Biotransformations of the racemic lactone with three fungal strains: Absidia cylindrospora AM336, Absidia glauca AM177 and Syncephalastrum racemosum AM105, were carried out. Microbial transformations afforded hydroxylactones with the hydroxy group in the allylic position.Racemic [(±)-4-isopropyl-1-methyl-7-oxa- Many of them, e.g., mintlactone, isomintlactone and wine lactone, are known for their interesting odoriferous properties . 1,. 1,2 was2 possesses a weak odour slightly resembling that of coumarin. Knowing that fragrance of compounds depends on their chirality, we decided to synthesize two optically active unsaturated lactones: (–)-2 and (+)-2. These compounds (–)--2 and (+)--2 were obtained from the corresponding iodolactones (–)--1 and (+)--1, respectively, similarly to the synthesis of racemic lactone (±)-2 -2 and (+)--2, as determined by chiral GC, were 98% and 96%, respectively (the same as the starting iodolactones). The odoriferous properties of the enantiomerically enriched lactones (–)-2 and (+)-2 were also evaluated. The lactone (–)--2 possesses an intense, dill odour, while the fragrance of (+)--2 is medium intensity and coumarin-like -ne (±)-2 . The enarin-like . The com2. Three of them: Absidia cylindrospora AM336, Absidia glauca AM177 and Syncephalastrum racemosum AM105 transformed the substrate into the same products – hydroxylactones 3 and 4 , was observed after 10 days of incubation.Six fungi strains were tested for their ability to transform racemic lactone (±)- 3 and 4 . No form4 was the major product in all biotransformations carried out. The enantioselectivity of its formation depended on the fungi strains applied. The composition of the products mixture of biotransformation of (±)-2 by selected microorganisms are presented in The progress of biotransformation was monitored by chiral gas chromatography. Hydroxylactone 2 in the culture of A. cylindrospora AM336 the product mixture contained 66% of unreacted substrate (2), 14% of hydroxylactone 3 and 20% of product 4. In the next days the amount of 2 decreased and after four days the formation of 39% of 3 and 61% of 4 was observed. The enantiomeric excess of hydroxylactone (+)-3 obtained in this way was the highest one and reached a value of 39%.After the first day of incubation (±)-2 (120 mg) in the shaken cultures of A. cylindrospora AM336 after four days afforded: 15 mg (12% isolated yield) of (+)-3-hydroxy-4-isopropyl-1-methyl-7-oxa-cis-bicyclo[4.3.0]non-4-en-8-one (3) , ee = 39%) and 28 mg (22% isolated yield) of (+)-4-(1'hydroxy-1'methylethyl)-1-methyl-7-oxa-cis-bicyclo[4.3.0]non-4-en-8-one (4) .The preparative biotransformation of unsaturated lactone (±)-3 and 4 was confirmed by absorption bands in the IR spectra at 3438 and 3450 cm−1, respectively. Furthermore, the absorption bands at 1760 and 1773 cm−1, respectively, indicated that the γ-lactone ring has been retained. The final proof of the structure of hydroxylactones 3 and 4 was delivered by NMR spectroscopy.The structure of these products was confirmed by their spectral data. The presence of the hydroxy group in products 1H-NMR spectrum of product 3 indicates that biohydroxylation had taken place at the C-3 position. The presence of multiplet at 4.37 ppm for proton H-3 and the change of multiplicity and chemical shift of protons CH2-2 (compared to the spectrum of the substrate) confirm this assignation. The signal from the pseudoequatorial H-2 proton (δ = 2.07) gave a doublet of doublets with a geminal coupling constant JH2a-H2e = 14.0 Hz and a small coupling constant J = 5.2 Hz with the pseudoequatorial H-3 proton. The pseudoaxial H-2 proton also gave a doublet of doublets (δ = 1.67) with the same geminal coupling constant (JH2a-H2e = 14.0 Hz) and with the vicinal coupling constant J = 7.1 Hz with the pseudoequatorial H-3 proton. On the basis of these data and according to model analysis (Dreiding models) a pseudoequatorial position was assigned to the H-3 proton. This analysis indicates also that the H-3 proton is situated close to the plane of the double bond, so it is deshielded and its signal is located at 4.37 ppm. Additionally, a significant increase in the chemical shift difference of the CH2-9 protons (0.22 ppm) in comparison to difference in the spectrum of (±)-2 (0.08 ppm) indicates the nearness of the hydroxy group to one of these protons and the cis-orientation of the OH group to the γ-lactone ring in product 3. The fungal strain A. cylindrospora AM336 is known for its allylic position hydroxylation ability. Our previous biotransformation of unsaturated lactones with A. cylindrospora AM336 afforded hydroxylactones with pseudoaxially oriented hydroxy groups in the allylic position non-4-en-8-one (3) , ee = 22%) and 21 mg (16% isolated yield) of (+)-4-(1'hydroxy-1'methylethyl)-1-methyl-7-oxa-cis-bicyclo[4.3.0]non-4-en-8-one (4) .The preparative biotransformation of lactone (±)-4 was also as the main product of transformation of (±)-2 in the culture of S. racemosum AM105. On the second day of biotransformation S. racemosum AM105 produced (+)-3 with 18% enantiomeric excess. In the next days the enantioselectivity of this process was increased and finally (+)-isomer of 3 was obtained with 38% enantiomeric excess. Six days of incubation of lactone (±)-2 (120 mg) afforded: hydroxylactones 3 , ee = 38%) and 4 ).Hydroxylactone 3 and (+)-4 obtained from the conversion of (±)-2, enantiomerically enriched substrate (–)-2 (ee = 98%), with known absolute configuration  was subjected to the biotransformation. Microbial transformation of (–)--2 was performed on analytical scale by A. cylindrospora AM336.Additionally, to confirm the absolute configuration of products (+)-3 obtained from the conversion of (–)--2 was tR = 115.45 min and the retention times of the two enantiomers of 3 formed during the biotransformation of (±)-2 were tR = 114.30 and 115.45 min, respectively. These results indicate that stereoisomer with tR = 115.45 min has R configuration at C-1 and C-6. Knowing that the hydroxy group at C-3 is cis-oriented with respect to the γ-lactone ring it was possible to ascribe the absolute configuration at this chirality centre. On the basis of these data 1S,3S,6S configuration of (+)-isomer of 3 with tR = 114.30 min and 1R,3R,6R configuration of the (–)-enantiomer of 3 with tR = 115.45 min were assigned.The retention time of product 4 by chromatographic methods using the chiral columns at our disposal. However, the analysis of the composition of the products mixture obtained from transformation of lactone (±)-2 -isomer of 4 has a 1R,6R configuration.Unfortunately, it was impossible to evaluate the enantiomeric excess of hydroxylactone ne (±)-2  indicate1H-NMR, 13C-NMR, DEPT 135 (distortionless enhancement by polarisation transfer), 1H–1H COSY (correlation spectroscopy) and HSQC (heteronuclear single quantum correlation) spectra were recorded in CDCl3 solution on a Bruker Avance™ 600 (600 MHz) spectrometer. Chemical shifts were referenced to the residual solvent signal . IR spectra were recorded for liquid films on a Thermo-Nicolet IR300 FT-IR spectrometer. HRCIMS was measured on a JEOL JMS-700 MS station. CH elemental analyses were performed on a CE Instruments EA-1110 elemental analyzer. Optical rotations were determined on an Autopol IV automatic polarimeter (Rudolph) in chloroform solutions, concentrations denoted in g/100 mL. Analytical TLC was performed on Fluka Kieselgel 60, F254 plates with the solvent system hexane/diethyl ether, 4:1. Compounds were detected by spraying the plates with 1% Ce(SO4)2, 2% H3[P(Mo3O10)4] in 10% H2SO4, followed by heating to 120 °C. Column chromatography was performed on silica gel  with a mixture of hexane and diethyl ether (in various ratios) as eluent. Gas chromatography analysis was carried out on an Agilent 6890N GC . Enantiomeric excesses were determined on a CP-Chirasil-DEX CB column (25 m × 0.25 mm × 0.25 μm) with the following temp. Program: 90 °C, 155 °C (0.5 °C/min), 200 °C (20 °C/min) (10 min). The total run time was 142.25 min; tR (–)--2 = 58.04 min, tR (+)--2 = 58.72 min, tR 4 = 109.37 min, tR (+)--3 = 114.30 min, tR (–)--3 = 115.45 min.Unless otherwise stated, all chemicals were purchased as the highest purity commercially available and were used without further purification. DBU  was purchased from Fluka . 2, (–)-2 and (+)-2 were synthesized from corresponding δ-iodo-γ-lactones (±)-1, (–)-1 and (+)-1, respectively, which were obtained starting from corresponding trans-piperitols as described earlier non-4-en-8-one [(±)-2]. Lactone (±)-2  was obtained from (±)-5-iodo-4-isopropyl-1-methyl-7-oxa-cis-bicyclo[4.3.0]nonan-8-one ; 1H-NMR (CDCl3) δ: 1.01 and 1.02 2CH-), 1.13 , 1.53 , 1.62 , 2.05 , 2.26 2CH-), 2.33 and 2.41 , 4.42 , 5.53 ; 13C-NMR (CDCl3) δ: 20.83 and 21.11 ((CH3)2CH–), 22.77 (C-3), 22.94 (CH3-1), 30.03 (C-2), 34.94 ((CH3)2CH-), 36.83 (C-1), 42.30 (C-9), 82.34 (C-6), 114.65 (C-5), 150.81 (C-4), 176.28 (C-8); IR : 2962 (m), 1778 (s), 1664 (w), 1384 (w), 1364 (w), 1157 (m); elemental analysis calcd (%) for C12H18O2 (194.28): C 74.19, H 9.34; found: C 73.51, H 9.59.(±)--4-Isopropyl-1-methyl-7-oxabicyclo[4.3.0]non-4-en-8-one [(-)-2]. Lactone (-)-2  was obtained from (–)--5-iodo-4-isopropyl-1-methyl-7-oxabicyclo[4.3.0]nonan-8-one ((-)-1) .  = −1.9° . IR and NMR spectra were identical with those of (±)-2.(-)--4-Isopropyl-1-methyl-7-oxabicyclo[4.3.0]non-4-en-8-one [(+)-2]. Lactone (+)-2  was obtained from (+)--5-iodo-4-isopropyl-1-methyl-7oxabicyclo[4.3.0]nonan-8-one ((+)-1) .  = +2.3° . IR and NMR spectra were identical with those of (±)-2.(+)Absidia cylindrospora AM336, Absidia glauca AM177, Fusarium culmorum AM3/1, Fusarium oxysporum AM145, Penicillium vinaceum AM110 and Syncephalastrum racemosum AM105. The microorganisms came from the Collection of the Institute of Biology and Botany Medical University, Wrocław. They were maintained at 4 °C on Sabouraud agar slants containing peptone (10 g), glucose (40 g) and agar (15 g) dissolved in water (1 L) at pH 5.7.The following fungal strains were used for screening: 2 by six microorganisms were carried out in 300 mL Erlenmeyer flasks. The corresponding microorganisms were cultivated at room temperature in Erlenmeyer flasks containing 100 mL medium, which had the following composition (per 1 L of distilled water): peptone (10 g) and glucose (30 g). Cultures were incubated in a rotary shaker at 150 rpm at 25 °C. After 3–5 days, 10 mg of substrate (±)-2 dissolved in 1 mL of acetone was added to the shaken cultures. The samples of biotransformation mixture were extracted with chloroform after 1, 2, 4 and 6 days. They were dried over anhydrous MgSO4, concentrated in vacuo and analyzed by TLC and GC.Screening scale biotransformations of lactone (±)-2 by Absidia cylindrospora AM336.The same procedure was applied during the transformation of (–)-Additionally, two control flask were used for each biotransformations. A culture control contained sterile culture medium with microorganism inoculum. This experiment used to define and exclude the secondary metabolites generated by fungi. The second control flask contained substrate and sterile growth medium incubated without fungi. The substrates used for the biotransformation were stable in these conditions. It can be concluded that all products isolated from the cultures were bioconversion products.2 (120 mg in 1 mL acetone) was added to the grown cultures of corresponding microorganisms  prepared as described in the screening procedure. After 4 or 6 days the products were extracted with chloroform (3 × 150 mL). The organic solutions were dried over anhydrous MgSO4 and concentrated in vacuo. Mixtures of products as well as metabolites produced by the fungi were separated by column chromatography . The spectral data of biotransformation products are given below:Preparative scale biotranasformations were carried out in 2 L flat bottomed flasks containing 400 mL of medium . Lactone (±)-3-Hydroxy-4-isopropyl-1-methyl-7-oxa-cis-bicyclo[4.3.0]non-4-en-8-one (3). Colourless oily liquid. 1H-NMR (CDCl3) δ: 1.07 and 1.10 2CH–), 1.29 , 1.62 , 1.67 , 2.07 , 2.22 and 2.44 , 2.63 2CH-), 4.37 , 4.60 , 5.55 ; 13C-NMR (CDCl3) δ: 20.93 and 22.23 ((CH3)2CH–), 26.39 (CH3-1), 29.72 ((CH3)2CH-), 37.52 (C-1), 39.91 (C-2), 40.70 (C-9), 64.95 (C-3), 82.46 (C-6), 117.59 (C-5), 150.98 (C-4), 176.02 (C-8); IR : 3438 , 2961 (s), 1760 (s), 1168 (m), 1014 (m), 956 (m). HRCIMS [M+H]+ m/z 211.1333 .4-(1'Hydroxy-1'methylethyl)-1-methyl-7-oxa-cis-bicyclo[4.3.0]non-4-en-8-one (4). Colourless oily liquid. 1H-NMR (CDCl3) δ: 1.15 , 1.34 and 1.35 2C(OH)–), 1.52 , 1.58 , 1.66 , 2.13 , 2.21  2.36 and 2.43 , 4.48 , 5.89 ; 13C-NMR (CDCl3) δ: 21.10 (C-3), 22.99 (CH3-1), 28.64 and 28.84 ((CH3)2C(OH)–), 30.21 (C-2), 36.57 (C-1), 42.12 (C-9), 72.61 ((CH3)2C(OH)–), 81.96 (C-6), 114.82 (C-5), 150.84 (C-4), 176.09 (C-8); IR : 3450 , 2972 (s), 1773 (s), 1199 (s), 956 (s). HRCIMS [M+H]+ m/z 211.1328 .2 and an enantiomeric pair of unsaturated γ-lactones with the p-menthane system (–)-2 and (+)-2 were synthesized and their odoriferous properties were evaluated. Comparison of the odour of racemic (±)-2 and optically active compounds (–)-2 and (+)-2 confirmed that their fragrance depends on the configuration of their chiral centers.Racemic (±)-2 by all fungi strains studied has taken place in the allylic positions C-3 and C-11. Similar to the literature data [The results obtained in this work indicate that hydroxylation of (±)-ure data  the prod


The structure of the natural dipyranocoumarin dipetalolactone has been confirmed by an unambiguous synthetic route from resorcinol. This sequence was initiated by a pyran ring formation step which introduced the 3-chloro-3-methylbut-1-yne moiety. Then, the expected product undergoes a Fremy’s salt-meditated oxidative addition followed by ring closure to yield dipetalolactone. Dipetalolactone was also found to have immunological activity in a mouse carcinoma S180-bearing mice cell line. Zanthoxylum dipetalum, Metrodorea flavido and Diplolaene mollis (Rutaceae) , 75.4 (≡CH), δ 29.2 (2CH3); EIMS (C11H12O2) (% int.) m/z 176 (100), 205 [M–H]+ (25), 190 [M–OH]+ (21), 176 [M–2CH3]+ (8) and 107 [M–HC≡C−C(CH3)2−O]+ (12); Analysis: calcd. %C, 74.98; %H, 6.83; %O, 18.18. Found: %C 74.88; %H, 6.81; %O 18.09.To a solution of resorcinol (0.01 mol) and potassium carbonate (0.01 mol) in acetone (30 mL), 3-chloro-2-methylbut-1-yne (0.01 mol) was added drop wise under nitrogen. The reaction mixture was heated and stirred at 70 °C for 72 h and monitored by TLC. After cooling to room temperature the solution was poured in water (100 mL) then acidified with dilute HCl (5%) to remove the residue of K2 (0.01 mole) was boiled in dry xylene for 8 h under a nitrogen atmosphere, the reaction mixture was cooled, and then extracted with dichloromethane. The residue after removing the solvent was subjected to column chromatography over silica gel (800 g) and eluted with mixtures of hexane/ethyl acetate (1:2 v/v) as eluent to produce compound 3, which was crystallized from ethanol to give white crystals (60%) with mp 155–156 °C; IR (KBr) νmax cm−1: 3,540, 3,352, 1,335, 1,129, 931, 841; 1H-NMR (CDCl3): δ 11.2 , 7.13 , 6.86 , 6.55 , 6.31 , 5.91 , 1.59 ; 13C-NMR (CDCl3): δ 156.7 (C-5), 155.5 (C-8a), 130.4 (C-7), 128.5 (C-3), 116.2 (C-4), 109.8 (C-4a), 109.7 (C-6), 106.5 (C-8), 85.7 (C-2), 28.5 ; EIMS (C11H12O2) (% int.) m/z 176 (100), 175 [M–H]+ (32), 160 [M–OH]+ (21) and 146 [M–2CH3]+ (12); Analysis: calcd. %C, 74.96; % H, 6.85; %O, 18.19. Found: %C 74.91; %H, 6.82; %O 18.12.Compound 2PO4 (10 g) in water (200 mL) was mechanically stirred in a 2 L round bottom flask and ice (200 g) was added compound 3 (0.01 mol). The flask was cooled in an ice-ethanol mixture and (KSO3)2NO  added, followed by a solution of 4-(methylmercapto)phenol  in diethyl ether (20 cm3). After 1 min the solution turned orange and after 5 min dark red. After 1 h the mixture was evaporated at 20 °C to remove the diethyl ether and extracted with chloroform (3 × 50 cm3). Evaporation gave a crimson solid  with mp 180–181 °C; IR (KBr) νmax cm−1: 1,750, 1,735, 1,343, 1,139, 951, 841; 1H-NMR (CDCl3): δ 6.93 , 6.44 , 5.81 , 1.42 ; 13C-NMR (CDCl3): δ 185.5 (C-5), 181.7 (C-8), 150.4 (C-8a), 136.2 (C-6), 135.5 (C-7), 132.8 (C-3), 119.7 (C-4a), 116.5 (C-4), 85.9 (C-2), 28.7 ; EIMS (C11H10O3) (% int.) m/z 190 (100), 175 [M–1CH3] + (22), 160 [M–2CH3] + (9), 162 [M–CO] (41); Analysis: calcd. %C, 69.46; %H, 5.30; %O, 25.22. Found: %C 69.41; %H, 5.22; %O 25.12.To a solution of KH4 (0.01 mol) was dissolved in refluxing p-dioxane (200 mL) and acetic anhydride (0.03 mol) for 8 h. After cooling, the acetylated compound solution was concentrated and dried under reduced pressure. The crude acetylated lignin was dissolved in chloroform (3 mL) and the solution was added with stirring to diethyl ether. The precipitated acetate was centrifuged, collected, and dried under high vacuum for 24 h. The precipitate formed after cooling the mixture on ice water solution was recrystallized from ethanol give compound 5 as white crystals  with mp 277–279 °C. IR (KBr) νmax cm−1: 1,763, 1,750 and 1,710 (3 C=O ester), 1,576, 1,345, 1,129, 911, 849; 1H-NMR (CDCl3): δ 6.88 , 6.42 , 5.88 , 2.28, 2.2, 2.1 , 1.49 ; 13C-NMR (CDCl3): δ 169.1 (3C=O), 153.2 (C-8a), 147.2 (C-5), 144.2 (C-7), 130.1 (C-8), 127.4 (C-3), 114.1 (C-4a), 116.5 (C-4), 103.5 (C-6), 86.7 (C-2), 28.2 , 20.5 (3CH3-ester); EIMS (C17H18O7) (% int.) m/z 334 (100), 319 [M–1CH3] + (34), 304 [M–2CH3] + (21) and 199 [M–3OAc] (14)”; Analysis: calcd. %C, 61.08; %H, 5.43; %O, 31.48. Found: %C 60.88; %H, 5.31; %O 31.59.Acetylation was carried out following literature method . In brie6 was obtained by dissolving (0.1 mol) of compound 5 in 10% NaOH solution, then by addition of HCl, we obtain a precipitate, which is filtered off , then transferred into a conical flask and water (30 mL) is added. The mixture is brought to its boiling point, FeCl3 (1.5 g) is added and boiled for 10 min, the hot solution is filtered, washed with boiling water and he precipitate formed dried in air. Recrystallization from toluene gives brown crystals (40%) with mp 254–255 °C; UV (MeOH) λmax: 331 (log ε 3.67), 292 (log ε 4.07), 262 (log ε 4.45); IR (KBr) νmax cm−1: 3,340, 1,752, 1,722, 1,576, 1,345, 1,129, 911, 849; 1H-NMR (CDCl3): δ 16.2 , 6.83 , 6.42 , 6.81 , 6.41 , 5.81 , 1.39 ; 13C-NMR (CDCl3): δ 182.5 (C-5), 196.2 (C-8), 176.5 (C-7), 141.2 (C-8a), 126.5 (C-3), 117.4 (C-4a), 115.5 (C-4), 109.5 (C-6), 86.7 (C-2), 29.5 ; EIMS (C11H10O4) (% int.) m/z 206 (100), 205 [M–H] + (21), 190 [M–OH] + (24) and 176 [M–2CH3] + (11); Analysis: calcd. %C, 64.08; %H, 4.83; %O, 31.88. Found: %C 64.12; %H, 4.81; %O 31.09.Compound 4 as faint brown crystals  with mp 187–189 °C; IR (KBr) νmax cm−1: 1,750, 1,720, 1,711, 1,581, 1,345, 1,099, 901, 869; 1H-NMR (CDCl3): δ 6.82 , 5.84 , 4.07 , 1.44; 13C-NMR (CDCl3): δ 176.5 (C-8), 196.2 , 141.2 (C-8a), 117.4 (C-4a), 126.5 (C-3), 115.5 (C-4), 45.8(C-6),86.7 (C-2), 29.5 ; EIMS (C11H10O4) (% int.) m/z 206 (100), 178 [M–CO] + (34) and 176 [M–2CH3] + (27); Analysis: calcd. %C, 64.06; %H, 4.88; %O, 31.04. Found: %C 63.92; %H, 5.81; %O 30.89.Compound 7 was obtained by the same procedure described for the synthesis of compound 8 was obtained by the same procedures described for the synthesis of compounds 2 and 3, respectively, by refluxing compound 7 (0.01 mol) with 3-chloro-3-methylbut-1-yne (0.01 mol), followed by ring closure to give the di pyrano derivative in dry xylene for 8 h, then the precipitate obtained was filtered off and crystallized from a mixture of cyclohexane and ethanol give compound 8 as faint brown crystals  with mp 292–294 °C; UV (MeOH) λmax: 462 (log ε 3.75), 302 (log ε 3.97), 271 (log ε 4.47), 261 (log ε 4.43); IR (KBr) νmax cm−1: 1,789, 1,754, 1,598, 1,375, 1,049, 911, 859; 1H-NMR (CDCl3): δ 6.75 , 6.48 , 5.89 , 5.84 , 1.34 ; 13C-NMR (CDCl3): δ 179.4 (C-1), 176.6 (C-2), 173.2 (C-6b), 131.3 (C-9), 130.1 (C-2a), 127.5 (C-6a), 125.5 (C-5), 97.1 (C-10a), 88.9 (C-8), 88.5 (C-4), 29.5 , 28.7 ; EIMS (C16H16O4) (% int.) m/z 272 (100), 257 [M–1CH3]+ (31), 244 [M–CO]+ (17) and 242 [M–2CH3]+ (14); Analysis: calcd. %C, 70.54; %H, 5.95; %O, 23.50. Found: %C 70.32; %H, 5.81; %O 23.39.Compound 8. 30% Aqueous NaOH solution (30 mL) was added to the Wittig reaction product and the mixture was boiled for 3 h, cooled, acidified with ice cold hydrochloric acid, and the reaction mixture refluxed for another 2 h. The precipitate obtained after cooling was filtered off, washed with water and crystallized from dil. ethanol to give compound 10 through the intermediate 9 as pale brown crystals  with mp 295–296 °C; UV (MeOH) λmax: 211 (log ε 4.27), 282 (log ε 3.75) and 232 (log ε 3.97); IR (KBr) νmax cm−1: 1,720, 1,711, 1,591, 1,345, 1,089, 901, 869; 1H-NMR (CDCl3): δ 7.75 , 6.88 , 6.84 , 5.97 , 1.84 ; 13C-NMR (CDCl3): 151.9 (C-12b), 149.8 (C-8b), 148.9 (C-4b), 139.5 (C-4), 128.5 (C-11), 128.2 , 116.4 (C-8), 113.6 (C-3), 108.2 (C-8a), 105.3 (C-4a), 102.5 (C-12a), 86.5 , 28.6  as lit.  + (32), 285 [M–1CH3] + (21) and 250 [M–4CH3] + (7); Analysis: calcd. %C, 73.54; %H, 5.85; %O, 20.64. Found: %C 73.32; %H, 5.81; %O 20.29.Wittig reaction was carried out as described in the discussion  using 0.ad libitum. Young adult (30 ± 5 g) ICR mice  were provided by the Egyptian Holding Company for Biological Products and Vaccines, Cairo, Egypt. Animals were maintained under standard conditions of ventilation, temperature (25 ± 2 °C), humidity (60%–70%) and light/dark condition (12/12 h). The rats were housed in stainless steel cages and provided with free access to food and drinking water 10 solution on tumor growth was estimated by evaluating body weight, tumor weight, and percentage of tumor inhibition. S180 tumor cell line was originally obtained from Cairo Institute of Oncology, Cairo, Egypt and maintained as the ascites form by serial passages intraperitoneal in ICR mice. For solid tumor development, S180 cell suspension  was inoculated subcutaneously into right armpits of mice under sterile condition. The mice were divided into six random groups (10 in each): S180-bearing control, normal control, compound 10 . Test doses were decided on the basis of findings from preliminary studies. Body weight of animals was recorded before the experiment. The doses administrated p.o. daily for 12 days. Normal control and S180-bearing control groups received the same volume of normal saline. On the 13th day, all animals were euthanized. Their body and tumor weights were obtained and documented [The effect of compound cumented .6/mL were prepared in phosphate buffer solution (PBS). A total of 1.0 mL of 0.4% SRBC and 1.0 mL of 10% guinea pig serum were mixed with cell suspension and incubated for 1 h at 37 °C. After a 3 min centrifugation at 3,000 rpm, the absorbance of the supernatant was measured at 413 nm using a spectrophotometer [The mice were injected i.p. with 3:5 (v/v) sheep red blood cells  prepared in normal saline on the 8th day of the experiment. QHS assay was performed in those animals following the immunization. Eyeballs were removed and single cell suspensions of 1 × 10otometer .6 cells/mL of culture media for subsequent experiments [For the assessment of cellular immune function, lymphocyte proliferation and Natural Killer (NK) cell cytotoxicity tests were performed. After the experiment was completed, their spleens were aseptically removed and filtered over a double layer of stainless-steel mesh to obtain single cell suspension. After these washes in Hanks’ balanced salt solution, the spleen cells were finally suspended in 10% FCS RPMI 1640 media supplemented with benzyl penicillin 100 U/mL, streptomycin 100 μg/mL. The cell number was adjusted to 3 × 10eriments .Canavalia ensiformis Type III, Sigma, Munich, Germany) and a polyclonal T cell mitogen. The micro plates were cultured at 37 °C for 72 h in the humidified 5% CO2 incubator. At 72 h, 1 μCi/well 3H-TdR  was added to each well. The cells were harvested 16 h later and the radioactivity incorporated was counted using a liquid scintillation counter [For the splenocyte proliferation assay, the spleen cell suspension was added to micro plate wells with 5 μg/mL of concanavalin A  solution (100 μL/well) and the amount of released LDH was determined. The OD value of each well was measured at 490 nm using a spectrophotometer. The percentage of cytotoxicity generated by NK cells was calculated according to the following formula:er  was the LDH release from co-cultures at an E/T ratio of 50:1; ODesr (ODeffectorspontaneousrelease) and ODtsr (ODtargetspontaneousrelease) were spontaneous LDH releases from effector and target cells incubated with medium alone, respectively; and ODtmr (ODtargetmaximumrelease) was the maximum release from target cells lysed with the lysis solution [The splenocyte prepared as described above were used as effector cells. YAC-1 cells, mice lymphoma sensitive to NK cells were used as target cells. Effectors and target cells resuspended in RPMI-1640 medium supplemented with 3% heat-inactivated fetal bovine serum were added to each well of a 96-well U-bottom micro culture plate in triplicate to obtain an effectors/target (E/T) ratio of 50:1, and incubated at 37 °C in the humidified 5% COsolution .Phagocytic activity of macrophages was used to assess the nonspecific immune function. The mice were injected i.p. with 0.5 mL 5% cock red blood cells (CRBC) 10 h prior to the last dose. On the 13th day of the experiment macrophages were obtained from the peritoneal exudates harvested by peritoneal lavage using sterile cold Hanks’ solution. The number of CRBC ingested by macrophages was counted in an optical microscope .p < 0.05 [Data were expressed as the mean ± standard deviation (S.D.) in tables. Statistical analyses were carried out using the analysis of variance (ANOVA) and post-hoc tests for multiple comparisons. Differences were considered statistically significant at p < 0.05 .in vivo Immunomodulatory activities in S180-bearing mice, where higher concentrations of compound 10 appeared to show more effective inhibition of tumor by increasing the antibodies secreted by spleen cells in mice. In summary we have developed a new simple route approach to naturally dipetalolactone starting from resorcinol and substituted-1-butyne via Fremy’s salt as mediator oxidizing agent and examined its effect on


The proposed structures of all the synthesized compounds were confirmed using elemental analysis, UV, IR, 1H-NMR and mass spectroscopy. All compounds were evaluated for antifungal activity against plant pathogens, larvicidal and biting deterrent activity against the mosquito Aedes aegypti L. and in vitro cytotoxicity and anti-inflammatory activity against some human cell lines. Phomopis species were the most sensitive fungi to these compounds. Compounds 1b, 1c, 3a and 4e demonstrated selectively good activity against Phomopis obscurans and only 1b and 4e showed a similar level of activity against P. viticola. Compound 3d, with a LD50 value of 67.9 ppm, followed by 1c (LD50 = 118.8 ppm) and 3e (LD50 = 165.6 ppm), showed the highest toxicity against Aedes aegypti larvae. Four of these compounds showed biting deterrent activity greater than solvent control, with the highest activity being seen for 1c, with a proportion not biting (PNB) value of 0.75, followed by 1e, 2b and 1a. No cytotoxicity was observed against the tested human cancer cell lines. No anti-inflammatory activity was observed against NF-κB dependent transcription induced by phorbol myristate acetate (PMA) in human chondrosarcoma cells.A series of novel thiourea and urea derivatives containing 1,2,4-triazole moieties were synthesized and evaluated for their antifungal and larvicidal activity. Triazole derivatives  Thioureas are important sulphur and nitrogen-containing compounds that have proved to be useful substances in drug research in recent years ,3,4,5,6.Penicillum expansum and Fusarium oxysporum + peaks in agreement with their molecular formulae.In the IR spectra of compounds Colletotrichum, Botrytis, Fusarium and Phomopsis using 96-well micro-dilution broth assays. Microbioassay results indicated that the thiourea derivatives 1b, 1c, 3a and 3d bearing 2,6-dichloro-, 2,4,6-trichloro-, 4-methysulfanyl- and 4-trifluoromethyl- groups on the phenylthiourea ringand the urea derivative 4e having a 4-nitro group were the most active against Phomopsis obscurans and P. viticola. Compounds 1b, 3a, and 1c and 4e at 30 μM inhibited the growth of P.obscurans by 100% and 80%, respectively, after 120 h exposure, whereas 1b, 3d and 4e showed 80%, 60% and 80% growth inhibition, respectively, at 30 μM, respectively  value of 0.75, followed by 1e, 2b and 1a having 4-nitro-, 2,6-dichloro- and 2,4,6-trichloro-groups, with PNB values of 0.71, 0.68 and 0.55, respectively, at 25 nmol/cm2. However, biting deterrent activity in all these compounds was significantly lower than that of the reference compound DEET (PNB = 0.89).In biting deterrent bioassays with activity  than the1c, 3d and 3e which showed larvicidal activity in preliminary screening were selected for further study in dose response bioassays. Dose mortality data of these thiourea compounds are presented in In order to discover new mosquito toxicants, synthesized compounds were also screened against 1 d old larvae. The three compounds 3d carrying a 4-trifluorophenyl group  showed the highest mortality, followed by 1c having a 4-methylsulfanylphenyl group on the thiourea moiety (LD50 = 118.8 ppm) and 3e with a 4-nitrophenyl moiety (LD50 = 165.6). The comparison of larvicidal activity of thiourea and urea derivatives showed that thiourea derivatives were much more potent than the corresponding urea derivatives. There was not not much increase in mortality at 48 h post treatment.Compound 11) or human solid tumor cells  up to a highest concentration of 25 μg/mL, except for compound 3e having 4-nitro group, which showed mild cytotoxicity with IC50 values in the range of 11.0–25.0 μg/mL towards most of the tested cell lines. All synthesized compounds were tested for their cytotoxicity and anti-inflammatory activity in cellular assays. No anti-inflammatory activity was observed against NF-κB mediated transcription induced by phorbol myristate acetate (PMA) in human chondrosarcoma cells up to the highest tested concentration of 25 μg/mL and no cytotoxicity was observed against mammalian kidney cells  in DMSO-d6. Chemical shifts were recorded in parts per million (ppm) downfield from TMS. The splitting patterns of 1H-NMR were designated as follows: s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet. Mass spectra were recorded on an Agilent 1100 LC-MS series system  in the electrospray mode. Elemental analysis was performed on a Leco CHNS-932 analyzer . All chemicals and solvents were procured from Merck or Aldrich . The thiourea derivatives 1a–e were prepared by reacting 4-(aminophenyl)acetic acid with the corresponding isothiocyanate. Spectroscopic characterisation of 1a–e has been previously reported amino}phenyl)acetic acid (2a). UV λmax. (EtOH) nm (log ε): 251 (4.45). IR : 3286 , 3286 (C-H), 1687, 1651 (C=O), 1543, 1510 (N-H). 1H-NMR δ: 3.48 , 6.99–7.83 , 8.22 , 8.94 , 12.24 . Anal. Calcd. for C15H11Cl3N2O3; C: % 48.22; H: % 2.97; N: % 7.50 Found: C: % 47.95; H: % 3.00; N: % 7.30. MS-ES (m/z): 374.62 (MH+). carbamoyl]amino}phenyl)acetic acid (2b). UV λmax. (EtOH) nm (log ε): 251 (4.41). IR : 3252 , 3016 (C-H), 1697, 1651 (C=O), 1573, 1545 (N-H). 1H-NMR δ: 3.49 , 7.09–7.60 , 8.18 , 8.91 , 12.24 . Anal. Calcd. for C15H12Cl2N2O3; C: % 53.12; H: % 3.57; N: % 8.26 Found: C: % 52.84; H: % 3.55; N: % 8.15. MS-ES (m/z): 340.18 (MH+). 4-{[(4-(Methylsulfanyl)phenyl)carbamoyl]amino}phenyl)acetic acid (2c). UV λmax. (EtOH) nm (log ε): 279 (3.12). IR : 3290 , 3007 (C-H), 1695,1629 (C=O), 1550 (N-H). 1H-NMR δ: 3.35 , 3.54 , 7.02–7.62 , 8.63 , 8.70 , 12.24 . Anal. Calcd. for C16H16N2O3S; C: % 60.74; H: % 5.10; N: % 8.85, S: 10.14 Found: C: % 60.54; H: % 5.09; N: % 8.73, S: 10.00. MS-ES (m/z): 317.37 (MH+).(4-{[(4-Trifluoromethylphenyl)carbamoyl]amino}phenyl)acetic acid (2d). UV λmax. (EtOH) nm (log ε): 261 (4.06). IR : 3304 , 3304 (C-H), 1697,1630 (C=O), 1552, 1512 (N-H). 1H-NMR δ: 3.48 , 6.81–7.42 , 8.43 , 8.53 , 12.21 . Anal. Calcd. for C16H13F3N2O3; C: % 56.81; H: % 3.87; N: % 8.28 Found: C: % 56.60; H: % 3.72; N: % 8.16. MS-ES (m/z): 339.28 (MH+).(4-{[(4-Nitrophenyl)carbamoyl]amino}phenyl)acetic acid (2e). Compound 2e was previously synthesized by Deny et al. phenyl}-3--thiourea (3a). UV λmax. (EtOH) nm (log ε): 258 (3.97). IR : 3252 (N-H), 3020 (C-H) 1651 (C=N), 1600, 1510 (N-H), 1345, 1246 (C=S), 1182 (C-N). 1H-NMR δ: 3.20 , 5.48 , 7.20–7.46 , 7.83 , 8.55 , 11.70 . Anal. Calcd. for C16H13Cl3N6S2; C: % 41.79; H: % 2.85; N: % 18.28; S: % 13.95 Found: C: % 41.64; H: % 3.09; N: % 18.25; S: % 13.72. MS-ES (m/z): 460.80 (MH+).1-{4-[methyl]phenyl}-3--thiourea (3b). UV λmax. (EtOH) nm (log ε): 278 (3.51). IR : 3194 (N-H), 3012 (C-H), 1650 (C=N), 1541 (N-H), 1350, 1240 (C=S), 1180 (C-N). 1H-NMR δ: 3.47 , 5.50 , 7.15–7.59 , 9.68 , 9.79 , 11.80 . Anal. Calcd. for C16H14Cl2N6S2; C: % 45.18; H: % 3.32; N: % 19.76; S: % 15.08. Found: C: % 45.10; H: % 3.30; N: % 19.66; S: % 15.00. MS-ES (m/z): 426.36 (MH+).1-{4-[methyl]phenyl}-3-(4-(methylsulfanyl)-phenyl)thiourea (3c). UV λmax. (EtOH) nm (log ε): 282 . IR : 3220 (N-H), 3026 (C-H), 1537 (N-H), 1345, 1234 (C=S), 1178 (C-N). 1H-NMR δ: 3.30 , 3.49 , 5.10 , 6.90–7.40 , 9.40 , 9.46 , 12.00 . Anal. Calcd. for C17H18N6OS2; C: % 50.72; H: % 4.51; N: % 20.88; S: % 23.90. Found: C: % 50.27; H: % 4.70; N: % 20.69; S: % 23.92. MS-ES (m/z): 403.56 (MH+).1-{4-[methyl]phenyl}-3-(4-(trifluoromethyl)-phenyl)thiourea (3d). UV λmax. (EtOH) nm (log ε): 257 (4.62). IR : 3288 , 1697 (C=N), 1627, 1552 (N-H), 1346, 1294 (C=S), 1188 (C-N). 1H-NMR δ: 3.41 , 7.05–7.52 , 8.45 , 8.69 , 12.30 . Anal. Calcd. for C17H15F3 N6S2; C: % 48.10; H: % 3.56; N: % 19.80; S: % 15.11 Found: C: % 50.29; H: % 4.12; N: % 22.50; S: % 17.17. MS-ES (m/z): 425.47 (MH+).1-{4-[methyl]phenyl}-3-(4-nitrophenyl)thiourea (3e). UV λmax. (EtOH) nm (log ε): 246 (4.06). IR : 3280 (N-H), 3023 (C-H), 1593 , 1494 (N-H), 1320, 1298 (C=S), 1170 (C-N). 1H-NMR δ:3.55 , 5.25 , 7.11–8.36 , 9.20 , 9.40 , 12.20 . Anal. Calcd. for C16H15N7O2S2; C: % 47.87; H: % 3.77; N: % 24.42; S: % 15.97. Found: C: % 47.80; H: % 3.74; N: % 24.40; S: % 15.96. MS-ES (m/z): 402.47 (MH+).1-{4-[methyl]phenyl}-3--urea (4a). UV λmax. (EtOH) nm (log ε): 253 (3.24). IR : 3271 , 1651 (C=O), 1595 (C=N), 1539, 1508 (N-H), 1284–1253 (C=S), 1182 (C-N). 1H-NMR δ: 4.96 , 7.15–7.79 , 9.12 , 9.63 . Anal. Calcd. for C16H13Cl3N6OS; C: % 43.31; H: % 2.95; N: % 18.94; S: % 7.23 Found: C: % 42.95; H: % 2.91; N: % 19.03; S: % 7.24. MS-ES (m/z): 444.74 (MH+).1-{4-[methyl]phenyl}-3--urea (4b). UV λmax. (EtOH) nm (log ε): 263 (4.60). IR : 3300 , 1699 (C=O), 1633 (C=N), 1591, 1541 (N-H), 1228 (C=S), 1089 (C-N). 1H-NMR δ: 3.47 , 7.10–7.58 , 9.29 , 9.48 . Anal. Calcd. for C16H14Cl2N6OS2; C: % 45.18; H: % 3.45; N: % 20.53; S: % 7.83 Found: C: % 47.00; H: % 3.42; N: % 20.50; S: % 7.92. MS-ES (m/z): 410.29 (MH+).1-{4-[methyl]phenyl}-3-(4-methylsulfanyl-phenyl)urea (4c). UV λmax. (EtOH) nm (log ε): 261 (4.06). IR : 3304 , 3304 (C-H), 1697, 1633 (C=O), 1552,1512 (N-H). 1H-NMR δ: 3.32 , 3.48 , 5.20 , 6.81–7.42 , 8.43 , 8.53 , 12.40 . Anal. Calcd. for C17H18N6OS; C: % 52.83; H: % 4.69; N: % 21.70; S: 16.59. Found: C: % 52.70; H: % 4.49; N: % 21.67; S: 16.50. MS-ES (m/z): 387.49 (MH+).1-{4-[methyl]phenyl}-3-(4-trifluoromethyl-phenyl)urea (4d). UV λmax. (EtOH) nm (log ε): 258 (4.33). IR : 3290 (N-H), 1699 (C=O), 1556 (C=N), 1510, 1494 (N-H), 1211-1186 (C=S), 1151, 1095 (C-N). 1H-NMR δ: 3.42 , 7.02–7.66 , 8.65 , 12.50 . Anal. Calcd. for C17H15F3 N6OS; C: % 50.00; H: % 3.70; N: % 20.58; S: % 7.85 Found: C: % 50.10; H: % 3.67; N: % 20.50; S: % 7.89. MS-ES (m/z): 409.40 (MH+).1-{4-[methyl]phenyl}-3-(4-nitrophenyl)urea (4e). UV λmax. (EtOH) nm (log ε): 253 . IR : 3290 , 1695 (C=O), 1629 (C=N), 1589, 1545 (N-H), 1296 (C=S), 1111 (C-N). 1H-NMR δ: 3.48 , 5.40 , 7.01–7.62 , 8.52 , 9.93 , 13.48 . Anal. Calcd. for C16H15FN7O3S; C: % 49.86; H: % 3.92; N: % 25.44; S: % 8.32 Found: C: % 50.08; H: % 4.08; N: % 25.37; S: % 8.26. MS-ES (m/z): 386.40 (MH+).Botrytis cinerea, Colletotrichum acutatum, C. fragariae, C. gloeosporioides, P.viticola, P. obscurans and Fusarium oxysporum. The fungicide captan was used as an internal fungicide standard in all assays. Each fungus was challenged in duplicate in a dose-response format using test compounds where the final treatment concentrations were 0.3, 3.0 and 30.0 μM. Microtiter plates  were covered with a plastic lid and incubated in a growth chamber as described previously [P. obscurans and P.viticola where the data were recorded at 144 h. Means for percent inhibition of each fungus at each dose of test compound relative to the untreated positive growth controls were used to evaluate fungal growth. The SAS Proc ANOVA module [A standardized 96-well micro-dilution broth assay as developed by Wedge and Kuhajek  was usedeviously . Fungal A module  was usedA module . Aedes aegypti (L.) used in these studies were from a laboratory colony maintained since 1952 at the Mosquito and Fly Research Unit at Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL, USA. This colony is maintained using standard procedures [A. aegypti were conducted by using the system described by Pridgeon et al. [A. aegypti were added to each well of 24-well plates in a droplet of water using a disposable 22.5-cm Pasteur pipette by placing on an illuminated light box. One mL deionized water was added to each well using a Finnpipette stepper . Fifty µL of larval diet  and brewer’s yeast  were then added to each well. All chemicals to be tested were diluted in dimethyl sulfoxide (DMSO). Eleven microliters of the test chemical was added to the labeled wells, and in control treatments 11 µL of DMSO alone was added. After the treatment, the plates were swirled in clockwise and counter clockwise motions and front and back and side to side five times to ensure even mixing of the chemicals. Larval mortality was recorded 24- and 48-h after treatment. Larvae that showed no movement in the well after manual disturbance of water by a pipette tip were recorded as dead. A series of concentrations were used in each treatment to get a range of mortality. Treatments were replicated 15 times for each compound. LD50 values for larvicidal data were calculated by using the SAS Proc Probit module [ocedures . Bioassan et al. . In briet module . Controlin vitro Klun & Debboun (K & D) module bioassay system as developed by Klun et al. [et al. [N,N-diethyl-meta-toluamide (DEET) was obtained from Sigma Aldrich  and used as a positive control. Molecular biology grade ethanol was obtained from Fisher Scientific Chemical Co. . All synthesized compounds . Briefl [et al. , a feediompounds  were scrThe temperature of the solution in the reservoirs was maintained at 37.5 °C by continuously passing the warm water through the reservoir using a circulatory bath. The reservoirs were covered with a layer of collagen membrane. The test compounds were randomly applied to six 4 cm × 5 cm areas of organdy cloth and positioned over the membrane-covered CPDA-1+ATP solution with a separator placed between the treated cloth and the six-celled module. A six celled K & D module containing five females per cell was positioned over cloth treatments covering the six CPDA-1+ATP solution membrane wells, and trap doors were opened to expose the treatments to these females. The number of mosquitoes biting through cloth treatments in each cell was recorded after a 3 min exposure and mosquitoes were prodded back into the cells. These mosquitoes were then squashed to determine the number which has actually engorged the solution. A replicate consisted of six treatments: four test compounds, DEET (a standard biting deterrent) and ethanol treated cloth as solvent control. A set of five replications was conducted on different days using a new batch of females in each replication. Treatments were replicated 15 times.In vitro cytotoxicity was determined against a panel of human cancer cell lines  as well as noncancerous pig kidney epithelial (LLC-PK11) and monkey kidney fibroblast (VERO) cells. The assay was performed in 96-well tissue culture-treated microplates. Cells were seeded to the wells of the plate  and incubated for 24 hours. Samples were added and again incubated for 48 h. The number of viable cells was determined using the supravital dye Neutral Red according to the procedure described earlier [ earlier . DoxorubκB mediated transcription was determined in human chondrosarcoma (SW1353) cells by a reporter gene assay as described earlier [κB reporter luciferase plasmid construct at 160 V and one 70-ms pulse in a BTX Electro Square Porator T 820. Transfected cells were plated in 96-well plates (1 × 105 cells/well) and incubated for 24 h. After 24 h, cells were treated with test samples and after 30 min treated with PMA (70 ng/mL) for the activation of NF-κB and incubated for 8 h. After removing medium, cells were lysed by adding 40 µL of a 1:1 mixture of LucLite reagent and PBS containing 1 mM calcium and magnesium. Luciferase activity was measured as light output on a SpectraMax plate reader. Sp-1 was used as a control transcription factor to evaluate the toxicity of tested compounds in the same assay and parthenolide was used as a positive control as described earlier [Inhibition of NF- earlier ,37. In bPhomopsis species. Based on the biological activity evaluation these compounds may not be useful as therapeutic or pharmaceutical agents. However, selective compounds exhibiting antifungal activity could have a potential to be developed as agrochemicals, and further work should be performed to modify their structure with an aim to increase their activity. Phomopsis appeared to be the most sensitive fungal species to the tested compounds. Phomopsis cane and leaf spot (P. viticola) causes serious economic losses to the vine grape production in the United States of America and Europe, while P. obscurans causes Phomopsis leaf blight and fruit rot of strawberry. Compound 3d was the most active derivative among the tested compounds in a larvicidal assay against A. aegypti. The comparison of antifungal and insecticidal activities of thiourea and urea derivatives showed that thiourea derivatives bearing 2,6-dichlorophenyl, 2,4,6-trichlorophenyl, 4-nitophenyl, 4-trifluoromethylphenyl substituents were the active compounds. Thiourea compounds, 1b, 1c, 3a, 3d, 3e, and urea compound 4e show sufficient potential to warrant further modification to discover new antifungal and insecticidal molecules.The molecules described in this study demonstrated no cytotoxicity and no anti-inflammatory activity, but some of them showed good antifungal activity against


The novel thioctic-oligonucleotides were used for the functionalization of gold nanoparticles and the surface coverage and stability of the resulting thioctic-oligonucleotide gold nanoparticles were assessed. In all cases gold nanoparticles functionalized with thioctic-oligonucleotides had higher loadings and higher stability in the presence of thiols than gold nanoparticles prepared with commercially available thiol-oligonucleotides. Furthermore, the thioctic derivative carrying the triglycine linker is sensitive to cathepsin B present in endosomes. In this way this derivative may be interesting for the cellular delivery of therapeutic oligonucleotides as these results provides the basis for a potential endosomal escape.Oligonucleotide gold nanoparticle conjugates are being used as diagnostic tools and gene silencing experiments. Thiol-chemistry is mostly used to functionalize gold nanoparticles with oligonucleotides and to incorporate DNA or RNA molecules onto gold surfaces. However, the stability of such nucleic acid–gold nanoparticle conjugates in certain conditions may be a limitation due to premature break of the thiol-gold bonds followed by aggregation processes. Here, we describe a straightforward synthesis of oligonucleotides carrying thioctic acid moiety based on the use of several thioctic acid- During the past decade colloidal gold nanoparticles (AuNPs) have become attractive materials due to an increasing interest in their potential applications in biotechnology, molecular diagnostics and biomedicine ,3,4,5,6.There are numerous examples of biomolecules that have been immobilized onto AuNPs such as peptides , proteinThe conjugation of AuNps with biomolecules such as oligonucleotides, proteins and antibodies can be carried out through chemisorption of the biomolecule to the particle surface or electrostatic interactions between the biomolecule. The use of heterobifunctional ligands allows the functionalization of AuNps with biomolecules through covalent attachments, or specific biomacromolecular interactions such as streptavidin/biotin ,18,19,20Due to the strong affinity of thiolated species for gold surfaces and the ease of modification of nucleic acids with thiolated derivatives, thiolated oligonucleotides have been extensively used in the preparation of DNA-functionalized AuNps since the pioneering publication of Mirkin and co-workers in 1996 .Although such oligonucleotide-AuNP conjugates have been demonstrated to be very stable under most DNA detection conditions, limited stability has been observed in certain biological media due to a partial desorption of thiolated oligonucleotides by ligand exchange in the presence of some additives , mercaptoethanol or biogenic thiols) ,23,24.et al. functionalized AuNPs with thiolated systems like poly(L-lysine)-graft-poly(ethyleneglycol (PLL-g-PEG graft copolymers). The conjugates were stable under extreme conditions, making this polymer a promising ligand to use in biotechnological applications +) 294.1199 found 294.1192; m/z calcd for C24H46N2NaO6S4 ([2M + Na]+) 609.2138 found 609.2131.The compound was obtained as a mixture of two diastereoisomers. A solution of (±)-α-lipoic acid , -2-aminobutane-1,3-diol (L-threoninol)  were dissolved in anhydrous DMF (15 mL) under argon and allowed to react at room temperature for 3 h. The solvent was removed under reduced pressure and the residue was dissolved in toluene and concentrated to dryness (×3). Crude compounds 7a and 7c were dissolved in 100 mL of DCM and the organic phase was washed with 50 mL 10% NaHCO3 aqueous solution (×2) and 50 mL of a saturated NaCl aqueous solution. The organic layers were dried with anhydrous MgSO4 and the corresponding crudes were purified by flash chromatography on silica gel using as solvent pure DCM to DCM with 4% MeOH. Crude of compound 7b once dried with toluene was directly purified by flash chromatography on silica gel .The corresponding carboxylic acid (2.11 mmol) along with 4-nitrophenol  were dissolved in anhydrous pyridine (15 mL) under argon and the solution was cooled at 0 °C. Then, 524 mg (2.54 mmol) of 1,3-dicyclohexylcarbodiimide (DCC) were added under argon and the reaction was stirred at 0 °C for 30 min and at room temperature overnight. The precipitate was filtered out and the solvent was removed under reduced pressure. The residue was dissolved in toluene and concentrated to dryness under reduced pressure (×3). The resulting p-nitrophenyl esters −1. 1H-NMR : δH= 6.75 , 5.07 , 4.14 , 3.84 , 3.75 , 3.65 , 3.59 , 3.55–3.47 , 3.35 , 3.21 , 2.61–2.54 , 1.75 , 1.43 3-C), 1.19  ppm.13C-NMR : δC = 173.54 (CO), 172.72 (CO), 172.62 (CO), 77.43 (CH3)3-C-O), 70.62 (O-CH2-CH2-O), 70.30 (O-CH2-CH2-O), 70.29 (O-CH2-CH2-O), 69.63 (O-CH2-CH2-O), 69.59 (CH2-O), 69.49 (CH-O-), 64.93 (CH2-O), 64.86 (CH2-O), 55.63 (N-CH-CH2), 55.62 (CH2-N), 51.03 (CH2-N), 32.44 (CH2-CO), 32.16 (CH2-CO), 29.93 (CH2-CH2), 28.98 (CH2-CH2), 28.62 (CH3-C), 20.57 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for C23H46N3O9 ([M + H]+) 508.3229 found 508.3231; m/z calcd for C23H45N3NaO9 ([M + Na]+) 530.3048 found 530.3050.Yield: 738 mg (69%). IR (film): 3325, 2930, 2871, 1690, 1654, 1365, 1170, 1103 cm1H-NMR : δH = 8.09–8.03 , 7.27 , 6.97 , 4.61–4.54 , 3.85–3.81 , 3.72–3.70 , 3.63–3.58 , 3.56 , 3.44–3.40 HH(B)-CH)), 3.33–3.28 (B)H-CH)),1.36 3-C), 0.97  ppm.13C-NMR : δC = 170.58 (NH-CO), 169.67 (NH-CO), 169.37 (NH-CO), 156.50 (O-CO-NH), 78.83 (CH3-C), 64.88 (CH3-CH), 61.12 (OH-CH2-CH), 56.43 (NH-CH-CH), 43.99 (NH-CH2-CO), 42.77 (NH-CH2-CO), 42.70 (NH-CH2-CO), 28.87 (CH3-C), 20.73 (CH3-CH). HRMS (ESI+): m/z: calcd. for C15H29N4O7([M + H]+) 377.2036 found 377.2031; m/z: calcd. for C15H28N4NaO7 ([M + Na]+) 399.1856 found 399.1856.Yield: 612 mg (78%). −1. 1H-NMR : δH = 6.30 , 4.56 , 4.19 , 3.83 , 3.07 , 2.26 , 1.66 , 1.44 3-C), 1.33 , 1.21  ppm. 13C-NMR : δC = 174.31 (CO-NH), 77.43 (CH3-C-O), 69.43 (CH-O), 65.62 (CH2-O), 54.61 (N-CH-CH2), 40.63 (CH2-CO), 36.86, 29.94, 29.04, 28.62, 26.54, 25.57 , 20.82 (CH3-CH-) ppm. HRMS (ESI+): m/z calcd for C17H35N2O5 ([M + H]+) 347.2540 found 347.2543; m/z calcd for C34H68N4NaO10 ([2M + Na]+) 715.4828 found 715.4824.Yield: 701 mg (96%). IR (film): 3323, 2974, 2930, 2858, 1689, 1652, 1525, 1366, 1251, 1171 cmp-nitrophenol  in anhydrous pyridine (15 mL) was prepared under argon. Then, the solution was cooled to 0 °C and DCC  was added under argon. The reaction was stirred at 0 °C for 30 min and at room temperature overnight. The precipitate was filtered out and the solvent was removed under reduced pressure. The residue was dissolved in toluene and concentrated to dryness under reduced pressure (×3). Then the crude was dissolved in DCM (75 mL) and the organic phase was washed with 5% aqueous NaHCO3 (2 × 30 mL) and saturated aqueous NaCl (30 mL). The organic phase was dried (MgSO4) and concentrated to dryness under reduced pressure and then dried in a desiccator. The p-nitrophenyl ester 6 (yellow solid) was used in the next step without further purification. Then, the corresponding protected amines 7a–7c (1.30 mmol) were subjected to acidic conditions (DCM/TFA 10%) in order to obtain the respective free amines as trifluoroacetate salts. Each protected amine was dissolved in DCM (20mL) and TFA (2 mL) was added slowly at room temperature. Stirring was continued until TLC showed the consumption of all starting material , then the mixture was concentrated to dryness under reduced pressure. The residue was dissolved in toluene and concentrated to dryness under reduced pressure. This operation was repeated three times to remove traces of TFA to yield the TFA salt of the deprotected amino compound (colorless oil). The residue was dried in vacuo and used in the next step without further purification. Then, each deprotected amino compound was dissolved in anhydrous DMF (10 mL) and anhydrous DIEA  was added under argon. The solution was added dropwise under argon to a prepared solution  of the activated lipoic acid derivative. The mixture was allowed to react at room temperature and under argon overnight. The solvent was removed under reduced pressure and the residue was dissolved in toluene (3 × 10 mL) and concentrated to dryness. Crudes of compounds 8a and 8c were dissolved into 75 mL of dichloromethane and were washed with 5% NaHCO3 (3 × 30 mL) and with a saturated aqueous solution NaCl (30 mL). The organic layers were dried with anhydrous MgSO4 and the corresponding crudes were purified by flash chromatography on silica gel using as solvent pure DCM to DCM with 4% MeOH. Crude of 8b once dried with toluene was directly purified by flash chromatography on silica gel .Using the same experimental procedure as described above for the activation of carboxylic acids, a solution of (±)-α-lipoic acid  and −1. 1H-NMR : δH = 6.73 , 6.65 , 6.43 , 4.16 , 3.88 , 3.75 , 3.65 , 3.61 , 3.55 , 3.35 , 3.18 , 3.14 , 2.64 , 2.49 , 2.17 , 1.74 , 1.44 , 1.38  ppm.13C-NMR : δC = 173.72 (CO), 173.35 (CO), 172.51 (CO), 70.73 (O-CH2-CH2-O), 70.62 (O-CH2-CH2-O), 70.20 (O-CH2-CH2-O), 70.19 (O-CH2-CH2-O), 70.18 (CH2-O), 70.08 (CH2-O), 68.89 (CH-O), 64.54 (CH2-O), 56.75 (CH2-CO), 55.71 (CH2-CO), 41.86 (CH2-CH-N), 40.43 (CH2-S), 38.62 (CH2-N), 38.16 (CH2-N), 37.82 (CH2-CH-S), 36.67 (CH2-CO), 34.88 (S-CH2-CH2-CH-S) 37.85 (S-CH-CH2), 36.63 (-CH2-CH2-), 34.85 (-CH2-CH2-), 32.31 (NH-CH2-CH2-), 32.21 (-CH2-CH2-CH2-O), 32.09, 29.34, 29.05 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for C26H50N3O8S2 ([M + H]+) 596.3034, found 596.3038.The compound was obtained as a mixture of two diastereoisomers. Yield: 565 mg (73%). IR (film): 3320, 2928, 1652, 1539, 1456, 1216, 1130 cm1H-NMR : δH = 3.91–3.86 , 3.82–3.81 , 3.79 , 3.74–3.70 , 3.58–3.53 HH(B)-CH), 3.51–3.46 (B)H-CH), 3.13–2.97 , 2.41–2.33 HH(B)-CH2), 2.20 , 1.84–1.76 (B)H-CH2), 1.68–1.50 , 1.42–1.35 , 1.05 and 1.04  ppm. 13C-NMR : δC = 173.30 (NH-CO), 170.36 (NH-CO), 169.70 (NH-CO), 169.41 (NH-CO), 64.91 (CH3-CH), 61.13 (OH-CH2-CH), 56.79 (NH-CH-CH2-), 56.45 (S-CH-CH2), 46.40 (S-CH2-CH2), 43.96 (NH-CH2-CO), 42.74 (NH-CH2-CO), 42.72 (NH-CH2-CO), 38.77 (S-CH-CH2), 35.63 (CH2-CH2-CO-NH), 34.81 , 29.01 , 25.5 , 20.72 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for C18H33N4O6S2 ([M + H]+) 465.1842, found 465.1848; m/z calcd for C18H32N4NaO6S2 ([M + Na]+) 487.1661 found 487.1658.The compound was obtained as a mixture of two diastereoisomers. Yield: 459 mg (76%). −1. 1H-NMR : δH = 6.37 , 5.59 , 4.18  3.84 , 3.57 , 3.23 , 3.13 , 2.47 , 2.27 , 2.17 , 1.91 , 1.67 , 1.48 , 1.33 , 1.21  ppm.13C-NMR : δC = 174.45 (CONH), 173.44 (CONH), 69.69 (CH-O-), 65.72 (CH2-O), 56.62 (NH-CH-CH2-), 54.65 (NH-CH2), 45.85 (CH2-CO), 40.47 (CH2-CO), 39.55 (CH2-S), 38.72 (CH2-CH-S), 36.81, 34.72, 29.42, 29.07, 28.76, 28.40, 28.39, 26.37, 25.72 , 20.72 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for C20H39N2O4S2 ([M + H]+) 435.2346, found 435.2351; m/z calcd for C40H76N4NaO8S4 ([2M + Na]+) 869.4619 found 869.4615.The compound was obtained as a mixture of two diastereoisomers. Yield: 231 mg (41%). IR (film): 3304, 2927, 2855, 1646, 1557, 1540, 1457, 1201 cm7b. In this case DMT-Cl , DIEA (109 µL 0.60 mmol) and DMAP  were dissolved in dry pyridine (5 mL) and were added to the mixture. The solution was stirred at room temperature for 6 h and quenched with methanol. The solvent was evaporated under reduced pressure. The residue was dissolved in toluene (3 × 10 mL) and concentrated to dryness. Then, the resulting crude was dissolved in dichloromethane (100 mL) and the organic phase was washed with 5% aqueous NaHCO3 (50 mL) and with saturated aqueous NaCl (50 mL). The solvent was evaporated and the residue was purified by flash chromatography on silica gel. The column was packed using a 1% triethylamine (TEA) solution in DCM. Compounds 2, 9a and 9c were eluted with a gradient of MeOH from 0% to 4% in DCM and compound 9b was eluted with a gradient of MeOH from 0% to 10% in DCM.Each diol (0.80 mmol) was dried by evaporation of anhydrous acetonitrile (ACN) under reduced pressure (×2). Then to a solution of the corresponding diol in anhydrous pyridine (15 mL) at 0 °C, DIEA , 4,4'-dimethoxyltritylchloride (DMT-Cl)  and dimethylaminopyridine (DMAP)  were added. After 15 min, the mixture was allowed to reach room temperature. Then the reaction was stirred for an additional 24 h and finally was quenched with methanol except for diol −1. 1H-NMR : δH = 7.67 , 7.36 , 7.27 , 6.84 , 6.7 , 4.07 , 3.92 , 3.79 , 3.72 , 3.56 , 3.41 , 3.30 , 3.17 , 3.00 , 2.45 , 2.23 , 1.90 , 1.69 , 1.49 , 1.13  ppm.13C-NMR : δC = 173.25 (CO), 158.84 (aromC), 150.06 (aromC), 144.51 (aromC), 136.14 (aromC), 135.73 (aromC), 135.66 (aromC), 135.53 (CHarom), 130.01 (CHarom), 128.20 (CHarom), 128.18 (CHarom), 127.23 (CHarom), 123.92 (CHarom), 113.57 (CHarom), 87.03 (C-Carom), 68.94 (CH-O-), 65.55 (CH2-O), 56.57 (N-CH-CH2-), 55.63 (OCH3), 55.49 (OCH3), 53.54 (COCH2), 40.43 (CH2-S), 38.64 (CH2-CH-S), 36.73, 34.93, 29.16, 25.76 , 20.18 (CH3-CH) ppm. HRMS (ESI+): m/z calcd for C66H82N2NaO10S4 ([2M + Na]+) 1213.4745, found 1213.4745.The compound was obtained as a mixture of two diastereoisomers. Yield: 295 mg (62%). IR (film): 3176, 2927, 1646, 1508, 1249, 1175, 1033 cm−1. 1H-NMR : δH = 7.39 , 7.29 , 7.21 , 6.84 , 6.69 , 6.64 , 6.49 , 4.07 , 3.91 , 3.79 , 3.64 , 3.59–3.54, 3.34 , 3.09 , 2.52 , 2.14 , 1.85–1.49 , 1.12  ppm. 13C-NMR : δC = 172.83 (CO), 172.74 (CO), 172.06 (CO), 158.55 (aromC), 145.36 (aromC), 135.87 (aromC), 135.79 (aromC), 130.06 (CHarom), 129.89 (Carom), 127.82 (CHarom), 127.79 (CHarom), 126.43 (CHarom), 113.44 (CHarom), 86.54 (C-O), 70.57 (CH-O-), 70.49 (CH-O-), 70.05 (CH-O-), 69.98 (CH-O-), 69.88 (CH-O-), 68.26 (CH-O-), 64.8 (CH-O-), 56.44 (OCH3), 55.23 (OCH3), 54.06 (CH-N), 53.64 (CH-N), 41.93 (CH2-S), 38.46 (CH2-S), 37.83 (CH2-CH-S), 36.37(COCH2), 34.68 (N-CH2), 31.95 (N-CH2), 31.72, 29.62, 29.08, 28.96, 28.86, 25.43, 19.82, 17.33 , 17.31 (CH3CH) ppm. HRMS (ESI+): m/z calcd for C26H50N3O8S2 ([M + H-DMT]+) 596.3033, found 596.3032.The compound was obtained as a mixture of two diastereoisomers. Yield: 467 mg (65%). IR (film): 2929, 2662, 1653, 1608, 1545, 1508, 1463, 1393, 1249, 1178, 1131, 1095, 1033, 753 cm1H-NMR : δH = 8.13 , 8.07–8.04 , 7.50 , 7.37–7.17 , 6.86 , 4.54 , 3.95–3.90 , 3.89–3.82 , 3.78–3.69 , 3.61–3.53 , 3.18–3.02 HH(B)-CH and S-C2H-CH2), 2.86–2.82 (B)H-CH), 2.41–2.34 HH(B)-CH2), 2.12 , 1.88–1.79 (B)H-CH2), 1.68–1.59 , 1.54–1.46 , 1.36–1.31 , 0.94  ppm. 13C-NMR : δC = 173.25 and 173.16 (NH-CO)(*), 170.31, 170.29, 170.23 and 170.21 (NH-CO)(*), 169.64, 169.62, 169.57 and 169.55 (NH-CO)(*), 169.29, 169.23 and 169.21 (NH-CO)(*), 158.65 (Carom), 145.76 (Carom), 136.49 (Carom), 136.45 (Carom), 130.37 (CHarom), 128.45 (CHarom), 128.39 (CHarom), 127.22 (CHarom), 113.80 (CHarom), 85.82 (C-O), 65.44 and 65.33 (CH3-CH)(*), 63.39 (DMTO-CH2-CH), 56.79 (S-CH-CH2), 55.69 (O-CH3), 54.74, 54.72, 54.66 and 54.63 (NH-CH-CH)(*), 46.24 (S-CH2-CH2), 42.77 and 42.75 (NH-CH2-CO)(*), 42.66 and 42.63 (NH-CH2-CO)(*), 42.47 and 42.45 (NH-CH2-CO)(*), 38.77 (S-CH-CH2), 35.64 and 35.59 (CH2-CH2-CO-NH)(*), 34.81 , 29 01 , 25.51 , 20.87 and 20.81 (CH3-CH)(*) ppm. HRMS (ESI+): m/z calcd for C39H50N4NaO8S2 ([M + Na]+) 789.2968, found 789.2960; m/z calcd for C18H33N4O6S2 ([M-DMT+H]+) 465.1842, found 465.1845; m/z calcd for C18H32N4NaO6S2 ([M-DMT + Na]+) 487.1661 found 487.1661. (*)Multiple peaks observed due to rotamers of the amide bond.The compound was obtained as a mixture of two diastereoisomers. Yield: 325 mg (53%). −1. 1H-NMR : δH = 7.67 , 7.39 , 7.27 , 6.83 , 6.1 , 4.08 , 3.94 , 3.78 , 3.56 , 3.39 , 3.23 , 3.10 , 2.42 , 2.21 , 2.14 , 1.89 , 1.64 , 1.45 , 1.32 , 1.12  ppm. 13C-NMR : δC = 173.63 (CO), 172.91 (CO), 158.82 (aromC), 149.93 (aromC), 144.66 (aromC), 136.24 (aromC), 135.84 (aromC), 135.66 (aromC), 130.21 (CHarom), 130.19 (CHarom), 128.20 (CHarom), 128.18 (CHarom), 127.21 (CHarom), 124.05 (CHarom), 113.57 (CHarom), 86.86 (C-Carom), 68.55 (CH-O-), 65.37 (CH2-O), 56.73 (N-CH-CH2-), 55.46 (OCH3), 53.63 (COCH2), 40.47 (CH2-S), 39.65 (CH2-S), 38.63 (CH2-CH-S), 36.94 (COCH2), 36.69 (NCH2), 34.83, 29.76, 29.20, 29.19, 29.08, 26.83, 25.88, 25.68 , 20.25 (CH3CH) ppm. HRMS (ESI+): m/z calcd for C41H56N2NaO6S2 ([M + Na]+) 759.3472, found 759.3475; m/z calcd for C82H112N4NaO12S4 ([2M + Na]+) 1495.7052, found 1495.7032.The compound was obtained as a mixture of two diastereoisomers. Yield: 277 mg (47%). IR (film): 3019, 2925, 1652, 1058, 1215 cm2, and 9a–9c) were incorporated on a long-chain alkylamine-controlled pore glass support (LCAA-CPG) as described +) 796.3583, found 796.3577.Compound 12)TA_ITA_III(a)-(c) were synthesized on a DNA/RNA synthesizer (Applied Biosystems) on a 0.2 µmol scale using the corresponding modified CPG solid supports. The solid support 4 was used to synthesize the oligonucleotide 12)TA_I(T and the corresponding solid supports 11(a)–(c) for oligonucleotides 12)TA_III(a)(T–(c). The last DMT group was removed at the end of the synthesis. The resulting oligonucleotides were deprotected and cleaved from the solid supports with aqueous concentrated ammonia using three different conditions: (i) at room temperature for 4 h; (ii) at 55 °C for 1 h and (iii) at 55 °C overnight. The mixtures were filtered and ammonia solutions were concentrated to dryness. The resulting products were analyzed by HPLC. Solvent A: 5% ACN in 100 mM triethylammonium acetate (TEAA) (pH = 7) and solvent B: 70% ACN in 100 mM TEAA (pH = 7). Column: XBridgeTM OST C18 column; . Flow rate: 1 mL/min. Conditions: 10 min of linear gradient from 5% to 35% of B. The isolated products were analyzed by mass spectrometry (MALDI-TOF) .The TA terminated dodecathymidine oligonucleotides TA_I, TA_II and TA_III(a)-(c)  were synthesized a 1 µmol scale using the corresponding modified CPG solid supports. Oligonucleotide TA_II was synthesized using the solid support 4 once the commercially available spacer 18-O-dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-] phosphoramidite was incorporated. Once the synthesis of TA_I, TA_II and TA_III(a)–(c) was completed, each solid support was divided in two. The last DMT group was removed at the end of the synthesis. One half was deprotected and the other half was used for the synthesis of the corresponding fluorescently labeled oligonucleotides (F)TA_I, (F)TA_II and (F)TA_III(a)–(c). Coupling yields were >97%. TA_I, TA_II and their corresponding fluorescently labeled versions were treated with aqueous concentrated ammonia at 55 °C for 12 h to cleave the products from the supports and remove the Bz and iBu groups. The TA_III series were treated with aqueous concentrated ammonia at room temperature for 4 h to cleave the products from the supports and remove the Bz and dmf groups. The mixtures were filtered and ammonia solutions were concentrated to dryness. The resulting oligonucleotides were desalted and purified by HPLC using the same solvents described above. Column: XBridgeTM OST C18 semipreparative column . Flow rate: 3 mL/min. Conditions: 10 min of linear gradient from 0% to 30% of B for non fluorescently labeled oligonucleotides and 10 min. of linear gradient from 6% to 40% for the corresponding fluorescently labeled oligonucleotides. The pure fractions were combined and evaporated to dryness. The resulting oligomers were analyzed by mass spectrometry, UV/Vis spectroscopy and HPLC as well using the conditions and gradients described above but using an analytic column . Flow rate: 1 mL/min), . The yields obtained ranged from 33% to 49%.The thioctic acid terminated 21mer oligonucleotides 5'TA_I,  was synthesized on a 0.2 µmol scale employing LV200® polystyrene supports. Phosphoramidite 12 was used to incorporate the TA derivative. The DMT determination showed that the efficiency of coupling of the phosphoramidite was >90%. The last DMT group was removed at the end of the synthesis. The solid support was treated with aqueous concentrated ammonia at 55 °C for 12 h. and oligonucleotide 5'TA_I purification by HPLC was using the same protocol as described above. . The purified oligonucleotide was obtained in 51% yield.The thioctic acid terminated 21mer oligonucleotide ALK_DS (5'-CGG AGG TAC ATT CGA CTT GA-Y-3')  was synthesized on a 1 µmol scale using the 3'-Thiol modifier C3 S-S CPG following the procedure described previously. The last DMT group was removed at the end of the synthesis. As well, once the synthesis was completed, half of the solid support was used to incorporate the fluorescent dye at the 5'-termini to obtain the fluorescently labeled (F)ALK_DS analogue. Each solid support was treated with aqueous concentrated ammonia at 55 °C for 12 h. Work-up was similar as described above. The oligonucleotides were analyzed by HPLC using the conditions described above and characterized by mass spectrometry (MALDI-TOF) and UV/Vis spectroscopy  and used without further purification. The yields obtained were 74% for ALK_DS and 72% for (F)ALK_DS. The 3'-thiol-modified oligonucleotide ALK_SH and its fluorescently labeled version (F)ALK_SH were obtained from the corresponding disulfide oligonucleotides ALK_DS and (F)ALK_DS respectively by thiol deprotection [260) were dissolved in 300 µL of an aqueous solution 0.1 M TEAA (pH = 7). Then, 34 L of an aqueous solution 0.1 M of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) were added to the solution and allowed to react at 55 °C overnight. Under these conditions, the disulfide group was completely removed and the deprotected thiol oligonucleotides (ALK_SH and (F)ALK_SH, respectively) were purified with Sephadex G-25 (NAP-5 column) and used in the next step without further purification.The 3'-disulfide-modified oligonucleotide otection ,45. The TA_I, TA_II, TA_III(a)–(c)), with 3'-alkylthiolated-modified oligonucleotides (ALK_DS and ALK_SH) and with their respectively fluorescently labeled versions as well. To prepare the conjugates, 5 nmol of desalted oligonucleotide were dissolved in 1 mL of MilliQ water and were added to a 1 mL of the gold nanoparticles solution. After 24 h, the solution was brought to a final concentration of 10 mM sodium phosphate (pH = 7.2). The mixture was then allowed to equilibrate for 30 min before bringing the concentration to 0.15 M NaCl over a 7 h 30 min period in a stepwise manner (0.05 M NaCl increments each 2 h 30 min). The solutions were sonicated for 10 s. before each addition to keep the particles dispersed during the salting procedure. The salting process was followed by an incubation period of 48 h at room temperature. Finally, to remove all unbound oligonucleotides, the solution was centrifuged at 13,200 rpm  for 30 min. This procedure was repeated three times. As we wanted to compare stabilities of the different DNA-AuNP conjugates, we dispersed the reddish residue in 500 µL of 0.15 M NaCl, 10 mM sodium phosphate (pH = 7.2), 0.01% NaN3 solution. Then, each solution was analyzed by UV-visible absorption spectroscopy and the concentration of gold nanoparticle conjugates was adjusted to 6.9–7.1 nM by adding the required volume of buffer. The conjugates obtained with the fluorescent oligonucleotides were directly resuspended in 1 mL of a 0.15 M NaCl, 10 mM sodium phosphate (pH = 7.2), 0.01% NaN3 solution. The extinction coefficient used was the same as that used for unmodified nanoparticles . Each solution was then stored in the fridge (4 °C) prior to use.Colloidal gold nanoparticles (9.7 nm diameter) were functionalized with the TA-modified oligonucleotides –(c) gold nanoparticle conjugates was assessed by treating the AuNP-conjugates with a DTT solution based on protocols described in the literature [ALK_DS and ALK_SH respectively) were studied as well for comparison purposes. Then, to a 500 µL of each solution were added 12.5 µL of a 4 M DTT solution . Prior to the addition of DTT, the solutions were allowed to equilibrate at 40 °C and were sonicated for 20 s to keep the particles dispersed. To give an indication of the durability of the oligonucleotide conjugates, absorbance at 675 nm was monitored against time at 40 °C. For AuNPs conjugated with TA-terminated oligonucleotides absorbance was recorded at 1 min intervals and for AuNPs conjugated with alkylthiolated oligonucleotides absorbance was recorded at 1 s intervals. The measures were performed with 0.5 cm quartz cuvettes. The cells used had a ground hole at the top to adapt a PTFE stopper to provide a more suitable seal to reduce evaporation of the sample. Then, the half-life  was calculated for each conjugate by fitting the data obtained to a Boltzmann equation using the Origin 8.0 software.The stability of the different TA-terminated oligonucleotides terature ,37. The Ex = 495 nm and λEm = 510 − 550 nm). For each oligonucleotide two different oligonucleotide-AuNP conjugates were prepared and two different samples were prepared for each conjugate. Fluorescent measurements were done in duplicate. Standard curves were prepared with known concentrations of the fluorescently labeled TA-terminated oligonucleotide (F)TA_I using the same buffer, salt and DTT concentrations. The average number of oligonucleotides per particle was obtained by dividing the measured oligonucleotide molar concentration by the original oligonucleotide-AuNP conjugate concentration. The average area that each oligonucleotide occupies on the nanoparticle surface (footprint) was calculated by dividing the calculated surface area (nm2) by the average number of oligonucleotides per particle.The number of oligonucleotides loaded onto gold nanoparticles was analyzed following procedures previously described ,47. FirsTA_I, TA_II, TA_III(a)–(c)) and 5 mM of reduced glutathione. The reaction buffer was 100 mM NaCl, 40 mM sodium acetate, 1 mM EDTA and 1% triton-100x, pH = 5. The reaction mixture was left at 37 °C, with soft stirring during 12, 24, 48, 72 and 168 h respectively. The reaction was quenched by heating the samples at 70 °C during 20 min. The resulting products were analyzed by HPLC. Solvent A: 5% ACN in 100 mM TEAA (pH = 7) and solvent B: 70% ACN in 100 mM TEAA (pH = 7). Column: Nucleosil 120‒10 C18; (250 × 4 mm). Flow rate: 1 mL/min. Conditions: 20 min of linear gradient from 10% to 40% of B. The isolated products were analyzed by mass spectrometry.Cathepsin B was dissolved in this buffer: 50 mM NaCl, 10 mM sodium phosphate pH = 6, 1 mM EDTA and 30% glycerol and stored at −20 °C until needed. In order to find the optimal conditions for the enzymatic reaction, different assays were carried out at different pH, reaction times and also changing the proportions of the agents in the reaction. The optimal conditions were as follow: 13.8 µL (0.02 unt/µL) of Cathepsin B were added to 200 µL of a solution containing 0.25 OD’s of different modified oligonucleotides ) or a TA-oligo(ethylene glycol) ligand (AuNP-TA_III(a)) showed a remarkable increase in probe stability probably due to the disposal of the spacers, covering and protecting the gold nanoparticle surface. This increase in the stability of the system may be also due to the position of the amide group, which may induce favorable interactions between the neighboring alkyl or oligo(ethylene glycol) chains. Finally, the thioctic derivative carrying the triglycine linker is shown to be the most sensitive to cathepsin B. These preliminary results may open the door to an interesting application for these oligonucleotide derivatives in cellular delivery applications.


N-substituted 2-pyrrolidone aldehyde 7 in satisfactory yields. Of these, 11 and 12 had the highest potency and, compared to sunitinib, showed: (1) significant increase in anti-proliferation of various cancer cells with a favorable selective index (SI); (2) higher inhibitory potency against both VEGFR-2 and PDGFRβ. The molecular modeling results showed that, in terms of VEGFR-2 binding, the synthesized products had a similar binding mode to sunitinib but with tighter interaction.Signaling pathways of VEGFs and PDGFs are crucial in tumor angiogenesis, which is essential in solid tumor progression and metastasis. This study reports our strategy for designing and synthesizing a series of novel 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives as potential multi-target tyrosine kinase receptor inhibitors. The target compounds were obtained by condensation of 5-substituted oxindoles with  To avoid apoptosis, necrosis and/or provide paths for metastasis to survive, tumor cells secrete several pro-angiogenesis factors inducing new blood vessel formation near tumor tissues . Th. Th5, Juruitless ,27,28,293 was hydrolyzed into carboxylic acid 4 with 1 N sodium hydroxide in 96% yield. Performing CDI-mediated lactamization of the resulting zwitterionic compound 4 in dry tetrahydrofuran under a nitrogen atmosphere proved effective for synthesizing the desired fused pyrrole 5 in 56% yield. Compound 5 was then subjected to hydrolytic decarboxylation with sulfuric acid under a reflux condition to provide 6. Without isolation and purification, crude 6 was smoothly formylated at C(2) by treatment with a Vilsmeier reagent in dichloromethane to furnish 7 in 91% yield. Condensation of 7 with 5-substitued oxindoles afforded target molecules 8–12 in 43–56% yields.Our laboratory finally found a way to circumvent the predicament in a stepwise manner. First, ester All target compounds were isolated as free bases. In most cases, crystallization or column chromatography was required for purification. The structures of most synthetic intermediates and products were established by spectroscopy and high-resolution mass spectrometry.8–12 against human colon cancer line HCT116, human non-small cell lung cancer cells NCI-H460, and human renal cell carcinoma 786-O. For comparison, sunitinib was employed as a positive control. The preliminary results showed that all synthetized compounds had significant inhibiting effects on HCT-116 cancer cell proliferation, but for 8 which bears C(5)-H. While the IC50 values of 9 (C(5)-F) and 10 (C(5)-Cl) were approximately equal to that of sunitinib (IC50 3.42 ± 0.57 μM), the values for 11 (C(5)-Br) (IC50 1.05 ± 0.18 M) and 12 (C(5)-I) (IC50 0.42 ± 0.16 μM) were much better. As to the anti-proliferation effect on the NCI-H460 cells, 8–10 showed poor inhibitory activity (IC50 > 10 μM); 11 was approximately equal in potency to sunitinib . Notably, 12 revealed prominent inhibitory activity (IC50 = 2.95 μM). As to the anti-proliferation effect on the 786-O cells, both 8 and 9 were once again void of activity (IC50 > 10 μM). The potencies of compounds 10–12 (IC50 approximately 7 μM) were comparable to that of sunitinib.8–12 and sunitinib. In 12 had high selectivity against HCT116 and NCI-H460 cancer cell lines.Potential anticancer drug candidates should have greater selectivity for tumor cells compared with normal cells. Therefore, human normal fibroblast cells Detroit 551 were used for evaluation so as to indicate the selective indexes of 8–12 were resulted from the acute cytotoxicity of indicated compounds, this study performed a WST-8 assay to assess their effects of acute cellular toxicity on Detroit 551, HCT116, NCI-H460 and 786-O cell lines. 8–12, together with sunitinib, were nearly free from acute cellular toxicity on all the tested cell lines.To exclude the possibility that the inhibitory responses of sunitinib and compounds 9 (C(5)-F) had approximately equal or slightly better potency in the anti-proliferation activity against HCT116 to that of sunitinib (IC50 2.94 μM vs. 3.42 μM). The corresponding 10 (C(5)-Cl), 11, (C(5)-Br) and 12 (C(5)-I) showed a similar trend. For example, compound 12 was strikingly potent, with approximately 8-fold stronger than sunitinib and 10-fold stronger than 8 (C(5)-H).In the (2-oxoindolin-3-ylidene)methylpyrrole system, the C(5) halogen substituent is known to have critical roles in the cytotoxicity and anti-proliferation of cancer cells ,19,30,31Apoptosis of human colon cancer cells, including HCT116 and HT29 cells, can be induced by VEGFR-2 inhibitors, such as regorafenib, crizotinib, sorafenib, and sunitinib, via p53 upregulated modulator of apoptosis (PUMA) ,33,34,358 and 11 for 24 h exhibited an increase phase of polyploid  of oxindole has a strong influence on both anti-proliferation activity and selectivity. The C(5)-halogen substituents of the title compounds seemed to be advantageous in general and had marked effect: the bulkier halogen the substituent is, the stronger anti-proliferation activity it has.In summary, the experiments in this study suggest that the structural skeleton constructed by fusion of (2-oxoindolin-3-ylidene)methylpyrrole with 8-12, compared to sunitinib. The IC50 values for 8, 10, 11, and, 12 against VEGFR-2 were 4- to 6-fold higher, and their IC50 values against PDGFRβ were approximately 2- to 4-fold higher and 11 was the most potent. Comparing with sunitinib, The results once again displayed the similar trend that different C(5) substitutions had significant influence on the biochemical activities against VEGFR-2 and PDGFRβ.8) and other C(5)-halogen analogs  showed that compound 9 (C(5)-F) had much lower inhibitory activity against both VEGFR-2 and PDGFRβ and, comparing to sunitinib, only little increase in inhibitory activity against VEGFR-2 but decrease against PDGFRβ. These results markedly differed from the results reported in experiments using 2-piperidinone fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives pyrrol-2-yl)methylene)indolin-2-one (8). Yield: 50%, pale orange solids. Mp: 167–168 °C. UV λmax (MeOH), nm (logɛ): 390 (4.12), IR (ATR), cm−1: 3172, 1651, 1568. 1H-NMR  δ, ppm: 7.75 , 7.73 , 7.18 , 7.03 , 6.98 , 4.59 , 3.55 , 2.65 , 2.55 2), 2.47 , 0.99 2), 13C-NMR  δ, ppm: 171.5, 170.0, 149.6, 139.3, 133.6, 126.3, 126.0, 125.2, 123.4, 120.9, 119.6, 118.6, 111.4, 51.9, 48.6, 48.2, 39.6, 9.9, 8.8. HRMS m/z (ESI): calcd., 379.2129 [M + H]+, found, 379.2133 [M + H]+.(Z)-3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolopyrrol-2-yl)methylene)-5-fluoroindolin-2-one (9). Yield: 56%, orange solids. Mp: 245–246 °C, UV λmax (MeOH), nm (logɛ): 291 (4.33). IR (ATR), cm−1: 3534, 3400, 1668, 1572 cm−1. 1H-NMR  δ, ppm: 7.62 , 7.38 , 6.96–6.90 , 4.59 , 3.97 , 3.49 , 3.37 2), 2.47 , 1.31 2). 13C-NMR  δ, ppm: 169.8, 166.1, 158.6, 148.3, 135.5, 132.4, 127.1, 125.9, 125.0, 121.0, 117.1, 113.7, 110.7, 107.0, 49.0, 46.7, 46.6, 37.3, 10.0, 8.7. HRMS m/z (ESI): calcd., 397.2034 [M + H]+; found, 397.2038 [M + H]+.(Z)-5-Chloro-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolopyrrol-2-yl)methylene)indolin-2-one (10). Yield: 54%, orange solids. Mp: 252–253 °C, UV λmax (MeOH), nm (logɛ): 393 (4.37). IR (ATR), cm−1: 3162, 1634, 1582 cm−1. 1H-NMR  δ, ppm: 7.64 , 7.62 , 7.16 , 6.96 , 4.59 , 3.97 , 3.49 , 3.37 2), 2.47 , 1.31 2). 13C-NMR  δ, ppm: 171.2, 169.7, 149.9, 137.7, 133.6, 128.6, 128.5, 127.9, 127.4, 126.6, 121.1, 119.6, 117.2, 112.5, 51.8, 48.6, 48.1, 39.5, 9.9, 8.7. HRMS m/z (ESI): calcd., 413.1744 [M + H]+; found, 413.1743 [M + H]+.(Z)-5-Bromo-3-((5-(2-(diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolopyrrol-2-yl)methylene)indolin-2-one (11). Yield: 56%, orange solids. Mp: 266–267 °C. UV λmax (MeOH), nm (logɛ): 394 (4.56), IR (ATR), cm−1: 3161, 1634, 1581 cm−1. 1H-NMR  δ, ppm: 7.74 , 7.59 , 7.30 , 6.90 , 4.55 , 3.96 , 3.50 , 3.38 2), 2.46 , 1.33 2). 13C-NMR  δ, ppm: 171.0, 169.6, 149.8, 138.0, 133.6, 130.7, 128.4, 127.3, 126.4, 122.4, 121.1, 117.0, 115.8, 112.9, 51.8, 48.5, 48.1, 39.5, 9.9, 8.7. HRMS m/z (ESI): calcd., 457.1239 [M + H]+; found, 457.1236 [M + H]+.(Z)-3-((5-(2-(Diethylamino)ethyl)-3-methyl-4-oxo-1,4,5,6-tetrahydropyrrolopyrrol-2-yl)methylene)-5-iodoindolin-2-one (12). Yield: 43%, brown solids, Mp: 267–268 °C. UV λmax (MeOH), nm (logɛ): 394 (4.36), IR (ATR), cm−1: 3174, 1689, 1584 cm−1. 1H-NMR  δ, ppm: 7.94 , 7.64 , 7.50 , 6.81 , 4.59 , 3.97 , 3.49 , 3.37 2), 2.47 , 1.31 2). 13C-NMR  δ, ppm: 171.6, 169.8, 149.9, 149.9, 149.8, 133.5, 127.5, 126.5, 121.1, 116.1, 114.6, 112.2, 112.1, 106.7, 51.6, 49.4, 48.2, 39.5, 9.9, 8.8. HRMS m/z (ESI): 505.1100 [M + H]+; found, 505.1094 [M + H]+.2 atmosphere.The HCT116  and Detroit 551  were maintained in DMEM  containing 10% FBS . NCI-H460 (BCRC 60373) and 786-O (BCRC 60243) were maintained in RPMI 1640  containing 10% FBS . They were incubated at 37 °C in a 5% COThe cells incubated as above were plated at a density of 2000 cells/well (cancer cells) ,41,42 or8–12 was determined by Cell-Counting-Kit-8  assay on HCT116, NCI-460, 786-O and Detroit 551 with the manufacturer’s protocol. Cells were seeded at 5000 cells/well on a 96-well plate for 24 h. The indicated compounds in different concentration (100 μL) were added to cells. Post 6 h, old medium was aspirated, and cells were washed three times with PBS. WST-8  (10 µL) was added to each well, and the absorbance of the plate was recorded at 450 nm on a microplate spectrophotometer .The acute cytotoxicity effect of sunitinib and compounds 8–10, 1.0 μM for 11 and 12) were added to cells. After incubation for 24 h, 100,000 cells were harvested and centrifuged for 5 min at 400 g at room temperature, washed once with PBS (50 μM) and resuspended with 50 μL of lysis buffer  containing 10 μg/mL of 2-(4-amidinophenyl)-1H-indole-6-carboxamidine . The cells were incubated at 37 °C for 5 min, 50 μL of stabilization buffer  was added to the solution, and the cellular fluorescence was measured with an NC-3000 image cytometer using NC-SlideA8 . The NC-3000 software  was used for image acquisition, image analysis and quantification and data visualization.Cell cycle profiles of HCT116 cells were determined with an NC-3000 image cytometer  in accordance with manufacturer’s protocol. Briefly, cells were seeded at 200,000 cells/well on a 6-well plate for 24 h. 2 mL of indicated compounds . The medium renewals with or without agent in cultures were conducted every 3 days. After 11 days HUVECs were fixed and stained with anti-CD31 antibody in line with the manufacturer’s protocols. The tube formation area were measured using Angiogenesis Image Analyzer  with suaramin (70 μM) as positive control.Angiogenesis Kit  was used in accordance with the manufacturer’s protocols. Briefly, HUVECs and fibroblasts were co-cultured in 24-well plates with basal medium or solvent control medium  or solvent control medium containing http://www.reactionbiology.com) HotSpot assay platform was used to determine the inhibitory activity of compounds 8–12 and sunitinib against VEGFR-2, PDGFR and aurora A, which was measured by quantifying the amount of 33P incorporated into the substrate in the presence of the test compound [33P ATP  was added to make a final concentration of 10.0 μM. Reactions were stood at room temperature for 120 min, and spot the reactions onto P81 ion exchange filter paper . Unbound phosphate was removed by extensive washing of filters in 0.1% phosphoric acid. Kinase activity data was reported as the percent remaining kinase activity in test compounds compared to solvent control (DMSO).The Reaction Biology Corporation methylpyrrole derivatives 8–12 were successfully prepared and examined for their anti-tumor activities. Several of them significantly improved inhibitory activity against VEGFR-2 and PDGFRβ over sunitinib. Compared to sunitinib, all our synthetic compounds showed a similar but tighter binding mode with VEGFR-2. The nature of C(5) halogens and the rigid conformation of the scaffold obviously were important determinants of both enzymatic and cellular anti-tumor activities. Among the products obtained, 11 (C(5)-Br) and 12 (C(5)-I) consistently displayed the most potent and favorable selective indexes and might be promising to be used as novel anti-angiogenic agents in the targeted therapy of cancer. The results gleaned from this investigation have important potential applications in the development of new anticancer drugs.A series of novel


These new compounds were elucidated byspectroscopic analysis, including 1D, 2D NMR, and HR-ESI-MS spectra. Additionally, all the isolates were evaluated for cytotoxicity against three human cancer cell lines  with the IC50 ranging from 2.25 to 49.44 µM in vitro with treatment 48 h and showed low cytotoxicity in human normal liver L-02 cells (IC50 > 50 µM). Compound 5 showed the most significant cytotoxic activity with the IC50 value of 2.25, 4.72, and 5.33 µM in 48 h, respectively.Five new cucurbitane-typetriterpenoid glycosides, named Xuedanoside F–J ( Hemsleya pengxianensis W.J. Chang, a native plant andwidely distributed in the south-west provinces of China, belongs to the genus of Hemsleya in Cucurbitaceae family + 687.3725in the HR-ESI-MS . Its IR data displayed absorptions for hydroxyl (3565–3340 cm-1) and carbonyl (1651 and 1687 cm-1) groups. Acid hydrolysis of 1 with HCl gave D-glucose as the constituent unit, which was tested by GC analysis. D-Glucose (tR = 25.5 min) was detected by comparison with authentic monosaccharide. The configuration of the glycosidic bond was β on the basis of the coupling constant of the anomeric proton at δH 4.80 . The 1H-NMR data (δH 1.26 (s), 1.21 (s), 1.44 (s), 1.91 (s), 1.34 (s), 1.28 (s), and 1.45 (s); four oxygenated methines at δH4.08 (m), 4.20 (m), 3.40 , 5.06 (m), and 5.19 ; and two olefinic proton signals at δH 5.68 (m) and 6.89 . The 13C APT NMR data , 6 methylenes, 9 methines and 8 quaternary carbons , of which 30 were assigned to the aglycon part, and the remaining 6 were ascribed to the sugar moiety. All assignments of proton signals achieved by 1H and 13C correlations in the HSQC spectrum. The IR and 1H and 13C-NMR spectra data identified that compound 1 is an oxygenated cucurbitane triterpenoid glycoside derivative + 671.3768 . An analysis of the 1H and 13C-NMR data  in 13C-NMR data of 2. Additionally, in the HMBC spectrum (H 2.54) to H-3 (δH 3.70), from H-3 (δH 3.70) to H3-29 (δH 1.13) confirmed the relative configurations of methyl groups and other protons in the tetracyclic rings. The coupling constant of J = 12.0 Hz further confirmed the antiperiplanar relationship between H-16 and H-17.Taken together with the analysis of NOE spectra between the two compounds, compound 2 was elucidated as 3β, 20β-dihydroxycucurbita-16α-23α-epoxy-5, 24(E)-diene-11-one-26-O-d- glucopyranoside and it was named Xuedanoside G.Compound NMR data  displayespectrum , correlaspectrum  correlat3 was determined to be a molecular formula of C36H58O8, as established with its HR-ESI-MS data at m/z [M + Na]+ 641.4021 . Comparing its 1H and 13C-NMR data  in 3 instead of the sugar group at C-26 (δC 67.1) in 2. Similarly, itlackedthe a hydroxyl group at C-20 (δC 36.4) and the loss of an ether bond between H-16 and C-23 in 3. In the HMBC spectrum (H 3.62 (H-3) to anomeric carbon at δC 107.8 (C-1′), and the signal for C-3 indicated a significant downfield shift to δ 87.7 (+11.7 ppm). Similarly, in comparison to 2, the signals for C-16, C-20, and C-23 revealed the powerful upfield shift to δ 28.5 (–42.7 ppm), δ 36.4 (–36.6 ppm), and δ 25.1 (–45.9 ppm), respectively, while a hydroxyl group and an ether bond were absent. Compound 3 was eventually determined to be 26-hydroxycucurbita-5, 24(E)-diene-11-one-3-O-β-d-glucopyranoside, and it was named Xuedanoside H.Compound NMR data  with thaspectrum , the sug4 had a molecular formula C36H60O8  on the basis of ion peak at m/z [M + Na]+ 643.4178 in HR-ESI-MS. The 1D NMR signals  in 3, where it was substituted for a hydroxy group at δC 78.6 in compound 4. This difference was verified by 2D NMR spectra (H 4.18 to C-8 (δC 43.9), C-10 (δC 37.3), and C-13 (δC 47.8) revealed that the hydroxyl group was located at C-11. Within the NOESY spectrum, from H3-19 to H-11 and from H-11 to H3-18 suggested that H-11 was β-oriented, and the structure of compound 4 was established as 11α, 26-dihydroxycucurbita-5, 24(E)-diene-3-O-β-d-glucopyranoside, and it was named Xuedanoside I.Compound  signals  were tig spectra . In the 5 possesses a molecular formula of C36H56O10on the basis of HR-ESI-MS at m/z [M + Na]+ 671.3779  and NMR spectra. Its 1H and 13C-NMR  and a hydroxyl group at C-27 (δC 58.9) in 5, respectively. In the HMBC spectrum (H 6.31) with the downfield carbon C-7 (δC 199.6) (compared with C-7 in 3) implied acarbonyl group at C-7. Furthermore, in comparison to 3, the signal for C-27 revealed a powerful downfield shift to δ 58.9 (+44.4 ppm), while a hydroxyl group was added at C-27. The form of 5 was confirmed by spectra of 1H-1H COSY, HSQC, HMBC, and NOESY .Furthermore, the cytotoxicity of all isolates was assessed with three human tumor cell lines  according to the MTT procedure, and doxorubicin was used as the positive control. The results of cytotoxicity were displayed in Hemsleya, which are the effective constituents and show potent anti-tumor cytotoxicity. As a result, we evaluated all the isolates for their cytotoxic activity against three human cancer cell lines. Compared to the doxorubicin positive control group, all compounds showed moderate cytotoxicity due to their 24-ethylenic linkage substituent + 687.3725; 1H and 13C-NMR spectra data, see Xuedanoside G (2). C36H56O10, c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1675, 1689, 3569-3254; UV λmax (MeOH) nm (log ε): 210.5 (5.68); HR-ESI-MS m/z [M + Na]+ 671.3768; 1H and 13C-NMR spectra data, see Xuedanoside H (3). C36H58O8, c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1170, 1661, 1723, 3633-3354; UV λmax (MeOH) nm (log ε): 202.8 (5.10); HR-ESI-MS m/z [M + Na]+ 641.4021; 1H and 13C-NMR spectra data, see Xuedanoside I (4). C36H60O8, c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1145, 1640, 3658-3385; UV λmax (MeOH) nm (log ε): 202.4 (5.08); HR-ESI-MS m/z [M + Na]+ 643.4178 ; 1H and 13C-NMR spectra data, see Xuedanoside J (5). C36H56O10, c 0.1, MeOH), white amorphous powder; IR (KBr) νmax cm-1: 1195, 1651, 1680, 3650-3460; UV λmax (MeOH) nm (log ε): 209.6 (5.60); HR-ESI-MS m/z 671.3779 [M + Na]+ ; 1H and 13C-NMR spectra data, see 1–5 were accomplished by the procedure described previously [Acid hydrolysis of Compounds eviously ,15.1–5 isolated from H. penxianensis were screened for cytotoxicity against three human cancer cell lines, including Hela, breast cancer MCF-7, lung cancer A-549, and the normal liver L-02 cells. This used the MTT method as described in previously published literature [5 cells/mL in 96-well microtiter plate, were cultured in DMEM medium with 10% fetal bovine serum at 37 °C in a 5% CO2 incubator overnight. Then, the cells were treated with the test compounds at five concentrations in triplicate. After 24 h and 48 h of treatment, the cells were incubated with 10 μL of MTT (4 mg/mL) for another 4 h. The residual liquid was removed, and 200 µL DMSO was added. The absorbance was tested using a microplate reader at a wavelength of 570 nm.Compounds terature ,17. Brie