Dengue NS2B/NS3 protease complex is essential for viral replication and is a primary target for anti-dengue drug development. In this study, a NS2B/NS3 protease inhibition assay was developed using AlphaScreen® donor and nickel chelate acceptor beads in 384-well format.The assay system utilized a known NS2B/NS3 peptide substrate, a recombinant of NS2B/NS3 protease with proprietary StrepTactin® assay revealed weak NS2B/NS3 inhibition activities.The optimized assay to screen for NS2B/NS3 protease inhibitors was demonstrated to be potentially useful with reasonable zʹ factor, coefficient variance and signal to background ratio. However, screening of synthesized thioguanine derivatives using the optimized AlphaScreen® assay to screen for NS2B/NS3 protease inhibitors is potentially applicable for high throughput screening.The AlphaScreen Dengue virus (DENV) is a member of the flavivirus genus within the family of Flaviviridae +224.3059.9-isopropyl-6-(isopropylthio)-9H-purin-2-amine (2). Compound 2-amino-9H-purine-6-thiol  and Cs2CO3  were dissolved in DMF (7 mL) and a mixture containing isopropyl bromide , tetra-butylammonium iodide (TBAI)  and DMF (7 mL) was added in small portions. The mixture was allowed to stand for 4 h at room temperature, then poured into water (70 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo. The residual crude product was chromatographed on silica gel with n-hexane: ethyl acetate (1:3) to obtain 2 as a colorless amorphous , 1H NMR (CDCl3): δH 1.45 , 1.55 , 4.28 , 4.67 , 4.84 , 7.69 ; 13C NMR (CDCl3): δC 22.5 (C-14 and C-17), 23.3 (C-11 and C-16), 30.9 (C-13), 34.2 (C-10), 46.5 (C-4), 125.9 (C-2), 137.7 (C-9), 158.3 (C-7), 167.3 (C-5); QTOF-MS m/z calcd for C11H17N5NaS [M+Na]+274.3404.6-(-2-ethylhexylthio)-9-(-2-ethylhexyl)-9H-purin-2-amine (3). Compound 2-amino-9H-purine-6-thiol  and Cs2CO3  were dissolved in DMF (7 mL) and a mixture containing 2-ethylhexyl bromide , tetra-butylammonium iodide (TBAI)  and DMF (7 mL) was added in small portions. The mixture was allowed to stand for 24 h at room temperature, then poured into water (70 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo. The residual crude product was chromatographed on silica gel with n-hexane: ethyl acetate (1:3) to obtain 3 as a colorless amorphous , Colorless amorphous, 1H NMR (CDCl3): δH 0.86–0.95 , 1.26–1.33 , 1.65–1.73 , 1.84–1.91 , 3.37–3.49 , 3.88–4.00 , 4.81 , 7.58 ; 13C NMR (CDCl3): δC 10.4 (C-27), 11.1 (C-25), 14.0 (C-22), 14.1 (C-15), 22.8 (C-21), 22.9 (C-14), 23.6 (C-24), 25.8 (C-26), 28.4 (C-20), 28.9 (C-13), 30.2 (C-12), 30.9 (C-19), 32.2 (C-11), 32.6 (C-18), 39.3 (C-17), 39.5 (C-10), 125.7 (C-4), 140.2 (C-2), 150.7 (C-9), 158.7 (C-7), 161.9 (C-5).2-amino-9-cyclopentyl-9H-purine-6-thiol (4). Compound 2-amino-9H-purine-6-thiol  and Cs2CO3  were dissolved in DMF (7 mL) and a mixture containing cyclopentyl bromide , tetra-butylammonium iodide (TBAI)  and DMF (7 mL) was added in small portions. The mixture was allowed to stand for 4 h at room temperature, then poured into water (70 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo. The residual crude product was chromatographed on silica gel with n-hexane: ethyl acetate (1:3) to obtain 4 as white powder , mp 160-162 °C, 1H NMR (CDCl3): δH 1.60–1.70 , 1.81 , 2.02 , 4.32 , 4.92 , 7.78 ; 13C NMR (CDCl3): δC 24.9 (C-13 and C-14), 33.6 (C-12 and C-15), 42.0 (C-11), 124.5 (C-4), 137.5 (C-2), 150.8 (C-9), 158.9 (C-7), 162.9 (C-5).9-cyclopentyl-6-(cyclopentylthio)-9H-purin-2-amine (5). Compound 2-amino-9H-purine-6-thiol  and Cs2CO3  were dissolved in DMF (3.5 mL) and a mixture containing cyclopentyl bromide , tetra-butylammonium iodide (TBAI)  and DMF (3.5 mL) was added in small portions. The mixture was allowed to stand for 24 h at room temperature, then poured into water (70 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo. The residual crude product was chromatographed on silica gel with n-hexane: ethyl acetate (1:3) to obtain 5 as white powder , mp 129-131 °C, 1H NMR (CDCl3) δH 1.67–1.70 , 1.70–1.80 , 1.91–2.21 , 2.21–2.26 , 4.71–4.76 , 4.77–4.98 , 4.83 , 7.67 ; 13C NMR (CDCl3): δC 23.8 (C-12 and C-13), 24. 9 (C-18 and C-19), 32.6 (C-11 and C-14), 33.6 (C-17 and C-20), 55.3 (C-16 and C-10), 126.0 (C-4), 138.0 (C-2), 150.5 (C-9), 158.7 (C-7), 162.5 (C-5); QTOF-MS m/z calcd for C15H22N5S [M+H]+304.4336.Methyl {[2-amino-9-(2-methoxy-2-oxoethyl)-9H-purin-6-yl]sulfanyl} acetate (6). Compound  and Cs2CO3  were dissolved in DMF (3.5 mL) and a mixture containing methyl bromoacetate , tetra-butylammonium iodide (TBAI)  and DMF (3.5 mL) was added in small portions. The mixture was allowed to stand for 24 h at room temperature, then poured into water (70 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo. The residual crude product was chromatographed on silica gel with n-hexane: ethyl acetate (1:3) to obtain 6 as white powder , mp 139-141 °C, 1H NMR (CDCl3) δH 3.75 , 3.79 , 4.09 , 4.82 , 4.89 , 7.69 ; 13C NMR (CDCl3): δC30.7 (C-19), 43.7 (C-13), 52.7 (C-16), 52.9 (C-10), 124.9 (C-9), 140.3 (C-2), 151.0 (C-4), 158.9 (C-6), 159.5 (C-9), 167.5 (C-11), 169.7 (C-17); QTOF-MS m/z calcd for C17H14N5O4S [M+H]+312.3249.6-{[(pyridin-2-yl)methyl]sulfanyl}oolpl.-9H-purin-2-amine (7). Compound 2-amino-9H-purine-6-thiol  and Cs2CO3  were dissolved in DMF (3.5 mL) and a mixture containing methyl pyridinium bromide hydrobromide , tetra-butylammonium iodide (TBAI)  and DMF (3.5 mL) was added in small portions. The mixture was allowed to stand for 24 h at room temperature, then poured into water (70 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo. The residual crude product was chromatographed on silica gel with n-hexane: ethyl acetate (1:3) to obtain 7 as white powder , mp 260-263 °C, 1H NMR (DMSO-d6) δH 4.65 , 6.46 , 7.26–7.91 , 8.51 ,12.57 ; 13C NMR (DMSO-d6): δC 33.6 (C-11), 122.7 (C-15), 123.9 (C-17), 137.2 (C-4), 137.6 (C-16), 139.3 (C-2), 141.9 (C-14), 149.6 (C-9), 152.3 (C-12), 158.3 (C-7), 160.0 (C-5).6-(benzylsulfanyl)-9H-purin-2-amine (8). Compound 2-amino-9H-purine-6-thiol  and Cs2CO3  were dissolved in DMF (3.5 mL) and a mixture containing benzyl bromide , tetra-butylammonium iodide (TBAI)  and DMF (3.5 mL) was added in small portions. The mixture was allowed to stand for 12 h at room temperature, then poured into water (70 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo. The residual crude product was chromatographed on silica gel with n-hexane: ethyl acetate (1:3) to obtain 8 as white powder , m.p. 193-195 °C +437.5593, found 437.2747.n-hexane: ethyl acetate (1:3) resulted in 11 -9H-purin-2-amine) as a brown liquid , 1H NMR (CDCl3): δH 4.45 , 7.21–7.38 ; 13C NMR (CDCl3): δC (CDCl3) 33.6 (C-18), 65.4 (C-26 and C-33), 72.1 (C-10), 127.6 , 127.8 (C-24 and C-20), 127.9 , 128. 4 , 128.7 (C-21 and C-23), 128.8 (C-13 and C-15), 129.7 (C-12 and C-16), 129.9 (C-4), 131.6 , 133.2 (C-19), 134.5 (C-2), 137.8 (C-9), 138.3 (C-7), 139.3(C-5); QTOF-MS m/z calcd for C33H29N5S [M ]+527.6818, found 527.3339.Continued elution with 2-(4-fluorobenzylideneamino)-9H-purine-6-thiol (12). Compound 2-amino-9H-purine-6-thiol  was mixed with 4-fluorobenzaldehyde , stirred for 15 min and the liquid mixture of ethanol and 10% (v/v) NaOH [3.0 mL (1:1)] was added in small portions. The mixture was allowed to stand for 2 h at 80 °C, then neutralized with HCl (6 M) until precipitated. The solid product was filtered, washed with water followed by ethyl acetate. The residual crude product was chromatographed on silica gel with CHCl3: methanol (2:2) to obtain 12 as yellow crystal from methanol , mp 149-151 °C, 1H NMR (DMSO-D6): δH 7.32 , 7.34 , 7.78 , 7.87 ; 13C NMR (CDCl3): δC 116.1 (C-15 and C-17), 116.3 (C-13), 125.8 (C-14 and C-18), 130.3 (C-4), 130.4 (C-2), 131.0 (C-7), 142.1 (C-9), 163.1 (C-12), 165.1 (C-16), 188.5 (C-5); QTOF-MS m/z calcd for C12H8FN5S [M+H]+274.2976.2-methoxy-4-{[(6-sulfanyl-9H-purin-2-yl)imino]methyl}phenol (13). Compound 2-amino-9H-purine-6-thiol  was mixed with 4-hydroxy-3-methoxybenzaldehyde , stirred up for 15 minutes and the liquid mixture of ethanol and NaOH 10% [3.0 mL (1:1)] was added in small portions. The mixture was allowed to stand for 2 h at room temperature, then neutralized with HCl (6 M) until precipitated. The solid product was filtered, washed with water followed by ethyl acetate. The residual crude product was chromatographed on silica gel with methanol (100%) to obtain 13 as white powder , decomps. 300 °C, 1H-NMR (DMSO-d6): δH 3.85 , 7.126 , 8.16 , 8.28 , 12.59 ; 13C NMR (DMSO-d6): δC 84.1 (C-20), 127.5 (C-17), 133.1 (C-14), 140.3 (C-18), 155.0 (C-4), 155.1 (C-2), 155.2 (C-16), 161.1 (C-15), 161.4 (C-7), 163.2 (C-9), 168.5 (C-12), 172.4 (C-5).5-{[(6-sulfanyl-9H-purin-2-yl)imino]methyl}benzene-1,3-diol (14). Compound 2-amino-9H-purine-6-thiol  was mixed with 3,5-dihydroxybenzaldehyde , stirred up for 15 minutes and the liquid mixture of ethanol and NaOH 10% [3.0 mL (1:1)] was added in small portions. The mixture was allowed to stand for 2 h at room temperature, then neutralized with HCl (6 M) until precipitated. The solid product was filtered, washed with water followed by ethyl acetate. The residual crude product was chromatographed on silica gel with CH2Cl2: methanol (2:2) to obtain 14 as brown powder , decomps. 300 °C, 1H NMR (DMSO-d6): δH 7.02 , 8.14 , 8.74 , 12.51 ; 13C NMR (DMSO-d6): δC 95.6 (C-16), 97.4 (C-14 and C-18), 121.0 (C-4), 141.0 (C-13), 147.6 (C-2), 148.9 (C-7), 154.6 (C-9), 160.2 (C-15 and C-20), 170.0 (C-12), 172.0 (C-5); QTOF-MS m/z calcd for C12H13N6O2S [M+NH4]+305.3356.2.5↓ SLT), and is a part of a sequence of dengue polyprotein precursor (NS3/NS4A) as reported by Khumtong and co-workers [®, an engineered streptavidin ® TruHits Detection Kit  consisting of StrepTactin® donor beads and biotinylated acceptor beads was carried out. On the other hand, to ensure that the peptide substrate is adequately tagged with polyhistidine on the C-terminal, competitive binding assay using Histidine (Nickel Chelate) Detection Kit  was carried out. This kit comprises of both StrepTactin® donor beads and nickel chelate acceptor beads.The peptide substrate used in this assay has a known NS2B/NS3 cleavage site between lysine (K), and serine (S)  in a total reaction volume of 25 μL under subdued light. All dilutions were initially performed in starting assay solution containing 25 mM HEPES buffer (pH 7.4), 100 mM NaCl and 0.01 % (w/v) BSA. StrepTactin® donor beads and nickel chelate acceptor beads were used at final concentration of 20 μg/mL. The 384-well plate was read using Envision 2104 Multilabel Plate Reader  according to AlphaScreen® settings . For optimization, peptide substrate (0.3 nM–3 μM) and NS2B/NS3 protease were cross-titrated to determine the concentrations that generate optimal AlphaScreen® signals. Briefly, in each well, 5 μL of peptide substrate and NS2B/NS3 protease enzyme were mixed with 5 μL assay buffer and incubated at 37 °C. After incubating for 1 h, 5 μL of donor beads and acceptor beads were added, followed by 1 h incubation at room temperature prior to reading of signal. To develop an assay that delivers high signal and low background, different buffer system, various concentrations of HEPES, NaCl and BSA, pH and incubation time were optimized. The optimum conditions were determined based on the maximum signal obtained from well containing peptide alone and minimum signal produced by well containing peptide substrate, enzyme and beads, indicating maximum cleavage of the NS2B/NS3 enzyme on the substrate and lowest background signal. Once the assay was optimized, it was used to screen the compounds for their potential NS2B/NS3 protease inhibition activity. Aprotinin and panduratin A were used as positive controls. Since the amount obtained for diversity 0713 was in minute quantity and was insufficient for subsequent testing, diversity 0713 was not used as control in this study. All compounds were dissolved in DMSO and the assay was performed in either 1% (v/v) or 10% (v/v) DMSO in final concentrations, depending on the solubility of the thioguanine derivatives.In general, AlphaScreen2.650 values were derived based on non-linear regression analysis computed using GraphPad Prism 5.0 . All data were presented as mean ± SD. The zʹ factor was calculated as described by Zhang and co-workers ® signal of the substrate in the absence  and presence  of the protease, and μH and μL represent the mean values of high signal and low signal, respectively. To evaluate the robustness of the assay, interday variations were evaluated in assay plate replicates and coefficient variation (CV) was calculated using the formula; % CV = (SD/√n)/average x 100.IC33.1i) for diversity 0713 was −6.64 kcal/mol and 13.49 μM, respectively . However, both compounds have hydrophobic interactions with all three catalytic triad residues  of dengue NS2B/NS3 protease. Nine out of fourteen compounds  have FEB and estimated Kprotease . Generalresidues . Based o3.2N-alkylation and S-alkylation) and Schiff base formation. The alkylation was carried out by reacting thioguanine and alkyl bromide. In this reaction, the chemo selective S-alkylation  was applied by using tetrabutyl ammonium iodide (TBAI) as phase transfer catalyst (PTC) while cesium carbonate (Cs2CO3) was used as the base catalyst. A polar aprotic solvent such as dimethylformamide (DMF) was employed as the solvent in the N-alkylation. Further alkylation at NH  as well as NH2 (10 and 11) required at least 2-fold of alkyl bromide mole number. The second series of the thioguanine derivatives (12-14) were synthesized based on the reaction between primary amine of thioguanine and a diverse aromatic aldehyde in a mixture of NaOH-ethanol solution. The structures of the synthesized compounds are shown in The synthesis of thioguanine derivatives was divided into 2 types of reaction, alkylation . Similarly, the extent of polyhistidine tagging on the substrate was determined and based on the titration curve, signal increased proportionally to the amount of peptide substrate present, as shown in 50 value at 0.11 μM).The NS2B/NS3 protease enzyme was expressed and purified as described previously . The speCross titration of peptide substrate and NS2B/NS3 protease revealed a typical bell-shaped curve illustrating the hook effect when the concentration of peptide substrate was greater than 300 nM. Beyond this point, saturation of the peptide substrate with the beads caused a decrease in signal intensity. In addition, increasing concentration of enzyme produced a reduction in signal, indicating proteolytic activity of the NS2B/NS3 enzyme. A significant decrease in signal was observed from 100 nM onwards. The peptide substrate was almost completely cleaved in the presence of 3 μM enzyme c. Based Initially, a starting buffer consisting of 25 mM HEPES, 100 mM NaCl, 0.01% (w/v) BSA, pH 7.4) was used. However, the assay buffer was re-optimized to increase signal/background (S/B) ratios. Eventually, 10 mM Hepes (pH 9.0), 20 mM NaCl and 0.2% BSA provided the most robust assay conditions. The addition of BSA improved the signal by a few hundred thousand counts per second d–g. To e3.4® assay, aprotinin exhibited dose dependent inhibition against NS2B/NS3 enzyme with an IC50 value of 0.35 μM    was used for compounds with low solubility such as compound 8, 9, 10, 11 and 12. There were no significant differences in the alpha signal obtained between assays run in 1% v/v and up to 10% v/v DMSO. Using the AlphaScreen 0.35 μM a, which 0.15 μM) b. As fornge used c. On theexpected d. Howeved (1 mM) e and f. The interday variations were evaluated in assay plate replicates, with each plate containing wells of minimum and maximum signal controls. The mean value for the maximum signal groups on day 1, 2, 3 for aprotinin were 1.14 million cps (CV = 2.27%), 1.03 million cps (CV = 0.15%) and 1.30 million cps (CV = 3.49%), respectively. On the other hand, the mean value for the minimum signal was 5688 cps (CV = 3.90%), 6024 cps CV = 5.41%), and 7256 cps (CV = 8.27%), respectively. The mean S/B ratio for aprotinin was 205:1. Similarly, the mean value for the maximum signal groups on day 1, 2, 3 for panduratin A were 1.67 million cps (CV = 3.15%), 1.27 million cps (CV = 2.61%) and 1.11 million cps (CV = 1.18%), respectively. Meanwhile, the mean value for the minimum signal was 6199 cps (CV = 6.22%), 3956 cps (CV = 4.22%), and 5191 cps (CV = 9.89%), respectively. The mean S/B ratio for panduratin A was 265:1. The interday variability of both maximum and minimum signals was generally less than 10%. Finally, the zʹ factor value for three independent experiments were calculated to determine the robustness and the quality of the developed assay. The individual zʹ factor of the optimized assay ranged between 0.36 to 0.65 for aprotinin, panduratin and compound 4, respectively, indicating that the assay are potentially useful for screening for anti-dengue activities , 41.%, and 724in vitro high-throughput screening assays and suitable in vivo models to screen for anti-dengue drugs Dengue infection is a global health problem and according to WHO, an approximately 40% of the world population are at risk of dengue infection ® by Sanofi Pasteur was registered in Mexico in December, 2015 but was reported to cause antibody dependent enhancement (ADE) and severe diseases in some cases [50 values ranging from 15 to 44 μM 50 values at 2.2 μM based on NS2B/NS3 protease assay 50 values of 15.22 and 16.23 μM There are intensive efforts to develop drugs targeted against structural and/or nonstructural proteins involved in dengue viral replication, along with the development of vaccine. The first dengue vaccine, Dengvaxiame cases , 20. Meame cases , 54, Bz-me cases , 21, 47.® based assay is robust and has been widely utilized in various aspects of cell signaling research, drug discovery and screening platform and biomarker quantification ® assay AlphaScreen50 value of 0.35 μM using AlphaScreen® assay, which was rather similar with the value derived using the protease assay. Interestingly, the published IC50 value of aprotinin was much lower (65 nM) in a different assay system on similar enzyme activity 50 value of more than 100 μM was expected based on the AlphaScreen® assay. In fact, protease assay produced an IC50 value of 211.5 μM when higher concentration range of the compound was tested, a value higher than previously reported Subsequently known compounds such as aprotinin and a natural compound reported to have some NS2B/NS3 protease inhibitory activities such as panduratin A was evaluated using the optimized assay. Aprotinin, a broad spectrum protease inhibitor, was found to inhibit dengue NS2B/NS3 protease with an IC® and protease assays have indicated that panduratin A activity on NS2B/NS3 protease was weak and thus, may not be suitable to be used as positive control inhibitor for NS2B/NS3 protease. Tan and co-workers initially determined the NS2B/NS3 inhibitory activities of hydroxypanduratin and panduratin A using protease assay and found that concentration as high as 200 μM (or 80 ppm) was needed to inhibit 65-80% of the protease activity. Ki values derived were 21 and 25 μM, respectively, indicating moderate to weak inhibitory effects 50 values in the in vitro protease assays 50 value of 0.91 ± 0.05 μM and Ki value of 2.36 ± 0.13 μM Appropriate and specific inhibitors that can be used as good controls in assays are currently lacking for the NS2B/NS3 enzyme. Studies by Leung and co-workers have found that even standard serine protease inhibitors such as 4-(2-aminoethyl) benzenesulfonyl fluoride.hydrochloride and N-Tosyl-L-phenylalanine chloromethyl ketone showed only 20–30% inhibition of this enzyme at 500 μM and 1 mM, respectively. Other serine protease inhibitors for which no enzyme inhibition was found included soybean trypsin inhibitor, 4-Amidinophenylmethanesulfonyl fluoride.hydrochloride, phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, benzamidine and N-Tosyl-L-lysine chloromethyl ketone.hydrochloride in silico simulation results suggested otherwise. Only compound 4 was found to weakly inhibit the NS2B/NS3 protease with an IC50 value probably near to 1 mM as determined using both AlphaScreen® and protease assays. Although the calculated FEB usually correlates with the binding affinity and that the lower energy obtained, the stronger the binding affinity and the more likelihood to exhibit inhibitory activity against the protease, this was not observed in majority of the synthesized compounds. Currently no DENV NS2B/NS3 protease inhibitors have advanced to clinical trials despite numerous efforts to find potent inhibitors Unfortunately, none of the fourteen derivatives showed significant inhibitory activity against dengue NS2B/NS3 protease despite earlier 50 values, although it was proved to be broad spectrum and not specific to NS2B/NS3 Recently, a study has attempted to develop a conformational switch assay based on split luciferase complementation to monitor conformational change of NS2B and to identify candidate allosteric inhibitors ® beads for screening of dengue NS2B/NS3 protease inhibitory activity. Although Takahashi and co-workers used similar method to examine the interaction between NS3 and NS5, these interactions could also serve as targets for the development of new antivirals against DENV ® assay to screen for NS2B/NS3 protease inhibitors is potentially applicable for use in high throughput screening [This is a first report on the utilization of AlphaScreencreening , 41. FurMuhammad Asyraf Abduraman, Maywan Hariono: Performed the experiments, Analyzed and interpreted the data, Wrote the paper.Rohana Yusof, Noorsaadah Abd Rahman: Conceived and designed the experiments.Habibah A. Wahab, Mei Lan Tan: Conceived and designed the experiments, Analyzed and interpreted the data, Wrote the paper.Ministry of Energy, Science, Technology, Environment and Climate Change, Malaysia (MESTECC) (Grant No 02-05-23-SF0006), which was awarded to the corresponding author (Mei Lan Tan).This work was supported by Sciencefund Grant from the The authors declare no conflict of interest.Data associated with this study will be made available upon request to the corresponding authors.


In general, compounds were more active against Plasmodium falciparum than Leishmania panamensis. Rotenone had an EC50 of 19.0 µM against P. falciparum, and 127.2 µM against L. panamensis. Although chemical transformation does not improve its biological profile against P. falciparum, three of its derivatives showed a significant level of action within an adequate range of activity with EC50 values < 50.0 µM. This antiplasmodial activity was not due to red blood cell hemolysis, since LC50 was >>400 µM. On the other hand, all derivatives displayed a non-specific cytotoxicity on several cell lines and primary human cell cultures.Six derivatives of the known biopesticide rotenone were prepared by several chemical transformations. Rotenone and its derivatives showed differential  Malaria and cutaneous leishmaniasis (CL) are tropical diseases that still remain a public health problem because of their high morbidity and mortality rates in many countries around the world. Unfortunately, the development of resistance to insecticides by insect vectors or decreased drug sensitivity by parasites have worsened the problem in those countries where therapeutic alternatives have become limited . The avaLeishmania and Plasmodium parasites, and is a pharmacological target that has not been very well explored furo-chromen-6(6aH)-one : A mixture of rotenone  and 3-chloroperbenzoic acid  in dichloromethane  was stirred at RT for 2 h; then, NaOH 2.0 M was added to the reaction mixture and extracted with DCM (2 × 15 mL). The combined organic layer was washed with brine solution (30 mL), dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexane/EtOAc 4:1) yielding compound 2  as a colorless oil corresponding to a mixture of epoxides.1H-NMR (CDCl3) δ (ppm): 1.8 , 2.1 , 2.4 , 2.9 , 3.3 , 3.8 , 3.8 , 3.9 , 4.5 , 4.7 , 4.9 , 5.3 , 6.4 , 6.8 , 6.6 , 7.3 . 13C-NMR (CDCl3) δ (ppm): 17.1 (CH3), 29.7 (CH2), 31.1 (CH2), 44.6 (CH), 55.8 (OCH3), 56.4 (OCH3), 63.8 (C), 67.5 (OCH2), 72.2 (CH), 88.0 (CH), 101.0 (CH), 104.9 (CH), 105.3 (C), 109.3 (CH), 112.7 (C), 113.2 (C), 130.1 (CH), 144.0 (C), 149.5 (C), 151.1 (C), 157.7 (C), 168.1 (C), 188.9 (C=O).2R,6aR,12aS-8,9-Dimethoxy-2-(prop-1-en-2-yl)-1,2,6,6a,12,12a-hexahydrochromenofurochromen-6-ol . Rotenone  in methanol (10 mL) was treated with NaBH4,  and stirred at 0 °C for 3 h; water was then added to the reaction mixture and extracted with DCM (2 × 15 mL). The combined organic layer was washed with brine solution (30 mL), dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexane/EtOAc 4:1) yielding compound 3 as a light yellow solid . Melting point (m.p.): 154–156 °C. IR (KBr): 3,445, 2,918, 2,865, 1,617. 1H-NMR (CDCl3) δ (ppm): 1.8 , 2.9 , 3.3 , 3.8 , 3.8 , 4.1 , 4.6 , 4.8 , 4.9 , 4.9 , 5.2 , 5.3 , 6.4 , 6.9 , 6.6 , 7.3 . 13C-NMR (CDCl3) δ (ppm): 17.1 (CH3), 32.0 (CH2), 38.1 (CH), 55.9 (OCH3), 56.4 (OCH3), 65.1 (OCH2), 66.4 (CH), 69.2 (CH), 86.6 (CH), 100.8 (CH), 102.8 (CH), 105.3 (C), 111.9 (CH), 112.1 (=CH2), 112.9 (C), 113.9 (C), 130.4 (CH), 143.8 (C), 143.9 (C), 149.5 (C), 151.1 (C), 149.7 (C), 161.9 (C).2R,12aS-8,9-Dimethoxy-2-(prop-1-en-2-yl)-1,2,12,12a-tetrahydrochromenofuro-chromene (4). A mixture of rotenone  and palladium on carbon  in DCM (5 mL) was treated with hydrogen and stirred at RT overnight. The reaction mixture was filtered through a pad of Celite, and the solvent was evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexane/EtOAc 4:1) yielding compound 4 as a white solid . Melting point (m.p.): 204–206 °C. IR (KBr): 3,450, 2,960, 1,682, 1,610. 1H-NMR (CDCl3) δ (ppm): 1.1 , 1.1 , 2.0 , 2.9 , 3.2 , 3.8 , 3.8 , 3.9 , 4.2 , 4.6 , 4.9 , 6.5 , 6.8 , 7.8 . 13C-NMR (CDCl3) δ (ppm): 17.6 (CH3), 17.9 (CH3), 29.4 (CH2), 33.2 (CH), 44.6 (CH), 55.8 (OCH3), 56.3 (OCH3), 66.3 (OCH2), 77.5 (CH), 90.8 (CH), 100.9 (CH), 104.8 (CH), 104.9 (C), 110.4 (CH), 113.1 (C), 113.4 (C), 129.8 (CH), 143.9 (C), 147.4 (C), 149.5 (C), 157.7 (C), 167.7 (C), 188.9 (C=O).2R,6aS,12aS-2-Isopropyl-8,9-dimethoxy-1,2,12,12a-tetrahydrochromeno-furochromen-6(6aH)-one (5). A mixture of compound 4  and palladium on carbon  in DCM (5 mL) was treated with hydrogen and stirred at RT overnight. The reaction mixture was filtered through a pad of Celite, and the solvent was evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexane/EtOAc 5:1) yielding compound 5 as a colorless oil . IR (KBr): 3,436, 2,958, 1,709, 1,620. 1H- NMR (CDCl3) δ (ppm): 1.0 , 1.1 , 2.04 , 2.9 , 3.1 , 3.4  3.8 , 3.9 , 4.3 , 4.6 , 4.8 , 4,9 , 6.4 , 6,5 , 6.7 , 7.1 . 13C-NMR (CDCl3) δ (ppm): 17.7 (CH3), 18.2 (CH3), 30.1 (CH2), 33.3 (CH), 38.2 (CH), 55.9 (OCH3), 56.6 (OCH3), 65.1 (CH2) 66.39 (OCH2), 69.2 (CH), 89.5 (CH), 100.8 (CH), 102.7 (CH), 110.2 (C), 111.5 (CH), 111.7 (C), 115.7 (C), 130.3 (CH), 143.3 (C), 148.3 (C), 148.7(C), 149.7 (C), 160.52 (C).2R,6aR,12aS-2-Isopropyl-8,9-dimethoxy-1,2,6,6a,12,12a-hexahydrochromenofuro-chromen-6-ol (6). This derivative was obtained by spontaneous decomposition of compound 4 after two days of exposure to daylight in chloroform. Once the solvent was evaporated under reduced pressure, a brown solid is produced. Melting point (m.p.): 167–169 °C. IR (KBr): 3,437, 2,924, 1,618, 1,508. 1H-NMR (CDCl3) δ (ppm): 1.8 , 2.9 , 3.3 , 3.8 , 3.9 , 4.1 , 4.6 , 4.9 , 5,1 , 5.2 , 5.3 , 6.4 , 6.6 (1H. s), 6.9 , 7.0 . 13C-NMR (CDCl3) δ (ppm): 17.2 (CH3), 31.6 (CH2), 55.9 (OCH3), 56.4 (OCH3), 67.9 (OCH2), 71.0 (CH), 86.6 (CH), 100.9 (CH), 102.8 (CH), 105.2 (CH), 110.8 (C), 112.1 (CH), 112.1 (=CH2), 112.8 (C), 116.8 (C), 123.2 (=C), 126.7 (=CH), 143.8 (C), 144.7 (=C), 149.1 (C), 149.4 (C), 150.3 (C), 161.3 (C).2R,6aS,12aS-2-Isopropyl-8,9-dimethoxy-1,2,6,6a,12,12a-hexahydrochromeno-furo-chromene (7). Compound 6   and palladium on carbon (Pd/C)  in DCM (5 mL) Celite, and the solvent was evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column (hexane/EtOAc 5:1) yielding compound 7 as a light yellow solid . Melting point (m.p.): 136–138 °C. IR (KBr): 3,434, 2,927, 2,854, 1,679, 1,609. 1H-NMR (CDCl3) δ (ppm): 0.9 , 1.1 , 1.9 , 2.8 , 3.0 , 3.1  3.2  3.8 , 4.2 , 4.5 , 4.7 , 6.3 , 6.4 , 6.7 , 6.8 . 13C-NMR (CDCl3) δ (ppm): 17.7 (CH3), 18.2 (CH3), 29.4 (CH2), 30.1 (CH2), 31.8 (CH), 33.2 (CH), 55.9 (OCH3), 56.6 (OCH3), 65.6 (OCH2), 68.7 (CH), 89.1 (CH), 100.6 (CH), 101.8 (CH), 111.2 (CH), 111.0 (C), 112.8 (C), 113.8 (C), 123.2 (CH), 143.8 (C), 147.8 (C), 149.4 (C), 150.3 (C), 160.1 (C).in vitro for cytotoxicity on mammalian cells, antiplasmodial activity on asynchronized P. falciparum culture forms and leishmanicidal activity on intracellular amastigotes of L. panamensis.The compounds were assayed TM) were isolated from human histiocytic lymphoma; Vero cells (CCL-81TM) were derived from the kidney of the African green monkey, Cercopithecus aethiops; HepG2 (HB-8065TM) cells were derived from human liver tissue, and CAD-R1 cells, a subclone, were derived from mouse neuronal cells [2. U-937 cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin 100 U/mL and streptomycin 0.1 mg/mL). Vero, HepG2 and CAD-R1 were cultivated in DMEM medium supplemented with 5% FBS and antibiotics.U937 cells  and human red blood cells (huRBCs) obtained from healthy donors after signing the informed consent were also used. The huMDM were obtained from peripheral mononuclear cells isolated by Ficoll-Hypaque 1077 gradient centrifugation. huMDMs were suspended at 3 × 10hodology ,18. On tin vitro using the MTT -2,5-diphenyltetrazolium bromide assay) as described previously [5 U-937 cells/mL, 2.5 × 105 Vero, HepG2 or CAD-R1 cells/mL and 5 × 105 huMDM cells/mL in RPMI 1640 supplemented with 10% SBF and the corresponding concentration of each compound. Six serial-based four-dilution concentrations from 200 to 0.19 μg/mL were tested. The amount of formazan produced by the viable cells was registered as optical densities (OD) obtained in an ELISA reader (Bio-Rad) at 570 nm. Cells exposed to amphotericin B, doxorubicin and chloroquine were used as positive (cytotoxicity) controls, while unexposed cells were used as negative (viability) controls. Additionally, hemolytic activity on huRBCs was assessed by the osmotic lysis method [Cytotoxic activity of rotenone and it derivatives on U-937, Vero, HepG2, CD-R1 and huMDM cells was evaluated eviously ,14,15. CPlasmodium falciparum (chloroquine sensitive 3D7 strain) cultured in standard conditions [P. falciparum were adjusted to a 1.5%–2% parasitemia and 5% hematocrit in RPMI medium supplemented with 10% human serum. Then, 500 µL of parasites and 500 µL of each concentration of compound were placed in each well of 24-well plates. Plates were incubated for 72 h at 37 °C in a N2 (90%), CO2 (5%) and O2 (5%) atmosphere. Parasites cultured in the absence of compounds were used as controls of viability (negative control), while parasites exposed to chloroquine were used as positive controls. After 72 h of exposure, DNA from parasites was extracted and purified using a lysis solution containing proteinase K. Each compound and each concentration were assessed in triplicate in at least two separate experiments.The antiplasmodial activity of rotenone and its derivatives was evaluated on nditions . The effnditions . BrieflyLeishmaniapanamensis by flow cytometry following the methodology previously described [L. panamensis transfected with the green fluorescent protein gene (MHOM/CO/87/UA140pIR-GFP) in a proportion of 35:1 (parasites:cells). Plates were incubated at 34 °C, 5% CO2, and after 3 h, cells were washed twice with phosphate buffer (PBS) and incubated again. After 24 h, the infected cells were exposed for 72 h to each concentration of the compounds. Cells were removed using trypsin/EDTA solution and washed twice with PBS by centrifuging 10 min at 1,100 rpm, 4 °C. Then, cells were analyzed in a flow cytometer  by reading at 488-nm excitation, 525-nm emissions over an argon laser and counting 10,000 events. The percentage of infected cells was determined by dot plot analysis, while the parasitic load was calculated by the mean fluorescence intensity using histogram analysis. In parallel, cells incubated in medium alone were used as the controls of infection (negative control), and cells exposed to amphotericin B were used as controls of leishmanicidal activity (positive control) [The leishmanicidal activity was tested on intracellular amastigotes of escribed ,15. Prev50) calculated by the probit analysis [Cytotoxicity was determined according to the percentages of viability and mortality obtained for each concentration and experimental condition , and the results are expressed as the median lethal concentration  calculated by probit analysis using the % of inhibition [Antiplasmodial activity was determined by the inhibition of parasite growth for each experimental condition according to the florescence emitted in arbitrary units (FAU), using Equation 2, where the FAU of unexposed cells (negative control) corresponds to 100% parasite growth. In turn, % inhibition of parasite growth corresponds to 100%–% parasite growth. Results are expressed as the median effective concentration (EChibition :% paras50) calculated by probit analysis using the % of inhibition of infection [Antileishmanial activity was determined according to the reduction of infected cells and their parasite load obtained for each experimental condition according to the fluorescence median intensity (FMI) emitted by the cytometer and using Equation 3, where the % of parasitemia in unexposed cells corresponds to 100% parasitemia. In turn, the percentage of inhibition of parasitemia corresponds to 100% infection. Results are expressed as the median effective concentration (ECnfection :% paras50 values using the following arbitrary scale: active, EC50 < 20.0 μM; moderately active, EC50 >20–<50.0 μM; and potentially non-active, EC50 > 50.0 μM. The index of selectivity was calculated with the equation: IS = LC50/EC50.The antiplasmodial and antileishmanial activity was graded according to the CEPlasmodium and Leishmania redox metabolism may occur by modulation of redox equilibrium, damaging the mitochondrial ultrastructure or targeting essential processes and pathways with different apoptogenic agents and inhibitors [Rotenone was selected because it is a product abundant in Nature, with a known effect on the mitochondrion of many organisms, and it has several functional groups, which can be quite easily functionalized to optimize the activity or to obtain a wider pharmacological profile. Results suggest that in spite of the toxicological background, rotenone and its derivatives have potential against malaria and cutaneous leishmania, possibly by inducing the production of ROS metabolites that are potent antiprotozoal products or modulating redox signaling in parasites. Targeting of hibitors ,24,25,26in vitro activities and the selectivity of rotenone, but also its pharmacokinetic parameters. In the specific case of antileishmanial activity, only compound 6 displays activity against intracellular amastigotes, suggesting that slight structural changes may modify pharmacodynamic aspects, showing or enhancing the biological activity of one inactive compound, such as rotenone. Due to the small number of obtained compounds, a clear relationship structure-activity was not established; however, in vitro assays seems to point towards the importance of the exocyclic double bond and the carbonyl group to modulate the antiparasite activity.Minor structural modifications in the rotenone molecule allow synthesizing a series of derivatives with antiplasmodial or antileishmanial activity that deserve further study to improve the 6, rotenone and derivatives 2, 3, 4 and 7 represent starting points in the search for new antileishmanial and antiplasmodial compounds, respectively, which would, however, require testing further structural modifications and thorough investigations on structure-activity relationships.The synthesis, antileishmanial, antiplasmodial and cytotoxicity screening of rotenone and six derivatives were reported. In spite of their toxicities, compound


Lunagariya et al., MedChemComm, 2018, DOI: ; 10.1039/c7md00472a.Correction for ‘Synthesis, characterization and biological application of 5-quinoline 1,3,5-trisubstituted pyrazole based platinum( The names are missing ‘4,5-dihydro’ as displayed in the experimental section. The correct names are as follows: 2-chloro-3-(3-(5-chlorothiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)quinoline (L1), 2-chloro-3-(3-(4-methylthiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)quinoline (L2), 3-(3-(5-bromothiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-2-chloroquinoline (L3), 3-(3-(3-bromothiophen-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-2-chloroquinoline (L4) and 2-chloro-3-(1-phenyl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)quinoline (L5).The authors regret that ligands L1–L5, is shown below.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


The absolute configuration of sohiracillinone (1) was unambiguously identified by NMR and electronic circular dichroism (ECD) data. The structures of other new compounds were elucidated on the basis of NMR and mass spectroscopy (MS), and the cytotoxic activities of all the isolated components were evaluated.A previously undescribed naphthalenone derivative, sohiracillinone ( Poria cocos, also named Fuling in Chinese, is the dried sclerotium of P. cocos (Schw.) Wolf, belonging to the Polyporaceae and grown in China, Japan, Korea, and North America. A well-known traditional Eastern Asian medicine, P. cocos extracts exert beneficial health effects and fight against illnesses − . The 1H-NMR data , δH 2.26 , δH 1.18 , and two mutually-coupled aromatic protons at δH 7.03 , δH 6.77 . Additionally, two pairs of equivalent secondary methyl signals were identified at δH 1.08 , δH 1.10  and δH 1.05 , H3-26 and H3-27). The 32 carbon signals observed in the 13C-NMR spectrum were sorted into eight methyls, indicating the presence of two isopropyl groups, eight methyl groups, seven methylene groups, and six methine groups, suggesting that 2 is a lanostane triterpene that is similar to daedaleanic acid A − indicated a molecular formula of C32H50O6. Its 1H-NMR , δH 1.02 , δH 1.24 , δH 1.24 , δH 0.89 , δH 0.91 , δH 1.13  and δH 2.03  and two oxygen-bearing methynes at δH 4.45  and δH 4.05 . Its 13C-NMR  and a pair of tetrasubstituted olefinic carbonds at δ 135.6  and δ 136.0  and three oxygenated carbons at δ 82.5 , δ 77.9 , δ 71.1 . In addition, the 13C NMR and HSQC spectra also exhibited two olefinic signals at δC 126.4  and δC 140.2 . There were 1H–1H COSY (δH 1.66) and H-3 (δH 4.45), between H-5 (δH 1.16) and H-6 (δH 1.66), between H-15 (δH 2.18), H-16 (δH 4.05), H-17 (δH 2.06), H-20 (δH 2.32), H-22 (δH 2.50), and H-23 (δH 5.62). Therefore, it can be concluded that 3 was a lanost-8-ene triterpenoid. On the basis of the HMBC (3-26 (δH 1.24) to CH3-27 (δC 29.8), H3-27 (δH 1.24) to CH3-26 (δC 29.8), H3-27 (δH 1.24) to C-25 (δC 71.1), and H3-26 (δH 1.24) to C-24 (δC 140.2) indicated the presence of a hydroxy group at C-25. The correlation from H-3 (δH 4.45) to C-1′ (δC 172.9) showed that the acetyl group was located at C-3. The NOE correlations between H-16 (δH 4.05) and H-20 (δH 2.32), and between H3-30 (δH 1.13) and H-17 (δH 2.06) indicated α-orientations for OH-16 and H-17. The correlation between H3-18 (δH 0.79) and H3-19 (δH 1.02), H-16 (δH 4.05) and H-20 (δH 2.32) showed that CH3-18, CH3-19 and H-20 have β-orientations. Thus, the structure of 3 was determined to be 3β-acetoxy-16α,25-dihydroxylanost-8,23-dien-21-oic acid, and it was named ceanphytamic acid A  and H-3 (δH 4.59), between H-5 (δH 1.04) and H-6 (δH 1.61), and between H-15 (δH 2.34), H-16 (δH 4.47), H-17 (δH 2.30), H-20 (δH 2.86), H-22 (δH 1.24), H-23 (δH 2.33), and H-24 (δH 1.41). The 1H-NMR showed nine methyl groups at H3-18 , H3-19 , H3-26 , H3-27 , H3-28 , H3-29 , H3-30 , H3-2′ , and H3-OMe . The 13C-NMR (δC 170.8) and three oxygenated carbons at C-3 (δC 80.7), C-16 (δC 76.6), and C-25 (δC 74.4). The signals observed in the 1H NMR and 13C NMR spectra  and CH3-27 (δC 25.0), H3-27 (δH 1.00) and CH3-26 (δC 25.0), H3-26 (δH 1.01) and C-25 (δC 74.4), and H3-OMe (δH 3.08) and CH3-25 (δC 74.4), the presence of a methoxy group at C-25 was indicated. The NOE correlations between H3-30 (δH 1.40) and H-17 (δH 2.30) indicated α-orientations for H-17. The correlations between H3-18 (δH 1.05) and H3-19 (δH 0.89), and between H-16 (δH 4.47) and H-20 (δH 2.86) indicated β-orientations for CH3-18, CH3-19 and H-20. Therefore, the structure of 4 , Calu-1 , HeLa , MDA-MB-231 , SW579 , SK-OV-3 , T84 , Hep G2 , Hep 3B2.1-7 , KATO III , MCF-7  by the CCK-8 method. None of compounds exhibited cytotoxicity against T84 cells, Hep G2 cells, Hep 3B2.1-7 cells, KATO III cells, or MCF-7 cells. Compound 79 cells . Compoun1H, 125/100 MHz for 13C, respectively, with tetramethylsilane as an internal standard. HRESIMS spectra were obtained on a UPLC Premier QTOF spectrometer . Reversed phase medium pressure liquid chromatography (RP-MPLC) was performed on a GRACE system . Preparative HPLC was performed on an 1260 series instrument  equipped with a Capcellpak C18 column . Column chromatography were performed with silica gel , Sephadex LH-20 , and YMC gel ODS-AQ . Thin-layer chromatography (TLC) and preparative TLC were carried out on HSGF254 plates .Optical rotations were obtained with an Autopol VI polarimeter . IR spectra were recorded with a Perkin Elmer FT-IR spectrometer . One-dimensional and two-dimensional NMR spectra were run on a AVANCE-III instrument  operating at 600/400 MHz for Poria cocos was purchased from Shanghai Kang Qiao Herbal Pieces Co., Ltd. , and identified by Professor Hong Xu from Shanghai University of Traditional Chinese Medicine. A voucher specimen (150910) was deposited at Shanghai R&D Center for standardization of Chinese Medicines, Shanghai, China.The sclerotia of P. cocos (100 kg) was extracted three times, each time with 95% EtOH (1000 L) to give a crude extract (2300 g), which was suspended in water and successively partitioned with petroleum ether (PE), CH2Cl2 and n-BuOH. The CH2Cl2 extracts (804 g) were loaded into silica gel column chromatography (200–300 mesh) and eluted with a stepwise gradient of PE-EtOAc (100:1 to 1:1) and EtOAc-MeOH (20:1 to 1:1) to obtain 13 fractions (Fr. A-Fr. M) on the basis of their TLC characteristics.Dried sclerotia of 2O , followed by Sephadex LH-20 (MeOH) and preparative HPLC  to give harzianone . Fr. B-3 (2.2 g) was chromatographed by preparative HPLC  to yield sorbicillin , 2′,3′-dihydrosorbicillin , sohirnone A . Fr. B-5 (4.0 g) was eluted repeatedly with MeOH-H2O  and preparative HPLC to yield sohiracillinone , 11β-ethoxydaedaleanic acid A , and daedaleanic acid A , respectively.Fraction B (18.4 g) was repeatedly chromatographed on silica gel (PE/EtOAc = 50:1 to 1:1) and Sephadex LH-20 (MeOH) columns to obtain six fractions (Fr. B-1 to Fr. B-6). Fr. B-2 (1.7 g) was subjected to an ODS preparative MPLC column eluted with MeOH-H2O , and followed by preparative HPLC to yield pachymic acid , dehypachymic acid , and polyporenic acid C , respectively. Fr. C-2 was also isolated by the same method as Fr.C-1 to yield deyhdrotrametenolic acid , trametenolic acid , eburicoic acid , and 3β-p-hydroxybenzoyl-dehydrotumulosic acid , respectively.Fr. C (21.0 g) was repeatedly chromatographed on silica gel (PE/EtOAc = 50:1 to 1:1) to obtain seven fractions (Fr. C-1 to Fr. C-7). Fr. C-1 (11.5 g) was subjected to an ODS preparative MPLC column eluted with MeOH-H2O , and followed by preparative HPLC  to yield ceanphytamic acid A . Fr. D-4 (2.2 g) was subjected to an ODS preparative MPLC column eluted with MeOH-H2O , and followed by preparative HPLC  to yield poricoic acid A , and poricoic acid G .Fr. D (9.5 g) was repeatedly chromatographed on silica gel (PE/EtOAc = 50:1 to 1:1) to obtain five fractions (Fr. D-1 to Fr. D-5). Fr. D-2 (1.5 g) was subjected to an ODS preparative MPLC column eluted with MeOH-H3CN in H2O, 16 mL/min) to yield ceanphytamic acid B . Fr. F-5 (0.8 g) was eluted by HPLC  to yield 3β,15α-dihtdroxylanosta-7,9(11),24-trien-21-oic acid .Fr. F (12.7 g) was repeatedly chromatographed on silica gel (PE/EtOAc = 50:1 to 1:1) to obtain seven fractions (Fr. F-1 to Fr. F-7). Fr. F-3 (0.3 g) was eluted by HPLC  to yield dehydrosulphurenic acid .Fr. G (7.1 g) was repeatedly chromatographed on silica gel (PE/EtOAc = 50:1 to 1:1) to obtain three fractions (Fr. G-1 to Fr. G-3). Fr. G-1 was eluted by HPLC  to yield tumulosic acid .Fr. H (5.1 g) was repeatedly chromatographed on silica gel (PE/EtOAc = 100:1 to 1:1) to obtain three fractions (Fr. H-1 to Fr. H-3). Fr. H-2 was subjected to an ODS preparative MPLC column eluted with MeOH-HSohiracillinones (1): Yellow oil; c = 0.033, CHCl3). IR: 2920, 2852, 1740, 1596, 1413, 1366, 1099, 779, 720, 616 cm−1. 1H and 13C NMR are shown in −, C17H19O4; calcd. 288.1362). More Spectroscropic data can be found in the 11β-Ethoxydaedaleanic acid A (2): White amorphous powder; c = 0.1, C5H5N). IR: 3395, 2919, 2849, 1645, 1438, 1069, 804, 760, 703 cm−1. 1H and 13C NMR are shown in −, C33H49O5; calcd. 525.3580). More Spectroscropic data can be found in the Ceanphytamic acid A (3): White amorphous powder; c = 0.03, C5H5N). IR: 3676, 2988, 2902, 1702, 1635, 1394, 1075, 1066, 1056, 892 cm−1. 1H and 13C NMR are shown in −, C32H49O6; calcd. 530.3607). More Spectroscropic data can be found in the Ceanphytamic acid B (4): White amorphous powder; c = 0.03, C5H5N). IR: 3676, 2988, 2902, 1707, 1620, 1407, 1394, 1251, 1066, 1057, 891 cm−1. 1H and 13C NMR are shown in +, C33H53O6; calcd. 546.3920). More Spectroscropic data can be found in the v/v) heat-inactivated fetal bovine serum (FBS) and 0.1% (v/v) penicillin-streptomycin in a humidified incubator with 5% CO2 at 37 °C. Calu-1 and SK-OV-3 were maintained in McCoy’s 5A supplemented with 10% (v/v) heat-inactivated FBS and 0.1% (v/v) penicillin-streptomycin in a humidified incubator with 5% CO2 at 37 °C. A549 was maintained in F12K supplemented with 10% (v/v) heat-inactivated FBS and 0.1% (v/v) penicillin-streptomycin in a humidified incubator with 5% CO2 at 37 °C. KATO III, MDA-MB-231, and SW579 were maintained in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FBS and 0.1% (v/v) penicillin-streptomycin in a humidified incubator with 5% CO2 at 37 °C. T84 was maintained in D-MEM/F-12 supplemented with 20% (v/v) heat-inactivated FBS and 0.1% (v/v) penicillin-streptomycin in a humidified incubator with 5% CO2 at 37 °C. All cell culture media and reagents were purchased from Thermo Scientific Hyclone .Nine human cancer cell lines, A549, Calu-1, HeLa, SK-OV-3, T84, Hep G2, Hep 3B2.1-7, KATO III, and MCF-7, were kindly provided by Stem Cell Bank, Chinese Academy of Sciences. Two human cancer cell lines, MDA-MB-231 and SW579, were kindly provided by Professor Hong Xu from Shanghai University of Traditional Chinese Medicine. A Cell Counting Kit-8 was purchased from Shanghai DOJINDO Co., Ltd. . Hep G2, Hep 3B2.1-7, HeLa, and MCF-7 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%  was added to each well of the plate. After removal from the medium, the cells were incubated at 37 °C for 1 h. Cell viability was calculated as a percentage of viable cells in the compound-treated group vs. the control group by the following equation: Cell viability (%) = [1 − OD (Compound)/OD (Blank)] × 100%.The compounds’ cytotoxicity was measured by the CCK-8 method. Compounds were dissolved with dimethylsulfoxide (DMSO). The cells were seeded onto 96-well microplates at a density of 1 × 101), and three new triterpenoids, 11β-ethoxydaedaleanic acid A (2), ceanphytamic acid A (3), and ceanphytamic acid B (4), were characterized from the EtOH extract of the sclerotia of Poria cocos. The skeleton of 1 was unique and the absolute configurations was determined by ECD. Moreover, a rare 4,5-seco-lanostane triterpenoid 2 and two unreported side-chain of lanostane triterpenoids 3 and 4 were also isolated. This adds new facets to the structural diversity of the P. cocos family. Our findings showed that 1 has stronger cytotoxicity against HeLa cells than the other tested compounds, and the other compounds showed selective inactive cytotoxicity against the testing cancer cell lines. Even though the level of cytotoxic activity was not significant, but these findings also suggest that P. cocos may have the potential to be a useful therapeutic natural source for cancer prevention. Besides, following the study of the compounds’ chemistry, we propose that lanostane triterpenoids is a reasonable index standard for P. cocos.In sum, one new naphthone derivative, sohiracillinone (


High affinity towards 1NFY with greater docking score was observed for the compounds  than the control ligand RPR200095. In addition, impressive inhibitory potential against factor Xa (F-Xa) was observed with higher docking scores (5612–6270) with Atomic Contact Energy (ACE) values  than the control ligand RPR200095 . In vitro, in vivo, and in silico results proposed that these newly synthesized compounds might be used as anticoagulant agents.A new series of 1,3,4-oxadiazoles derivatives was synthesized, characterized, and evaluated for their in vitro and in vivo anti-thrombotic activity. Compounds ( Nowadays cardiovascular diseases (CD) like coronary heart disease (CHD), atherosclerosis, hypertension, and acute myocardial infarction are the main causes of death in humans. Thrombosis is one of the frequently cause of CD . Almost Thrombolysis is the breakdown of blood clots by pharmacological action of drugs and reduces the risk of CVDs . ThrombuThe 1,3,4-oxadizole moiety is a versatile pharmacore for designing potentially biological active agents because of its significant role in medicinal chemistry and wide range of applications as a pharmacological and pharmaceutical agent . These hKeeping in mind the biological activities of 1,3,4-oxadiazoles, various new derivatives were synthesized and characterized. The in vitro and in vivo antithrombotic efficacy of all derivatives, along with their evaluation by molecular docking and computational studies, were explored. Novel compounds and their inhibitory effect on F-Xa were investigated by molecular docking for visualizing the efficiency of clot lysis agents.3a–3i) (1) and N-substituted-2-bromoacetamides (2a–2i) were prepared according to a previously reported procedure [1 with 2a–2i was carried out using lithium hydride as a base under ultrasonic radiations at room temperature to obtain the N-substituted 5-{[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-ylthio}acetamides (3a–3i). This modified method gave a better yield in less time than the conventional reported method where only stirring was used to procede the reaction [The targeted compounds (3a–3i)  were synrocedure . The nucreaction . The sup3a was obtained as white shining white fluffy amorphous flakes. The electron ionization/mass spectroscopy (EI/MS) spectra showed molecular ion peak at m/z 403 M+[C19H19ClN3O3S]+. The IR spectrum demonstrated the absorption band at 3138 (N–H stretching), 3000 (C–H aromatic stretching), 1671 (C=O amide stretching), and 1552 (C=C aromatic ring stretching). In 1H-NMR spectrum the appearance of singlet at δ10.42 represents one proton of NH. In aromatic region, the appearance of two doublets one at δ7.57, J = 8 Hz having integration for two protons (H-3′ & H-5′) and the other at δ6.75, J = 9 Hz for two protons (H-2′ & H-6′) revealed that aromatic ring is disubstituted at para  positions. Similarly, the appearance of triplet at δ7.33, J = 7.8 Hz for two protons (H-2″ & H-6″), a doublet at δ7.28, J = 8.5 Hz for two protons (H-3″ & H-5″), and a triplet at δ7.08, J = 7.3 Hz integrate one proton (H-4″) can be assigned to the unsubstituted benzene ring of amide. A singlet at δ4.33 was due to methylene group (CH2) linking the amide and sulfanyl group. In aliphatic region of the spectrum a singlet at δ1.71 was assigned to 6H of two methyl groups. Therefore on the basis of above cumulative evidences the structure of 3a was confirmed as 2-{5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-ylthio}-N-phenylacetamide. The structures of other N-substituted acetamides were also characterized in a similar manner. All the signals in 1H-NMR spectrum of (3a–3i) thoroughly affirmed the successful substitutions on the parent 1,3,4-oxadiazole core.The compound 3i has impressive clot lysis activity (41%) compared to SK (38%). While compound 3c depicted less clot lysis activity (11%), and the order of the in vitro clot lysis effect of all tested compounds is 3i>SK>3e>3a>3d=3f>3h>3g>3b>1>3c. The results of all synthesized compounds are presented in Results demonstrated that compound 3a–3i) with increasing the time after their exposure to rats. Heparin was used as reference compound.Rats were used to evaluate the anticoagulation activity of 1,3,4-oxadiazole derivatives. Clotting time (CT) was used to measure the anticoagulation activity of synthesized compounds. Increasing trend in CT values was found for each compounds (1. Compounds 3a (342 s), 3i (214 s), 3e (167 s), 3d (132 s), and 3h (130 s) showed CT values greater than heparin (110 s). The order CT values of all the tested oxadiazole derivatives is 3a>3i>3e>3g>3d>3h>3b>3f>3c>1. The CT results of all tested compounds are presented in All oxadiazole derivatives showed prominent prolongation in clotting time except compound The serine protease F-Xa plays a crucial role in blood coagulation process by converting prothrombin to thrombin. This F-Xa is located at the conjunction point of extrinsic and intrinsic pathway. In coagulation process one molecule of F-Xa activates many molecules of prothrombin to thrombin by signal amplification ,19. Ther243, Arg25, and Met242.There are four binding pockets labeled as S1 to S4 within the active site. The most important are the S1 and S4 binding pockets that are exploited by Factor Xa inhibitors. The S1 pocket is in the form of a narrow cleft and it usually favors positively charged moieties such as benzamidine, amine, and guanidine . S4 is t3a–3i), five ligands 3a, 3d, 3e, 3h, and 3i showed significant better interaction patterns when compared with RPR200095. These were found to bind near the entrance of active site gorge , Leu123 and nitrogen of 1,3,4-oxadiazole (3.03 Å), Pro124 and the NH adjacent to the carbonyl and 2,3-dimethylphenyl groups (3.17 Å). Similarly, ligand 3e exhibited hydrophobic contact potential with pocket amino acids Lys236, Ala24, Arg25, Leu123, and Cys44 respectively. Ligand 3e has shown potential for pi-stack interaction with Leu235 and the 2,3-dimethylphenyl moiety , Leu123, and nitrogen of 1,3,4-oxadiazole (3.25 Å) and also exhibited hydrophobic contact potential with pocket amino acids Cys44, Pro120, Arg25, Leu235, and Lys236 and can play an important part to give this ligand good binding affinity than other ligands of this series. Ligand 3h has shown potential for van der Waals contact and such interactions involve the hydrophobic contact with Pro43, Leu123, and Phe114. This ligand depicted arene-cation contact with Arg25, also exhibited pi-sulfur contact with Met242, and 1,3,4-oxadiazoleand was unable to show polar interactions with the pocket amino acids.Ligands 3a bound to protein was assessed by Hyde utility of Lead IT software. Hyde allows visual approximation of favorable and unfavorable contributions due to the structure/bound conformation of inhibitor with the neighboring amino acids. The favorably contributing structural elements (atoms and torsions) to the overall binding energy colored blue, similarly the structural elements that are not contributing favorably are colored in red, and neutral elements are in white  measures the kinetic stability of the molecule -1,3,4-oxadiazol-2-thiol 1 [N-substituted-2-bromoacetamides (2a–2i) were synthesized by previously reported methods with slight modifications [Chemicals were purchased from Sigma Aldrich  and Alfa Aesar . Melting points were taken on Griffin and George melting point apparatus using the open capillary tube method and were reported as uncorrected. Infrared spectra were recorded in KBr on a Jasco-320-A spectrophotometer. -thiol 1  and arom1  and lithium hydride  was dissolved in DMF (10 mL) and the contents were subjected to ultrasound irradiations for 15 min followed by addition of N-substituted-2-bromoacetamide (1 mmol) with further irradiation till completion of the reaction as indicated by TLC . Reaction times for different N-substituted 5-{[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-ylthio}acetamides varied from 1 to 1.5 h. After completion, the reaction mixture was poured on crushed ice. The precipitates were filtered, washed with distilled water, and dried to afford N-substituted 5-{[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-ylthio}acetamide. The products were recrystallized from 30% ethanol.A mixture of 5-[2-(4-Chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 3a was obtained from the reaction of 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo N-phenylacetamide  after 60 min. Shining white fluffy amorphous flakes; yield: 75% (0.302 g); m.p. 84–86 °C; IR : 3138 (N–H str.), 3000 (C–H aromatic str.), 1671 (C=O amide str.), 1552 (C=C aromatic ring str.); 1H-NMR : δ (ppm) 10.42 , 7.57 , 7.33 , 7.28 , 7.08 , 6.75 , 4.33 , 1.71 2) : 168.85 (C=O), 165.17 (C-2), 164.90 (C-5), 153.29 (C-1′), 139.10 (C-1″), 129.93 (C-4′), 129.76 (C-3′ & C-5′), 129.32 (C-3″ & C-5″), 128.56 (C-4″), 124.18 (C-2″ & C-6″), 119.62 (C-2′ & C-6′), 75.99 (C(CH3)2), 37.32 (S-CH2-CO), 25.70 (C(CH3)2) 2) ; 13C-NMRC(CH3)2) ; EIMS: m[M+ + 2] .3b was synthesized by the reaction of 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo-N-(2-methylphenyl)acetamide  after a period of 1 h and 20 min. Light yellow color powder; yield: 87% (0.362 g); m.p. 140–142 °C; IR : 3272 , 2965 , 1644 (C=O amide str.), 1383 ; 1H-NMR : δ (ppm) 10.48 , 7.38–7.32 , 7.22 , 6.94 , 4.26 , 4.19 , 2.18 , 1.47, 1.46 2) : 171.80 (C=O), 169.58 (C-2), 164.64 (C-5), 154.10 (C-1′), 136.48 (C-1″), 134.57 (C-2″), 131.26 (C-3″), 129.71 (C-5″), 129.46 (C-3′ & C-5′), 129.15 (C-4′), 127.31 (C-4″), 126.40 (C-6″), 121.91 (C-2′ & C-6′), 80.83 (C(CH3)2), 33.08 (S-2CH-CO), 25.47, 25.44 (C(CH3)2), 17.56 (Ar-CH3) 2) ; 13C-NMR(Ar-CH3) ; EIMS: m[M+ + 2] .3c was achieved from the reaction of 5-[2-(4-chlorophenoxy) propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo-N-(3-methylphenyl)acetamide  after a period of 1 h and 15 min. Lemon yellow color powder; yield: 78% (0.325 g); m.p. 71–72 °C; IR : 3155 , 2989 , 1655 (C=O amide str.), 1488 ; 1H-NMR : δ (ppm) 8.93 , 7.35 , 7.27 , 7.19 , 7.11–7.10 , 6.93 , 6.65–6.63 , 3.97 , 2.32 , 1.77 2) : 169.88 (C=O), 166.17 (C-2), 165.02 (C-5), 152.84 (C-1′), 139.04 (C-3″), 137.43 (C-1″), 129.41 (C-3′ & C-5′), 128.87 (C-5″), 125.61 (C-4′), 122.98 , 120.46 (C-4″), 116.99 (C-2″), 75.55 (C(CH3)2), 36.23 (S-CH2-CO), 25.86 (C(CH3)2), 21.44 (Ar-CH3) 2) ; 13C-NMR(Ar-CH3) ; EIMS: m[M+ + 2] .3d was obtained from the reaction of 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo-N-(4-methylphenyl)acetamide  after 60 min. Dirty white color powder; yield: 84% (0.350 g); m.p. 68–70 °C; IR : 3313 , 2979 , 1676 (C=O amide str.), 1483 ; 1H-NMR : δ (ppm) 10.33 , 7.46 , 7.27 , 7.12 , 6.75 , 4.31 , 2.25 , 1.71 2) : 168.84 (C=O), 164.91 (C-2), 164.90 (C-5), 153.29 (C-1′), 136.60 (C-4″), 133.13 (C-1″), 129.75 (C-3′ & C-5′), 129.68 (C-3″ & C-5″), 128.57 (C-4′), 124.18 (C-2″ & C-6″), 119.64 (C-2′ & C-6′), 75.98 (C(CH3)2), 37.31 (S-CH2-CO), 25.70 (C(CH3)2), 20.91 (Ar-CH3) 2) ; 13C-NMR(Ar-CH3) ; EIMS: m[M+ + 2] .3e was obtained from the reaction of 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo-N-acetamide  after 1 h and 30 min. Creamy white color powder; yield: 86% (0.371 g); m.p. 186–188 °C; IR : 3300 , 2952 , 1645 (C=O amide str.), 1392 ; 1H-NMR : δ (ppm) 10.45 , 7.34 , 7.27 , 7.21 , 7.04 , 6.94 , 4.26 , 4.18 , 2.31 , 2.03 , 1.47, 1.46 2) : 171.87 (C=O), 169.49 (C-2), 164.72 (C-5), 154.07 (C-1′), 138.23 (C-6″), 134.94 (C-1″), 134.58 (C-3″), 130.93 (C-4′), 129.45 (C-3′ & C-5′), 126.68 (C-2″), 126.60 (C-4″), 126.38 (C-5″), 121.90 (C-2′ & C-6′), 80.82 (C(CH3)2), 33.03 (S-CH2-CO), 25.45 (C(CH3)2), 20.36 (Ar-CH3), 14.22 (Ar-CH3) 2) ; 13C-NMR(Ar-CH3) ; EIMS: m[M+ + 2] .3f was obtained from the reaction of 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo-N-acetamide  after 1 h and 20 min. Golden yellow color powder; yield: 81% (0.349 g); m.p. 78–80 °C; IR : 3260 , 2980 , 1632 (C=O amide str.), 1381 ; 1H-NMR : δ (ppm) 8.59 , 7.69 , 7.11–7.09 , 6.98–6.97 , 6.65–6.63 , 4.01 , 2.27 , 2.16 , 1.77 2) : 169.93 (C=O), 166.13 (C-2), 165.32 (C-5), 152.87 (C-1′), 135.19 (C-4″), 132.85 (C-2″), 131.24 (C-1″), 129.39 (C-3′ & C-5′), 129.17 (C-3″), 127.23 (C-5″), 122.83 , 122.66 (C-4′), 75.51 (C(CH3)2), 35.97 (S-CH2-CO), 25.88 (C(CH3)2), 20.85 (Ar-CH3), 17.85 (Ar-CH3) 2) ; 13C-NMR(Ar-CH3) ; EIMS: m[M+ + 2] .3g was obtained from the reaction of 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo-N-acetamide  after 1 h. Creamy white color powder; yield: 89% (0.383 g); m.p. 98–100 °C; IR : 3330 , 2958 , 1661 (C=O amide str.), 1482 ; 1H-NMR : δ (ppm) 9.71 , 7.26 , 7.09–7.06 , 6.76 , 4.34 , 2.13 , 1.73 2) : 168.9 (C=O), 164.98 (C-2), 164.94 (C-5), 153.31 (C-1′), 135.60 (C-2″ & C-6″), 135.01 (C-1″), 129.76 (C-3′ & C-5′), 128.54 (C-4″), 128.18 (C-3″ & C-5″), 127.14 (C-4′), 124.15 (C-2′ & C-6′), 75.98 (C(CH3)2), 36.25 (S-CH2-CO), 25.75 (C(CH3)2), 18.47 (Ar-2CH3) 2) ; 13C-NMRAr-2CH3) ; EIMS: m[M+ + 2] .3h was obtained from the reaction of 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo-N-acetamide  after 1 h and 10 min. Off-white color powder; yield: 91% (0.392 g); m.p. 80–82 °C; IR : 3357 , 2904 , 1640 (C=O amide str.), 1380 ; 1H-NMR : δ (ppm) 10.22 , 7.33 , 7.26–7.22 , 7.04 , 6.75–6.71 , 4.27 , 2.16 , 1.69 2) : 168.37 (C=O), 164.38 (C-2), 164.35 (C-5), 152.79 (C-1′), 136.42 (C-3″), 136.32 (C-1″), 131.47 (C-4″), 129.63 (C-5″), 129.24 (C-3′ & C-5′), 128.08 (C-4′), 123.74 (C-2′ & C-6′), 120.39 (C-2″), 116.72 (C-6″), 75.51 (C(CH3)2), 36.79 (S-CH2-CO), 25.22 (C(CH3)2), 19.75 (Ar-CH3), 18.75 (Ar-CH3) 2) ; 13C-NMR(Ar-CH3) ; EIMS: m[M+ + 2] .3i was obtained from the reaction of 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol 1  with 2-bromo-N-acetamide  after 1 h and 30 min. Off-white color powder; yield: 85% (0.366 g); m.p. 122–124 °C; IR : 3299 , 2995 , 1680 (C=O amide str.), 1580 ; 1H-NMR : δ (ppm) 10.27 , 7.27 , 7.20 , 6.75 , 6.72 , 4.29 , 2.23 , 1.70 2) : 168.3 (C=O), 165.03 (C-2), 164.90 (C-5), 153.27 (C-1′), 138.96 (C-1″), 138.29 (C-3″ & C-5″), 129.73 (C-3′ & C-5′), 128.59 (C-4″), 125.69 (C-4′), 124.27 (C-2″ & C-6″), 11.39 (C-2′ & C-6′), 76.00 (C(CH3)2), 37.31 (S-CH2-CO), 25.68 (C(CH3)2), 21.53 (Ar-2CH3) 2) ; 13C-NMRAr-2CH3) ; EIMS: m[M+ + 2] .A 100 mg each of each synthetic compound was mixed in 10 mL distilled water and shaken vigorously then stirred overnight. The resulting solution was then filtered by using 0.22 μm syringe filter. One-hundred micro liters of this aqueous preparation was added to the pre-weighed Eppendorf tube containing the clots to check thrombolytic activity.In vitro antithrombotic activity was carried as reported earlier . BrieflySprague Dawley (SD) rats (100–120 g) were used to determine the in vivo anticoagulation effects of tested compounds and kept under control temperature (25 ± 5 °C) and humidity (50 ± 10%) in an animal house, with free access of pathogen and autoclave tap water for 24 h. Experiments were approved by Institutional Ethical Committee, University of the Punjab, Lahore  and international ethical guidelines were also followed for the care of laboratory animals to provide them with a healthy environment.3a–3i) and suspended in 0.5% CMC orally (25 mg/kg). Blood was drawn from the tail of each rat to check the CT. The time course effect was determined CT and measured at regular intervals of one hour.Blood CT determination was used to evaluate the in vivo antithrombotic activity of synthetic compounds. Previously established method with slight modifications was used to estimate the CT ,8. SD RaIn silico analysis of the newly designed 1,3,4-oxadiazoles derivatives against F-Xa protein were carried out. The crystal structure of the F-Xa protein was retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB ID 1NFY). The experimental (in vivo) studies were carried out against Factor Xa (Ec: 3.4.21.6) from Sprague Dawley rats and the docking study against human F-Xa (PDB ID: 1NFY at 2.1 Å resolution).1, RPR200095, and 3a–3i were docked with F-Xa (1NFY) by Patch Dock (http://bioinfo3d.cs.tau.ac.il/PatchDock/) to find the docking transformations that produced a good molecular shape complementarily based on shape complementarily principles -1,3,4-oxadiazol-2-thiol derivatives calls for further comprehensive pharmacological research into their long term toxic effects and their protective effects.The carboxamide group is present in many F-Xa inhibitor agents; a set of novel compounds 5-[2-(4-chlorophenoxy)propan-2-yl]-1,3,4-oxadiazol-2-thiol derivatives (approach ,19. The rombosis ,23,31. D


Cistanche tubulosa aqueous extract (CTE) is already used as a botanical prescription drug for treating dementia in China. Our previous studies reported that phenylethanoid glycosides of CTE have anti-Alzheimer’s disease (AD) activity by inhibiting amyloid β peptide (Aβ) aggregation and deposition. However, recent studies considered that the phenylethanoid glycosides may be metabolized by intestinal bacteria, because all analysis results showed that the bioavailability of phenylethanoid glycosides is extremely low. In this study we demonstrate how iron chelation plays a crucial role in the Aβ aggregation and deposition inhibition mechanism of phenylethanoid glycosides of CTE. In addition, we further proved phenylethanoid glycosides (1–3) could reach brain. Active CTE component and action mechanism confirmation will be a great help for product quality control and bioavailability studies in the future. At the same time, we provide a new analysis method useful in determining phenylethanoid glycosides (1–3) in plants, foods, blood, and tissues for chemical fingerprint and pharmacokinetic research. Dementia is one of the most common chronic aging diseases. In 2015 the World Alzheimer Report estimated that about 46.8 million people suffered from dementia, and the number is expected to be 74.7 million in 2030 and 131.5 million in 2050  and accuracy (95.6–104.2%) were acceptable by UPLC for 1–3. UPLC quantified the 1, 2, and 3 of CTE contents to be 254, 38, and 41 mg/g, respectively  were mixed and spiked with rat brain tissue homogenate and analyzed using Ultra performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS) with solid phase extraction. The lower limit of detection (LLOQ) of 1–3 was 0.2 ng/mL. The phenylethanoid glycosides recovery spiked with brain tissue homogenate (10 ng/mL) at 91.39% for 1, 89.54% for 2, and 96.81% for 3, respectively (The phenylethanoid glycosides (ectively .1–3) were observed in the hippocampus , AD-like rat brain tissues were collected and divided into two parts, hippocampus and striatum, and were both homogenized . Becausepocampus A and strpocampus B. There pocampus C. In theanalysis . Therefo1–3) were added into a 10 μg/mL solution of copper (Cu), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe) or rat serum respectively. Each compound with each metal solution or serum was analyzed by UPLC. The phenylethanoid glycosides (1–3) exhibted metal chelating activity with iron and serum  oral administration (100 mg/kg) and analyzed by UPLC. In order to understand the distribuion of echinacoside in rat serum, the serum was collected from the initial time to 720 min after echinacoside , acteoside (2), and isoacteoside (3) would protect neurons from damage by Aβ, decrease Aβ oligomerization in vitro, and significantly ameliorate cognitive dysfunction induced by Aβ in vivo. The in vitro study indicated the active doses of 1–3 were up to 50 μg/mL −; 1H NMR : δ 1.07 , 2.79 , 3.56 , 3.79 , 3.91 , 4.29 , 4.38 , 5.00 , 5.17, 6.27 , 6.58 , 6.67 , 6.70 , 6.77 , 6.94 , 7.05 , δ 7.59 ; 13C-NMR : δ 18.5 (Rha-C-6), 36.6 (C-7), 62.6 (Glc-C-6), 69.4 (C-6′), 70.5 (C-4′), 70.6 (Rha-C-5), 71.5 (Glc-C-4), 72.0 (Rha-C-3), 72.3 (C-8), 72.4 (Rha-C-2), 73.8 (Rha-C-4), 74.7 (C-5′), 75.1 (Glc-C-2), 76.1 (C-2′), 77.8 (Glc-C-5), 77.9 (Glc-C-3), 81.7 (C-3′), 103.1 (Rha-C-1), 104.2 (C-1′), 104.7 (Glc-C-1), 114.7 (C-2″), 115.3 (C-8″), 116.3 (C-5″), 116.5 (C-2), 117.1 (C-5), 121.3 (C-6), 123.3 (C-6″), 127.6 (C-1″), 131.5 (C-1), 144.7 (C-3), 146.1 (C-4), 146.9 (C-4″), 148.2 (C-7″), 149.8 (C-3″), 168.5 (C-9″) (Echinacoside (5 (C-9″) .2): white powder; ESI−MS m/z: 623 [M − H]−; 1H NMR : δ 0.94 , 2.68 , 4.33 , 4.70 , 5.01 , 6.18 , 6.48 , 6.62 , 6.73 , 6.75 , 6.97 , 7.01 , 7.44 ; 13C NMR: : δ 18.2 (C-6‴), 35.1 (C-7), 60.8 (C-6″), 68.8 (C-5‴), 69.2 (C-4″), 70.3 (C-3‴), 70.3 (C-8), 70.4 (C-2‴), 70.6 (C-4‴), 71.7 (C-5″), 74.5 (C-2″), 79.2 (C-3″), 101.3 (C-1‴), 102.3 (C-1″), 113.6 (C-8′), 114.7 (C-2′), 115.5 (C-5), 115.8 (C-5′), 116.3 (C-2), 119.6 (C-6), 121.5 (C-6′), 125.6 (C-1′), 129.2 (C-1), 143.6 (C-4), 145.0 (C-3), 145.0 (C-3′), 145.6 (C-7′), 148.5 (C-4′), 165.7 (C-9′) (Acteoside (7 (C-9′) .3): white powder; ESI−MS m/z: 623 [M − H]−; 1H NMR : δ 1.07 , 2.65 , 4.35 , 5.10 , 6.27 , 6.45 , 6.58 , 6.73 , 6.74 , 6.94 , 7.04 , 7.46 ; 13C NMR : δ 17.9 (C-6‴), 35.2 (C-7), 63.5 (C-6″), 68.2 (C-5‴), 70.4 (C-4″), 70.6 (C-3‴), 71.7 (C-8), 72.1 (C-2‴), 73.7 (C-4‴), 74.1 (C-5″), 74.5 (C-2″), 80.9 (C-3″), 100.7 (C-1‴), 102.7 (C-1″), 113.9 (C-2′), 114.9 (C-8′), 115.5 (C-5), 115.8 (C-5′), 116.3 (C-2), 119.6 (C-6), 121.5 (C-6′), 125.5 (C-1′), 129.2 (C-1), 143.5 (C-4), 145.0 (C-3), 145.3 (C-3′), 145.6 (C-7′), 148.5 (C-4′), 165.6 (C-9′) (Isoacteoside (6 (C-9′) .C. tubulosa powder was extracted twice by refluxing with water for 1.5 h, and the extract solution was filtered. The filtered aqueous extract (CTE) was stored at 4 °C before use.Stem of The AD-like rat model and experimental schedule used in this study were as prescribed in our previous study . After iMethanol (HPLC grade) was purchased from Merck . Formic acid  was purchased from Panreac . Acetonitrile (HPLC grade) was purchased from J.T. Baker . Pure water was prepared using the ELGA system . Hesperidin was purchased from Chromadex and used as internal standard (IS) for UPLC analysis.v/v) (A) and formic acid-acetonitrile  (B). The time program was as follows: 0–5 min, A–B  changed to A–B ; 5–7 min, A-B ; 7-7.1 min, A-B  changed to A–B ; 7.1–9 min, . The injection volume was 5 μL into the UPLC/MS/MS system with a negative electrospray ionization (ESI) ion source. The total ion chromatograms (TIC) for 1–3 were carried out using the multiple-reaction monitoring (MRM) mode. The mass parameters were optimized to obtain the clearest, high intensity signals. The best choice of parameters were described as 3000 V capillary voltage, 20 V cone voltage, 125 °C source temperature, 400 °C desolvation temperature, 50 L/h cone gas flow, 900 L/h desolvation gas flow, and 38 eV collision energy. Data was processed by the MassLynx 4.1  software.Waters Acquity TQD ultra performance liquid chromatography (UPLC)/MS/MS System  was used for UPLC/MS/MS analysis. The chromatographic experiments were conducted on an Acquity UPLC BEH C18 column  under gradient elution at 0.4 mL/min flow rate. The mobile phase was composed of formic acid-water . The homogenate centrifuged at 10,000 rpm at 4 °C for 10 min. The supernatant (50 μL) with 10 μL IS (100 μg/mL) added pure water to 1 mL. The mixture was carefully loaded onto the Oasis HLB  cartridge , which had already been eluted with 1mL of methanol and then equilibrated with 1 mL of water under vacuum conditions. After 1 mL of 10% methanol was used for washing the cartridge, 1 mL of methanol was used to elute to obtain the analyzed sample which was transferred into another 1.5 mL Eppendorf tube with 10 μL of 1% ascorbic acid solution and dried by evaporation. Finally, the residue from the sample analyzed was dissolved in 200 μL of 50% methanol for analysis.1–3 were used to prepare calibration standards respectively added to brain tissue homogenate and hesperidin as internal standard to obtain the final calibration standard concentrations which were 1.0, 5.0, 10.0, 50.0, and 100.0 ng/mL.Compounds 1–3, in the ranges 1.0, 5.0, 10.0, 50.0, and 100.0 ng/mL, respectively, and was regarded as linear with over 0.99 of coefficient of determination (R-squared). The lower limit of detection (LLOQ), defined as ± 20% of the concentration of a test sample, produced a signal peak rather than noise. The credibility and accuracy of the analysis method was evaluated using a recovery test at 10 ng/mL of 1–3.The calibration standard curve was obtained using the linearity test using five different concentrations of 1–3 containing 10 μg/mL of metal solution of copper, calcium, magnesium, zinc, iron or rat serum. Each compound with each metal solution or serum was analyzed by UPLC. The metal chelating effect would change the retention time for 1–3 in liquid chromatography. The percentage of metal chelating effect was calculated as below and the data represented mean ± S.D. (n = 3).UPLC was used to analyze the metal chelating effect using the 50 μg/mL solution of CTE phenylethanoid glycosides 1–3 are the active components of CTE for anti-AD activity. Iron chelation has become the new concept for designing a new generation of drugs for the treatment of AD [1–3 would reach the brain though blood–brain barrier (BBB). Active components and underlying mechanism confirmation of CTE will greatly help quality control and the bioavailability of product studies in the future. At the same time, the advanced and efficient analysis method presented will be useful in determining 1–3 in plants, foods, blood, and brain tissues for chemical fingerprint and pharmacokinetic research.This study demonstrated that nt of AD . CTE is


Their structures were established by IR, HR-ESI-MS, 1D and 2D NMR techniques. Among the isolated angucyclines, saquayamycin B (4) displayed potent cytotoxic activity against hepatoma carcinoma cells HepG-2, SMMC-7721 and plc-prf-5, with IC50 values 0.135, 0.033 and 0.244 μM respectively, superior to doxorubicin. Saquayamycin B (4) also induced apoptosis in SMMC-7721 cells as detected by its morphological characteristics in 4′,6-diamidino-2-phenylindole (DAPI) staining experiment.Four angucycline glycosides including three new compounds landomycin N ( Streptomycetes species, in which a decaketide initially derived from acetyl-CoA is catalytically cyclized to four-ring core of angucycline by polyketide cyclase [2 [Angucycline is a group of aromatic polyketides containing a benz[a]anthraquinone framework of the aglycone which is mostly attached with C-glycosidic moiety . Natural cyclase . The str cyclase ,2,3,4. I cyclase , grincam cyclase , and vinclase + . The 1H and 13C NMR spectra showed that its aliphatic proton and carbon signals were similar to those of 1, suggesting the presence of the disaccharide α-l-cinerulose B--β-d-olivosyl moiety , 7.87 , 7.73 , 7.52 (brs) and 6.95 (brs), where the singlet at δH 8.39 (s) has higher frequency than the corresponding singlet of 1. The 13C NMR spectrum  was isolated as a yellow powder. Its HR-ESI-MS displayed the quasimolecular ion at m/z 838.3292  and m/z 843.2838 , indicating the same molecular formula (C43H48O16) as saquayamycin B (4). Similar to that of saquayamycin B, the 1H NMR of 3 also showed two pairs of coupling protons signals at δH 7.94  and 7.80 , and δH 7.84  and 7.75 , along with a pair of olefinic protons signals of α,β-conjugated carbonyl group at δH 7.03  and 6.02  /δC 96.0 (CH-1D), δH 5.26 /δC 92.2 (CH-1B), and δH 5.20 (brs)/δC 92.0 (CH-1C), and one C-glycosidic anomeric proton and carbon signals at δH 5.01 /δC 72.1 (CH-1A). The most obvious difference in 13C NMR spectra of 3 and 4 is the absence of a signal above δC 200 in 3, and the presence of a signal at δC 172.2, characteristic of a carboxylic acid or ester group. Accordingly, 3 was suggested to have a tricyclic system with a side chain, probaly due to the opening of the cyclohexanone ring of saquayamycin B (4) [H 7.94 (H-10) to C-8 (δC 159.6) and C-11a (δC 133.0), δH 7.80 (H-11) to C-7a (δC 116.3), C-9 (δC 138.8) and C-12 (δC 189.2), δH 7.84 (H-5) to C-6a (δC 132.5) and C-12b (δC 162.4), δH 7.75 (H-6) to C-4a (δC 136.4), C-7 (δC 189.1) and C-12a (δC 116.4) were observed (H 1.43 (H-13) to C-2 (δC 44.6), C-3 (δC 78.0) and C-4 (δC 39.0), along with the correlations from the methylene protons appearing as a couple of AB system at δH 2.72 and 2.63 (H-2) to C-1 (δC 172.1), confirmed the side chain. The linkage between the anthraquinone and side chain was deduced to be at C-4a by the HMBC correlations from methylene protons at δH 3.23 and 3.19 (H-4) to C-4a (δC 136.4), C-5 (δC 140.9) and C-12b (δC 162.4). The presence of two disaccharides α-L-cinerulose B--β-d-olivosyl and α-l-aculose-(1→4)-α-l-rhodinosyl groups were further deduced by COSY, HMBC and NOESY correlations (H 5.01) to C-8 (δC 159.6), C-9 (δC 138.2) and C-10 (δC 134.3) suggested that the α-l-cinerulose B--β-d-olivosyl group was linked to C-9 through C-1 of d-olivose moiety. The HMBC correlation from H-3A (δH 5.20) to C-3 (δC 78.0) indicated that α-l-aculose-(1→4)-α-l-rhodinosyl group was linked to C-3. In general, tricyclic angucyclines are derived from typical angucyclines with the same tetracyclic core structure under acidic conditions [4) and other tricyclic angucyclines, e.g., grincamycin B, vineomycin B2 and fridamycin D [3 was established and named vineomycin D  . The 1H in B (4) . The skeobserved . The corelations . The HMBamycin D . Thus, tomycin D .1 etc., have been reported to exhibit remarkable antitumor activity against a series of tumor cell lines [1–4 were assayed for their cytotoxic activity against normal liver cell LO2, hepatoma carcinoma HepG-2, SMMC-7721 and plc-prf-5 cell lines by 3--2,5-diphenyltetrazolium bromide (MTT) method (1–3 displayed no cytotoxicity against any of the tested cell lines. Saquayamycin B (4) displyed potent cytotoxic activity against HepG-2, SMMC-7721 and plc-prf-5 cells, with IC50 values 0.135, 0.033 and 0.244 μM, respectively, which are less than the IC50 of doxorubicin. Treatment of SMMC-7721 cells with saquayamycin B at concentrations ranging from 0.025 to 0.100 μM for 24 h, SMMC-7721 cells resulted in chromatin dispersion and formation of apoptotic body in DAPI staining test  method . At the ing test a. The aping test b.254 (10–40 µm) used in thin layer chromatography (TLC) were supplied by Qingdao Marine Chemical Factory in China.Optical rotations were measured with an Anton Paar MCP 200 polarimeter with a sodium lamp (589 nm) . UV spectra were obtained on Genesys 10S UV-Vis spectrometer ; IR spectra were recorded with a Nicolet IS5 FT-IR spectrometer ; NMR spectra were recorded on Bruker AVANCE III 500 spectrometer . HPLC-MS were acquired on Agilent 1200HPLC/6520QTOFMS . Semi-preparative HPLC isolation was performed on Agilent 1260 Infinity II  with an ODS column . Silica gel (200–300 and 300–400 mesh) used in column chromatography (CC) and silica gel GF3, 0.5 g/L NaCl, 0.5 g/L K2HPO4·H2O, 0.5 g/L MgSO4·H2O, 0.01 g/L FeSO4·H2O, and 3.0% sea salt) containing potassium dichromate (6 μg/mL) and nalidixic acid (20 μg/mL) as antifungal and antibacterial agents. The procedures of DNA extraction and PCR amplification of 16S rRNA were same as described in reference [The intertidal sediment was collected after the tide has ebbed in Xiaoshi Island, Weihai, China in September 2016. The strain OC1610.4 was isolated from this sediment using Gause’s synthetic medium . Total 30 L medium was shaking-cultured at 140 rpm and 28 °C for 9 days. The fermentation broth including mycelia was extracted with equal volume of EtOAc five times to give 4.6 g crude extract. The extract was subjected to silica gel CC  eluting with n-hexane-acetone  to give four fractions F1–F4. Part (72 mg) of fraction F1 (n-hexane-acetone 10:1) was isolated by semi-preparative HPLC eluting with CH3OH-H2O  to give 5 (5.6 mg). Fraction F2  was further purified by silica gel CC  eluting with n-hexane-acetone (10:1) to give sub-fractions F2a and F2b. Sub-fractions F2a (67 mg) was purified by semi-preparative HPLC eluting with CH3OH-H2O  to give 6 (4.6 mg). The sub-fractions F2b (26 mg) was a mixture presenting two brown spots on TLC, and was isolated by semi-preparative HPLC eluting with CH3CN-H2O  to give 1 (4.2 mg) and 2 (3.4 mg). Fraction F3  was subjected to a silica gel CC  eluting with CH3Cl-CH3OH (20:1) to give two subfractions F3a and F3b. From F3a (220 mg), compound 4 (18 mg) was purified using a low pressure silica gel CC  eluting with n-hexane-acetone (4:1). Subfractions F3b (67 mg) was isolated by semi-preparative HPLC eluting with CH3CN-H2O  to give 3 (5 mg).The spore and mycelia suspension of strain OC1610.4 was inoculated in Erlenmeyer flasks (500 mL) each of which contains 100 mL S-medium : brown amorphous powder; c 0.002, MeOH); UV (MeOH) λmax (log ε) 225 (2.99), 327 (2.65) nm; IR (KBr) νmax 3203, 2974, 2916, 1726, 1629, 1607, 1578, 1433, 1295, 1111, 1075, 852, 791 cm−1; 1H NMR  and 13C NMR  data, m/z 561.1753 .Landomycin N (2): reddish-brown amorphous powder; c 0.003, MeOH); UV (MeOH) λmax (log ε) 265 (2.40), 340 (2.07) nm; IR (KBr) νmax 3383, 2917, 2879, 1727, 1657, 1608, 1584, 1525, 1471, 1286, 1247, 1108, 1017, 872, 836, 716 cm−1; 1H NMR  and 13C NMR  data, m/z 561.1752 .Galtamycin C (3): yellow amorphous powder; c 0.050, MeOH); UV (MeOH) λmax (log ε) 230 (3.56), 259 (3.28), 295 (2.83) nm; IR (KBr) νmax 3557, 2978, 2935, 1731, 1702, 1625, 1581, 1431, 1259, 1080, 1014, 899, 808 cm−1; 1H NMR  and 13C NMR  data, m/z 838.3292  and m/z 843.2838 .Vineomycin D -2,5-diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as positive control drug and deionized H2O with the same DMSO concentration was used as parallel control. DAPI staining test was employed to qualitatively observe apoptosis, and the apoptotic ratio was measured by flow cytometric analysis . These tests were conducted using the methods as previously described [The cytotoxicity evaluations of escribed ,26.1), galtamycin C (2), vineomycin D (3) and saquayamycin (4), along with two alkaloids 1-acetyl-β-carboline (5) and indole-3-acetic acid (6), were isolated from the fermentation broth of strain Streptomyces sp. OC1610.4, obtained from the intertidal sediment. Galtamycin C (2) and vineomycin D (3) are rearranged angucycline derivatives respectively possessing a linear tetracyclic and a tricyclic framework of angucycline. Vineomycin D (3) and saquayamycin B (4) are isomers, comprising the same two disaccharides in the structures. Among the isolated angucycline glycosides, saquayamycin B (4) displayed the most potent cytotoxic activity against hepatoma carcinoma HepG-2, SMMC-7721 and plc-prf-5 cells. Although saquayamycin B was shown to induce an apoptosis in SMMC-7721 cell, its antineoplastic mechanism needs to be further investigated.Four angucycline glycosides including landomycin N (


The new triterpene glycosides were identified as 23-O-methyl-24-deoxy-2'-O--cimiaceroside B (1) and 2'-O-actein (2) based on analysis of their spectral data and chemical reactions.Two new 9,19-cycloartane triterpene glycosides  C. foetida from different geographic regions, we have separated more than 40 triterpene glycosides  27D -25.0° ); negative FABMS m/z (%) 717 [M-H]– (8), 689 (25), 118 (43), 601 [M-117]– (4) and 117 [C3H7O4]– (44); negative HRFABMS ;IR (KBr) νmax 3454, 2957, 2927, 2870, 1728, 1631, 1442, 1381, 1366, 1339, 1211, 1172, 1072, 1052, 971, 897, 820, 633, 583, 537 cm−1. 1H-NMR  and 13C-NMR data  see Cimifoside E (2). white powder; m.p. 168–179 °C; [α] 27D -45.5° ); negative FABMS m/z 775 [M-H]–, 662 [M-103]– and 104 [C3H4O4]–; negative HRFABMS ; IR (KBr) νmax 3455, 2953, 1760, 1737, 1457, 1440, 1369, 1348, 1244, 1204, 1163, 1075, 1042, 1024, 984, 831, 726, 633, 604, 540 cm–1. 1H-NMR  and 13C-NMR data  see 1 and 2 (6 mg of each) were refluxed with 5% HCl in MeOH (7 mL) for 8 h. Each mixture was diluted with H2O and neutralized with NaHCO3. The neutral hydrolysate revealed the presence of only xylose by TLC  upon comparison with the authentic sample.Compounds Cimicifuga species have been extensively studied by several groups [Cimicifuga were identified.Due to their medicinal uses, chemical constituents of l groups ,12,17. Tl groups ,18. In o


A click chemistry approach based on the reaction between alkynylflavins and mono(6-azido-6-deoxy)-β-cyclodextrin has proven to be a useful tool for the synthesis of flavin-cyclodextrin conjugates studied as monooxygenase mimics in enantioselective sulfoxidations. Mono(6-azido-6-deoxy)-β-cyclodextrin 7 was readily available using standard procedures starting from commercial β-cyclodextrin amine (TBTA) ligand which is known to stabilize Cu(I) towards disproportionation and oxidation (method B) + calcd for C14H10N4NaO2 289.0696, found 289.0696.1-(But-3-ynyl)-3-methylalloxazine (5b): Synthesis according to general procedure with 3-methylalloxazine , 4-brombut-1-yn  and potassium carbonate . 5b was obtained  as yellow solid. M.p. 260–261 °C. 1H-NMR  δ 8.39–8.32 , 8.09–8.02 , 7.92 , 7.78 , 4.69 , 3.61 , 2.78 , 1.97 , 13C-NMR  δ 159.96, 150.50, 146.94, 145.56, 143.42, 140.41, 134.19, 131.04, 129.56, 128.14, 81.41, 70.56, 41.09, 29.45, 17.67. HRMS-ESI+m/z: [M + Na]+ calcd for C15H12N4NaO2 303.0858, found 303.0855.3-Methyl-1-(pent-4-ynyl)alloxazine (5c): Synthesis according to general procedure with 3-methylalloxazine , 5-brompent-1-yn  and potassium carbonate . 5c was obtained  as yellow solid. M.p. 262–264 °C. 1H-NMR  δ 8.35 , 8.04 , 7.95–7.87 , 7.81–7.72 , 4.60 , 3.61 , 2.38 , 2.18–2.02 , 1.95 , 13C-NMR  δ 150.83, 145.28, 143.70, 140.41, 134.06, 131.02, 129.42, 128.14, 98.52, 83.50, 68.99, 42.26, 34.87, 29.40, 26.46, 16.55. HRMS-ESI+m/z: [M]+ calcd C16H14N4NaO2+ 317.1014, found 317.1011.1-Methyl-3-propargylalloxazine (6): Synthesis according to general procedure with 1-methylalloxazine , propargylbromide  and potassium carbonate . 6 was obtained  as yellow solid. M.p. 186–187 °C. 1H-NMR  δ 8.43–8.27 , 8.11–7.99 , 7.92 , 7.78 , 4.97 , 3.85 , 2.26–2.23 . 13C-NMR  δ 159.01, 149.96, 145.55, 143.39, 140.07, 134.13, 130.85, 129.69, 129.36, 127.89, 77.58, 71.43, 31.69, 29.70. HRMS-ESI+m/z: [M + Na]+ calcd for C14H10N4NaO2 289.0696, found 289.0696.5 or 6 and 6-azido-6-deoxy-β-cyclodextrin 7 (1.2 equiv.) was stirred with copper (I) iodide (3 mol %) and N,N-diisopropylethylamine (2 equiv.) in dry N,N-dimethylformamide (DMF) at room temperature for 24 h. Conjugate was precipitated after addition of acetone and separated by centrifugation. Crude product was purified by flash chromatography on the column with reversed phase (gradient MeOH/H2O 1:9→MeOH/H2O 2:3). The obtained solid product 8 or 9 was dried in vacuo.The mixture of alkynyl alloxazine 5 or 6 and 6-azido-6-deoxy-β-cyclodextrin 7 (1.2 equiv.) was stirred with TBTA methyl]amine) (20 mol %), sodium ascorbate (20 mol %) and copper (II) sulfate (10 mol %) in dry N,N-dimethylformamide (DMF) at room temperature for 24 h. Conjugate was precipitated after addition of acetone and separated by centrifugation. Crude product was purified by flash chromatography on the column with reversed phase (gradient MeOH/H2O 1:9→MeOH/H2O 2:3). The obtained solid product 8 or 9 was dried in vacuo.The mixture of alkynyl alloxazine 1-{methyl}-3-methylalloxazine (8a): Synthesis according to general procedure A for click reactions using 3-methyl-propargylalloxazine , 6-azido-6-deoxy-β-cyclodextrin , copper (I) iodide , N,N-diisopropylethylamine  and 4 mL of dry DMF. Product 8a  was obtained as yellowish powder.8a  was obtained as yellowish powder. M.p. 238–239 °C. 1H-NMR see 13C-NMR see +m/z: [M + Na]+ calcd for C56H79N7NaO36 1448.4458, found 1448.4456. c = 0.107 g/100 mL, H2O).Synthesis according to general procedure B for click reactions using 3-methyl-propargylalloxazine , 6-azido-6-deoxy-β-cyclodextrin , TBTA , sodium ascorbate , copper (II) sulfate , 4 mL of dry DMF. Product 1-{ethyl}-3-methylalloxazine (8b): Synthesis according to general procedure B for click reactions using 1-but-3-ynyl-3-methylalloxazine , 6-azido-6-deoxy-β-cyclodextrin 7 , TBTA , sodium ascorbate , copper (II) sulfate , 4 mL of dry DMF. Product 8b  was obtained as yellowish powder. M.p. 241–242 °C. 1H-NMR see 13C-NMR see +m/z: [M + Na]+ calcd for C57H81N7NaO36 1462.4615, found 1462.4615. c = 0.108 g/100 mL, H2O).1-{propyl}-3-methylalloxazine (8c): Synthesis according to general procedure A for click reactions using 3-methyl-1-pent-4-ynylalloxazine , 6-azido-6-deoxy-β-cyclodextrin 7 , copper (I) iodide , N,N-diisopropylethylamine  and 2 mL of dry DMF. Product 8c  was obtained as yellowish powder.7 , TBTA , sodium ascorbate , copper (II) sulfate , 4 mL of dry DMF. Product 8c  was obtained as yellowish powder. M.p. 272–274 °C. 1H-NMR see 13C-NMR see +m/z: [M + H]+ calcd for C58H84N7O36 1454.4952, found 1454.4958. c = 0.110 g/100 mL, H2O).Synthesis according to general procedure B for click reactions using 3-methyl-1-pent-4-ynylalloxazine , 6-azido-6-deoxy-β-cyclodextrin 3-{methyl}-1-methylalloxazine (9): Synthesis according to general procedure A for click reactions using 1-methyl-3-propargylalloxazine , 6-azido-6-deoxy-β-cyclodextrin 7 , copper (I) iodide , N,N-diisopropylethylamine  and 1 mL of dry DMF. Yellowish product 9  was obtained.7 , TBTA , sodium ascorbate , copper (II) sulfate , 4 mL of dry DMF. Product 9  was obtained as yellowish powder. M.p. 288–290 °C. 1H-NMR see 13C-NMR see +m/z: [M + Na]+ calcd for C56H79N7NaO36 1448.4458, found 1448.4434. c = 0.102 g/100 mL, H2O).Synthesis according to general procedure B for click reactions using 1-methyl-3-propargylalloxazine , 6-azido-6-deoxy-β-cyclodextrin 8 or 9 was mixed with acetaldehyde (300 equiv.), palladium on carbon (10% w/w), ethanol, water and 0.1 M perchloric acid. Reaction mixture was stirred in autoclave for 21 h under hydrogen atmosphere (0.6 MPa). Palladium was filtered off and organic solvents were evaporated in vacuo. Residue was diluted with water to adjust the volume to exactly 2 mL.Conjugate Despite our great efforts, we were unable to record clear NMR spectra of the quaternized conjugates due to peak broadening as a result of the presence of radical species ,30,31. N-{methyl}-3-methylalloxazinium perchlorate (3a): Synthesis according to general procedure for quarternisation of conjugates using conjugate 8a , acetaldehyde , palladium on carbon , ethanol (800 µL), perchloric acid , water (600 µL). HRMS-ESI+m/z: [M + H]+ calcd for C58H84N7O36 1454.4952, found 1454.4955, UV-VIS : 382 nm, 441 nm.11-{ethyl}-3-methylalloxazinium perchlorate (3b): Synthesis according to general procedure for quarternisation of conjugates using conjugate 8b , acetaldehyde , palladium on carbon , ethanol (570 µL), perchloric acid  and water (430 µL). HRMS-ESI+m/z: [M + H]+ calcd for C59H86N7O36 1468.5109, found 1468.5122. UV-VIS : 382 nm, 448 nm.1-{+ calcd for C60H88N7O36 1482.5265, found 1482.5272. UV-VIS : 382 nm, 448 nm.3-{methyl}-1-methylalloxazinium perchlorate (4): Synthesis according to general procedure for quarternisation of conjugates using conjugate 9 , acetaldehyde , palladium on carbon , ethanol (2.4 mL), perchloric acid  and water (1.8 mL). HRMS-ESI+m/z: [M + H]+ calcd for C58H84N7O36 1454.4952, found 1454.4954, UV-VIS : 385 nm, 448 nm.−5 mol), appropriate volume of catalyst solution (1 mol %–5 mol %) and finally aqueous hydrogen peroxide (2.3 mol equiv.). Stirring of the reaction mixture was provided by wrist action shaker (900 rpm) for 1 h at 25 °C. Then the reaction was quenched by addition of a solution of sodium dithionite in water . The mixture was extracted with CDCl3 (3 × 0.5 mL). Collected organic layers were dried with molecular sieves (3Å) and used for 1H-NMR determination of the conversion. Enantiomeric excess was determined by HPLC analysis on column with chiral stationary phase of the sample obtained by evaporation of CDCl3 and dissolving a residue in heptane (0.5 mL).Catalytic oxidations of alkyl or aryl methyl sulfides were performed in 1 mL thick-walled screw-capped vial (Supelco). The reaction mixtures were prepared by addition of sodium-phosphate buffer , liquid substrate (3 × 101, which possesses an amide linker. However, 3a is comparable with the other chiral flavin catalysts [1 are rather exceptional. The extreme sensitivity to small structural changes observed among the flavin-cyclodextrin conjugates is a typical phenomenon that also occurs in the natural enzymes and its elucidation is currently being investigated in our laboratory.We have demonstrated the CuAAC concept is useful for the preparation of flavin-cyclodextrin conjugates. The click chemistry represents alternative approach to the original procedure based on amide coupling and seems to be slightly more advantageous especially by reducing the number of reaction steps and improving the overall yield. Recently, Rotello found the isoalloxazine ring tolerates “click chemistry” when preparing flavin-polymer conjugates ,38; we hatalysts ,3 and ev