BF showed the highest total phenolic content , total flavonoid content  and total proanthocyanidin content , accompanied with the highest antioxidant activity compared to other fractions through 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assays. Five flavonol glycosides, namely hyperoside (1), isoquercitrin (2), quercetin-3-O-β-d-glucuronide (3), isorhamnetin-3-O-β-d-galactoside (4) and syringetin-3-O-β-d-glucoside (5) were isolated from the Receptaculum Nelumbinis. Compounds 2–5 were isolated for the first time from the Receptaculum Nelumbinis. The five isolated flavone glycosides, particularly compounds 1–3, demonstrated significant DPPH and ABTS radical scavenging activity, with IC50 values of 8.9 ± 0.2, 5.2 ± 0.2, 7.5 ± 0.1 for DPPH and 114.2 ± 1.7, 112.8 ± 0.8, 172.5 ± 0.7 μg/mL for ABTS, respectively. These results suggest that Receptaculum Nelumbinis has strong antioxidant potential and may be potentially used as a safe and inexpensive bioactive source of natural antioxidants.The antioxidant activities of ethanolic crude extract (ECE) and its four different solvent sub-fractions , ethyl acetate fraction (EAF),  Nelumbo nucifera Gaertn. (lotus) is an economically important aquatic plant grown in lakes, swamps, pools and rice fields. China is the leading lotus producer in the world; it has been cultivated for more than 2,000 years and is widely cultivated in almost all provinces including Hunan, Zhejiang, Fujian, Hubei, Anhui and Jiangxi −; 1H NMR : δ 5.15 , 6.20 , 6.39 , 6.86 , 7.58 , 7.83 . 13C NMR : δ 179.8 (C-4), 163.3 (C-5), 159.1 (C-7), 159.1 (C-9), 158.8 (C-2), 150.3 (C-4′), 146.1 (C-3′), 136.1 (C-3), 123.2 (C-1′), 123.2 (C-6′), 118.1 (C-5′), 116.4 (C-2′), 105.9 (C-10), 105.7 (C-1″), 100.3 (C-6), 95.1 (C-8), 77.5 (C-5″), 75.4 (C-3″), 73.5 (C-2″), 70.3 (C-4″), 62.5 (C-6″). These data are identical with those for Hyperin reported elsewhere −; 1H NMR  δ 3.20–3.78 , 5.24 , 6. 23 , 6.42 , 6.88 , 7.58 , 7.72 . 13C NMR  δ: 179.8 (C-4), 167.0 (C-7), 163.4 (C-5), 159.1 (C-2), 158.7 (C-9), 149.8 (C-4′), 145.9 (C-3′), 135.7 (C-3), 123.2 (C-1′), 123.1 (C-6′), 117. 6 (C-5′), 116.0 (C-2′), 105.8 (C-10), 104.4 (C-1″), 99.9 (C-6), 94.8 (C-8), 78.8 (C-5″), 78.2 (C-3″), 75.8 (C-2″), 71.3 (C-4″), 62.4 (C-6″). These data are identical with those for isoquercitrin reported elsewhere −; 1H NMR  δ: 8.08 , 7.43 , 6.83 , 6.37 , 6.19 , 5.33 . 13C NMR  δ: 177.6 (C-4), 172.7 (C-6″), 164.7 (C-7), 161.0 (C-5), 157.1 (C-2), 156.5 (C-9), 148.5 (C-4′), 144. 9 (C-3′), 133. 8 (C-3), 121.1 (C-1′), 120.6(C-6′), 117.3 (C-2′), 115.4 (C-5′), 103.7 (C-10), 102.3 (C-1″), 99.0 (C-6), 93.7 (C-8), 76.5(C-3″), 74.7 (C-5″), 74.0 (C-2″), 71.8 (C-4″). These data are identical with those for quercetin-3-O-β-d-glucuronide reported elsewhere −; 1H NMR : δ 8.04, 7.51, 6.90 , 6.46 , 6.22 , 5.50 . 13C NMR : δ 177.6 (C-4), 164.4 (C-7), 161.4 (C-5), 156.5 (C-2), 156.4 (C-9), 149.6 (C-4′), 147.2 (C-3′), 133.2 (C-3), 122.0 (C-1′), 121.2 (C-6′), 115.3 (C-5′), 113.6 (C-2′), 104.1 (C-10), 101.8 (C-1″), 98.9 (C-6), 93.9 (C-8), 76.0 (C-5″), 73.3 (C-3″), 71.4 (C-2″), 68.1 (C-4″), 57.0 (3′-OCH3), 60.5 (C-6″). These data are identical with those for isorhamnetin-3-O-β-d-galactoside reported elsewhere −; 1H NMR : δ 7.51 , 6.14 , 6.00 , 5.23 , 3.91 . 13C NMR : δ 178.4 (C-4),165.1 (C-7), 162.7 (C-5), 159.5 (C-9), 157.4 (C-2), 149.3 , 141.0 (C-4′), 135.4 (C-3), 121.4 (C-1′), 108.3 , 104.7 (C-6), 103.6 (C-1″), 103.1(C-10), 97.5 (C-8), 78.8 (C-5″), 78.5 (C-3″), 76.3 (C-2″), 71.8 C-4″), 62.8 (C-6″), 57.4 . These data are identical with those for syringetin-3-O-β-d-glucoside reported elsewhere [Syringetin-3-lsewhere .The total phenolic content of the crude extract and its fractions were determined using Folin-Ciocaleteu assay according to the previously described method . Gallic sample/Ablank) × 100%, where Ablank is the absorbance of the blank reaction (using 2 mL of methanol instead of the tested sample) and Asample is the absorbance of the tested sample. For each sample, its concentration with 50% scavenging ability on the DPPH radicals (IC50) was determined by interpolation of linear regression analysis. BHT was used as positive control.The DPPH radical scavenging activities of the crude extract of Receptaculum Nelumbinis and its fractions were determined according to the previously described method . The DPP50 were calculated as described in the DPPH radical scavenging assay. BHT was used as positive control.The ability of the crude extract and fractions of Receptaculum Nelumbinis to scavenge ABTS radicals were determined according to ABTS decolorization assay as previously described . The abip < 0.05 to detect significant differences among groups.All the experiments were conducted in triplicates, and the data were presented as mean ± SD. SPSS  and Origin  were used to process the results, which were analyzed by one-way ANOVA and Tukey-HSD at 1), isoquercitrin (2) and quercetin-3-O-β-d-glucuronide (3) showed effective activity and the structure-activity relation investigation indicated that the catechol group in the B ring affected the antioxidant activity. Being the by-products of lotus food production, the utilization of Receptaculum Nelumbinis is still low: it is usually discarded as waste. The obvious antioxidant activity in Receptaculum Nelumbinis confirmed its important role in the bioactivity of whole lotus and suggested that the Receptaculum Nelumbinis removal may induce a more significant nutrient loss. Until now, the Receptaculum Nelumbinis has not received much attention with respect to recycling it for the antioxidants instead of disposing it due to the lack of commercial application of these wastes. Our findings not only provide an added value to this regional bio-resource, but could also facilitate its development as a safe and inexpensive bioactive source of natural antioxidants, especially for the fast growing functional food industry nowadays.This study was designed to evaluate the total phenolic, flavonoid and proanthocyanidin contents and antioxidant activity of ECE and its sub-fractions of Receptaculum Nelumbinis. The BF sub-fraction showed the highest total phenolic and flavonoids contents, exhibited the highest free-radical scavenging activities. Five flavonol glycosides were isolated and their antioxidant activities were evaluated using DPPH and ABTS models. Hyperoside (


This short-lived intermediate disclosed its occurrence through a reversible proton transfer which competed with an oxygen transfer from DMSO that created dimethyl sulfide. The presumably resultant transitory ketene incorporated KOH to afford the potassium salt of 1,1,3,3-tetramethylindan-2-carboxylic acid . The lithium salt of this key acid is able to acylate aryllithium compounds, furnishing one-sidedly overcrowded ketones along with the corresponding tertiary alcohols. The latter side-products (ca. 10%) were formed against a substantially increasing repulsive resistance, as testified through the diminished rotational mobility of their aryl groups. As a less troublesome further side-product, the dianion of the above key acid was recognized through carboxylation which afforded 1,1,3,3-tetramethylindan-2,2-dicarboxylic acid. Brominative deoxygenation of the ketones furnished two one-sidedly overcrowded bromoalkenes. Some presently relevant properties of the above Cl,K-carbenoid are provided in  In the outlined mechanism, the formal “hydrolysis” of 6 to produce the acid 10 can be seen to consist of an initial reduction of 6 generating the carbenoid 12, followed by the oxidation of 12 by DMSO that affords the precursors of 10.In a rapid oxygen-transfer reaction that is known –32 for slic acid ; this is readily ; thus, tisolated   which can more efficiently be cleaned from the last traces of DMSO through washing with water. The procedure can also be made colleague-friendly by an early deodorizing of the Me2S contamination through a short treatment of the aqueous solution of 17 with NaOCl (but not KMnO4), to be terminated soon through the addition of NaHSO3. Gratifyingly, all side-products did not contaminate the acid 10 because they assembled in the nonacidic fraction as described in the sequel.Although the isolated yield of acid 23 (24) 10 or with solid CO2 and subsequent isolation of the highly symmetric diacid 40 . The constitution of 40 followed from its ready decarboxylation in CDCl3 solution at rt to regenerate acid 10 within four days.As a second side-reaction, deprotonation of 2-H in 38a into bromoalkene 42a through brominative deoxygenation DMSO; IR (KBr) ν: 2971, 2959, 2920, 2860, 1627 (w), 1484, 1363, 1032, 1022, 968, 748, 677, 505 cm−1; anal. calcd for C15H20OS (248.39): C, 72.53; H, 8.12; S, 12.91; found: C, 72.75; H, 8.25; S, 12.94.analogue ; 1H NMR 2-(p-Methylbenzoyl)-1,1,3,3-tetramethylindane (38a): A round-bottomed Schlenk flask (50 mL) was charged with 4-bromotoluene , anhydrous Et2O (10 mL), and a magnetic stirring bar. The contents were stirred and cooled at −78 °C under argon gas cover during the dropwise addition of t-BuLi (9.16 mmol) in pentane (6.10 mL), then stirred without cooling for 30 min. After the dropwise addition of acid 10  in anhydrous Et2O (10 mL) to this solution of p-methylphenyllithium (34a) and further stirring at rt for 18 hours, the mixture was poured onto solid CO2, warmed up, and diluted with aqueous NaOH . The aqueous layer was shaken with Et2O (3 × 20 mL) and the combined four Et2O layers were washed with distilled water until neutral, dried over Na2SO4, and concentrated to leave the crude nonacidic material (455 mg) consisting mainly of 38a, 39a, and toluene (9:1:9). Repeated crystallizations from pentane afforded white needles of 38a (isolated yield up to 35%); mp 95.5–96.5 °C (methanol); 1H NMR  δ 1.34 and 1.39 , 2.42 , 4.10 , 7.17 and 7.24 , 7.28 , 7.88  ppm; 1H NMR  δ 1.29, 1.35, 2.39, 3.99, 7.06 , 7.18 (d), 7.80 (d) ppm; 13C NMR  δ 21.56 (p-CH3), 28.02 and 30.98 (2 × 1-/3-CH3), 47.17 (C-1/-3), 64.32 (C-2), 122.34 (C-4/-7), 127.12 (C-5/-6), 128.28 and 129.34 (2 × C-m and 2 × C-o), 138.36 (C-ipso), 143.50 (C-p), 149.99 (C-3a/7a), 201.25 (C=O) ppm; IR (KBr) ν: 2967, 2925, 2862, 1664 (s), 1606, 1480, 1368, 1228, 1209, 1186, 869, 759 cm−1; anal. calcd for C21H24O (292.42): C, 86.26; H, 8.27; found: C, 86.50; H, 8.41.2O (3 × 10 mL). These combined Et2O extracts were washed with distilled water until neutral, dried over Na2SO4, and evaporated to leave a white powder containing acid 10, p-methylbenzoic acid, p-cresol, and diacid 40 (ca. 4:2:2:1).The above aqueous NaOH layer was acidified with concd. hydrochloric acid and was shaken with Et1,1,3,3-Tetramethylindan-2,2-dicarboxylic acid (40): The mixture of acids obtained above (see 38a) was leached with pentane, which left the insoluble diacid 40 behind . One recrystallization from hot toluene afforded clean 40 (30 mg) but led to the decarboxylation of a portion that remained dissolved. The transparent needles of pure 40 had a mp of 195–197.5 °C (dec.), decomposed slowly on standing at rt in CDCl3 solution, and were weakly soluble in CCl4 only as long as they were a part of the original mixture with the other carboxylic acids. 1H NMR  δ 1.58 , 7.21  ppm; 13C NMR  δ 28.83 (2 × 1-/3-CH3), 48.98 (C-1/-3), 122.28 (C-4/-7), 125.79 (C-2), 127.59 (C-5/-6), 150.30 (C-3a/7a), 171.73 (2 × CO2H) ppm, assigned through comparison with the acid 10; IR (KBr) ν: 3400–2500 (very broad H–O), 1713 (s), 1291, 759 cm−1; anal. calcd for C15H18O4 (262.3): C, 68.69; H, 6.92; found: C, 69.23; H, 6.81.40 was not formed from acid 10 with lithium N,N-diisopropylamide in THF as the solvent at rt: final quenching with solid CO2 afforded only starting material 10.2-(α-Bromo-p-methylbenzylidene)-1,1,3,3-tetramethylindane (42a): A dry NMR tube (5 mm) was charged under argon cover gas with 2-bromo-1,3,2-benzodioxaphosphole 10, 33, 39a, 39b, 42b, and 43; FBW ring expansion of carbenoid 12; SNV reactions of 12 with PhCH2K and with KN(SiMe3)2.Alternative synthesis of ketone


Aim. Ducrosia anethifolia is used as flavoring additive. There have been little detailed phytochemical reports on this genus and the antidiabetic activity of this plant is not yet evaluated.  Method. Structure of compounds was deduced by spectroscopic analyses. Preliminary in vitro evaluation of the antidiabetic activity of crude extract and its furanocoumarins was carried out . The in vivo activity was investigated by measuring some oxidative stress markers. Biomarkers of liver injury and kidney were also determined. Results. Eight linear furanocoumarins, psoralen, 5-methoxypsoralen, 8-methoxypsoralen, imperatorin, isooxypeucedanin, pabulenol, oxypeucedanin methanolate, oxypeucedanin hydrate, and 3-O-glucopyranosyl-β-sitosterol, were isolated. All compounds were reported for the first time from the genus Ducrosia except pabulenol. The blood glucose level, liver function enzymes, total protein, lipid, and cholesterol levels were significantly normalized by extract treatment. The antioxidant markers, glucolytic, and gluconeogenic enzymes were significantly ameliorated and the elevated level of kidney biomarkers in the diabetic groups was restored. The compounds showed inhibitory activity in a concentration dependant manner. Imperatorin and 5-methoxypsoralen showed the most potent inhibiting power. Conclusion. D. anethifolia extract showed hypoglycemic, hypolipidemic, and antioxidant effect as well as ameliorating kidney function. This extract and some linear furanocoumarins exhibited carbohydrate metabolizing enzymes inhibitory effect. Furanocoumarins are well-known natural products that occur in most evolved genera of the Apiaceae family. These compounds were found to have antitumor, phytotoxic, photosensitizing, antibacterial, and high antifungal effects , 2. db/db mice , 158 , 130 , 102 ; 1H NMR : δ ppm 7.78 , 7.68 , 7.66 , 7.43 , 6.82 , 6.35 ; 13C NMR : δ ppm 160.9 (C-2), 156.3 (C-7), 151.9 (C-9), 146.8 (C-2′), 144.0 (C-4), 124.8 (C-6), 119.8 (C-5), 115.3 (C-10), 114.5 (C-3), 106.3 (C-3′), 99.7 (C-8).Compound  2 : C12H8O4; whitish yellow needle crystals; m.p. 189-190°C; Rf: 0.73 (S2); UV: λmax (MeOH): 230, 246, 266, 309; MS m/z (100%): 216 , 201 , 188 , 173 , 145 , 89 ; 1H NMR : δ ppm 8.16 , 7.60 , 7.15 , 7.03 , 6.28 , 4.28 ; 13C NMR : δ ppm 161.2 (C-2), 158.4 (C-7), 152.7 (C-9), 149.6 (C-5), 144.8 (C-2′), 139.2 (C-4), 112.7 (C-6), 112.6 (C-3), 106.5 (C-10), 105.0 (C-3′), 93.9 (C-8), 60.1 (OCH3-5).Compound  3 : C12H8O4; white crystals; m.p. 146-147°C; Rf: 0.63 (S2); UV λmax (MeOH): 247, 299 nm; MS m/z (100%): 216 , 201 , 188 , 173 , 158 , 145 , 89 ; 1H NMR : δ ppm 7.76 , 7.69 , 7.34 , 6.82 , 6.36 , 4.29 ; 13C NMR : δ ppm 160.4 (C-2), 147.6 (C-7), 146.6 (C-2′), 144.3 (C-4), 143.0 (C-9), 106.7 (C-3′′), 132.8 (C-8), 126.1 (C-6), 116.4 (C-10), 114.7 (C-3), 112.9 (C-5), 106.7 (C-3′), 61.3 (OCH3-8).Compound  4 (imperatorin): C16H14O4; whitish yellow needle crystals; m.p. 102–104°C, Rf: 0.53 (S2); UV λmax (MeOH): 247, 264, 301 sh; MS m/z (100%): 271 , 270 , 202 , 174 , 145 , 89 ; 1H NMR : δ ppm 7.75 , 7.68 , 7.35 , 6.80 , 6.34 , 5.60 , 5.00 , 1.73 and 1.71 ; 13C NMR : δ ppm 160.4 (C-2), 148.6 (C-7), 146.5 (C-2′), 144.3 (C-4), 143.8 (C-9), 139.6 (C-3′′), 131.6 (C-8), 125.8 (C-6), 119.7 (C-2′′), 116.4 (C-10), 114.6 (C-3), 113.1 (C-5), 70.1 (C-1′′), 25.7 and 18.1 (2CH3-3′′).Compound  5 (isooxypeucedanin): C16H14O5; white powder; m.p. 149°C; Rf: 0.60 (S3); UV λmax (MeOH): 252.5, 269 sh, 278, 329 nm; MS m/z (100%): 286 , 287 , 215 , 202 , 187 , 174 , 145 , 71 , 43 ,; 1H NMR (CD3OD-d6 300 MHz): δ ppm 8.29 , 7.58 , 7.15 , 6.82 , 6.29 , 5.06 , 2.84 , 1.16 ; 13C NMR : δ ppm 208.8 (C-2′′), 160.9 (C-2), 158.0 (C-7), 152.6 (C-9), 147.9 (C-5), 145.5 (C-2′), 139.2 (C-4), 113.6 (C-6), 113.2 (C-3), 107.4 (C-10), 104.1 (C-3′), 95.0 (C-8), 75.0 (C-1′′), 37.4 (C-3′′), 17.9 (2CH3-4′′/5′′).Compound  6 (pabulenol): C16H14O5; white powder; m.p. 128°C; Rf: 0.55 (S3); UV λmax (MeOH): 247, 331 nm; MS m/z (100%): 287 , 286 , 215 , 202 , 174 , 145 ; 1H NMR (CD3OD-d6 300 MHz): δ ppm 8.15 , 7.57 , 7.10 , 6.94 , 6.23 , 5.17 , 5.04 , 4.40 , 1.81 ; 13C NMR : δ ppm 161.2 (C-2), 158.0 (C-7), 152.5 (C-9), 148.5 (C-5), 145.1 (C-2′), 143.3 (C-3′′), 139.2 (C-4), 114.0 (C-6), 113.3 (C-4′′), 112.8 (C-3), 107.2 (C-10), 104.7 (C-3′), 94.6 (C-8), 75.5 (C-1′′), 74.1 (C-2′′), 18.7 (C-5′′).Compound  7 (oxypeucedanin methanolate): C17H17O6; white powder; m.p. 126°C; Rf: 0.49 (S3); UV λmax (MeOH): 254, 268, 310 nm; MS m/z (100%): 318 , 319 , 215 , 202 , 174 , 145 , 73 ; 1H NMR : δ ppm 8.18 , 7.55 , 7.08 , 6.97 , 6.22 , 4.55 , 4.35 , 3.91 , 3.24 , 1.24 , 1.21 ; 13C NMR : δ ppm 161.2 (C-2), 158.1 (C-7), 152.5 (C-9), 148.8 (C-5), 145.0 (C-2′), 139.2 (C-4), 114.0 (C-6), 112.7 (C-3), 107.2 (C-10), 104.9 (C-3′), 94.4 (C-8), 76.1 (C-2′′), 75.9 (C-3′′), 74.2 (C-1′′), 49.2 (C-OCH3-3′′), 20.8 (CH3-4′′), 20.6 (CH3-5′′).Compound  8 (oxypeucedanin hydrate): C16H16O6; white shiny crystal; m.p. 128°C; Rf: 0.44 (S3); UV λmax (MeOH): 253, 268, 311 nm; MS m/z (100%): 304 , 305 , 289 , 286 , 202 , 174 , 145 , 118 , 89 , 59 ; 1H NMR (CD3OD-d6 300 MHz): δ ppm 8.25 , 7.69 , 6.95 , 7.10 , 6.16 , 4.74 and 4.35 , 3.84 , 1.34 , 1.29 ; 13C NMR : δ ppm 164.0 (C-2), 160.3 (C-7), 154.3 (C-9), 151.2 (C-5), 147.5 (C-2′), 142.3 (C-4), 115.6 (C-6), 113.4 (C-3), 108.7 (C-10), 107.0 (C-3′), 95.1 (C-8), 78.8 (C-2′′), 76.3 (C-1′′), 73.5 (C-3′′), 28.0 (CH3-4′′), 25.8 (CH3-5′′).Compound  9 (3-O-glucopyranosyl-β-sitosterol): C35H60O6; white powder; Rf: 0.62 (S4); m.p. 280-281°C; HRESI-MS m/z (100%): 1150.6 [2M-H]−, 575 [M-H]−; 1H NMR : δ ppm 5.32 , 4.20 , 3.61 , 3.40–2.90 ; 13C NMR : δ ppm 140.4 (C-5), 121.1 (C-6), 100.8 (C-1′), 77.0 (C-3′/5′), 76.7 (C-3), 73.4 (C-2′), 70.0 (C-4′), 61.0 (C-6′), 56.2 (C-14), 55.0 (C-17), 49.6 (C-9), 45.1 (C-24), 41.8 (C-13), 39.7 (C-12), 38.3 (C-4), 36.8 (C-1), 36.2 (C-10), 35.8 (C-20), 33.8 (C-22), 31.4 (C-7/8), 29.5 (C-25), 29.2 (C-2), 28.7 (C-16), 26.1 (C-23), 24.8 (C-15), 22.6 (C-28), 20.9 (C-11), 19.6 (C-26), 18.9 (C-27), 18.6 (C-21), 19.0 (C-19), 11.7 (C-29), 11.6 (C-18).CompoundThe spectral data of the isolated furanocoumarins were illustrated as follows.1)α-Amylase Inhibitory Activity. Determination of  The inhibitory activity of α-amylase was determined according to the method described by Ali et al. [α-amylase activity was determined by measuring the absorbance at 540 nm. The α-amylase inhibitory activity (%) was defined as the percent decrease in the maltose production rate over the control. Acarbose was used as positive control. The α-amylase inhibition was expressed as a percentage of inhibition and calculated from the equation A540  control − A540  sample/A540  control × 100, where A is the absorbance.β-Galactosidase Inhibitory Activity Determination of . The inhibitory activity of β-galactosidase was measured by the method of Sanchez and Hardisson [ O-nitrophenolate ions was measured at 420 nm. A standard curve was performed using different concentrations of O-nitrophenol.(ardisson  and the 3)α-Glucosidase Inhibitory Activity Determination of . The α-glucosidase inhibitory activity was determined according to the method of Kim et al. [ p-nitrophenol from p-nitrophenyl α-D-glucopyranoside at 405 nm. A standard curve was carried out using different concentrations of p-nitrophenol. were selected for this study. They were obtained from the Animal House, National Research Centre, Egypt. All animals were housed in a temperature- (26–29°C) and humidity- (50–60%) controlled environment, in steel mesh cages of ten rats, each on wood-chip bedding, with a fixed light/dark cycle (12 hours), for one week as an acclimatization period with free access of water and food D. anethifolia for 45 days according to LD50 which revealed that the extract is safe till 1.5 g/kg BW, and group 3: diabetic group; where diabetes was induced by streptozocin (STZ). Each rat was injected intraperitoneally with a single dose of STZ (60 mg/kg BW) dissolved in 0.01 M citrate buffer immediately before use [Fifty male albino rats were selected for this study and divided into five groups (ten rats each) as follows: group 1: normal healthy control rats, group 2: normal rats orally treated with 500 mg/kg body weight (BW) total ethanolic extract offore use . D. anethifolia total extractfor 45 days and group 5: diabetic rats orally administered the antidiabetic reference drug glibenclamide at a dose of 5 mg/kg BW d for 45 days [After injection, animals had free access for food and water and given 5% glucose solution to drink overnight to encounter hypoglycemic shock. Animals were checked daily for the presence of glycosuria. Animals were considered to be diabetic if glycosuria was present for 3 consecutive days. After 3 days of STZ injection fasting blood samples were obtained and fasting blood sugar was determined (>300 mg/dL). Hyperglycemic rats were used for the experiment and classified as follows: group 4: diabetic animal oral administered 500 mg/kg BW of 45 days .After 45 days of treatments, rats were fasted overnight (12–14 hours), anesthetized by diethyl ether and blood collected by puncture of the sublingual vein in clean and dry test tube, left 10 minutes to, and centrifuged at 3000 rpm for serum separation. The separated serum was used for biochemical analysis of liver function enzymes, blood glucose level, lipid profile, and total protein content.After blood collection, rats of each group were sacrificed; the livers were removed immediately, weighed and homogenized in 5–10 volumes bidistilled water by a ratio of 1 : 10 w/v using electrical homogenizer, and centrifuged at 4000 rpm for 15 min. The supernatants were collected and placed in Eppendorf tubes, stored at −20°C, and used for determination of oxidative stress markers (nitric oxide (NO) and malondialdehyde (MDA)), antioxidant (reduced glutathione (GSH) and superoxide dismutase (SOD)), and carbohydrate metabolizing enzymes (hexokinase (HK), pyruvate kinase (PK), and phosphoenolpyruvate carboxy kinase (PEPCK)). The homogenization was carried out as described by Newsholme .μmol/mL.Glucose was determined in serum by colorimetric assay method kits . Alkaline phosphatase (ALP) enzyme activity was measured by the method of Belfield and Goldberg . The libμmol/mL. Total protein was assayed in serum according to Bradford [Aspartate and alanine aminotransferases (AST and ALT) enzyme activities were measured by the method of Javier Gella et al. . ColorimBradford . Total pCarbohydrates Metabolizing Enzymes. Hexokinase enzyme assay in liver tissue homogenate was assayed in liver tissue homogenate according to Abrahao-Neto et al. [2, and 30 Mm mercaptoethanol (pH 7.5) to yield 10% homogenate and the supernatant was used for enzyme assays. Glucose 6-phosphate formed by the hexokinase reaction is measured by adding glucose-6-phosphate dehydrogenase and nucleoside derived amino acids NADP following NADPH formation. This method minimizes inhibition due to glucose 6-phosphate by oxidizing it to 6-phosphogluconic acid. The rate of change in optical density is measured at 340 nm. The change in optical density should be between 0.005 and 0.020/minute and measured from the second to the tenth minute after adding adenosine triphosphate (ATP). Pyruvate kinase (PK) and phosphoenolpyruvate carboxy kinase (PEPCK) enzyme activities were determined in liver tissue homogenate using the previous method [3 serving as the source of CO2 and was determined by measuring the oxidation of NADH in the presence of malate dehydrogenase (MDH) at 340 nm. Lactate dehydrogenase (LDH) enzyme activity was determined in liver tissue homogenate according to the method of Bergmeyer et al. [p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT) with phenazine methosulfate (PMS) serving as an intermediate election carrier, the result is the formation of formazan of INT. The developed colour was measured at 503 nm. All enzyme activities are expressed inμmol/mg protein/min.o et al. . An aliqs method  and the r et al. . LDH catOxidative Stress Markers and Antioxidant Enzymes. Lipid peroxide (LPO)/malondialdehyde (MDA) [μmol/min/g tissue. Nitric oxide (NO) in acid medium and in the presence of nitrite formed nitrous acid diazotize with sulphanilamide and the product is coupled with N-(1-naphthyl) ethylenediamine. The resulting azo-dye has a bright reddish-purple colour which can be measured at 540 nm. NO is expressed inμg/g tissue.de (MDA)  and nitrde (MDA)  were detμg glutathione/mg protein. Superoxidedismutase (SOD) was assayed according to Paoletti et al. [2 reduction. The increase in absorbance with time was read at 560 nm. SOD is expressed in μmol/mg protein/min.Reduced glutathione (GSH) was assayed according to Moron et al. . The meti et al.  in the tLipid Profile and Kidney Markers. Profiles of the following serum lipids were established: total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), very-low-density lipoprotein cholesterol (VLDL-c), and total lipid using diagnostic kits . Total urea and creatinine were carried out using diagnostic kits .Statistical Analyses. Data were analyzed by comparing values for different treatment groups with the values for individual controls. Results are expressed as mean ± SD. The significant differences among values were analyzed using analysis of variance (one-way Anova) coupled with CoStat Computer Program. Unshared letters indicate significant correlation at P < 0.05. D. anethifolia was fractionated on a Si column, followed by consecutive purification steps on Si columns to yield eight linear furanocoumarins (1–8) and one sterol glycoside 9 , 5-methoxypsoralen (2), 8-methoxypsoralen (3), imperatorin (4), isooxypeucedanin (5), pabulenol (6), oxypeucedanin methanolate (7), oxypeucedanin hydrate (8), and 3-O-glucopyranosyl-β-sitosterol (9).The isolated compounds were identified as psoralen   . D. anethifolia total extract. On the other hand, diabetic rats showed significant increase in lipid profile, total cholesterol, triglycerides, LDL-cholesterol, and total lipids with percentages of increase amounting to 222.90, 171.05, 658.42, and 95.54%, respectively, while a significant decrease in HDL-cholesterol was observed (−69.71%), as compared to the normal control group. Treatments with D. anethifolia ethanolic extract significantly reversed these elevations and controlled the reduced LDH-c level with percentages of improvement 147.17, 111.65, 30.01, 465.49, and 58.20%, respectively, for total cholesterol, triglycerides, HDL-c, LDL-c, and total lipid. In addition, standard antidiabetic reference glibenclamide drug declared more or less similar results as illustrated in The data obtained in  D. anethifolia total extract ) was recorded in STZ-induced diabetic rats  or with antidiabetic standard drug , as compared to the normal control.With respect to glucolytic and gluconeogenic enzymes, an insignificant change was observed in all enzyme levels as a result of treatment of normal control rats withtic rats . A signi D. anethifolia total extract as compared to normal control group  [2) [3) [4) [5) [6) [7) [8) [O-glucopyranosyl-β-sitosterol (9) [The structures of isolateswere identified on the basis of interpretation of their chemical physicochemical analyses , compariing with the corresponding published data in the literature , 22. Bas1) , 5-metho1) [2) , 8-metho [2) [3) , imperat [5) [6) , oxypeuc [7) [8) . The steerol (9) . 1–8 exhibited UV absorption characteristic of linear furanocoumarins [ 1–8 showed absorption maxima at range of 247–269 and 294–334 nm which were characteristic of linear type of furanocoumarins [ m/z 201, 202, 174, and 145 in the mass spectra suggests a furanocoumarin moiety [1H NMR spectral data for compounds 2, 5–8 were characteristic of a 5-O-substituted furanocoumarin and compounds 3 and 4 were of 8-substituted one. They all confirmed the typical pattern of a linear furanocoumarins [ J = 9.6–9.8 Hz; the signal due to H-3 was observed at δ 6.16–6.36. The chemical shift of H-4 distinguished the C-8 and C-5 substituted furanocoumarins. In the spectra of compounds 2, 5–8, the resonance signal due to H-4 appeared at δ 8.15–8.29, indicating that they were substituted at C-5 [δ 6.95–7.15. On the other hand, 1H NMR spectra of compounds 3 and 4 contained resonance signals due to H-4, relatively up field at δ 7.75–7.76, indicating that these compounds were substituted at C-8 [δ 7.34–7.35. The furanoprotons appeared as doublets with J = 2.1–2.4 Hz. The resonance signals due to H-2′ and H-3′ were observed at δ 7.68-7.69 and 6.80–6.82, respectively. With respect to the compounds 2, 5–8, the resonance signals due to H-2′ and H-3′ were observed at δ 7.55–7.69 and 6.82–7.03, respectively. A downfield chemical shift of compounds 2, 5–8, for C-5, appeared at δ 147.9–151.2 indicating that these compounds were substituted at C-5, whereas compounds 3 and 4 contained resonance signals relatively upfield at δ 112.9–113.1 indicating that these compounds were substituted at C-8. Furthermore, compounds 2, 5–8 were shown upfield of C-8 at δ 93.9–95.1 indicating that these compounds were unsubstituted at C-8. On the other hand, compounds 3 and 4 were shown downfield of C-8 at δ 131.6–132.8 indicating that these compounds were substituted at C-8 [ 9 was identified as 3-O-glucopyranosyl-β-sitosterol from its mass spectrum and NMR spectroscopic measurements [ 9 showed the existence of 35 signals; the sugar moiety gives rise to one methylene group and five methine groups, whereas the aglycone part exhibits twenty-nine carbon signals, arising from six methyl groups, eleven methylene groups, nine methine groups, and three quaternary carbon atoms. The aglycone moiety was identified as β-sitosterol from its 1H and APT spectra. The sugar moiety was connected to C-3 atom as shown from its HMBC spectrum cross peaks H-1′/C-3.Compoundsoumarins . UV specn moiety . The 1H d at C-5 ; the sigd at C-8 ; the ched at C-8 . Compoun Ducrosia genus except pabulenol.All compounds are reported here for the first time in theD. enthifolia is an herb used as additives in foods. Chemoprevention by dietary means continues to attract major attention for the management of chronic degenerative diseases primarily due to the dramatic rise of diabetes mellitus as major healthcare problem [ problem .Cell damage has been implicated in a variety of chronic diseases. Oxidative stress and alterations in glucose metabolism are important risk factors for diabetes and its related complications. Advanced glycation end products (AGEs) and their carbonyl derivatives contribute to the pathogenesis of diabetes by their interaction with specific cell membrane receptors triggering to induce the expression of proinflammatory mediators and elicit oxidative stress, which exacerbate diabetic complications . There iα-amylase, α-glucosidase, and β-galactosidase, in the digestive tract. Inhibition of these enzymes delays carbohydrate digestion and prolongs overall carbohydrate digestion time, causing a reduction in the rate of glucose absorption and consequently blunting the postprandial plasma glucose rise [One therapeutic approach for treating diabetes is to decrease the postprandial hyperglycemia by retarding the absorption of glucose through the inhibition of the carbohydrate hydrolyzing enzymes, ose rise . Ionidium suffruticosum at the dose of 10 mg/kg BW/day along with high-fat diet significantly (P < 0.001) prevented the rise in the plasma total and LDL-c, triglycerides, and phospholipids and also showed cardioprotective effect against hyperlipidemia [Furanocoumarins provide examples of their versatility in a range of applications in the fields of biology and pharmacology. The administration of coumarin-rich fraction ofipidemia .4) showed the highest activity against carbohydrate metabolizing enzyme  and 5-methoxypsoralen (2) showed the highest activity, while a moderate activity for psoralen (1), oxypeucedanin hydrate (8), and isooxypeucedanin (5) was recorded , which is in agreement with the reported data of Iyer and Patil [In the present study, a significant reduction in total cholesterol (TC) and triglycerides (TGs) after the treatment with extract  as compand Patil .The present results indicate significant elevation in NO and MDA levels in liver of diabetic rats . These eLipid peroxidation can damage protein, lipid, carbohydrates, and nucleic acids and is one of the risk factors of protein glycation. Moustafa  reportedConcerning glucolytic  and gluconeogenic (PEPCK) enzymes, a significant decrease in enzyme pathways was noticed in diabetic group as compared to normal control . SherlocIn streptozotocin-induced hyperglycemic rats, glomerular alterations such as microalbuminuria, increase in urea levels, and decrease in creatinine clearance, as well as tubular disorders, were observed . However D. anethifolia total extract could reverse the above-mentioned diabetic effects. This may be through potentiating the pancreatic secretion of insulin from islet β-cells or the transport of blood glucose to the peripheral tissue. In addition D. anethifolia might increase the levels of insulin and C-peptide in diabetic [Oral administration ofdiabetic . D. anethifolia may be due to its inhibition of endogenous synthesis of lipids [ D. anethifolia extract was found to have therapeutic potential due to its antioxidant activity in several areas, including the capacity of preventing and decreasing the damage caused by hyperlipidemia and hyperglycemia [One of the possible actions off lipids . Furtherglycemia .The enhanced hexokinase and other glucolytic enzyme activities , with suppression in gluconeogenic enzyme PEPCK in plant extract treated rats , suggest4) was reported to be an active constituent increasing in vitro insulin release to 170.3% [In addition, the antihyperglycemic effect of different isolated furanocoumarin compounds could inform their stimulatory action on intracellular glucose transport by causing an increase in glucose uptake by different cells in absence of insulin and thus could attenuate cytokine-induced toxicity which reduce the oxidative damage of the pancreas and hence may ameliorate the endocrine function of this gland . Also, so 170.3% . D. anethifolia showed a decrease in the blood glucose in rats with hyperglycemia when compared to the standard antidiabetic drug glibenclamide [The crude extract ofnclamide . These rnclamide . D. anethifolia offers an attractive potential strategy to regulate postprandial hyperglycemia toward an overall dietary support for type 2 diabetes management.This research suggests that in vitro inhibitory effects against carbohydrate metabolizing enzymes  in a concentration dependent relationships. Imperatorin and 5-methoxypsoralen showed the most potent inhibitory power, while psoralen, oxypeucedanin hydrate, and isooxypeucedanin showed moderate inhibitory activities. The biological activity of in vivo D. anethifolia crude extract showed hypoglycemic, hypolipidemic, and antioxidant effects as well as an amelioration in kidney function through improvement in total urea and creatinine levels.It could be concluded that the crude extract and its major isolated furanocoumarins exhibited


The rAd5 expressed a clade B Gag-Pol fusion protein and secreted gp140s of HIV-1 strains 92RW020 (clade A), HxB2/Bal-V3/ V1V2 (clade B) and 97ZA012 (clade C). The NYVAC-B expressed a clade B Gag-Pol-Nef polyprotein and the secreted gp120 of Bx08 (clade B). A total of 80 participants were randomized into a placebo group (P) and four treatment groups: T1, 2x NYVAC-B/1x rAd5  followed by SF162.LS (42.1%/54), BaL.26 (18.4%/15.5), MW965.26 (14.5%/31) and Bx08.16 (11.8%/19.5). Five subjects neutralized all 5 tier 1 viruses, 5 subjects neutralized 4 viruses, 7 subjects neutralized 3 viruses, 14 subjects neutralized 2 viruses (MN.3 and SF162.LS) and 18 subjects neutralized 1 virus (MN.3). Aggregate magnitude-breadth scores across the tier 1 panel were strongest for T4 followed by T3, T1 and T2. Differences were significant for T1 vs. T3 (p=0.048) and T1 vs. T4 (p=0.004). Responses against tier 2 viruses were weak and sporadic in the A3R5 assay and were nearly absent in the TZM-bl assay.10 dose of rAd5 was superior to the two lower doses of 109 and 108 for both binding and neutralizing antibodies. At the highest rAd5 dose tested, rAd5/NYVAC-B was superior to NYVAC-B/rAd5 for neutralizing antibodies.A 10


The 1H NMR spectra of complexes 1–4 proved that the Sn atoms adopt octahedral configurations. The nanoparticles of the complexes were also prepared by ultrasonication, and their SEM micrographs indicated identical spherical morphologies with particles sizes about 20–25 nm. The fluorescence spectra exhibited blue shifts for the maximum wavelength of emission upon complexation.Four novel organotin(IV) complexes containing phosphoric triamide ligands were synthesized and characterized by multinuclear ( In recent years, the increasing progress in the preparation of nanomaterials has led to characterization of a great number of nanostructures . NanoscaUltrasonic vibration has the potential to be a simple and effective process to produce homogenous nanomaterials. Some investigations have proved that ultrasonication can be applied to prepare nanosized coordination compounds –29. The 2(CH3)2L12 (1), SnCl2(OH2)2L12 (2), SnCl(C6H5)3L12 (3) and SnCl2(CH3)2L22 (4), where L1 = C6H5C(O)NHP(O)[NC4H8N(C6H5)]2 (5) and L2 = P(O)[NC4H8N(C6H5)]3 (6), were synthesized and characterized. The results of NMR, IR, UV and fluorescence spectroscopy of complexes 1–4 were compared with each other and their related phosphoric triamide ligands 5, 6. The spherical nanoparticles of complexes 1–4 were obtained by ultrasonication with particle sizes of about 20–25 nm.In this work, following on from our previous works to prepare organometallic compounds including phosphoric triamide ligands –18, nove1–4 were synthesized from the reaction of SnClR3 with phosphoric triamide ligands (1–4 and their corresponding ligands C6H5C(O)NHP(O)[NC4H8N(C6H5)]2 (5) [4H8N(C6H5)]3 (6) 2 (5) , P(O)[NC5)]3 (6)  and also earlier –1517–18 31P), of compounds 1–4 demonstrates that it is the most deshielded atom in 4 (containing three N-phenylpiperazinyl substituents on the P atom with δ(31P) = 17.33 ppm). The 31P NMR of complexes 1–3, each containing identical phosphoric triamide ligands, show that the phosphorus atom is at the most upfield region in 1 with M = SnCl2(CH3)2. Further, the δ(31P) shifts downfield from 1 to 3. The complexes 1 and 4 both contain SnCl2Me2, and they differ in the phosphoric triamide ligands. The 31P NMR reveals that the phosphorus atom in 1 appears at a much more upfield region than that of 4, which is due to the presence of more electron-donating phosphoric triamide ligands in 1. The δ(31P) in complex 1 and its corresponding ligand 5 are observed at about 9.00 ppm, while those of complex 4 and its ligand 6 are at about 18.00 ppm. These downfield shifts in compounds 4 and 6 show that the N-benzoyl substituents cause more electron donation to the phosphorus atoms than do the 4-phenylpiperazinyl moieties.Comparing the phosphorus chemical shift, δ3, in which the Sn atoms indicate distorted trigonal bipyramidal geometries. The 1H NMR spectra of compounds 1 and 4 exhibit the 2J = 55.1, 83.4 Hz for the geminal coupling of the 119Sn atom with the hydrogen atoms of the CH3 groups. The 2J coupling constants were obtained in the range of 114.0/110.8 Hz (for 10) to 71.2/68.2 Hz (for 11), see It can be deduced from the urations . The coo1–4 are weaker than their corresponding ligands 5 and 6 due to the formation of Sn–O–P bonds. Also, the ν(C=O) of complexes 1–3 are nearly equal to (or slightly weaker than) the ν(C=O) value in ligand 5 (1675 cm−1). The bands at about 560 and 520 cm−1 correspond to asymmetric and symmetric stretching frequencies of Sn–C bonds. Also, the bands at ≈450 cm−1 are for the stretching frequencies of Sn–O bonds.The IR spectra indicate that the ν(P=O) of complexes 1–4 were prepared by ultrasonication. In this way, the synthesis of these compounds was performed in an ultrasonic bath at 30 °C for about 1–2 h. The SEM micrographs of the nanoparticles are shown in Figures 3–6 indicating that the particle sizes are about 20–25 nm with identical spherical morphologies of the nanoparticles. It is notable that SEM micrographs could be obtained from both dissolved and powdered samples. For the dissolved samples, a few droplets were placed on a small piece of foil, whereas the powdered compounds were directly placed on the sample holder. Here, the complexes were dissolved in methanol, and after evaporation of the solvent, the SEM images were obtained from the nanoparticles prepared on aluminum foil. The fluorescence spectra of complexes 1–4 and their related phosphoric triamide ligands 5 and 6 are represented in Figures 7–12 (see below), respectively. Also, a summary of the UV–vis and fluorescence spectra of compounds 1–6 is given in The nanoparticles of complexes 5 and 6 while it decreases in their corresponding complexes (blue shift). This observation shows that the band gap (ΔE) increases upon complexation. Moreover, the peak intensities are very much smaller in complexes 1–3 relative to that of their corresponding ligand 5. However, the λ(max) of emission and the peak intensity are very close to each other in compound 4 and its phosphoric triamide ligand 6.The fluorescence spectra of each compound were repeated at various wavelengths from about 220 to 360 nm to find the maximum emission intensity. The emission signals with maximum intensities are shown in Figures 7–12. It can be seen that the λ(max) of emission appears at 360 nm for both ligands 1H, 13C and 31P spectra were recorded on a Bruker Avance DRS 500 spectrometer. 1H and 13C chemical shifts were determined relative to internal TMS and 31P chemical shifts relative to 85% H3PO4 as an external standard. Infrared (IR) spectra were recorded on a Shimadzu model IR-60 spectrometer. Elemental analysis was performed by using a Heraeus CHN-O-RAPID apparatus. Melting points were obtained with an Electrothermal instrument. The scanning electron microscopy (SEM) micrographs were obtained with a Philips instrument (XL30), under vacuum, accelerated at 30 kV. The fluorescence and UV–visible spectra were measured by using a Perkin Elmer LS55 instrument and a Genway spectrophotometer, respectively.The N-benzoyl-bis(4-phenylpiperazin-1-yl)phosphinic amide  at room temperature. After two hours stirring, the solution flask was placed in an ultrasonic bath at 30 °C for two hours. Then, the solution was evaporated and the nanoparticles of the product were dried. The product was further purified by washing with diethylether and CCl4. mp 122–124 °C; yield: 55%; anal. calcd for C56H70Cl2N10O4P2Sn: C, 56.11; H, 5.88; N, 11.68; found: C, 56.08; H, 5.87; N, 11.69; 119Sn{1H} NMR (DMSO-d6): δ −166.77 (s); 31P{1H} NMR (DMSO-d6): δ 9.33 (s); 1H NMR (DMSO-d6): δ 0.91  = 55.1 Hz, Sn-CH3), 1.02 , 3.11 , 3.29 , 6.78  = 8.3 Hz, 4H, Ar-H, phenylpiperazinyl), 6.92  = 8.3 Hz, 8H, Ar-H, phenylpiperazinyl), 7.20  = 8.3 Hz, 8H, Ar-H, phenylpiperazinyl), 7.47  = 7.6 Hz, 4H, Ar-H, N-benzoyl), 7.58  = 7.6 Hz, 2H, Ar-H, N-benzoyl), 7.92  = 7.6 Hz, 4H, Ar-H, N-benzoyl), 9.41  = 7.2 Hz, 2H, NH); 13C NMR (DMSO-d6): δ 22.36 , 40.10 (s), 48.85  = 5.3 Hz, CH2), 115.66 (s), 119.07 (s), 128.19 (s), 128.27 (s), 128.88 (s), 132.18 (s), 133.60  = 7.9 Hz, ipso-C), 151.14 (s), 168.37 ; FTIR (KBr): 3437 (NH), 3071 (CH), 2906 (CH), 2840 (CH), 1671 (C=O), 1595, 1441, 1210, 1137 (P=O), 972 (P-Namine), 742 (P-Namide), 708, 532 (Sn-C)s cm−1.A solution of dimethyltin(IV) dichloride  in dry methanol (20 mL) was added dropwise to a solution of N-benzoyl-bis(4-phenylpiperazin-1-yl)phosphinic amide  in methanol (15 mL), dichlorotin(IV) dihydrate  was added at room temperature, and the mixture was stirred for one hour. Then the solution flask was placed in an ultrasonic bath at 30 °C for two hours. After evaporation of the solvent, the nanoparticles of the product were obtained. The powder product was further purified by washing with diethylether and CCl4. mp 143–145 °C; yield: 58%; anal. calcd for C54H68Cl2N10O6P2Sn: C, 53.84; H, 5.69; N, 11.63; found: C, 53.85; H, 5.70; N, 11.62; 31P{1H} NMR (CD3OD): δ 11.21 (s); 1H NMR (CD3OD): δ 3.29 , 3.48 , 6.92  = 8.0 Hz, 4H, Ar-H, phenylpiperazinyl), 7.05  = 8.0 Hz, 8H, Ar-H, phenylpiperazinyl), 7.27  = 8.0 Hz, 8H, Ar-H, phenylpiperazinyl), 7.50  = 7.5 Hz, 4H, Ar-H, N-benzoyl), 7.59  = 7.5 Hz, 2H, Ar-H, N-benzoyl), 7.91  = 7.5 Hz, 4H, Ar-H, N-benzoyl); 13C NMR (CD3OD): δ 45.80 , 51.97, 118.40 (s), 122.58 (s), 129.23 (s), 129.74 (s), 130.29 (s), 133.93 (s), 134.74  = 8.9 Hz, ipso-C), 152.09 (s), 171.49 ; FT-IR (KBr): 3424 (NH), 3062 (CH), 2833 (CH), 1675 (C=O), 1593, 1491, 1447, 1230, 1137 (P=O), 973 (P-Namine), 753 (P-Namide), 690, 465 (Sn-O) cm−1.To a solution of N-benzoyl-bis(4-phenylpiperazin-1-yl)phosphinic amide  was dissolved in methanol (15 mL), and triphenyltin(IV) chloride  was added at room temperature and the mixture stirred for half an hour. Then the solution flask was placed in an ultrasonic bath at 30 °C for one hour. The evaporation of the solvent yielded the nanoparticles of the product, which was further purified by washing with diethylether and CCl4. mp 114–115 °C; yield: 54%; anal. calcd. for C72H79ClN10O4P2Sn: C, 63.37; H, 5.84; N, 10.26; found: C, 63.35; H, 5.83; N, 10.25; 31P{1H} NMR (CD3OD): δ 11.32 (s); 1H NMR (CD3OD): δ 3.32 , 3.43 , 6.84  = 8.1 Hz, 4H, Ar-H, phenylpiperazinyl), 6.96  = 8.1 Hz, 8H, Ar-H, phenylpiperazinyl), 7.23  = 8.1 Hz, 8H, Ar-H, phenylpiperazinyl), 7.44  = 5.9 Hz, 6H, Ar-H, Sn-Ph), 7.49  = 5.9 Hz, 3H, Ar-H, Sn-Ph), 7.60  = 5.9 Hz, 6H, Ar-H, Sn-Ph), 7.83  = 7.0 Hz, 2H, Ar-H, N-benzoyl), 7.70  = 7.0 Hz, 4H, Ar-H, N-benzoyl), 7.86  = 7.0 Hz, 4H, Ar-H, N-benzoyl); 13C NMR (CD3OD): δ 46.01 , 51.34  = 5.1 Hz, CH2), 117.92 (s), 121.46 (s), 128.90 (s), 129.17 (s), 129.66 (s), 130.08 (s), 130.46 (s), 130.77 (s), 133.74 (s), 133.90 (s), 134.93  = 10.2 Hz, ipso-C), 152.99 (s), 171.37 ; FT-IR (KBr): 3431 (NH), 3064 (CH), 2852 (CH), 2820 (CH), 1667 (C=O), 1596, 1454, 1377, 1326, 1190 (P=O), 1132 (P-Namine), 965(P-Namide), 759, 688, 572 (Sn-C)as, 532 (Sn-C)s cm−1.4. mp 145–147 °C; yield: 61%; anal. Calcd for C62H84Cl2N12O2P2Sn: C, 58.13; H, 6.61; N, 13.12; found: C, 58.14; H, 6.60; N, 13.13; 31P{1H} NMR (DMSO-d6): δ 17.33 (s); 1H NMR (DMSO-d6): δ 0.97  = 83.4 Hz, Sn-CH3), 1.02 , 3.09 , 3.19 , 6.79  = 8.0 Hz, 6H, Ar-H), 6.96  = 8.0 Hz, 12H, Ar-H), 7.22  = 8.0 Hz, 12H, Ar-H); 13C NMR (CD3OD): δ 22.94 , 45.71 , 49.07  = 5.7 Hz, ring-CH2), 115.73 (s), 119.20 (s), 128.91 (s), 151.21 (s), 165.57 ; FT-IR (KBr): 2914 (CH), 2822 (CH), 1597, 1499, 1450, 1231, 1135 (P=O), 968 (P-N), 759, 635 (Sn-C)as, 529 (Sn-C)s cm−1.Tris(4-phenylpiperazin-1-yl)phosphinic amide  was added to a solution of dimethyltin(IV) dichloride  in dry methanol (15 mL) at room temperature, and the mixture was stirred for two hours. Then the solution flask was placed in an ultrasonic bath at 30 °C for one hour. The evaporation of the solvent gave the nanoparticles of the product, which were further purified by washing with diethylether and CCl1H, 31P, 13C NMR, IR, UV, fluorescence spectroscopy and elemental analysis. According to the 1H NMR spectra, it was concluded that the Sn atoms adopt octahedral conformations. The geminal Sn, H coupling constants 2J = 55.1, 83.4 Hz, were measured in the 1H NMR spectra of complexes 1 and 4. Using ultrasonication, spherical nanoparticles of complexes 1–4 were prepared, and their SEM micrographs indicate that the nanoparticle sizes are about 20–25 nm. The fluorescence spectra illustrate blue shifts for the λ(max) of emission and a decrease in the peak intensities upon complexation.Four new organotin(IV) complexes with phosphoric triamide ligands were synthesized and characterized by


R,6Z)-dendrolasin-5-acetate, which was fully characterized by 2D NMR studies, was isolated from the nudibranch Hypselodoris jacksoni, along with the sesquiterpenes (+)-agassizin, (−)-furodysinin, (−)-euryfuran, (−)-dehydroherbadysidolide and (+)-pallescensone. A synthetic sample ([α]D −8.7) of the new metabolite was prepared by -Wittig rearrangement of a geranylfuryl ether followed by acetylation of purified alcohol isomers. The absolute configuration at C-5 was established as R by the analysis of MPA ester derivatives of (Z)-5-hydroxydendrolasin obtained by preparative enantioselective HPLC.A small sample of (−)-(5 Marine organisms have been proven a prolific source of natural products that potentially can be used as lead compounds or which have inspired synthetic chemists as synthetic targets . SimilarHypselodoris is generally characterized by sesquiterpene chemistry, as evidenced by a number of studies on European D −53 , displayed a (+)-HRESIMS ion adduct at m/z 299.1624 [M + Na]+ consistent with the molecular formula C17H24O3. The 1H NMR spectrum  of 1 showed diagnostic signals for a furan  along with two alkene signals . Signals for three methyl groups were observed at δH 1.72, 1.67, and 1.59 and were linked to signals at δC 23.3, 25.7, and 17.6, respectively, by HSQC. There were also signals characteristic of an acetate group . These partial NMR assignments were comparable to those of the known dendrolasin (3), a sesquiterpene previously isolated from Hypselodoris cantabrica -Wittig rearrangement of geranyl 3-furylmethyl ether (6) to produce 7a/b, and acetylation to obtain target 1. The overall synthesis  of natural material available, a synthetic study was undertaken to provide stereochemical characterization of ynthesis  was adap4) was prepared in 78% yield by reduction of 2-furoic acid with LiAlH4 in dry diethyl ether, whereas geranyl bromide (5) was obtained through bromination of geraniol with carbon tetrabromide and triphenylphosphine in 90% yield. The 1H NMR spectrum of the geraniol sample used indicated the presence of traces of nerol, the Z-isomer of geraniol. Etherification of furan-3-ylmethanol (4) with the geranyl/neryl bromide mixture (5) in the presence of NaH furnished the 3-furylmethyl ether 6 (65% yield) as an E/Z mixture (3:1). The H-5 signal for the minor Z-isomer of 6 resonated at δH 3.90  compared to that of the E-isomer . Irradiation of the H-5 signal of the Z-isomer increased the intensity of the signal associated with the adjacent methyl (CH3-14), confirming the Z double bond at C6/C7. The isomeric mixture of 6 was immediately subjected to -Wittig rearrangement with t-BuLi (4 equiv) and LDA at −78 °C to afford racemic 5-hydroxydendrolasin (39% yield), again as an E/Z mixture (7a/b) for which a 3:1 ratio was determined by 1H NMR comparison with data for the individual stereoisomers (see below). The 3:1 ratio of E/Z-isomers was confirmed via analytical HPLC.Furan-3-ylmethanol  or -rearranged products such as 9 or 10 depending on which site adjacent to the ether oxygen is preferentially deprotonated  was next subjected to HPLC  to provide the individual E- (7a) and Z-isomers (7b) that were each carefully characterised by 1H NMR and 1D nOe experiments. Assignment of the upfield signals corresponding to the three methyl groups attached to the double bonds (3-14 in the Z-isomer resonated downfield (δH 1.74) compared to that in the E-isomer (δH 1.65) while the signal for H-5 appeared slightly upfield (δH 4.48) in the Z-isomer compared to that in its E counterpart (δH 4.51). 1D NOESY irradiation of the CH3-14 signal for the Z-isomer resulted in enhancement of the alkene signal at δH 5.24 (H-6), fully consistent with the Z configuration; there was no corresponding nOe between CH3-14 and H-6 in the E-isomer, although such data must be interpreted with care.Racemic 5-hydroxydendrolasin -5-hydroxydendrolasin (7b) was next undertaken (7b and (−)-7b isomers, respectively. Treatment of each enantiomer of 7b with acetic anhydride and pyridine gave their acetate derivatives. Surprisingly, (+)-7b ([α]D +9.3) gave an acetate derivative with [α]D −8.7 and vice versa ([α]D –12 for (−)-7b and [α]D +13 for its acetate derivative). As anticipated, the 1H NMR spectrum  of the synthetic sample of (−)-(6Z)-1 was fully superimposable with that of the sample of (6Z)-dendrolasin-5-acetate isolated from H. jacksoni -MPA acid in the presence of DMAP and DCC at room temperature to give a mixture of diastereomeric -11 and -11, which was separated by reversed phase (RP) HPLC  -11 and -11 resonated at δH 5.04 and at δH 5.13, respectively, in agreement with H-6 of the -isomer positioned in the shielding cone of the aryl ring of the MPA moiety D between the alcohol and its acetate derivative, these data verified that the naturally-occurring (−)-acetate 1 had a 5R configuration.It remained to determine the absolute configuration of C-5 in the natural acetate derivative. A portion of the racemic mixture of ; 63:35) . The H-6A moiety . The dia1 were measured as [α]D −53 for the natural sample and −8.7 for the synthetic sample. Despite having the same negative sign of specific optical rotation, the [α]D values of the natural sample was considerably larger than that of the synthetic sample. Although a partial explanation for the difference in values was apparent, namely that the natural sample size was small (0.1 mg) compared to the synthetic sample size (0.9 mg), better experimental evidence of their stereochemical uniformity was desirable. Enantioselective HPLC analyses run under identical conditions using 5% isopropanol in n-hexane were carried out on the synthetic sample of racemic 1, the two synthetically prepared R and S enantiomers of 1, and the natural sample as shown in 1 (shown in trace (a)), and that the (+)-enantiomer elutes before the (−)-enantiomer (traces (b) and (c)). Finally, the natural sample has the same retention time as the (−)-enantiomer (trace (d)). A co-injection experiment performed on a mixture of the synthetic (+)-enantiomer and the natural sample consistently displayed two peaks in which the former compound eluted before the natural sample.Specific rotation values for H. jacksoni collected from Queensland yielded a new sesquiterpene -dendrolasin-5-acetate (1) whose structure and stereochemistry were confirmed by means of a five-step synthesis involving -Wittig rearrangement of geranyl furyl ether (6) followed by acetylation of the purified alcohol products. The absolute configuration in 1 was established as 5R by detailed comparison with MPA ester derivatives prepared from individual enantiomers of synthetic (6Z)-5-hydroxydendrolasin purified by preparative enantioselective HPLC.Chemical investigation of Isolation of (−)--dendrolasin-5-acetate (1) from Hypselodoris jacksoni: Eight individuals of H. jacksoni (21–40 mm in length) were collected from various dive sites in South East Queensland  between March and October of 2012. The whole animals were chopped, macerated in acetone (20 mL), sonicated for 2 min before being filtered through a cotton plug. The resulted filtrate was combined, concentrated under reduced pressure and partitioned between Et2O (3 × 20 mL) and water. The Et2O crude fraction (39.2 mg) was resolved by NP flash column chromatography eluted with gradient solvent from 100% hexanes→100% EtOAc to give ten fractions. The first and the second fraction (coded as HJF1–2) (21.3 mg) were combined based on their TLC profile, and yielded the major metabolites (−)-furodysinin (3.8 mg), (+)-agassizin (2.5 mg) and (−)-euryfuran (0.8 mg) following NP HPLC separation using 100% hexanes. The fourth fraction coded as HJF4 (1.8 mg) was subjected to NP HPLC eluting with 2.5% EtOAc/hexanes to give (−)-dehydroherbadysidolide (0.1 mg), (+)-pallescensone (0.2 mg), and the new sesquiterpene (−)--dendrolasin-5-acetate .  −53 ; 1H NMR  δH 7.33 , 7.23 , 6.27 , 5.64 , 5.15 , 5.07 , 2.73 , 2.63 , 2.15–1.95 , 1.72 , 1.67 , 1.59 ; 13C NMR  δC 142.7 (C-1), 141.5 (C-7), 140.1 (C-15), 132.0 (C-11), 123.9 (C-10), 123.7 (C-6), 120.2 (C-3), 111.8 (C-2), 70.7 (C-5), 32.5 (C-8), 30.7 (C-4), 26.5 (C-9), 25.7 (C-12), 23.3 (C-14), 17.6 (C-13); (+)-HRESIMS m/z: [M + Na]+ calcd. for C17H24NaO3, 299.1618; found, 299.1624.Procedure for Wittig rearrangement of 3-furylmethyl ether (6) +.-ol (7a) : Colorle(±)-(Z)-1-(furan-3-yl)-4,8-dimethylnona-3,7-dien-2-ol (7b): Colorless glass; 1H NMR  δH 7.37 , 7.29 , 6.32 , 5.24 , 5.07 , 4.48 , 2.65 , 2.58 , 2.12–2.01 , 1.74 , 1.69 , 1.60 ; 13C NMR  δC 142.9 (C-1), 140.3 (C-15), 139.5 (C-7), 132.6 (C-11), 128.0 (C-3), 124.0 (C-10), 121.1 (C-6), 111.7 (C-2), 68.1 (C-5), 33.2 (C-8), 32.5 (C-4), 26.6 (C-9), 25.8 (C-12), 23.5 (C-14), 17.8 (C-13); (+)-HRESIMS m/z: [M + Na]+ calcd for C15H22NaO2, 257.1512; found, 257.1515.Procedure for acetylation of (±)-(Z)-1-(furan-3-yl)-4,8-dimethylnona-3,7-dien-2-ol (7b): A portion of 7b (8.4 mg) was dissolved in dry pyridine (0.5 mL) and cooled in an ice bath. After the addition of acetic acid anhydride (1.5 mL) the mixture brought to rt and stirred for 1.5 hours. Toluene (0.5 mL) was added and the resulting azeotrope was evaporated under reduced pressure to give (±)-(6Z)-dendrolasin-5-acetate (1) (6.4 mg); 1H and 13C NMR data  are identical to those of 1. (+)-LRESIMS m/z 299 [M + Na]+.Procedure for acetylation of the (+) isomer of 7b: A portion of the (+) isomer of 7b (1.3 mg) was acetylated using the same procedure as for 7b to obtain (−)-(6Z)-dendrolasin-5-acetate ; Colorless glass; [α]D −8.7 ; 1H and 13C NMR data  are the same as those of the 1. (+)-LRESIMS m/z: 299 [M + Na]+.Procedure for acetylation of the (−)-isomer of 7b: A portion of the (−)-isomer of 7b (1.3 mg) was acetylated using the same procedure as for 7b to obtain (+)-(6Z)-dendrolasin-5-acetate ; Colorless glass; [α]D +12.9 ; 1H and 13C NMR  are identical to those of 1. (+)-LRESIMS m/z: 299 [M + Na]+.Procedure for preparation of (R)- and (S)-MPA esters of racemic (6Z)-5-hydroxydendrolasin (7b): (±)-(6Z)-5-Hydroxydendrolasin (2.0 mg) was dissolved in dry DCM (0.2 mL) to which (R)-methoxyphenylacetic acid (MPA) (0.83 mg in 0.1 mL of DCM) was added, followed by DCC  and DMAP . The reaction was stirred at rt for 16 h and then filtered through a cotton wool plug. The solvent was removed by rotary evaporation and the residue passed through a pipette silica column eluted with hexanes/EtOAc (95:5) to give a racemic MPA ester (0.8 mg). The racemic MPA ester was subsequently subjected to RP HPLC  to give the R,R (0.2 mg) and R,S diastereomers (0.2 mg).-(6Z)-dendrolasin MPA ester (11a): Colorless glass; [α]D +10.1 ; 1H NMR  δH 7.38–7.30 , 7.28 , 7.14 , 6.20 , 5.66 , 5.04 , 5.02 , 4.70 , 3.37 , 2.73 , 2.63 , 2.16–2.10 , 1.98–1.86 , 1.64 , 1.56 ; (+)-HRESIMS m/z: [M + Na]+ calcd for C24H30NaO4, 405.2036; found, 405.2038.-(6Z)-dendrolasin MPA ester (11b): Colorless glass; [α]D +10.7 ; 1H NMR  δH 7.38−7.36 , 7.17 , 6.83 , 5.95 , 5.68 , 5.12 , 5.04 , 4.68 , 3.36 , 2.58 , 2.51 , 2.24–2.20 , 2.06–1.92 , 1.71 , 1.67 , 1.58 ; (+)-HRESIMS m/z: [M + Na]+ calcd for C24H30NaO4, 405.2036; found, 405.2043.Procedure for preparation of MPA ester of the (+)-isomer of 7b: A portion of the (+)-isomer of 7b (1.3 mg) was treated with (R)-MPA (0.83 mg in 0.1 mL of DCM) using the same procedure as for the preparation of MPA ester of the racemic (6Z)-5-hydroxydendrolasin (7b) to obtain the (+)-MPA ester (1.2 mg). The 1H NMR data  were identical to those for the -dendrolasin MPA ester (11a). [α]D −93 ; (+)-LRESIMS m/z: 405 [M + Na]+.Procedure for preparation of MPA ester of the (−)-isomer of 7b: A portion of the (−)-isomer of 7b (1.3 mg) was treated with (R)-MPA (0.83 mg in 0.1 mL of DCM) using the same procedure as for the preparation of MPA ester of the racemic (6Z)-5-hydroxydendrolasin (7) to obtain the (−)-MPA ester (1.0 mg). The 1H NMR data  are identical to those of the -dendrolasin MPA ester (11b). [α]D −22 ; (+)-LRESIMS m/z: 405 [M + Na]+.Enantioselective HPLC experiments: Enantioselective HPLC analysis of the (±)-(6Z)-dendrolasin-5-acetate (1) racemic mixture, the individual 5R- and 5S-enantiomers of (6Z)-dendrolasin-5-acetate, and the natural sample of (−)--dendrolasin-5-acetate, were performed using an Agilent 1200 series liquid chromatography system  equipped with UV (220 nm) and ALP  detectors and a Chiralpak OJ column . Retention times were: (+)-enantiomer, >97% ee 8.3 min; (–)-enantiomer, >98.5% ee 9.4 min; natural sample 9.4 min.6, spectroscopic data and other relevant information for compounds 1, 7b, 11a, and 11b.File 1Experimental details and spectroscopic data.


Buddleja davidii is widely distributed in the southwestern region of China. We have undertaken a systematic analysis of B. davidii as a Chinese traditional medicine with anticancer activity by isolating natural products for their activity against the human gastric cancer cell line Mgc-803 and the human breast cancer cell line Bcap-37.B. davidii, among which colchicine was identified in B. davidii for the first time. The inhibitory activities of these compounds were investigated in Mgc-803, Bcap-37 cells in vitro by MTT  assay, and the results showed that luteolin and colchicine had potent inhibitory activities against the growth of Mgc-803 cells. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in Mgc-803 cells. The results also showed that the percentages of early apoptotic cells  and late apoptotic cells (Annexin V+/PI+) increased in a dose- and time-dependent manner. After 36 h of incubation with luteolin at 20 μM, the percentages of cells were approximately 15.4% in early apoptosis and 43.7% in late apoptosis; after 36 h of incubation with colchicine at 20 μM, the corresponding values were 7.7% and 35.2%, respectively.Ten compounds were extracted and isolated from B. davidii have potential applications as adjuvant therapies for treating human carcinoma cells. These compounds could also induce apoptosis in tumor cells.Colchicine and luteolin from  Buddleja belongs to the Loganiaceae family and has a pantropical distribution across South Asia, Africa, and America method. Luteolin had more potent inhibitory activities against the growth of Mgc-803 cells than the other compounds, and colchicine exhibited high activities against the growth of these cells as well. Further investigations of luteolin and colchicine were thus carried out in Mgc-803 and Bcap-37 cells. Their IC50 values were determined. Fluorescent staining and flow cytometry analysis indicated that both compounds could induce apoptosis in Mgc-803 cells. To the best of our knowledge, this is the first study to report on the apoptosis-inducing and antitumor activities of luteolin and colchicine in Mgc-803 and Bcap-37 cells.The following 10 compounds were isolated from B. davidii samples collected from Guizhou were studied, and the following compounds were isolated from n-butanol extracts, which were identified based on their physicochemical as well as spectroscopic data as luteolin (1), naringenin (2), puerarin (3), rutin (4), quercetin (5), hesperetin (6), acacetin-7-O-α-L-rhamno-pyranosyl (1–6)-β-D-glucopyranoside , stigmasterol , ferulic acid ;, and colchicine . Among them, colchicine (10) was obtained from the plants for the first time. All the isolated compounds were shown in FigureDried luteolin , naringe1  δ: 7.43 , 7.40 , 6.89 , 6.68 , 6.45 , 6.19 ; 13C NMR  δ: 182.2 (C-4), 164.6 (C-7), 164.4 (C-3), 161.9 (C-9), 157.8 (C-5), 150.2 (C-4’), 146.2 (C-3’), 122.0 (C-1′), 119.5 (C-6’), 116.5 (C-5’), 113.8 (C-2’), 104.2 (C-2), 103.4 (C-10), 98.3 (C-8), 94.3 (C-6).Compound luteolin), yellow2  δ: 7.28 , 6.79 , 5.86 , 5.31 , 3.12 , 3.06 , 2.65 ; 13C NMR  δ: 196.5 (C-4), 167.0 (C-7), 164.1 (C-5), 163.6 (C-9), 157.7 (C-4′), 129.7 (C-1′), 127.7 , 114.9 (C-3/), 1 (C-5′), 102.0 (C-10), 95.7 (C-6), 94.8 (C-8), 79.1 (C-2), 42.7 (C-3).Compound ringenin), yellow3  δ: 8.03 , 6.98 , 7.35 , 6.83 , 8.16 , 5.09 ; 13C NMR  δ: 176.9 (C-4), 161.7 , 157.4 (C-4′), 153.2 (C-2), 130.1 , 126.8 , 124.2 (C-3), 122.9 (C-10), 117.1 , 114.9 (C-6), 111.8 (C-8), 81.4 (C-5′′), 78.7 (C-3′′), 74.3 (C-1′′), 71.6 (C-4′′), 70.4 (C-2′′), 61.4 (C-6′′).Compound puerarin,23), whi4  δ: 7.56 , 7.54 , 6.86 , 6.39 , 6.20 , 5.36 , 4.54 ; 13C NMR  δ: 177.8 (C-4), 164.6 15 (C-7), 161.7 (C-5), 157.1 (C-9), 156.9 (C-2), 148.9 (C-4′), 145.3 (C-3′), 133.8 (C-3), 122.1 (C-1′), 121.7 (C-6′), 116.8 (C-5′), 115.8 (C-2′), 104.5 (C-10), 101.7 (C-1′′), 101.3 (C-1′′′), 99.2 (C-6), 94.1 (C-8), 76.3 (C-3′′), 76.1 (C-5′′), 74.5 (C-2′′), 72.3 (C-4′′′), 71.0 (C-3′′′), 70.9 (C-2′′′), 70.5 (C-4′′), 68.8 (C-5′′′), 67.4 (C-6′′), 18.3 (C-6′′′).Compound 4 rutin,25), yel, yel4  δ: 7.64 , 7.53 , 6.78 , 6.28 , 6.20 ; 13C NMR  δ: 176.4 (C-4), 164.4 (C-7), 161.2 (C-5), 156.7 (C-9), 148.2 (C-2), 147.3 (C-3′), 145.6 (C-4′), 136.3 (C-3), 122.5 (C-1′), 120.5 (C-6′), 116.1 (C-5′), 115.6 (C-2′), 103.5 (C-10), 98.7 (C-6), 93.9 (C-8).Compound uercetin), yellow6  δ: 6.91 , 6.90 , 6.88 , 5.87 , 5.86 , 5.38 , 3.73 (s), 3.16 , 2.68 ; 13C NMR  δ: 196.7 (C-4), 167.2 (C-7), 164.0 (C-5), 163.3 (C-9), 148.4 (C-4′), 146.9 (C-5′), 131.7 (C-1′), 118.2 (C-2′), 114.6 (C-6′), 112.4 (C-3′), 102.3 (C-10), 96.3 (C-6), 95.5 (C-8), 78.8 (C-2), 56.2 (C-7′), 42.6 (C-3).Compound speretin), white 7 (acacetin-7-O-α-L-rhamnopyranosyl(1–6)-β-D-glucopyranoside+, 615 [M + Na]+, 631 [M + K]+; 1 H NMR  δ: 12.9 , 8.05 , 7.15 , 6.93 , 6.79 , 6.45 , 5.07 , 4.55 ; 13C NMR  δ: 182.6 (C-4), 19 164.5 (C-2), 163.5 (C-7), 162.9 (C-5), 161.7 (C-4′), 157.1 (C-9), 129.0 (C-2′), 128.5 20 (C-6′), 123.2 (C-1′), 115.3 (C-3′), 115.3 (C-5′), 105.9 (C-10), 104.4 (C-3), 101.0 (C-1′′′), 21 100.4 (C-1′′), 100.2 (C-6), 95.3 (C-8), 76.8 (C-3′′), 75.7 (C-5′′), 73.6 (C-2′′), 72.6 22 (C-4′′′), 71.3 (C-3′′′), 70.9 (C-2′′′), 68.4 (C-5′′′), 67.7 (C-4′′), 66.1 (C-6′′), 55.6 (OMe), 1 17.8 (C-6′′′).Compound ranoside), 14 yel8  δ: 5.34 , 5.14 , 5.08 , 3.49–3.53 , 0.7–2.7 ; 13C NMR  δ: 140.8 (C-5), 138.4 (C-22), 129.3 (C-23), 121.8 (C-6), 71.9 (C-3), 56.9 (C-14), 56.0 (C-17), 51.3 (C-24), 50.2 (C-9), 42.4 (C-4), 42.3 (C-13), 40.6 (C-20), 39.8 (C-12), 37.3 (C-1), 37.3 (C-10), 32.7 (C-7), 31.9 (C-8), 31.7 (C-25), 31.7 (C-2), 29.0 (C-16), 25.5 (C-28), 24.5 (C-15), 21.3 (C-11), 21.3 (C-21), 21.3 (C-26), 19.5 (C-19), 19.1 (C-27), 12.4 (C-29), 12.1 (C-8).Compound masterol), white 9  δ: 3.89 , 6.33 , 6.99 , 7.11 , 7.18 , 7.56 ; 13C NMR 15  δ: 168.5 (C-1), 149.6 (C-3′), 148.4 (C-4/), 145.1 (C-3), 126.3 (C-1′), 123.4 (C-6′), 116.1 (C-5′), 116.0 (C-2), 111.6 (C-2′), 56.2 (−OCH3).Compound lic acid), yellow10  δ: 7.63 , 7.35 , 6.89 , 6.55 , 4.65 , 4.12 , 3.94 , 3.91 , 3.66 , 2.38–2.01 ;13C NMR  δ: 179.6 (C-9), 170.2 (C-13), 164.1 (C-10), 153.6 (C-3), 152.6 (C-7a), 151.3 (C-1), 141.7 (C-2), 137.0 (C-12a), 135.7 (C-12), 134.4 (C-4a), 130.5 (C-8), 125.7 (C-12b), 113.0 (C-11), 107.4 (C-4), 61.7 (OCH3-1), 61.5 (OCH3 -2), 56.5 (OCH3-10), 56.2 (OCH3-3), 52.8 (C-7), 36.5 (C-6), 29.9 (C-5), 22.9 (C-14).Compound lchicine), yellowB. davidii on the viability of Mgc-803 and Bcap-37 cells were investigated using MTT assay at 5 and 20 μM, with adriamycin[The potential effects of the extracts from riamycin used as riamycin,33. MTT 50 values of luteolin and colchicine on Mgc-803 cells were 19.87±1.0 and 18.79±1.6 μM, respectively, and IC50 values of luteolin and colchicine on Bcap-37 cells were 41.78±2.2 and 76.01±0.6 μM, respectively. The results also showed that luteolin and colchicine had more potent activities against Mgc-803 cells. Besides, the inhibitory effect on Bcap-37 cells of luteolin was stronger than that of colchicine.Further experiments also found that proliferation of Mgc-803 cells was significantly 4 inhibited by luteolin and colchicine, as shown in FiguresApoptosis is a physiological pattern of cell death characterized by morphological features and extensive DNA fragmentation, the frequency and time of appearance of which depend on the cell line and the apoptosis-inducing signal. It has been well studied that luteolin is capable of inducing cell cycle arrest or apoptosis in various human cancer cells-40, suchSince AO is a crucial dye and can stain nuclear DNA across an unbroken cell membrane, whereas EB can only stain cells that had lost an intact cell membrane. Thus, tMembrane-permeable Hoechst 33258 (a blue fluorescent dye with low cytotoxicity) staining was also performed to investigate the apoptosis-inducing activities of colchicine and luteolin in Mgc-803 cells. Live cells with homogeneous light blue nuclei could be observed under fluorescence microscopy after staining with Hoechst 33258, and the apoptotic cells displayed bright blue due to karyopyknosis and chromatin condensation after treating with Hoechst 33258, and the nuclei of dead cells could not be stained. As showTUNEL staining was further carried out to confirm the cell apoptosis-inducing activities of colchicine and luteolin. TUNEL staining is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. ApoptotFrom the figure, it could be found that most nuclei in the treatment groups with VP-16 . PI does not penetrate whole cells with intact membranes and was used to differentiate between necrotic cells , late apoptotic cells , and early apoptotic cells . The results are shown in FigureMoreover, the apoptosis ratios induced by colchicine and luteolin caused apoptosis in the Mgc-803 cells were analyzed by flow cytometry with Annexin V/propidium iodide (PI) double staining after treating with colchicine and luteolin for 12, 24, and 36 h. Exposure of the membrane phospholipid phosphatidylserine to the external cellular environment is one of the earliest markers of apoptotic cell death. Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine expressed on the cell surface used to differentiate intact cells  and Q2 (Annexin V+/PI+) were approximately 0.6% and 4.6% at 5 μM, 3.1% and 11.7% at 10 μM, and 15.0% and 44.4% at 20 μM, respectively after 12 h treating with luteolin. While the percentages that were treated for 24 h were approximately 3.5% and 12.7% at 5 μM, 2.2% and 12.7% at 10 μM, and 3.3% and 45.1% at 20 μM for Q4 and Q2 cells, respectively. In addition, the corresponding values of treating for 36 h were approximately 2.4% and 20.8% at 5 μM, 1.3% and 11.3% at 10 μM, and 15.4% and 43.7% at 20 μM, respectively. When the experiments were treated with colchicine, the percentages (after treating 12 h) of Q4 and Q2 cells were approximately 4.4% and 6.9% at 5 μM, 7.0% and 12.9% at 10 μM, and 3.9% and 15.7% at 20 μM, respectively. When the time was extended to 24 h, the corresponding percentages were approximately 2.8% and 12.8% at 5 μM, 7.7% and 13.0% at 10 μM, and 3.5% and 19.6% at 20 μM, respectively. Moreover, the corresponding percentages (after treating for 36 h) were approximately 2.0% and 14.9% at 5 μM, 5.9% and 19.7% at 10 μM, and 7.7% and 35.2% at 20 μM, respectively. These results showed that luteolin and colchicine may exert their anticancer activities in Mgc-803 cells by interference of cell proliferation via apoptosis in a dose- and time-dependent manner.As indicated in FigureIn summary, the inhibitory effects observed in response to colchicine and luteolin were associated with the induction of apoptotic cell death.B. davidii, a class of Chinese traditional medicine, which showed variety of biologically activity and is widely distributed in southwestern of China. Studies on the chemical constituents of B. davidii and their biological activities have focused on the rational development and utilization of this plant.B. davidii grown in Guizhou, and their cell growth inhibitory effects on Mgc-803 and Bcap-37 cells were evaluated by MTT assay. Among these ten compounds, colchicine 10 was extracted from B. davidii firstly. Both of colchicines and luteolin showed potent anticancer activities on Bcap-37 and BGC-823 cells in a dose-dependent manner. And the IC50 values of luteolin and colchicine on Mgc-803 cells were 19.87±1.0 and 18.79±1.6 μM, respectively, The IC50 values of luteolin and colchicine on Bcap-37 cells were 41.78±2.2 and 76.01±0.6 μM, respectively. This is the first study to have extracted colchicine from B. davidii. Moreover, the apoptotic activities induced by colchicine and luteolin in Mgc-803 cells were investigated through AO/EB staining, Hoechst 33258 staining, TUNEL assay, and flow cytometry analysis. The results demonstrated that both compounds are promising adjuvant therapies for treating human carcinoma cells. Further studies are needed to clarify the action mechanism of B. davidii on the inhibition of human malignant tumor cell proliferation.In the current study, 10 compounds were extracted and identified from B. davidii samples were collected from Bijie, Guizhou, in 2008. The plant was identified by Professor Deqing Long . A voucher specimen was deposited in the Botany and Pharmacognosy Department, School of Pharmacy, Guiyang Medical University.Fresh B. davidii (20 kg) samples were powdered and extracted with ethanol at room temperature four times for 7 days each. After filtration, the extract was evaporated under reduced pressure for ethanol removal to obtain a water suspension that was sequentially extracted at room temperature with petroleum ether (5000 mL), ethyl acetate (5000 mL), n-butanol (5000 mL), and water (5000 mL).Dried n-butanol extract of B. davidii (195 g) was chromatographed on a Si gel column  eluted with ethyl acetate/methanol mixtures. The fractions eluted with a 10:1 ethyl acetate/methanol ratio yielded 10.2 g of extract A, whereas those eluted with a 5:1 ethyl acetate/methanol ratio afforded 15.4 g of extract B. Extract A was subjected to passage over a Si gel column (250 mL) and eluted with trichloromethane, trichloromethane/methanol, and methanol. It was fractionated into three parts (A1–A3): The A1 fraction was purified on Merck Silica gel 60RP-18  to obtain compound 10 (18 mg) and compound 8 (60 mg). The A2 fraction was purified on a Sephadex LH-20 column (methanol/water) and then a Si gel column  to obtain compound 9 (43 mg). The A3 fraction was purified on Si gel column  to obtain compound 2 (30 mg).The 6 (25 17 mg) and compound 5 (63 mg). The B2 fraction was purified on Merck Silica gel 18 60RP-18  to obtain compound 3 (31 mg) and compound 1 (24 mg). The B3 fraction was purified on a Si gel column  to obtain compound 7 (160 mg). The B4 fraction was purified on a Sephadex LH-20 column (methanol) to obtain compound 4 (42 mg).Extract B was fractionated into four parts (B1–B4) over a Si gel column (350 mL) eluting with trichloromethane/methanol (30:1): The B1 fraction was purified on Merck Silica gel 60RP-18  to obtain compound 2; All cell lines were purchased from Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences.The human gastric cancer cell line Mgc-803, human breast cancer cell line Bcap-37 were grown in RPMI  1640 and supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% COin vitro using MTT assay. All cell lines (2000 cells/well) were incubated for 24 h at 37°C in an atmosphere of 5% CO2 before the compounds were added. After treatment with various concentrations of each extract, the cells were incubated for an additional 72 h at 37°C in 5% CO2. Subsequently, the medium was removed and the cells in each well were incubated with 100 μL of MTT solution (0.5 mg/mL) for 4 h at 37°C. Finally, the mixture was supplemented with sodium dodecyl sulfate and the cells were incubated for 12 h at 37°C.The activities of the compounds in Mgc-803, Bcap-37 were evaluated All experiments were performed in triplicate. The inhibition rate was calculated as follows:Apoptotic morphology was investigated by double staining with AO and EB as described by Baskic et al.. In the Mgc-803 cells grown on sterile cover slips in six-well tissue culture plates were treated with puerarin and colchicine for a certain range of time. The culture medium containing compounds was removed, and the cells were fixed with 4% paraformaldehyde for 10 min. After washing twice with PBS, the cells were stained with 5 μg/mL of Hoechst 33258 for 5 min. After rewashing twice with PBS, the percentage of apoptotic cells was determined using an IX71SIF-3 fluorescence microscope. As some of the dead cells were rinsed off as the experiment progressed, the apoptotic ratios could have been underestimated in comparison with those from the MTT assay.2O2 in methanol at room temperature for 20 min to inactivate the endogenous peroxidases, after which the cells were washed three times with PBS. Thereafter, the cells were incubated with 2 μL of terminal deoxynucleotidyl transferase enzyme and 48 μL of biotin-dUTP per specimen for 60 min at 37°C. After termination for 10 min, the cells were incubated with 50 μL of streptavidin-HRP for 30 min at room temperature after being washed thrice with PBS.TUNEL assay identifies apoptosis by tagging the 3’-OH ends of DNA fragments with fluorescein. In the 5 cells/mL for 24 h and then treated with puerarin and colchicine at 5, 10, and 20 μM. After 12, 24, or 36 h, the cells were collected, washed twice with PBS, and centrifuged at room temperature. Subsequently, the Mgc-803 cells were gently resuspended in 500 μL of binding buffer. Thereafter, the cells were stained in 5 μL of Annexin V/FITC and shaken well. Finally, 5 μL of PI was added to these cells; the reaction was incubated for 40 min in the dark and analyzed using FACSCalibur (Becton Dickinson).For measuring apoptosis, Mgc-803 cells were seeded in six-well plates at a density of 5 × 10HCPT: Hydroxycamptothecine; VP-16: Etoposide; MTT: 3--2,5diphenyltetrazolium bromide; AO: Acridine orange; EB: Ethidium bromide; TUNEL: Terminal deoxynucleotidyl transferase-UTP nick end labeling assay.The authors declare that they have no competing interests related to the work described in this article.WY, JW performed the experiments, analyzed the data and wrote the paper. L-HJ, DH planned and analyzed the data and B-AS, JW planned the experiments, analyzed the data and wrote the paper. All authors contributed to this study, read and approved the final manuscript.


Helicobacter pylori is recognized as the main cause of gastritis and gastroduodenal ulcers and classified as class 1 carcinogen pathogen. Different 1,3,4-thiadiazole derivatives bearing 5-nitroaryl moiety have been shown considerable anti- H. pylori activity. In attempt to find new and potent derivatives of described scaffold, a new series of 1-(substituted benzyl)-4-(5-(5-nitroaryl-2-yl)-1,3,4-thiadiazol-2-yl)piperazine derivatives were synthesized and evaluated against three metronidazole-resistant isolates of H. pylori using paper disk diffusion bioassay test.5a-b and substituted benzyl chloride in DMF. The inhibitory activity of the new derivatives 6a-q against three metronidazole-resistant isolates of H. pylori was evaluated by the disc diffusion method and compared with the commercially available standard drug metronidazole.The title compounds were prepared through the reaction of 1-(5-(5-nitroaryl-2-yl)-1,3,4-thiadiazol-2-yl) piperazine H. pylori activity profile of synthesized derivatives is mainly attributed to the substituted nitroaryl moiety at the C-5 position of 1,3,4-thiadiazole ring. Most of 1,3,4-thiadiazole derivatives bearing 5-nitrofuran moiety at C-5 position of central thiadiazole ring, demonstrated more promising anti-H. pylori than the 5-nitrothiophen counterpart.The results of SAR study indicated that the potency and anti-6i), demonstrated strong anti-H. pylori potential at studied concentrations 100-25 μg/disk (IZD > 20 mm) against all studied metronidazole- resistant isolates of H. pylori.The most potent nitrofuran derivative containing 3-methoxybenzyl piperazine pendant at the C-2 position of 1,3,4-thiadiazole ring (compound  Helicobacter pylori, an spiral-shaped Gram-negative bacterium, has been considered as the leading cause of gastritis and gastroduodenal ulcer in developing countries. H. pylori is also classified as the class 1 carcinogen pathogen because of its epidemiological relationship to gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma [Helicobacter pylori requires targeted therapeutic strategy.lymphoma . TherefoH. pylori infection resulted to ulcer healing and reduced prevalence of gastric cancer [Different studies show that eradication of c cancer . Howeverc cancer ,5,6.H. pylori infection, emergence of resistance strains is a growing global concern. Clinical evaluation of current therapeutic agents indicated the incidence of drug-drug interaction, infection relapses and side effects of common drugs [Helicobacter pylori drugs and search for novel therapeutic molecules that offer better protection and decreased relapse towards resistant strains.Although several combination therapy regimes using various anti-bacterial agents through different duration of therapy are proposed for eradication of on drugs ,8. TheseHelicobacter pylori[-H. pylori agents, herein, we describe the synthesis and anti-Helicobacter evaluation of a new series of 5-(nitrothienyl) and 5-(nitrofuryl)-1,3,4-thiadiazoles containing different piperazine side chain at 2-position of 1,3,4-thiadiazole ring system  are reported in ppm relative to internal tetramethylsilane. The mass spectra were run on a Agilent 1100/Bruker Daltonic (Ion trap) VL instrument. at 70 eV. Fross- Heraeus CHN-O rapid analyzer was used for elemental analysis of synthesized compounds and the results are within ± 0.4% of the theoretical values. Analytical thin-layar chromatography (TLC) on Merck silicagel 60 F254 plates using various mobile phases of different polarities was performed in order to confirm the purity of final products.A Kofler hot stage apparatus was used for the measurement of reported melting. The IR spectra were recorded on a Nicollet FT-IR Magna 550 spectrometer. The 5a-b (1.0 mmol) and different benzyl chloride derivatives (1.0 mmol) in DMF (15 mL), NaHCO3 (0.21 mmol) was added and the resulted mixture was stirred at room temperature overnight. After completion of the reaction, water was added, and the resulted precipitate was filtered off, washed with water, and crystallized from ethanol.To a mixture of 1-(5-(5-nitroaryl-2-yl)-1,3,4-thiadiazol-2-yl)piperazine -1 (NO2); 1H-NMR(CDCl3) δ: 2.48-2.64 , 3.48-3.63 , 3.48-3.63 , 7.16 , 7.45 , 7.52-7.60 , 7.80-7.92 ; MS: m/z (%) 432 , 415 (21), 241 (15), 191 (81), 172 (15), 136 (100), 120 (33), 91 (34), 69 (93), 42 (25). Anal. Calcd. For C17H16N6O4S2: C, 47.21; H, 3.73; N, 19.43; Found: C, 47.03; H, 3.51; N, 19.67.Yield 40%; m.p. 219-221°C; IR(KBr): 1343, 1527 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.26-2.69 , 3.60-3.72 , 7.16 , 7.58 , 7.68 , 7.86 , 8.15 , 8.24 ; MS: m/z (%) 432 , 241 (14), 191 (88), 172 (14), 155 (16), 136 (100), 111 (16), 90 (60), 73 (18), 56 (47). Anal. Calcd. For C17H16N6O4S2: C, 47.21; H, 3.73; N, 19.43; Found: C, 47.54; H, 3.61; N, 19.83.Yield 33%; m.p. 205-206°C; IR(KBr): 1344, 1528 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.35-2.50 , 3.40-3.60 , 7.02 , 7.37 , 7.70 , 8.02 ; MS: m/z (%)432 , 415 (16), 395 (46), 368 (11), 313 (11), 296 (14), 241 (32), 191 (100), 172 (33), 155 (12), 136 (77), 106 (31), 78 (37), 56 (30). Anal. Calcd. For C17H16N6O4S2 C, 47.21; H, 3.73; N, 19.43; Found: C, 47.56; H, 3.99; N, 19.07.Yield 60%; m.p. 232-234°C; IR(KBr): 1340, 1509 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.64-2.76 , 3.60-3.71 , 3.79 , 6.92 , 7.14 , 7.85 ; MS: m/z (%) 423 , 182 (100), 166 (30), 127 (100), 111 (15), 83 (21), 57 (31), 41 (23). Anal. Calcd. For C17H15F2N5O2S2: C, 48.22; H, 3.57; N, 16.54; Found: C, 48.49; H, 3.34; N, 16.16.Yield 31%; m.p. 198-199°C; IR(KBr): 1343, 1503 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.64 , 3.59 , 3.65 , 6.90-7.00 , 7.12 , 7.20-7.38 , 7.87 ; MS: m/z (%) 441 , 241 (13), 200 (86), 182 (19), 145 (100), 128 (11), 69 (11), 42 (14). Anal. Calcd. For C17H14F3N5O2S2: C, 46.25; H, 3.20; N, 15.86; Found: C, 45.88; H, 3.44; N, 16.03.Yield 33%; m.p. 213-215°C; IR(KBr): 1342, 1513 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.65-2.74 , 3.60-3.74 , 7.13-7.32 , 7.50 , 7.86 ; MS: m/z (%) 459 , 457 , 455 , 214 (93), 192 (18), 159 (100), 123 (18). Anal. Calcd. For C17H15Cl2N5O2S2: C, 44.74; H, 3.31; N, 15.35; Found: C, 44.51; H, 3.70; N, 15.67.Yield 93%; m.p. 210-211°C; IR(KBr): 1344, 1504 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.51-2.63 , 3.52 , 3.60-3.68 , 7.13-7.24 , 7.40 , 7.85 ; MS: m/z (%) 459 , 457 , 455 , 241 (18), 214 (81), 159 (100), 124 (16), 89 (12), 56 (19). Anal. Calcd. For C17H15Cl2N5O2S2: C, 44.74; H, 3.31; N, 15.35; Found: C, 44.46; H, 3.62; N, 15.14.Yield 36%; m.p. 183-185°C; IR(KBr): 1343, 1508 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.53-2.68 , 3.52 , 3.60-3.69 , 7.14-7.20 , 7.44-7.50 , 7.85 ; MS: m/z (%) 467 , 465 , 296 (16), 280 (14), 265 (10), 254 (41), 239 (100). Anal. Calcd. For C17H16BrN5O2S2: C, 43.78; H, 3.46; N, 15.02; Found: C, 43.94; H, 3.74; N, 14.78.Yield 50%; m.p. 207-208°C; IR(KBr): 1343, 1509 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.63 , 3.57 , 3.67 , 3.83 , 6.84-6.87 , 6.92-6.95 , 7.17 , 7.21-7.23 , 7.44 ; MS: m/z (%) 401 , 176 (86), 147 (13), 121 (100), 91 (24), 57 (18), 40 (14). Anal. Calcd. For C18H19N5O4S: C, 53.85; H, 4.77; N, 17.45; Found: C, 53.97; H, 4.54; N, 17.79.Yield 54%; m.p. 126-127°C; IR(KBr): 1355, 1536 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.62 , 3.62 , 3.89 , 7.17 , 7.44 ,7.45-7.48 , 7.56-7.59 , 7.85 ; MS: m/z (%) 416 , 399 (46), 381 (13), 225 (18), 191 (86), 166 (14), 136 (100), 105 (12), 78 (44), 56 (19). Anal. Calcd. For C17H16N6O5S:C, 49.03; H, 3.87; N, 20.18; Found: C, 49.28; H, 3.64; N, 20.42.Yield 43%; m.p. 197-199°C; IR(KBr): 1353,1529 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.66 , 3.69 , 3.71 , 7.18 , 7.44 , 7.54 , 7.71 , 8.17 , 8.26 ; MS: m/z (%) 416 , 399 (12), 225 (19), 191 (100), 166 (20), 136 (86), 90 (37), 57 (21), 40 (18). Anal. Calcd. For C17H16N6O5S: C, 49.03; H, 3.87; N, 20.18; Found: C, 48.88; H, 3.56; N, 20.44.Yield 50%; m.p. 144-146°C; IR(KBr): 1353, 1529 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.65 , 3.69, 3.70 , 7.18 , 7.44 , 7.56 , 8.23 ; MS: m/z (%) 416 , 399 (13), 225 (21), 191 (100), 166 (12), 136 (87), 106 (36), 78 (49), 60 (11), 42 (40). Anal. Calcd. For C17H16N6O5S: C, 49.03; H, 3.87; N, 20.18; Found: C, 49.31; H, 3.58; N, 20.36.Yield 50%; m.p. 212-213°C; IR(KBr): 1348, 1505 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.69 , 3.67 , 3.79 , 6.93 , 7.16 , 7.28-7.38 , 7.43 ; MS: m/z (%) 407 , 182 (85), 149 (11), 127 (100), 69 (11), 42 (11). Anal. Calcd. For C17H15F2N5O3S:C, 50.12; H, 3.71;N, 17.19; Found: C, 50.35; H, 3.45; N, 17.54.Yield 50%; m.p. 176-179°C; IR (KBr): 1353, 1540 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.65 , 3.60 , 3.68 , 6.84-6.86 , 7.18 , 7.25-7.26 , 7.74 ; MS: m/z (%) 425 , 225 (13), 200 (94), 166 (11), 145 (100), 82 (12). Anal. Calcd. For C17H14F3N5O3S: C, 48.00; H, 3.32; N, 16.46; Found: C, 48.35; H, 3.44; N, 16.18.Yield 41%; m.p. 174-175°C; IR(KBr): 1355, 1517 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.71 , 3.67 , 3.68 , 7.17 , 7.22 , 7.32 , 7.45 , 7.51 ; MS: m/z (%) 443 , 441 , 439 , 313 (12), 236 (16), 214 (95), 192 (16), 159 (100), 123 (16), 99 (13), 82 (16), 57 (20), 40 (16). Anal. Calcd. For C17H15Cl2N5O3S: C, 46.37; H, 3.43; N, 15.91; Found: C, 46.65; H, 3.67; N, 15.69.Yield 92%; m.p. 165-167°C; IR(KBr): 1353, 1549 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.62 , 3.53 , 3.67 , 7.17 , 7.19 , 7.41-7.45, 7.47 ; MS: m/z (%) 443 , 441 , 439 , 273 (12), 238 (11), 214 (93), 159 (100), 123 (16), 100 (7), 82 (13), 56 (15). Anal. Calcd. For C17H15Cl2N5O3S: C, 46.37; H, 3.43; N, 15.91; Found: C, 46.68; H, 3.16; N, 16.09.Yield 62%; m.p. 161-162°C; IR(KBr): 1353, 1503 cm-1 (NO2); 1H-NMR(CDCl3) δ: 2.61 , 3.54 , 3.66 , 7.17 , 7.23 , 7.44 , 8.25 ; MS: m/z (%) 451 , 449 , 210 (20), 169 (100), 90 (44), 56 (22), 40 (21). Anal. Calcd. For C17H16BrN5O3S: C, 45.34; H, 3.58; N, 15.55; Found: C, 45.59; H, 3.92; N, 15.27.Yield 28%; m.p. 192-193°C; IR(KBr): 1353, 1508 cmH. pylori were obtained from 160 dyspeptic patients consisted of 78 men and 82 women whose mean ages were 48 and 43 years, respectively. Based on the endoscopic diagnosis, the patients were classified into three groups: gastritis , ulcers  and cancer.Different isolates of 2 incubator; Heraeus, Germany). After 3–5 days, all cultures were examined for observation of pinpoint (1–2 mm) glistening colonies. Identification of H. pylori isolates was carried out according to the spiral microscopic appearance, Gram negative stain and some biochemical examinations such as urease, oxidase and catalase positive test and negative activities of nitrate and H2S.Antral biopsies demonstrating positive urease tests were transported to the microbiology lab in semisolid (0.1% agar) normal saline. The biopsies were cultured using selective medium containing brucella agar (Merck), 7% defibrinated sheep blood, vancomycin (5 mg/L), trimethoprim (5 mg/L), polymyxin B (50 mg/L), and amphotericin B (4 mg/L). Incubation of cultured isolates was performed at 37°C under microaerobic conditions .Written informed consent was obtained from the patient for the publication of this report.H. Pylori isolates were recruited for susceptibility testing with metronidazole  and with 2, 1, and 0.5 μg/mL of tetracycline. The susceptibility tests were repeated twice for the resistant strains. Bacterial suspensions were prepared in normal saline with the turbidity of Mac-Farland standard No.2 . 100 μl of each bacterial suspension were inoculated in the surface of non- selective blood agar plates and the culture plates were allowed to dry at room temperature (10 min). Sterile blank disks were deposited on the surface of inoculated plates. 10 μL of each antibiotic dilution was poured into a blank disk. Moreover, control plates with growth positive bacterial culture were prepared using the introduction of 10 μl of the antibiotic solvent into the blank disks.Antimicrobial susceptibility test was performed using disk diffusion method (DDM). Recruited antibiotics included metronidazole, tetracycline. In the first step of the susceptibility evaluation, one hundred and ten strains were recruited. As a result of remarkable resistance of different studied bacterial isolates to recruited antibiotics and in attempt to increase the accuracy of the metronidazole resistant rates, in the second step of our study, an additional fifty strains of H.pylori demonstrated IZDs ≥20 mm and ≤10 mm for metronidazole, respectively. The antibacterial activities of target compounds were evaluated against three metronidazole-resistant isolates of H. pylori. All experiments were performed in triplicate and the mean of IZDs produced by test compounds in four concentrations  was considered as antibacterial activity.Plates were incubated at described condition and the inhibition zone diameters (IZD) were examined after 3–5 days. Susceptible and resistant isolates of H. pylori by the filter paper disk diffusion method at 37°C, under microqerophilic condition on selective Brucella agar with 7% defibrinated horse blood. Four concentrations of titled compounds in dimethylsulfoxide (DMSO) were used for evaluation of anti Helicobacter activity assay. Blank standard disks (6 mm in diameter) were deposited on the surface of test plates and impregnated with 10 μL of different concentrations of target compounds. Test plates were incubated at 37°C for 3–5 days and the inhibition zone around each disk (average diameter) was measured. The control disks were impregnated with 10 μL of DMSO. All antibacterial activity experiments were performed in triplicate and the antibacterial activity was expressed as the mean of IZDs (mm) produced by the test compounds at each evaluated concentration.As mentioned earlier, the growth inhibitory potential of test compounds was evaluated against three metronidazole resistant isolates of 6a-q is depicted in the Scheme 1a-b with thiosemicarbazide was refluxed in ethanol to afford thiosemicarbazone 2a-b. Amino-1,3,4-thiadiazoles 3a-b, were synthesized through the oxidative cyclization of 2a-b in presence of ammonium ferric sulfate. In the next step, diazotization of 3a-b in hydrochloric acid and in the presence of copper powder yielded chloro-1,3,4-thiadiazole 4a-b. 1-(5-(5-nitroaryl-2-yl)-1,3,4-thiadiazol-2-yl)piperazine 5a-b were prepared through the reaction of chloro-1,3,4-thiadiazole derivatives 4a-b with piperazine hydrate in stirred ethanol.The synthetic pathway for the target compounds 5a-b were further reacted with different substituted benzyl chlorides in refluxing DMF to give the corresponding 1-substituted-benzyl-4-(5-(5-nitroaryl-2-yl)-1,3,4-thiadiazol-2-yl)piperazine 6a-q. The structures of compounds 6a-q were determined using spectroscopic methods including mass spectrometry,1H NMR, IR, and elemental analysis. The chemical structure of target compounds are shown in Figure The prepared key intermediates in vitro anti-Helicobacter activity of synthesized derivatives was determined by paper disk diffusion bioassay against three metronidazole resistant H. pylori isolates. The average of inhibition zone diameters (IZD) of compounds against three isolates at four different concentrations  is summarized in Figure H. pylori activity of target derivatives could be simply categorized as follows: strong response, zones range diameter >20 mm; moderate response, zone diameter 16–20 mm; weak response, zone diameter 11–15 mm; and little or no response, zone diameter <10 mm [The H. pylori activity varied from little (IZD <10 mm) to strong (IZD >20 mm) response at concentration of 100 μg/disk against metronidazole resistant strains. In view of the obtained data, the following structure-activity relationship might be developed:Investigation of the IZD of studied compounds revealed that the target derivatives demonstrated a wide spectrum of anti-6a-h and nitrofuran 6i-q derivatives. The results of anti-H. pylori activity indicated that the inhibitory responses of test compounds is mainly attributed to the substituted nitroaryl moiety at the C-5 position of 1,3,4-thiadiazole ring. While all of nitrothiophene derivatives 6a-h demonstrated weak (IZD = 11–15 mm) to little (IZD <10 mm) inhibitory response at concentration of 100 μg/disk against three metronidazole resistant isolates, most of nitrofurane derivatives 6i-q showed strong (IZD > 20 mm) to moderate (IZD = 16–20) growth inhibitory potential at the same concentration.Based on the substituted nitroaromatic group, the studied 1,3,4-thiadiazole derivatives could be classified into two groups: Nitrothiophen 6i-q derivatives of 1,3,4-thiadiazole scaffold, are more potent than the nitrothiophen 6a-h counterpart.It could be concluded that nitrofuran 6i-q, 3-methoxybenzyl piperazine derivative 6i, demonstrated strong anti-H. pylori potential at studied concentrations 100–25 μg/disk (IZD > 20 mm) against studied isolates. Investigation of the growth inhibitory potential of the nitrofuran series 6i-q revealed that substitution of nitro group at the meta position of the benzyl piperazine side chain, resulted in compound with strong (IZD = 20 mm) to moderate (IZD = 16–20 mm) growth inhibitory potential at 100 and 50–12.5 μg/disk, respectively. However, introduction of nitro substitute at ortho or para position of the benzyl piperazine pendant, resulted in compound with diminished inhibitory potential against resistant strains of H. pylori isolates  and 6l  respectively). Moreover, substitution of fluorine groups at different positions of benzyl pieperzine side chain influenced the growth inhibitory potential of compounds which is mainly dependent on the position and number of substituted fluorine groups; compound 6n containing 2,4,5-triflouro benzyl piperazine pendant at C-2 position of 5-nitrofuran-1,3,4-thiadiazole scaffold, produced strong inhibitory response at 100 and 50 μg/disk ; while the anti-H. pylori potential of 2,5-difluoro benzyl piperazine counterpart was diminished to weak (IZD = 12 mm) to no response (IZD = 5 mm) at 100 and 50 μg/disk, respectively.In order to find the structural requirement of substituted moiety at C-2 position of 1,3,4-thiadiazole scaffold, different benzyl piperazine derivatives were substituted at the described position. Among the nitrofuran derivatives H.pylori using paper disk diffusion bioassay test. Structure-activity relationship study of these derivatives indicated that 1,3,4-thiadiazole derivatives bearing 5-nitrofuran moiety at C-5 position of central thiadiazole ring, demonstrated more promising anti-H. pylori than the 5-nitrothiophen counterpart. The most potent nitrofuran derivative had 3-methoxybenzyl piperazine pendant at the C-2 position of 1,3,4-thiadiazole ring. The results indicated that the anti-H. pylori potential of the nitrofurane derivatives of 1,3,4-thiadiazole scaffold is mainly attributed to the type and position of the substituted group at the benzyl piperazine pendant. Future studies may be aimed at designing more potent derivatives of these series in order to investigate the structure-activity relationship of cyclic amine derivatives of 5-(nitroaryl)-1,3,4-thiadiazole derivatives as Anti-H. pylori agents.A novel series of 5-(5-nitroaryl)-1,3,4-thiadiazole derivatives containing various benzyl piperazine moiety at C-2 position of 1,3,4-thiadiazole ring were synthesized and evaluated against three metronidazole-resistant isolates of The authors declare that they have no competing interests.NM: Synthesis of some target compounds. PS: Evaluation of the antibacterial activities (10%). AG: Evaluation of the antibacterial activities. HA: Evaluation of the antibacterial activities (10%). FA: Synthesis of the intermediates and some target compounds. NE: Collaboration in identifying of the structures of target compounds. FS: Evaluation of the antibacterial activities. AF: Collaboration in design and identifying of the structures of target compounds, manuscript preparation. AS: Design of target compounds and management of the synthetic and pharmacological parts. All authors read and approved the final manuscript.