solvent. The highest yield was obtained using 1,4-dioxane and sodium carbonate. Glycerol , is one of the most versatile and valuable chemicals known to man. Glycerol has over 1,500 known end uses, including applications as an ingredient or processing aid in cosmetics, toiletries, personal care products, pharmaceutical formulations and food + C9H16Cl2O2Na calculated: 249.0425, found: 249.0518.2-Chloro-1-(chloromethyl)ethyl-2-methyl-2-phenylpropanoate(1d). 1H-NMR δ: 7.46–7.21, m, 5H, CHar), 5.16 , 3.73–3.59 , 1.63 2-C). 13C- NMR δ: 175.6 (C=O), 143.8 (Car), 128.4, 126.9, 125.7 (CHar), 72.0 (O-CH), 46.7 ((CH3)2-C), 42.3 (CH2-Cl), 26.3 ((CH3)2-C). IR (KBr) νmax: 3083.9, 3053.7, 3023.5, 2976.1, 2928.6, 2872.5, 1729.0, 1241.4, 1133.5 cm−1. HRMS (ESI+) [M+Na]+ C13H16Cl2O2Na calculated: 297.0425, found: 297.0531.4, and the solvent was evaporated. The crude compound was analyzed by 1H-NMR to determine the yield of the corresponding product using N,N-dimethylformamide (DMF) as an internal standard. Further purification by dry flash column chromatography on silica gel (hexane/ethyl acetate) gave the desired product.A solution of 2-chloro-1-(chloromethyl)ethyl ester (1 mmol), water  and either sodium carbonate  or 1-butylimidazole  in dried solvent ) was heated at 115 °C for 48 h in a capped reaction vial. After cooling, dichloromethane was added, and the mixture was washed three times with water. The organic layer was dried over anhydrous MgSO3-Chloro-2-hydroxy-1-propyl hexadecanoate . 1H-NMR δ: 0.90 , 1.20–1.40 12), 1.66 , 2.30 , 3.59 and 3.65 , 4.00 , 4.15 . 13C-NMR δ: 177 (C=O), 70 (CH-OH), 66 (O-CH2), 45 (CH2-Cl), 34 (CH2-COO), 32–22 , 17 (CH3). GC-MS m/z: 348 [M]+, 299 [M-CH2Cl]+, 269 [M-C2H2ClO]+, 255 [M-C3H6ClO]+, 239 [M-C3H6ClO2]+, 152 [C5H9O3Cl]+ (McLafferty rearrangement) + C8H15ClO3Na calculated: 217.0607, found: 217.0701.3-Chloro-2-hydroxy-1-propyl 2,2-dimethylbutanoate (2c). 1H-NMR δ: 4.20 , 4.05 , 3.65–3.54 , 1.56 , 1.16 2-C), 0.83 . 13C-NMR δ: 178.1 (C=O), 69.8 (CH-OH), 65.0 (O-CH2), 45.9 (CH2-Cl), 42.8 ((CH3)2-C), 33.3 (CH2-CH3), 24.6 ((CH3)2-C), 9.2 (CH2-CH3). IR (KBr) νmax: 3480.9, 2967.4, 2937.2, 2876.8, 1724.7, 1241.4, 1150.8 cm−1. HRMS (ESI+) [M+Na]+ C9H17ClO3Na calculated: 231.0764, found: 231.0860.3-Chloro-2-hydroxy-1-propyl 2-methyl-2-phenylpropanoate (2d). 1H-NMR δ: 7.39–7.23 , 4.21 , 3.97 , 3.46–3.36 , 1.62 2-C). 13C-NMR δ: 176.8 (C=O) 144.2 (Car), 128.5, 126.9, 125.5 (CHar), 69.5 (CH-OH), 65.2 (O-CH2), 46.6 ((CH3)2-C), 45.7 (CH2-Cl), 26.3 ((CH3)2-C). IR (KBr) νmax: 3480.9, 3083.9, 3053.7, 3019.2, 2971.7, 2924.3, 1729.0, 1245.7, 1142.2 cm−1. HRMS (ESI+) [M+Na]+ C13H17ClO3Na calculated: 279.0764, found: 279.0873. 4, and the solvent was distilled. The crude compound was analyzed by 1H-NMR to determine the yield of the corresponding product using DMF as an internal standard. Further purification by dry flash column chromatography on silica gel (hexane/ethyl acetate) gave the desired product.A solution of 2-chloro-1-(chloromethyl)ethyl ester (1 mmol), water  and a carbonate (1 mmol) in dried 1,4-dioxane (3 mL) was heated at 115 °C for 48 h in a capped reaction vial. After cooling, dichloromethane was added and the mixture was washed three times with water. The organic layer was dried over anhydrous MgSO4, and the mixture was distilled obtaining 2.9 g of the desired compound (62% yield) b.p.: 70 °C/ 40 Pa. The compound was identified by 1H-NMR and 13C-NMR.A solution of 2-chloro-1-(chloromethyl)ethyl 2,2-dimethylpropanoate , water  and sodium carbonate  in dried 1,4-dioxane (72 mL) was heated at 115 °C for 48 h in a capped reactor. After cooling, dichloromethane was added and the mixture was washed three times with water. The organic layer was dried over anhydrous MgSO1,3-Dichloro-2-propyl esters prepared from glycerol can be transformed into the corresponding 3-chloro-2-hydroxy-1-propyl esters. These dissymmetric chlorohydrin esters are potentially valuable compounds and can be used as building blocks. With the methodology described above, different 3-chloro-2-hydroxy-1-propyl esters were synthesized with all studied compounds; better reaction conditions were obtained when using sodium carbonate as base and 1,4-dioxane as solvent. In addition, when different inorganic carbonates were used, sodium carbonate gave the best results. The best procedure developed allows to prepare 3-chloro-2-hydroxy-1-propyl 2,2-dimethylpropanoate on a multigram-scale.


Smilax china L. stems (SCS) showed significant in vitro anti-glycation and rat lens aldose reductase inhibitory activities. Bioactivity-guided isolation was performed with SCS and four solvent fractions were obtained, which in turn yielded 10 compounds, including one phenolic acid, three chlorogenic acids, four flavonoids, one stilbene, and one phenylpropanoid glycoside; their structures were elucidated using nuclear magnetic resonance and mass spectrometry. All solvent fractions, isolated compounds, and stem extracts from plants sourced from six different provinces of South Korea were next tested for their inhibitory effects against advanced glycation end products, as well as aldose reductase. α-Glucosidase, and lipase assays were also performed on the fractions and compounds. Since compounds 3, 4, 6, and 8 appeared to be the superior inhibitors among the tested compounds, a comparative study was performed via high-performance liquid chromatography with photodiode array detection using a self-developed analysis method to confirm the relationship between the quantity and bioactivity of the compounds in each extract. The findings of this study demonstrate the potent therapeutic efficacy of SCS and its potential use as a cost-effective natural alternative medicine against type 2 diabetes and its complications.The search for natural inhibitors with anti-diabetes properties has gained increasing attention. Among four selected Smilacaceae family plants,  Type 2 diabetes mellitus (T2DM), a disease caused by insulin resistance, currently represents a major health issue concerning both the governments of countries where patients live as well as affected individuals. Approximately 415 million people worldwide suffer from T2DM and the number is forecast to rise to 642 million by 2040 . This elSmilax china L., a member of the Smilacaceae family, is widely distributed worldwide in tropical and temperate regions, especially in East Asia −.5-O-Caffeoylquinic acid (2): Amorphous green powder; 1H-NMR (D2O in DMSO-d6): δ 7.47 , 7.02 , 6.94 , 6.78 , 6.18 , 5.21 , 3.93 , 3.55 , 2.02 , 1.91 , 1.83 ; 13C-NMR: δ 176.1 (C-7), 166.1(C-9′), 148.2 (C-4′), 145.6 (C-7′), 144.4 (C-3′), 125.7 (C-1′), 121.1 (C-6′), 115.8 (C-5′), 115.1 (C-2′), 114.6 (C-8′), 72.9 (C-1), 71.2 (C-5), 71.0 (C-4), 67.3 (C-3), 35.1 (C-2), 34.9 (C-6); (ESI) m/z = 354.31 [M − H]−.3-O-Caffeoylquinic acid (3): Amorphous white powder; 1H-NMR (D2O in DMSO-d6): δ 7.38 , 7.02 , 6.97 , 6.76 , 6.10 , 5.01 , 3.88 , 3.54 , 2.10 , 1.93 , 1.76 ; 13C-NMR: δ 173.6 (C-7), 165.4 (C-9′), 148.5 (C-4′), 145.6 (C-7′), 145.1 (C-3′), 125.4 (C-1′), 121.3 (C-6′), 115.9 (C-5′), 114.6 (C-2′), 113.9 (C-8′), 73.0 (C-1), 71.0 (C-5), 69.3 (C-4), 66.9 (C-3), 37.3 (C-2), 35.1 (C-6); (ESI) m/z = 354.31 [M − H]−.4-O-Caffeoylquinic acid (4): Amorphous green powder; 1H-NMR (D2O in DMSO-d6): δ7.48 , 7.04 , 6.98 , 6.77 , 6.26 , 4.69 , 4.10 , 3.88 , 1.86 , 1.78 , 1.22 ; 13C-NMR: δ175.7 (C-7), 166.4(C-9′), 148.2 (C-4′), 145.5 (C-7′), 144.9 (C-3′), 125.7 (C-1′), 121.4 (C-6′), 115.8 (C-5′), 114.8 (C-2′), 114.6 (C-8′), 76.6 (C-1), 69.7 (C-5), 65.9 (C-4), 64.7 (C-3), 38.9 (C-2), 36.2 (C-6); (ESI) m/z = 354.31 [M − H]−.d-glucopyranosyl-7-O-α-l-rhamnopyranosideKaempferol 3-O-β- (5): Amorphous yellow powder; 1H-NMR: δ 8.07 , 6.89 , 6.82 , 6.44 , 5.54 , 5.47 , 3.17-4.02 , 1.11 ; 13C-NMR: δ 177.7 (C-4), 161.6(C-7), 160.9 (C-4′), 160.2 (C-5), 156.8 (C-2), 156.0 (C-9), 133.5 (C-3), 131.0 , 120.7 (C-1′), 115.2 , 105.7 (C-10), 100.8 (C-1′′′-glc), 99.4 (C-1′′-rham), 77.6 (C-5′′′), 76.4 (C-3′′′), 74.2 (C-2′′′), 71.6 (C-4′′), 70.27 (C-4′′′), 70.09 (C-3′′), 69.93 (C-2′′), 69.8 (C-5′′), 60.9 (C-6′′′), 17.9 (C-6′′); (ESI) m/z = 594.52 [M − H]−.l-rhamnosideQuercetin 3-O-α- : Amorphous yellow powder; 1H-NMR (D2O in DMSO-d6): δ 7.29 , 7,24 , 6.85 , 6.36 , 6.18 , 5.24 , 3.96 - 3.13 , 0.80 ; 13C-NMR: δ177.6 (C-4), 164.7 (C-7), 161.2 (C-5), 157.2 (C-2), 156.5 (C-9), 148.5 (C-4′), 145.2 (C-3′), 134.1 (C-3), 121.1 (C-6′), 120.7 (C-1′), 115.6 (C-5′), 115.4 (C-2′), 103.8 (C-10), 101.8 (C-1′′-rham), 98.8 (C-6), 93.6 (C-8), 71.2 (C-4′′), 70.6 (C-3′′), 70.3 (C-2′′), 70.0 (C-5′′), 17.5 (C-6′′); (ESI) m/z = 448.38 [M − H]−.l-rhamnosideKaempferol 3-O-α- : Amorphous yellow powder; 1H-NMR (D2O in DMSO-d6): δ 7.73 , 6.90 , 6.37 , 6.17 , 5.28 , 3.97–3.07 , 0.78 ; 13C-NMR: δ 177.5 (C-4), 165.0 (C-7), 161.2 (C-5), 160.0 (C-4′), 157.0 (C-2), 156.5 (C-9), 134.1 (C-3), 130.5 , 120.5 (C-1′), 115.4 , 103.8 (C-10), 101.8 (C-1″-rham), 98.9 (C-6), 93.8 (C-8), 71.1 (C-4″), 70.6 (C-3″), 70.3 (C-2″), 70.1 (C-5″), 17.5 (C-6″); (ESI) m/z = 432.38 [M − H]−.3,5,4′-Trihydroxy-trans-stilbene : dark brown needle shaped crystals; 1H-NMR (D2O in DMSO-d6): δ 7.37 , 6.92 , 6.78 , 6.74 , 6.38 , 6.09 ; 13C-NMR: δ 158.6 , 157.3 (C-4′), 139.7 (C-1), 128.5 (C-1′), 128.3 (C-b), 128.2 , 125.9 (C-a), 115.8 , 104.6 , 102.0 (C-4); (ESI) m/z = 229.24 [M + H]+.d-glucopyranoyl-(12-Feruloyl-O-α-→d-fructofuranoside2)-3,6-O-feruloyl-β- : Amorphous white powder; 1H-NMR (D2O in DMSO-d6): δ 3.46–4.50 , 5.20 , 5.43 , trans-p-feruloyl units: R1: 3.80 , 6.42 , 6.80 , 7.15 , 7.32 , 7.57 , R2: 3.80 , 6.50 , 6.80 , 7.13 , 7.29 , 7.61 ; 13C-NMR: δ 167.1 (C-9′′), 166.6 (C-9′′′), 149.7 (C-4′′), 148.0 (C-4′′′), 145.8 (C-3′′′), 145.5 (C-3′′), 145.3 (C-7′′′), 145.2 (C-7′′), 125.4 (C-1′′), 123.4 (C-1′′′), 123.2 (C-6′′), 123.0 (C-6′′′), 115.7 (C-5′′), 115.5 (C-5′′′), 114.1 (C-8′′), 114.0 (C-8′′′), 111.6 (C-2′′), 111.3 (C-2′′′), 104.3 (C-2′), 91.7 (C-1), 79.6 (C-5′), 76.9 (C-3′), 73.2 , 73.0 (C-5), 72.8 (C-2), 71.5 (C-4), 65.6 (C-6′), 65.1 (C-1′), 63.0 (C-6), R1: 55.7 (-OCH3), R2: 55.7 (-OCH3); (ESI) m/z = 694.63 [M − H]−.Isoscutellarein 8-O-rhamnoside (10): Amorphous yellow powder; 1H-NMR (D2O in DMSO-d6): δ 8.11 , 6.93 , 6.80 , 6.40 , 5.53 , 3.17–3.85 , 1.13 ; 13C-NMR: δ 181.4 (C-4), 165.9 (C-2), 160.6 (C-4′), 159.1 (C-5), 155.8 (C-7), 139.7 (C-9), 132.0 (C-3), 129.6 , 127.5 (C-8), 121.9 (C-1′), 115.5 , 109.7 (C-10), 106.1 (C-1′′′-rham), 98.4 (C-6), 71.6 (C-4″), 70.2 (C-3″), 70.1 (C-2″), 69.8 (C-5″), 18.0 (C-6″); (ESI) m/z = 432.38 [M − H]−.1–10 were dissolved in DMSO for the AGEs assay at five different final concentrations (10–500 µg/mL for extracts and fractions or 10–500 µM for isolated constituents). After 48 h of incubation at 60 °C, 200 μL of the reaction products was taken to 96-well plates and its fluorescence was measured using a spectrofluorometric detector  at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. DMSO was used as negative control instead of the sample. The IC50 values and 95% confidence intervals were obtained via regression analysis. The percentage of inhibition (%) was calculated by the following equation, where Ac stands for the fluorescence of the control and As stands for the fluorescence of the sample:In accordance with a reported protocol  with sli dl-glyceraldehyde as substrate. All extract samples, including hexane, water, EA, and NB fractions, as well as compounds 1–10, were dissolved in DMSO for the AR assay at five different final concentrations (10–100 µg/mL for extracts and fractions or 0.1–100 µM for isolated constituents). IC50 values were determined using the least-squares regression line of log concentration plotted against residual activity. TMG, a typical AR inhibitor, was used as the positive control. A negative control was fulfilled by using DMSO instead of the sample.All experimental protocols for animal care and use were approved by the local ethics board . Animal husbandry and procedures were performed in accordance with institutional guidelines. Lenses (one lens per 0.5 mL sodium buffer) were removed from Sprague-Dawley rats (weight 250–280 g) and stored in a deep freezer at −80 °C until use. The rat lenses were homogenized and centrifuged at 10,000 rpm  and the supernatant was used as an enzyme source . AR actip-nitrophenyl α-d-glucopyranoside (p-NPG), and sample dissolved in 10% DMSO at five different final concentrations (5–100 µg/mL for extracts and fractions or 1–1000 µM for isolated constituents). A chromogenic substance was used for quantification purposes. The mixture solution was added to each well, then, 20 μL of 10 mM phosphate buffer (pH 6.8) containing 0.2 U/mL α-glucosidase was added. The plate was incubated at 37 °C for 15 min, then, 80 μL of 0.2 mol/L sodium carbonate solution was added to stop the reaction. Absorbance was measured at 405 nm immediately after reaction cessation by using a SUNRISE microplate reader . The negative control contained the same reaction mixture with phosphate buffer substituted for the sample solution. Acarbose was dissolved in 10% DMSO and used as the positive control. The inhibition rate of α-glucosidase activity was calculated using the same equation (1) used for inhibition of AGEs formation, but absorbance was measured instead of fluorescence.This enzyme inhibition assay was carried out by using a 96-well microplate reader and a spectrophotometer. The reaction volume (60 μL) contained 100 mM phosphate buffer (pH 6.8), 2.5 mM p-nitrophenyl butyrate (p-NPB), which is converted to p-nitro-phenol in the presence of lipase, giving a yellow color that can be used to quantify the extent of the reaction. Type II porcine pancreatic lipase was used at a concentration of 5 mg/mL. The substrate (p-NPB) was prepared in DMSO to achieve a concentration of 5 mM. Tested samples, dissolved in 10% DMSO at five different final concentrations (0.05–5.0 mg/mL for extracts and fractions or 10–2000 µM for isolated constituents), were mixed with Tris–HCl buffer (pH 7.4), and enzyme solution, with a total reaction mixture volume of 840 μL. After incubation at 37 °C for 15 min, p-NPB was added, and the solution was further incubated at 37 °C for 25 min. Then, the mixture was cooled for 2 min, and 200 μL was transferred to a 96-well plate. Absorbance was measured using the SUNRISE microplate reader. Blanks, in which enzymes were replaced with Tris–HCl buffer, were prepared for background corrections. The negative control consisted of all solutions except for the samples, and the common lipase inhibitor, orlistat, was used as the positive control. The inhibition rate of lipase activity was also calculated using Equation (1).Lipase inhibition activity was evaluated according to a slightly modified method of de Camargo et al. . The subO-caffeoylquinic acid, 4-O-caffeoylquinic acid, quercitrin and trans-resveratrol, from the SCS extract, reversed-phase HPLC was performed using a Waters 600 pump  equipped with a Waters 996 photodiode array detector (Waters) for detection, a Waters 717 plus (Waters) autosampler, a YL CTS30  column oven, and an ERC-3415  degasser. Samples were separated using a RPAQUEOUS C30 column  at 30 °C. Solvent A  and solvent B (acetonitrile) were used as gradient linear mobile phases  at a flow rate of 1.0 mL/min. The injection volume was 10 µL and absorbance wavelength was 320 nm. For preparation of extract stock solutions, plant powders were sonicated with 70% MeOH for 90 min and dried under vacuum by using a rotary evaporator at 50°C. Then, they were dissolved in 70% MeOH to a concentration of 10,000 ppm. Standard compound stock solutions were dissolved in 100% MeOH. All analyzed solutions were strained using a 0.45-µm PVDF membrane filter prior to injection. The standard calibration curve was constructed using five concentrations. The linear relationship between peak area and concentration is described in .Statistical significance was analyzed via a one-way ANOVA and Student’s 3, 4, 6, and 8 possessing the highest inhibition capabilities. Therefore, we investigated the relation between the inhibition potency and the quantities of these four compounds in extracts from six SCS samples collected from different provinces of Korea. Samples from Gyeongsangnam-do Province possessed both the strongest inhibitory activity and the highest levels of the aforementioned four compounds. Isolated compounds from SCS also demonstrated powerful inhibitory activities against α-glucosidase and lipase enzymes. Consequently, SCS extracts could possess inhibitory effects against oxidative stress-related diabetic complications. 3-O-Caffeoylquinic acid, 4-O-caffeoylquinic acid, quercitrin, and trans-resveratrol obviously played vital roles in mediating these effects. Additionally, we also figured out that when using SCS for medicinal purposes, it is best to collect or cultivate the plant from Gyeongsangnam-do Province of Korea. Further studies are required to evaluate if the activity is sufficient to be used in clinical applications.In this study, with the aim of searching for novel therapies for diabetic complications, we evaluated the capacities of four stem extracts from the family Smilacaceae to inhibit AGEs formation and AR activity. Since we identified that SCS extracts exhibited the most potent inhibitory capacities, we isolated the 10 major bioactive compounds of SCS and characterized their structures. All solvent fractions and isolated compounds were investigated for their antidiabetic potential, with most of the isolated compounds showing an inhibitory potency, with compounds


Compound 2 was also reacted with NaCN to give 2-cyano-1,4,6-androstatriene-3,17-dione (5) and 2β-cyano-1α-hydroxy-4,6-androstadiene-3,17-dione (6). 6α,7α-Epoxy-1,4-androstadiene-3,17-dione (8) was reacted with HCl, HBr and NaN3 to form the corresponding 7β-chloro-, 7β-bromo- and 7β-azido-6α-hydroxy-1,4-androstadiene-3,17-diones. The cytotoxic activity of these compounds towards T47D (estrogen-dependent) and MDA-MB231 (estrogen-independent) breast cancer cell lines was evaluated. The 6α-hydroxy-7β-substituted analogs were more active than the 2-substituted analogs on both cell lines. Compound 2 showed the highest selective activity against the T47D (IC507.1 μM) cell line and 5 showed good cytotoxic activity on MDA-MB231 (IC5018.5 μM) cell line, respectively. The 6α,7α-epoxy analog 8 also showed high cytotoxic activity on both cell lines (IC50 17.3 μM on T47D and IC50 26.9 μM on MDA-MB231).2-Chloro-, 2-bromo- and 2-azido-1,4,6-androstatriene-3,17-diones were synthesized from 1α,2α-epoxy-4,6-androstadiene-3,17-dione ( Breast cancer is the most commonly diagnosed cancer among women and continues to be a major cause of cancer related deaths. The medical treatments available for breast cancer include endocrine therapy, cytotoxic chemotherapy and adjuvant treatments. Estrogens are involved in numerous physiological processes, including the development and maintenance of the female sexual organs, the reproductive cycle, reproduction, and various neuroendocrine functions. On the other hand, estrogens enhance growth and proliferation of certain target cells, such as breast epithelial cells and estrogen-dependent mammary carcinoma cells. Tamoxifen is an estrogen receptor antagonist in breast tissue and has been the standard endocrine (antiestrogen) therapy for hormone-sensitive early breast cancer in post-menopausal women, however, prolonged treatment may cause endometrial cancer . Estroneet al. determined and characterized the expression of aromatase mRNA in the breast carcinoma cell lines T47D and MCF-7 + at m/z 307. The 1H-NMR spectrum of 6 showed peaks at 6.28 (H-7), 6.19 (H-6) and 5.68 (H-4) ppm for three double bond hydrogens and at δ 4.17 and 3.51 ppm for H-1 and H-2, respectively. Peaks at 155.8, 143.0, 132.2 and 123.7 ppm in the 13C-NMR spectrum were assigned to four double bond carbons and the CN carbon was identified at 121.3 ppm. The stereochemistry of H-1 and H-2 were determined with the 1D-NOESY spectrum. Thus, the β-configuration of H-1 was confirmed by irradiation of the H-1 proton which showed a NOE at H-19. The α-configuration of H-2 was also identified by irradiation of the H-2 proton which did not show a NOE at H-19. 2-Azido-1,4,6-androstatriene-3,17-dione (7) was synthesized and identified by the same method. When 8) was stereoselectively obtained by reacting 1 with m-chloroperoxybenzoic acid + at m/z 334. Compound 8 was reacted with sodium azide in ethanol/H2O (1:8) at room temperature to form 7β-azido-6α-hydroxy-1,4-androstadiene-3,17-dione (11) in 68% yield. The stereochemistry of H-6 and H-7 were determined with the aid of the corresponding 1D-NOESY spectrum. The β-configuration of H-6 was confirmed by irradiation of the H-6 proton which showed a NOE on the H-19 protons and the α-configuration of H-7 was also identified by irradiation of the H-19 protons which did not show a NOE on the H-19 protons. 4,6-Androstadiene-3β,17β-diol (12) was obtained from the reaction of 1 and sodium borohydride + at m/z 316 (C19H24O4). The stereochemistry of H-6 and H-7 were determined from the 1D-NOESY spectrum. The α-configuration of OH-6 was confirmed by irradiation of the H-6 proton, which showed a NOE on the H-19 protons. The β-configuration of OH-7 was also verified by irradiation of the H-7 proton which did not show a NOE on the H-19 protons.Unexpectedly, when compound 17-dione . Two hyd8 was treated with sodium cyanide and ammonium chloride in ethanol/H2O (8:1) solution, the reaction did not proceed at room temperature, but when 8 was reacted under reflux conditions, 2α-cyano-6α,7α-epoxy-4-androstene-3,17-dione (14) was obtained in 77% yield. The structure of 14 was determined by the presence of peaks at δ 6.29 ppm for only one double bond hydrogen (H-4) and at 3.17 (H-2) ppm and at δ 3.56 (H-7) and 3.49 (H-6) ppm for two epoxy hydrogens on the 1H-NMR spectrum and two double bond carbons at 158.6 (C-5), 131.8 (C-4) and a CN carbon at 118.5 ppm, and two epoxy carbons at 53.3 and 52.5 ppm in the 13C-NMR spectrum. The mass spectrum of compound 14 showed a molecular ion [M]+ at m/z 325 (C20H23NO3). The configuration and position of cyano group were determined from the 1D-NOESY and 1H-1H COSY data. The α-configuration of the CN substituent was identified by irradiation of the H-2 proton which showed a NOE on the H-19 protons in the 1D-NOESY spectrum (1H-1H COSY spectrum (When spectrum . We acquspectrum .1-5 and 7-14 on T47D and MDA-MB231 breast cancer cells. The results are shown in We assessed the cytotoxicity of synthesized compounds 2) showed a selective dose dependent and high cytotoxicity on T47D cells (IC507.1 μM), which was better than that of tamoxifen  +. Compound 6: yield: 13% (30 mg); IR : 3358 (OH), 2945, 2249 (CN), 1729 (17-C=O), 1644 (3-C=O); 1H-NMR (DMSO-6d) δ: 6.28 , 6.19 , 5.68 , 4.17 , 3.51 , 0.98 , 0.83 ; 13C-NMR (DMSO-6d) δ: 224.0 (C-17), 180.1 (C-3), 155.8 (C-5), 143.0 (C-7), 132.2 (C-6), 123.7 (C-4), 121.3 (CN), 77.2 (C-1), 53.8 (C-2), 52.8, 52.5, 41.7, 41.0, 40.5, 36.3, 36.2, 25.2, 20.0, 18.7, 13.4; GC-MS (EI) m/z: 325 [M]+, 307 [M-H2O]+.2-Azido-1,4,6-androstatriene-3,17-dione (7): Compound 2  was dissolved in 8:1 ethanol-H2O (9 mL) and NH4Cl  and NaN3  were added and the mixture refluxed for 24 h. The reaction mixture was cooled down and treated with NH4Cl to obtain a crude brown compound, which was extracted with dichloromethane (30 mL × 3) and washed with water (30 mL). The organic layer was dried with anhydrous magnesium sulfate, filtered, and concentrated to crude oil, which was purified by column chromatography (ethyl acetate-n-hexane=1:3) to give pure white compound 7. Yield: 58% (126 mg), mp: 248–249 °C; IR (cm-1): 2948, 1721 (17-C=O), 1640 (3-C=O); 1H-NMR (CDCl3) δ: 6.50 , 6.33 , 6.16 , 6.06 , 1.24 , 1.00 ; 13C-NMR (CDCl3) δ: 218.9 (C-17), 181.0 (C-3), 162.6 (C-5), 136.8 (C-1), 134.9 (C-2), 134.4 (C-7), 127.8 (C-6), 123.0 (C-4), 48.9, 48.8, 47.8, 41.6, 37.5, 35.5, 31.2, 21.3, 21.2, 13.8; GC-MS (EI) m/z: 295 [M-N2]+. 1α,2α-Epoxy-4,6-androstadiene-3,17-dione (8): According to the previously reported procedure +.7β-Bromo-6α-hydroxy-1,4-androstadiene-3,17-dione (10): Compound 8  was dissolved in acetone (2 mL), HBr (0.2 mL) was added at 0 °C and the mixture stirred at room temperature for 2 h. and then treated by the same method of compound 9 to give a pure white product 10. Yield: 58% (75 mg), m.p: 225–227 °C; IR (cm-1): 3356 (OH), 2950, 1739 (17-C=O), 1651 (3-C=O); 1H-NMR (CDCl3) δ: 7.24 , 6.38 , 6.15 , 4.91 , 3.99 , 1.46 , 0.90 ; 13C-NMR (CDCl3) δ: 219.7(C-17), 185.4 (C-3), 160.9 (C-5), 157.6 (C-1), 130.7 (C-2), 126.5 (C-4), 72.0 (C-6), 54.2 (C-7), 47.3, 43.7, 41.7, 35.8, 34.3, 31.4, 24.4, 21.5, 21.3, 13.9, 45.3; GC-MS (EI) m/z: 379 [M]+.7β-Azido-6α-hydroxy-1,4-androstadiene-3,17-dione (11): Compound 8  was dissolved in 8:1 ethanol-H2O (9 mL), NH4Cl  and NaN3  were added and the mixture refluxed for 24 h. The reaction mixture was then neutralized with 10% HCl to give a yellow precipitate which was filtered, washed with water and purified by column chromatography (ethyl acetate-n-hexane=1:3) to give a pure pale yellow compound 11. Yield: 68% (155 mg), m.p: 275–277 °C, IR (cm-1): 3348 (OH), 2959, 1730 (17-C=O), 1649 (3-C=O); 1H-NMR (CDCl3) δ: 7.21 , 6.29 , 6.13 , 5.29 , 4.47 , 3.65 , 1.31 , 0.83 ; 13C-NMR (CDCl3) δ: 224.6 (C-17), 190.0 (C-3), 164.6 (C-5), 162.3 (C-1), 135.9 (C-2), 131.6 (C-4), 74.0 (C-6), 52.1, 49.8, 47.8, 47.2, 40.5, 39.8, 36.1, 26.2, 26.1, 25.1, 18.6; GC-MS (EI) m/z: 313 [M-N2]+.4,6-Androstadiene-3β,17β-diol (12): A solution of compound 8  in ethanol (30 mL) and sodium borohydride  was stirred at room temperature for 4 h. The reaction mixture was evaporated to form an oily residue which was treated with H2O and then with 10% HCl, H2O and saturated NaHCO3 and extracted with dichloromethane. The organic layer was dried with anhydrous magnesium sulfate, filtered, and concentrated to a crude oil, which was purified by column chromatography (ethyl acetate-n-hexane=1:3) to give pure white compound 12. Yield: 69% (695 mg), m.p : 157–158 °C : Compound 8  was dissolved in acetone (2 mL) and HF (0.3 mL) was added at 0 °C and stirred at room temperature for 2 h and treated as the same method of compound 9 to give a pale yellow product which was purified by column chromatography (ethyl acetate-n-hexane=1:1) to give pure white compound 13. Yield: 68% (71 mg), m.p: 280–282 °C, IR (cm-1): 3350 (OH), 2952, 1734 (17-C=O), 1653 (3-C=O); 1H-NMR (CDCl3) δ: 7.11 , 6.04 , 5.97 , 5.43 , 4.72 , 4.08 , 3.69 , 1.29 , 0.82 ; 13C-NMR (CDCl3) δ: 220.1 (C-17), 185.9 (C-3), 166.0 (C-5), 158.2 (C-1), 128.1 (C-2), 126.6 (C-4), 77.6 (C-6), 71.6 (C-7), 55.6, 47.5, 45.2, 43.7, 43.6, 35.9, 34.6, 31.5, 31.4, 21.7, 21.6, 20.9, 13.8; GC-MS (EI) m/z: 316 (M)+.-Cyano-6α,7α-epoxy-4-androstene-3,17-dione (14): Compound 8  was dissolved in 8:1 ethanol-H2O (9 mL), NaCN  was added and the mixture was stirred at room temperature for 4 h. The reaction mixture was cooled down and treated like compound 11 to give a pure pale yellow product 14. Yield: 77% (167 mg), m.p : 286–288 °C, IR (cm-1): 2951, 2250 (CN), 1733 (17-C=O), 1649 (3-C=O), 1H-NMR (CDCl3) δ: 6.29 , 3.56 , 3.49 , 3.17 , 1.24 , 0.96 ; 13C-NMR (CDCl3) δ: 218.7 (C-17), 192.4 (C-3), 158.6 (C-5), 131.8 (C-4), 118.5 (CN), 53.3 (C-6), 52.5 (C-7), 47.9, 46.8, 38.4, 38.2, 36.8, 35.9, 35.8, 34.6, 31.0, 21.6, 19.3, 18.3, 13.8; GC-MS (EI) m/z: 325 [M]+.2αT47D (estrogen receptor dependent cell line) and MDA-MB231 (estrogen receptor independent cell line) breast cancer cells were purchased from the Korean Cell Line Bank (Seoul). Formestane and tamoxifen were purchased from Aldrich and were used as positive controls. RPMI medium 1640, fetal bovine serum , penicillin-streptomycin, phosphate buffered saline (PBS) pH 7.4, 0.25% trypsin EDTA, dimethylsulfoxide , thiazolyl blue tetrazolium bromide  were also all commercially available reagents.3) were plated in 96 well plates and incubated in 5% CO2 incubator (NAPCO Water-Jacketed CO2 Incubator) for 24 h. Then cells were treated with 0, 1, 5, 10, 20, 50, 100 μM concentrations of sample (5 μL/well) and incubated in 5% CO2 incubator for 48 h at 37 °C. Triplicate wells were used for each concentration. After incubation for 48 h, 50 μL/well of MTT (2 mg/mL in PBS) was added and further incubated for 4 h in the dark. After removal of the MTT solution, 150 μL/well DMSO was added to dissolve the purple crystal for 15 minutes, after which the absorbance of the solution was measured at 540 nm with an ELISA reader . The inhibition effect of T47D and MDA-MB231 cell growth was expressed as relative cell survival percentage. Experiments were performed in triplicate.T47D and MDA-MB231 breast cancer cells were grown in RPMI medium 1640 containing 10% fetal bovine serum (FBS) supplemented with 1% penicillin-streptomycin. Cells (2 × 10


N,N'-bis(4-hydroxy)-benzylidene-o-toluidine (1). The structures of these compounds were confirmed by CHN, FT-IR, 1H-NMR, and 13C-NMR spectrophotometer. Their thermotropic liquid crystalline behavior was studied using differential scanning calorimetry (DSC) and polarizing optical microscope (POM). 4,4'-di(4-Hydroxybutoxy)-N-benzylidine-o-tolidine (2a) does not exhibit liquid crystalline properties. A nematic texture was observed for mesogenic diols 2b, and 2d, whereas the diol 2c exhibits a smectic mesophase. The increase of terminal alkyl chain in these mesogenic diols leads to a decrease in the transition temperature. A series of new mesogenic azomethine diols were successfully synthesized by condensation reactions between various chloroalkanols and  Organic compounds exhibit thermotropic liquid crystalline (LC) properties when the molecules possess rod- or disc-like structure. The stabilization and polarizability are two of the most important factors in liquid crystal devices. Researchers have discovered that small molecules are also capable of exerting a significant impact on the molecular polarizability as well as the stabilization in the liquid crystalline compounds. Azomethine (CH=N) linkages present in the backbone provide an attractive class of high performance material. High thermal stability, excellent mechanical strength, and good optoelectronic properties are also attributed to the resonance stabilization of the Schiff’s base unit . HoweverN,N' bis(4-hydroxyl)benzylidine-o-tolidine derivatives and to investigate the liquid crystalline properties of the new diols.Small molecules  with geometrical anisotropy and high polarizability often exhibit one or more liquid crystalline phases besides the crystalline and isotropic phases . Therefo2(a–d) is explained in 1 and the diols 2(a–d) were confirmed by elemental analysis (CHN), FT-IR, 1H-NMR and 13C-NMR spectrophotometry. 1, 2a, 2b and 2d. In the obtained FT-IR spectra the disappearance of the NH2 and carbonyl absorption bands at  cm-1and 1,667 cm-1 and the appearance of characteristic absorption bands at 1,620 cm-1 assigned to the presence of a -CH=N bond could be observed. This correlates with the data obtained by Issam [et al. [-1. The sharp absorption band at  cm-1 is attributed to the -OH group [-1 and 1,592 cm-1, which is corresponds to aromatic –C=C–, indicates that an aromatic ring exists in the synthesized compound. The FT-IR data for the diols 2(a–d) and the reactants are given in Experimental section. Preparation of diols  3,336–3,72 cm-1an [et al. , where t1H-NMR and 13C-NMR spectra, corresponding to diol 2b. In the 1H-NMR spectrum, the methylene protons in (HO-CH2-) and (Ph-O-CH2-) appear at 3.57 ppm and 4.05 ppm, respectively. This is similar to the data that obtained by Stamatoiu et al. [-1. 1H-NMR data for the diols 2(a–d) are given in the Experimental section and the properties are summarized in u et al. . The sinu et al. . The azo2(a–d) were studied by polarizing optical microscope (POM) and differential scanning calorimetry (DSC). 2(b–d). DSC analysis was carried out for all diols obtained and 2b. The crystal phase to nematic phase transition temperature (Cr-N) and nematic phase to isotropic phase transition (N-I) temperature is observed under POM. When the diols are observed under POM at 5 °C/min (to correlate with the DSC rate), the results revealed that the diols 2b, and 2d exhibit a nematic liquid crystal phase, diol 2c exhibits a smectic liquid crystal phase, whereas diol 2a do not show any liquid crystalline behavior, but simply changes from a solid to an isotropic fluid. Compounds that exhibit liquid crystalline behavior are due to their high aspect ratio (length to breadth ratio). The POM images for all obtained compounds are shown in The mesophases of the diols et al. [o-tolidine) possesses two methyl groups where each one is bonded to the aromatic ring in the meta position will increase molecular broadening of the rigid core, such that intermolecular interactions are responsible for mesomorphism decreased as the intermolecular separation increases. This would in turn increase the entropy (ΔS) of the nematic to isotropic phase transition. As explained earlier, 4-4'-di(6-hydroxyhexoxy)-N-benzylidene-o-tolidine (diol 2b), 4-4'-di(8-hydroxyoctoxy)-N-benzylidene-o-tolidine (2c) and 4-4'-di(10-hydroxydecoxy)-N,N'-benzylidene-o-tolidine (2d), only show marbled texture when heated above its melting point. The isotropic points for diols 2(b–d) are 275.2 °C, 231.0 °C and 251.0 °C, respectively. Diols 2b and 2d exhibit typical nematic texture and no trace of smectic structure was seen in these diols. Diol 2a do not show liquid crystalline behavior, but only melts at 242.11 °C. The reason that this compound does not exhibit any liquid crystal phase because is presumably because the carbon chain is not long enough. The effect of a flexible substituent in depressing the mesophase stability is greater in more ordered phases such as smectics than nematics . The alket al. . Chemicaet al. . Neverthet al. . The linet al. . This wio-tolidine , 4-hydroxybenzaldehyde , 4-chloro-1-butanol , 6-chloro-1-hexanol , 8-chloro-1-octanol , 10-chloro-1-decanol , potassium carbonate , absolute ethanol 99.5% , 1-butanol 99.5% , N,N-dimethylformamide Unless otherwise stated, all chemicals were commercially obtained and used without further purification or distillation: -1 and 400 cm-1 with a resolution of 2.0 cm-1. The 1H-NMR and 13C-NMR spectra were obtained using a Bruker 400 MHz and a 300 MHz spectrometer. The samples were prepared at 100 mg/mL in CDCl3 with tetramethylsilane (TMS) as the internal reference. CHN microanalyses were performed using a Perkin Elmer 2400 LS Series CHNS=O analyzer. Differential scanning calorimetry (DSC) studies were carried out with a Perkin-Elmer DSC7 series at heating rate of 5 °C/min in nitrogen, Melting points of LC monomers were obtained based on the peak of the melting temperatures from DSC analysis. Polarizing optical microscope (POM), a Carl Zeiss Axioskop equipped with 40 Linkam LTS350 hot stage, Linkam TMS94 temperature controller and Linkam LNP cooling system (pump) was used to determine the liquid crystalline mesophase. Thermal stability was studied using Perkin-Elmer TGA-7 under N2 atmosphere. The heating rate was programmed to 5 °C/min and it was performed at the temperature range of 30–900 °C.FT-IR spectra were measured using a Perkin-Elmer 2000 FTIR equipped with a potassium bromide (KBr) beam splitter. Thirty-two (32) scans were collected between 4000 cmp-Hydroxybenzaldehyde  was added dropwise into a solution of o-tolidine  in absolute ethanol (60 mL) in a 500 mL reaction flask. The mixture was refluxed for 6 h with magnetic stirring. The product was then filtered, washed several times with diethyl ether and dried in the vacuum oven at 70 °C. Final purification was carried out by re-crystallization using 1-butanol to give yellow crystals. Yield was 74% with a melting point of 281–282 °C. FT-IR (KBr disc): 3,414 cm-1 (O-H stretch), 1,608 cm-1 (C=N stretch), 1,516 cm-1 (C=C stretch). 1H-NMR (400 MHz): δ 8.56 , 6.05 , 7.79–6.85 , 7.35 , 7.27 , 7.76 , 2.34  ppm. 13C-NMR (CDCl3): δ 18.64, 116.21, 122.68, 126.34, 129.12, 130.62, 131.08, 132.14, 141.24, 151.36, 160.58, 161.23 ppm.1 (0.20 mol) in DMF (60 mL) was added in the above mixture in a 500 mL reaction flask. The mixture was refluxed at 130 °C for 12 h. The product was precipitated by pouring the obtained solution into cold distilled water (2 L). The precipitate was then filtered, washed several times with diethyl ether and dried in a vacuum oven at 90 °C. Final purification was carried out by re-crystallization using the mixture solvent of DMF-butanol (1:1).Chloroalkanol (0.40 mol) and sodium carbonate (0.45 mol) were dissolved in DMF (400 mL). Bisphenol 4,4'-di(4-Hydroxybutoxy)-N-benzylidine-o-tolidine (2a). Colour: yellow. Yield: 58%. Melting point: 242–244 °C. FT-IR (KBr disc): 3,414 cm-1 (O-H stretch), 1,607 cm-1 (C=N stretch), 1,509 cm-1 (C=C stretch). 1H-NMR (400 MHz): δ 8.37 , 7.87–6.98 , 7.50 , 7.45 , 7.85 , 4.03 , 3.65 , 2.41 , 1.89–1.49  ppm. 13C-NMR (CDCl3): δ 18.5 (Ph-CH3), 25.57–32.31 (-CH2-), 62.00 (-O-CH2-), 69.54 (-CH2-OH), 114.0–160.74 (aromatic carbons), 162.24 (-CH=N-) ppm. Elemental analysis: found: C, 76.43; H, 7.14; N, 5.06, C36H40N2O4.Calcd: C, 76.59; H, 7.09; N, 4.96. 4,4'-di(6-Hydroxyhexoxy)-N-benzylidine-o-tolidine (2b). Colour: Brown. Yield: 70%. Melting point: 160–162 °C. FT-IR (KBr disc): 3,419 cm-1 (O-H stretch), 1,621 cm-1 (C=N stretch), 1,509 cm-1 (C=C stretch). 1H-NMR (400 MHz): δ 8.35 , 7.90–7.02 , 7.51 , 7.49 , 7.87 , 4.05 , 3.65 , 2.41 , 1.90-1.30  ppm.13C-NMR (CDCl3): δ 18.52 (Ph-CH3), 25.57-32.31 (-CH2-), 62.65 (-O-CH2-), 68.64 (-CH2-OH), 114.43–158.74 (aromatic carbons), 161.24 (-CH=N-) ppm. Elemental analysis: found: C, 77.30; H, 7.79; N, 4.50, C40H48N2O4.Calcd: C, 77.14; H, 7.74; N, 4.51. 4,4'-di(8-Hydroxyoctoxy)-N-benzylidine-o-tolidine (2c). Colour: yellow. Yield: 68%. Melting point: 119–120 °C. FT-IR (KBr disc): 3,419 cm-1 (O-H stretch), 1,621 cm-1 (C=N stretch), 1,509 cm-1 (C=C stretch). 1H-NMR : δ 8.35 , 7.90–6.96 , 7.49 , 7.46 , 7.88 , 4.02 , 3.66 , 2.42 , 1.86–1.29  ppm. 13C-NMR : δ 18.5 (Ph-CH3), 25.57–32.31 (-CH2-), 62.00 (-O-CH2-), 69.54 (-CH2-OH), 114.50–160.45 (aromatic carbons), 162.40 (-CH=N-) ppm. Elemental analysis: found: C, 78.28; H, 8.11; N, 4.20, C44H56N2O4.Calcd: C, 78.1; H, 8.28; N, 4.14. 4,4'-di(10-Hydroxydecoxy)-N-benzylidine-o-tolidine (2d). Colour: yellow. Yield: 81%. Melting point: 108–109 °C. FT-IR (KBr disc): 3,353 cm-1 (O-H stretch), 2931 cm-1, 2,862 cm-1, 1,620 cm-1 (C=N stretch), 1509 cm-1 (C=C stretch). 1H-NMR : δ 8.39 , 7.92–6.98 , 7.51 , 7.49 , 7.88 , 4.07 , 3.68 , 2.46 , 1.89–1.25  ppm. 13C-NMR (CDCl3) δ 18.5 (Ph-CH3), 25.57–32.31 (-CH2-), 62.00 (-O-CH2-), 69.54 (-CH2-OH), 114.0–160.74 (aromatic carbons), 162.24 (-CH=N-) ppm. Elemental analysis: found: C, 78.60; H, 8.90; N, 3.80, C48H64N2O4.Calcd: C, 78.68; H, 8.74; N, 3.82.N,N'-bis(4-hydroxyl)benzylidine-o-tolidine compounds with various alkyl chain lengths , was successfully synthesized. The diols were found to demonstrate enantiotropic nematic phase based on the POM and DSC observations. Diols 2b and 2d exhibit enantiotropic nematic liquid crystal phase (marble texture and shredded texture respectively) but diol 2a did not exhibit any liquid crystalline phase, while the diol 2c exhibits a smectic mesophase. DSC analyses showed that the increase in alkyl chain length in diols will result in a the decrease in the transition temperature for this series of compounds. A series of


Their structures were elucidated on the basis of spectral and chemical evidence. Four new dammarane-type triterpene saponins named floranotoginsenosides A ( Panax notoginseng (Burk) F. H. Chen , commonly referred to notoginseng and “San-Qi”, is a well-known medicinal herb, used historically in traditional Chinese medicine as a hemostatic agent that both invigorates and builds blood. The flowers of P. notoginseng have been used for treating hypertension, vertigo, tinnitus and laryngopharyngitis -20-O-[β-D-xylopyrano-syl(1→6)]-β-D-glucopyranoside (6) D25: -4.93o (MeOH). The molecular formula of 1 was determined to be C53H90O23 on the basis of its negative FAB-MS  and high-resolution ESI-MS data . The 1H-NMR spectrum of 1 showed signals assignable to eight singlet methyls  together with four anomeric proton signals at δΗ 4.94 (s), 4.82 , 4.66 , 4.60 . In the 13C-NMR spectrum of 1, 30 carbon signals due to the aglycone moiety were in good agreement with those of 5, especially the olefinic carbon signals (δC 123.8 and 141.9), and the oxygenated carbon signal (δC 71.5) of the side chain, suggesting that the aglycone of compound 1 is the same as that of 5, in which a double bond located at C-23, 24 and a hydroxyl group attached to C-25. The signals at δC 91.3 and 84.6 indicated that 1 is a bisdesmoside. Besides the aglycone’s 13C-NMR signals, the remaining carbon signals of compound 1 were found to be superimposable on those of compound 5 except for the presence of signals derived from an α-L-arabinofuranosyl moiety instead of β−D-xylopyranosyl moiety in 5, suggesting that oligosaccharide chain of α-L-arabinofuranosyl-(1→6)-β-D-glucopyranosyl attached to 20-OH of the aglycone. This was further supported by chemical shift comparison of these sugar signals with those of compound 8. On the basis of these results, the structure of floranotoginsenoside A was assigned to formula 1 (Compound ormula 1 . (2) was also obtained as a white powder, whose negative FAB-MS showed a quasimolecular ion peak at m/z 1,091 ([M-H-H2O]-). The molecular formula C53H90O24 was determined by high-resolution ESI-MS . The 1H-NMR spectra of 2 showed four anomeric proton signals at δΗ 5.38 , 5.05 , 5.00 , 4.92 , a vinyl methyl [δΗ 1.96 ], and a methine proton bearing a hydroperoxyl group [δΗ 4.79 ] which was supported by chemical shift comparison with those of related hydroperoxylated triterpenes - peak at m/z 1,091 in the negative FAB-MS and [M-H]- at m/z 1,109.5747 in negative HR-ESI-MS. Its 1H-NMR and 13C-NMR spectral data again suggested a side chain with a hydroperoxyl group at C-24, as evidenced by the signals ascribed to the vinyl methyl [δΗ 1.97 ], and methine group bearing hydroperoxyl group . Comparison of the 1H-NMR and 13C-NMR spectra of 3 with those of 2 revealed that the only difference was the replacement of the terminal β-D-xylopyranosyl group in C-20 position in 2 by an α-L-arabinofuranosyl moiety in 3. Based on these evidences, structure 3 was assigned to floranoto-ginsenoside C (Floranotoginsenoside C (noside C . 4) has the molecular formula C53H90O23 which is one oxygen less than 3, on the basis of the [M-H]- peak at m/z 1,093 in the negative FAB-MS and m/z 1,093.5792 for [M-H]- in negative HR-ESI-MS spectra. It’s 1H-NMR and 13C-NMR spectra closely resembled those of 3, especially the signals arising from the sugar moiety and the rings A-D, indicating that 4 is a also dammaranes bidemoside. In comparison of the 13C-NMR spectra of 4 with that of 3, the upfield shift of C-24 (ΔδC -13.7) and C-26 (ΔδC -2.9), as well as the downfield shift of C-25 (ΔδC +3.4) were observed, indicating that a hydroxyl group is located at C-24 position in 4. This was supported by chemical shift comparison of H-24 (δH 4.42) and C-24 (δC 76.4) with that of 9 and related compounds with the same side chain structure D25: -4.93º (MeOH). Negative FAB-MS (m/z):1,093 [M-H]-; Negative HR-ESI-MS (m/z): 1,093.5790 [M-H]- ; 1H-NMR : δ 0.86, 0.91, 0.93, 1.02, 1.07, 1.33, , 1.29 , 4.60 , 4.66 , 4.82 , 4.94  5.71 ; 13C-NMR data are listed in Floranotoginsenoside B (2): C53H90O24, white powder, [α]D25: -9.50º  (MeOH); Negative FAB-MS (m/z):1,091[M-H-H2O]-; Negative HR-ESI-MS (m/z) 1,109.5748 [M-H]- ; 1H-NMR : δ0.81, 0.94, 0.96, 1.11, 1.28, 1.64, 1.96 , 3.27 , 4.74 , 4.92 , 5.00 , 5.05 , 5.38 ; 13C-NMR data are listed in Floranotoginsenoside C (3): C53H90O24, white powder, [α]D25: -14.68º (MeOH); Negative FAB-MS (m/z):1,091[M-H-H2O]-; Negative HR-ESI-MS (m/z) 1,109.5747 [M-H]- ; 1H-NMR : δ 0.80, 0.94, 0.94, 1.11, 1.28, 1.62, 1.97,, 3.27 , 4.79 , 4.91 , 5.10 , 5.39 , 5.66 ; 13C-NMR data are listed in Floranotoginsenoside D (4): C53H90O23, white powder, [α]D25: -14.04º (MeOH); Negative FAB-MS (m/z):1,093[M-H]-; Negative HR-ESI-MS (m/z) 1,093.5792 [M-H]- ; 1H-NMR : δ0.80, 0.93, 0.99, 1.10,1.28, 1.64, 1.95, , 3,25 , 4.90 , 5.10 , 5.09 , 5.40 , 5.66 ; 13C-NMR data are listed in Reduction of2and3: Triphenylphosphine (10 mg) was added to a solution of 2 (10 mg) in MeOH (5 ml). After stirring at room temperature for 4 hr, the solution was evaporated to dryness under reduced pressure, then subjected to silica gel chromatography (CHCl3-MeOH-Water: 8:2:0.2) to afford 9 (6.1 mg) whose 1H- and 13C-NMR spectral data are completely identical with those of gypenoside LXXI. Reduction of 3 (10 mg) in a manner similar to that described for 2 yielded floranotoginsenoside D .2 and 3 have a hydroperoxyl group. Although hydroperoxydammarane triterpene saponins have been previously isolated from the roots of Panax notoginseng [Among four new darmmarane-type triterpene saponins isolated from notoginseng flowers, compounds oginseng , this wa


Allium macrostemon Bunge. Their structures were elucidated from their spectral data as (25R)-26-O-β-D-glucopyranosyl-5α-furostane-3β,12β,22,26-tetraol-3-O-β-D-glucopyranos-yl (1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galactopyranoside (1), (25R)-26-O-β-D-glucopyranosyl-5α-furostane-3β,12α,22,26-tetraol-3-O-β-D-gluco- pyranosyl (1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galacto- pyranoside (2) and (25R)-26-O-β-D-glucopyranosyl-5β-furostane-3β,12α,22,26-tetraol-3-O-β-D-glucopyranosyl (1→2)-β-D-galactopyranoside (3), respectively. The inhibition effect of all compounds on CD40 ligand (CD40L) expression on the membrane of activated platelets stimulated by ADP was tested. Compounds 1 and 2 exhibited significant inhibitory activities in a dose dependent manner (P < 0.05), suggesting their potential application as CD40L inhibitors.Three new compounds were isolated from the dried bulbs of  The positive reaction to the Ehrlich reagent suggests a furostanol glycoside structure for 1 -β-D-glucopyranosyl (1→4)-β-D-galactopyranoside  and carbon signal at δC 71.5, proton signal at δH 3.98 (H-12) and carbon signal at δC 29.5 (C-11) and 45.6 (C-13) revealed the presence of hydroxyl group at C-12. The NOESY correlation between δH 3.98 (H-12) and δH 0.99 (H-18) suggested its α-orientation. The presence and configuration of C-12 hydroxyl group was also supported by carbon signals of C-11, C-12, C-13, C-14 and C-17 of 2 shifted higher field by approximately – 2.1, - 7.8, - 1.2, - 6.7 and - 9.2 ppm, respectively, while the carbon signal of C-18 shifted to down field by +6.1 ppm comparing with those of 1. Therefore, the structure of 2 was established as (25R)-26-O-β-D-glucopyranosyl-5α-furostane-3β,12α,22,26-tetraol-3-O-β-D-gluco- pyranosyl (1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galactopyranoside  and 1.02 (H-19). The 1H-NMR and 13C-NMR spectra of 3 showed three anomeric protons at δH 4.80 , 4.84  and 5.25  and three anomeric carbons at δC 104.9, 102.3 and 106.0, suggesting that there were only three sugar units in 3 and the same large constants  for anomeric protons revealed the β-configuration of all these sugars. Acid hydrolysis of 3 yielded D-glucose and D-galactose in a ratio of 2:1. In the HMBC spectrum, long-range correlations from δH 4.84  to δC 75.1 (C-3 of aglycone), from δH 5.25  to δC 81.7  and from δH 4.80  to δC 75.3 (C-26 of aglycone) showed that two sugar chains were separately attached to C-3 and C-26 of the aglycone, and two glucoses were linked at C-2 of the inner galactose attached at C-3 and C-26 of aglycone respectively. Thus, the structure of 3 was established as (25R)-26-O-β-D- glucopyranosyl-5β-furostane-3β,12α,22,26-tetraol-3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside (Compound ranoside .1-3 of CD40 ligand (CD40L) expression on the membrane of activated platelets stimulated by ADP was measured by real time PCR. Compounds 1 and 2 exhibited significantly inhibit expressions of CD40L in ADP induced active platelets in a dose dependent manner (P < 0.05) (The inhibitory effect of compounds  < 0.05)  which su3 micro-melting point apparatus and are uncorrected. Optical rotations were obtained on a P-1020 digital polarimeter (JASCO Corporation). IR spectra were measured on a Shimadzu FT/IR-8400 spectrometer. 1D and 2D NMR spectra were taken on a Bruker AV-400 (400 MHz for 1H-NMR) spectrometer in C5D5N solution. ESIMS spectra were acquired using a Bruker Esquire 2000 mass spectrometer. Column chromatography was carried out on Diaion HP-20 , silica gel  and ODS . TLC analyses were taken on Silica gel 60F254  and the spots were detected spraying with Ehrlich reagent and heating. Preparative HPLC was performed using an ODS column . Quantitative real-time PCR was performed using the PE5700 PCR system . The Qiagen RNeasy minikit system  was used to collect mRNA from ADP induced active platelets. The mRNA was converted to cDNA using the AMV Reverse Transcription System .Melting points were determined with a Yanaco MP-SAllium Macrostemon Bunge were purchased from Liaoning Province of China and identified by Professor Qi-Shi Sun . The voucher specimen has been deposited at the Department of Natural Product Chemistry, Shenyang Pharmaceutical University, Shenyang (110016), P. R. China.The bulbs of Allium Macrotemon Bunge (5 kg) were extracted twice with 60% ethanol (10 L) for 2 h each time. The ethanol extraction was concentrated under reduced pressure, suspended in water and then passed through Diaion HP-20 column using EtOH/H2O gradient system (0–100%). The 60% EtOH eluted fraction (90 g), which was subjected to silica gel column chromatography with CHCl3/MeOH/H2O  and MeOH finally, gave nine fractions. Fraction 2 was further purified by ODS column chromatography eluting with MeOH/H2O  to afford five sub fractions. The third subfraction was also further purified by repeated Rp-18 HPLC preparation, eluting with MeOH/H2O , to yield 1 (8.1 mg), 2 (8.5 mg) and 3 (30.6 mg). The dried bulbs of Compound1((25R)-26-O-β-D-glucopyranosyl-5α-furostane-3β,12β,22,26-tetraol-3-O-β-D-gluco- pyranosyl(1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galactopyranoside). White amorphous powder, m.p. 216–217 ºC, -26.4° . HR-ESIMS (positive mode) at m/z 1283.5862 [M+ Na]+ . ESI-MS (positive mode) at m/z 1283 [M+ Na]+, 1265 [M+ Na- H2O]+, 1121 [M+ Na- 162]+, 1103 [M+ Na- H2O- 162]+, 941 [M+ Na- H2O- 162× 2]+, 779 [M+ Na- H2O- 162× 3]+; ESI-MS (negative mode) at m/z 1259 [M- H]-, 1097 [M- H- 162]-, 935 [M- H- 162× 2]-, 773 [M- H- 162× 3]-, 611 [M- H- 162× 4]-; IR νmax (KBr) cm-1: 3419 (OH), 2937 (CH), 1000-1100; 1H-NMR (C5D5N) data see 13C-NMR, see 2 Compound ((25R)-26-O-β-D-glucopyranosyl-5α-furostane-3β,12α,22,26-tetraol-3-O-β-D-gluco- pyranosyl (1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galactopyranoside). White amorphous powder, m.p. 177–178 ºC, -3.5° . HR-ESIMS (positive mode) at m/z 937.5075 [M+ H]+ . ESI-MS (positive mode) at m/z 959 [M+ Na]+, 941 [M+ Na- H2O]+, 919 [M+ H- H2O]+, 797 [M+ Na- 162]+, 617 [M+ Na- H2O- 162× 2]+, 595 [M+ H- H2O- 162× 2]+, 433 [M+ H- H2O- 162×2- 162]+; ESI-MS (negative mode) at m/z 935 [M- H]-, 773 [M- H- 162]-, 611 [M- H- 162× 2]-; IR νmax (KBr) cm -1: 3415 (OH), 2925 (CH), 1045; 1H-NMR (C5D5N) data see 13C-NMR, see Compound3 ((25R)-26-O-β-D- glucopyranosyl-5β-furostane-3β,12α,22,26-tetraol-3-O-β-D- glucopyranosyl(1→2)-β-D-galactopyranoside).White amorphous powder, m.p. 215–216 ºC,  -28.7° . HR-ESIMS (positive mode) at m/z 1283.5891 [M+Na]+ . ESI-MS (positive mode) at m/z 1283 [M+ Na]+, 1121 [M+ Na- 162]+, 959 [M+ Na- 162× 2]+, 797 [M+ Na- 162× 3]+, 779 [M+ Na- 162× 3- H2O]+; ESIMS (negative mode) at m/z 1259 [M- H]-, 1097 [M- H- 162]-, 935 [M- H- 162× 2]-, 773 [M- H- 162× 3]-, 611 [M- H- 162× 4]-; IR νmax (KBr) cm -1: 3429 (OH), 2933 (CH), 1039; 1H-NMR (C5D5N) data see 13C-NMR, see 2OH·HCl (2 mg) were added to the residue, and the mixture was heated at 100 ºC for 1 h. After cooling, Ac2O (0.5 mL) was added and the mixtures were heated at 100 ºC for 1 h. The reaction mixtures were evaporated under reduced pressure, and the resulting aldononitrile peracetates were analyzed by GC-MS using standard aldononitrile peracetates as reference samples. D-glucose and D-galactose were identified in a ratio of 4:1 for compound 1 and 2 and in a ratio of 2:1 for compound 3. Each glycoside (5 mg) was heated in an ampoule with 15% aq. HCl (5 mL) at 110 ºC for 2 h. The aglycone was extracted with dichloromethane three times and the aqueous residue was evaporated under reduced pressure. Then, pyridine (1 mL) and NH9 platelets/mL. PRP (400 μL) was added to the wells of a tissue-culture plate  and pretreated with compounds  or AR-C67085MX (10 μM) at 37 ºC for 10 min, and then stimulated with 20 μM ADP for 5 min. After obtaining consent, blood samples were collected from healthy volunteers  and 3.8% sodium citrate  was added as an anticoagulant. All subjects avowed that they had not taken any anti-platelet drug at least 2 weeks prior to the extraction. Platelet-rich plasma (PRP) was prepared by centrifugation at 125×g for 10 min at room temperature. Platelet count was adjusted to 5 × 10β-actin primers for PCR analysis were based on the human platelet sequences and designed using the Primer 5 software. The forward and reverse primers were as follows:The mRNA was collected from ADP induced active platelets following the manufacturers' recommended protocol. The mRNA was converted to cDNA and cDNA samples were kept at -20 ºC until needed. The CD40L and housekeeping gene CD40L mRNA: 5′ACATACAACCAAACTTCTCCC 3′ and 5′AGATGTTGTTTTACTGCTGGC 3′, (799 bp)β-actin mRNA: 5’-AAGGATTCCTATGTGGGC-3’ and 5’-CATCTCTTGCTCGAAGTC-3, (362 bp)The reaction mixture contained TaqMan universal PCR Master Mix (25 μL), 10 μM (2 μL) of each primer, TaqMan probe (2.5 μL) and cDNA (2 μL). It was adjusted to 22.5 μL with DNase/RNase free water. Conditions of the real-time-PCR were as follows: 50 ºC for 2 min, 95 ºC for 15 min, and for 40 cycles, 95 ºC for 15 s and 60 ºC for 1 min. The Ct value is defined as the number of fractional cycles in which the fluorescence emitted by the reaction mixture exceeds a preset threshold and marks the beginning of an exponential growth of the fluorescence signal. The results were analyzed with PE 5700 software.p values less than 0.05 were considered to be significantly.All results were expressed as the mean±standard error (SE). Analyses were performed with SPSS software Version 13.0 . ANOVA was used to determine if significant variation existed between group means. Two-side


Alternaria alternata JS0515 was isolated from Vitex rotundifolia (beach vitex). Twelve secondary metabolites, including one new altenusin derivative (1), were isolated. The isolated metabolites included seven known altenusin derivatives (2–8), two isochromanones , one perylenequinone (11), and one benzocycloalkanone (12). Their structures were determined via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and computational electronic circular dichroism (ECD) analysis. Compounds 3 and 11 increased pyruvate dehydrogenase (PDH) activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. The IC50 values of 3 and 11 were 32.58 and 27.82 μM, respectively.The fungal strain  Endophytes are microorganisms that live within the internal tissues of plants, and they form symbiotic relationships with their host plants . The funVitex rotundifolia Linne fil. (Verbenaceae) is a halophyte that is widely distributed along the coast of East Asia +, calcd for C16H14O8Na, 357.0581; 1H-NMR  δ 11.12 , 7.25 , 7.23 , 7.17 , 6.81 , 3.92 , 3.61 , 1.51 ; 13C-NMR  δ 196.5 (C-10), 168.0 (C-14), 166.0 (C-5), 165.5 (C-1), 163.8 (C-3), 163.5 (C-8), 131.5 (C-7), 125.8 C-9), 106.0 (C-6), 105.0 (C-4), 99.6 (C-2), 88.8 (C-12), 87.5 (C-11), 56.4 (5-OCH3), 53.0 (14-OCH3), 24.3 (C-13); 1H- and 13C-NMR  spectroscopic analysis  H-4→C-2, C-3, C-5, and C-6; H-6→C-2, C-4, C-5, and C-8; H-9→C-7, C-8, C-10, C-11, and C-12; H-13→C-8, C-11, and C-12; 11-OH→C-10, C-11, C-12, and C-14; 5-OCH3→C-5; 14-OCH3→C-14.Alternatiol  was added and incubated overnight. Next, the cells were washed and Alexa fluor 488, goat anti-rabbit ab  was added with Hoechst 33258  and incubated for two hours. Finally, cells were washed and the plates were measured in Operetta . The raw data were normalized for the pharmacological inhibitory control (5 mM dichloroacetate (DCA)) and percent effect values using the software package Harmony High-Content Imaging and Analysis Software 3.1. The Deose response curves were generated by plotting the percent effect values and calculated ICp < 0.05 for all analyses) was assessed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons using Prism 5.01 software .All values are expressed as means ± standard error of the mean. The statistical significance threshold (