The synthesis was initiated by the reaction of 1 with 4-methylbenzenesulfonyl chloride (2) in aqueous sodium carbonate solution at pH 9 to yield N-(4-methoxyphenethyl)-4-methylbenzensulfonamide (3).This parent molecule 3 was subsequently treated with various alkyl/aralkyl halides, (4a–j), using N,N-dimethylformamide (DMF) as solvent and LiH as activator to produce a series of new N-(4-methoxyphenethyl)-N-(substituted)-4-methylbenzenesulfonamides (5a–j). The structural characterization of these derivatives was carried out by spectroscopic techniques like IR, 1H-NMR, and 13C-NMR. The elemental analysis data was also coherent with spectral data of these molecules. The inhibitory effects on acetylcholinesterase and DPPH were evaluated and it was observed that N-(4-Methoxyphenethyl)-4-methyl-N-(2-propyl)benzensulfonamide (5c) showed acetylcholinesterase inhibitory activity 0.075 ± 0.001 (IC50 0.075 ± 0.001 µM) comparable to Neostigmine methylsulfate (IC50 2.038 ± 0.039 µM).The docking studies of synthesized ligands 5a–j were also carried out against acetylcholinesterase (PDBID 4PQE) to compare the binding affinities with IC50 values. The kinetic mechanism analyzed by Lineweaver-Burk plots demonstrated that compound (5c) inhibits the acetylcholinesterase competitively to form an enzyme inhibitor complex. The inhibition constants Ki calculated from Dixon plots for compound (5c) is 2.5 µM. It was also found from kinetic analysis that derivative 5c irreversible enzyme inhibitor complex. It is proposed on the basis of our investigation that title compound 5c may serve as lead structure for the design of more potent acetylcholinesterase inhibitors.The present study comprises the synthesis of a new series of sulfonamides derived from 4-methoxyphenethylamine ( Sulfonamides are derivatives of sulfonic acids and are the basis of several groups of drugs. The original antibacterial sulfonamides (sulfa drugs) are synthetic. A general method for the synthesis of sulfonamides involves the coupling of sulfonyl chloride with primary or secondary amine or a substituted amine. A sulfonyl group plays a very important role as a key constituent of number of biologically active molecules . Sulfonapara position is designated as N4 while nitrogen of −SO2NH2 is designated as N1. Systemic sulfa drugs are evolved by substitution at N1 position whereas gut active sulfa drugs are produced by substituting N4 position. Research data showed that by substitution at N1 and N4 positions about 5,000 compounds have been synthesized which depicts the significance of these positions in designing of novel compounds  is a primary cholinesterase in the body which catalyzes the breakdown of acetylcholine and some other choline esters functioning as neurotransmitters. Acetylcholinesterase belongs to carboxylesterase family of enzymes and its activity serves to terminate synaptic transmission. Cholinestrases are potential target for the symptomatic treatment of Alzheimer’s disease and related dementias . Therefon-hexane as mobile phase. Spots were detected by UV254.All the chemicals, along with analytical grade solvents, were purchased from Sigma-Aldrich , Alfa Aesar  or Merck  through local suppliers. Pre-coated silica gel Al-plates were used for TLC with ethyl acetate and ν, cm−1) were recorded by KBr pellet method in the Jasco-320-A spectrometer. 1H-NMR spectra  were recorded at 600 MHz  in DMSO-d6 using the Bruker Advance III 600 Ascend spectrometer using BBO probe. The coupling constant (J) is given in Hz and chemical shift (δ) in ppm. The abbreviations used in interpretation of 1H NMR spectra are as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; br.t, broad triplet; q, quartet; quint, quintet; sex, sextet; sep, septet; m, multiplet.Gallonkamp apparatus was used to detect melting points in capillary tubes. IR spectra (1) was added in 40 mL of distilled water at room temperature and solution was stirred for 30 min. Ten percent Aqueous Na2CO3 solution was added in the reaction mixture to maintain pH up to 8–9. When the mixture was stirred for half an hour, 4-methylbenzenesulfonyl chloride  was added in the reaction mixture gradually. The mixture was stirred again for 2–3 h, and was monitored by TLC until completion in n-hexane: ethyl acetate . After completion of reaction, concentrated HCl was added drop wise till pH 5 to obtain the precipitates which was filtered, precipitates were washed with distilled water thoroughly to remove any impurities, and dried to yield the parent molecule, N-(4-methoxyphenethyl)-4-methylbenzenesulfonamide (3), as off-white powder in 91% yield.In 250mL round bottom flask, 4-methoxyphenethylamine -4-methylbenzensulfonamide  dissolved in 5 mL N,N-dimethyl formamide (DMF) was taken in 50 mL round bottom flask. Catalytic amount of lithium hydride  as an activator was added in the reaction mixture and was stirred for 30 min at room temperature. Then, different alkyl/aralkyl halides  were added into the reaction mixture and was stirred again for 4–5 h. The reaction was monitored by TLC until completion in n-Hexane: ethyl acetate . When the reaction was completed, iced distilled water was poured into the reaction mixture and was shaken thoroughly till precipitates were formed. Precipitates obtained were filtered, washed and air-dried to get the respective pure products (5a–j).18H23NO3S; Mol. Mass: 333 g/mol: iR υ: 3,398 (Secondary amide N-H stretching), 3,045 (C-H str. of aromatic ring), 2,856 , 1,536 (C=C aromatic str.), 1,248 (C-O-C stretching of aromatic ether), 1,036 (C-N). 1H-NMR : δ 7.67 , 7.39 , 7.12 , 6.85 , 3.72 , 3.24 , 3.16 , 2.71 , 2.38 , 1.00 . 13C-NMR : δ 158.32 (C-4), 143.38 (C-4′), 137.15 (C-1′), 130.86 (C-1), 130.22 (C-2 & C-6), 130.12 (C-3′ & C-5′), 127.24 (C-2′ & C-6′), 114.28 (C-3 & C-5), 55.44 (C-9), 49.32 (C-8), 42.96 (C-1″), 34.46 (C-7), 21.37 (C-7′), 14.28 (C-2″). Anal. Calc. for C18H23NO3S (333.15): Calculated: C, 64.84; H, 6.95; N, 4.20. Found: C, 64.79; H, 6.87; N, 4.12.Light yellow gel; yield: 72%; Mol. Formula: C19H25NO3S; Mol. Mass: 347 g/mol: IR υ: 3,399 (secondary amide N-H stretching), 3,085 (C-H str. of aromatic ring), 2,889 , 1,553 (C=C aromatic str.), 1,254 (C-O-C stretching of aromatic ether), 1,050 (C-N). 1H-NMR : δ 7.68 , 7.40 , 7.11 , 6.84 , 3.72 , 3.21 , 3.04 , 2.69 , 2.39 , 1.45 , 0.80 . 13C-NMR : δ 158.32 (C-4), 143.40 (C-4′), 137.06 (C-1′), 130.85 (C-1), 130.24 (C-2 & C-6), 130.09 (C-3′ & C-5′), 127.28 (C-2′ & C-6′), 114.31 (C-3 & C-5), 55.48 (C-9), 50.13 (C-1″), 50.02 (C-8), 34.47 (C-7), 21.89 (C-2″), 21.40 (C-7′), 11.45 (C-3″). Anal. Calc. for C19H25NO3S (347.16): Calculated: C, 65.68; H, 7.25; N, 4.03. Found: C, 65.53; H, 7.19; N, 3.95.Off-white solid; yield: 75%; m.p.: 79 °C, Mol. Formula: C19H25NO3S; Mol. Mass: 347 g/mol: IR υ: 3,408 (secondary amide N-H stretching), 3,087 (C-H str. of aromatic ring), 2,894 , 1,579 (C=C aromatic str.), 1,258 (C-O-C stretching of aromatic ether), 1,059 (C-N). 1H-NMR : δ 7.72 , 7.40 , 7.16 , 6.87 , 3.96 , 3.73 , 3.18 , 2.82 , 2.38 , 0.93 . 13C-NMR : δ 158.34 (C-4), 143.33 (C-4′), 138.20 (C-1′), 131.23 (C-1), 130.23 (C-2 & C-6), 130.20 (C-3′ & C-5′), 127.22 (C-2′ & C-6′), 114.31 (C-3 & C-5), 55.48 (C-9), 49.62 (C-8), 46.34 (C-2″), 37.58 (C-7), 21.39 (C-7′), 20.94 (C-1″ & C-3″). Anal. Calc. for C19H25NO3S (347.16): Calculated: C, 65.68; H, 7.25; N, 4.03. Found: C, 65.56; H, 7.15; N, 3.90.White solid; yield: 81%; m.p.: 67 °C , Mol. Formula: C20H27NO3S; Mol. Mass: 361g/mol: IR υ: 3,414 (secondary amide N-H stretching), 3,088 (C-H str. of aromatic ring), 2,898 , 1,584 (C=C aromatic str.), 1,260 (C-O-C stretching of aromatic ether), 1,064 (C-N). 1H-NMR : δ 7.67 , 7.40 , 7.11 , 6.84 , 3.72 , 3.22 , 3.06 , 2.68 , 2.38 , 1.41 , 1.23 , 0.84 . 13C-NMR : δ 158.33 (C-4), 143.40 (C-4′), 137.01 (C-1′), 130.23 (C-2 & C-6), 130.10 (C-3′ & C-5′), 130.08 (C-1), 127.29 (C-2′ & C-6′), 114.31 (C-3 & C-5), 55.47 (C-9), 50.02 (C-8), 48.18 (C-1″), 34.48 (C-7), 30.66 (C-2″), 21.39 (C-7′), 19.74 (C-3″), 13.97 (C-4″). Anal. Calc. for C20H27NO3S (361.17): Calculated: C, 66.45; H, 7.53; N, 3.87. Found: C, 66.39; H, 7.47; N, 3.75.Dull yellow gel; yield: 69%; Mol. Formula: C21H29NO3S; Mol. Mass: 375 g/mol: IR υ: 3,417 (secondary amide N-H stretching), 3,090 (C-H str. of aromatic ring), 2,901 , 1,586 (C=C aromatic str.), 1,262 (C-O-C stretching of aromatic ether), 1,067 (C-N). 1H-NMR : δ 7.67 , 7.39 , 7.10 , 6.84 , 3.72 , 3.22 , 3.05 , 2.68 , 2.38 , 1.41 , 1.22 , 1.17 , 0.81 . 13C-NMR : δ 158.33 (C-4), 143.39 (C-4′), 137.00 (C-1′), 130.87 (C-1), 130.22 (C-2 & C-6), 130.10 (C-3′ & C-5′), 127.29 (C-2′ & C-6′), 114.29 (C-3 & C-5), 55.45 (C-9), 50.03 (C-8), 48.46 (C-1″), 34.51 (C-7), 28.70 (C-3″), 28.18 (C-2″), 22.15 (C-4″), 21.38 (C-7′), 14.24 (C-5″). Anal. Calc. for C20H27NO3S (361.17): Calculated: C, 66.45; H, 7.53; N, 3.87. Found: C, 66.38; H, 7.47; N, 3.74.Off-white gel; yield: 81%; Mol. Formula: C23H33NO3S; Mol. Mass: 403 g/mol: IR υ: 3,419 (secondary amide N-H stretching), 3,093 (C-H str. of aromatic ring), 2,904 , 1,589 (C=C aromatic str.), 1,267 (C-O-C stretching of aromatic ether), 1,069 (C-N). 1H-NMR : δ 7.66 , 7.38 , 7.10 , 6.83 , 3.71 , 3.22 , 3.05 , 2.68 , 2.37 , 1.38 , 1.25-1.15 , 0.84 . 13C-NMR : δ 158.34 (C-4), 143.34 (C-4′), 137.07 (C-1′), 130.86 (C-1), 130.17 (C-2 & C-6), 130.07 (C-3′ & C-5′), 127.27 (C-2′ & C-6′), 114.27 (C-3 & C-5), 55.41 (C-9), 49.99 (C-8), 48.47 (C-1″), 34.54 (C-7), 31.62 (C-5″), 28.72 (C-4″), 28.48 (C-2″), 26.47 (C-3″), 22.45 (C-6″), 21.35 (C-7′), 14.31 (C-7″). Anal. Calc. for C23H33NO3S (403.22): Calculated: C, 68.45; H, 8.24; N, 3.47. Found: C, 68.37; H, 8.17; N, 3.41.Off-white gel; yield: 70%; Mol. Formula: C25H29NO3S; Mol. Mass: 423 g/mol; IR υ: 3,434 (Secondary amide N-H stretching), 3,094 (C-H str. of aromatic ring), 2,924 , 1,604 (C=C aromatic str.), 1,266 (C-O-C stretching of aromatic ether), 1,104 (C-N). 1H-NMR : δ 7.64 , 7.39 , 7.28 , 7.15 , 7.20 , 7.09 , 6.83 , 3.71 , 3.24 , 3.10 , 2.68 , 2.52 , 2.38 , 1.70 . 13C-NMR : δ 158.32 (C-4), 143.47 (C-4′), 141.76 (C-1″), 136.86 (C-1′), 130.80 (C-1), 130.25 (C-2 & C-6), 130.11 (C-3′ & C-5′), 128.73 (C-3″ & C-5″), 128.66 (C-2″ & C-6″), 127.30 (C-2′ & C-6′), 126.25 (C-4″), 114.29 (C-3& C-5), 55.45 (C-9), 50.04 (C-8), 48.03 (C-9″), 34.37 (C-7), 32.65 (C-7″), 30.17 (C-8″), 21.40 (C-7′). Anal. Calc. for C25H29NO3S (423.19): Calculated: C, 70.89; H, 6.90; N, 3.31. Found: C, 70.84; H, 6.87; N, 3.25.Off-white solid; yield: 80%; m.p.: 73 °C , Mol. Formula: C24H27NO3S; Mol. Mass: 409 g/mol; IR υ: 3,465 , 2,945 , 1,649 (C=C aromatic str.), 1,265 (C-O-C stretching of aromatic ether), 1,159 (C-N). 1H-NMR : δ 7.79 , 7.45 , 7.34 , 7.20 , 7.10 , 6.87 , 6.80 , 6.75 , 4.31 , 3.67 , 3.09 , 2.81 , 2.41 , 2.32 . 13C-NMR : 158.24 (C-4), 145.65 (C-2″), 143.72 (C-4′), 137.33 (C-1′), 136.68 (C-1″), 130.78 (C-1), 130.36 (C-2 & C-6), 130.18 (C-3″), 130.00 (C-3′ & C-5′), 128.27 (C-6″), 127.11 (C-5″), 126.24 (C-4″), 127.53 (C-2′ & C-6′), 114.28 (C-3 & C-5), 55.42 (C-9), 50.96 (C-7″), 50.12 (C-8), 34.28 (C-7), 21.44 (C-7′), 19.13 (C-8″). Anal. Calc. for C24H27NO3S (409.17): Calculated: C, 70.39; H, 6.65; N, 3.42. Found: C, 70.31; H, 6.57; N, 3.37.Dull white gel; yield: 80%; Mol. Formula: C24H27NO3S; Mol. Mass: 409 g/mol; IR υ: 3,465 (secondary amide N-H stretching), 3,096 (C-H str. of aromatic ring), 2,946 , 1,653 (C=C aromatic str.), 1,266 (C-O-C stretching of aromatic ether), 1,164 (C-N). 1H-NMR : δ 7.81 , 7.45 , 7.23 , 7.03 , 7.11-7.08 , 6.88 , 4.29 , 3.69 , 3.18 , 2.47 , 2.40 , 2.26 . 13C-NMR : 158.27 (C-4), 143.61 (C-4′), 137.18 (C-1″), 137.13 (C-1′), 136.70 (C-3″), 130.65 (C-1), 130.36 (C-2 & C-6), 130.32 (C-3′ & C-5′), 130.17 (C-2″), 129.26 (C-4″), 128.26 (C-6″), 127.37 (C-2′ & C-6′),126.31 (C-5″), 114.29 (C-3 & C-5), 55.44 (C-9), 50.51 (C-7″), 49.88 (C-8), 33.96 (C-7), 21.42 (C-7′), 21.30 (C-8″). Anal. Calc. for C24H27NO3S (409.17): Calculated: C, 70.39; H, 6.65; N, 3.42. Found: C, 70.30; H, 6.59; N, 3.39.White solid; yield: 80%; m.p.: 30 °C , Mol. Formula: C24H27NO3S; Mol. Mass: 409 g/mol; IR υ: 3,468 (Secondary amide N-H stretching), 3,096 (C-H str. of aromatic ring), 2,946 , 1,654 (C=C aromatic str.), 1,267 (C-O-C stretching of aromatic ether), 1,167 (C-N). 1H-NMR : δ 7.80 , 7.73 , 7.45 , 7.41 , 7.15 , 6.88 , 4.29 , 3.69 , 3.16 , 2.82 , 2.40 , 2.29 . 13C-NMR : 158.28 (C-4), 143.58 (C-4′), 137.23 (C-1′), 137.17 (C-1″), 136.70 (C-4″), 130.61 (C-1), 130.36 (C-2 & C-6), 130.17 (C-3′ & C-5′), 129.46 (C-3″ & C-5″), 128.27 (C-2″ & C-6″), 127.36 (C-2′ & C-6′), 114.29 (C-3 & C-5), 55.43 (C-9), 50.52 (C-7″), 49.8 (C-8), 33.85 (C-7), 21.56 (C-7′), 21.14 (C-8″). Anal. Calc. for C24H27NO3S (409.17): Calculated: C, 70.39; H, 6.65; N, 3.42. Found: C, 70.29; H, 6.51; N, 3.35.White solid; yield: 67%; m.p.: 97 °C , Mol. Formula: C50 values were calculated by nonlinear regression using GraphPad Prism 5.0.The inhibitory activities of synthesized compounds were determined spectrophotometrically using acetylthiocholine iodide as substrate by following the method of . BrieflyB and S are the absorbance’s for the blank and samples.The % of Inhibition of Acetylcholinesterase was calculated as following: 50 value) was analyzed as the same method described in acetylcholinesterase inhibition assay section. The reaction mixture consisted of 180 µL of 50 mM Tris-HCl buffer (pH 7.7); 10 µL of 5c at the different concentrations  and 20 µL enzyme AChE (50 U per well). We added 20 µL of DTNB 0.3 mM and 20 µL of ATCI substrate at the different concentrations  and mixed, pre-incubated time was same as acetylcholinesterase assay. We measured the absorbance at 412 nm up to 5 min. Lineweaver-Burk plot of the inverse of velocities vs. the inverse of substrate concentration was used for assessment of the type of inhibition AChE kinetically. The EI dissociation constant Ki was calculated from the secondary plot of 1/V vs. inhibitor concentration. The results (change in absorbance per sec) were processed by using SoftMaxPro software.The kinetic inhibition of 5c (selected ligand based upon most potent IChttps://www.rcsb.org/structure/4pqe). UCSF Chimera 1.10.1 tool was employed for energy minimization by using conjugate gradient algorithm and amber force field (The protein structure of human acetylcholinesterase (PDBID: 4PQE) was accessed form Protein Data Bank (PDB)  of synthesized compounds, 5a–j, were predicted and justified, respectively using online computational tools such as Molinspiration (http://www.molinspiration.com/) and Molsoft (http://www.molsoft.com/).The synthesized ligands, 5a–j) against targeted protein through PyRx docking tool  values and hydrogen/hydrophobic interactions pattern using UCSF Chimera 1.10.1 tool. The 2D graphical depiction of all other docked complexes were evaluated by Discovery Studio tool.Docking experiment was performed on all synthesized compounds (ing tool . In dock5a–j), were synthesized with 4-methoxyphenethylamine (1) as starting material according to the outline illustrated in 1) and 4-methylbenzenesulfonyl chloride (2) in aqueous alkaline medium with 4–5 h stirring at room temperature to afford parent molecule; N-(4-methoxyphenethyl)-4-methylbenzensulfonamide (3), which was isolated by acidification of the reaction mixture to pH 2–3 with concentrated HCl in good yield as off-white powder. In the second step 3, was subjected to nucleophilic substitution using different alkyl/aralkyl halides  as electrophiles in polar aprotic solvent, i.e., DMF, using LiH as a base to achieve target N-(4-methoxyphenethyl)-4-methyl-N-(substituted)benzensulfonamides (5a–j). These synthesized derivatives were subjected to structural analysis using spectroscopic techniques like IR, 1H-NMR and 13C-NMR, along with elemental analysis. The structural characterization of one of the molecules is discussed hereby in detail for the expediency of the readers. The molecule 5b was obtained as white solid in 90.3% yield having melting point 99 °C. The molecular formula of this compound was established by counting the number of protons in its 1H-NMR spectrum and number of carbon resonances in 13C-NMR spectrum. The CHNS analysis data was also in agreement with its molecular formula, C19H25NO3S. Various functionalities in the molecule were affirmed by its IR data. Absorption bands were observed at 3,399 (secondary amide N-H stretching), 3,085 (C-H stretching of aromatic ring), 2,889 , 1,553 (C=C aromatic stretching), 1,254 (C-O-C stretching of aromatic ether), 1,050 (C-N). In its 1H-NMR spectrum . Similarly, another di-ortho coupled pattern was observed for 4-methoxyphenethylamino moiety at δ 7.11  and δ 6.84 , along with a singlet at δ 3.72 for –OCH3 group at 9 position. Triplets were observed for two connected methylenes, resonating one at δ 3.21 (CH2-8) and other at δ 2.69 (CH2-7). The chemical shifts of these were consistent that former methylene was attached with a nitrogen atom while latter was linked with an aromatic ring. The 1-Propyl group, substituted at nitrogen atom, displayed three signals, a triplet at δ 3.04 (CH2-1″), a sextet at δ 1.45 for CH2-2″ and a triplet for terminal methyl group at δ 0.80 (CH3-3″). The 13C-NMR spectrum (δ 143.40 (C-4′), 137.06 (C-1′), 130.09 (C-3′ & C-5′) and 127.28 (C-2′ & C-6′) along with a signal of para-positioned methyl group at δ 21.40 (C-7′). Similarly, the signals of 4-methoxyphenethylamino unit were pragmatic at δ 158.32 (C-4), 130.85 (C-1), 130.24 (C-2 & C-6), 114.31 (C-3 & C-5), 55.48 (C-9), 50.02 (C-8) and 34.47 (C-7). The 1-Propyl substituent attached to the nitrogen atom in the molecule was corroborated by three peaks at δ 50.13 (C-1″), 21.89 (C-2″) and 11.45 (C-3″). So, on the basis of aforementioned cumulative spectral evidences, the structure of 5b was confirmed and it was named as N-(4-methoxyphenethyl)-4-methyl-N-(1-propyl)benzensulfonamide. A similar protocol was exercised for the structural characterization of other derivatives.In the presented work, 10 sulfonamide derivatives, -N-(substituted)-4-methylbenzenesulfonamides (5a–j) have been designed to evaluate their inhibitory effects on acetylcholinesterase enzyme. Neostigmine methylsulfate, which is a competitive acetylcholinesterase inhibitor, was used as standard for comparison purpose. The targeted sulfonamides as described in the preceding section are of keen interest because most of the molecules exhibited lower IC50 values as compared to the standard used. The most active compound was 5c which exhibited better IC50 value of 0.075 ± 0.001 µM, relative to the standard having an IC50 value of 2.038 ± 0.039 µM. The inhibitory potential of 5c might be attributed to the substitution of a branched alkyl (isopropyl) group in this molecule. Similarly, the molecules 5b and 5d also exhibited good inhibitory potentials against acetylcholinesterase with IC50 values of 0.119 ± 0.701 µM and 0.124 ± 0.021 µM, respectively. The suitable inhibitory potential of 5b and 5d might be an outcome of the substitution of 1-propyl and 1-butyl groups, respectively. Therefore, it has been exposed from our bioassay results  against acetylcholinesterase was analyzed using kinetic assay. Based upon our IC50 results, we select our most potent compound 5c to determine their inhibition type and inhibition constant. The kinetic results of the enzyme by the Lineweaver-Burk plot of 1/V versus substrate acetylthiocholine iodide 1/[S] in the presence of different inhibitor concentrations gave a series of straight lines, the result of Lineweaver-Burk plot of compound 5c showed that Vmax remains the same without significantly effecting the slopes. Km increases with increasing concentration while Vmax remains the same with insignificant difference. This behavior indicates that 5c compound inhibits the enzyme in competitive manner  and psi (ψ) angles among the coordinates of receptor and most of residues were plunged in acceptable region.Human acetylcholinesterase is a class of hydrolase protein having single chain and comprises 543 amino acids. The VADAR 1.8 structure analysis of human acetylcholinesterase depicted that, it consists of 33% d region , 4B. The5a–j) were predicted by using computational tools. The synthesized compounds were validated through RO5 analysis. It has been observed that log P and molecular mass values should be less than 5 and 500 (g/mol), respectively. Moreover, compounds should possess no greater than 10 HBA and 5 HBD, respectively. Literature data exposed that the exceed values of HBA and HBD results in poor permeation   . Howeverlue (>5) .5a–j) were analyzed on the basis of lowest binding energy values  and hydrogen/hydrophobic bonding analyses. The docking energy values and their binding pattern inside the active region of target protein is mentioned in The docked complexes of synthesized compounds (in-vitro results 5c was most active compounds enzyme inhibition experiment. Therefore, 5c was most active in the in-vitro analysis therefore, selected to view conformational pose in the target protein. The SAR analysis shows that 5c interacted with protein residues Leu289 and Trp286 by hydrophobic and π–π interaction, respectively. The methyl group of benzene ring form hydrophobic interaction against Leu289 having bond length 4.26 Å. Moreover, single π–π interaction was observed between benzene of 5c and Trp286 residue having distance 5.38 Å. Literature study also justified that these interacted residues are significant in downstream signaling pathways and justify the significance of our docking results (All synthesized compounds directly interact within the active region with different conformational positions. Based on  results . The gra5c inhibits acetylcholinesterase via competitive inhibition mode having a Ki value 2.5 µM. Molecular docking studies also found in good correlation with the experimental results. Both bioactivity and computational studies results depicted that these newly synthesized molecules can serve a structural template in designing novel drugs against Alzheimer’s disease.In this research article, a novel series of sulfonamides derived from 4-methoxyphenethylamine were synthesized and the synthesized compounds were characterized through FT-IR, 1H NMR, 13C NMR. All the synthesized compounds showed significant activity against acetylcholinesterase. Kinetic studies were explored to find the binding mode of inhibition, and it was found that compound 10.7717/peerj.4962/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4962/supp-2Data S1Click here for additional data file.10.7717/peerj.4962/supp-3Data S2Click here for additional data file.10.7717/peerj.4962/supp-4Supplemental Information 2Click here for additional data file.10.7717/peerj.4962/supp-5Data S3Click here for additional data file.10.7717/peerj.4962/supp-6Supplemental Information 3Click here for additional data file.


C-glycoside, isovitexin 6″-O-E-p-coumarate (1) and two known flavonoid glycosides—quercetin 3-O-β-D-glucuronopyranoside (2) and isoorientin (3)—were isolated from an ethanol extract of aerial parts of Clematis rehderiana. Their structures were determined by spectroscopic methods. The antioxidant effects of the two flavone C-glycosides were evaluated by both the MTT and DPPH assays. Compound 1 showed potent activities against H2O2-induced impairment in PC12 cells within the concentration range tested, whereas compound 3 scavenged DPPH radical strongly, with an IC50 value of 13.5 μM.A new flavone  Clematis (Ranunculaceae), which comprises about 300 species, is widespread throughout the World. About 147 species (93 endemic ones) are distributed in China, and 56 of these are distributed in Yunnan province −, calcd: 577.1346.Compound 2:  = −8.6 ; UV (MeOH) λmax (log ε) 404, 270, 210 nm; IR (KBr): 3,408, 2,922, 1,653, 1,604; HRFABMS (neg): m/z 477.0669 [M-H]−, calcd: 477.0669; 1H-NMR (DMSO-6d) δ: 12.35 , 8.05 , 7.42 , 6.83 , 6.39 , 6.19 , 5.32 , 3.39 1H, m, H-5″), 3.11-3.25 ; 13C-NMR (DMSO-6d) δ: 157.2 (C-2), 133.8 (C-3), 177.4 (C-4), 161.0 (C-5), 98.9 (C-6), 164.5 (C-7), 93.7 (C-8), 156.4 (C-9), 103.7 (C-10), 120.6 (C-1′), 115.3 (C-2′), 144.8 (C-3′), 148.5 (C-4′), 117.5 (C-5′), 120.9 (C-6′), 102.4 (C-1″), 74.1 (C-2″), 76.4 (C-3″), 71.7 (C-4″), 74.7 (C-5″), 171.7 (C-6″).2O2-induced impairment in PC12 cells was conducted according to the reported protocol [1 and 3 together with freshly prepared H2O2 were added and incubation continued for 1 hour. Then MTT -2,5-diphenyltetrazolium bromide) solution was added and the incubation continued for 4 hours. Finally, solution (100 μL) containing 5% iso-butanol, 10% SDS (Sigma) and 0.004% HCl was added. The mixtures were kept overnight and the index of cell viability (% of control) was calculated by measuring the optical density of the color produced by MTT dye reduction with a microplate reader at 570 nm.The antioxidant assay against Hprotocol . Briefly50 values denote the concentration of sample required to scavenge 50% DPPH free radicals.DPPH radical-scavenging activity assays were performed according to our previously reported procedures . Each coC-glycoside, isovitexin 6″-O-E-p-coumarate (1) and two known flavonoid glycosides—quercetin 3-O-β-D-glucuronopyranoside (2) and isoorientin (3)—were isolated from an ethanol extract of aerial parts of C. rehderiana. The antioxidant effects experiments of these compounds by both the MTT and DPPH assays suggested that flavone glycosides were major constituents with antioxidant activities in this plant. A new flavone


Cistanches deserticola and purified by preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water . A total of 30.9 mg acteoside, 13.0 mg isoacteoside, 12.5 mg syringalide A 3'-α-L-rhamnopyranoside and 7.2 mg 2'-acetylacteoside with purity of higher than 95%, as determined by HPLC-ELSD, were obtained in one-step separation from 297 mg of Cistanche deserticola extract, respectively. Their structures were identified by HR-MS, 1H-NMR and 13C-NMR.Following a constituent enrichment step on a silica gel column, four phenyl-ethanoid glycosides were successfully isolated from  Cistanches deserticola Y.C. Ma, a species of Cistanches which belongs to the Orobanchaceae family, is a well-known Traditional Chinese Medicine for the treatment of kidney deficiency, female infertility, morbid leucorrhea, neurataxia and senile constipation [Cistanches genus and others by HSCCC [α-L-rhamnopyranoside and 2'-acetylacteoside in one biphasic system.tipation . It is atipation . Some oftipation ,4,5,6. Htipation ,8,9. Altby HSCCC ,12,13,14C. deserticola. Finally, 30.9 mg of acteoside, 13.0 mg of isoacteoside, 12.5 mg of syringalide A 3'-α-L-rhamnopyranoside and 7.2 mg of 2'-acetylacteoside were obtained in a one-step separation from 297 mg of fraction with purities of 99%, 95%, 99% and 98%, respectively. Their structures were characterized on the basis of 1H- and 13C-NMR spectral data and HR-MS spectra. The structures of the four PhGs identified in this investigation are shown in In this paper, we report a simple method for the separation and purification of four PhGs from n-butanolic extract from C. deserticola was analyzed by HPLC-UV. The column used was an Accurasil C18  , the mobile phase was methanol-water . The flow rate was 1 mL/min. Detector wavelength was set at 254 nm. The HPLC chromatogram is shown in 1), isoacteoside (2), syringalide A 3'-α-L-rhamnopyranoside (3) and 2'-acetylacteoside (4), respectively.The constituent enriched fraction of K) of each target component and an optimum range of K should be from 0.5 to 2.Successful separation by HSCCC largely depends upon the selection of a suitable two-phase solvent system, the two phase solvent system was selected according to the partition coefficient values  gave suitable partition coefficients for the target compounds. Eventually, the solvent system composed of ethyl acetate–n–butanol– ethanol–water  was used to isolate and purify four compounds of C. deserticola, as shown in α-L-rhamnopyranoside 99% and 2′-acetylacteoside 98%) could be obtained from the crude fraction.In the experiment, the R) was 5 cm, and the β value varied from 0.5 at the internal terminal to 0.8 at the external terminal . An HX 105 constant-temperature circulation implement  was used to control temperature in the experiment. The HSCCC system was equipped with a model TBP-5002 middle pressure constant-flow pump, a model TBP-2000 UV detector operating at 254 nm, and a model V4.0 workstation .The HSCCC equipment employed in the present study was a TBE-300B high-speed counter-current chromatography system  with three preparative multilayer coil separation columns connected in series  and a 20 mL sample loop. The revolution radius or the distance between the holder axis and central axis of the centrifuge  were measured at room temperature (22 °C/295.1 K).The high-performance liquid chromatography (HPLC) equipment used was an Agilent 1200 system which consists of a G1312A binary pump, a G1329A autosampler, a G1314A variable wavelength detector (VWD), a G1316A thermostatted column compartment, a G1379B degasser and Agilent LC workstation. An alltech 3300 ELSD was used for detection of the purities. Agilent 6520 Q-TOF and Bruker AVANCE III 500-NMR spectrometers were used for compounds’ structural identification. n-butanol and ethanol were analytical grade and methanol was HPLC grade. All the solvents were purchased from Tianjin Concord Technology Company . Milli-Q water (18.2 MΩ)  was used for all solutions and dilutions. The rhizome of C. deserticola was collected from the Inner-Mongolia Province, People’s Republic of China, in June 2009. The plant was identified by Prof. Lijuan Zhang, and a voucher specimen (No. 20091001) was deposited in our laboratory.Ethyl acetate, C. deserticola (1 Kg) were refluxed twice with aqueous ethanol  for 2 h each time. The extract was evaporated under reduced pressure and at a temperature of 60 °C until total evaporation of the ethanol. The residue was suspended in water and extracted successively three times with chloroform, ethyl acetate and n-butanol, affording 5.16 g of n-butanolic extract after evaporation to dryness under reduced pressure. The n-butanolic extract was then separated on a silica gel column (200–300 mesh) using progressive gradient elution  to give eight fractions. Fraction 6 (297 mg) was used for further HSCCC isolation and separation.Powdered air dried fleshy stems of K) of the target components in the fraction of C. deserticola. The K values were determined by LC analysis as follows: Suitable amount of crude extract powder (fraction 6) was dissolved in the lower phase of the solvent system and analyzed by HPLC. The areas of the peaks were recorded as A1. Then equal volume of the upper phase was added to the solution and mixed thoroughly to reach partition equilibrium. The lower phase was then analyzed by HPLC again. The latter peak areas were recorded as A2. The K-values were calculated according to the following equation: K = (A1 − A2)/A2 [The two-phase solvent systems were selected according to the partition coefficient (− A2)/A2 ,16.n-butanol–ethanol–water  was used for HSCCC separation. Each solvent was added to a separatory funnel and thoroughly equilibrated at room temperature. The upper phase and the lower phase were separated and degassed by sonication for 30 min shortly before use. The sample solution was prepared by dissolving 297 mg of Fraction 6 in 15 mL of the lower phase of ethyl acetate–n-butanol–ethanol–water .In the present study, the two-phase solvent system composed of ethyl acetate–HSCCC was performed as follows. The multilayer coiled column was first entirely filled with the upper organic stationary phase. The lower aqueous mobile phase was then pumped into the head end of the column at a flow-rate of 1.5 mL/min, and at the same time, the HSCCC apparatus was run at a revolution speed of 900 rpm. After hydrodynamic equilibrium was established, as indicated by a clear mobile phase eluting at the tail outlet ( about two hours later ), 15 mL sample solution containing 297 mg of the crude extract (fraction 6) was injected through the injection valve. The effluent of the column was continuously monitored under 254 nm. Four peak fractions were collected according to the chromatogram and then evaporated under reduced pressure. The temperature of the apparatus was set at 25 °C.v/v). The flow rate was 1 mL/min. ELSD was operated under the following conditions: Temp 45 °C, gas 1.6 L/min. Structural identification of the four HSCCC peak fractions was carried out by HR-ESI-MS, 1H and 13C-NMR spectroscopy.The peak fractions from HSCCC were analyzed by HPLC-ELSD. The column used was an Accurasil C18 , the mobile phase was methanol–water −, calcd for C29H35O15, 623.1981. 1H-NMR  δ ppm: 1.09 , 2.79 , 4.37 , 5.18 , 6.27 , 7.59 , 6.5–7.1 . 13C-NMR  δ ppm: 131.5 (C-1), 117.2 (C-2), 146.2 (C-3), 144.7 (C-4), 116.4 (C-5), 121.3 (C-6), 72.4 (C-α), 36.6 (C-β), 127.7 (Caf-1), 115.3 (Caf-2), 146.9 (Caf-3), 149.8 (Caf-4), 116.6 (Caf-5), 123.2 (Caf-6), 168.3 (Caf-α), 114.8 (Caf-β), 148.1 (Caf-γ), 104.3 (G-1), 76.1 (Glc-2), 81.7 (Glc-3), 70.5 (Glc-4), 76.3 (Glc-5), 62.4 (Glc-6), 103.1 (Rha-1), 72.3 (Rha-2), 72.1 (Rha-3), 73.9 (Rha-4), 70.7 (Rha-5), 18.5 (Rha-6). Compared with the data given in literature [Fraction II: HR-ESI-MS observed at m/z 623.1987 (M−H)−, calcd for C29H35O15, 623.1981. 1H-NMR  δ ppm: 1.25 , 2.78 , 4.33 , 5.17 , 6.28 , 7.56 , 6.5–7.0 . 13C-NMR  δ ppm: 131.5 (C-1), 117.2 (C-2), 146.2 (C-3), 144.7 (C-4), 116.5 (C-5), 121.3 (C-6), 72.4 (C-α), 36.7 (C-β), 127.8 (Caf-1), 115.2 (Caf-2), 146.8 (Caf-3), 149.7 (Caf-4), 116.6 (Caf-5), 123.2 (Caf-6), 169.2 (Caf-α), 115.0 (Caf-β), 147.3 (Caf-γ), 104.5 (G-1), 75.5 (Glc-2), 84.2 (Glc-3), 70.1 (Glc-4), 75.7 (Glc-5), 64.7 (Glc-6), 102.8 (Rha-1), 72.4 (Rha-2), 72.4 (Rha-3), 74.1 (Rha-4), 70.5 (Rha-5), 17.9 (Rha-6). The 1H-NMR and 13C-NMR spectral data were in agreement with those of isoacteoside as reported in the literature [Fraction terature .III: HR-ESI-MS observed at m/z 607.2032 (M−H)−, calcd for C29H35O14, 607.2032. 1H-NMR  δ ppm: 1.09 , 2.84 , 4.37 , 5.19 , 6.27 , 7.59 , 6.6–7.1 . 13C-NMR  δ ppm: 130.8 (C-1), 116.2 (C-2), 130.9 (C-3), 156.8 (C-4), 130.8 (C-5), 116.2 (C-6), 72.4 (C-α), 36.4 (C-β), 127.8 (Caf-1), 114.8 (Caf-2), 149.8 (Caf-3), 146.9 (Caf-4), 116.6 (Caf-5), 123.2 (Caf-6), 168.3 (Caf-α), 115.4 (Caf-β), 148.0 (Caf-γ), 104.3 (G-1), 76.3 (Glc-2), 81.7 (Glc-3), 70.4 (Glc-4), 76.1 (Glc-5), 62.5 (Glc-6), 103.0 (Rha-1), 72.3 (Rha-2), 72.2 (Rha-3), 73.9 (Rha-4), 70.7 (Rha-5), 18.4 (Rha-6). According to the literature [α-L-rhamnopyranoside.Fraction IV: HR-ESI-MS observed at m/z 665.2100 (M−H)−, calcd for C31H37O16, 665.2087. 1H-NMR  δ ppm: 1.09 , 2.00 , 2.72 , 4.55 , 4.90 , 6.29 , 7.62 , 6.5–7.0 .13C-NMR  δ ppm: 131.9 (C-1), 117.3 (C-2), 146.1 (C-3), 144.7 (C-4), 116.4(C-5), 121.4 (C-6), 72.7 (C-α), 36.4 (C-β), 127.7 (Caf-1), 115.4 (Caf-2), 146.9 (Caf-3), 149.9 (Caf-4), 116.6 (Caf-5), 123.2 (Caf-6), 168.1 (Caf-α), 114.7 (Caf-β), 148.2 (Caf-γ), 101.8 (G-1), 75.3 (Glc-2), 80.3 (Glc-3), 70.8 (Glc-4), 76.2 (Glc-5), 62.3 (Glc-6), 103.3 (Rha-1), 72.0 (Rha-2), 71.8 (Rha-3), 73.7 (Rha-4), 70.8 (Rha-5), 18.5 (Rha-6), 171.5 (C=O), 20.9 (OAC). The 1H-NMR and 13C-NMR spectral data were in agreement with those of 2'-acetylacteoside [Fraction α-L-rhamnopyranoside and 2'-acetylacteoside from Cistanches deserticola Y.C. Ma was established. The present study indicates that HSCCC is a very powerful technique for the preparative separation and purification of bioactive components from plant materials. Also, the compounds can be isolated on a sufficiently large scale with high purities and may then be used as reference substances for chromatography or for bioactivity studies. The method is a feasible, economical, and efficient technique for rapid preparative isolation of complicated natural products.An HSCCC method for the preparative separation and purification of acteoside, isoacteoside, syringalide A 3'-


Cremanthodium stenactinium (Asteraceae) were collected in Sichuan Province, China. From the ethyl acetate extracts of the roots, three new eremophilane-type sesquiterpenoids and one new trinoreremophilane compound were isolated, together with other known eremophilanes. Their structures were determined based on the spectroscopic data. This is the first report of isolation of eremophilane-type compounds from the genus Cremanthodium.Two samples of  Cremanthodium (Asteraceae) are known to grow in high mountain areas and to be very small in size  -12000 at 225 nm (EtOH) which was similar to that of the known compound, 7 extract . Therefo3 exhibited a quasi-molecular ion peak at m/z 319 and the molecular formula was determined to be C20H30O3 by HRMS. The 1H-NMR spectrum indicated the presence of three doublet methyls, one singlet methyl, an exomethylene, a trisubstituted olefin, as well as two oxymethylene protons  ) D22 +99.5 ; FT-IR (KBr) 1695, 1676 cm−1; CD [θ] (EtOH) -5323 (319 nm), +27023 (237 nm); MS (CI) m/z 232 [M]+, 150, 135 (base); HRMS (CI) Obs m/z 232.1456 .4S,5R,7R-8-Oxoeremophila-9,11(13)-dien-12-al (2): [α]D22 −72 ; FT-IR (KBr) 1693, 1679 cm−1; CD [θ] (EtOH) +667 (328 nm), −12025 (225 nm); MS (CI) m/z 233 [M+H]+ (base), 150, 135; HRMS (CI) Obs m/z 233.1540 .4S,5R,7S-12-(3′-Methylbutyryloxy)eremophila-9,11(13)-dien-8-one (3): [α]D21 +178 ; FTIR (KBr) 1737, 1677 cm−1; CD [θ] (EtOH) −7240 (319 nm), +43635 (235 nm); MS (CI) m/z 319 [M+H]+ (base), 217; HRMS (CI) Obs m/z 319.2275 .4S,5R-Trinoreremophil-9-en-8-one (4): [α]D22 +143 ; FTIR (KBr) 1680 cm−1; CD [θ] (EtOH) -2768 (319 nm), +34310 (238 nm); MS (CI) m/z 179 [M+H]+, 89, 61 (base); HRMS (CI) Obs m/z 179.1422 .1 and 2, and sample 2 compounds 3 and 4. This is the first report of the isolation of eremophilane-type sesquiterpenoids from Cremanthodium spp. A trinoreremophilane compound, dendryphilellin A, has been isolated from the marine deuteromycete Dendryphiella salina [1, 2, 5, or 6. The present results show that eremophilane-type sesquiterpenoids are common compounds both in Ligularia and Cremanthodium, implying that the genus Cremanthodium is quite close to Ligularia or Parasenecio [Sample 1 afforded compounds a salina , but thea salina ,23,24,25asenecio . More sa


The structures of the new compounds were elucidated based on the analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. All compounds from the plants were measured for antifungal activities against Microsporum gypseum and Trichophyton rubrum. Glinusopposide B (6), glinusopposide Q (21), glinusopposide T (24), and glinusopposide U (25) showed strong inhibitory activities against M. gypseum  and T. rubrum . For those active compounds with an oleanane skeleton, glycosylation (21–26) or oxidation (3) of 3-OH was helpful in increasing the activity; replacement of the 30-methyl group (29) by a carboxymethyl group (26) enhanced the activity; the presence of 11,13(18) double bonds (20) decreased the activity.Four new triterpenoids, 3β,12β,16β,21β,22-pentahydroxyhopane ( Epidermophyton, Microsporum and Trichophyton + . By comparing its NMR data (O-β-d-xylopyranoside (31) [5 might be combined by a modified hopane and a β-xylopyranose [δH 4.87 d (J = 7.6 Hz)]. The configuration of xylopyranose in the plant was determined as the d-configuration by acidic hydrolysis of 31 followed by acetylation to yield 1,2,3,4-tetra-O-acetyl-d-xylopyranose. The genin was deduced as 16-deoxyspergulagenin A by 1H–1H COSY, HMBC, and ROESY experiments. The ROESY correlations .The HRESIMS of glinusopposide A  and C (7) have the same molecular formula, C41H68O11, based on 13C-NMR data ]-β-d-xylopyranoside (glinusopposide B) and 16-deoxyspergulagenin A 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide C), respectively.Both glinusopposides B  were deduced to be C43H70O13, C45H72O13, C47H74O14, and C44H70O13, respectively. By comparing their NMR data (O-β-d-xylopyranoside (31)  spergulagenin A (glinusopposide D), 3-O-[α-l-rhamnopyranosyl-(1→3)-4-O-trans-2-butenoyl-β-d-xylopyranosyl] spergulagenin A (glinusopposide E), 3-O-[α-l-rhamnopyranosyl-(1→3)-4-O-trans-2-butenoyl-β-d-xylopyranosyl] 12-O-acetylspergulagenin A (glinusopposide F), and 3-O-[β-d-xylopyranosyl-(1→3)-4-O-trans-2-butenoyl-β-d-xylopyranosyl] spergulagenin A (glinusopposide G), respectively.Based on NMR data  and HRESNMR data  with tho′/H-4′′′ . The clo spectra , the str12) was assigned the molecular formula C36H58O8, with eight degrees of unsaturation as determined by 13C-NMR data  in the HRESIMS. The 1H and 13C-NMR data indicated the compound might be a hopane triterpenoid saponin with eight methyl groups , one tetrasubstituted double bond (δC 152.5 and 146.8), and one β-glucopyranose . In addition to the signals for the sugar, signals  for four oxygenated carbon atoms were observed. The sugar was attached to C-12 based on the HMBC correlations from H-12 to C-1′ and from H-1′ to C-12, and the 17(21)-double bond was confirmed by the correlations from H3-28 to C-17 and from H3-29 and H3-30 to C-21  and C-22 (δC 84.2) to downfield compared with the analogues 1, 2, and 22,24,28-trihydroxy-hop-17(21)-ene -β-d-xylopyranoside (glinusopposide J), and 3β,12β-dihydroxy-16β,22-epoxyhop-17(21)-ene 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide K), respectively.Based on NMR data  and HRESof 13–15  with tho3–15 (12  which ar3–15 (12 . Therefo16) exhibited an ion peak at m/z 731.4353 [M + Na]+ , implying a molecular formula of C39H64O11. The NMR data of 16 (35) [16 was elucidated to be spergulatriol 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide L).The HRESIMS of glinusopposide L (ta of 16  were hig 16 (35) , indicat13C-NMR data , glinusopposide M (17) had the same molecular formula, C39H64O11, as saponin 16. The 1D and 2D-NMR spectra  double bond was identified based on the HMBC correlations from H3-28 to C-17 and from H3-22 to C-17 and C-21. Therefore, the structure of 17 was determined to be 29,30-bisnor-3β,12β,16β-trihydroxyhop-17(21)-ene 3-O-[α-l-rhamnopyranosyl-(1 → 3)]-β-d-xylopyranoside (glinusopposide M).According to NMR data  and the  spectra  indicate13C-NMR data (18) and O (19) were deduced to be C33H52O6 and C39H62O10, respectively. Comparison of their NMR data -diene 3-O-β-d-xylopyranoside (glinusopposide N) and 29,30-bisnor-3β,12β-dihydroxyhopa-15,17(21)-diene 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide O), respectively.According to NMR data  and HRESNMR data  with tho12/H3-28 . The 15, spectra , 18 and 20) was determined to be C44H66O15 based on 13C-NMR data  in the HRESIMS. The NMR data (4), an α-rhamnopyranosyl group , and a 6-O-methyl-β-glucuronopyranosyl group . The linkage of the sugar chain was determined to be Rha-(1→3)-[6-O-methyl-GlcA]-O-C-3 based on the key HMBC correlations from H-1″ to C-3′, from H-3′ to C-1″, from H-1′ to C-3, and from H-3 to C-1′ (20 was elucidated to be 3-O-[α-l-rhamnopyranosyl-(1→3)-6-O-methyl-β-d-glucuronopyranosyl]-3β-hydroxyoleana-11,13(18)-diene-28,30-dioic acid 30-methyl ester (glinusopposide P).The molecular formula of glinusopposide P  were deduced to be C39H61NO10, C44H68O15, C45H70O15, C36H56O9, and C42H66O13, respectively. By comparing their NMR data with those of 30-O-methyl spergulagenate (27) -30-O-methyl spergulagenate (glinusopposide R), 3-O-[α-l-rhamnopyranosyl-(1→3)-6-O-ethyl-β-d-glucuronopyranosyl]-30-O-methyl spergulagenate (glinusopposide S), 30-O-methyl spergulagenate 3-O-β-d-xylopyranoside (glinusopposide T), and 30-O-methyl spergulagenate 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide U), respectively.Based on NMR data  and HRESate (27) , these s spectra , saponin26 in methanol-d4 (O-β-d-glucuronopyranoside-6′-O-methyl ester   . Based oidine-d5 , especiaO-methyl spergulagenate (27) +; HRESIMS m/z 515.3718 [M + Na]+ .3β,12β,16β,21β,22-Pentahydroxyhopane  Å, b = 17.7389(6) Å, c = 20.4841(7) Å, α = 90°, β = 90°, γ = 90°, V = 2847.26(17) Å3, T = 100.(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.653 mm−1, 51,068 reflections measured, 5642 independent reflections (intR = 0.0514). The final 1R values were 0.0363 (I > 2σ(I)). The final wR(F2) values were 0.1081 (I > 2σ(I)). The final 1R values were 0.0371 . The final wR(F2) values were 0.1102 . The goodness of fit on F2 was 0.998. Flack parameter = 0.02(5). The supplementary crystallographic data can be obtained free of charge from the Cambridge Crystallographic Data Centre (CCDC) (deposition number CCDC 1917520) via http://www.ccdc.cam.ac.uk.Crystal data for 2). White powder; c 0.12, MeOH); UV (MeOH) λmax (logε) 280 (1.98), 219 (2.73), 203 (2.91) nm; ECD  λmax (Δε) 289 nm (+0.17), 232 (+0.28), 208 (–0.29) nm; IR νmax (KBr) 3443, 3427, 1690, 1452, 1385, 1084, 1047, 879 cm−1; 1H and 13C-NMR data, see m/z 513 [M + Na]+; HRESIMS m/z 513.3551 [M + Na]+ .12β,16β,21β,22-Tetrahydroxyhopan-3-one (3). White powder; c 0.26, MeOH); UV (MeOH) λmax (logε) 371 (1.73), 252 (2.75), 239 (2.72) nm; 1H and 13C-NMR data, see m/z 507 [M + Na]+; HRESIMS m/z 507.3084 [M + Na]+ .The 3-Oxo-olean-12-ene-28,30-dioic acid (4). White powder; c 0.15, MeOH); UV (MeOH) λmax (logε) 250 (2.65), 242 (2.59) nm; ECD  λmax (Δε) 250 (–3.92) nm; 1H and 13C-NMR data, see m/z 521 [M + Na]+; HRESIMS m/z 521.3234 [M + Na]+ .The 3β-Hydroxyoleana-11,13(18)-diene-28,29-dioic acid 29-methyl ester (5). White powder; c 0.05, MeOH); 1H and 13C-NMR data, see m/z 613 [M + Na]+; HRESIMS: m/z 613.4068 [M + Na]+ .Glinusopposide A (6). White powder; c 0.1, MeOH); 1H and 13C-NMR data, see m/z 759 [M + Na]+; HRESIMS m/z 759.4650 [M + Na]+ .Glinusopposide B (7). White powder; c 0.35, MeOH); 1H and 13C-NMR data, see m/z 759 [M + Na]+; HRESIMS m/z 759.4656 [M + Na]+ .Glinusopposide C (8). White powder; c 0.16, pyridine); UV (MeOH) λmax (logε) 275 (2.77), 245 (3.02), 204 (3.59) nm; ECD  λmax (Δε) 284 (+1.00), 249 (–0.17), 218 (+1.03), 197 (–1.11) nm; 1H and 13C-NMR data, see m/z 833 [M + K]+, 817 [M + Na]+; HRESIMS m/z 817.4709 [M + Na]+ .Glinusopposide D (9). White powder; c 0.09, MeOH); UV (MeOH) λmax (logε) 415 (2.39), 206 (4.09) nm; ECD  λmax (Δε) 284 (+0.48), 206 (+0.73), 200 (–1.80) nm; IR νmax (KBr) 3442, 3428, 1677, 1644, 1449, 1431, 1385, 1202, 1144, 1086, 1047, 879 cm−1; 1H and 13C-NMR data, see m/z 843 [M + Na]+; HRESIMS m/z 843.4867 [M + Na]+ .Glinusopposide E (10). White solid; c 0.04, MeOH); 1H and 13C-NMR data, see m/z 885 [M + Na]+; HRESIMS m/z 885.4930 [M + Na]+ .Glinusopposide F (11). White powder; c 0.15, MeOH); UV (MeOH) λmax (logε) 252 (3.21), 206 (3.81) nm; ECD  λmax (Δε) 201 (–1.59), 197 (+1.29) nm; IR νmax (KBr) 3448, 3427, 1639, 1447, 1383, 1084, 1046, 879 cm−1; 1H and 13C-NMR data, see m/z 829 [M + Na]+; HRESIMS m/z 829.4711 [M + Na]+ .Glinusopposide G (12). White powder; c 0.28, MeOH); 1H and 13C-NMR data, see m/z 641 [M + Na]+; HRESIMS m/z 641.4021 [M + Na]+ .Glinusopposide H (13). White powder; c 0.07, MeOH); 1H and 13C-NMR data, see m/z 611 [M + Na]+; HRESIMS m/z 611.3898 [M + Na]+ .Glinusopposide I (14). White powder; c 0.11, MeOH); 1H and 13C-NMR data, see m/z 757 [M + Na]+; HRESIMS m/z 757.4486 [M + Na]+ .Glinusopposide J (15). White powder; c 0.13, MeOH); 1H and 13C-NMR data, see m/z 757 [M + Na]+; HRESIMS m/z 757.4510 [M+ Na]+ .Glinusopposide K (16). White powder; c 0.1, MeOH); UV (MeOH) λmax (logε) 203 (3.82) nm; ECD  λmax (Δε) 196 (–6.89) nm; IR νmax (KBr) 3425, 1632, 1454, 1385, 1129, 1094, 1047, 974 cm−1; 1H and 13C-NMR data, see m/z 731 [M + Na]+; HRESIMS m/z 731.4353 [M + Na]+ .Glinusopposide L (17). White powder; c 0.05, MeOH); UV (MeOH) λmax (logε) 203 (3.54) nm; ECD  λmax (Δε) 206 (–4.38), 196 (+4.32) nm; IR νmax (KBr) 3443, 3426, 1639, 1453, 1421, 1384, 1084, 1047, 879 cm−1; 1H and 13C-NMR data, see m/z 731 [M + Na]+; HRESIMS m/z 731.4347 [M + Na]+ .Glinusopposide M (18). White powder; c 0.18, MeOH); UV (MeOH) λmax (logε) 250 (3.76), 213 (3.38) nm; 1H and 13C-NMR data, see m/z 567 [M + Na]+; HRESIMS m/z 567.3655 [M + Na]+ .Glinusopposide N (19). White powder; c 0.13, MeOH); UV (MeOH) λmax (logε) 250 (3.76), 218 (3.43) nm; 1H and 13C-NMR data, see m/z 729 [M + K]+, 713 [M + Na]+; HRESIMS m/z 713.4200 [M + Na]+ .Glinusopposide O (20). White powder; c 0.1, MeOH); UV (MeOH) λmax (logε) 250 (4.07), 217 (3.68) nm; 1H and 13C-NMR data, see m/z 857 [M + Na]+; HRESIMS m/z 857.4300 [M + Na]+ .Glinusopposide P (21). White powder; c 0.13, MeOH); 1H and 13C-NMR data, see m/z 726 [M + Na]+; HRESIMS m/z 726.4201 [M + Na]+ .Glinusopposide Q (22). White powder; c 0.15, MeOH); ECD  λmax (Δε) 221 (–1.44) nm; 1H and 13C-NMR data, see m/z 859 [M + Na]+; HRESIMS m/z 859.4452 [M + Na]+ .Glinusopposide R (23). White powder; c 0.1, MeOH); 1H and 13C-NMR data, see m/z 873 [M + Na]+; HRESIMS m/z 873.4606 [M + Na]+ .Glinusopposide S (24). White powder; c 0.22, MeOH); 1H and 13C-NMR data, see m/z 655 [M + Na]+; HRESIMS m/z 655.3820 [M + Na]+ .Glinusopposide T (25). White powder; c 0.1, MeOH); 1H and 13C-NMR data, see m/z 801 [M + Na]+; HRESIMS m/z 801.4393 [M + Na]+ .Glinusopposide U (O-(6-O-Methyl-β-d-glucuronopyranosyl)-olean-12-ene-28,30-dioic acid 30-methyl ester (26). White powder; c 0.12, MeOH); 1H and 13C-NMR data, see m/z 713 [M + Na]+; HRESIMS m/z 713.3871 [M + Na]+ .The 3β-31 (15.5 mg) was dissolved in 2 M HCl (1 mL) and stirred at 90 °C for 4 h. After cooling, the solution was evaporated to dryness under reduced pressure. The reaction mixture was purified by silica gel column chromatography  to afford xylose (1.9 mg). The sugar was dissolved in pyridine (0.1 mL) and acetic anhydride (0.1 mL) and stirred for 21 h at room temperature. Then, water (5 mL) was added to the reaction mixture, followed by extraction with EtOAc (5 mL). The organic layer was dried under reduced pressure to yield 1,2,3,4-tetra-O-acetyl-d-xylopyranose (0.9 mg), which was identified based on its 1H-NMR spectrum and optical rotation value: c 0.08, CHCl3) [Compound n-butanol-soluble part (20.0 g) was subjected to D101 resin column chromatography, eluted using water (discarded) and 60% EtOH to yield the saponin mixture (4.0 g). The latter (1.0 g) was dissolved in 2 M HCl (3 mL) and stirred at 90 °C for 5 h. The reaction mixture was dried and purified by silica gel column chromatography  to yield rhamnose (97.9 mg) and glucose (9.4 mg). The glucose was identified as d-glucose based on its 1H-NMR spectrum and optical rotation value: c 0.22, H2O) [O-acetyl-α-l-rhamnopyranose (1.4 mg), which was identified based on its 1H-NMR spectrum and optical rotation value: c 0.14, CHCl3) [The  +40 c 0., H2O [22, CHCl3) .T. rubrum ATCC 4438 and M. gypseum CBS118893) were purchased from the Institute of Dermatology and Hospital for Skin Diseases, Chinese Academy of Medical Sciences. An antifungal assay was performed according to modified versions of the clinical and laboratory standards institute (CLSI), formerly national committee for clinical laboratory standards (NCCLS) methods, as described previously [50) was calculated by the Reed-Muench method [The fungi strains eviously ,25. Terbh method .1−4), 21 new triterpenoids glycosides (5−25), and 12 known compounds were isolated from G. oppositifolius. However, we cannot exclude the possibility that some of the isolated compounds might be artifacts resulted from the extraction treatment, for example compound 23 might be artifacts of ethanol extraction. The triterpenoids and their glycosides were hopane-type and oleanane-type which have been proofed to be existing in this plant [6), glinusopposide Q (21), glinusopposide T (24), and glinusopposide U (25) showed considerable inhibitory activities against M. gypseum and T. rubrum. According to the study of SARs, sugars at 3-hydroxy, 30-carboxymethyl group, and the double bond at C-12 play a key role in oleanane type compounds for antifungal activities. The SARs of hopane type compounds for antifungal activities remain for further research. This study provides a scientific evidence of traditional practice on applying G. oppositifolius to treat dermatophytosis.In this study, four new triterpenoids (is plant ,13. Four


Complexes 1–4 are stable in the dark in aqueous solution over 48 h and photolysis studies indicate that they can photodissociate the monodentate m/p-COOMe-Py ligands selectively with yields lower than 1%. DFT and TD-DFT calculations (B3LYP/LanL2DZ/6-31G**) performed on singlet and triplet states pinpoint a low-energy triplet state as the reactive state responsible for the selective dissociation of the monodentate pyridyl ligands.New Ru(II) arene complexes of formula [( Fuelled by the success of photodynamic therapy (PDT) , photoacNevertheless, photochemotherapy is not the only field where photoactivatable complexes have been applied and other research areas have benefitted from the development of light-triggerable systems. Examples are metal-based caged compounds for the controlled delivery of neurotransmitters ,8 Ru(N-N)(X)]2+ (where for example arene = para-cymene and N-N = bidentate chelating ligand) were reported by Sadler and co-workers to be promising systems for their anticancer properties [η6-arene)Ru(N-N)(OH2)]2+ can be generated in situ, potentially triggering the biological activity of the complexes.Among the various classes of photoactivatable complexes, Ru(II) arene derivatives of the type [Ru(N-N)(X)]2+ (1–4) and discuss their structural, chemical and photochemical properties, including the X-ray structure for complexes 3 and 4. The ester group on the pyridyl ligand plays a key role in conferring dark stability to all derivatives, a fundamental requirement for photoactivatable prodrugs. DFT-based computational methods were successfully used to obtain insights on the singlet and triplet excited states, allowing individuation of the dissociative state responsible for the photochemical behaviour of 1–4. In this contribution, we report on new complexes of the family [(N)(X)]2+  (1–4) anη6-p-cym)Ru(N-N)(X)]2+ studied in this work were synthesized as PF6 salts in good to moderate yields and are shown in 2 was previously reported by some of us [η6-p-cym)Ru(N-N)Cl][PF6] complex [3 in a 1:1 mixture of MeOH/H2O to generate the corresponding aqua species [(η6-p-cym)Ru(N-N)(OH2)]2+, to which an excess of the appropriate ligand X and KPF6 were added. All the synthesized complexes were fully characterized by spectroscopic and analytical methods and are consistent with the structures depicted in The complexes 2+ is observed. The reaction progression is easily monitored by UV-Vis following the decrease in intensity of the bands in the UV spectrum and the increase of the absorption at λ > 400 nm (1 (η6-p-cym)Ru(N-N), there are no contributions from the antibonding orbital L+1 to low energy transitions (over 330 nm).As shown by the TD-DFT calculations, the selective photochemical dissociation of the methyl nicotinate ligand is consistent with the presence of σ-antibonding orbitals that participate in several singlet transitions. In particular, the antibonding orbitals L+1 and L+3 participate in all the transitions between 300 and 440 nm of 1–4 in their T0 and T1 states, can be ascribed to the population of σ-antibonding h-SOMOs, involving one Ru–N bond. In the lowest-lying triplet state T0, the dissociation of the chelating ligand is prevented by the strong coordination of the other ring to the Ru atom, which makes this state less likely to prompt release of ligands 2 was prepared based on literature methods [η6-p-cym)Ru(bpm)Cl][PF6] and [(η6-p-cym)Ru(bpy)Cl][PF6] were synthesised following a method previously described [PF6] and a 1 mol equivalent of AgNO3 in a 1:1 mixture of MeOH/H2O (10 mL) and the reaction mixture stirred at 323 K (4 h). Precipitated AgCl was then removed by filtration. To the clear filtrate up to 10 mol equivalents of the appropriate pyridine derivative was added and the reaction mixture left stirring overnight at ambient temperature. The volume of the reaction mixture was reduced by rotary evaporation and up to 5 mol equivalents of KPF6 was added. The precipitate that formed was filtered and redissolved by the addition of acetone and the clear reaction mixture was left to evaporate slowly at ambient temperature. The crystalline materials that were formed were collected by filtration, washed with methanol and ether and dried in air. Quantitative details of the preparations and the nature of the product of the individual reactions are described below.The complexes reported . An alum3 , MeOH/H2O , [(η6-p-cym)Ru(bpm)Cl][PF6] . To the clear solution of [(η6-p-cym)Ru(bpm)H2O]2+, methyl-nicotinate  and KPF6  was added. Red crystals were obtained. Yield: . 1H-NMR (D2O/DMSO-d6 (5%), ppm) δ: 10.02 , 9.24 , 8.59 , 8.11 , 7.85 , 6.47 , 6.16 , 3.85 , 2.42 , 1.79 , 0.88 . 13C-NMR  δ: 164.2, 164.1, 161.5, 160.7, 155.1, 140.0, 126.1, 125.8, 107.6, 106.7, 90.8, 86.2, 53.8, 30.4, 22.2, 17.8. Elemental Analysis: Calculated for C26H29F12N3O2P2Ru.0.5(CH3)2CO: H 3.73, C 39.52, N 5.03; found H 3.35, C 39.48, N 4.99.The complex was prepared as described above: AgNO3 , MeOH/H2O , [(η6-p-cym)Ru(bpm)Cl][PF6] . To the clear solution of [(η6-p-cym)Ru(bpm)H2O]2+, methyl-isonicotinate  and KPF6  was then added. Reddish yellow crystals were obtained. Yield: . 1H-NMR (D2O/DMSO-d6 (5%), ppm) δ: 10.07 , 9.25 , 8.85 , 8.55 , 8.42 , 8.13 , 7.53 , 6.48 , 6.18 , 3.87 , 2.43 , 1.80 , 0.88 .13C-NMR  δ: 164.0, 161.6, 160.9, 157.3, 153.9, 140.5, 128.9, 127.4, 126.0, 107.5, 106.5, 90.6, 86.2, 56.5, 53.5, 30.4, 22.2, 19.0, 17.8. Elemental Analysis: Calculated for C26H29F12N3O2P2Ru. 0.5(CH3)2CO: H 3.73, C 39.52, N 5.03; found H 3.34, C 39.44, N 5.01.The complex was prepared as described above: AgNO3 , MeOH/H2O , [(η6-p-cym)Ru(bpy)Cl][PF6] . To the clear solution of [(η6-p-cym)Ru(bpy)H2O]2+, methyl-nicotinate  and KPF6  was then added. The yellow needles obtained were collected by filtration and washed with methanol and ether and dried in air in the dark. Some of the crystals were used for single crystal X-ray analysis. Yield: . 1H-NMR (D2O/DMSO-d6 (5%), ppm) δ: 9.70 , 8.80 , 8.55 , 8.37 , 8.30 , 8.23 , 7.90 , 7.47 , 6.44 , 6.05 , 3.85 , 2.42 , 1.77 , 0.83 . 13C-NMR  δ: 163.5, 156.9, 156.6, 155.4, 152.8, 141.8, 140.7, 129.5, 128.9, 127.5, 125.2, 108.6, 103.8, 92.3, 85.1, 53.5, 30.6, 22.2, 17.9. Elemental Analysis: Calculated for C24H27F12N5O2P2Ru. 0.5(CH3)2CO: H 3.60, C 36.56, N 8.36; found H 3.29, C 36.32, N 8.16.The complex was prepared as described above: AgNO3. Red crystals were collected by filtration and washed with methanol and ether and dried in air in the dark. Some of the crystals were used for single crystal X-ray analysis. Yield: . 1H-NMR (D2O/DMSO-d6 (5%), ppm) δ: 9.66 , 8.57 , 8.30 , 8.23 , 7.88 , 7.80 , 6.43 , 6.02 , 3.84 , 2.41 , 1.75 , 0.83 . 13C-NMR  δ: 164.0, 156.7, 155.3, 154.4, 141.7, 140.3, 129.6, 125.9, 125.1, 108.7, 104.1, 92.5, 85.1, 53.7, 30.6, 22.2, 17.9. Elemental Analysis: Calculated for C24H27F12N5O2P2Ru. 0.5(CH3)2CO: H 3.60, C 36.56, N 8.36; found H 3.27, C 36.56, N 8.42.The complex was prepared in an identical manner as described for F2 using SHELXL-97 2+ have been synthesised and characterized in this work, including the X-ray structure of two of them, namely [(η6-p-cym)Ru(bpy)(m-COOCH3-Py)]2+ (3) and[(η6-p-cym)Ru(bpy)(p-COOCH3-Py)]2+ (4). Pyridyl ester derivatives (1–4) are stable in aqueous solution and do not hydrolyze in the dark within the monitored period (48 h). On the contrary, their carboxylic analogues are not stable in the dark and readily dissociate the pyridyl ligand affording the [(η6-p-cym)Ru(N-N)(OH2)]2+ complex. This is a crucial discovery for the design of novel photoactivatable Ru(II) arene complexes which are required to be substitutionally inert in the dark since there are evidences that aqua species play a fundamental role in their cytotoxicity mechanism [vs COOH) can affect the overall stability of the complex.Several new Ru(II) arene complexes of formula [ arene complexes with dyes  or targe1–4, pinpointing for the first time that the dissociative ll-T1 triplet state is likely to be responsible for the selective light-induced ejection of the monodentate pyridyl ligand.Finally, DFT calculations provided a valuable rationalization of the photochemistry of complexes


A single-crystal X-ray analysis showed a pseudo-octahedral piano-stool geometry of [Ru(η6-p-cym)(dpa)I]PF6 (3), with a η6-coordinated p-cymene, bidentate N-donor dpa ligand and iodido ligand coordinated to the Ru(II) atom. The results of the 1H-NMR solution behaviour studies proved that the complexes 1–5 hydrolyse were in the mixture of solvents used (10% MeOD-d4/90% D2O). Complexes 1–5 were in vitro inactive against the A2780 human ovarian carcinoma cell line, up to the highest tested concentration (IC50 > 100 μM).Halogenido and carboxylato Ru(II) half-sandwich complexes of the general composition [Ru(η It has been reported that the in vitro cytotoxicity of these complexes can be improved when the chlorido ligand is replaced by different halogenido ligand, as exemplified on the [Ru(η6-p-cym)(L1)Cl]PF6 and [Ru(η6-p-cym)(L1)I]PF6 complexes, showing the IC50 values of 16.2, and 3.0 µM, respectively, against the A2780 human ovarian carcinoma cells; L1 = N,N-dimethyl-N′-(2-pyridinylmethylene)  (IC50 = 1.3 µM) significantly exceeded potency of trans-[Pt(dach)(OH)2(ox)] (IC50 = 11.4 µM) against the A2780 cells PF6 complex (50 = 40.8 µM) 2 complex, or as η6-coordinated 4-phenylbutyric acid (PBA) in the [Ru(η6-PBA)(L3)Cl]+ or [Ru(η6-ar)(η6-PBA)]2+ complexes; L2 = pyridine or triphenylphosphane, L3 = e.g., 1,10-phenanthroline or benzaldehyde monothiosemicarbazone, and ar = e.g., 1,2,3,4,5,6-hexamethylbenzene , re, re6-p-c40.8 µM) . From thlbenzene ,13,14. H6-p-cym)(dpa)Cl]PF6 were reported. In this work, its structure was modified by either different halogenido ligands (bromido or iodido) or monodentate O-coordinated carboxylato ligands . The obtained products were thoroughly characterized, their solution chemistry was studied by 1H-NMR and their in vitro cytotoxicity against the A2780 cells was assessed.Recently, the preparation ,11,15,166-p-cym)(dpa)Cl]+ was reported in the literature, but with different reaction conditions (4 h/ambient temperature 2 and [Ru(µ-I)(η6-p-cym)I]2 [6-p-cym)(dpa)Cl]Cl (1*) was dechlorinated by 2 molar equivalents of silver triflate, followed by the addition of the appropriate potassium halogenide to form [Ru(η6-p-cym)(dpa)X]+ . In the case of carboxylato complexes 4 and 5, a one-step replacement of the chlorido ligand of 1 by the VP or PB ones was achieved using the silver carboxylates Ag(VP) (for 4) or Ag(PB) (for 5).The synthesis of the herein used complex [Ru. In thi-cym)I]2 , the eas6-p-cym)(dpa)X]PF6 aliph (2930–2980 cm−1) of p-cymene, as well as ν(C–H)ar (3000–3200 cm−1), and ν(C–C)ar and ν(C–N)ar  of the dpa and p-cymene ligands +, of the studied complexes 1–5 were detected in the ESI+ mass spectra (1–3) or carboxylato (for 4 and 5) ligands under the used electrospray ionization conditions led to the [Ru(p-cym)(dpa–H)]+ species, whose peaks appeared at 406.2 m/z uniformly for all complexes 1–5.The prepared complexes of the general formula [Ru(ηpa)X]PF6  were tho ligands . The maxbrations . The peaar 3000–30 cm−1, a ligands  clearly  spectra . A relea1H- and 13C-NMR spectra of the complexes 1–5 (measured in DMSO-6d) contained all the signals of both the p-cym and dpa ligands  was overlapped by the signal of the used solvent for complexes 1–3, but this signal was clearly detected in the 2D NMR spectra, as well as in the 1H-NMR spectra obtained in different solvents . The signals of C4–H, C5–H and C6–H shifted downfield as a consequence of the coordination of the dpa ligand to the Ru(II) atom, while the signals detected for C3–H showed strong upfield shifts  were detected at ca. 181 ppm (for the VP-containing complex 4) and 179 ppm (for the PB-containing complex 5), and they were shifted by 4.0 ppm, and 4.7 ppm, respectively, as compared with the free carboxylic acids . The positions of these characteristic 13C-NMR signals of the complexes 4 and 5 were consistent with those reported for trans-[Pt(NH3)(py)(VP)2] (182.1 ppm), containing valproate(1−) as the monodentate O-donor ligand [2(L2)]2, containing PB as the O-donor bridging ligand, and [Ru(η6-PBA)(L3)Cl]+ or [Ru(η6-ar)(η6-PBA)]2+, containing PBA as η6-coordinated ligand, showed different 13C-NMR δ values of their COO− signals. In particular, the δCOO– value for [Ru(µ-PB)(CO)2(L2)]2 equals 186.5 ppm [6-PBA)(L3)Cl]+ + . Czomple6-p-cym)(dpa)I]PF6 (3) was characterized by a single crystal X-ray analysis. The crystal data and structure refinement are given in The complex [Ru(dpa)Cl]PF6  complexes  Å (the range of 2.025–2.219 Å) of 30 ruthenium complexes, such as [Ru(η6-bz)(dpa)Cl]PF6 [6-p-cym)(dpa)Cl]BF4 [6-hmbz)(dpa)Cl]PF6∙CH2Cl2 PF6 , [Ru(η6-a)Cl]BF4  or PF6 [6-p-cym)(en)I]I [6-p-cym)(L5)I] -4-methylbenzenesulfonamide. The average Ru–I bond length of these three complexes is 2.74(3) Å. However, only [Ru(η6-p-cym)(L4)I]PF6 is structurally similar to complex 3 with a heterocyclic chelating ligand coordinated to the Ru(II) atom through the two nitrogen atoms PF6 , [Ru(η6-)(en)I]I  and [Ru(m)(L5)I] , were cren atoms .p-cymene ring equal 29.46(8)° (formed by the ring containing the N1 atom) and 27.40(8)° (formed by the ring containing the N1A atom). The crystal structure of the complex 3 is stabilized by a variety of non-covalent contacts of the N–H···F and C–H···F types °, while the dihedral angles between theses rings and ·F types .As it is known for the anticancer complexes of various transition metals   or ruthe1–3 in a mixture of 10% MeOD-d4/90% D2O, the new sets of 1H-NMR signals of both the dpa and p-cym ligand were detected . The formation of these new signals is most likely connected with the hydrolysis of the original complexes, showing in the formation of the [Ru(η6-p-cym)(dpa)(H2O)]2+ and/or [Ru(η6-p-cym)(dpa)(OH)]+ species 2+ and free carboxylates  were consistent with those of the hydrolyzed halogenido complexes (see above), which strongly suggested the same composition of the complex species (most likely aqua complexes [Ru(η6-p-cym)(dpa)(H2O)]2+) formed by the hydrolysis of all the complexes 1–5. Further, the signals of the released carboxylato ligands appeared in the 1H-NMR spectra with the δ values well-correlating with free carboxylate anions  of the VP ligand showed at 0.62 ppm in the 1H-NMR spectra recorded on the fresh 10% MeOD-d4/90% D2O solutions. Together with this signal, another triplet was detected at 0.78 ppm, whose integral intensity increased in time and whose position correlated with that of free valproate(1–) anion (0.78 ppm in the same mixture of solvents), thus this signal can be unambiguously assigned to the released VP ligand. Regarding the 4-phenylbutyrato complex 5, the 1H-NMR multiplets of the aliphatic hydrogens of the PB ligand appeared at 1.65, 2.17 and 2.25 ppm, for the fresh 10% MeOD-d4/90% D2O solutions of complex 5 and its mixture with GSH. A hydrolytic release of the PB ligand led to the changes of these positions to 1.77, 2.10 and 2.54 ppm, which correlated well with the positions of these signals detected in the spectrum of free 4-phenylbutyrate (the experiment performed with PBA in 10% MeOD-d4/90% D2O).Regarding the carboxylato complexes oxylates . The pose anions . As for 4 and 5 differed markedly one from another, in connection with the hydrolysis process. It has been observed that ca. 90% of the PB-containing complex 5 hydrolyzed after 48 h of standing at ambient temperature, while the valproato complex 4 was more stable and only ca. 25% hydrolyzed (dpa)Cl]PF6 (1 in this work) PF6 containing 2-pyridylpropylimine (L6), which was inactive against the A2780 cells (IC50 > 200 µM)  is more potent (IC50 = 2.9 µM) than its chlorido analog [Ru(η6-p-cym)(L7)Cl] (IC50 = 4.6 µM) against the NCI-H460 non-small cell lung carcinoma; HL7 = 5,7-diiodo-8-quinoline [6-p-cym)(L1)Cl]PF6 complex against the A2780 cells (IC50 = 16.2 µM) was lower than for the iodido complex [Ru(η6-p-cym)(L1)I]PF6 (IC50 = 3.0 µM) 2 was prepared according to the reported synthetic procedure performed in the microwave reactor [[Ru(µ-Cl)(η reactor . Silver 6-p-cym)Cl]2 (0.5 mmol) and dpa (1.0 mmol) in methanol (15 mL) reacted in a microwave reaction system for 1 min at 100 °C, leading to the color change from orange-red to yellow. The obtained solution of [Ru(η6-p-cym)(dpa)Cl]Cl (1*) was cooled to the ambient temperature and NH4PF6 (2.5 mmol) was added. The yellow precipitate of the complex 1 : δ 10.87 , 8.55 , 7.97 , 7.22 , 5.73 , 5.56 , 2.50 (C17–H), 1.84 , 1.13 . 1H-NMR : δ 8.63 , 7.93 , 7.21 , 5.67 , 5.57 , 2.62 , 2.10 , 1.22 . 13C-NMR : δ 154.6, 152.4, 140.5, 119.2, 113.8, 105.4, 99.7, 85.1, 83.8, 30.3, 21.7, 17.7. ESI + MS : 442.0 (dpa)Cl]+), 406.2 (dpa–H)]+). FTIR (νATR/cm−1): 448w, 463w, 533m, 641w, 677w, 762s, 827vs, 878m, 966w, 1026w, 1058w, 1092w, 1125w, 1161m, 1233w, 1279w, 1341m, 1376w, 1392w, 1437s, 1464s, 1491w, 1523w, 1582m, 1625s, 2961m, 2980m, 3025m, 3088m, 3134m, 3192m, 3223m, 3262m.The mixture of [Ru(µ-Cl)(ηomplex 1  was coll6-p-cym)(dpa)Cl]Cl , prepared as described above for 1 and further used in situ, was left to interact with silver triflate (0.2 mmol) at ambient temperature in the dark  for 1 h. The white precipitate of AgCl was filtered off and 0.25 mmol of KBr was added to the obtained pure yellow filtrate. NH4PF6 (0.5 mmol) was added to the obtained orange solution after 3 h of stirring at ambient temperature. The light orange precipitate of the complex 2 : δ 10.88 , 8.62 , 7.96 , 7.20 , 5.75 , 5.68 , 2.50 (C17–H), 2.06 , 1.13 . 13C-NMR : δ 155.7, 152.4, 140.5, 119.2, 113.8, 106.1, 99.5, 85.5, 83.7, 30.5, 21.7, 18.1. ESI + MS : 488.0 (dpa)Br]+), 406.2 (dpa–H)]+). FTIR (νATR/cm−1): 464w, 532m, 555s, 639w, 677w, 762s, 828vs, 877m, 1024w, 1057w, 1161m, 1233w, 1342m, 1376w, 1392w, 1436s, 1465s, 1582m, 1625m, 2978w, 3023w, 3088m, 3133m, 1392m, 3223m, 3260w.+), 406.2 (dpa–H)]+). FTIR (νATR/cm−1): 452w, 462w, 530w, 555s, 636w, 672w, 745m, 831vs, 880m, 1033w, 1059w, 1122w, 1160m, 1233w, 1348m, 1382w, 1433m, 1468s, 1523w, 1584m, 1626m, 2933w, 2967w, 3045w, 3092w, 3224w, 3253w, 3371s.The complex 3  was prep3 from the dichloromethane/n-hexane mixture of solvents provided the crystals suitable for a single-crystal X-ray analysis.Recrystallization of the complex 1 (0.1 mmol) was dissolved in methanol (50 mL) and 0.35 mmol of Ag(VP) was added. The mixture of the formed AgCl and unreacted Ag(VP) was filtered off after the overnight stirring in the dark  at ambient temperature, and NH4PF6 was added (0.5 mmol). Then the reaction mixture was evaporated (using the nitrogen gas) until the light yellow solid precipitated. The product (1). Anal. Calcd. for RuC28H38N3O2PF6: C, 48.41; H, 5.51; N, 6.05%; found: C, 48.03; H, 5.81; N, 6.11%. 1H-NMR : δ 10.83 , 8.73 , 7.97 , 7.23 , 5.84 , 5.79 , 2.40 , 2.04 , 1.93 , 1.25 , 1.08 , 0.97 , 0.62 . 13C-NMR : δ 181.1, 153.7, 152.0, 140.3, 128.6, 125.9, 118.7, 113.7, 103.8, 99.4, 83.7, 83.3, 46.7, 35.1, 30.0, 23.8, 21.6, 19.9, 17.3, 13.7. ESI + MS : 549.8 (dpa)(VP)]+), 406.2 (dpa–H)]+). FTIR (νATR/cm−1): 467w, 552w, 614w, 699w, 771m, 833vs, 881w, 1006w, 1029w, 1058w, 1165w, 1229w, 1242w, 1306w, 1362m, 1384m, 1437m, 1475s, 1533m, 1579s, 1619m, 1644m, 2872m, 2935s, 2957s, 3081w, 3192w, 3241w, 3299w.The complex  product  was coll5  used instead of Ag(VP). A yield was ca. 60% (related to the starting complex 1). Anal. Calcd. for RuC30H34N3O2PF6: C, 50.42; H, 4.80; N, 5.88%; found: C, 50.05; H, 4.80; N, 5.48%. 1H-NMR : δ 8.77 , 7.95 , 7.20 , 7.11 , 6.92 , 5.86 , 5.75 , 2.40 , 2.31 , 2.07 , 1.89 , 1.67 , 1.09 . 13C-NMR : δ 179.0, 153.8, 152.3, 142.0, 140.5, 128.1, 125.6, 119.0, 114.1, 103.8, 99.6, 84.0, 83.7, 36.4, 34.6, 28.1, 24.0, 21.8, 17.5. ESI + MS : 569.8 (dpa)(PB)]+), 406.2 (dpa)]+). FTIR (νATR/cm−1): 439w, 494w, 538w, 555s, 648w, 663w, 701m, 759m, 781m, 830vs, 874w, 1029w, 1063w, 1082w, 1158w, 1224m, 1303m, 1349m, 1387s, 1413w, 1434m, 1467s, 1490w, 1530w, 1571m, 1615w, 1639w, 2867m, 2930m, 2967m, 3005w, 3065w, 3183w, 3184w, 3232w, 3287w.The complex 5  was prep1H, 13C, 1H–1H gs-COSY, 1H–13C gs-HMQC and 1H–13C gs-HMBC spectra were acquired for DMSO-6d solutions at 298 K on a JEOL JNM-ECA 600II device at 600.00 MHz (1H) and 150.86 MHz (13C); gs = gradient selected, COSY = correlation spectroscopy, HMQC = heteronuclear multiple quantum coherence, HMBC = heteronuclear multiple bond coherence. The spectra were calibrated against the residual signals of the used solvent at 2.50 ppm (1H-NMR) and 39.52 ppm (13C-NMR) PF6 (3) was selected and placed on an D8 QUEST monocrystal diffractometer  with PHOTON 100 CMOS detector, using the Mo–Kα radiation (λ = 0.71075 Å). The APEX3 software package was used for data collection and reduction PF6 (1–5), showing a pseudo-octahedral piano-stool geometry, has been prepared and fully characterized. The halogenido complexes 1–3 contain the chlorido, bromido, and iodido ligand, respectively. For the first time, we report on the organometallic ruthenium(II) complexes 4 and 5 containing the monodentate O-donor valproato (VP), and 4-phneylbutyrato (PB) ligand, respectively. The obtained results of the solution behavior in a water-containing solution clearly proved a release of the halogenido (for 1–3) and carboxylato (for 4 and 5) ligands used. The rate of release of the ligands is increased in the presence of the reduced glutathione. The complexes 1–5 did not show any cytotoxic effect in vitro up to the highest tested concentration (IC50 > 100 μM) against the A2780 human ovarian carcinoma cells.A series of the half-sandwich ruthenium(II) complexes of the general formula [Ru(η


O-tricosanoylphorbol-20-acetate (hop-8), a novel ester of phorbol which was isolated from Ostodes katharinae of the family Euphorbiaceae, that inhibited the replication of wild-type HIV-1 and HIV-2 strains and drug-resistant strains broadly both in C8166 cells and PBMCs with low cytotoxicity and the EC50 values ranged from 0.106 μM to 7.987 μM. One of the main mechanisms of hop-8 is to stimulate A3G expressing in HIV-1 producing cells and upregulate the A3G level in progeny virions, which results in reducing the infectivity of the progeny virus. This novel mechanism of hop-8 inhibition of HIV replication might represents a promising approach for developing new therapeutics for HIV infection.APOBEC3G is a member of the human cytidine deaminase family that restricts Vif-deficient viruses by being packaged with progeny virions and inducing the G to A mutation during the synthesis of HIV-1 viral DNA when the progeny virus infects new cells. HIV-1 Vif protein resists the activity of A3G by mediating A3G degradation. Phorbol esters are plant-derived organic compounds belonging to the tigliane family of diterpenes and could activate the PKC pathway. In this study, we identified an inhibitor 12- Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). The highly active antiretroviral therapy (HAART) reduces the plasma viral load and prolongs the lifespan of people with HIV . HoweverThe replication of HIV-1 in host cells cannot be completed without utilizing host cell receptors and factors, such as CD4, CCR5, CXCR4, cyclophilin A, and LEDGF/p75 . ConversO-tricosanoylphorbol-20-acetate (hop-8) , the most common phorbol ester, strongly induces A3G expression in proliferating H9 cells through the protein kinase C (PKC) α/βI/MEK/ERK pathway ,39. Howe (hop-8)  which wac 0.12, CH3Cl). Its molecular formula was determined as C45H74O8 based on the HREIMS m/z 742.5022  as well as ESIMS m/z 765 [M + Na]+, indicating nine degrees of unsaturation. The structure of hop-8 could be identified to be phorbol ester by its typical 1H-NMR signals of phorbol-type diterpenes as follows: δH 7.59 , 5.70 , 5.41 , 4.47, 4.42 , 2.52 , 2.42 , 1.05 , 0.85 , and 1.75 . However, there is an additional fatty acid moiety as well as an acetic acid moiety in hop-8 compared with phorbol. The absorption bands of its IR spectrum also indicated the presence of ester groups . Considering its molecular formula, fatty acyl could be a tricosanoyl, which further demonstrated by the fragments of EI+ MS spectrum m/z: 403 (13), 387 (15), 310 (100), and 282 (30). Compared 1H-NMR data of hop-8 with phorbol  and H-12 [δH 5.41 ] indicated the tricosanoyl linked at 12-OH, while the acetyl group of hop-8 then linked at 20-OH. The relative configuration was established by its NOESY correction spectrum of NMR. The structure of hop-8 was thus identified as 12-O-tricosanoylphorbol-20-acetate.Hop-8 was obtained as colorless oil, 50 values 0.106 ± 0.003 and 0.390 ± 0.038 μM, respectively  less efficiently with an EC50 7.987 ± 0.481 μg/mL, and compared to the EC50 of HIV-1IIIB, it represented a fold change of 9.1  A,B. The ectively B. Hop-8 e of 9.1 C. The an6.915 μM D. Prostr6.915 μM . The antTM flow cytometer. The results showed that in the absence of hop-8 and Dox the MFI was 403 ± 21, and 223 ± 7 when Dox was present. The EYFP intensity was picked up significantly with the addition of hop-8 and was dose dependent  expression was recovered with the addition of hop-8, and this recovery was dose-dependent A. To demependent B.To elucidate the mechanism underlying the recovery of A3G in cells treated with hop-8, protein expression levels were determined with western blots. The results showed that in cells treated with hop-8, it could upregulate A3G expression and maintain A3G at high levels compared to the cells that were not treated with hop-8. The A3G expression levels were 6.2-, 2.8-, and 1.3-fold higher in cells treated with 6.75, 1.35, and 0.27 μM hop-8, respectively, compared to untreated cells under induced expression of Vif. Meanwhile, the A3G level was significantly higher in hop-8-treated cells with Dox than in cells without Dox, indicating that hop-8 enhanced the A3G expression when Vif was present. .Ostodes katharinae were collected from Xishuangbanna, Yunnan Province, P.R. China in August, 2011.The twigs and leaves of Ostodes katharinae (17 kg) were refluxed with 95% methanol for three times. The methanol distillate was concentrated in vacuum to obtain a crude residue. After suspending in water, the crude extract was extracted successively with petroleum ether and ethyl acetate, respectively. The combined these two fractions (270 g) was subjected to silica gel column chromatography, eluted with petroleum ether/ethyl acetate (from 1:0 to 1:1), and finally eluted with MeOH yielding six fractions A–F. Fr. B (25 g) was further separated over a MCI to give seven subfractions (B1–B7), which was purified by Sephadex LH-20 eluted with MeOH and a series of silica gel column chromatography to afford the 12-O-tricosanoylphorbol-20-acetate (22 mg) and other analogs with the different fatty acid moieties.The dried leaves and twigs of Colourless oil; ESIMS m/z 765 [M + Na]+; HREIMS m/z 742.5022 ; c 0.12, CH3Cl); EIMS m/z 742 (M+), 531 (15), 403 (13), 387 (15), 343 (20), 310 (100), 282 (30). IR (KBr) νmax 3424, 2925, 1748, 1730, 1629, 1377, 1261 cm-1; UV (CH3Cl) λmax 242 nm (11.14); 1H-NMR : 7.59 , 5.70 , 5.41 , 4.47 , 4.42 , 2.52 , 2.42 , 1.05 , 0.85 , 1.75 , 2.08 ; 13C-NMR : 160.8 (C-1), 132.7 (C-2), 208.8 (C-3), 73.4 (C-4), 39.2 (C-5), 135.7 (C-6), 132.7 (C-7), 39.2 (C-8), 78.0 (C-9), 55.9 (C-10), 42.7 (C-11), 76.3 (C-12), 65.4 (C-13), 35.9 (C-14), 25.5 (C-15), 16.7 (C-16), 23.7 (C-17), 14.5 (C-18), 9.2 (C-19), 69.0 (C-20), 173.7, 21.1 (OAc), 173.5 (C-1′), 34.1 (C-2′), 24.8 (C-3′), 28.8-29.5 (C-4′-19′), 31.5 (C-20′), 14.2 (C-21′).ΔVifNL4-3 was kindly gifted by Prof. Yong-Hui Zheng (Michigan State University). The plasmids pcDNA3.1-APOBEC3G-HA and pNL4-3, the cell lines 293T and TZM-bl, and the HIV-1 drug-resistant strains HIV-1A17, HIV-174V, HIV-1RF/V82F/184V, HIV-1NL4-3gp41 (36G) V38A, N42T, and HIV-2CBL-20 were obtained from NIH AIDS Reagent Program. The human T cell line, C8166, and the laboratory-adapted HIV-1 strain HIV-1IIIB were obtained from Medical Research Council, AIDS Reagent Project and maintained in RPMI 1640 Medium  containing 10% fetal bovine serum , 100 units/mL penicillin  and streptomycin . The cell lines 293T, TZM-bl, and TREX-hvif-15 were maintained in Dulbecco’s modified Eagle medium  containing 10% FBS. PBMCs were isolated from peripheral blood of healthy donors using density-gradient centrifugation with Ficoll standard operating procedure  . PBMCs were stimulated with 5 μg/mL phytohemagglutinin  for 72 h and were cultured in RPMI-1640 containing 10% FBS and 50 units/mL IL-2. HIV-1IIIB, HIV-1A17, HIV-174V, HIV-1RF/V82F/184V, pNL4-3gp41 (36G) V38A, N42T, and HIV-2CBL-20 were propagated in H9 cells. The clinically isolated strains HIV-1KM018 and HIV-1TC-1 were isolated from local AIDS patients  and propagated by co-culture with healthy PBMCs  ; mouse anti-HA antibody [HA-7] ; rabbit anti-HA antibody ; anti-beta-actin antibody ; MYC-tag antibody [2D11A8] ; anti-FLAG M2 antibody ; anti-CBFb antibody [EPR6322] ; mouse anti-p24 antibody (produced in-house); anti-mouse IgG (H + L) antibody ; anti-rabbit IgG (H + L) antibody .To identify the possible target of hop-8, the inhibition of HIV-1 reverse transcriptase, protease, integrase and gp41 was determined. The inhibition of reverse transcriptase was measured by using Reverse Transcriptase Assay, colorimetric  followed the manufacturer’s instructions. The integrase inhibition assay was determined with the HIV-1 Integrase Wild-Type Assay  and the protease inhibitory assay was measured by using a SensoLyt™ 490 HIV-1 Protease Assay Kit * Fluorimetric * (ANASPEC). The inhibition of gp41 6-helix bundle formation was determined by ELISA, as described previously .50 and CC50 values of the inhibitor and other statistical tests were carried out using Origin 8.5  and GraphPad Prism 6.0 .The ECO-tricosanoylphorbol-20-acetate , which inhibits the replication of clinical isolates in PBMCs and lab-adapted and drug-resistant strains of HIV-1 as well as HIV-2 with different tropisms in C8166 cells. Hop-8 inhibits HIV replication in PBMC mainly by upregulating A3G expression in HIV producing cells and enhancing A3G incorporation in progeny virions. The results of this study might indicate a novel strategy for treating HIV infection.We have identified and characterized a novel phorbol ester, 12-


Compounds 1 and 2 represent two unusual nitrogen-containing compounds, which are composed of a sterol portion condensed via two bonds to phenalenone derivatives. Compound 6, which contains unprecedented imine functionality between two carbonyl groups to form a oxepane -imine-dione ring, exhibited a moderate cytotoxicity against K562, U266, and SKM1 cancer cell lines. Moreover, molecular docking studies were done on estrogen receptor α-ligand binding domain (ERα-LBD) to compounds 1 and 2 to correlate with binding energies and affinities calculated from molecular docking to the anti-proliferative activity.The new phenalenone metabolites  Coniothyrium cereale was isolated from the Baltic Sea algae Enteromorpha sp. Cultivation of the fungus C. cereale in media supplemented with sea salt led to structurally unusual nitrogenous phenalenone derivatives 1, 2, and 6. In this paper, we report the unusual heterodimer phenalenone metabolites 1 and 2 which are constructed from sterol and polyketidic phenalenone derivatives -Ergosta-4,6,8(14)22-tetraen-3-one (3), and entatrovenetinone (5) from the same fungus. As a preliminary test for biological activity, products were screened for their antimicrobial and cytotoxic activities and the results were correlated with molecular docking studies on estrogen receptor α-ligand binding domain (ERα-LBD).Phenalenone derivatives have been reported both from higher plants and microbial sources. They possess significant biological and chemical importance . Marine ivatives . We repoC. cereale was subjected to RP-HPLC separation to give the yellowish brown compounds 1 and 2. At first glance, from their 1H- and 13C-NMR spectra, they were suspected to be a mixture of a sterol and a phenalenone like compounds, though the HPLC chromatogram showed only one peak for each compound. Further elucidation of the mass gave a signal at 733 Da correspondent to a molecular formula C47H59NO6, suggesting that compounds 1 and 2 are adducts of a sterol and phenalenone derivatives. Further investigation of the 1D and 2D NMR spectra confirmed the adduct formation as follows.Subfraction 11 of the ethyl acetate extract of the mycelium of 1 showed a molecular formula of C47H59NO6 on the basis of the number of signals in both 1H- and 13C-NMR spectra and accurate mass measurement  with a carbonyl group (IR 1711/3354 cm−1), and the second is weakly chelated (δH 9.69 for 9-OH), in addition to a characteristic NH resonance at δH 3.93 which has no correlation in the HSQC (Compound spectrum  of the pthe HSQC .1H- and 13C-NMR spectra showed an aromatic methyl (δH/C 2.78/25.9 for CH3-12) and two aromatic protons . A UV maximum at 395 nm clearly evidenced that compound 1 has an extended aromatic system. Further two 1H-NMR singlet resonance signals arose from aromatic protons (δH 6.38 for H-1 and δH 6.81 for H-10). These aromatic protons (H-1 and H-10), each had a distinctive set of correlations in the 1H-13C HMBC spectrum suggesting that each of these protons is attached to a different benzene ring. In the 1H-13C HMBC spectrum, H-1 showed cross peak correlations with C-2, C-3, C-5, C-13, and C-14, whereas H-10 had correlations with C-7, C-8, C-9, C-12, and C-13. H3-12 had heteronuclear couplings to C-10, C-11, and C-13. 2-OH showed cross peak correlations with C-1, C-2, and C-3; and 9-OH with C-8, C-9, and C-10. This pattern of heteronuclear correlations, together with the 1H- and 13C-NMR data indicated for a naphthalene-type compound of two connected penta-substituted benzene rings, substituted at C-2 and C-9 with phenolic groups and at C-11 with a methyl group. The presence of the 3-methyl-2-butenyl group in 1 was proven as follows: the 1H- and 13C-NMR spectrum contained two singlet resonances at δH/C 1.81/25.8 for CH3-18 and δH/C 1.76/18.3 for CH3-19 due to a geminal dimethyl group attached to an olefinic carbon. This was corroborated by the HMBC cross peak correlations between H3-18 and H3-19 and C-22. The downfield shifted doublet at δH/C 4.69/66.0 is assigned for the methylene protons CH2-20 which is attached to oxygen and the methine triplet at δH/C 5.56/118.4 is assigned for CH-21. The 1H-1H COSY spectrum showed cross peak correlations for a 1H-1H-spin system ranging from both terminal methyl protons via H-21 to H2-20. The prenylation occurred at the oxygenated carbon C-14 due to the HMBC correlation of H2-20 to C-14 as depicted in The 1 gave rise to 1H- and 13C-NMR signals  which has the same structure as -ergosta-4,6,8(14)-22-tetraen-3-one +, calc 341.1025 [M + 1]+, using additionally prepared extract prepared under nitrogen atmosphere and without subjecting the extract to acetone during the extraction process (The structures of compounds ) on C-6 . The posanone A′ . Also co) on C-6 . The pos spectra . The comabietina . The gen process .6a was elucidated based on extensive spectroscopic measurements . The position of the imine at C-6 is supported by the structure of compounds 1 and 2 where the nitrogen atom is at the same position which is the mid carbonyl group. This is also chemically the most plausible position because the mid carbonyl group of a triketone system is the most active one for a transamination reaction. In addition, the respective carbon chemical shifts were in agreement with the values calculated by the ACD NMR predictor software® (ACD laboratories) for 6a. We report here the complete spectroscopic data (6 (6a).Compound urements . Compounated one . The lat spectra . The attspectrum . The lat -methyl-Δ22-sterol side chain of known steroids + and m/z calcld = 756.4240 [M + Na]+.S-Dehydroazasirosterol (2): Yellowish brown powder ; UV λmax MeOH (log ε): 395 (4.81), 240 (4.50) nm; 3); IR νmax (ATR): 3382, 2919, 2850, 2358, 1737, 1711, 1608, 1461, 1371, 1295, 1193, 1064, 1033, 977, 861 cm−1; 1H- and 13C-NMR see supp. info. m/z found = 756.4235 [M + Na]+ and m/z calcld = 756.4240 [M + Na]+.-Ergosta-4,6,8(14)22-tetraen-3-one (3): Yellowish brown powder ; 13C-NMR : δ 34.0 (CH2-1), 18.9 (CH2-2), 200.5 (C-3), 122.6 (CH-4), 165.6 (C-5), 124.3 (CH-6), 134.6 (CH-7), 124.3 (C-8), 44.2 (CH-9), 36.8 (C-10), 25.4 (CH2-11), 34.0 (CH2-12), 44.0 (C-13), 156.7 (C-14), 35.5 (CH2-15), 27.7 (CH2-16), 55.6 (CH-17), 18.9 (CH3-18), 16.6 (CH3-19), 39.3 (CH-20), 21.2 (CH3-21), 134.9 (CH-22), 132.2 (CH-23), 42.8 (CH-24), 33.1 (CH-25), 19.6 (CH3-26), 19.9 (CH3-27), 17.6 (CH3-28); (−)ESIMS m/z = 391.6 [M − H]−.The acetone adduct of c4ompound  (4a): Yellowish brown powder ; UV λmax MeOH (log ε): 331 (4.01), 255 (3.91), 216 (3.81) nm; IR νmax (ATR): 2919, 2850, 2359, 1708, 1608, 1460, 1379, 1206, 836, 720, 668, 534 cm−1; 1H- and 13C-NMR see supp. info. m/z found = 421.1258 [M + Na]+ and m/z calcld = 421.1264 [M + Na]+.The acetone adduct of c5ompound  (5a): Yellowish brown powder ; 1H- and 13C-NMR see supp. info. m/z found = 421.1260 [M + Na]+ and m/z calcld= 421.1264 [M + Na]+.The hydrated form of c6ompound  (6a): Yellowish green powder ; UV λmax MeOH (log ε): 385 (4.91), 242 (4.21) nm; IR νmax (ATR): 3380, 2920, 2853, 2458, 1740, 1717, 1610, 1465, 1373, 1300, 1195, 1065, 1030, 970, 862 cm−1; 1H- and 13C-NMR see supp. info. m/z found = 396.1057 [M + Na]+ and m/z calcld = 396.1059 [M + Na]+.2 in RPMI 1640 medium  supplemented with 10% FCS  completed with 50 units/mL penicillin, 50 mg/mL streptomycin, and 1 mM sodium pyruvate.Cell Lines. The human cancer cell lines K562 (chronic myelogenous leukemia), U266 (myeloma), SKM1 (myelodysplastic symdrom), and Kasumi (acute myeloid leukemia) were provided by ATCC and were grown at 37 °C under 10% CO4/mL) were incubated with 1 or 2 for the times indicated. 50 µL of XTT kit (sodium 39--bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) was added to each well, which contain 100 µL of medium. Absorbance of the formazan dye produced by metabolically active cells was measured at 490 nm. Each assay was performed in quadruplicate.Measurement of Cell Metabolism (XTT). Cells  with MM force field and saved in Tripos mol2 file format. MolDock score functions were used with a 0.3 Å grid resolution. Prior to the calculations of the subject compounds, the MVD software was benchmarked docking the Estrogen.All the modeling studies were carried out on a laptop PC, Intelode 1A52  were dowode 1A52  was usedConiothyrium cereale. The unusual heterodimer compounds 1 and 2 are composed of a sterol portion condensed to phenalenone derivatives. Compound 6 contains unprecedented imine functionality between two carbonyl groups to form an oxepane-imine ring.Marine fungi are promising inspiring sources for new chemical skeletons. This work introduced new and promising alkaloidal phenalenone derivatives from the marine-derived endophytic fungus