The cytotoxic potentiality of novel compounds 4–16 was assessed by the MTT assay method on colon, lung and breast tumour cell lines. Compound 5 gave the most significant specificity anticancer activity with safety response on normal cell lines. In vitro enzyme assay and several apoptotic parameters were examined to elucidate the mode of action of compound 5. Molecular docking studies also were simulated to put insight and give better understanding to its structural features.A series of  It is generally known that basic reaction media enhance racemization. However, under the reaction conditions employed in this work, especially short reaction times and temperatures below (°C), only negligible racemization was observed.Melting points were determined in an “Electro Thermal” Digital melting point apparatus  (model: IA9100). Elemental analysis was found within the acceptable limits of the calculated values . Infrared spectra (KBr) were recorded on a Nexus 670 FTIR Nicolet, Fourier Transform infrared spectrometer . Proton nuclear magnetic resonance (1H-NMR) spectra were run in [d6] DMSO on Jeol 270 MHz or 500 MHz instruments . Chemical shifts d are given in ppm. Mass spectra were run on a MAT Finnigan SSQ 7000 spectrometer , using the electron impact technique (EI). Analytical thin layer chromatography (TLC) was performed on silica gel aluminium sheets, 60 F254 (E. Merck**). The following solvent systems (by volume) were used as eluents for the development of the plates: S: chloroform–methanol–acetic acid (85:10:5); S1: S-petroleum ether (b.p. 40–602.1.1.2) prepared according to previously reported methodsThe titled compound  was added to a DCM solution of the isophthaloyl chloride (2) . And then the reacted mixture was stirred for , then for (24 h at room temperature). It was then washed with , followed by water again and then dried over sodium sulphate. Next, the solvent was evaporated and the obtained compound was solidified by petroleum ether (boiling point 40–60 °C). The solid was filtered off, dissolved in methanol (MeOH) and precipitated by petroleum ether to give compound 3.A dichloromethane (DCM) solution of free bis-(azanediyl)) dipropionate (3):Dimethyl 2, 2'-: (KBr): νIR (cm = 3330 (NH stretching), 3060 , 2950 , 1744 , 1675, and 1610 .1H-NMR : δ = 8.90, 8.85 , 8.50–8.30 , 4.35–4.25 , 3.70–3.50 , 1.50–1.44 . MS : m/z (%) = 336 , 222 (3.06), 78 (100), 63. Molecular formula (molecular weight): C16H20N2O6 (336.3). Calculated analysis: C, 57.14; H, 5.99; N, 8.33; Found: C, 57.10; H, 5.90; N, 8.8.2.1.3.3) (1 mmol), hydrazine hydrate 99%  was added. The mixture was then refluxed for 3 h, the solvent was evaporated and the obtained residue was triturated with diethylether, filtered off, and precipitated from MeOH/diethylether to give 4.To a stirred methanolic solution (50 mL) of the corresponding amino acid methyl ester : (KBr):νIR (cm = 3293 (2NH2), 3131 , 2972 , 1650 (4CO), 1528 (2 C = N). 1H-NMR : δ = 1.36–1.49 , 4.24 , 4.57, 5.38 , 7.24–8.25 , 8.74, 9.70 . MS : m/z (%) = 336 , 331 (10), 323 (25), 316 (23), 310 (35), 304 (69), 300 (25), 299 (15), 285 (24), 281 (19), 277 (11), 266 (21), 258 (31), 124 (13), 93 (12), 77 (18), 42 (100). Molecular formula (molecular weight): C14H20N6O4 (336.3). Calculated analysis: C, 49.99; H, 5.99; N, 24.99; Found: C, 49.92; H, 5.97; N, 24.88.bis (1-hydrazinyl-1-oxopropan-2-yl) isophthalamide (4)  in DMF (10 ml) was added isatin and/or chloroisatin (0.2 mol) refluxed for 4 h, cold poured on to crushed ice. The product formed was filtered off and crystallized from ethanol to give the target compounds.To a solution of compound 1-(1-oxo-1-(-2-(2-oxoindolin-3-ylidene) hydrazinyl) propan-2-yl)-N3-(1-oxo-1-(-2-(2-oxoindolin-3-ylidene) hydrazinyl) propan-2-yl) isophthalamide (N5)Yield: 75%; melting point: 271–273 °C, −1): (KBr): νIR , 2923 , 1698, 1647 (4CO), 1524 (2 C = N). 1H-NMR : δ = 1.49–1.51 , 4.64, , 6.92–8.49 , 10.00 , 11.25, 13.50 . MS : m/z (%) = 594 , 531 (11), 487 (13), 419 (16), 389 (17), 371 (27), 362 (13), 346 (11), 317 (22), 308 (13), 288 (21), 160 (98), 143 (16), 132 (43), 129 (39), 118 (20), 103 (100), 88 (23), 76 (74), 62 (28). Molecular formula (molecular weight): C30H26N8O6 (594.5). Calculated analysis: C, 60.60; H, 4.41; N, 18.85; Found: C, 60.64; H, 4.45; N, 18.82.1-(1-(-2-(5-chloro-2-oxoindolin-3-ylidene)hydrazinyl)-1-oxopropan-2-yl)-N3-(1-(-2-(5-chloro-2-oxoindolin-3-ylidene) hydrazinyl)-1-oxopropan-2-yl)isophthalamide (N6)Yield: 70%; melting point: 271–273 °C, −1): (KBr): νIR , 2922 , 1698 (2CO) 1647 , 1521 (2 C = N). 1H-NMR : δ = 1.49–1.53 , 4.65 , 6.95–8.59 , 10.30 , 11.35, 13.45 . MS : m/z (%) = 663 , 637 (19), 586 (24), 486 (21), 446 (83), 433 (31), 418 (48), 413 (16), 401 (21), 387 (24), 378 (58), 194 (52), 142 (13), 132 (14), 116 (18), 109 (20), 85 (100).Molecular formula (molecular weight): C30H24Cl2N8O6: (663.47). Calculated analysis: C, 54.31; H, 3.65; Cl, 10.69; N, 16.89; Found: C, 54.36; H, 3.59; Cl, 10.61; N, 16.79.1, N3-bis (1-hydrazinyl-1-oxopropan-2-yl) isophthalamide  and carbon disulphide (6 ml) in pyridine (10 ml) and DMF (5 ml) was heated under reflux on water bath for 8 h. After cooling, the solvent was evaporated under reduced pressure and residue was triturated with an ice-water mixture and neutralized with diluted HCl. the solid precipitate formed was filtered off and recrystallized from ethanol to afford 7 as pale yellow crystals.A mixture of N1,N3-bis  ethyl) isophthalamide (N7)Yield: 80%; melting point: 132–134 °C, −1): (KBr): νIR , 2919 , 1635 (2CO), 1520 (2 C = N), 1114, 1057 (2 C = S). 1H-NMR : δ = 1.17–1.41 , 3.35–4.14 , 7.71–8.39 , 873, 9.25 , 7.00 . MS : m/z (%) = 420 , 407 (10), 393 (10), 392 (16), 97 (19), 70 (100), 57 (59). Molecular formula (molecular weight): C16H16N6O4S2: (420.07). Calculated analysis: C, 45.71; H, 3.84; N, 19.99; S, 15.25; Found: C, 45.75; H, 3.82; N, 19.89; S, 15.28.2.1.6.1,N3-bis(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide  in absolute ethanol (20 ml) containing glacial acetic acid, the respective monosaccharide in water (2 ml). The mixture was heated under reflux for 3 h and the resulting solution was concentrated and left to cool to room temperature. The formed precipitate was filtered off, washed with water and ethanol, then dried and recrystallized from ethanol to afford 8 and 9 as white crystals.A mixture of N1,N3-bis -2, 3, 4, 5-tetrahydroxypentylidene) hydrazinyl) propan-2-yl) isophthalamide (N8)Yield: 60%; melting point: <300 °C, −1): (KBr):νIR (cm = 3427 (OH), 3365, 3307 (2NH), 2922 , 1639 (4CO), 1527 (2 C = N). 1H-NMR : δ = 1.17–1.41 , 3.35 , 4.11 , 4.45 , 4.59 , 5.30 , 7.62–8.52 ,839, 8.80 . Molecular formula (molecular weight): C25H37N5O12: (599.59). Calculated analysis: C, 50.08; H, 6.22; N, 11.69; Found: C, 50.14; H, 6.27; N, 11.54.1,N3-bis -2, 3, 4, 5-tetrahydroxypentylidene) hydrazinyl) propan-2-yl) isophthalamide (N9):Yield: 60%; melting point: < 300 °C, −1): (KBr):νIR (cm = 3423 (OH),, 2922 , 1645 (4CO), 1531 (2 C = N). 1H-NMR : δ = 1.18–1.42 ,3.78 , 4.13 , 4.45–4.52 , 4.86 , 5.31 , 7.62–8.52 , 872,8.83 . Molecular formula (molecular weight): C25H37N5O12: (599.59). Calculated analysis: C, 50.08; H, 6.22; N, 11.69; Found: C, 50.18; H, 6.24; N, 11.66.bis hydrazide compound  in absolute ethanol (20 ml) containing glacial acetic acid (5 ml) was added different aromatic aldehydes (0.02 mol) the reaction mixture was refluxed for 6–8 h. The formed precipitate was filtered off and crystallized form acetic acid to afford compounds 10–13.A solution of 1,N3-bis-(1-(-2-(4-chlorobenzylidene) hydrazinyl)-1-oxopropan-2-yl) isophthalamide (N10):Yield: 80%; melting point: 296–298 °C, −1): (KBr): νIR , 2983 , 1639 (2CO), 1537 (2 C = N). 1H-NMR : δ = 1.34–1.45 , 4.55, 5.34 , 7.23–8.25 , 8.50 , 10.00 N = CH), 8.72, 8.81 . 11.31, 11.40 . MS : m/z (%) = 581 , 575 (11), 523 (26), 519 (10), 494 (14), 488 (28), 460 (17), 425 (14), 434 (31), 421 (40), 418 (15), 401 (24), 390 (39), 368 (100), 365 (21), 350 (31), 348 (15), 336 (16), 293 (12), 133 (10), 95 (20).Molecular formula (molecular weight): C28H26Cl2N2O4 (581.14). Calculated analysis: C, 57.84; H, 4.51; Cl, 12.19; N, 14.45; Found: C, 57.83; H, 4.52; Cl, 12.09; N, 14.47.1,N3-bis-(1-(-2-(4-fluorobenzylidene) hydrazinyl)-1-oxopropan-2-yl) isophthalamide (N11)Yield: 85%; melting point: 285–287 °C, −1): (KBr): νIR , 2981 , 1645 (2CO), 1537 (2 C = N). 1H-NMR : δ = 1.37–1.47 , 4.56, 5.32 , 7.27–8.27 , 8.55 , 8.74, 8.82 . 11.30, 11.42 . MS : m/z (%) = 548 , 516 (20.5), 487 (10), 477 (37.9), 463 (11.2), 433 (42), 419 (13), 406 (53), 394 (17), 394 (17), 376 (24), 365 (75), 339 (100), 321 (59), 147 (19), 74 (63). Molecular formula (molecular weight): C28H26F2N6O4 (548.55). Calculated analysis: C, 61.31; H, 4.78; F; 6.93, N, 15.32; Found: C, 61.32; H, 4.76; F; 6.95, N, 15.34.1,N3-bis-(1-(-2-(4-methoxybenzylidene) hydrazinyl)-1-oxopropan-2-yl) isophthalamide (N12)Yield: 80%; melting point: 273–275 °C, −1): (KBr): νIR , 2981 , 1641 (2CO), 1533 (2 C = N). 1H-NMR : δ = 1.43–1.47 , 3.08 , 4.56, 5.34 , 7.00–8.21 , 8.45 , 8.69, 8.78 , 11.30, 11.46 . MS : m/z (%) = 572 , 544 (28), 504 (25), 496 (19), 487 (22), 462 (16), 431 (36), 419 (25), 395 (33), 389 (22), 347 (24), 331 (24), 312 (100), 295 (34), 84 (64), 59 (77). Molecular formula (molecular weight): C30H32N6O6 (572.62). Calculated analysis: C, 62.93; H, 5.63; N, 14.93; Found: C, 62.95; H, 5.64; N, 14.98.1,N3-bis- hydrazinyl)-1-oxopropan-2-yl) isophthalamide (N13)Yield: 85%; melting point: 277–279 °C, −1): (KBr): νIR , 2985 , 1647 (2CO), 1536 (2 C = N). 1H-NMR : δ = 1.43–1.46 , 3.08 , 4.55, 5.35 , 7.00–8.42 , 8.50 , 8.71,8.74 ,11.32, 11.467 . MS : m/z (%) = 632 , 607 (19), 592 (10), 582 (16), 54713), 558 (21), 547 (9), 493 (13), 488 (32), 462 (35), 390 (24), 318 (16), 300 (100), 206 (17), 76 (10), 40 (18). Molecular formula (molecular weight): C32H32N6O8 (632.26). Calculated analysis: C, 60.75; H, 5.74; N, 13.28; Found: C, 60.74; H, 5.76; N, 13.38.2.1.8.10–12 (0.01 mol) in dry benzene (20 ml), and then refluxed for 7–9 h. After completion of the reaction, excess solvent was evaporated under reduced pressure and the residue was neutralized with cold dilute sodium bicarbonate solution, the formed product was filtered off, washed with water and then re-crystallized from methanol to give compounds 14–16.Thioglycolic acid (0.01 mol) was added to a well stirred solution of Schiff’s bases 1,N3-bis-(1-(2-(4-chlorophenyl)-4-oxothiazolidin-3-ylamino)-1-oxopropan-2-yl) isophthalamide (N14)Yield: 65%; melting point: 218–220 °C, −1): (KBr): νIR , 2925 , 1724, 1677 (4CO). 1H-NMR : δ = 1.15, 1.29 , 3.70, 3.83 , 4.42 , 5.80, 5.93 , 7.15–7.97 , 8.33, 8.75 . 10.29 . MS : m/z (%) = 731 , 730 , 385 (1.4), 355 (9), 314 (2.5), 286 (2.5), 245 (11), 229 (7), 212 (22), 202 (14), 174 (23), 166 (13), 158 (15), 148 (18), 130 (17), 111 (30), 102 (18), 97 (100), 71 (53), 67 (16). Molecular formula (molecular weight): C32H30Cl2N6O6S2 (729.65). Calculated analysis: C, 52.68; H, 4.14; Cl, 52.68 N, 11.52; S, 8.79; Found: C, 52.69; H, 4.16; Cl, 52.69 N, 11.57; S, 8.76.1,N3-bis-(1-(2-(4-fluorophenyl)-4-oxothiazolidin-3-ylamino)-1-oxopropan-2-yl) isophthalamide (N15)Yield: 64%; melting point: 168–170 °C, −1): (KBr): νIR , 2921 , 1721, 1679 (4CO). 1H-NMR : δ = 1.13, 1.28 , 3.69, 3.85 , 4.42 , 5.80, 5.93 , 7.15–7.96 , 8.31,8.73 ,10.27 .13C-NMR : δ = 19.32 (2 CH3), 29.77 (2S-CH2), 49.75 (2 CH-CH3), 61.60 (N-CH), 115.69–158.00 (Ar-C), 166.03, 168.00, 172.14 (3 C = O).MS : m/z (%) = 696 , 601 (4), 578 (8), 461 (5.6), 414 (21), 382 (10), 366 (13), 351 (28), 333 (28), 324 (23), 294 (39), 285 (51), 281 (95), 267 (19), 256 (23), 130 (4), 80 (10), 78 (22), 56 (18), 40 (100). Molecular formula (molecular weight): C32H30N6O6S2 (696.16). Calculated analysis: C, 55.16; H, 4.34; F, 5.45; N, 12.06; S, 9.20; Found: C, 55.19; H, 4.36; F, 5.49 N, 12.16; S, 9.25.1,N3-bis -(1-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-ylamino)-1-oxopropan-2-yl) isophthalamideN(16)Yield: 65%; melting point: 242–244 °C, −1): (KBr): νIR , 2923 , 1725.1678 (4CO). 1H-NMR : δ = 1.16, 1.30 , 3.72, 3.76 , 3.84–3.88 , 4.46, 4.52 , 5.72, 5.79  7.33–8.23 , 8.061, 8.68 . 1023 . MS : m/z (%) = 721 , 721 , 710 (19), 687 (11), 659 (12), 650 (27), 627 (20), 593 (16), 589 (45), 564 (34), 535 (41), 503 (52), 477 (100), 465 (16), 422 (25), 381 (30), 353 (12), 336 (17), 236 (11), 97 (25), 73 (29). Molecular formula (molecular weight): C34H36N6O8S2 (720.20). Calculated analysis: C, 56.65; H, 5.03; N, 11.66; S, 8.90; Found: C, 56.62; H, 5.05; N, 11.68; S, 8.92.2.2.2.2.1.50 values were performed using SPSS computer program .Human colon carcinoma (HCT-116 cell line), human lung carcinoma (A-549), human breast carcinoma (MCF-7 cell line) and human skin normal cell line (BJ-1) were obtained from Karolinska Center, Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden. IC3 cells per well in case of MCF-7, 20 × 103 cells/well in case of HCT-116 cell lines using 96-well plates at 37 °C. After 48-h incubation, the medium was aspirated and 40 μl MTT salt (2.5 mg/ml) were added and further incubated for 4 h. About 200 μl 10% sodium dodecyl sulphate (SDS) was added. The absorbance was measured at 595 nm.The procedure was done in laminar air flow cabinet bio safety class II level. Culturing and sub culturing were carried out according to Thabrew et al.2.2.2.in vitro enzyme inhibition assessment for compound 5 was performed at confirmatory diagnostic unit, Vacsera, Egypt. The screening achieved profiling of the compound 5 against a range of four protein kinases  by ELISA assay method using staurosporine as a standard according to the previously reported methodsThe 2.2.3.Bax protein levels were evaluated according to Onur et al. BCL-2 in the samples and standards were estimated according to Barbareschi et al. Human p53 present in the sample or standard binds to antibodies adsorbed to the microwells. A biotin-conjugated is added. After incubation and dispense of unbound biotin-conjugated streptavidin HRP is added. The reaction is terminated by addition of acid and absorbance is measured at 450 nm2.2.4.The micro ELISA plate provided in this kit pre-coated with CASP7-specific antibody. A biotinylated CASP7 antibody and Avidin–HRP conjugate was added. Aspire the excess components. The substrate solution was added wells that contain CASP7, biotinylated detection antibody and Avidin–HRP conjugate will appear blue in colour. The colour turns yellow followed the addition of sulphuric acid solution. The optical density (OD) was measured at a wavelength of 450 nm ± 2 nm2.2.5.MCF-7 cells were inoculated at concentration 1.2–1.8 × 10,000 cells/well using DMEM (supplemented with 10% FBS and 1% penicillin–streptomycin). Tested compound added for 18–24 h before the enzyme assay for Tubulin. Avidin conjugated to HRP is added to each microplate well and incubated. After TMB substrate solution is added, wells that contain TUBb, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in colour. Sulphuric acid solution added to terminate enzymatic reaction. Colour change is measured spectrophotometrically at a wavelength of 450 nm ± 10 nm2.2.6.,g, to separate intact chromatin from DNA fragments. The amount of DNA was measured by using a diphenylamine reagent. The optical density is measured at 600 nm.DNA fragmentation of the cells was assayed, as previously described2.3.5 was carried out using molecular operating environment  software5.The molecular modelling of the compound 3.3.1.,l-alanine and isophthalic acid. The novel candidates 3–16 were synthesized based on N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide (4) which may be expected to possess promising anticancer properties. Accordingly, a rational design, synthesis and structural characterization of N1, N3-bis (1-hydrazinyl-1-oxopropan-2-yl) isophthalamide, were synthesized via synthetic peptide coupling methods, (in solution). The compound N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide (4) was synthesized by the reaction of isophthalic acid (1) with thionyl chloride, to give isophthaloyl chloride (2), while compound 2 was then coupled with a free l-Alanine methyl ester to give dimethyl 2,2'-) dipropionate (3). IR of compound 3 showed absorption bands at 3330 cm−1 due to NH in addition to absorptions of carbonyl groups at 1744 cm−1 (C = O ester). 1H-NMR of ester 3 revealed two signals in the region δ 3.7–3.5 of 2CH3(CH3 ester) in addition to the D2O exchangeable signals of amidic. Hydrazinolysis of dimethyl 2,2'-) dipropionate (3) with hydrazine hydrate led to the corresponding hydrazide 4 in 75% yield  isophthalamide (4) with isatin or chloroisatin took place through refluxing in DMF to produced hydrazide substituted candidates 5 and 6 (1H-NMR of hydrazide candidates 5 and 6 in highly yield (75% and 70%), respectively. showed the characteristic signals of NH, pyrrole ring, at 10.00 and 10.30, respectively, as well as signals of NH in the range δ 13.50–11.25. The mass spectrum of hydrazide derivatives 5 and 6 displayed a characteristic peaks equal to its molecular weights at m/z = 594 and 663, respectively. On the contrary, hydrazide 4 was reacted with carbon disulphide in pyridine and DMF which produced oxadiazolederivative 7  isophthalamide (4) was reacted with the respective monosaccharide in ethanol containing glacial acetic acid which produced sugar hydrazone derivatives 8 and 9  and dupletes at δ 1.17–1.41 .The condensation of the  5 and 6 . 1H-NMR vative 7 . The str 8 and 9 . 1H-NMR 4 with 4-chlorobenzaldehyde or 4-fluorobenzaldehyde took place through refluxing in ethanol containing glacial acetic acid. This produced substituted hydrazides 10 and 11  and base beak at m/z = (368 and 339), respectively. On the contrary, 1,NN3-bis (1-hydrazinyl-1-oxopropan-2-yl) isophthalamide (4) was reacted with 4-Anisaldehyde or Veratraldehyde to give the corresponding hydrazide derivatives 12 and 13, respectively , 4.42 (2CH of α-l-Ala), and the mass spectrum of 14 and 15 exhibited a characteristic peaks equal to its molecular weights at m/z = (731 and 696) and base beak at m/z = (97 and 40), respectively.The compounds ectively . 1H-NMR 3.2.3.2.1.50) and doxorubicin was used as the reference drug as shown in 5, 7, 12 and 14 showed moderate to excellent % of inhibition ranging from 72.5 to 98.3 towards the three tested cancer cell lines.Cancer is ranked as the second cause global lifelessness; therefore, there is a need for developing an antitumor candidate with minimal side effects to increase the efficacy of chemotherapy. In this study thirteen compounds were preliminary screened for their cytotoxicity utilizing three human carcinoma cell lines, namely human colon carcinoma (HCT-116), lung carcinoma (A-549) and human breast carcinoma (MCF-7) . The com50 data, it was observed that only the compounds 5 and 14 exhibited higher potency against all tested cell lines in comparison with the standard drug.Concerning IC5, 7, 12 and 14 were also screened for their cytotoxic effects on human normal skin fibroblasts cell line BJ-1 at 100 μM to test their safety on normal cells . Cyclization with 2-oxoindoline moiety gave the most potent derivative 5 against the three tested cell lines  in comparison with the reference drug, doxorubicin . On the one hand, substitution of 2-oxoindoline with chlorine atom at p-5 in compound 6 displayed drastic decrease in the activity towards all tested cell lines . On the other hand, cyclization with 1,3,4-oxodiazole scaffold in compound 7 increased the sensitivity only towards MCF-7 cell line (% inhibition = 77.5 and IC50 = 0.073 ± 2.91 µM). A dramatic drop in the anticancer effect was detected upon attachment of isophthalamide with 2,3,4,5-tetrahydroxypentyl sugars in 8 and 9. Insertion of phenyl ring substituted at p-4 with electron withdrawing groups as Cl or F in compounds 10 and 11, or with electron donating group as methoxy in compound 12 showed variable and noticeable increase in the cytotoxic activity, while substitution with two methoxy groups at p-3,4 led to reduction in the activity due to steric hindrance in compound 13. The 4-oxothiazolidine derivative linked at p-2 to 4-chlorophenyl 14, exhibited excellent anticancer potency  and it displayed higher percentage of inhibition at 100 μM than the 4-fluorophenyl and 4-methoxyphenyl congeners 15 and 16.Depending on the previous results, it was noticed that the open chain isophthalamide derivative 3.2.3.in vitro cytotoxicity studies of the newly synthesized isophthalamide derivatives, the highly active compound 5 was selected for evaluation of its inhibitory activities versus a panel of four different kinases: epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor-2 (VEGFR-2), cyclin-dependent kinase-2 (CDK-2) and c-kit (receptor tyrosine kinase type III) using staurosporine as multitarget inhibitor.Based on observing the results of 50 values depicted in 5 potently inhibited EGFR rather than the reference . In contrast, compound 5 seemed to have weak inhibitory activities against VEGFR-2, CDK-2 and c-Kit kinases with IC50 values ranging from 2.85 ± 0.04 to 6.24 ± 1.21 μM.From IC3.2.4.5, which has the highest activity on all tested cancer cells including breast carcinoma cells, was considered the safest one among other promising compounds on normal skin cells with weak effect of cytotoxicity (32.6%). Therefore, compound 5 was chosen to elucidate its possible apoptotic mode of action on breast carcinoma cells through investigation of its effect on Bax, BCL-2, Bax/BCL2 ratio, P53, CASP-7, Tubulin polymerization (TubB) and % of DNA fragmentation.According to our results, compound 5 at IC50 for 24 h resulted in significant up-expression of p53 and Bax, with consequent down-expression in the level of BCL-2 compared to the control  and the mitochondrial (intrinsic) pathwayslchicine . In conc3.3.in vitro assay and proved that compound 5 is the most active and promising chemotherapeutic agent. Accordingly, the inhibitory evaluation of compound 5 was carried out against different human proteins which expressed in most of tumour cells especially breast carcinoma namely, EGFR, VEGFR-2, CDK-2 and c-Kit, to validate and specify the mechanism of action or proteins responsible for the anticancer activity.Cytotoxic activity of all synthesized derivatives has been realized by using 5 through investigating its binding mode and interaction with the key amino acids (hot spots) in the active site of the EGFR. Docking simulations were done using Molecular Operating Environment software 10.2008 (MOE), Chemical Computing Group Inc., Montreal, Quebec, Canada5 in the same binding site.Consequently, molecular docking was performed with the aim of explaining the promising EGFR inhibitory activity of compound Met769 as erlotinib, through the N atom and the carbonyl oxygen localized in its oxoindoline moiety . Moreover, the other 2-oxoindoline moiety exhibited a third hydrogen bond acceptor between its carbonyl oxygen and the sidechain of Arg817 (distance: 2.74 Å). Additionally, a hydrophobic interaction (arene–arene interaction) with EGFR binding site was introduced through the central phenyl moiety of compound 5 and Phe699.The obtained results are pictured in 5 and EGFR, as presence of two 2-oxoindoline moieties in its structure let it to get good fitting with the binding site.This helped us to find an explanation for the excellent binding affinity between compound 4.4–16 were synthesized using solution phase method in peptide synthesis. All novel compounds were evaluated for their cytotoxic activity upon HCT-116, A-549 and MCF-7 cell lines using MTT assay. Compound 5 exhibited the most significant suppression of the proliferation of the cancer cells. Furthermore, compound 5 was tested against four kinases, and showed significant inhibition of EGFR enzyme. Antitumor potency of compound 5 was further confirmed by studying its effect upon different apoptotic parameters in breast cancer cells. The docking studies also support the results concluded from the enzyme assay and the anti-proliferation screening. It was clear that our ligand 5 that bearing 2-oxoindoline moiety may be considered as a suitable lead for further development of anticancer drugs in future.In the current study, novel isophthalamide based derivatives incorporated to different hetercycles


The sugar-annulated PYOX ligand formed a stable metal complex with Pd(II), which was fully characterized by NMR spectroscopy and X-ray crystallography. NMR and X-ray analysis revealed a change of the conformation in the sugar moiety upon complexation with the palladium(II) species. Both glycosylated ligands resulted in high asymmetric induction (up to 98% ee) upon application as chiral ligands in the Pd-catalyzed allylic alkylation of rac-1,3-diphenylallyl acetate with dimethyl malonate (Tsuji-Trost reaction). Both ligands provided mainly the (R)-enantiomer of the alkylation product.Two novel carbohydrate-derived pyridyl (PYOX)- and cyclopropyl (CYBOX)-substituted oxazoline ligands were prepared from  During the past decades, the use of carbohydrates as chiral auxiliaries, chiral reagents, organocatalysts and, most notably, as chiral ligands for metal-catalyzed stereoselective reactions has become a powerful tool for organic chemists ,2. This glucoPHOX ligand -catalyzed allylic substitution of various symmetrically and non-symmetrically 1,3-disubstituted 2-propenyl acetates with dimethyl malonate 2 and 11 mol % of the chiral ligand. The C1-symmetrical PYOX ligand 2 showed preparative yields for (R)-9 of up to 92% with an enantiomeric excess of 47% (R)-9 in a somewhat lower yield (53%–71%) but increased the stereoselectivity of the reaction  but yielded (R)-9 at only 9%  and enantioselectivity ]2 and 11 mol % of PYOX ligand 2, we decided to conduct further studies with CYBOX ligand 5. However, cyclopropane-based ligand 5 led to full conversions in 24 h and afforded nearly exclusively linear substituted product 11l (11l:11b ≥ 99:1).As very low conversions (5%–7%) were found within 24 h of reaction time with 5 mol % of malonodinitril (DCTB) as the matrix. Elemental analyses were performed on a HEKAtech Euro 3000 CHN  analyzer. Optical rotations were measured with a Perkin-Elmer Polarimeter 341  in a 10 cm cuvette at 20 °C. Melting points were determined with a Büchi Melting Point M-560 apparatus . Reactions were monitored by TLC on Polygram Sil G/UV silica gel plates from Machery & Nagel . Detection of spots was effected by charring with H2SO4 (5% in EtOH), staining by spraying the plates with an alkaline aqueous solution of potassium permanganate or by inspection of the TLC plates under UV light. Preparative chromatography was performed on silica gel (0.032–0.063 mm) from Machery & Nagel (Macherey & Nagel GmbH & Co. KG) with different mixtures of solvents as eluent. Racemic samples of 9 and 11 were synthesized according to the general procedure described in 9 was determined by chiral HPLC analysis on a Sykam S 1121 chromatograph  equipped with a Reprosil Chiral-NR column : tR = 6.8 min for (R)-9, tR = 8.6 min for (S)-9. All yields given below are isolated yields determined after purification of the product either by silica gel column chromatography or crystallization and were not optimized unless noted otherwise.All solvents were dried according to standard methods, distilled and stored over molecular sieves 3 Å under an atmosphere of nitrogen prior to their use. Petroleum ether (PE) refers to the fraction boiling in the 60–90 °C range. All non-aqueous reactions were performed in oven-dried glassware under an atmosphere of Nd-glucopyranose1,3,4,6-Tetra-O-acetyl-2-deoxy-2-picolinamido- (1): To an ice cold solution of d-glucosamine hydrochloride  and Hünig’s base   in dry DMF (15 mL) was added in the following order: HOBt (1-hydroxybenzotriazole) , picolinic acid  and HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) . The resulting mixture was stirred for 1 h at 0 °C followed by rt until TLC  showed complete consumption of starting material (14 h). The solution was evaporated in vacuo, re-dissolved in pyridine (10 mL) and treated with Ac2O  at 0 °C. The resulting mixture was stirred at rt until TLC (eluent EtOAc) indicated complete O-acetylation (12 h). The solution was poured into ice/water mixture (50 mL) and extracted twice with CH2Cl2 (20 mL). The combined organic phases were washed with 0.5 M citric acid, sat. aqueous NaHCO3-sol., H2O and brine, dried over MgSO4, filtered and concentrated. Chromatography of the residue  afforded 1  as an amorphous foam . Spectroscopic data were in accordance with literature values -2-oxazoline2-(2-Pyridyl)-: HBr  was added dropwise to an icecold solution of 1  in CH2Cl2 (20 mL). After complete addition the mixture was allowed to warm to rt and stirring at this temperature was continued for 2 h until all starting material was consumed . The solution was hydrolyzed by addition of ice/water and the phases were separated. The organic layer was washed with sat. aqueous NaHCO3-sol., H2O and brine, dried over Na2SO4, filtered and concentrated. The residue was re-dissolved in MeCN (20 mL) and Bu4NBr  and NaHCO3  were added. After stirring for additional 12 h at rt the suspension was evaporated in vacuo and the yellowish residue re-dissolved in EtOAc (15 mL), washed with H2O and brine, dried over Na2SO4, filtered and concentrated. Chromatography of the residue  afforded 2  as a colorless foam. 3). Rf = 0.43 . 1H-NMR : δ = 8.72–8.71 , 8.04 , 7.82–7.80 , 7.44–7.41 , 6.23 , 5.41 , 4.97–4.94 , 4.42–4.39 , 4.15–4.13 , 3.70–3.66 , 2.10, 2.03, 1.98 . 13C-NMR : δ = 170.6, 169.6, 169.2 (C=O), 164.2 (OCN), 150.2, 145.4, 136.9, 126.4, 124.3 (pyridine-C), 100.4 (C-1), 70.3 (C-3), 68.4 (C-4), 67.8 (C-5), 65.4 (C-2), 63.2 (C-6), 21.0, 20.8, 20.8 (CH3). HRMS-ESI: m/z calcd for C18H20N2O8Na [M + Na]+: 415.111187; found: 415.111056. Anal. calcd for C18H20N2O8 (392.4): C, 55.10; H, 5.14; N, 7.14; found: C, 55.06; H, 5.45; N, 6.67.d-glucopyranos-2-yl)-cyclopropane-1,1-dicarboxamideN,N′-Bis: To an ice cold solution of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-d-glucopyranose hydrochloride (3) +: 811.23796; found: 811.23806. Anal. calcd for C33H44N2O20 (788.7): C, 50.25; H, 5.62; N, 3.55; found: C, 50.28; H, 5.72; N, 3.37.ride (3)  -2-oxazoline]2,2′-Cyclopropylidenbis+: 691.19570; found: 691.19644. Anal. calcd for C29H36N2O16 (668.6): C, 52.10; H, 5.43; N, 4.19; found: C, 52.35; H, 5.66; N, 4.10.d-glucopyranoso)--2-oxazoline]palladium(II)Dichloro+. Anal. calcd for C18H20Cl2N2O8Pd (569.7): C, 37.95; H, 3.54; N, 4.92; found: C, 37.66; H, 3.45; N, 4.93. Crystal Data for C18H20Cl2N2O8Pd (M = 569.66 g/mol): orthorombic, space group P212121 (no. 19), a = 8.6185(6) Å, b = 14.6441(10) Å, c = 16.5033(11) Å, α = β = γ = 90°, V = 2082.9(2) Å3, Z = 4, T = 100(2) K, μ(MoKα) = 1.197 mm−1, Dcalc = 1.817 g/cm3, 44885 reflections measured (2.666° ≤ 2θ ≤ 30.526°), 6370 unique (Rint = 0.0287) which were used in all calculations. The final R1 was 0.0144 (I > 2σ(I)) and wR2 was 0.0362 .6 for X-ray analysis were obtained by slow diffusion of Et2O into a sat. solution of 6 in MeCN. X-ray data were collected on a Bruker SMART APEX II DUO  diffractometer using a graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). Corrections for absorption effects were applied using SADABS 2  in dry CH2Cl2 (1 mL) were stirred for 30 min at rt. To this catalyst solution allyl acetate , dimethyl malonate (8) , N,O-bis(trimethylsilyl)acetamide  and KOAc  were added and stirring was continued for 12 h at rt. The reaction mixture was diluted with Et2O (3 mL) and NH4Cl (2 mL) and the aqueous layer was extracted with Et2O (3 × 5 mL). The combined organic phases were washed with water and brine, dried over Na2SO4 and concentrated. Chromatography of the residue  afforded the alkylated product 9 or 11, respectively, as a colorless oil. Spectroscopic data were in accordance with literature [General Procedure for the addition of dimethyl malonate  and CYBOX (5) from d-glucosamine hydrochloride and readily available 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-d-glucopyranose hydrochloride in two steps, respectively. PYOX ligand 2 formed a stable Pd(II) complex upon reaction with PdCl2(cod) and was fully characterized by means of NMR, CHN, MS and crystal structure analysis. NMR and X-ray studies revealed a conformational change of the carbohydrate scaffold in ligand 2 upon complexation with Pd(II). Both ligands were used as pre-catalysts in the Pd-catalyzed allylic substitution of rac-1,3-diphenylallyl acetate with dimethyl malonate (Tsuji-Trost reaction). CYBOX ligand 5 provided the (R)-enantiomer of the alkylation product with high enantiomeric excess (up to 98% ee). Further ligand optimizations and substrate scope evaluations of allylic substitution are currently under investigation and will be published soon.In summary, we have synthesized the novel sugar-annulated ligands PYOX (


Their structures were elucidated based on High resolution electrospray ionization mass spectrometry (HR-ESI-MS), Infrared Spectroscopy (IR), 1D and 2D Nuclear Magnetic Resonance (NMR) data. Both constituents 1 and 2 exhibited moderate cytotoxicity against colon cancer HCT-116, gastric cancer MKN-45 and breast cancer MCF-7.In this research, a new triterpenoid, tirucalla-8,24-diene-3β,11β-diol-7-one ( Euphorbia contain more than 2000 species spread all over the world, and about 80 species distribute in China [Euphorbia kansui has long been used for the treatment of asthma, edema and ascites in traditional Chinese medicine. The structure type of the compounds in Euphorbia kansui are diterpenes, triterpenes, flavonoids, phenolic and acids [Euphorbia kansui, which show a wide range of pharmacological activities, such as antiviral, skin irritating and modulation of multidrug resistance effects [1) was first isolated from natural plants, and an euphane-type triterpene named eupha-8,24-diene-3β,11β-diol-7-one (2) (Euphorbia kansui for the first time in our present study. The two compounds were identified by 1D and 2D NMR including Heteronuclear Single Quantum Coherence (HSQC), Heteronuclear Multiple-Bond Correlation (HMBC), COrrelation SpectroscopY (COSY), Nuclear Overhauser Effect Spectroscopy (NOESY) and HR-ESI-MS data.The plants of in China ,2. The dnd acids ,4,5. Amo effects ,7,8,9. A-one (2)  was isol1 was obtained as a white powder. The molecular formula C30H48O3 was established by HR-ESI-MS   with C-8 (δC 140.46), C-9 (δC 161.25) and C-12 (δC 42.79) in HMBC spectrum  δH 0.88, H-C(22) δH 1.08–1.13 (m), 1.56–1.62 (m), C-20 δC 35.61, C-22 δC 35.51) and compound 1: H-C(21) δH 0.94, H-C(22) δH 1.03–1.12 (m), C-20 δC 36.03, C-22 δC 36.24. In the HSQC plot (H 1.03–1.12 and 1.40–1.47 showed correlations with C(22), C(20) at δC 36.24, 36.03 respectively, which indicated compound 1, H-C(22) δH 1.03–1.12 (m). The relative configuration of 1 was determined by the 1H-NMR  confirmed that compound 1 was classified as the tirucallane series [H 3.31, J = 9.5, 6.4 Hz) obviously indicated that the 3-OH group was in equatorial β-position [H 3.31)/H-C(5) (δH 1.65–1.68), H-C(3) (δH 3.31)/CH3-28 (δH 1.00), H-C(11) (δH 4.70)/CH3-18 (δH 0.71), CH3-30 (δH 1.15)/H-C(17) (δH 1.54–1.61), CH3-29 (δH 0.92)/CH3-19 (δH 1.28), CH3-18 (δH 0.71)/CH3-19 (δH 1.28), showed that H-C(3), H-C(5), H-C(17), CH3-28, were all in α-orientation, whereas 11-OH, CH3-19, CH3-30 and CH3-29 were all in β-orientation. Furthermore, compound 1 showed NOESY correlations between CH3-18 and H-C(20) (δH 1.40–1.47) and CH3-21, between CH3-21 and H-12β (δH 2.38–2.46). These correlations were consistent with those of tirucallane-type triterpenes [1 was identified as tirucalla-8,24-diene-3β,11β-diol-7-one.Compound 57.3682) , IR  with C-8 (δC 140.44), C-9 (δC 161.30) and C-12 (δC 42.83) in HMBC spectrum  obviously indicated that the 3-OH group was in equatorial β-position [H 3.31)/H-C(5) (δH 1.62–1.68), H-C(3) (δH 3.31)/CH3-28 (δH 1.01), H-C(11) (δH 4.69)/CH3-18 (δH 0.73), CH3-30 (δH 1.16)/H-C(17) (δH 1.55–1.60), CH3-29 (δH 0.93)/CH3-19 (δH 1.27), CH3-18 (δH 0.73)/CH3-19 (δH 1.27), showed that H-C(3), H-C(5), H-C(17), and CH3-28 were all in α-orientation, whereas 11-OH, CH3-19, CH3-30 and CH3-29 were in β-orientation, and no correlations between CH3-21/CH3-18 [1H-NMR chemical shift of CH3-21  further confirmed that compound 2 pertained to the euphane rather than the tirucallane series [2 was elucidated to be eupha-8,24-diene-3β,11β-diol-7-one.The comparison of  13C-NMR  and NOES 13C-NMR  showed tmpound 1  were semspectrum . The lar1/CH3-18 ,15 indic3–14) were identified according to the spectroscopic data  [O--20-O-acetylingenol (4) [O--20-O-acetylingenol (5) [O-benzoyl-20-deoxyingenol (6) [O-benzoyl-20-deoxyingenol (7) [8) [epi-kansenone (9) [10) [11) [12) [13) [14) [All 12 of the known compounds (e the 3) , 3-O-2′3–1 were ide (7) [8) , epi-kannone (9) , kansuin(9) [10) , kansuin12) [13) , and tir13) [14) .1 and 2 were assessed for their inhibitory effects on HCT-116, MKN-45 and MCF-7 cell lines  less than cancer cells , and compounds 1 and 2 presented definite anticancer activities with IC50 values of 20.84 ± 1.28 and 33.97 ± 2.15 µΜ against HCT-116 cells, 10.18 ± 1.36 and 14.95 ± 1.82 µΜ against MKN-45 cells, and 10.82 ± 1.18 and 20.11 ± 2.16 µΜ against MCF-7 cells, respectively. kansenone induces apoptosis through both the death receptor and mitochondrial pathways [1 and 2 were similar with kansenone in structure, excluding that a Hydrogen at C-11 of kansenone was superseded by a hydroxyl group in compounds 1 and 2, thus compounds 1 and 2 may induce apoptosis in the same way as kansenone. Further research will be conducted on the anticancer mechanism of compounds 1 and 2.Compounds ll lines , as wellll lines . The res18 , . IR spectra were gained on a Nicole Is5 of Thermo Fisher spectrophotometer . The NMR spectra were measured on Avance 400 spectrometers , with TMS as an internal standard. The UV spectra were measured on a Shimadzu UV-2401 UV-Vis spectrophotometer . The HR-ESI-MS data were obtained by using a LTQ Orbitrap MS . Column chromatography (CC): silica gel .HPLC: Hanbon NP 7000  Serials pump with an NU 3000 Serials UV-Vis detector , Phecda Si ; Waters 1525 with a 2996 Diode Array Detector (DAD) , XBrige-Prep CEuphorbia kansui was collected from Red River valley of Baoji, Shaanxi Province of China, in October 2015 and was identified by Prof. Yu Ping Tang . The voucher specimen (20151020) has been deposited in the Herbarium of college of pharmacy, Nanjing University of Chinese Medicine .The dried root of Euphorbia kansui (12.2 kg) were extracted twice (each time for 2 h) with 95% EtOH under reflux to give the 95% EtOH extract (871.9 g) by evaporation of the solvent under reduced pressure, and then the 95% EtOH extract was extracted with ethyl acetate (EtOAc) to obtain ethyl acetate extract (530.8 g). Finally, the fraction of EtOAc was subjected to silica gel column chromatography (14 × 59 cm) with a gradient elution  to get fractions A-T. Fr. G (2 g) was eluted with Pet:EtOAc (100:20). Compound 1 (40.8 mg) was isolated by HPLC , and further purified by reversed-phase HPLC  flow rate 16 mL/min (tR 15.342 min). Compound 2 (70.3 mg) was isolated by HPLC , and further purified by reversed-phase HPLC  flow rate 16 mL/min (tR 16.060 min). Fr. A (18 g) was eluted with Pet:EtOAc (100:3). Compound 13 (6.082 g) was isolated by HPLC  with  flow rate 16 mL/min (tR 35.452 min). Compound 14 (782.5 mg) was isolated by HPLC  with  flow rate 16 mL/min (tR 33.645 min). Fr. B (3 g) was eluted with Pet:EtOAc (100:5). Compound 6 (320 mg) was isolated by HPLC  flow rate 16 mL/min (tR 24.003 min). Compound 7 (46 mg) was isolated by HPLC  flow rate 16 mL/min (tR 22.209 min). Fr. C (5 g) was eluted with Pet:EtOAc (100:6). Compound 3 (920 mg) was isolated by HPLC  flow rate 16 mL/min (tR 24.128 min). Fr. D (2 g) was eluted with Pet:EtOAc (100:8). Compound 8 (161 mg) was isolated by HPLC  flow rate 16 mL/min (tR 23.218 min). Compound 9 (28 mg) was isolated by HPLC  flow rate 16 mL/min (tR 20.674 min). Fr. E (2 g) was eluted with Pet:EtOAc (100:13). Compound 4 (80 mg) was isolated by HPLC  flow rate 16 mL/min (tR 21.097 min). Compound 5 (40 mg) was isolated by HPLC  flow rate 16 mL/min (tR 24.298 min). Fr. H (10 g) was eluted with Pet:EtOAc (100:55). Compound 10 (186 mg) was isolated by HPLC , and further purified by reversed-phase HPLC  flow rate 16 mL/min (tR 10.502 min). Compound 11 (60 mg) was isolated by HPLC , and further purified by reversed-phase HPLC  flow rate 16 mL/min (tR 9.702 min). Compound 12 (80 mg) was isolated by HPLC , and further purified by reversed-phase HPLC  flow rate 16 mL/min (tR 11.302 min).The dried roots of 4 cells/mL, two normal cells L-O2 and GES-1 were seeded in 96-well plate at a concentration of 1 × 104 cells/mL and 1 × 105 cells/mL respectively [2 at 37 °C. One hundred microliters of cells were treated with compound 1 and 2 at different doses of 1.25, 2.5, 5, 10, 20 and 40 μg/mL in dimethyl sulfoxide (DMSO) in triplicate for 48 h at 37 °C with 5% CO2. Then, each of them were added with 20 μL of MTT (5.0 mg/mL) and incubated for further 4 h, the growth medium was removed from all the wells. Finally, 150 μL DMSO were added to every sample. Cisplatin  and 5-fluorouracil (5-Fu)  served as positive control. Absorbance was determined by a microplate spectrophotometer at 490 nm.Cytotoxicity of two compounds against HCT-116, MKN-45 and MCF-7 cancer cell lines , normal liver cell L-O2  and gastric epithelial cell GES-1  were evaluated with the MTT -2,5-diphenylt-etrazolium bromide) method as described in the literature ,21. All 1 and 2, which are triterpenes, as well as twelve other known compounds (3–14). Compound 1 is a new tirucallane-type triterpene named tirucalla-8,24-diene-3β,11β-diol-7-one. Compound 2 was isolated from Euphorbia kansui for the first timeand named eupha-8,24-diene-3β,11β-diol-7-one. They also display effective anticancer activities against HCT-116, MKN-45 and MCF-7 cells. As we all know, Euphorbia kansui has pharmacological activities including tumor inhibition and antiviral effects [1 and 2 may be the main material basis of anticancer for Euphorbia kansui.The article reported two compounds,  effects ,23; this1, 2, 8, 9, 13 and 14 may be drawn based on their chemical structures. Above all, the CH3-21 of compounds 9 and 14 were all in β-orientation and their 1H-NMR chemical shift of CH3-21 at , whereas the CH3-21 of compounds 8 and 13 were all in α-orientation and their 1H-NMR chemical shift of CH3-21 at  [9 had a chemical shift greater than compound 8. The C-20 and C-22 of compound 14 was larger than compound 13  ,19. The pound 13 , which w


Bacillus stearothermophilus α-glucosidase inhibitors were isolated from the flowers of Musa spp. (Baxijiao). Using NMR spectroscopy analysis they were identified as vanillic acid (1), ferulic acid (2), β-sitosterol (3), daucosterol (4) and 9-(4′-hydroxyphenyl)-2-methoxyphenalen-1-one (5). The half maximal inhibitory concentration (IC50) values of compounds 1–5 were 2004.58, 1258.35, 283.67, 247.35 and 3.86 mg/L, respectively. Compared to a known α-glucosidase inhibitor , compounds 3, 4 and 5 showed a strong α-glucosidase inhibitory effect. A Lineweaver-Burk plot indicated that compound 5 is a mixed-competitive inhibitor, while compounds 3 and 4 are competitive inhibitors. The inhibition constants (Ki) of compounds 3, 4 and 5 were 20.09, 2.34 and 4.40 mg/L, respectively. Taken together, these data show that the compounds 3, 4 and 5 are potent α-glucosidase inhibitors.The use of α-glucosidase inhibitors is considered to be an effective strategy in the treatment of diabetes. Using a bioassay-guided fractionation technique, five  Acanthopanax senticosus Harm leaves ) ) .1), ferulic acid (2), β-sitosterol (3), daucosterol (4) and 9-(4ꞌ-hydroxyphenyl)-2-methoxyphenalen-1-one (5), were isolated from PE and EA fractions. Among these compounds, β-sitosterol, daucosterol and 9-(4ꞌ-hydroxyphenyl)-2-methoxyphenalen-1-one showed excellent α-glucosidase inhibition in mixed-competitive and competitive modes, and their IC50 values are smaller than that of acarbose. Thus, α-sitosterol, daucosterol and 9-(4ꞌ-hydroxyphenyl)-2-methoxyphenalen-1-one have great potential to prevent hyperglycemia caused by α-glucosidase, and are thus potential candidates to treat diabetes mellitus and should be further evaluated in vivo studies.The ethanol extract of banana flowers displayed α-glucosidase inhibitory activity, and the PE and EA fractions had the highest such activity. Five α-glucosidase inhibitors, including vanillic acid (


The structures of the compounds were established by spectroscopic techniques. The compounds were evaluated for their inhibitory effects on polymorphonuclear leukocyte (PMN) chemotaxis by using the Boyden chamber technique and on human whole blood and neutrophil reactive oxygen species (ROS) production by using a luminol-based chemiluminescence assay. Among the compounds tested, compounds 1–4, 6 and 9 exhibited strong inhibition of PMN migration towards the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) with IC50 values of 6.8; 2.8; 2.5; 4.1; 3.7 and 3.6 μM, respectively, comparable to that of the positive control ibuprofen (6.7 μM). Compounds 2–4, 6, 7 and 9 exhibited strong inhibition of ROS production of PMNs with IC50 values of 0.9; 0.9; 1.3; 1.1; 0.5 and 0.8 μM, respectively, which were lower than that of aspirin (9.4 μM). The bioactive compounds might be potential lead molecules for the development of new immunomodulatory agents to modulate the innate immune response of phagocytes.Three new triterpenoids; namely 28,28,30-trihydroxylupeol ( Andrographis paniculata, Allium sativum, Trigonella foenum graecum, Pouteria cambodiana, Centella asiatica, Asparagus racemosus, Baliospermum montanum, Curcuma longa, Panax ginseng, Phyllanthus debilis, Tinospora cordifolia, and Picrorhiza scrophulariiflora have been reported to be able to modulate the immune system, including both adaptive and innate arms of the immune responses, exhibiting either immunostimulant or immunosuppressive effects +, corresponding to a molecular formula of C30H50O4 (M = 474.1465). The 1H-NMR spectrum exhibited singlets indicating the presence of five methyl groups, deshielded methine protons at δH 2.04 (H-18) and 2.38 (H-19) and two olefinic protons at δH 4.69 and 4.57. The corresponding 13C-NMR spectrum displayed 30 carbon signals, including five methyls, twelve methylenes, seven methines and six quaternary carbons. Two methine carbons at δC 80.1 (C-3) and 95.1 (C-28) were typical for C-OH and two sp2 carbons at δC 150.9 (C-20) and 109.3 (C-29), indicated the presence of one double bond. Another methylene carbon at δC 69.2 (C-30) was characteristic for hydroxyl (C-OH). The 1H- and 13C-NMR data of 1 were very similar to those reported for lupeol +, 479.1023 [M − 23]+, 453.0885 [M − CH3OH − 17]+ and 311.1563 [M − side chain (C8H14O5) − 1]+. Based on HRESIMS data its molecular formula was determined to be C30H46O6 and the molecular weight was established as 502.3290 [M]+. The 1H-NMR spectrum displayed characteristic signals for methyl protons at δH 0.80 , 0.85 , 0.92  and 1.04 . One proton downfield signal appeared at δH 4.79  and was attributed to the vinylic H-6 proton. Another proton resonating as a triplet at δH 3.44  was ascribed to a β-oriented H-3 carbinol proton. The 13C-NMR spectrum showed thirty carbon signals, including four methyls, ten methylenes, six methines and eight quaternary carbon signals. One methylene carbon at δC 69.2 and two methine carbons at δC 80.1 and δC 95.1 were typical for C-OH moieties. Important signals were observed for a carboxylic acid carbon at δC 173.8 (C-27), vinylic carbons at δC 151.0 (C-5) and δC 109.3 (C-6) carbinol carbons at δC 80.1 (C-3) and methyl carbons between δC 17.7–14.1. The 1H- and 13C-NMR data of 2 were compared to the reported data of other lanostanoic acids + and 463.3474 [M + Na]+ while ESIMS (negative mode) showed a molecular ion at m/z 439.3759 [M − H]– and 879.7791 [2M − H]−. HRESIMS (positive mode) displayed a molecular ion at m/z 457.2734 [M + OH]+. Its molecular formula was thus deduced to be C30H48O2 . The 1H-NMR spectrum of 3 revealed signals for five tertiary methyls at δH 0.83 , 0.90 , 0.76 , 0.89 , 0.73 , a vinyl methyl at δH 1.10 ppm  and an exo-methylene proton at δH 5.32 ppm .Compound 13C-NMR spectrum showed six methyl groups at δC 22.1 (C-23), 16.6 (C-24), 15.4 (C-25), 17.6 (C-26), 13.9 (C-27), and 20.1 ppm (C-30), an exo-methylene group at δC 150.9 (C-20) and 116.3 (C-29) and a carboxylic acid group at δC 183.5 (C-28). In addition the 13C-NMR spectrum showed twelve methylene carbons, five methine carbons and six quaternary carbon signals. The 1H- and 13C-NMR data of 3 were very similar to those reported for betulinic acid +, 931.7436 [2M + H − 18]+, HRESIMS m/z: 474.1465 [M]+, calculated 474.1465 for molecular formula C30H50O4.3,21,21,26-Tetrahydroxylanostanoic acid (2): White powder (17.2 mg), melting point 150–155 °C, UV νmax (C2H5OH) nm: 272, 207 . IR νmax (CHCl3) cm−1: 3215, 2915, 2850, 2134, 1985, 1735, 1678, 1642, 1472, 1120. ESIMS (negative mode) m/z: 502.3290 [M]+, 479.1023 [M − 23]+, 453.0885 [M − CH3OH − 17]+, and 311.1563 [M − side chain (C8H14O5) − 1]+, HRESIMS (positive mode) m/z: 503.3570 [M + H]+, calculated 502.3290 for molecular formula C30H50O. 1H-NMR (CDCl3): δH 0.80 , 0.85 , 0.92 , 0.94 , 0.99 , 1.01, 1.03 , 1.18 , 1.31 , 1.34 , 1.35 , 1.48 , 1.57 , 1.63 , 1.68 , 1.77 , 3.44 , 4.03 , 4.57 , 4.79 , 4.80 , 2.25 (C-21), 2.30 (C-21), 4.68 (C-3), 4.69 (C-26). 13C-NMR (CDCl3): δC 29.8 (C-1), 25.7 (C-2), 80.1 (C-3), 43.0 (C-4), 151.0 (C-5), 109.3 (C-6), 31.9 (C-7), 50.3 (C-8), 48.2 (C-9), 39.9 (C-10), 23.7 (C-11), 22.7 (C-12), 42.8 (C-13), 40.9 (C-14), 34.5 (C-15), 29.4 (C-16), 55.6 (C-17), 27.4 (C-18), 17.7 (C-19), 35.3 (C-20), 95.1 (C-21), 35.5 (C-22), 25.0 (C-23), 150.9 (C-24), 109.3 (C-25), 69.2 (C-26), 173.8 (C-27), 14.1 (C-28), 15.9 (C-29), 16.5 (C-30).Dehydroxybetulinic acid (3): White powder (17.4 mg) with a melting point 285–290 °C. UV λmax (C2H5OH) nm: 195. IR νmax(CHCl3) cm−1: 2924, 2238, 2158, 1732 1648, 1463, 1248 and 971 cm−1. ESIMS (positive mode) m/z: 441.3663 [M + H]+ 463.3474 [M + Na]+and ESIMS (negative mode) m/z: 439.3759 [M − H]−, 879.7791 [2M − H]. HRESIMS (positive mode) m/z: 457.2734 [M + OH]+, calculated 440.3787 for molecular formula C30H48O2. 1H-NMR (CDCl3): δH 1.41 , 1.31 , 1.42 , 1.32 , 1.42 , 1.33 , 1.34 , 1.65 , 1.46 , 1.56 , 1.54 , 1.29 , 1.55 , 1.29 , 1.24 , 1.49 , 1.00 , 1.80 , 1.25 , 1.68 , 2.09 , 1.60 , 1.75 , 0.83 , 0.90 , 0.76 , 0.89 , 0.73 , 5.32 , 1.10 . 13C-NMR (CDCl3): δC 36.1 (C-1), 28.2 (C-2), 36.7 (C-3), 30.8 (C-4), 59.6 (C-5), 18.6 (C-6), 37.4 (C-7), 40.1 (C-8), 51.9 (C-9), 37.7 (C-10), 20.6 (C-11), 25.2 (C-12), 37.7 (C-13), 42.9 (C-14), 39.7 (C-15), 40.4 (C-16), 59.6 (C-17), 46.5 (C-18), 37.2 (C-19), 150.9 (C-20), 29.2 (C-21), 23.0 (C-22), 22.1 (C-23), 16.6 (C-24), 15.4 (C-25), 17.6 (C-26), 13.9 (C-27), 183.5 (C-28), 116.3 (C-29), 20.1 (C-30).Saccharomyses cerevisiae suspensions and serum), luminol , phosphate buffer saline (PBS), Hanks Balance Salt Solution (HBSS++), ficoll, N-formyl-methionylleucyl-phenilalanine (fMLP), tryphan blue, phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO), acetylsalicylic acid (purity 99%) and ibuprofen (purity 99%) were purchased from Sigma. Haematoxylin and xylene were obtained from BDH . Chemiluminscence measurements were carried out on a Luminoscan Ascent luminometer . A Boyden chamber with a 3 and 5 μm polycarbonate membrane filter separating the upper and lower compartments was purchased from Neuro probe .Serum opsonized zymosan A  were incubated with 6.25 or 100 μg/mL of pure compounds in triplicate at 37 °C for 1 to 2 h. The cell death was indicated by the blue dye uptake. The percentage of cell viability was considered from the total cell counts.Cell viability tests were performed using trypan blue dye exclusion method with the modification of a published procedure . The neu++ into each well of 96-well microplate. The plate was incubated with 25 μL of tested compounds and aspirin at five different concentrations  of each sample for 50 min at 37 °C in a luminoscan while 25 μL of HBSS++ was used as the negative control. The cells were induced with 25 μL of serum opsonized zymosan (SOZ) followed by 25 μL of luminol into each well. Then HBSS++ solution was added into each well to make the final volume of 200 μL. Aspirin was used as a positive control while the negative control contained zymosan, luminol, DMSO (0.5%), HBSS++ and cells. The percentage of inhibition was calculated by the measurement of RLU (reading luminometer unit) of peak and total integral values with repeated scans.Luminol enhanced chemiluminescence assay was carried out using the modification of the published method . In brie−8 M, diluted with chemo attractant buffer solution) was added to the lower compartment of the Boyden chamber. PMN cell suspension (45 μL) with 5 μL of test compounds and ibuprofen at five different concentrations  were added to the upper compartment of the chamber whereas the negative control contained 45 μL of PMN cell suspension and 5 μL of chemo attractant buffer. Ibuprofen was used as a positive control. The final concentrations of test compounds and ibuprofen in the wells were 0.625, 1.25, 2.5, 5 and 10 μg/mL. The chamber was incubated in 5% carbon dioxide incubator for 1 h at 37 °C. After incubation the polycarbonate membrane (where the migrated cells were remained) was stained with PBS, methanol (99.5%), haematoxylin, distilled water, ethanol (70% and 95.8%) and xylene respectively. The distance of cell’s migration was measured using a low power microscope with magnifications 40×.The assay was carried out using the modified 48-well Boyden chamber method . The Boypost hoc Tukey’s test. p < 0.05 was considered to be statistically significant using the SPSS 17.0  statistical software. All values were characterized as mean ± SEM. Probit programme was used to determine the IC50 values for active compounds.Data were analyzed using One-way ANOVA, F. aurantiaca afforded three new triterpenoids—28,28,30-trihydroxylupeol (1), 3,21,21,26-tetrahydroxylanostanoic acid (2) and dehydroxybetulinic acid (3) together with five known triterpenoids: taraxerone (4), taraxerol (5), stigmasterol (8), ursolic acid (9), acetyl ursolic acid (10), one coumarin, herniarin (7) and one unsaturated hydrocarbon, ethyl palmitate (6). This is the first report on the phytochemical investigation and biological effects of compounds from F. aurantiaca on PMN chemotaxis and ROS inhibitory activity of human whole blood and PMNs. Compounds 1–3, 6, 7 and 9 showed the strongest inhibitory activities for both chemiluminescence and chemotaxis whereas the IC50 values were lower than those of the standard drugs used. This study provided evidence that F. aurantiaca is a source of new immunomodulatory compounds to modulate the innate immune response of phagocytes and further studies are needed to investigate the mechanisms of their immuno-modulatory responses.In conclusion, a phytochemical investigation of the stem of


Vellozia gigantea is a rare, ancient, and endemic neotropical plant present in the Brazilian Rupestrian grasslands. The dichloromethane extract of V. gigantea adventitious roots was phytotoxic against Lactuca sativa, Agrostis stolonifera, and Lemna paucicostata, and showed larvicidal activity against Aedes aegypti. Phytotoxicity bioassay-directed fractionation of the extract revealed one new isopimaradiene, 8(9),15-isopimaradien-1,3,7,11-tetraone, and three new cleistanthane diterpenoids, 7-oxo-8,11,13-cleistanthatrien-3-ol, 3,20-epoxy-7-oxo-8,11,13-cleistanthatrien-3-ol, and 20-nor-3,7-dioxo-1,8,11,13-cleistanthatetraen-10-ol. These new structures are proposed based on interpretation of 1H, 13C, COSY, NOESY, HSQC, and HMBC NMR data. 8(9),15-isopimaradien-1,3,7,11-tetraone was especially phytotoxic with an IC50 value (30 μM) comparable to those of commercial herbicides clomazone, EPTC, and naptalam. In addition, 7-oxo-8,11,13-cleistanthatrien-3-ol provided 100% mortality at a concentration of 125 ppm against one-day-old Ae. aegypti larvae. Our results show that ancient and unique plants, like the endangered narrowly endemic neotropical species V. gigantea present in the Rupestrian grasslands, should also be protected because they can be sources of new bioactive compounds. Brazil has a rich biodiversity of plant species, which includes rare and endemic taxa. Among the typical biomes from Brazil, the Espinhaço mountain range has the Rupestrian grasslands (“campo rupestre”) that shelter different plant species with a high richness of endemism ,2,3. TheVellozia gigantea N. L. Menezes & Mello-Silva, known as “canela de ema”, is a rupicolous monocot species that is at least 6 m tall and endemic to the Rupestrian grasslands in Brazil, specifically present in the Serra do Cipó National Park in Minas Gerais state − acquired in the negative ion mode. The presence of four methyl singlets  a two-proton olefinic signal at δ 5.04 and an olefinic signal at δ 5.79 in the 1H-NMR spectrum combined with olefinic carbons at δ 147.11, 144.18, and 143.28 in the 13C-NMR spectrum indicated compound 1 possesses an isopimaradiene skeleton. Four 13C-NMR spectral shifts at δ 208.98, 202.51, 199.69, and 198.87 suggested a tetraone configuration. Inspection of the literature revealed similar isopimaradiene diones + acquired in positive ion mode. The presence of a methyl triplet (δ 1.19), four methyl singlets , and two aromatic proton doublets  in the 1H-NMR spectrum combined with information from the 13C-NMR spectrum  indicated that compound 2 possesses a cleistanthatriene skeleton. Inspection of the literature indicated similarities to 7-oxo-8,11,13-cleistanthatriene with differences in the A ring carbon shifts + acquired in positive ion mode. The presence of a methyl triplet (δ 1.16), three methyl singlets , and two aromatic proton doublets  in the 1H-NMR spectrum combined with information from the 13C-NMR spectrum  indicated that compound 4 possesses a cleistanthatetraene skeleton. C-3 and C-7 ketone positions were easily established using HMBC correlation data. Positioning of the hydroxyl at C-10 was unequivocally confirmed with HMBC correlations between H-11 and C-10, among others. NOESY correlation data was unable to establish the relative configuration of the hydroxyl at C-10 due to the lack of definitive correlation data. Complete structure determination was accomplished using COSY, HSQC, HMBC, and DEPT spectroscopic data ,15-isopimaradien-1,3,7,11-tetraone (1), 7-oxo-8,11,13-cleistanthatrien-3-ol (2) and 20-nor-3,7-dioxo-1,8,11,13-cleistanthatetraen-10-ol (4) were moderately active (2–4 ranking) on both species, whereas 3,20-epoxy-7-oxo-8,11,13-cleistanthatrien-3-ol (3) had no activity on lettuce at this concentration, but was moderately active on A. stolonifera. We can observe that after the fractionation of fraction G to obtain compound 4, there was a reduction of the activity initially expressed. This fact may suggest that the initial activity of fraction G (4/5 effect) could be related not only to compound 4 (showed 2/3 effect), but may also be acting synergistically between this compound and some other compound present in the fraction. Additionally, all four compounds were active as growth inhibitors of L. paucicostata, with the order of activity being 1 > 4 > 3 > 2 , EPTC (62 μM), and naptalam (128 μM) in the same bioassay .3.The root crude extract, fractions, and pure compounds were analyzed by NMR spectroscopy on a Varian INOVA 500 MHz spectrometer . NMR spectra were recorded in CDCl1), C′ (2), and E′ (3) peaks were characterized and identified.Fractions D and G, which are phytotoxic, were purified by HPLC. An Agilent 1200 system equipped with a quaternary pump, autosampler, diode-array detector, and vacuum degasser was used with a Zorbax RX-SIL  5 µm 9.4 mm × 250 mm HPLC column. A total of 150.9 mg of fraction D was dissolved in methanol and purified. The manual injection for each sample was 20 µL. The method was isocratic 99% hexane: 1% IPA for 10 min ramp to gradient 97% hexane: 3% IPA over 40 min. After running two samples, the column was washed with hexane:IPA for 5 min and re-equilibrated. Analytes were detected at 254 and 280 nm by diode array detector. Five major peaks were detected and collected . However, only the A′ (4) was characterized and identified.A total of 22 mg of fraction G was dissolved in methanol and purified by normal phase HPLC System. The manual injection for each sample was 20 µL. The method was 80% hexane:20% EtOAc to 62% hexane:38% EtOAc over 30 min. For each running the column was re-equilibrated. Analytes was detected at 254 and 280 nm by diode array detector. Two major peaks were detected and collected . However, only the A″ peak (8(9),15-Isopimaradien-1,3,7,11-tetraone (1). High-resolution ESI-MS: m/z 327.1597 [M − H]−; calculated for C20H23O4, 327.1596. [α]c 0.275, CHCl3). 1H-NMR  δ 5.79 , 5.04 , 3.70 , 3.39 , 2.76 , 2.67 , 2.61–2.54 , 2.40 , 2.33 , 2.15 , 1.61 , 1.26 , 1.12 , 1.07 . 13C-NMR  δ 208.98 (C-3), 202.51 (C-1), 199.69 (C-11), 198.87 (C-7), 147.11 (C-9), 144.18 (C-15), 143.28 (C-8), 113.06 (C-16), 53.46 (C-10), 50.60 (C-2), 49.68 (C-12), 47.00 (C-4), 45.33 (C-5), 38.08 (C-13), 34.75 (C-14), 34.55 (C-6), 28.52 (C-18), 25.79 (C-17), 21.55 (C-19), 15.59 (C-20).7-oxo-8,11,13-Cleistanthatrien-3-ol (2). High-resolution ESI-MS: m/z 301.2173 [M + H]+; calculated for C20H29O2, 301.2168. [α]c 0.33, CHCl3). 1H-NMR  δ 7.24 , 7.05 , 3.31 , 3.00–2.89 , 2.87–2.79 , 2.72–2.66 , 2.31 , 2.26 , 1.88 , 1.83 , 1.75 , 1.19 , 1.14 , 1.04 , 1.01 . 13C-NMR  δ 201.83 (C-7), 153.89 (C-9), 144.38 (C-14), 135.60 (C-13), 134.83 (C-12), 131.00 (C-8), 120.68 (C-11), 78.44 (C-3), 47.32 (C-5), 39.04 (C-4), 38.11 (C-6), 37.98 (C-10), 36.77 (C-1), 27.74 (C-2), 27.39 (C-18), 23.71 (C-15), 23.36 (C-20), 19.29 (C-17), 14.91 (C-19), 14.64 (C-16).3,20-Epoxy-7-oxo-8,11,13-cleistanthatrien-3-ol (3). High-resolution ESI-MS: m/z 337.1797 [M + Na]+; calculated for C20H26O3Na, 337.1779. [α]c 0.56, CHCl3). 1H-NMR  δ 7.25 , 7.09 , 4.21 , 3.99 , 3.02–2.82 , 2.78 , 2.51 , 2.49–2.42 , 2.30 , 2.25 , 2.20–2.11 , 1.94 , 1.15 , 1.10 , 1.02 . 13C-NMR  δ 201.87 (C-7), 144.28 (C-14), 142.96 (C-9), 136.48 (C-13), 134.83 (C-12), 131.80 (C-8), 124.40 (C-11), 99.02 (C-3), 71.14 (C-20), 47.30 (C-5), 40.55 (C-4), 39.27 (C-6), 37.11 (C-10), 34.80 (C-1), 29.46 (C-2), 26.76 (C-19), 23.52 (C-15), 19.42 (C-17), 18.14 (C-18), 14.51 (C-16).20-nor-3,7-dioxo-1,8,11,13-Cleistanthatetraen-10-ol (4). High-resolution ESI-MS: m/z 299.1669 [M + H]+; calculated for C19H23O3, 299.1647. [α]c 0.065, CHCl3). 1H-NMR  δ 7.34 , 7.24 , 7.17 , 6.07 , 3.09 , 3.07 , 2.86 , 2.73 , 2.50 , 2.38 , 1.24 , 1.16 , 0.63 . 13C-NMR  δ 203.12 (C-3), 199.77 (C-7), 150.04 (C-1), 144.46 (C-14), 143.48 (C-9), 138.76 (C-13), 135.00 (C-12), 131.75 (C-8), 127.44 (C-2), 123.41 (C-11), 70.17 (C-10), 49.93 (C-5), 45.54 (C-4), 39.09 (C-6), 24.67 (C-19), 23.11 (C-15), 21.28 (C-18), 19.61 (C-17), 14.47 (C-16).20. Twenty microliters of sample (approximately 0.1 mg/mL) were injected manually at 0.5 min while mass drift compensation standards  were injected at 1.5 min over the course of a 2 min run.High-resolution mass spectra (ESI-MS) were obtained using an Agilent 1100 HPLC coupled to a JEOL AccuTOF (JMS-T100LC) . All isolated compounds were prepared in MeOH and injected directly into a 0.3 mL/min stream of either MeOH or 80% MeOH/20% DI HLactuca sativa—Iceberg A Crisphead cultivar from Burpee Seeds, Warminster, PA, USA) and bentgrass  were surface sterilized with a 0.5% to 1% (v/v) sodium hypochlorite solution for approximately 10 min, rinsed with deionized water and dried in a sterile environment. The DCM crude extract of the adventitious roots of V. gigantea, column chromatography fractions, and pure compounds were tested for phytotoxicity on the monocot bentgrass and dicot lettuce according to published methods [−1∙m−2 average photosynthetically active radiation (PAR). A qualitative estimate of phytotoxicity was made by assigning a rating of 0 for no effect , 2 for less than 50% germination inhibition, 3 for about 50% germination inhibition, 4 for more than 50% germination inhibition, and 5 for no germination of the seeds. Each experiment was repeated three times.Seeds of lettuce ( methods . A filteLemna paucicostata (duckweed) was carried out using the method of Michel et al [−1) with added iron  in sterile jars with vented lids in a Percival Scientific CU-36L5 incubator under continuous light conditions at 26 °C and 120 μmol∙s−1∙m−2 average PAR. The iron solution (1000×) contained 18.36 g∙L−1 of Fe-EDTA. Media were changed every two to three days or new stocks were prepared in fresh media. Plant doubling time was approximately 24 to 36 hours. Both screening and replicate series tests were conducted using non-pyrogenic polystyrene and sterile six-well plates . Each well contained 4950 μL of the Hoagland’s media plus 50 μL of water, or the solvent, or the compound dissolved in the appropriate solvent (at a concentration of 100×). The final concentration of the solvent was therefore approximately 1% by volume. A graphic template of the six-well plates was used for LemnaTec  image analysis software. Each well was inoculated with two three-frond plants of the same age (four to five days old) and approximate size and incubated in the Percival incubator as described above. Each treatment was replicated three times. IC50 (concentration inhibiting growth by 50%) values were determined with R statistics software [The bioassay with el et al . Duckweesoftware . Plant fV. gigantea against Aedes aegypti. Five one-day-old larvae were transferred to each well of 24-well tissue culture plates in a 30–40 µL droplet of water. An aliquot of 50 µL of larval diet . All the compounds were diluted in dimethyl sulfoxide (DMSO). Larval mortality was recorded 24 h post-treatment. Larvae that showed no movement in the well after manual disturbance were recorded as dead. Larval mortality was converted into a percentage.Bioassays were conducted by using the bioassay system described by Ali et al.  to deterVellozia gigantea is a unique and endemic species living only in the Rupestrian grasslands biome in Brazil. From the extract of adventitious roots of V. gigantea, we isolated four new diterpenes with phytotoxic and larvicidal activity. Other diterpenes from plants have been found to have pesticidal activities in the past [V. bilocor [V. flavicans [S,17-cyclolabdadiene, and 17-hydroxy-8(9),15-isopimaradien from V. compacta [V. gigantea until now. Morales et al. [V. gigantea.the past ,18,19. O bilocor , pimaranlavicans  and 19-hcompacta . Few coms et al.  describeV. gigantea. These compounds might be used as scaffolds to develop new herbicides and other pesticides. Additionally, our results show that ancient and unique plants, like the endangered, narrowly endemic neotropical species V. gigantea present in the Rupestrian grasslands, should be protected because of their intrinsic value as sources of new, bioactive compounds. As V. gigantea is an endangered species, further studies will be necessary to obtain micropropagated plants and isolate these new pesticidal diterpenes, which may be modified to increase their activity and modify their selectivity.In conclusion, our study proposes the structures of four new natural diterpene compounds produced by the adventitious root tissues of


Saprolegnia activities of chalconic compounds, nine dialkoxychalcones 2–10, along with their key building block 2′,4′-dihydroxychalcone 1, were evaluated for their potential oomycide activities against Saprolegnia australis strains. The synthesis afforded a series of O-alkylated derivatives with typical chalcone skeletons. Compounds 4–10 were reported for the first time. Interestingly, analogue 8 with the new scaffold demonstrated remarkable in vitro growth-inhibitory activities against Saprolegnia strains, displaying greater anti-oomycete potency than the standard drugs used in the assay, namely fluconazole and bronopol. In contrast, a dramatic loss of activity was observed for O-alkylated derivatives 2, 3, 6, and 7. These findings have highlighted the therapeutic potential of the natural compound 1 scaffold to be exploitable as a drug lead with specific activity against various Saprolegnia strains.To investigate the anti- Saprolegnia ..17].The use of a polar aprotic solvent, such as DMF in this synthesis, decreased the intensity of the hydrogen bond, allowing the formation of the dialkylated products ,19. In o2–10 were confirmed by standard spectroscopic techniques. The 1H NMR data for the derivatives 2–10 confirms that the hydroxyl peaks of compound 1 are no longer present and that there are additional alkoxy peaks depending on the alkyl groups added, confirming the dialkylation. The spectra of these compounds showed two doublets with J ~ 15.5 Hz in the ~7.6 ppm range, confirming the existence of the trans double bond in all the analogs. The presence of carbonyl groups was supported by the signals at δ 191.8–190.5 in 13C NMR and 1636–1632 cm−1 in the IR spectra, respectively.The structure of dialkoxychalcones 2–10 and their precursor compound 1 were evaluated for their anti-oomycete activities against Saprolegnia australis strains according to the method described in the literature -3-phenylprop-2-en-1-one (3). Yellow oil. Yield: 80.6%. 1H NMR : 7.77 ; 7.69 ; 7.61 ; 7.52 ; 7.43 ; 6.53 ; 6.06 ; 5.41 ; 4.55 ; 4.51 . Spectroscopic data and physical properties of compound 3 were consistent with those reported in the literature phenyl}-3-phenylprop-2-en-1-one (4). Brown oil. Yield: 78.2%. Refractive index (n): 1.571. IR υmax (KBr) cm−1: 2957 (C-H), 1636 (C=O), 1521 (C=C), 1245 (Ar-O-R), 1132 (C-O). 1H NMR : 7.84 ; 7.69 ; 7.64 ; 7.57 ; 7.43 ; 6.51 ; 5.13 ; 5.04 ; 4.57 ; 4.49 ; 1.83 . 13C NMR : 191.8 (C-9); 166.6 (C-2′); 165.4 (C-4′); 144.8 (C-7); 139.9 (C-2″ and C-2′′′); 134.8 (C-1); 131.2 (C-60); 130.6 (C-4); 129.0 (C-3 and C-5); 128.7 (C-2 and C-6); 122.4 (C-10); 120.4 (C-8); 113.4 (C-3″ and C-3′′′); 108.2 (C-5′); 102.0 (C-3′); 72.3 (C-1″); 71.9 (C-1′′′); 19.3 (C-4″ and C-4′′′). HRMS: M + H ion m/z 349.4347 .(2E)-1-{2,4-Bis[(2-methylprop-2-en-1-yl)oxy]phenyl}-3-phenylprop-2-en-1-one (5). Brown solid. Yield: 75.3%. m.p.: 108–110 °C. IR υmax (KBr) cm−1: 2958 (C-H), 1635 (C=O), 1519 (C=C), 1243 (Ar-O-R), 1131 (C-O). 1H NMR : 7.84 ; 7.69 ; 7.64 ; 7.57 ; 7.43 ; 6.49 ; 5.95 ; 5.75 ; 4.57 ; 4.52 ; 1.77 . 13C NMR : 191.5 (C-9); 166.6 (C-2′); 165.4 (C-4′); 144.8 (C-7); 134.8 (C-1); 131.6 (C-2″ and C-2′′′); 131.1 (C-6′); 130.6 (C-4); 129.0 (C-3 and C-5); 128.5 (C-2 and C-6); 125.0 (C-1′); 120.4 (C-8); 114.1 (C-3″ and C-3′′′); 108.3 (C-5′); 101.8 (C-3′); 69.4 (C-1″); 68.9 (C-1′′′); 17.9 (C-4″ and C-4′′′). HRMS: M + H ion m/z 349.4441 .(2E)-1--3-phenylprop-2-en-1-one (6). Brown solid. Yield: 79.2%. m.p.: 95–97 °C. IR υmax (KBr) cm−1: 2950 (C-H), 1636 (C=O), 1524 (C=C), 1248 (Ar-O-R), 1130 (C-O). 1H NMR : 7.84 ; 7.68 ; 7.66 ; 7.57 ; 7.43 ; 6.49 ; 5.92 ; 5.19 ; 4.19 ; 4.12 ; 2.59 . 13C NMR : 191.9 (C-9); 166.7 (C-4′); 165.4 (C-2′); 144.4 (C-7); 134.8 (C-1); 133.9 (C-3″ and C-3′′′); 131.2 (C-6′); 130.6 (C-4); 129.0 (C-3 and C-5); 128.5 (C-2 and C-6); 120.4 (C-8); 117.4 (C-4″ and C-4′′′); 114.1 (C-1′); 108.1 (C-5′); 101.6 (C-3′); 67.9 (C-1″ and C-1′′′); 33.4 (C-2″ and C-2′′′). HRMS: M + H ion m/z 349.4347 .(2E)-1-{2,4-Bis[(3-methylbut-2-en-1-yl)oxy]phenyl}-3-phenylprop-2-en-1-one (7). Brown solid. Yield: 80.0%. m.p.: 103–104 °C. IR υmax (KBr) cm−1: 2958 (C-H), 1633 (C=O), 1517 (C=C), 1240 (Ar-O-R), 1130 (C-O). 1H NMR : 7.84 ; 7.69 ; 7.65 ; 7.56 ; 7.43 ; 6.48 ; 5.87 ; 5.08 ; 4.07 ; 4.01 ; ; 2.27 ; 1.94 ; 13C NMR : 191.6 (C-9); 166.7 (C-4′); 165.8 (C-2′); 144.3 (C-7); 137.4 (C-4″ and C-4′′′); 134.8 (C-1); 131.2 (C-6′); 130.6 (C-4); 129.0 (C-3 and C-5); 128.5 (C-2 and C-6); 120.4 (C-8); 115.5 (C-5″ and C-5′′′); 114.0 (C-1′); 108.1 (C-5′); 101.6 (C-3′); 67.9 (C-1″ and C-1′′′); 30.0 (C-2″ and C-2′′′); 28.1 (C-3″ and C-3′′′). HRMS: M + H ion m/z 377.4881 .(2E)-1--3-phenylprop-2-en-1-one (8). Yelow oil. Yield: 73.3%. Refractive index (n): 1.573. IR υmax (KBr) cm−1: 2959 (C-H), 1633 (C=O), 1524 (C=C), 1245 (Ar-O-R), 1120 (C-O). 1H NMR : 7.84 ; 7.91 ; 7.30 ; 7.57 ; 7.42 ; 6,51 ; 5.53 ; 5.49 ; 4.67 ; 4.59 ; 1.81 ; 1.79 . 13C NMR : 190.5 (C-9); 166.7 (C-2′); 165.6 (C-4′); 144.3 (C-7); 139.2 (C-3′′′); 138.6 (C-3″); 134.9 (C-1); 131.2 (C-6′); 130.6 (C-4); 129.0 (C-3 and C-5); 128.5 (C-2 and C-6); 120.4 (C-8); 118.7 (C-2″); 114.0 (C-1′); 108.3 (C-5′); 101.7 (C-3′); 64.6 (C-1″); 64.1 (C-1′′′); 25.8 (C-5″ and C-5′′′); 18,2 (C-4″ and C-4′′′). HRMS: M+H ion m/z 377.4709 (C25H28O3: 376.4712).(2E)-1--3,7-dimethylocta-2,6-dien-1-yl]oxy}phenyl)-3-phenylprop-2-en-1-one (9). Brown oil. Yield: 69.8%. Refractive index (n): 1.553. IR υmax (KBr) cm−1: 2940 and 2865 (C-H), 1633 (C=O), 1532 (C=C), 1241 (Ar-O-R), 1129 (C-O); 1H NMR : 7.91 ; 7.84 ; 7.65 ; 7.57 ; 7.43 ; 6.50 ; 5.48 ; 5.09 ; 4.60 ; 4.49 ; 2.12 , 1.75 ; 1.68 , 1.61 . 13C NMR : 190.8 (C-9); 166.7 (C-2′); 166.0 (C-4′); 144.3 (C-7); 141.2 (C-3″ and C-3′′′); 134.9 (C-1); 132.1 (C-6′); 131.2 (C-7″ and C-7′′′); 130.6 (C-4); 129.0 (C-3 and C-5); 128.5 (C-2 and C-6); 123.7 (C-6″ and C-6′′′); 120.4 (C-8); 119.6 (C-2″); 118.5 (C-2′′′); 114.0 (C-1′); 108.3 (C-5′); 101.8 (C-3′); 66.4 (C-1″); 65.3 (C-1′′′); 39.5 (C-4″ and C-4′′′); 26.3 (C-5″ and C-5′′′); 25.6 (C-10″ and C-10′′′); 17.7 (C-9″ and C-9′′′); 16.7 (C-8″ and C-8′′′). HRMS: M + H ion m/z 513.7119 .(2E)-1--3,7,11-trimethyldodeca-2,6,10-trien-1-yl]oxy}phenyl)-3-phenylprop-2-en-1-one (10). Brown oil. Yield: 60.4%. Refractive index (n): 1.547. IR υmax (KBr) cm−1: 2955 (C-H), 1632 (C=O), 1543 (C=C), 1243 (Ar-O-R), 1140 (C-O). 1H NMR : 7.84 ; 7.69 ; 7.65 ; 7.58 ; 7.35 ; 6.57 ; 6.52 ; 5.55 ; 5.50 ; 5.09 ; 4.60 ; 2.07 , 1.97 , 1.76 ; 1.74 ; 1.68 ; 1.59 . 13C NMR : 190.5 (C-9); 163.7 (C-2′); 160.1 (C-4′); 142.0 (C-7); 142.0 (C-3″ and C-3′′′); 135.8 (C-7″ and C-7′′′); 135.6 (C-1); 133.1 (C-6′); 131.4 (C-11″ and C-11′′′); 129.7 (C-4); 128.7 (C-3 and C-5); 128.2 (C-2 and C-6); 124.3 (C-6″ and C-6′′′); 123.6 (C-10″ and C-10′′′); 122.1 (C-8); 118.9 (C-2″ and C-2′′′); 118.7 (C-1′); 106.1 (C-5′); 100.3 (C-3′); 65.6 (C-1″ and C-1′′′); 39.7 (C-4″ and C-4′′′); 39.6 (C-8″ and C-8′′′); 26.2 ; 25.7 (C-15″ and C-15′′′); 17.7 (C-14″ and C-14′′′); 16.7 (C-12″ and C-12′′′); 16.0 (C-13″ and C-13′′′). HRMS: M + H ion m/z 649.9563 .S. australis were isolated from Salmo salar carp eggs [The strains of arp eggs .The minimum inhibitory concentrations (MIC) were assessed by the procedure previously described in reference . BronopoThe minimum oomycidal concentrations (MOC) were assessed by the procedure previously described in reference . BronopoThe percentage of cellular leakage was assessed by the procedure previously described in reference . Sodium Determinations of MIC and MOC were performed in triplicate and the results expressed as mean values ± SD. The results were analyzed using the standard method .1 and solvent (acetone and DMF) was optimized using DFT-B3LYP-6-31G incorporated into Gaussian 03 program [The solvent influence in the reaction of compound  program , obtaineOn the other hand, the dialkoxychalcones presented in As well as natural bonding orbital charge of chalcone skeleton C1, C2, C3, C4, C5, C6, C1′, C2′, C3′, C4′, C5′, C6′, Cα, Cβ, and CO (NBO-Charge). In addition, topological and steric descriptors, such as molecular weight (MW), the logarithm of the partition coefficient between n-octanol and water (CLogP), molar refractivity (MR), molecular area (MA), molecular volume (MV), hydrogen bond acceptor (HBA), hydrogen bonding donor (HBD), Balaban index (BI), molecular topological index (MTI), rotatable bonds (RB), topological diameter (TD), and Wiener index (WI), were obtained from Chem-Draw software.For this analysis, multiple linear regressions were used to check the influence of chemical features and anti-oomycete activity. The analysis was performed selecting different descriptors, such as MW, CLogP, MR, MA, MV, HBA, HBD, BI, MTI, RB, topological diameter TD, and WI. In addition, HOMO, LUMO, ΔLH, µ, η, S, and ω NBO-Charge on C1, C2, C3, C4, C5, C6, C1′, C2′, C3′, C4′, C5′, C6′, Cα, Cβ, and CO were used in gas and solvent phases. Model optimization was carried out by Statistica 7.0 Package Software.2 values are equal or greater than 0.5. q2 values are obtained by following formula:obs is pMIC observed, ycalc is pMIC calculated by QSAR model gas or solvent phase, and yave is pMIC average of all compounds in the training set.Cross-validation of QSRR model was carried out using the Golbraikh method . Accepta2–10 with typical chalcone cores have been successfully synthesized and found to display strong to weak oomycidal agents against Saprolegnia australis strains. Compound 8 with two allyloxy chains (C5) was found to exhibit a more potent anti-oomycete activity than the other alkoxy chalcone derivatives and the standard drugs, bronopol and fluconazole, used in the assays, revealing the structural framework to have application advantage associated with strong anti-oomycete effect elicited on Saprolegnia australis strains. Additionally, a study of structure–activity relationship was carried out using the calculations of all compounds in gas phase and solvent phase (water) using various topological, steric, and quantum descriptors in conjunction with multivariate regression models, obtaining that the model in gas phase possesses better statistical values than the model in solvent phase . In this context, the global electrophilicity index (ω) is the most important descriptor for explaining anti-oomycete activity.In conclusion, a series of chalcone derivatives


Eucalyptus perriniana was studied. Vanillin was converted into vanillin 4-O-β-D-glucopyranoside, vanillyl alcohol, and 4-O-β-D-glucopyranosylvanillyl alcohol by E. perriniana cells. Incubation of cultured E. perriniana cells with 8-nor- dihydrocapsaicin gave 8-nordihydrocapsaicin 4-O-β-D-glucopyranoside and 8-nordihydro- capsaicin 4-O-β-D-gentiobioside.Glycosylation of vanilloids such as vanillin and 8-nordihydrocapsaicin by cultured plant cells of  Biotransformations are considered to be an important method for converting inexpensive and plentiful organic compounds into costly and scarce ones ,2,3,4,5.Eucalyptus perriniana.Vanilloids including capsaicinoids have been widely used as food additives and have been studied for their broad range of physiological properties such as antioxidative, antimicrobial, analgesic, antigenotoxic, antimutagenic, and anticarcinogenic effects ,17,18,19i.e., vanillin and 8-nordihydrocapsaicin, was individually investigated using cultured cells of E. perriniana. After addition of substrates to the cultured suspension cells in Murashige and Skoog’s (MS) medium, the incubation was continued for five days, after which products in each medium and MeOH extracts of cells were detected by HPLC. No products were obtained in the control experiments undertaken without substrate.The biotransformation of vanilloids, 1) with the cultured E. perriniana cells for five days yielded compounds 2–4 which were identified as vanillyl alcohol (2%), vanillin 4-O-β-D-glucopyranoside (20%), and 4-O-β-D-glucopyranosylvanillyl alcohol (70%), respectively, by comparison of spectroscopic data, such as HRFABMS, 1H- and 13C-NMR (O-β-D-glucopyranoside (3) was formed, followed by formation of 4-O-β-D-glucopyranosylvanillyl alcohol (4) (O-β-D-glucopyranoside (3) decreased, accompanied by an increase of that of 4-O-β-D-glucopyranosylvanillyl alcohol (4). These findings suggest that vanillin 4-O-β-D-glucopyranoside (3) was converted into 4-O-β-D-glucopyranosylvanillyl alcohol (4) by E. perriniana cells.Incubation of vanillin   because E. perriniana were incubated with 8-nordihydrocapsaicin (5) for five days, compounds 6 and 7 were obtained as the products. They were identified as 8-nordihydrocapsaicin 4-O-β-D-glucopyranoside (62%)  and expressed as a relative percentage to the total amount of the whole reaction products extracted.To the 500 mL flask containing 200 ml of MS medium and the suspension cultured cells 100 g) of 0 g of E. methods ,12,13. T8-Nordihydrocapsaicin 4-O-β-D-gentiobioside (2 mg): HRFABMS: m/z 640.2920 [M+Na]+ ; 1H-NMR (CD3OD): δ 0.89 , 1.31 , 1.63 , 2.24 , 3.10–4.11 , 3.85 , 4.31 , 4.35 , 4.92 , 6.85 , 6.94 , 7.15 ; 13C-NMR (CD3OD), see E. perriniana are able to convert vanilloids, including capsaicinoid, into the corresponding β-glucosides and β-gentiobioside. Recently, several attempts have been made to glycosylate vanillin by cultured plant cells that resulted in the production of only vanillin 4-O-β-D-glucopyranoside [E. perriniana had high potential to glycosylate exogenous compounds to their glycosides including gentiobiosides [Catharanthus roseus cells [C. roseus cells are more active rather than E. perriniana cells in the capsaicin glycosylations. Further studies on the pharmacological activities of vanilloid glycosides are now in progress.The results of this experiment revealed that the cultured suspension cells of ranoside ,22. We hbiosides ,12,13. Hbiosides . The bious cells , and on us cells . Culture


S)-perillyl alcohol were designed and synthesized using (S)-perillaldehyde as the starting material. These derivatives showed increased antiproliferative activity in human lung cancer A549 cells, human melanoma A375-S2 cells and human fibrosarcoma HT-1080 cells comparing with that of (S)-perillyl alcohol. Among these derivatives, compounds 5VI and 7VI were the most potent agents, with the IC50s below 100 μM. It was demonstrated that the antiproliferative effect of 5VI was mediated through the induction of apoptosis in A549 cells.Two series of amino-modified derivatives of ( Most of the drugs on the clinic market today are inspired by or derived from natural sources . PerillyIV, VI were synthesized. Their activity of inhibiting tumor cell growth and the potential mechanism were studied in a few of cancer cell lines.Amino-modification has been proved to be an efficient approach to increase water solubility and/or antitumor activity of several natural products, such as that of comptothecin ,16, β-elS)-perillaldehyde is outlined in S)-Perillyl alcohol (I) was obtained from (S)-perillaldehyde via reduction with sodium borohydride.The synthetic route of the perillyl alcohol derivatives starting with (S)-perillyl alcohol, acetylation of the hydroxyl group was carried out. The resulted perillyl acetate (II) was reacted with hypochloric acid, affording the intermediate III. The nucleophilic substitution of III with a heterocyclic amine or an aromatic amine and subsequent hydrolysis gave the target compound IV. The selective chlorination from (S)-perillyl alcohol (I) to the intermediate V was achieved in a mild condition via Appel reaction, avoiding the undesired chlorination of olefins by other reagents. The substitution reaction of V with an aliphatic amine or a heterocyclic amine gave the target compound VI.For a selective chlorination at the terminal allyl group of (50s of (S)-perillyl alcohol in the three cells were more than 1,000 μM (the highest concentration used in this experiment). All the synthesized derivatives except 6IV displayed much more potent cytotoxicity than (S)-perillyl alcohol. Among them, the two secondary aliphatic amines, 5VI and 7VI, were the most effective agents with the IC50s below 100 μM in the three tumor cells. Introduction of a substituted piperazinyl moiety  was of benefit to antiproliferative activity to some extent, but it did not show the same enhanced effect as that in the modification of β-elemene + 265.2280, found: 265.2274.(S)-(4-(3-(4-Isopropylpiperazin-1-yl)prop-1-en-2-yl)cyclohex-1-enyl)methanol (2IV): Yield: 57.5%; mp: 52–53 °C; Rf = 0.39 (CH2Cl2/MeOH/Et3N: 200/10/1). 1H-NMR δ: 5.71 , 4.97, 4.89 , 4.02 , 2.95 , 2.76–2.30 , 2.27–2.22 , 2.17–2.09 , 2.05–1.84 , 1.57–1.50 , 1.09 2); HRMS: m/z calcd. for C17H31N2O [M+H]+ 279.2436, found: 279.2430.(S)-(4-(3-(4-(4-Methoxyphenyl)piperazin-1-yl)prop-1-en-2-yl)cyclohex-1-enyl)methanol (3IV): Yield: 54.6%; mp: 58–59 °C; Rf = 0.49 (CH2Cl2/MeOH: 22/5). 1H-NMR δ: 6.93–6.79 , 5.70 , 5.00, 4.92 , 4.00 , 3.76 , 3.18–2.93 , 2.65–2.45 , 2.37–1.86 , 1.60–1.49 ; HRMS: m/z calcd. for C21H31N2O2 [M+H]+ 343.2385, found: 343.2381.(S)-(4-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)prop-1-en-2-yl)cyclohex-1-enyl)methanol (4IV): yield: 50.2%; pale yellow oil; Rf = 0.47 (CH2Cl2/MeOH: 22/5). 1H-NMR δ: 7.04–6.79 , 5.71 , 5.01, 4.92 , 4.01 , 3.86 , 3.19–2.89 , 2.79–2.40 , 2.38–1.84 , 1.61–1.48 ; HRMS: m/z calcd. for C21H31N2O2 [M+H]+ 343.2385, found: 343.2380.(S)-(4-(3-(4-Benzylpiperazin-1-yl)prop-1-en-2-yl)cyclohex-1-enyl)methanol (5IV): Yield: 61.3%; pale yellow oil; Rf = 0.44 (CH2Cl2/MeOH: 22/5). 1H-NMR δ: 7.34–7.21 , 5.69 , 4.94, 4.88 , 4.00 , 3.51 , 2.98–2.88 , 2.63–2.31 , 2.30–2.07 , 2.01–1.83 , 1.56–1.44 ; HRMS: m/z calcd. for C21H31N2O [M+H]+ 327.2436, found: 327.2431.(S)-(4-(3-(Piperidin-1-yl)prop-1-en-2-yl)cyclohex-1-enyl)methanol (6IV): Yield: 51.3%; pale yellow oil; Rf = 0.49 (CH2Cl2/MeOH/Et3N: 200/10/1). 1H-NMR δ: 5.70 , 4.96, 4.87 , 4.00 , 2.90 , 2.41–2.08 , 2.01–1.84 , 1.59–1.41 ; HRMS: m/z calcd. for C15H26NO [M+H]+ 236.2014, found: 236.2009.(S)-(4-(3-(Pyridin-2-ylamino)prop-1-en-2-yl)cyclohex-1-enyl)methanol (7IV): Yield: 40.1%; pale yellow oil; Rf = 0.39 (CH2Cl2/MeOH: 22/5). 1H-NMR δ: 8.07 , 7.46–7.39 , 6.65–6.49 , 6.36 , 5.71 , 5.03, 4.92 , 4.01 , 3.95–3.85 , 2.34–1.88 , 1.62–1.51 ; HRMS: m/z calcd. for C15H21N2O [M+H]+ 245.1654, found: 245.1648.V  in acetonitrile (10 mL); potassium carbonate  and amine (5.5 mmol) were added. The mixture was stirred and refluxed for 6–8 h. Then the solvent was evaporated in vacuo. Brine (15 mL) was added to the residue and the mixture was extracted with dichloromethane (3 × 10 mL). The combined organic extracts were washed with brine; dried over anhydrous sodium sulfate; and filtered. The filtrate was concentrated in vacuo. The residue was purified on a silica gel column with dichloromethane–methanol (100:1 → 50:1 → 20:1) as eluent to afford the target product VI.To a solution of compound (S)-3,5-cis-Dimethyl-1-((4-(prop-1-en-2-yl)cyclohex-1-enyl)methyl)piperazine (1VI): Yield: 54.1%; colorless oil; Rf = 0.48 (CH2Cl2/MeOH/Et3N: 300/10/1). 1H-NMR δ: 5.59 , 4.72, 4.71 , 3.04–2.96 , 2.81–2.73 , 2.38–2.13 , 1.99–1.93 , 1.85–1.79 , 1.74 , 1.49–1.42 , 1.10 ; HRMS: m/z calcd. for C16H29N2 [M+H]+ 249.2331, found: 249.2325.(S)-1-Isopropyl-4-((4-(prop-1-en-2-yl)cyclohex-1-enyl)methyl)piperazine (2VI): Yield: 53.8%; pale yellow oil; Rf = 0.55 (CH2Cl2/MeOH/Et3N: 300/10/1). 1H-NMR δ: 5.60 , 4.77–4.63 , 2.86–2.76 , 2.69–2.42 , 2.15–1.91 , 1.87–1.78 , 1.73 , 1.50–1.38 , 1.10 2); HRMS: m/z calcd. for C17H31N2 [M+H]+ 263.2487, found: 263.2482.(S)-1-Methyl-4-((4-(prop-1-en-2-yl)cyclohex-1-enyl)methyl)piperazine (3VI): Yield: 48.2%; pale yellow oil; Rf = 0.50 (CH2Cl2/MeOH/Et3N: 300/10/1). 1H-NMR δ: 5.59 , 4.70 , 2.82, 2.54–2.29 , 2.28 , 2.25–1.79 , 1.73 , 1.54–1.33 ; HRMS: m/z calcd. for C15H27N2 [M+H]+ 235.2174, found: 235.2169.(S)-1-((4-(Prop-1-en-2-yl)cyclohex-1-enyl)methyl)piperidine (4VI): Yield: 34.2%; pale yellow oil; Rf = 0.44 (CH2Cl2/MeOH/Et3N: 300/10/1). 1H-NMR δ: 5.59 , 4.72 , 2.86–2.76, 2.38–2.23 , 2.18–1.82 , 1.75 , 1.60–1.41  [m/z calcd. for C15H26N [M+H]+ 220.2065, found: 220.2060.  ; HRMS: m(S)-N-((4-(Prop-1-en-2-yl)cyclohex-1-enyl)methyl)amantadine (5VI): Yield: 36.2%; mp: 223–224 °C; Rf = 0.32 (CH2Cl2/MeOH/Et3N: 300/10/1). 1H-NMR δ: 5.98 , 4.71, 4.67 , 3.49–3.40 , 2.29 , 2.17–2.05 , 1.96–1.82 , 1.73–1.65 , 1.51–1.45 ; HRMS: m/z calcd. for C20H32N [M+H]+ 286.2535, found: 286.2529.(S)-N-((4-(Prop-1-en-2-yl)cyclohex-1-enyl)methyl)diethanolamine (6VI): Yield: 38.4%; colorless oil; Rf = 0.34 (CH2Cl2/MeOH/Et3N: 300/10/1). 1H-NMR δ: 5.71–5.55 , 4.79–4.64 , 3.63 , 3.03 , 2.64 , 2.17–1.81 , 1.74 , 1.53–1.39 ; HRMS: m/z calcd. for C14H26NO2 [M+H]+ 240.1963, found: 240.1957.(S)-N-((4-(Prop-1-en-2-yl)cyclohex-1-enyl)methyl)cyclohexanamine (7VI): Yield: 35.4%; pale yellow oil; Rf = 0.39 (CH2Cl2/MeOH/Et3N: 300/10/1). 1H-NMR δ: 5.63 , 4.72, 4.70 , 3.20 , 2.50–2.45 , 2.15–2.06 , 1.96–1.81 , 1.73 , 1.70–1.43 , 1.28–1.13 ; HRMS;: m/z calcd. for C16H28N [M+H]+ 234.2222, found: 234.2216.Cell culture: Human lung cancer A549 cells, human melanoma A375-S2 Cells and human fibrosarcoma HT-1080 cells were obtained from American Type Culture Collection . A549 cells and HT1080 were cultured in DMEM medium with 10% fetal bovine serums , 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. A375-S2 cells were cultured in MEM medium with 10% fetal bovine serums, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in 5% CO2.MTT assay [3 cells per well. After 24 h incubation, they were treated with the tested agents for the indicated times. After washing the plates with PBS, a 20 μL aliquot of 3--2,5-diphenyltetrazolium (MTT) solution (5.0 mg/mL) was added to each well and incubated for 3 h. The resulting crystal was dissolved in dimethyl sulfoxide. Optical density was detected by ELISA reader . The percentage of cell viability inhibition was calculated as follows:TT assay : Cells wObservation of morphologic changes: A549 cells were treated with 0, 120, 160 and 180 μM 5VI for 24 h on 24-well flat bottom plates. Then changes in cellular morphology were examined using phase contrast microscopy .Acridine orange (AO) staining [5VI for 24 h on 24-well flat bottom plates. Then cells were washed with PBS, followed by incubation at room temperature with PBS containing 20 μg/mL AO for 15 min. The fluorescence of cells was observed using fluorescence microscopy.staining : A549 ceFlow cytometric analysis using propidium iodide (PI) [5VI for 24 h on 6-well flat bottom plates. The cells were harvested and washed by PBS and fixed by 70% cold ethanol at 4 °C for more than 18 h. After stained with 50 μg/mL PI and 1 mg/mL DNaseA-free RNaseA on ice in dark for 1 h, cells were analyzed on FACScan flow cytometer .ide (PI) : A549 ceWestern blot analysis [5VI for 24 h, both adherent and floating cells were collected and lysed by Ultrasonic Cell Disruptor  in whole cell lyse buffer (50 mM HEPES (pH 7.4), 1% Triton-X 100, 2 mM sodium orthovanadate, 100 mM sodiumfluoride, 1 mM edetic acid, 1 mM PMSF, 10 μg/mL aprotinin and 10 μg/mL leupeptin). The protein extracts was separated by 12% SDS-PAGE and transferred to PVDF membranes . After blocked with 5% skim milk, incubated with primary antibodies against procaspase-3 and caspase-3 at 4 °C overnight.analysis : After tStatistical assay: All the presented data were confirmed at least three independent experiments. The data were analyzed by ANOVA using Statistics Package for Social Science SPSS software .S)-perillyl alcohol were designed and synthesized. The target compounds showed improved antiproliferative activity against A549, A375-S2 and HT-1080 cells. The structure-activity relationships revealed that the replacement of the hydroxyl group of (S)-perillyl alcohol with an amino moiety was more favorable to improving cytotoxic activity than the introduction of an amino moiety at the terminal allyl group. The antiproliferative effect of 5VI was proved to be mediated through the induction of apoptosis in A549 cells.Two series of amino-modified derivatives of (


Acanthus montanus (Nees). T. Anderson, along with decaffeoylverbascoside, verbascoside, isoverbascoside, leucosceptoside A, (2R)-2-O-β-D-glucopyranosyl-2H-1,4-benzoxazin-3(4H)-one (HBOA-Glc), (2R)-2-O-β-D-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA-Glc), (3R)-1-octen-3-ol-3-O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside and ebracteatoside B. The structure elucidations were based on physical data and spectroscopic analyses including 1D- and 2D-NMR.A new phenylethanoid glycoside acylated with syringic acid, namely acanmontanoside, was isolated from the aerial portions of  Acanthus of the family Acanthaceae, we reported the constituents of Acanthus ilicifolius L., − .Amorphous powder, 1-5, benzoxazinoid glucosides 6, 7, and aliphatic alcohol glycosides 8, 9 from the aerial parts of A. montanus. These types of compounds have previously been reported in Acanthus species [The present study isolated phenylethanoid glycosides  species ,3,4,5. I