Belamcanda chinensis (L.) DC. The ethyl acetate extract showed greater antitumor activities than the other extracts, consequently leading to the isolation of 18 compounds identified as β-sitosterol (1), dausterol (2), quercetin (3), kampferol (4), shikimic acid (5), gallic acid (6), ursolic acid (7), betulin (8), betulonic acid (9), betulone (10), tectoridin (11), irisflorentin (12), 4′,5,6-trihydroxy-7-methoxyisoflavone (13), tectorigenin (14), irilins A (15), iridin (16), irigenin (17), and iristectongenin A (18). Compounds 3–10, 13, and 15 were isolated from B. chinensis for the first time. Compounds 4 and 7–10 showed potent cytotoxic activities against PC3, MGC-803, Bcap-37, and MCF-7 cell lines. The mechanism of the antitumor action of compound 7 was preliminarily investigated through acridine orange/ethidium bromide (AO/EB) staining, Hoechst 33258 staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which indicated the growth inhibition of MGC-803 cells via the induction of tumor cell apoptosis.An activity-directed fractionation and purification process was used to isolate antitumor compounds from the roots of  Belamcanda chinensis (L.) DC from the Iridaceaefamily consists of about 60 genera and 800 species worldwide and mainly two genera (Iridacea and Belamcanda) in China. B. chinensis is a shrub that is mainly found distributed in the southwestern part of China, particularly in Guizhou, Yunnan, and Sichuan provinces [B. chinensis is a perennial herbaceous plant with fan-shaped leaves that reach 2 to 3 feet in length on branching stems. The root of B. chinensis is a widely used important medicine in China for curing pulmonary diseases, acute and chronic pharyngitis, asthma, and cancer [B. chinensis as an official drug, which showed that the plant could be used as an anticancer herb. However, so far the constituents with anticancer activity of the plant remain unclear. Thus it is necessary to identify the potent antitumor compounds from this plant.rovinces ,2. B. chB. chinensis had discovered the main chemical components of B. chinensis, including isoflavonoids [B. chinensis systematically, and we tested the antitumor activity of the extracts and compounds. The current study was conducted to validate the medicinal use of B. chinensis. Finally, the isolation and structural elucidation of 18 compounds from the ethyl acetate extracts, which included eight is of lavones, two flavones, four triterpenes, were performed. Ten of the compounds were isolated from B. chinensis for the first time. During the screening for the antitumor agents from tropical medicinal plants, the ethyl acetate extract of B. chinensis exhibited great antitumor activity, and some of the compounds isolated from the ethyl acetate extract showed moderate antitumor activity against PC3, MGC-803, Bcap-37, MCF-7 cell lines and less cytotoxic activities toNIH3T3 cells which suggesting that these compounds can be used as lead compounds for further research.Previous chemical investigations on avonoids ,5,6,7 anavonoids ,9,10. WeB. chinensis (7.5 kg) were refluxed for 6 h with petroleum ether, ethyl acetate, and methanol, respectively. Each solvent extraction process was performed twice, and the extracts were combined and concentrated using a vacuum rotary evaporator at 50 °C to afford the petroleum ether extract (40 g), ethyl acetate extract (289 g), and methanol extract (321 g), respectively, which were evaluated for their antitumor activity using an MTT assay. The results suggest that the antitumor agents were mainly contained in the ethyl acetate extract. Further experiments were performed on the ethyl acetate extract to separate its antitumor compounds. The ethyl acetate extract was subjected to successive chromatographic fractionation and purification to yield compounds 1–18, as shown in The dried roots of 1H- and13C-NMR spectroscopy. The results of the analyses were compared with the NMR and IR data reported in literature to identify the chemical structures of the isolates. Compounds 3–10, 13, and 15 were isolated from B. chinensis for the first time.The isolated compounds were identified via spectroscopic analyses, including in vitro cytotoxic activity of all the compounds was evaluated against PC3, MGC-803, Bcap-37 and NIH3T3 cell lines. Adriamycin (ADM) and the compounds were dissolved using DMSO (negative control). The negative control cells were treated with culture medium containing 0.1% DMSO, while ADM, which showed very high antitumor activity against many kinds of tumor cells, was used as positive control. The inhibitory percentage of cells was treated with 20 μmol/L or 50 μg/mL of each compound or extract for 72 h (7–10) from the ethyl acetate extract exhibited moderate inhibitory activities against the growth of human carcinoma cell lines, which are higher than their activities against normal cell line NIH3T3. However the is of lavonoids (compounds 11–13) did not show obvious antitumor activities. The results suggest that the ethyl acetate extract of B. chinensis exhibited greater antitumor activities mainly because of the pentacyclic triterpene constituents. At the same time, compared with the crude extracts, the inhibitory activities of the isolated compounds were markedly reduced. The decrease in antitumor activity may be attributed to the synergistic effect of the chemical compounds of the ethyl acetate extract. The for 72 h . The resB. chinensis for the first time. UA was also found to have the greatest potency against the growth of human carcinoma cell lines and little cytotoxic effect on NIH3T3 cells among the isolated constituents. The IC50 of ursolic acid against MGC-803 cells was determined to be 22.32 ± 2.7 μmol/L using the MTT assay. Therefore, the morphological change in MGC-803 cells treated by UA was investigated through acridine orange/ethidium bromide (AO/EB) staining and Hoechst 33258 staining under fluorescence microscopy to determine whether the growth inhibitory activity of UA is related to the induction of apoptosis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was also conducted to confirm cell apoptosis. Hydroxy camptothecine (HCPT) was used as positive control, and the results are shown in Ursolic acid (UA) was obtained from AO is a vital dye that could stain nuclear DNA across an intact cell membrane, where as EB could only stain cells that have lost membrane integrity. The stained cells revealed the following four different types under a fluorescence microscope: green coloration of living cells with normal structure; red coloration of non-apoptotic dead cells with normal structure; green coloration of early apoptotic cells with morphous in the form of pycnosis; and orange coloration of late apoptotic cells with morphous in the form of pycnosis. The cytotoxicity of UA at a concentration of 10 μmol/L against MGC-803 cells for 48 h was detected via AO/EB staining, with hydroxyl camptothecine (HCPT) as positive control.As can be seen in Live cells with uniformly light blue nuclei were treated with Hoechst 33258 and observed under a fluorescence microscope. The apoptotic cells exhibited bright blue nuclei because of karyopyknosis and chromatin condensation, and the nuclei of dead cells could not be stained. The UA induced apoptosis at 10 μmol/L against MGC-803 cells for 48 h as detected using Hoechst 33258 staining. The HCPT concentration was also 10 μmol/L against the MGC-803 cells for 48 h.As can be seen in in situ via the detection of DNA fragmentation. The cells were observed under a fluorescence microscope, where brown precipitate was the result of positive apoptosis. UA and HCPT were at concentrations of 10 μmol/L against MGC-803 cells for 24 h. As can be seen in TUNEL is a popular method for identifying apoptotic cells 1H-NMR and 13C-NMR were recorded using a JEOL-ECX500 spectrometer at 22 °C, with tetramethylsilane as the internal standard and CDCl3, DMSO-d6, CD3COCD3, or CD3OD as the solvent. Column chromatography was performed using silica gel (200–300 meshes)  and silica gel H , Sephadex LH-20 , Amberlite XAD-7HP , HP-20 , and MCI-gel CHP 20 P . All other chemicals were of analytical reagent gradeand used without further purification.The melting points of the products were determined using an XT-4 binocular microscope . Infrared spectra were recorded on a Bruker VECTOR22 spectrometer in KBr disks. B. chinensis were collected from Bijie, Guizhou Province in China, in August 2011. Prof. Qing-De Long, Department of Medicine, Guiyang Medical University, identified the plant material. A voucher specimen was deposited at Guiyang Medical University, Guiyang, China.Fresh samples of B. chinensis (7.5 kg) were refluxed with petroleum ether (20 L), ethyl acetate (20 L), and methanol (30 L), respectively (3 times). Petroleum ether extract (40 g), ethyl acetate extract (289 g), and methanol extract (321 g) were obtained. The viscous dark mass of the ethyl acetate extract was adsorbed on silica gel (200–300 mesh) for column chromatography over a silica gel column packed in petroleum ether. The column was eluted successively with petroleum ether, a mixture of petroleum ether, and ethyl acetate , chloroform, and finally, a mixture of chloroform and methanol . Ten fractions were obtained. Fraction 1 (12 g) was applied to qsilica gel (200–300 mesh) column eluted with petroleum ether–EtOAc (10:1 to 0:1) to yield five fractions. Each fraction was separated via repeated silica gel chromatographyeluted with petroleum ether–EtOAc (10:1 to 0:1) to yield β-sitosterol  anddaucosterol . Fraction 2 (17 g) was applied to a silica gel (200–300 mesh) column eluted with petroleum ether–EtOAc and CHCl3–MeOH (10:1 to 0:1) to yield seven fractions of A–G. Fraction A was applied to asilica gel H columneluted with petroleum ether–EtOAc (20:1 to 0:1) to yield a yellow powder (4 g), which was applied to an Amberlite XAD7HP column first and then to a Sephadex LH-20 column using MeOH as elutant to obtain quercetin  and kampferol . Fraction B was separated using asilica gel H column eluted with petroleum ether–EtOAc (20:1 to 0:1) to yield a viscous dark mass (230 mg). Fraction B was then decolorized and separated on MCI geleluted with 50%–90% MeOH-H2O to yield shikimic acid  and gallic acid . Fractions Cand D were separated using HP-20 resineluted with 50%–90% MeOH–H2O, decolorized on Amberlite XAD-7HP first and then on MCI gel, and purified using a Sephadex LH-20 column eluted with MeOH to yield ursolic acid . Fractions 3 and 4 were applied to asilica gel (200–300 mesh) column eluted with petroleum ether–EtOAc (15:1 to 0:1) and EtOAc–MeOH (10:1 to 0:1) to yield a brown gum (208 mg), and then they were decolorized on MCI gel to yield two fractions, namely, Fractions 1′ and 2′. Fraction 2′ was purified using silica gel (100–200 mesh) eluted with petroleum ether–EtOAc (20:1 to 0:1) to yield betulin , betulonic acid , and betulone . Fractions 5 and 6 were applied to asilica gel (200–300 mesh) column eluted with petroleum ether–EtOAc (10:1 to 0:1) and CHCl3–MeOH (20:1 to 0:1) to afford five fractions, namely, Fractions 1″–5″. Fractions 1″–3″ were purified using a Sephadex LH-20 column eluted with MeOH to yield tectoridin  and irisflorentin . Fractions 4″ and 5″ were purified using PTLC to yield 4′,5,6-trihydoxy-7-methoxyisoflavone  and tectorigenin . Fractions 7–10 were applied to asilica gel (200–300 mesh) column eluted with petroleum ether–EtOAc (15:1 to 0:1) and EtOAc–MeOH (10:1 to 0:1) to yield three fractions, namely, Fraction 1–3′′′. Fraction 3′′′ was separated using silica gel (100–200 mesh) and purified through Sephadex LH-20 to yield irigenin  and a yellow powder (27 mg), which was consequently separated using MCI gel eluted with MeOH–H2O (20%–90%) to yield irilins A , iridin , and iristectorigenin A .The dried roots of Quercetin (3): C15H10O7, yellow powder, mp 306–308 °C, IR  v: 3346, 1653, 1616, 1506; 1H-NMR (CD3OD) δ: 7.70 , 7.62 , 6.85 , 6.34 , 6.08 . 13C-NMR (CD3OD) δ: 175.9 (C-4), 164.1 (C-7), 161.1 (C-5), 156.7 (C-9), 147.4 (C-2), 146.6 (C-3′), 144.5 (C-4′), 122.8 (C-1′), 120.3 (C-6′), 114.7 (C-5′′), 114.6 (C-2′), 103.0 (C-10), 97.8 (C-6), 93.1 (C-8). The above data were identical to the literature data [Kampferol (4): C15H10O6, yellow powder, mp 281–283 °C. IR  v: 3419, 1653, 1521. 1H-NMR (CD3OD) δ: 12.05 , 8.01 , 6.92 , 6.40 , 6.14 ; 13C-NMR (CD3OD) δ: 175.3 (C-4), 164.1 (C-7), 161.3 (C-5), 159.2 (C-4′), 146.0 (C-2), 135.8 (C-3), 129.6 (C-2′), 122.4 (C-1′), 115.5 (C-5), 103.3 (C-10), 98.3 (C-6), 93.7 (C-8). The above data were identical to the literature data [Shikimic acid (5): C7H10O5, white powder, mp 180–182 °C. IR  v: 3383, 1681, 1647, 677, 603. 1H-NMR (CD3OD) δ: 6.78 , 4.35, 3.97 , 3.70 , 2.68 , 2.18 . 13C-NMR (CD3OD) δ: 168.7 (COOH), 137.5 (C-2), 129.3 (C-1), 71.3 (C-4), 67.1 (C-5), 66.0 (C-3), 30.3 (C-6). The above data were identical to the literature data [Gallic acid (6): C7H10O5, white powder, mp 246–248 °C. IR  v: 3402, 3261, 2362, 1621, 1228, 1033. 1H-NMR (CD3OD) δ: 7.11 . 13C-NMR (CD3OD) δ: 170.5 (COOH), 146.3 (C-1), 141.5 (C-2), 105.3 (C-4), 125.6 (C-5). The Rf is the same as the standard sample in different solvents and the above data were identical to the literature data [Ursolic acid (7): C30H48O3, white powder, mp 257–259 °C. IR  v: 3444, 2928, 2868, 1683, 1458, 1028. 1H-NMR (CD3OD) δ: 0.78 , 0.84 , 0.90 , 0.92 , 0.97 , 1.09 , 3.20 , 5.2 . 13C-NMR (CD3OD) δ: 180.1 (C-28), 138.3 (C-13), 125.6 (C-12), 78.1 (C-3), 55.4 (C-5), 53.1 (C-18), 41.9 (C-14), 39.5 (C-8), 39.1 (C-19), 38.9 (C-1), 38.7 (C-4), 38.5 (C-20), 36.8 (C-10), 36.7 (C-22), 33.0 (C-7), 30.4 (C-21), 27.9 (C-23), 27.4 (C-15), 26.6 (C-2), 24.2 (C-16), 23.0 (C-11), 22.7 (C-27), 21.2 (C-30), 18.3 (C-6), 16.5 (C-26), 16.2 (C-29), 14.7 (C-25), 14.6 (C-24). The Rf is the same as the standard sample in different solvents and the above data were identical to the literature data [Betulin (8): C30H50O2, white powder, mp 234–236 °C. IR  v: 3498, 2941, 1456, 1033. 1H-NMR (CDCl3) δ: 3.79 , 4.67 , 4.56 , 3.77 , 3.31 , 3.18 , 1.66 . 13C-NMR (CDCl3) δ: 150.5 (C-20), 109.7 (C-29), 79.0 (C-3), 60.6 (C-28), 55.3 (C-5), 50.4 (C-9), 48.8 (C-18), 47.8 , 42.7 (C-14), 40.9 (C-8), 38.9 (C-4), 38.7 (C-1), 37.4 (C-13), 37.2 (C-10), 34.2 (C-7), 34.0 (C-22), 29.8 (C-21), 29.2 (C-16), 28.0 (C-23), 27.4 (C-2), 27.1 (C-15), 25.3 (C-12), 20.8 (C-11), 19.1 (C-30), 18.4 (C-6), 16.1 (C-25), 16.0 (C-26), 15.4 (C-24), 14.8 (C-27). The above data were identical to the literature data [Betulonic acid (9): C30H46O3, white powder, mp 291–293 °C. IR  v: 3444, 2947, 1699, 1683. 1H-NMR (CDCl3) δ: 3.01–2.96 , 4.71 , 4.58 , 1.68 . 13C-NMR (CDCl3) δ: 218.5 (C-3), 182.4 (C-28), 150.4 (C-20), 109.8 (C-29), 56.4 (C-17), 54.9 (C-5), 49.9 (C-9), 49.2 (C-18), 47.4 (C-4), 47.0 (C-19), 42.5 (C-14), 40.6 (C-8), 39.6 (C-1), 38.5 (C-13), 37.1 (C-22), 36.9 (C-10), 34.1 (C-2), 33.6 (C-7), 32.1 (C-16), 30.6 (C-21), 29.7 (C-15), 26.7 (C-23), 25.5 (C-12), 21.4 (C-11), 21.0 (C-24), 19.6 (C-6), 19.4 (C-30), 16.0 (C-26), 15.8 (C-25), 14.6 (C-27). The above data were identical to the literature data [Betulone (10): C30H48O2, white powder, mp 201–203 °C. IR  v: 3460, 2945, 2868, 1701, 1458, 1375, 1026. 1H-NMR (CDCl3) δ: 4.68 , 4.57 , 3.80 , 3.34 , 2.47 , 2.40 , 1.95 , 1.95 , 1.90 , 1.90 , 1.67 , 1.42 , 1.38 , 1.24 , 1.06 , 1.05 , 1.01 , 0.98 , 0.91 . 13C-NMR (CDCl3) δ: 218.5 (C-3), 150.4 (C-20), 109.8 (C-19), 60.6 (C-28), 54.9 (C-5), 49.8 (C-9), 48.7 (C-18), 47.8 , 47.4 (C-4), 42.8 (C-14), 40.9 (C-8), 39.6 (C-1), 37.4 (C-13), 36.9 (C-10), 34.2 (C-2), 34.0 (C-22), 33.5 (C-7), 29.7 (C-21), 29.1 (C-16), 27.0 (C-15), 26.7 (C-23), 25.2 (C-12), 21.4 (C-11), 21.1 (C-24), 19.7 (C-6), 19.1 (C-30), 16.0 (C-25), 15.8 (C-26), 14.7 (C-27). The above data were identical to the literature data [Tectoridin (11): C16H12O6, yellow powder, mp 273–274 °C. IR  v: 3367, 1646, 1635, 1080. 1H-NMR (DMSO-6d) δ: 12.91 , 9.58 , 8.41 , 7.36 , 6.86 , 6.79 , 5.41 , 5.13 , 3.73 , 3.68–3.65 , 3.43–3.40 , 3.28–3.24 , 3.16–3.12 . 13C-NMR (DMSO-6d) δ: 181.3 (C-4), 158.0 (C-4′), 157.1 (C-7), 155.2 (C-2), 153.4 (C-5), 153.0 (C-9), 132.9 (C-6), 130.7 , 122.5 (C-1′), 121.5 (C-3), 115.6 , 106.9 (C-10), 100.6 (Glc-1″), 94.5 (C-8), 77.8 (C-5″), 77.2 (C-3″), 73.6 (C-2″), 70.1 (C-4″), 61.1 (C-6″), 60.8 (6-OCH3). The above data were identical to the literature data [Irisflorentin (12): C20H18O8, white needle, mp 163–165 °C. IR  v: 3462, 1653, 1627, 1583, 1055. 1H-NMR (CD3COCD3) δ: 8.13 , 6.90 , 6.80 , 6.18 , 3.97 , 3.85 , 3.7 . 13C NMR (CD3COCD3) δ: 173.9 (C-4), 154.4 (C-5), 153.1 (C-3′), 153.1 (C-5′), 153.0 (C-9), 151.5 (C-2), 141.4 (C-7), 138.2 (C-4′), 136.3 (C-6), 127.9 (C-1′), 125.0 (C-3), 113.9 (C-10), 106.9 (C-2′), 106.9 (C-6′), 102.7 , 93.3 (C-8), 60.5 (5-OCH3), 59.7 (4′-OCH3), 55.6 (5′-OCH3), 55.6 (3′-OCH3). The above data were identical to the literature data [4′,5,6-Trihydroxy-7-methoxyisoflavone (13): C16H12O6, yellow powder, mp 281–283 °C, IR  v: 3460, 1670, 1609, 1448, 1062. 1H-NMR (CD3OD) δ: 7.89 , 7.31 , 6.81 , 6.26 , 3.80 . 13C-NMR (CD3OD) δ: 180.5 (C-4), 162.8 (C-7), 157.3 (C-4′), 154.3 (C-9), 152.9 (C-2′), 152.7 (C-5), 132.9 (C-6), 130.1 , 122.4 (C-3), 122.3 (C-1′), 114.9 , 103.5 (C-10), 95.0 (C-8), 59.4(7-OCH3). The above data were identical to the literature data [ure data .Tectorigenin (14): C16H12O6, yellow powder, mp 229–231 °C. IR  v: 3479, 1639, 1627, 1508, 1062. 1H-NMR (CD3OD) δ: 8.07 , 7.35 , 6.82 , 6.41 , 3.85 . 13C-NMR (CD3OD) δ: 181.3 (C-4), 157.6 (C-4′), 157.5 (C-7), 153.7 (C-9), 153.6 (C-2), 153.2 (C-5), 131.5 (C-6), 130.0 , 122.8 (C-3), 121.9 (C-1′), 114.9 , 105.3 (C-10), 93.6 (C-8), 59.0 (6-OCH3). The above data were identical to the literature data [ure data .Irilins A (15): C17H14O6, yellow powder, mp 254–257 °C. IR  v: 3421, 1639, 1610, 1580, 1064, 842, 813. 1H-NMR (CD3OD) δ: 8.04 , 7.35 , 6.41 , 3.84 , 3.79 . 13C-NMR (CD3OD) δ: 181.2 (C-4), 157.4 (C-2′), 154.2 (C-7), 153.8 (C-2), 153.6 (C-5), 153.2 (C-9), 131.4 (C-6), 130.0 (C-6′), 122.8 (C-4′), 121.9 (C-3), 121.5 (C-1′), 114.5 (C-5′), 109.8 (C-3′), 105.3 (C-10), 93.6 (C-8), 59.5 (6-OCH3), 55.1 (7-OCH3). The above data were identical to the literature data [Iridin (16): C24H26O12, yellow powder, mp 210–213 °C. IR  v: 3404, 1649, 1610, 1589, 1047. 1H-NMR (CD3OD) δ: 8.19 , 6.83 , 6.70 , 6.69 , 5.08 , 3.79 , 3.87 , 3.85 , 3.71–3.37 , 3.28 . 13C-NMR (CD3OD) δ: 181.1 (C-4), 156.6 (C-7), 154.8 (C-2), 153.3 , 153.1 (C-5′), 150.2 (C-3′), 136.6 (C-4′), 132.8 (C-6), 126.5 (C-1′), 123.1 (C-3), 109.8 (C-2′), 107.0 (C-10), 104.6 (C-6′), 100.5 (Glc-1″), 94.1 (C-8), 77.1 , 73.3 (C-2″), 69.8 (C-4″), 61.1 (C-6″), 60.1 (6-OCH3), 59.6 (4′-OCH3), 55.1 (5’-OCH3). The above data were identical to the literature data [Irigenin (17): C16H12O6, yellow powder. mp 184–186 °C. IR  v: 3462, 1635, 1579, 1060, 815, 709. 1H-NMR (DMSO-6d) δ: 13.0 , 10.75 , 9.23 , 8.33 , 6.67 , 6.62 , 6.47 , 3.75 , 3.71 , 3.65 . 13C-NMR (DMSO-6d) δ: 180.8 (C-4), 158.0 (C-5), 155.3 (C-2), 153.8 (C-7), 153.4 (C-9), 153.1 (C-5′), 150.7 (C-3′), 136.8 (C-4′), 131.9 (C-6), 126.6 (C-1′), 122.2 (C-3), 110.8 (C-2′), 105.3 (C-10), 105.0 (C-6′), 94.4 (C-8), 60.5 (6-OCH3), 60.4 (4′-OCH3), 56.3 (5′-OCH3). The above data were identical to the literature data [Iristectongenin A (18): C17H14O7, yellow powder, mp 232–235 °C. IR  v: 3446, 1653, 1558, 1193, 813, 669. 1H-NMR (DMSO-6d) δ: 13.0 , 9.1 , 8.32 , 7.09 , 6.95 , 6.79 , 6.46 , 3.75 , 3.70 . 13C-NMR (DMSO-6d) δ: 181.0 (C-4), 158.0 (C-9), 154.8 (C-2), 153.8 (C-7), 153.2 (C-5), 147.7 (C-4′), 147.1 (C-3′), 131.9 (C-6), 122.3 (C-1′), 122.2 (C-6), 122.1 (C-3), 115.7 (C-2′), 113.7 (C-5′), 105.3 (C-10), 94.3 (C-8), 60.4 (6-OCH3), 56.2 (4′-OCH3). The above data were identical to the literature data [2 at 37 °C.MGC-803, Bcap-37, MCF-7, PC3 and NIH3T3cell lines were obtained from the Institute of Biochemistry and Cell Biology, China Academy of Science. MGC-803 is stomach cancer cell line, Bcap-37 and MCF-7 are breast cancer cell lines, PC3 is prostate cancer cell line, and NIH3T3 is mouse fibroblast cell line. The entire cancer cell lines were maintained in the RPMI 1640 medium and NIH3T3 was maintained in the DMEM medium. They were supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere of 5% CO3 cells/well/100 μL of the proper culture medium and treated with the compounds at 1 μmol/L to 20 μmol/L for 72 h. In parallel, the cells treated with 0.1% DMSO served as negative controls, and the ADM served as positive control. Finally, 100 μL of MTT  was added, and the cells were incubated for 4 h. The MTT-formazan formed by metabolically viable cells was dissolved in 100 μL of SDS for 12 h. The absorbance was then measured at 595 nm using a microplate reader , which is directly proportional to the number of living cells in culture [All tested compounds were dissolved in DMSO and subsequently diluted in the culture medium prior to the treatment of the cultured cells. The tested cells were plated in 96-well plates at a density 2 × 10 culture . The per4 cell/mL in a volume of 0.6 mL on a sterile cover slip in 6-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS and then supplemented with compounds (10 μmol/L). After the treatment period, the cover slip with monolayer cells was inverted on the glass slide with 20 μL of AO/EB  stain (100 μg/mL). The fluorescence was read using an IX71SIF-3 fluorescence microscope .Thecells were seeded at a concentration of 5 × 10The cells grown on the sterile cover slip in 6-well tissue culture plates were treated with compounds for a certain range of treatment time. The culture medium containing compounds was removed, and the cells were fixed in 4% paraformaldehyde for 10 min. The cells were washed twice with PBS, and were consequently stained with 0.5 mL of Hoechst 33258 staining  for 5 min. The stained nuclei were washed twice with PBS, and were consequently observed under an IX71SIF-3 fluorescence microscope at 350 nm excitation and 460 nm emissions.2O2 in methanol at room temperature for 20 min to inactivate the endogenous peroxidases, after which the cells were washed thrice with PBS. Thereafter, the cells were incubated with 2 μL of TdT-enzyme and 48 μL of Biotin-dUTP per specimen for 60 min at 37 °C. The cells were terminated for 10 min, and were consequently incubated with streptavidin-HRP (50 μL per specimen) conjugate diluted at 1:50 in sample diluents for 30 min. The cells were washed three times with PBS, and were consequently incubated with diaminobenzidine solution (200 μL per specimen) for 10 min. Thereafter, the cells were rewashed twice with PBS, and were consequently imaged under an XDS-1B inverted biological microscope .TUNEL assays were performed using a colorimetric TUNEL apoptosis assay kit according to the manufacturer’s instructions (Beyotime). The cells grown in 6-well culture clusters were treated as mentioned in mitochondrial depolarization assay. TheMGC-803 and Bcap-37 cells grown in 6-well tissue culture plates were washed with PBS and fixed in 4% paraformaldehyde for 40 min. The cells were washed once with PBS, and were consequently permeabilized with immunol staining wash buffer (Beyotime) for 2 min on ice. The cells were rewashed once with PBS, and were consequently incubated in 0.3% HAll statistical analyses were performed using SPSS 10, and the data were analyzed using one-way ANOVA. The mean separations were performed using the least significant difference method. Each experiment was performed in triplicate, and all experiments were run thrice and yielded similar results. Measurements from all the replicates were combined, and the treatment effects were analyzed.B. chinensis was shown to exhibit greater antitumor activities compared with the other two extracts, leading to the isolation of 18 compounds from this extract. The compounds were identified as β-sitosterol (1), dausterol (2), quercetin (3), kampferol (4), shikimic acid (5), gallic acid (6), ursolic acid (7), betulin (8), betulonic acid (9), betulone (10), tectoridin (11), irisflorentin (12), 4′,5,6-trihydroxy-7-methoxyisoflavone (13), tectorigenin (14), irilins A (15), iridin (16), irigenin (17), and iristectongenin A (18). Compounds 3–10, 13, and 15 were isolated from B. chinensis for the first time. All compounds were tested for their antitumor activities and pentacyclic triterpenes (compounds 7–10) from the ethyl acetate extract exhibited moderate antiproliferative activities against human carcinoma cell lines. The cell apoptosis-inducing activity of ursolic acid was then further investigated against MGC-803 cell line via AO/EB staining, Hoechst 33258 staining, and TUNEL assay. The results showed that ursolic acid can induce cell apoptosis of MGC-803 cells. The studies of chemical modification of ursolic acid and antitumor bioassay of UA derivatives are currently underway.Using the bioassay-guided technique, the ethyl acetate extract of the roots of


Aralia chinensis var. dasyphylloides is widely distributed in China and used as a traditional herbal medicine for the treatment of digestive and immune system diseases. The present study aimed to search for novel oleanolic-type triterpenoids in low-polarity fractions. Six new triterpene derivatives (1–6), together with two known compounds were isolated from the barks of A. chinensis var. dasyphylloides. Their structures were elucidated by 1D- and 2D-NMR spectroscopic analysis and chemical methods. They were identified as 3-oxo-oleana-11,13(18)-dien-28,30-dioic acid (1), 30-hydroxy-3-oxo-oleana-11,13(18)-dien-28-oic acid (2), 3β-hydroxy-oleana-11,13(18)-dien-28-oic acid-28-O-β-d-glucopyranoside (3), 3β,30-dihydroxy-oleana-11,13(18)-dien-28-oic acid-28-O-β-d-glucopyranoside (4), 3β-hydroxy-oleana-11,13(18)-dien-28-oic acid-3-O-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (5), 3β,29-dihydroxy-oleana-9(11),12-dien-28-oic acid-28-O-β-d-glucopyranoside (6), namely, araliachinolic acids I and II and araliachinosides I–IV. The cytotoxicity of the isolated compounds was tested against HepG2, A549, SGC7901, and MCF7 cell lines, but no apparent activity was observed at a concentration of 50 μM. Aralia chinensis Linn. var. dasyphylloides Hand.-Mazz. Symb.  is distributed in the Sichuan, Guizhou, Guangxi, and Hubei provinces of China + at m/z 482.3033  observed in its HR-EI-MS and NMR spectroscopic data. The 1H-NMR spectrum revealed six methyl group signals at δ (ppm) 1.09 , 1.04 , 1.04 , 0.86 , 0.93 , and 1.17 , as well as two cis olefinic protons at δ (ppm) 5.64  and 6.73 . The 13C-NMR and DEPT spectra revealed six methyls, nine methylenes, two sp3 methines at δ (ppm) 55.6 (C-5) and 55.1 C-9), six sp3 quaternary carbon signals, four olefinic signals at δ (ppm) 127.8 (C-11), 126.6 (C-12), 137.1 (C-13), and 133.7 (C-18), and three carbonyl signals at δ (ppm) 220.3 (C-3), 180.2 (C-28), and 181.9 (C-30). The 13C-NMR signals, especially signals at δ (ppm) 55.6 C-5), 55.1 (C-9), 127.8 (C-11), 126.6 (C-12), 137.1 (C-13), and 133.7 (C-18), indicated the presence of an oleana-11,13(18)-diene-type triterpene, as confirmed by comparison with 3-oxo-11,13(18)-oleanadien-28-oic acid − at m/z 615.3895  evident in its HR-ESI-MS and its NMR spectroscopic data. The NMR data of 3  and further signals at δ (ppm) 3.27, 3.37, 3.30, 3.31, 3.66, and 3.85 revealed the presence of one sugar which could be identified as β-d-glucopyranoside by acid hydrolysis, derivatization, and HPLC analysis. In the HMBC spectrum, the β-d-glucose was linked to the carboxyl group at C-28, based on the up-field shift from δ 180.2 ppm to δ 176.9 ppm. The correlation between Me-24 protons at δ 0.77 ppm and C-3 (δ 79.7 ppm) confirmed the presence of one hydroxyl group at C-3. This hydroxyl group was β-oriented by the NOESY correlation of H-3 (δ 3.18 ppm) with H-5 (δ 0.83 ppm)  observed in its HR-ESI-MS and its NMR spectroscopic data. A comparison of the NMR data between 4 and 3 indicated that the two compounds possessed the same structure, with the only difference being the substitution of the methyl group at C-30 in 3 by a hydroxymethylene group in compound 4. This was confirmed by the correlation between Me-29 protons at δ 1.17 ppm and C-30 (δ 66.4 ppm) in the HMBC spectrum. β-d-Glucose was identified via HPLC analysis after acid hydrolysis and derivatization. These data indicated that compound 4 was a new compound, which was named as araliachinoside II. Compound 5 was obtained as a white amorphous powder with the molecular formula determined to be C41H64O12 from the pseudo-molecular ion peak [M + Cl]− at m/z 783.4086  evident in its HR-ESI-MS and its NMR spectroscopic data. A comparison of the NMR data of 5 with 3β-hydroxy-oleane-11,13(18)-dien-28-oic acid-3-O-β-d-glucopyranosyl-(1 → 2)-β-d-xylopyranoside − at m/z 631.3847  observed in its HR-ESI-MS and its NMR spectroscopic data. The 1H-NMR data  1.25 , 1.07 , 1.24 , 1.47 , 1.22 , and 1.09 , two methine signals at δ (ppm) 0.99  and 3.51 , and two olefinic signals at δ (ppm) 5.76  and 5.80 . In addition, the anomeric signal observed at δ 6.41 ppm , with further signals at δ (ppm) 4.25, 4.29, 4.40, 4.04, 4.42, and 4.48, revealed that compound 6 contained a β-d-glucose moiety, which was confirmed after acid hydrolysis, derivatization, and HPLC analysis. The 13C-NMR and DEPT spectra showed six methyls, nine methylenes, two sp3 methines at δ (ppm) 51.7 (C-5) and 39.8 (C-18), six quaternary sp3 carbons, one oxygenated methine , one carboxyl group , and one hydroxymethylene group . Moreover, four olefinic signals were observed at δ (ppm) 155.8 (C-9), 116.1 (C-11), 121.3 (C-12), and 145.9 (C-13), as well as a group of β-d-glucose signals at δ (ppm) 96.0 (glc-1), 74.2 (glc-2), 78.9 (glc-3), 71.0 (glc-4), 79.4 (glc-5), and 62.4 (glc-6). Taken together, these signals were similar to those of oleana-9(11), 12-diene-28-oic acid-28-O-β-d-glucopyranoside (7) c = 0.45, MeOH), HR-EI-MS: m/z 482.3033 [M]+ , EI-MS: m/z 482 [M]+ (100), 483 [M + 1]+ (40), 437 [M − CO2H]+ (30), 315 (15), 285 (13), 245 (16), 219 (25), 173 (43). 1H-NMR  and 13C-NMR  (see MHz) see .Araliachinolic acid II (2): White amorphous powder. [α]c = 0.35, MeOH), HR-ESI-MS: m/z 467.3159 [M − H]− , ESI-MS: m/z 467 [M − H]−, 1H-NMR  and 13C-NMR  (see MHz) see .Araliachinoside I (3): White amorphous powder. [α]c =0.18, MeOH), HR-ESI-MS: m/z 615.3895 [M − H]− , ESI-MS: m/z 615 [M − H]−, 1H-NMR  and 13C-NMR  (see MHz) see .Araliachinoside II (4): White amorphous powder. [α]c = 0.31, C5H5N), HR-ESI-MS: m/z 667.3616 [M + Cl]− , ESI-MS: m/z 667 [M + Cl]−. 1H- NMR  and 13C-NMR  (see MHz) see .Araliachinoside III (5): White amorphous powder. [α]c = 0.22, C5H5N), HR-ESI-MS: m/z 783.4086 [M + Cl]− , ESI-MS: m/z 783 [M + Cl]−. 1H-NMR  and 13C-NMR  (see MHz) see .Araliachinoside IV (6): White amorphous powder. [α]c = 0.15, C5H5N), HR-ESI-MS: m/z 631.3847 [M − H]− , ESI-MS: m/z 631 [M − H]−, 1H-NMR  and 13C-NMR  (see MHz) see .2Cl2 , and evaporated under reduced pressure to remove TFA. l-cysteine methyl ester hydrochloride (2.5 mg) was dissolved in anhydrous pyridine (1.0 mL) and added to the sugar residue. The solution was refluxed at 60 °C in the water bath for 2.5 h. O-Tolyl-isothiocyanate (10 μL) was added to the refluxed solution and heated for another 1 h. The reaction mixture was analyzed by HPLC on an Agilent HC-C18, 250.0 × 4.6 mm, 5 μm column at 30 °C with an isocratic elution of CH3CN–H2O . The flow was 1.0 mL/min and detection was at 250 nm. The retention times of standard monosaccharides derivatized using the same procedure were 14.3 min (d-glucose) and 16.2 min (d-xylose). Comparison of the retention times of standards and samples enabled to establish the absolute configuration of monosaccharides in each hydrolysate.Each compound (2.5 mg) was hydrolyzed with 4N aqueous trifluoroacetic acid  for 3.5 h at 95 °C in a water bath. The mixture was diluted with water (10 mL), extracted with CH2 for 24 h. 10 μL of MTT  was added to each well, followed by incubation for 2–4 h. The medium was aspirated and formazan crystals were dissolved with 100 μL of DMSO. Optical density at 492 nm was determined with a microplate reader. Cells viability in response to treatment was calculated as percentage of control cells treated with DMSO. The cell lines  were purchased from the cell bank of Chinese Academy of Sciences  and seeded in 96-well plates, incubated for 24 h. After incubation, cells were treated with compounds (50 μM) at 37 °C in 5% CO


Morus alba L. are an important herbal medicine in Asia. The systematic isolation of the metabolites of the leaves of Morus alba L. was achieved using a combination of liquid chromatography techniques. The structures were elucidated by spectroscopic data analysis and the absolute configuration was determined based on electronic circular dichroism (ECD) spectroscopic data and hydrolysis experiments. Their biological activity was evaluated using different biological assays, such as the assessment of their capacity to inhibit the aldose reductase enzyme; the determination of their cytotoxic activity and the evaluation of their neuroprotective effects against the deprivation of serum or against the presence of nicouline. Chemical investigation of the leaves of Morus alba L. resulted in four new structures 1–4 and a known molecule 5. Compounds 2 and 5 inhibited aldose reductase with IC50 values of 4.33 μM and 6.0 μM compared with the potent AR inhibitor epalrestat (IC50 1.88 × 10−3 μM). Pretreatment with compound 3 decreased PC12 cell apoptosis subsequent serum deprivation condition and pretreatment with compound 5 decreased nicouline-induced PC12 cell apoptosis as compared with control cells (p < 0.001).The leaves of  Morus alba L., known as white mulberry, belongs to the genus Morus of the family Moraceae, is native to China and now is cultivated throughout the world , one terminal double bond , one ester carbonyl (δC 168.8), two oxygenated methines , three methyls , one methylene , two methines , one tertiary carbon group [δC 29.5 (C-1)] and a glucopyranosyl unit . In combination with analysis of 1H-1H COSY spectrum, the NMR date displayed there were two spin systems C2-C3-C4 and C6-C7-C8 in 1. In the HMBC spectrum, the correlations of H-12, H-13/C-1, C-2, C-6; H-4/C-5, C-14; H-6/C-4; H-11/C-9; and H-15/C-8, C-10 determined the monocyclofarnesane carbon skeleton containing a 14,7-olide ring. In addition, the correlations of H-1′/C-3 indicated the glucopyranosyl unit was connected to C-3 of the monocyclofarnesane-type sesquiterpenoid aglycone.Moralsin . The D-configuration of the glucose was determined by GC analysis of the trimethylsilyl l-cysteine derivatives after acid hydrolysis of 1. The E-configuration of the 8,9-double bond was demonstrated by the nuclear Overhauser effect (NOE) effect of H-7/H-15 and H-8/H-10 in the ROESY 1D experiment. NOE effect of H-3/H-2β, H-7/H-12, and H-6/H-8, H-2β, and H-13 showed that H-2β, H-3, H-6, C-13, and C-8 were cofacial, assigned as the β-orientation, and H-7 and C-12 were α-oriented. The absolute configuration of aglycone moiety was assigned by analysis of the electronic circular dichroism (ECD) spectroscopy using excitation chirality method -2,6-dihydroxy-benzoate.Compound e 1H-NMR  spectrumbonyl 172 cm−1 andR)-hydroxyl--octadecatrienoic acid (5) by NMR analysis and comparison with literature data + .-7-Hydroxy-6,10-dimethyl-pentadecatrien-2,14-dione (2): yellow oil; UV(MeOH): λmax (log ε) 207 (4.31) nm, 247 (0.11) nm; IR νmax 3347, 2926, 1726, 1606, 1466, 1051, 1025 cm−1; 1H-NMR  and 13C-NMR  see m/z 561.1583 [M + Na]+ .2′-Hydroxy-4′-methoxyl-2H--pyran--flavane (3): yellow oil; UV(MeOH): λmax (logε) 206 (3.90) nm, 280 (2.80) nm; IR νmax 3372, 1718, 1602 cm−1; 1H-NMR  13C-NMR  m/z 357.16873 [M + H]+ .Benzyl 2-O-[d-apiofuranosyl(1→6)-β-d-glucopyranosyl]-2,6-dihydroxybenzoateβ- (4): yellow oil; UV(MeOH): λmax (log ε) 207 (4.32) nm, 247 (0.1) nm; IR νmax 3347, 2926, 1726, 1606, 1466, 1051, 1025 cm−1; 1H-NMR  and 13C-NMR  see m/z 561.1583 [M + Na]+ .(9R)-Hydroxy--octadecatrienoic acid (5): Yellow powder, 3); ESIMS m/z 317.2 [M + Na]+, 1H-NMR  δ: 2.18 , 1.47 , 1.38 , 2.04 , 0.92 , 3.97 , 5.66 , 6.44 , 5.26-5.40 , 5.96 , 2.88 , 1.24 . 13C-NMR  δ: 174.5 (C-1), 138.6 (C-10), 131.7 (C-16), 128.7 (C-13), 128.3 (C-12), 126.7 (C-15), 123. 5 (C-11), 70.44 (C-9), 37.2 (C-8), 33.7 (C-2), 28.5, 28.8, 28.9 (C-4-6), 25.5 (C-14), 24.9 (C-7), 24.5 (C- 3), 20.0 (C-17), 14.1 (C-18).1 (2 mg) was hydrolyzed in 2 M HCl/H2O at 80 °C for 2 h. The residue was reacted sequentially with l-cysteine methyl ester hydrochloride and N-trimethylsilylimidazole. The resulting monosaccharide N-trimethylsilylimidazole derivatives were analyzed by GC. d-Glucose was confirmed by comparison of the retention time of the derivatives with those of authentic sugars derivatized in a similar way, which showed retention times of 27.93 min. The constituent sugars of compounds 4 were identified by the same method as 1. Retention times of authentic samples were detected at 17.87 min (d-apiofuranose) and at 27.93 (d-glucose). The reaction and GC conditions were as described in the literature [Compound terature .The assay was operated in 96 well culture plate. A 100 μL mixture that contained 10 mM DL-glyceraldehyde, 0.16 mM NADPH-Na4 and aldose reductase in 0.1 M sodium phosphate buffer (pH 6.2), with or without test compounds was prepared at 0 °C. Appropriate blank were employed for corrections. The assay mixture was incubated at 25 °C. After 10 min of incubation, the plate was immediately cooled at −20 °C for 5 min to stop the reaction. The change in the absorbance at 340 nm due to NADPH oxidation was measured in a plate reader .4 cells/mL was seeded in each well of a poly-L-lysine-coated, 96-well culture plate and precultured for 24 h. The medium was then replaced by different fresh medium including the control (complete medium), the model (complete medium with 4 μM rotenone or serum free medium), and the sample , and the cells were cultured for 48 h. Then, 10 μL of MTT (5 mg/mL) was added to each well. After incubation for 4 h, the medium was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. The optical density (OD) of the PC12 cells was measured on a microplate reader at 570 nm [The PC12 cells were cultured in DMEM medium supplemented with 5% horse serum and 5% fetal bovine serum. Then, 100 μL of cells with an initial density of 5 × 10t 570 nm . Morus have been extensively investigated for their medicinal constituents and a series of unique structures were characterized [5 was isolated for the first time from the genus Morus and its neuroprotective activity reported for the first time. The compounds reported here provide new potential aldose reductase inhibitory or neuroprotective agents for further research.The plants of genus cterized ,35,36,37


R)-(+)-1-(5-bromopentyl)-1-methyl-7-methoxy-2-tetralone (3a), a key intermediate of dezocine, 17 cinchona alkaloid-derived catalysts were prepared and screened for the enantioselective alkylation of 1-methyl-7-methoxy-2-tetralone with 1,5-dibromopentane, and the best catalyst (C7) was identified. In addition, optimizations of the alkylation were carried out so that the process became practical and effective.In order to prepare asymmetrically ( The preparation of enantiomerically pure compounds has become a stringent requirement for pharmaceutical synthesis . In thisR,11S,13S)-13-amino-5-methyl-5,6,7,8,9,10,11,12-octahydro-5-methyl-5,11-methanobenzocyclodecen-3-ol  with 1,5-dibromopentane gave the designed (R)-(+)-1-(5-bromopentyl)-1-methyl-7-methoxy-2-tetralone (3a) and an equal amount of the S-isomer 3b, both 3a and 3b underwent the following cyclization, oximation and reduction, and then, -3-methoxy-5-methyl-5,6,7,8,9,10,11,12-octahydro-5,11-methanobenzocyclodecen-13-amine (6a) and -3-methoxy-5-methyl-5,6,7,8,9,10,11,12-octahydro-5,11-methanobenzocyclodecen-13-amine (6b) were separated by two times of resolution with L-tartaric acid and D-tartaric acid cinchonidinium bromide in a two-phase system gave the enantioselective product 3a, although the ee value of the product was 60%, determined by 1H NMR. And so far, no further report on the stereoselective alkylation of 2a was found. . In 1Br) in THF to obtain catalysts C1–C11 +.To a stirred mixture of 6a, and then 6a was transformed to dezocine with 23.0% overall yield and 100% purity. The mp, optical rotation value, MS and 1H NMR of the product were consistent with those in the literature [The above-obtained product underwent subsequent cyclization, oximation and reduction according to the literature  (withoutterature ,10.File 1C1–C17, synthesis of dezocine, 1H NMR and MS spectra of catalysts C1–C17 and chiral HPLC diagrams of 3. 1H NMR, 13C NMR, MS spectra of 3. 1H NMR, MS spectra HPLC diagrams of dezocine.Synthesis of catalysts


Both isoforms hCA I and II were inhibited by the quinolines reported here in variable degrees: hCA I was inhibited with KIs in the range of 0.966–9.091 μM, whereas hCA II in the range of 0.083–3.594 μM. The primary 7-chloro-6-flouro substituted sulphfonamide derivative 6e (KI = 0.083 μM) proved to be the most active quinoline in inhibiting hCA II, whereas, its secondary sulfonamide analog failed to inhibit the hCA II up to 10 μM, confirming the crucial role of the primary sulphfonamide group, as a zinc-binding group for CA inhibitory activity.Carbonic anhydrases  are crucial metalloenzymes that are involved in diverse bioprocesses. We report the synthesis and biological evaluation of novel series of benzenesulfonamides incorporating un/substituted ethyl quinoline-3-carboxylate moieties. The newly synthesised compounds were  Just to name a few, antimalarial6a–6f, 11, in vitro for their inhibitory activity towards the physiologically relevant hCA isoforms I and II, using stopped-flow CO2 hydrase assay.In the present study, we report a new series of primary benzenesulfonamides incorporating un/substituted ethyl quinoline-3-carboxylate 6a–6f,  as well 1H and 101 MHz 13 C NMR). 1H NMR spectra were referenced to tetramethylsilane (δ = 0.00 ppm) as an internal standard and were reported as follows: chemical shift, multiplicity . Column chromatography was performed on Merck Silica Gel 60 (230–400 mesh) and eluting solvents for all of these chromatographic methods were noted as appropriated-mixed solvent with given volume-to-volume ratios. TLC was carried out using glass sheets pre-coated with silica gel 60 F254 purchased by Merk. High-resolution spectra were performed on Waters ACQUITY UPLC BEH C18 1.7 μ–Q-TOF SYNAPT G2-Si High Definition Mass Spectrometry. Compounds 3a-f, 4a-f10All reaction and manipulations were performed in nitrogen atmosphere using standard Schlenk techniques. All reaction solvents and reagents were purchased from commercial suppliers and used without further purification. Microwave-assisted synthesis was carried out in a Biotage Initiator + apparatus operating in single mode, the microwave cavity producing controlled irradiation at 2.45 GHz . The reactions were run in sealed vessels. These experiments were performed by employing magnetic stirring and a fixed hold time using variable power to reach (during 1 − 2 min) and then maintain the desired temperature in the vessel for the programed time period. The temperature was monitored by an IR sensor focused on a point on the reactor vial glass. The IR sensor was calibrated to internal solution reaction temperature by the manufacturer. The NMR spectra were obtained on Bruker Avance 400 (400 MHz 3a–f (1.0 mmol) in POCl3 (6 ml) was refluxed for 1 h. The mixture was evaporated in vacuo and the residue was extracted with methylene chloride, crushed ice and aqueous NH3. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by column chromatography : n-Hex 10: 1) to get key intermediates 4a-f,A solution of compounds 4a-f (0.21 mmol), 3-aminobenzenesulfonamide 5  or 3-amino-N-methylbenzenesulfonamide 10 , and ethanol (12 ml) at room temperature. The MW vial was sealed and heated under MW conditions for 30 min at 150 °C. The mixture was evaporated in vacuo and the residue was extracted with EA and NaHCO3 (aq). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by column chromatography , in a gradient elution with 1:5 (EA: n-hex) ratio, to furnish quinolines 6a–f and 11, respectively.To a MW vial, were successively added the appropriate ethyl 4-chloroquinoline-3-carboxylate derivative 1H NMR  δ ppm: 1.16 , 4.05 , 7.16–7.18 , 7.34 , 7.45–7.56 , 7.80–7.84 , 8.01 , 8.10 , 9.01 , 9.75 ; 13 C NMR  δ ppm: 14.31 (CH3), 61.48, 111.07 (quinoline C-3), 116.42 (benzenesulfonamide C-2), 119.82 (benzenesulfonamide C-4), 121.40 (quinoline C-10), 121.92 (benzenesulfonamide C-6), 124.88 (quinoline C-5), 126.55 (quinoline C-6), 130.05 (quinoline C-8), 130.22 (benzenesulfonamide C-5), 131.95 (quinoline C7), 144.44 (benzenesulfonamide C-3), 145.60 (quinoline C-4), 148.12 (benzenesulfonamide C-1), 150.36 (quinoline C-2), 151.38 (quinoline C-9), 166.81 (C = O); HRMS (ESI) for C18H18N3O4S: calcd 372.1018, found: 372.1017 [M + H]+.White solid, yield: 49%, mp: 183.6 − 185.0 °C; 1H NMR  δ ppm: 1.09 , 2.40 , 3.89 , 7.25 , 7.39 , 7.47–7.55 , 7.73–7.74 , 7.96 , 8.26 , 8.88 , 10.25 ; 13 C NMR  δ ppm: 14.22 (CH2 CH3), 21.73 (CH3), 61.57 (CH2), 111.34 (quinoline C-3), 116.98 (benzenesulfonamide C-2), 120.58 (quinoline C-10), 121.17 (benzenesulfonamide C-4), 122.55 (benzenesulfonamide C-6), 123.97 (quinoline C-6), 127.10 (quinoline C-5), 130.19 (benzenesulfonamide C-5), 134.72 (quinoline C-8), 136.89 (quinoline C-7), 143.66 (benzenesulfonamide C-3), 145.60 (benzenesulfonamide C-1), 144.74 (quinoline C-2), 148.89 (quinoline C-9), 166.02 (C = O); HRMS (ESI) for C19H20N3O4S: calcd 386.1175, found: 386.1170 [M + H]+.Yellow solid, yield: 97%, mp: 223.0 − 224.5 °C; 1H NMR  δ ppm: 1.13 , 3.73 , 3.99 , 7.14–7.16 , 7.33 , 7.42–7.48 , 7.96 , 7.91–7.93 , 8.84 , 9.95 ; 13 C NMR  δ ppm: 14.31 (CH2 CH3), 21.73 (CH3), 55.92 (OCH3) 61.40 (CH2), 103.51 (quinoline C-4), 111.54 (quinoline C-3), 116.36 (benzenesulfonamide C-2), 119.51 (benzenesulfonamide C-4), 121.78 (quinoline C-10), 122.35 (benzenesulfonamide C-6), 123.78 (quinoline C-7), 130.17 (benzenesulfonamide C-5), 131.56 (quinoline C-8), 144.36 (benzenesulfonamide C-3), 145.55 (quinoline C-9), 146.16 (benzenesulfonamide C-1), 146.59 (quinoline C-2), 148.90 (quinoline C-4), 157.51 (quinoline C-6), 166.90 (C = O); HRMS (ESI) for C19H20N3O5S: calcd 402.1124, found: 402.1126 [M + H]+.White solid, yield: 61%, mp: 214.9 − 216.3 °C; 1H NMR  δ ppm: 1.07 , 3.88 , 7.17 , 7.36 , 7.45–7.51 , 7.94 , 8.53 , 8.92 , 9.71 ; 13 C NMR  δ ppm: 14.22 (CH3), 61.49 (CH2), 111.61 (quinoline C-3), 116.40 (benzenesulfonamide C-2), 119.81 (quinoline C-10), 119.95, (benzenesulfonamide C-4), 121.47 (benzenesulfonamide C-6), 123.08 (quinoline C-6), 126.60 (quinoline C-5), 130.21 (benzenesulfonamide C-5), 132.13 (quinoline C-8), 134.76 (quinoline C-7), 144.01 (benzenesulfonamide C-3), 145.68 (benzenesulfonamide C-1), 146.37 (quinoline C-2), 148.91 (quinoline C-9), 152.06 (quinoline C-4), 166.33 (C = O); HRMS (ESI) for C18H17BrN3O4S: calcd 450.0123, found: 450.0127 [M + H]+.Yellow solid, yield: 71%, mp: 235.6 − 237.2 °C; 1H NMR  δ ppm: 1.08 , 3.91 , 7.18–7.20 , 7.45–7.49 , 7.36 , 8.18 , 8.25 , 8.91 , 9.68 ; 13 C NMR  δ ppm: 14.22 (CH3), 61.58 (CH2), 110.16, 110.40 (quinoline C-5), 111.47 (quinoline C-3), 116.52 (benzenesulfonamide C-2), 120.18 (quinoline C-10), 121.24 (benzenesulfonamide C-4), 121.85 (quinoline C-10), 125.21 (benzenesulfonamide C-6), 124.41 (benzenesulfonamide C-5), 130.29 (quinoline C-7), 131.58 (quinoline C-8), 143.76 (benzenesulfonamide C-3), 145.69 (benzenesulfonamide C-1), 147.05, 147.52 (quinoline C-6), 152.27 (quinoline C-9), 153.76 (quinoline C-2), 156.21 (quinoline C-4), 166.26 (C = O); HRMS (ESI) for C18H16ClFN3O4S: calcd 424.0534, found: 424.0525 [M + H]+.Yellow solid, yield: 55%, mp: 190.0 − 191.0 °C; 1H NMR  δ ppm: 1.21 , 4.17 , 6.97–6.99 , 7.28–7.37 , 7.72 , 8.08 , 9.09 , 9.83 ; 13 C NMR  δ ppm: 14.22 (CH3) 62.08 (CH2), 114.54 (quinoline C-3), 117.87 (benzenesulfonamide C-2), 118.79 (quinoline C-10), 120.52 (benzenesulfonamide C-4), 127.44 (benzenesulfonamide C-6), 129.58 (quinoline C-6), 130.43 , 131.42 (benzenesulfonamide C-5), 136.15 (quinoline C-7), 136.62 (benzenesulfonamide C-1), 145.22 (quinoline C-4), 148.09 (quinoline C-9), 152.40 (quinoline C-2), 166.39 (C = O); HRMS (ESI) for C18H16Cl2N3O4S: calcd 440.0239, found: 440.0237 [M + H]+.Yellow solid, yield: 98%, mp: 228.7 − 230.3 °C; 1H NMR  δ ppm: 1.07 , 2.40 , 3.90 , 7.27 , 7.41 , 7.49–7.53 , 8.19 , 8.26 , 8.91 , 9.79 ; 13 C NMR  δ ppm: 14.19 (CH2CH3), 29 (NHCH3), 61.52 (CH2), 110.18 (quinoline C-5), 110.42 (quinoline C-5), 111.56 (quinoline C-3), 117.44 (benzenesulfonamide C-2), 121.17 (benzenesulfonamide C-4), 121.25 (quinoline C-10), 121.33 (quinoline C-10), 122.63 (benzenesulfonamide C-6), 125.22 (quinoline C-7), 125.43 (quinoline C-7), 130.61 (benzenesulfonamide C-5), 131.58 (quinoline C-8), 140.89 (quinoline C-8), 144.09 (benzenesulfonamide C-3), 146.97 (benzenesulfonamide C-1), 147.02 (quinoline C-9), 147.54 (quinoline C-2), 152.26 (quinoline C-4), 153.76 (quinoline C6), 156.21 (quinoline C-6), 166.24 (C = O); HRMS (ESI) for C19H18ClFN3O4S: calcd 438.0691, found: 438.0693 [M + H]+.Yellow solid; yield: 40%, 2 hydration activity2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionised water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were pre-incubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlierAn Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO6a–f and 11 are depicted in 1a–f were heated with diethyl ethoxymethylenemalonate in refluxing ethanol to furnish diethyl 2-((phenylamino)methylene)malonate derivatives 2a–f which thermally cyclised to the corresponding ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylates 3a–fvia heating in diphenyl ether. Next, chlorination of quinolinones 3a–f was carried out under anhydrous condition through heating with excess of phosphorus oxychloride to afford the key intermediates ethyl 4-chloroquinoline-3-carboxylates 4a–f. The target primary 3-(quinolin-4-ylamino)benzenesulfonamides 6a–f were obtained through a MW assisted nucleophilic substitution reaction of 3-aminobenzenesulfonamide 5 with the appropriate key intermediate 4a–f in ethyl alcohol .The structures of the newly prepared quinolines 1H NMR spectra of quinolines 6a-f showed new characteristic signals at δ 7.33– 7.37 ppm, and 9.68–10.25 ppm corresponding to NH2 and NH groups, respectively, that distinguished the target quinolines 6a-f from the key intermediates chloroquinolines 4a-f. Also, the 1H NMR of 7-chloro-6-fluoro-4–(3-methanesulphonylaminophenyamino)-quinoline-3-carboxylic acid ethyl ester (11) displayed three significant signals at δ 2.99, 9.63 and 9.79 ppm assigned to -NHCH3, -SO2NH- and -NH- protons, respectively.6a–f and 11 were evaluated for their ability to inhibit the physiologically relevant hCA cytosolic isoforms, hCA I and II, by a stopped-flow CO2 hydrase assayAAZ (as a standard inhibitor) against the two examined isoforms are summarised in The newly prepared 3-(quinolin-4-ylamino)benzenesulfonamides 11) failed to inhibit the tested hCA isoforms (hCA I and hCA II) up to 10 μM, which confirmed the crucial role of the primary sulfonamide as a zinc-anchoring group, with the additional two hydrogen bonds with Thr199 and Thr200 residues within the enzyme active site.The secondary sulfonamide reported here (6a-6e) weak potency in inhibiting the ubiquitous cytosolic isoform hCA I with inhibition constants (KIs) in the micromolar range, in detail, between 4.233 and 9.091 μM, except for the 6-methoxy substituted analog 6c which arose as a submicromolar hCA I inhibitor with a KI equals 0.966 μM, which represents 3.8-fold decreased efficacy to the reference drug AAZ . On contrary, the 5,7-dichloro substituted primary sulfonamide 6f failed to inhibit the hCA I up to 10 μM.The data presented in 6c; KI = 0.966 μM) resulted in 4.4-fold efficacy enhancement in comparison to the unsubstituted analogue 6a (KI = 4.233 μM). Regarding the impact of substitution of the quinoline moiety within the primary sulfonamides series 6a-6f; the inhibitory activities were decreased in the order of 6-OCH3 >6-CH3 >7-Cl-6-F > 6-Br >5,7-(Cl)2.Noteworthy, the SAR outcomes highlighted that grafting the strong electron-donating 6-metoxy group (compound 6a-6e) effectively interfere with hCA II catalytic activities in submicromolar/micromolar concentration range , whereas, no significant inhibition towards hCA II was revealed for quinoline 6f (KI >10 μM). Nevertheless, among the tested quinolines, 7-chloro-6-flouro substituted compound 6e (KI = 0.083 μM) proved to be the most active quinoline in inhibiting hCA II. Moreover, grafting a 6-methoxy group within the quinoline scaffold  was advantageous for the inhibitory activity toward hCA II, similarly to the SAR for hCA I inhibition. Regarding the substitution effect for the quinoline moiety; the inhibitory activities towards hCA II were decreased in the order of 7-Cl-6-F > 6-OCH3 > 6-CH3 > 6-Br > 5,7-(Cl)2.The second ubiquitous cytosolic isoform examined here was hCA II. It was apparent from the displayed results  that the6a-f and 11), which were evaluated for their inhibition of hCA I and hCA II. Both the examined isoforms were inhibited by the quinolines reported here in variable degrees; hCA I was inhibited with KIs in the range of 0.966–9.091 μM, whereas hCA II in the range of 0.083–3.594 μM. Among the tested compounds, the primary 7-chloro-6-flouro substituted sulfonamide derivative 6e (KI = 0.083 μM) proved to be the most active quinoline in inhibiting hCA II, whereas, its secondary sulfonamide analogue 11 failed to inhibit the hCA II up to 10 μM, confirming the crucial role of the primary sulphonamido group, as a ZBG, for CA inhibitory activity.In summary, we successfully synthesised new benzenesulfonamides, bearing un/substituted ethyl quinoline-3-carboxylate scaffold (


S,4'S)-cis-khellactone derivatives 3a–o is reported for the first time. Their structures were confirmed by 1H-NMR, 13C-NMR and MS. Their cytotoxic activity was evaluated by the MTT assay against three selected human cancer cell lines: HEPG-2 (human liver carcinoma), SGC-7901 (human gastric carcinoma), LS174T (human colon carcinoma). Some compounds showed high inhibitory activity against these human cancer cell lines. Among them, compound 3a exhibited strong cytotoxicity, with IC50 values ranging from 8.51 to 29.65 μM. The results showed that 4-methyl-cis-khellactone derivatives with S,S configuration could be a potential antitumor agents. An asymmetric synthesis of a series of novel 4-methyl-(3' Then, hydroquinidine 2,5-diphenyl-4,6-pyrimidinediyl diether [(DHQD)2 -PYR], 4.4 mg, 0.005 mmol) and K2OsO2(OH)4  were added to the solution. The mixture was stirred for 15 min. Then, the solution was cooled to 0 °C and methane-sulfonamide  added under stirring. When the solution turned from a light yellow to an orange color, compound 1  was added. The mixture was stirred at 0 °C for 1 day. The reaction was monitored using TLC, and at completion, Na2S2O5 (1 g), water (2.5 mL), and CH2Cl2 (2.5 mL) were added. After being stirred for 4 h at room temperature, the mixture was extracted with CH2Cl2 three times. The combined organic layer was dried over Na2SO4, and then solvent was removed to afford crude compound 2. The crude compound was crystallized from petroleum ether/acetone to give pure 2. Molecular formula (MW): C15H16O5 (276.28 g/mol); white solid; 70% Yield; mp: 213–215 °C; [α]D20 = –59.5 ; 74% ee. HPLC: Chiralpak AS-H , tR (minor) = 26.71 min, tR (major) = 68.88 min; 1H-NMR (DMSO-d6) δ 1.35 , 1.37 , 2.37 , 3.61 , 4.90 , 5.14 and 5.21 , 6.19 , 6.77 , 7.57 ; 13C-NMR (DMSO-d6) δ 18.17, 20.98, 26.86, 60.23, 71.23, 78.68, 110.55, 111.71, 112.56, 113.42, 125.66, 153.28, 153.52, 155.58, 160.03; MS (ESI) m/z 277.09 ([M+H]+).K2  in anhydrous CH2Cl2 (10 mL) tiglic acid (6 mmol), angelic acid (6 mmol), crotonic acid (6 mmol), isovaleric acid (4 mmol), 4-methoxybenzoic acid (3 mmol), 3,4-dimethoxybenzoic acid (3 mmol), 4-methoxycinnamic acid (2 mmol), o-toluic acid (3 mmol), 3-nitrobenzoic acid (3 mmol), 2-bromobenzoic acid (3 mmol), 3-bromobenzoic acid (3 mmol), 4-bromobenzoic acid (3 mmol), 2,3-dichlorobenzoic acid (2 mmol), 2,4-dichlorobenzoic acid (2 mmol) or 2,5-dichlorobenzoic acid (2 mmol) were added, respectively, followed by N,N’-dicyclo-hexylcarbodiimide  and 4-dimethylaminopyridine . The mixture was heated to reflux until the reaction was complete as monitored by TLC. After cooling to room temperature, filtered, and the filtrate was separated and purified by column chromatography  to give pure target compounds 3a–o.To a solution of crude compound -Di-O-tigloyl-4-methyl-(+)-cis-khellactone (3a). Molecular formula (MW): C25H28O7 (440.49 g/mol); white solid; 23% Yield; mp: 84–86 °C; +).-Di-O-angeloyl-4-methyl-(+)-cis-khellactone (3b). Molecular formula (MW): C25H28O7 (440.49 g/mol); white solid; 31% Yield; mp: 151–153 °C; +). -Di-O-crotonoyl-4-methyl-(+)-cis-khellactone (3c). Molecular formula (MW): C23H24O7 (412.43 g/mol); white solid; 45% Yield; mp: 63–65 °C; +).-Di-O-isovaleryloxy-4-methyl-(+)-cis-khellactone (3d). Molecular formula (MW): C25H32O7 (444.52 g/mol); light yellow oil; 52% Yield; +).-Di-O-p-methoxybenzoyl-4-methyl-(-)-cis-khellactone (3e). Molecular formula (MW): C31H28O9 (544.55 g/mol); white solid; 19% Yield; mp: 177–179 °C; +).-bis-O--4-methyl-(-)-cis-khellactone (3f). Molecular formula (MW): C33H32O11 (604.60 g/mol); white solid; 28% Yield; mp : 207–209 °C; +).-bis-O-p-Methoxycinnamoyl-4-methyl-(-)-cis-khellactone (3g). Molecular formula (MW): C35H32O9 (596.62 g/mol); white solid; 34% Yield; mp: 120–122 °C; +).-Di-O-o-methylbenzoyl-4-methyl-(-)-cis-khellactone (3h). Molecular formula (MW): C31H28O7 (512.55 g/mol); white solid; 47% Yield; mp: 196–198 °C; +).-Di-O-m-nitrobenzoyl-4-methyl-(-)-cis-khellactone (3i). Molecular formula (MW): C29H22N2O11 (574.49 g/mol); white solid; 34% Yield; mp: 187–189 °C; +).-Di-O-o-bromobenzoyl-4-methyl-(-)-cis-khellactone(3j). Molecular formula (MW): C29H22Br2O7 (642.29 g/mol); white solid; 42% Yield; mp: 178–180 °C; +).-Di-O-m-bromobenzoyl-4-methyl-(-)-cis-khellactone(3k). Molecular formula (MW): C29H22Br2O7 (642.29 g/mol); white solid; 36% Yield; mp: 201–203 °C; +).-Di-O-p-bromobenzoyl-4-methyl-(-)-cis-khellactone (3l). Molecular formula (MW): C29H22Br2O7 (642.29 g/mol); white solid; 40% Yield; mp: 192–194 °C; +).-bis-O--4-methyl-(-)-cis-khellactone (3m). Molecular formula (MW): C29H20Cl4O7 (622.28 g/mol); white solid; 29% Yield; mp: 197–199 °C; +).-bis-O--4-methyl-(-)-cis-khellactone (3n). Molecular formula (MW): C29H20Cl4O7 (622.28 g/mol); white solid; 41% Yield; mp: 140–142 °C; +).-bis-O--4-methyl-(-)-cis-khellactone (3o). Molecular formula (MW): C29H20Cl4O7 (622.28 g/mol); white solid; 28% Yield; mp: 116–118 °C; +).1  was added to a solution of osmium tetroxide  and N-methylmorpholine-N-oxide monohydrate  in t-BuOH-THF-H2O  and the mixture was stirred for 1 day at r.t. Saturated NaHSO3 solution (80 mL) was added, the mixture kept stirring for 2 h and extracted with CH2C12 (2 × 40 mL) and purified by column chromatography  to afford the pure compound 2'. Molecular formula (MW) : C15H16O5 (276.28 g/mol); white solid; 56% Yield; MS (ESI) m/z 277.09 ([M+H]+).Compound 2 at 37 °C. The MTT assay was used to evaluate the cytotoxic activities in vitro. Briefly, cells were harvested during logarithmic growth phase and seeded in 96 well plates at a density of 2 × 104 cells per well. After 24 h incubation at 37 °C in 5% CO2 atmosphere, cells were exposed to title compounds at concentrations from 5 to 200 μM for 48 h. Subsequently, MTT  was added to each well and the plates were incubated for a further 4 h at 37 °C. Then, the media were removed from all the wells and the resultant MTT formazan was solubilized in DMSO (200 μL). The absorbance values at 490 nm were measured using a microplate reader . The IC50 was calculated from the semi-logarithmic dose-response curves.All the cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator aerated with 5% COS,4'S)-cis-khellactone derivatives were designed and asymmetrically synthesized. Antitumor evaluation indicated that some of these new compounds displayed high inhibitory activity against three human cancer cell lines. Among them, compound 3a with a tigloyl group at the 3',4' positions on the pyran ring exhibited the most potent cytotoxic activity and these results could be useful for the further modification to obtain more potent antitumor agents.In conclusion, a series of novel 4-methyl-(3'


In addition, in silico ADME assay demonstrated favorable physicochemical properties for the new designed compounds. Thus, synthesized structures could be introduced as a novel template for designing safe anti-HIV compounds with integrase inhibitory potential. Some new diazo incorporated coumarin compounds were designed and synthesized to evaluate their anti-HIV activity. Overall, compounds were active against HIV at 100 μM. Additionally, no cytotoxic effect was observed at this concentration. The compound with 4-chlorobenzyl group indicated the best anti-HIV activity (52%). Docking studies using the later crystallographic data available for PFV integrase showed similar binding modes to HIV-1 integrase inhibitors. On the basis of these data, nitrogen atoms of 1,3,4-oxadiazole ring have been involved in the Mg Acquired Immunodeficiency Syndrome (AIDS) is the second cause of death from infectious diseases in low income countries . Human I2+ cofactors which are critical for the catalytic activity  bind in the interface of the viral DNA and catalytic DDE motif. For this reason, they could be classified as interfacial inhibitors. The DDE motif contains a triad of three conserved acidic residues  coordinating 2 Mgl moiety -19. 1, 2, H)-one derivatives possessing 1,3,4-oxadiazole ring as metal-chelating moiety along with a benzyl part necessary for HIV-1 IN inhibitory activity. Among them, compound 3 (50 = 50 µM and no considerable cytotoxicity (CC50 > 100 µM)  . SAR ana 100 µM) . In this 100 µM)  to prese1 and 3, suggesting that our novel designed compounds possess ability to be promising anti-HIV agents.The overall shape and functionalities of designed structure particularly resemble those of compounds ChemistryH-chromen-2-onederivatives were synthesized according to the 4) and ethanol in a routine Fischer esterification reaction under sulfuric acid catalysis. Ethyl 4-aminobenzoate (5) was converted to 4-aminobenzohydrazide (6) by refluxing in absolute ethanol and excess of hydrazine hydrate. The 2-mercaptho-1,3,4-oxadiazole ring  were prepared in 6 M hydrochloric acid and 10% sodium nitrite aqueous solution at 0-5 °C. The final derivatives 11a-h were obtained through coupling reaction of the diazonium salts with 4-hydroxycoumarin (10) in aqueous sodium carbonate solution at neutral pH.3--1,3,4-oxadiazol-2-yl) phenyl) diazenyl)-4-hydroxy-2ole ring  was closole ring , 27. S-aMaterialsAll reagents purchased from the Aldrich (USA) or Merck (Germany) Chemical Company and were used without further purifications.General1HNMR; Chloroform-D used as solvent. Coupling constant (J) values are estimated in hertz (Hz) and spin multiples are given as brs (broad singlet), s (singlet), d (double), t (triplet), q (quartet), m (multiplet), and br (broad). The mass spectral measurements were performed on an 6410 Agilent LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI) interface.Melting points (mp) were determined using a Thomas Hoover capillary apparatus . Infrared spectra were acquired on a Perkin-Elmer 1420 ratio recording spectrometer. A Bruker FT-500 MHz instrument  was used to acquire Ethyl 4-aminobenzoate (5) -1) 3424- 3348 (NH2), 1682 (C=O); LC-MS (ESI) m/z: 166.1 . Yield 74%; White crystalline powder; mp 89.2-91.0 °C; IR (KBr disk): υ (cm4-Aminobenzohydrazide (6)-1) 3432- 3350 (NH2), 3307-3278 (-CO-NHNH2), 3239 (-CO-NHNH2), 1603 (C=O); LC-MS (ESI) m/z: 152.0 . Yield 82%; White crystalline powder; mp 216.8-218.3 °C; IR (KBr disk): υ (cm5-(4-Aminophenyl)-1,3,4-oxadiazole-2-thiol (7)6 (6 mmol) and potassium hydroxide (1.23 g) in absolute ethanol (50 mL), carbon disulfide (8.2 mL) was added in an ice bath. The mixture was refluxed at 50-60 °C for 4 hours. Then, it was cooled to room temperature and the solvent was evaporated to dryness. The crude suspended in 7 mL water and acidified with 3 M hydrochloric acid until pH 4.5. The yellow precipitate was filtered and washed with water. Yield 73%; Yellow powder; mp 197-199 °C; IR (KBr disk): υ (cm-1) 3356- 3440 (NH2); LC-MS (ESI) m/z: 194.0 .To a solution of compound General procedure for preparation of 4-(5-(substituted thio)-1,3,4-oxadiazol-2-yl) aniline (8a-h)7 (1.55 mmol) in methanol (4 mL) sodium hydroxide  was added. The mixture was cooled in an ice bath and substituted benzyl/alkyl halide (1.55 mmol) was added dropwise and stirred at room temperature. After completion of the reaction, the precipitate was filtered, rinsed with water, and crystallized from ethanol.To a solution of compound 4-(5-(Methylthio)-1,3,4-oxadiazol-2-yl)aniline (8a)-1) 3327- 3425 (NH2); LC-MS (ESI) m/z: 207.9 .Yield 87%; Yellow crystalline powder; mp 105.8-107.6 °C; IR (KBr disk): υ (cm4-(5-(Ethylthio)-1,3,4-oxadiazol-2-yl)aniline (8b)-1) 3374-3444 (NH2); LC-MS (ESI) m/z: 221.9 . Yield 73%; Yellow crystalline powder; mp 80.4-83.2 °C; IR (KBr disk): υ (cm4-(5-(Propylthio)-1,3,4-oxadiazol-2-yl)aniline (8c)-1) 3333-3478 (NH2); LC-MS (ESI) m/z: 236.9 .Yield 67%; Yellow crystalline powder; mp 105.1-106.8 °C; IR (KBr disk): υ (cm4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)aniline (8d)-1) 3359-3493 (NH2); LC-MS (ESI) m/z: 284.9 .Yield 61%; Yellow crystalline powder; mp 120.8-124 °C; IR (KBr disk): υ (cm4-(5-(2-Chlorobenzylthio)-1,3,4-oxadiazol-2-yl)aniline (8e)-1) 3337-3399 (NH2); LC-MS (ESI) m/z: 318.9 .Yield 59%; Yellow crystalline powder; mp 117.8-118.9 °C; IR (KBr disk): υ (cm4-(5-(4-Chlorobenzylthio)-1,3,4-oxadiazol-2-yl) aniline (8f)-1) 3349-3417 (NH2); LC-MS (ESI) m/z: 318.9 .Yield 65%; Yellow crystalline powder; mp 141.8-144.3 °C; IR (KBr disk): υ (cm4-(5-(2-Fluorobenzylthio)-1, 3, 4-oxadiazol-2-yl) aniline (8g)-1) 3373-3506 (NH2); LC-MS (ESI) m/z: 302.9 .Yield 55%; Yellow crystalline powder; mp 121.8-123.8 °C; IR (KBr disk): υ (cm4-(5-(4-Fluorobenzylthio)-1, 3, 4-oxadiazol-2-yl) aniline (8h)-1) 3353-3422 (NH2); LC-MS (ESI) m/z: 302.9 .Yield 59%; Yellow crystalline powder; mp 104-106 °C; IR (KBr disk): υ -4-hydroxy-3-((4-(5-(substitutedthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-2H-chromen-2-one (11a-h) 8a-h (1 mmol) in 6 M hydrochloric acid (2.7 mL), 0.7 mL cooled solution of 10% sodium nitrite was added dropwise in order to keep the temperature less than 5 °C. Then, the mixture was poured into a solution of 4-hydroxy coumarin (10) (1 mmol) and sodium carbonate (0.05 g) in water (1.85 mL). The pH of the mixture was adjusted to 7 by addition of sodium acetate. The reaction mixture was stirred in an ice bath. After completion of the reaction, the precipitate was filtered, washed with water and crystallized from acetonitrile. To a cooled solution of substituted compounds 4-Hydroxy-3-((4-(5-(methylthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-2H-chromen-2-one (11a)-1) 1666-1744 (C=O); 1HNMR : δ (ppm) 2.59 ; 7.32  7.34-7.39 , 7.68-7.74 , 7.98 , 8.09 ; LC-MS (ESI) m/z: 381.2 . Anal. Calcd. for C18H12N4O4S; C, 56.84; H, 3.18; N, 14.73. Found: C, 56.99; H, 3.32; N, 14.52.Yield 81%; Orange powder; mp 211.6-212.1 °C; IR (KBr disk): υ (cm3-((4-(5-(Ethylthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-4-hydroxy-2H-chromen-2-one (11b)-1) 1760 (C=O); 1HNMR : δ (ppm) 1.53 , 3.34 , 7.30 , 7.34 , 7.70 , 7.78 , 8.09-8.14 ; LC-MS (ESI) m/z: 395.0 . Anal. Calcd. for C19H14N4O4S; C, 57.86; H, 3.58; N, 14.21. Found: C, 57.66; H, 3.41; N, Yield 78%; Orange powder; mp 290-291 °C (decomposed); IR (KBr disk): υ (cm14.01. 4-Hydroxy-3-((4-(5-(propylthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-2H-chromen-2-one (11c)-1) 1732 (C=O); 1HNMR : δ (ppm) 1.09 , 1.89 , 3.30 , 7.30 , 7.34 , 7.70 , 7.78 , 8.09-8.1 ; LC-MS (ESI) m/z: 409.2 . Anal. Calcd. for C20H16N4O4S; C, 58.82; H, 3.95; N, 13.72. Found: 58.61; H, 3.77; N, 13.55.Yield 71%; Orange powder; mp 205.5-206.8 °C; IR (KBr disk): υ (cm3-((4-(5-(Benzylthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-4-hydroxy-2H-chromen-2-one (11d)-1) 1737 (C=O); 1HNMR : δ (ppm) 4.58 , 7.35-7.39 , 7.50 , 7.74 , 7.82 , 8.13-8.15 ; LC-MS (ESI) m/z: 284.1 . Anal. Calcd. for C24H16N4O4S; C, 63.15; H, 3.53; N, 12.27. Found: C, 62.95; H, 3.41; N, 12.57.Yield 75%; Orange powder; mp 243.1-243.8 °C; IR (KBr disk): υ (cm3-((4-(5-(2-Chlorobenzylthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-4-hydroxy-2H-chromen-2-one (11e)-1) 1755 (C=O); 1HNMR : δ (ppm) 4.69 , 7.30-7.45 , 7.67-7.73 , 7.82 , 8.13-8.4 , 16.26 ; LC-MS (ESI) m/z: 491.2 . Anal. Calcd. for C24H15ClN4O4S; C, 58.72; H, 3.08; N, 11.41. Found: C, 58.62; H, 3.28; N, Yield 71%; Orange powder; mp 236.8-237.5 °C; IR (KBr disk): υ (cm11.25. 3-((4-(5-(4-Chlorobenzylthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-4-hydroxy-2H-chromen-2-one (11f)-1) 1748 (C=O); 1HNMR : δ (ppm) 4.49 , 7.30-7.36 , 7.41 , 7.70 , 7.78 , 8.09 ; LC-MS (ESI) m/z: 514.3 . Anal. Calcd. for C24H15ClN4O4S; C, 58.72; H, 3.08; N, 11.41. Found: C, 58.98; H, 3.23; N, 11.30.Yield 74%; Orange powder; mp 260.8-263.8 °C; IR (KBr disk): υ (cm3-((4-(5-(2-Fluorobenzylthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-4-hydroxy-2H-chromen-2-one (11g)-1) 1752 (C=O); 1HNMR : δ (ppm) 4.61 , 7.11-7.17 , 7.34-7.51 , 7.60 , 7.74 , 7.83 , 8.14-8.16 , 16.31 ; LC-MS (ESI) m/z: 475.0 . Anal. Calcd. for C24H15FN4O4S; C, 60.76; H, 3.19; N, 11.81. Found: C, 60.90; H, 3.03; N, 11.97.Yield 58%; Orange powder; mp 240.1-242.2 °C; IR (KBr disk): υ (cm3-((4-(5-(4-Fluorobenzylthio)-1, 3, 4-oxadiazol-2-yl) phenyl)diazenyl)-4-hydroxy-2H-chromen-2-one (11h)-1) 1763 (C=O); 1HNMR : δ (ppm) 4.56 , 7.07 , 7.35-7.41 , 7.49-7.51 , 7.75 , 7.83 , 8.14-8.15 , 16.31 ; LC-MS (ESI) m/z: 475.0 . Anal. Calcd. for C24H15FN4O4S; C, 60.76; H, 3.19; N, 11.81. Found: C, 61.01; H, 3.37; N, 11.69.Yield 64%; Orange powder; mp 246.6-251.6 °C; IR (KBr disk): υ . Thus, it could be considered as a good model for the development of INSTIs  of p24 expression in Hela cells cultures. Azidothymidine (AZT) was the positive control in the test. The cytotoxic properties were measured in the same cells by XTT proliferation assay and reported as percentage of cell viability. Results are summarized in 11a with 51% cell viability. Hence, it can be concluded that the anti-HIV-1 activity of the compounds was not a result of their cytotoxic effects. As it was expected, compounds containing halobenzyl substitutions exhibited better anti-HIV activity as compared to alkyl substitutions. Although compound 11a containing methyl substitution displayed inhibition rate of 79%, its cell viability was 51% which indicated that its anti-HIV-1 activity might be derived from cytotoxic property. Among alkyl substitutions, propyl (compound 11c) was reasonably well tolerated, giving similar results to the 2-chlorobenzyl substitution. Introduction of an unsubstituted benzyl group (compound 11d) was not well tolerated, resulting in reduction in anti-HIV-1 activity (inhibition rate = 6%). Introduction of fluorine atom in position 2 and 4 of benzyl ring  gave the similar inhibition rates . However, compound 11g with fluorine atom in position 2 showed less %cell viability. The best anti-HIV-1 activity was observed with compounds containing chlorobenzyl substitution (compounds 11e and 11f). The shift of the chlorine atom from position 4 (compound 11f) to position 2 (compound 11e) was associated with decrease in anti-HIV activity (inhibition rate from 52 to 33%) and increase in cytotoxicity (cell viability from 100 to 75%). It can be concluded that 4-chlorobenzyl moiety at C-5 position of 1,3,4-oxadiazol ring was optimal in terms of anti-HIV activity and cell viability.Compounds Molecular Modeling and Docking Study A molecular docking study was conducted in order to evaluate synthesized compounds interactions with the integrase active site. To validate the docking study, the co-crystalized ligand, raltegravir was redocked under the same condition and superimposed on co-crystalized ligand pose (RMSD = 0.001), and displayed high affinity binding .11f showed high binding affinity . The 3D and 2D alignment of compound 11f in the active site was shown in 2+ essential cofactors and was involved in metal acceptor interaction with the Mg2+ ions. Phenyl and 1,3,4-oxadiazole motifs of the structure have been interacted via π-stacking bonding with deoxy adenosine (DA-17) presenting in the active site and could be expected to displace it. In addition, the 4-hydroxycoumarin motif was able to participate in π-stacking bonding with the Tyr212 residue.Docking results revealed that all the docked compounds accommodated well in the active site with affinity binding energy range from -9.0 to -9.8 kcal/mol . Compoun11f and co-crystalized raltegravir in the active site shown in 11f and chelating motif of raltegravir are nearly superimposable. 4-Chlorobenzyl group of compound 11f and 4-florobenzyl group of raltegravir have been similarly oriented towards the hydrophobic pocket. 4-Hydroxycoumarin motif of compound 11f and oxadiazole ring of raltegravir occupy the same area as well.Orientation of compound Collectively, the docking results of tested compounds were in a good agreement with IN inhibitors, suggesting that their anti-HIV activity may be due to IN inhibition. In silico evaluation of drug-likenessOral bioavailability is considered as an important factor in design of new molecules as therapeutic agents. Thus, in drug design it is valuable to gain sufficient information about the molecular properties that limit oral bioavailability. There are some analyses that have correlated physical properties of molecules with successful drug development. Among them, Lipinski′s rule of 5  and VebeP values ranged from 3.32-5.41, MW ranging from 380.38-490.93, HBA value of 8, HBD value of 1, nROTB values ranging from 3-7 (<10), TPSA values lower than 140 Å2 (114.09 Å2) which all suggesting that the evaluated compounds have acceptable flexibility and are expected to possess good permeability and oral bioavailability (All compounds showed loglability . Accordilability . As can 11f containing 4-chlorobenzyl ring exhibited best anti-HIV-1 activity among synthesized compounds with inhibition rate of 52% near inhibition rate of AZT (58%). Docking studies showed that designed compounds bind to IN with a mode similar to co-crystalized raltegravir. Moreover, in silico computation of ADME properties of designed compounds showed non-violations of Lipinski′s rule of 5 and Veber′s criteria, suggesting that the designed compounds possess favorable physicochemical properties near to those of FDA-approved INSTIs, RAL, ELV, and DTG. However, the designed compounds displayed low aqueous solubility as compared to FDA-approved INSTIs which may be the main reason of their moderate anti-HIV-1 activity. So, these compounds could undergo further structural modifications to improve their anti-HIV-1 activity.The principal objective of this study was to investigate a new chemical scaffold with anti-HIV activity. A novel structure was constructed by hybridization of fragments from well-known anti-HIV-1 lead compounds. Key to the design was the introduction of a 1,3,4-oxadiazole ring as a chelating motif associated with a halobenzyl moiety to mimic pharmacophore of INSTIs. SAR analysis revealed that compound


In addition, 5,7-dihydroxy-2-methylchomanone (4) was isolated from a natural source for the first time. Their structures were established on the basis of spectroscopic data as well as direct comparison with authentic samples. A new phenolic, bismethane ( Diospyros maritima Blume (Ebenaceae), a medium-sized shrub, is widely distributed throughout Southern Asia. The genus Diospyros is well-known to produce various naphthoquinone derivatives, some of which exhibit cytotoxic, ichthyotoxic, germination inhibitory, and antifungal activities , and two quaternary carbons . A butoxyl group was elucidated by the COSY correlations between H-1΄ (δH 4.07) and H-2΄ (δH 1.57); H-2΄ and H-3΄ (δH 1.34); H-3΄ and H-4΄ (δH 0.88). From the above observations, compound 2 was considered as a butanedioate derivative with both methyl and butyl groups. The chemically equivalent signals of two methylene groups  were assigned to be located between C-1 (δC 172.3) and C-4 (δC 172.8) according to their HMBC relationship. The proposed structure of 2 was also supported by the molecular ion peak at m/z 188 and the fragmental ion peak at m/z 157 [M-OMe]+, 129 [M-COOMe]+, 115 [M-OCH2 CH2 CH2 Me]+, 101 [M-CH2CH2COOMe]+, and 73 [M-COCH2CH2COOMe] + in the EI-MS spectrum. Compound 2 was accordingly identified as butylmethyl succinate.The IR spectrum of 3 showed absorption bands at 3,360 (OH), 1,640 (conjugated ketone), and 1,580 (aromatic) cm-1. The 1H-NMR and 1H-1H COSY spectra displayed the signals for a set of aromatic ABX coupling system  and a phenolic proton with strong intramolecular hydrogen bond [δH 12.35 ]. In addition, the NMR signals for a complex coupling system attributing to a methine [δH 2.30 ], which was coupled with methyl [δH 1.17 ], oxymethine {δH 4.49  coupled with 4-OH [δH 1.92 ]}, and methylene  was observed. The above features were almost identical to those of the known compound, epi-isoshinanolone  coupling to both methyl [δH 1.48 ] and methylene [δH 2.84 ], a phenolic proton with strong hydrogen-bonding [δH 11.19 ] and a free phenolic proton [δH 5.90 ], as well as two meta-positioned aromatic protons . The above spectral information was similar to that of 5,7-dihydroxy-2-methylchromanone previously reported in the literature , 2 , 3 , 4 , 5 , 6 , and 7 . Fraction 1 (8.5 g) was further purified through a silica gel column eluted with n-hexane/EtOAc (95/5) to yield 16 (6.2 mg). Fraction 3 (27.2 g) was further chromatographed on a silica gel column eluted with n-hexane/EtOAc (8/2) and semipreparative HPLC eluted with CH2Cl2/n-hexane/EtOAc (8/3/1) to obtain 8 (5.3 mg), 9 (3.0 mg), 10 (3.6 mg), 11 (5.1 mg), and 15 (4.2 mg). Fraction 4 (38.5 g) was subjected to column chromatography over silica gel eluted with n-hexane/EtOAc (7/3) and semipreparative HPLC eluted with CH2Cl2/n-hexane/EtOAc (3/3/1) to yield 3 (3.8 mg), 5 (3.5 mg), 6 (6.1 mg), 7 (5.0 mg), 12 (12.4 mg), 17 (3.0 mg), 18 (6.3 mg), 19 (4.0 mg), 20 (10.1 mg), 21 (4.7 mg), 22 (5.1 mg), 27 (4.1 mg), and 30 (3.0 mg). Fraction 5 (32.1 g) was further chromatographed on silica gel eluted with n-hexane/EtOAc (5/5) and semipreparative HPLC eluted with CH2Cl2/n-hexane/EtOAc (2/3/1) to yield 1 (5.2 mg), 2 (10.5 mg), 4 (3.2 mg), 13 (3.1 mg), 14 (6.2 mg), 23 (3.1 mg), 24 (3.2 mg), 25 (3.0 mg), 26 (3.5 mg), 28 (4.7 mg), 29 (2.8 mg), and 31 (4.0 mg). The stems of Bismethane (1): Gum; UV (logε) (MeOH) λmax: 283 (3.4) nm; IR νmax cm-1: 3337, 1619, 1500, 1208, 1082, 897; 1H- and 13C-NMR (CDCl3): see m/z (rel. int. %): 380 [M]+ (38), 211 (23), 197 (39), 184 (100), 169 (42), 57 (25); HR-EI-MS m/z 380.1476 .Butylmethyl succinate (2): Colorless oil; IR νmax cm-1: 1735, 1155, 1016; 1H-NMR (CDCl3): δ 0.88 , 1.34 , 1.57 , 2.60 , 3.67 , 4.07 ; 13C-NMR (CDCl3): δ 13.7 (C-4’), 19.0 (C-3’), 28.9 , 30.6 (C-2’), 51.8 (-OCH3), 64.6 (C-1’), 172.3 (C-1), 172.8 (C-4); EI-MS (70 eV) m/z (rel. int. %): 188 [M]+(1), 157 (8), 129 (38), 115 (78), 101 (100), 87 (38), 73 (9), 55 (58); HR-EI-MS m/z 188.0940 .epi-Isoshinanolone (3): Amorphous solid; IR (dry film) νmax cm-1: 3360, 1640, 1580; 1H-NMR (CDCl3): δ1.17 , 1.92 , 2.30 , 2.42 , 2.90 , 4.49 , 6.89 , 7.09 , 7.49 , 12.35 ; EI-MS (70 eV) m/z [M]+ 192 (90), 177 (20), 150 (45), 121 (100).5,7-Dihydroxy-2-methylchromanone (4): Amorphous solid; IR (dry film) νmax cm-1: 3400, 1668, 1620, 1600; 1H-NMR (CDCl3): δ1.48 , 2.84 , 4.66 , 5.90 , 6.18 , 6.29 , 11.19 ; EI-MS (70 eV) m/z 194 [M]+ (40), 185 (100), 149 (69).D. maritima. Among them, bismethane (1) and butylmethyl succinate (2) are new compounds and epi-isoshinanolone (3) was isolated in pure form for the first time. In addition, 5,7-dihydroxy-2-methylchromanone (4) was isolated from the natural source for the first time. This investigation of secondary metabolites may contribute to better understanding on the chemical characteristics of D. maritima.Thirty-one compounds were isolated from the stems of


These compounds may serve as leads for the synthesis of more potent anti-inflammatory compounds for future drug discovery.The natural product phloroglucinol-based derivatives representing monoacyl-, diacyl-, dimeric acyl-, alkylated monoacyl-, and the nitrogen-containing alkylated monoacylphloro- glucinols were synthesized and evaluated for inhibitory activities against the inflammatory mediators such as inducible nitric oxide synthase (iNOS) and nuclear factor kappaB (NF-κB). The diacylphloroglucinol compound  MS ESI-(−) m/z = 363.0/365.2 [M − H]− for C17H17BrO4.4 (346 mg) and K2CO3 (2.76 g) in acetone (35 mL) was stirred for 30 min, then CH3OCH2Cl (480 mg) was added. After stirring at room temperature for 48 h, the reaction mixture was filtered. The filtrate was concentrated, and the residue was purified by chromatography on silica gel with acetone:hexane (1:12) to give 5 .A suspension of compound 1--2-methyl-propan-1-one (5). 1H-NMR  δ: 14.0 , 7.35 , 7.21 , 6.46 , 5.28 , 5.24 , 3.93 , 3.82 , 3.55 , 3.41 , 1.21 . 13C-NMR  δ: 210.7 (C-7), 164.0 (C-5), 160.5 (C-1), 158.9 (C-3), 140.6 (C-12), 131.0 , 130.4 , 119.2 (C-15), 110.7 (C-2), 106.0 (C-4), 94.9 (-OCH2), 94.0 (-OCH2), 91.4 (C-6), 56.8 (-OCH3), 56.5 (-OCH3), 39.8 (C-8), 27.7 (C-11), 19.4 . MS ESI-(−) m/z = 451.0/453.2 [M − H]− for C21H25BrO6.3 (98 mg) and NaOH (22 mg) in H2O (7.5 mL) was stirred about 30–40 min until it became clear. To this solution was added TBAI (9.8 mg) and p-xylylene dibromide (144 mg) in toluene (4.5 mL) dropwise. After stirring at room temperature for 6 h, the reaction mixture was extracted with ethyl acetate (EtOAc) (3 × 20 mL). The combined EtOAc layer was concentrated under reduced pressure to afford a residue, which was purified by chromatography on silica gel using acetone:hexane (1:2) to give 6 .A suspension of compound 2-Methyl-1-{2,4,6-trihydroxy-3-[4--benzyl]-phenyl}-propan-1-one (6). 1H-NMR  δ: 14.19 , 7.17 , 6.10 , 4.02 , 3.85 , 1.14 . 13C-NMR  δ: 210.1 , 164.8 , 162.0 , 159.7 , 138.9 , 128.1 , 107.3 , 103.4 , 94.3 , 38.7 , 28.5 , 18.8 . MS ESI-(−) m/z = 493.2 [M − H]− for C28H30O8.2Cl2 (100 mL) was added K2CO3 (5.46 g). After stirring at room temperature for 1 h, p-xylenedibromide (7.86 g) in CH2Cl2 (100 mL) was added dropwise, and the reaction continued for 3 h. The reaction mixture was filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by chromatography on silica gel using acetone:hexane (1:7) to yield the intermediate 1-(4-(bromomethyl) benzyl)piperidine (2.0 g).To a solution of piperidine (2.55 g) in CH3 (196 mg) and NaOH (44 mg) in H2O (60 mL) was added the intermediate (707 mg) in MeOH (40 mL) dropwise. After stirring at room temperature for 4 h, the reaction mixture was extracted with EtOAc (3 × 80 mL) and concentrated under reduced pressure. The residue was purified by chromatography on silica gel using CHCl3:MeOH:acetone (10:1:1) to yield 7 .To a solution of compound 2-Methyl-1--propan-1-one (7). 1H-NMR  δ: 7.74 , 7.68 , 6.61 , 4.37 , 4.35 , 4.23 , 2.97 , 1.77 , 1.29 , 1.28 . 13C-NMR  δ: 211.2 (C-7), 166.4 (C-1), 164.6 (C-3), 162.5 (C-5), 145.2 (C-12), 132.16 , 130.3 , 127.89 (C-15), 107.5 (C-6), 104.8 (C-2), 95.9 (C-4), 61.0 (C-16), 52.99 , 39.7 (C-8), 29.2 (C-11), 23.62 , 22.78 (C-19), 20.3 . HRTOF-MS ESI-(+) m/z = 384.2170 [M + H]+ for C23H30NO4 .1H-NMR for the isomer of 7  δ: 7.78 , 7.63 , 6.61 , 4.97 , 4.37 , 4.35 , 2.97 , 1.77 , 1.36 , 1.29 . 13C-NMR for the isomer of 7  δ: 211.2 (C-7), 166.4 (C-1), 164.6 (C-3), 162.5 (C-5), 145.2 (C-12), 132.23 , 130.3 , 128.04 (C-15), 107.5 (C-6), 104.8 (C-2), 95.9 (C-4), 64.3 (C-16), 53.25 , 39.7 (C-8), 29.2 (C-11), 23.84 , 23.01 (C-19), 20.3 .2 and 95% humidity. Cells were seeded in 96-well plates  and incubated for 24 h for a confluency of 75% or more. Test samples diluted in serum free medium were added, and after 30 min of incubation, LPS (5 μg/mL) was added and cells were further incubated for 24 h. The concentration of nitric oxide (NO) was determined by measuring the level of nitrite in the cell culture supernatant by using Griess reagent. Percent inhibition of nitrite production by the test compound was calculated in comparison to the vehicle control. IC50 values were obtained from concentration response curves. Parthenolide was used as a positive control [The assay was performed in mouse macrophages ) cultured in phenol red free RPMI medium supplemented with 10% bovine calf serum and 100 U/mL penicillin G sodium, and 100 μg/mL streptomycin at 37 °C in an atmosphere of 5% CO control .2 and 95% humidity. Cells (1.2 × 107) were washed once in an antibiotic and FBS-free DMEM/F12, and then resuspended in 500 μL of antibiotic-free DMEM/F12 containing 2.5% FBS. NF-κB luciferase plasmid construct was added to the cell suspension at a concentration of 50 μg/mL and incubated for 5 min at room temperature. The cells were electroporated at 160 V and one 70-ms pulse using BTX disposable cuvettes model 640 (4-mm gap) in a BTX Electro Square Porator T 820 . The transfected cells were plated to the wells of 96-well plates at a density of 1.25 × 105 cells per well. After 24 h, cells were treated with different concentrations of test compound for 30 min before the addition of PMA (70 ng/mL) and incubated for 8 h. Luciferase activity was measured using the Luciferase Assay kit (Promega). Light output was detected on a SpectraMax plate reader. Percent inhibition of luciferase activity was calculated as compared to vehicle control, and IC50 values were obtained from concentration response curves. Parthenolide was used as positive control [The assay was performed in human chondrosarcoma cells , Manassas, VA, USA). Cells were cultured in 1:1 mixture of DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin G sodium, and 100 μg/mL streptomycin at 37 °C in an atmosphere of 5% COCytotoxicity was determined against the mammalian cell line Vero (African green monkey kidney fibroblast) which was obtained from ATCC. The detailed assay procedure has been described previously . In brie