Structures of the isolated compounds were elucidated by extensive analysis of spectroscopic data, especially 2D NMR data and comparison with literatures. All isolates were evaluated for anti-inflammatory activity against nitric oxide (NO) production in vitro. Compounds 3 and 7 exhibited moderate inhibitory effects on NO production with IC50 values of 12.2 ± 0.8 and 18.9 ± 0.3 μM, respectively.One new monoterpene glycoside ( Scindapsus officinalis (Roxb.) Schott. is a large, epiphytic, and perennial liana in the family Araceae, which usually climbs up to trees and rocks using aerial roots and is mainly distributed in the south-central regions of China and the tropical parts of India. The whole plant of S. officinalis has been used in Guizhou, Yunnan and Guangxi Provinces in China as ethnomedicine to treat bone fractures, arthralgia, bronchitis, and pertussis + in the HRESIMS (positive ion mode) spectrum , representing four degrees of unsaturation. The 1H-NMR spectrum of 1  and 5.09 , and three methyls at δH 1.61 , 1.54  and 1.22 . In addition, one obvious anomeric proton at δH 4.13  which correlated with carbon signal at δC 105.1 (C-1′) observed in the HSQC spectrum, indicated one pyranose moiety. The relatively large coupling constant suggested that the pyranose moiety possessed a β-glycosidic bond. The 13C-NMR data of 1 exhibited a total of sixteen carbon signals (δC 61.7 (C-6′)), nine methines , and two quaternary carbons (including one olefinic carbon at δC 130.8 (C-7) and one mono-oxygen-connected carbon at δC 79.4 (C-1)), with ten assigned to the aglycon part, and six to a pyranose moiety. The sugar moiety which was consistent with d-glucose underwent GC analysis of the derivatives of its hydrolysis product and was compared with a referenced d-glucose.Compound rum of 1  indicate signals  assignab1 was analyzed in detail to determine the 2D structure of the aglycone part. Starting from the typical olefinic proton of H-2, the 1H 1H-COSY cross-peaks of H-2/H-3/H-4 (δH 1.96)/H-5  on the basis of NMR data and the correlations from H-6 to C-7, Me-8 and Me-9; from Me-8 to C-6, C-7 and Me-9; from Me-9 to C-6, C-7 and Me-8 in the HMBC spectrum. Additionally, 1H 1H-COSY cross-peak of H-5/H-6 and HMBC correlations between: H-5 to C-6 and also H-6 to C-4 (1 was determined as in glc to C-1 (δC 79.4) (The 2D NMR spectroscopic data of .96)/H-5 , coupled6 to C-4 , demonst6 to C-4 . Thus, tδC 79.4) .1 at C-1 and C-5 positions were unambiguously assigned by interpretation of the observed NOESY correlations (1 was identified as (rel)--1-methyl-5-(2-methylprop-1-en-1-yl)cyclopent-2-en-1-O-β-d-glucopyranosideby using a molecular modeling program called Chem 3D Ultra 14.0,which used MM2 force field calculations for energy minimization to provide a detailed computer-modeled 3D structure.The relative configurations of elations . The rel2 was obtained as light-yellow amorphous powder. Its molecular formula was established as C28H42O12 from its 13C-NMR data and the protonated ion peak at m/z 571.2798 [M + H]+ in the HRESIMS (positive ion mode) spectrum , representing eight degrees of unsaturation. The 1H-NMR spectrum of 2 , 6.67 , and 7.00 , one terminal double bond at δH 5.94  and 5.03–5.08 , one methoxy at δH 3.74  and one methyl at δH 0.87 . In addition, two obvious anomeric protons at δH 4.84  and 4.09  which correlated with carbon signals at δC 100.2 (C-1′) and 102.8 (C-1″) observed in the HSQC spectrum, indicated two pyranose moieties. The relatively large coupling constants of anomeric protons indicated that the two pyranose moieties possessed β-linkages. The 13C-NMR and HSQC spectra of 2 showed twenty-eight carbon signals (δC 55.6 (OMe-2)), eight methylenes (including two oxygenated methylenes at δC 60.8 (C-6′) and 61.1 (C-6″), one terminal double bond at δC 115.49 (C-9), and six high field methylenes), fourteen methines (including one olefinic carbon at δC 137.9 (C-8), three phenyl carbons at δC 112.9 (C-3), 120.8 (C-5), 115.53 (C-6), eight oxygenated methines, and two anomeric carbons at δC 100.2 (C-1′) and 102.8 (C-1″)), and four quaternary carbons (including three phenyl carbons at δC 144.86 (C-1), 144.87 (C-2) and 133.4 (C-4)), with twelve to two pyranose units. The two pyranose units were identified as d-glucose by means of the same method described above. Analysis of the 1H and 13C-NMR data of 2 revealed a strong similarity to that of 2,6-dimethoxy-4-(prop-2-enyl) phenyl O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside + , representing nine degrees of unsaturation. With the aid of 13C-NMR and HSQC spectra, the seventeen carbons (δC 52.3 (OMe-7), one methylene, nine methines (including three aromatic carbons at δC 115.3 (C-2′), 116.2 (C-5′) and 121.9 (C-6′), three olefinic methines at δC 140.1 (C-6), 145.9 (C-7′), and 114.4 (C-8′), and three oxygenated methines at δC 70.2 (C-1), 68.0 (C-2), and 65.8 (C-5)), six quaternary carbons (including two ester carbonyls at δC 166.7 (C-7) and 166.5 (C-9′), three aromatic carbons at δC 125.9 (C-1′), 146.0 (C-3′), and 148.9 (C-4′), and one olefinic carbon at δC 140.1 (C-6)). The proton and carbon NMR data of 3 were very similar to those of 1--cyclopenta-2,5-diol  from Fraction 5-3-9 . Fraction 6  was chromatographed on a Flash C18 column with a MeOH/H2O gradient (5%→100%) to give nine subfractions (F6-1-F6-9). Compound 7  was purified using preparative HPLC system  from fraction 6-1 . Fraction 7  was separated using Flash C18 CC with a MeOH/H2O gradient (5%→100%) to give six subfractions (F7-1-F7-6). Fraction 7-1  was further chromatographed by CC over Sephadex LH-20 with MeOH as mobile phase to give five subfractions (F7-1-1-F7-1-5). Compound 3  was purified using preparative HPLC system ; flow rate: 3 mL min−1; UV detection at 285 nm) from fraction 7-1-5 (80 mg).Air-dried and pulverized stems of escribed . The EtOd-glucopyranoside(rel)--1-Methyl-5-(2-methylprop-1-en-1-yl)cyclopent-2-en-1-O-β- (1): brown, gum; c 0.067, MeOH). 1H  and 13C-NMR , see m/z 337.1802 [M + Na]+ .d-glucopyranosyl-(4→4)-β-d-glucopyranoside2-Methoxy-4-(prop-2-enyl)-1″-(1-ethylcyclopropyl) methanol phenyl O-β- (2): light yellow, amorphous powder; c 0.09, MeOH). 1H  and 13C-NMR , see m/z 571.2798 [M + H]+ .(Z)--2,5-Dihydroxy-3-(2-methoxy-2-oxoethylidene)cyclopentyl 3- acrylate (3): brown, gum; c 0.27, MeOH). 1H  and 13C-NMR , see m/z 373.1013 [M + Na]+ .1 and 2 were subjected to acid hydrolysis the same as described in our previous study [3-CH3OH-H2O (15:9:2) as a developing solvent and 10% H2SO4 in 95% EtOH as a detection reagent. Then, the methylated thiazolidine derivatives of the hydrolyzed sugar moieties and the corresponding authentic glucose were prepared, and their absolute configurations were further identified by comparing the retention times of the hydrolyzed sugar derivatives with that of the authentic glucose derivative in the GC analysis  raised up to 300 °C in 24 min by 10 °C/min, and 300 °C kept for 10 min. tR d-glucose 21.10 min).Compounds us study , as well1–7) with six concentrations of 0.63, 1.25, 2.5, 5.0, 10 and 20 μM and incubated for 2 h firstly, and then stimulated by adding lipoplysaccharide . After being stimulated for 24 h, 50 μL of cell supernatants were taken out from each well and mixed with an equal volume of Griess reagents I and II to detect the NO production levels by measuring absorbance at 570 nm. All experiments were performed in triplicate. The NO inhibition is calculated as the percentage reduction of LPS stimulation.The anti-inflammatory activity assay against NO production in RAW 264.7 mouse peritoneal macrophages was conducted mainly using the procedures described previously . The macThe cytotoxicity against macrophage Raw 264.7 cells was investigated using the MTT assay . Briefly1), one new phenyl glycoside (2), one new caffeoyl derivative (3), and four known compounds (4–7) were isolated from S. officinalis. This is the first time that these kinds of compounds are reported in the title plant. Compounds 3 and 7 exhibited moderate inhibitory effects on NO production, and neither showed any cell toxicity against macrophage Raw 264.7 cells. Meanwhile, none of the other five compounds showed any anti-inflammatory activities. These definite pharmacological findings indicated limited structure–activity relationships as hydroxyl substituted benzene rings and conjugated ester moieties might take significant effect for anti-inflammatory activity, supported the application of the S. officinalis in Miao ethnomedicine system to treat inflammation.In the present study, one new monoterpene glycoside (


A quantitative structure-activity relationship (QSAR) study was performed and correlated with the experimental DPPH• scavenging data. We used structural, geometrical, topological and quantum-chemical descriptors selected with Genetic Algorithms in order to determine which of these parameters are responsible of the observed DPPH• radical scavenging activity. We constructed a back propagation neural network with the hydrophilic factor (Hy) descriptor to generate an adequate architecture of neurons for the system description. The mathematical model showed a multiple determination coefficient of 0.9196 and a root mean squared error of 0.0851. Our results shows that the presence of hydroxyl groups on the ring structure of 3-carboxy-coumarins are correlated with the observed DPPH• radical scavenging activity effects.The  Antioxidants play important roles in preventing diseases induced by reactive oxygen species, which result in oxidative damage, including protein denaturation, mutagenesis and degenerative or pathological events, such as aging, asthma, and cancer. The diversity of structural characteristics in the natural and synthetic coumarins offers a vast field of research for new biological properties of these compounds.Coumarin derivatives constitute an important class of compounds with varied biological properties such as anti-inflammatory, antibacterial, cytotoxic, anxiolytic, antitumor, anticoagulant, antiemetic and antioxidant activity ,4,5,6,7.in vitro assay of eight 3-carboxycoumarin derivatives with different structural variations for modular replication by the quantitative 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging activity method. These is the first time that this measurement has been performed on these compounds, although a similar type of coumarins was reported by Lin et al. in 2008 [Here we proposed to measure the antioxidant activity Quantitative Structure-Activity Relationship (QSAR/QSPR) methodologies are one of the most powerful tools for describing the relationships between biological activity and the physicochemical characteristics of molecules. Current literature demonstrates that almost every area of chemical and life sciences, as well as technology, utilizes quantitative structure-activity/property relationships (QSAR/QSPR) to accelerate product development and increase efficiency. The designs of pharmaceuticals, agrochemicals, and consumer products as well as the assessment of their toxicity and environmental impact have become major areas of application of QSAR/QSPR techniques, whose methods also penetrate into relatively new applications such as materials science and nanotechnology. In terms of methodology development the new trend is the integration of QSAR/QSPR with related computational methods such as virtual screening and molecular dynamics. Such a synergy offers unique opportunities and heralds a new era of computer-aided molecular design . QSAR/QSMolecular descriptors are formal mathematical representations of a molecule, obtained by a well-specified algorithm, and applied to a defined molecular representation or a well-specified experimental procedure: the molecular descriptor is the final result of a logic and mathematical procedure which transforms chemical information encoded within a symbolic representation of a molecule into a useful number or the result of some standardized experiment. A general consideration about the use of molecular descriptors in modeling problems concerns their information content. This depends on the type of molecular representation used and the defined algorithm for their calculation. There are simple molecular descriptors derived by counting some atom types or structural fragments in the molecule, as well as physicochemical and bulk properties such as, for example, molecular weight, number of hydrogen bond donors/acceptors, number of OH-groups, and so on. Other molecular descriptors are derived from algorithms applied to a topological representation. These are usually termed topological, or 2D-descriptors. Other molecular descriptors are derived from the spatial  coordinates of the molecule, usually called geometrical, or 3D-descriptors; another class of molecular descriptors, called 4D-descriptors, is derived from the interaction energies between the molecule, imbedded into a grid, and some probe. Single indexes derived from a molecular graph are called topological indexes. These are numerical quantifiers of molecular topology that are mathematically derived in a direct and unambiguous manner from the structural graph of a molecule, usually an H-depleted molecular graph. On the other hand many of those descriptors are based directly on the results of quantum-mechanical calculations or can be derived from the electronic wave function or electrostatic field of the molecule . Since tGenetic Algorithms (GA) are powerful computational tools that have been used in many areas of investigation because of their reliable mathematical models. This method is based on the mechanism of evolution of species, the higher descriptor weights (genes) the more preserved in the mathematic model, while the lower weights are eliminated. In this manner, the best mathematical models which represent the observed biological activity (phenotype) are obtained ,21. FurtH-1-benzopyran-2-one-3-carboxylic acids A1–D1 and 3-acetyl-6-R-2H-1-benzopyran-2-ones A2–D2  is an anisometry descriptor calculated as a function of the eigenvalues of the covariance matrix calculated from the molecular matrix:The SPH , nC represents the number of carbon atoms and nSK stands for all atoms excluding Hydrogen [et al. [iC term is the contribution of atom i to the molecular surface, in is the frequency of the atom i in the molecule and the sum runs over all types of polar fragments. The TPSA calculation takes into account the contribution of the functional groups containing oxygen and nitrogen atoms to the polarization of the molecular surface as implemented in the DRAGON code [The spherosity index varies from zero for flat molecules, such as benzene, to one for totally spherical molecules . The Ui ectively . The Hy Hydrogen . The AMRHydrogen . The ALOHydrogen . The TPS [et al.  is calcuGON code .η), electrophilicity index (ω), chemical potential (µ), softness (S) and gap HOMO-LUMO. In this work E, corresponds to the ground state energy of our coumarin molecules and the dipole moment was calculated as implemented in Gaussian 09 [E is the energy of the system and N is the number of electrons [I is the vertical ionization potential defined as the difference of total energy between cationic structures in the optimized geometry of the neutral compounds and the optimized neutral structures:Additionally we calculated quantum chemical descriptors from DFT  as totalssian 09 . The chelectrons . Here weA is the vertical electron affinity defined as the difference of the total energy between the optimized neutral structures and the corresponding anions in the optimized geometry of the neutral compounds:(η) is a global property of the molecular system and measure the resistance imposed by it to any change in its electron distribution:The hardness In the finite difference approximation the above equation is:(S) is the inverse of hardness:The softness (ω) can be determined from chemical potential (μ) and hardness (η) [ω represents the stabilization energy of the molecular system when it is saturated by electrons coming from the surroundings [The electrophilicity index ness (η)  as:(12)oundings .We introduced all 13 descriptors into the Neuroshell Predictor program code . Accordiexp  and log Ypred calculated by GA analysis (predicted). We obtained a coefficient of multiple determinations (Rsquared) of 0.9313, a correlation factor (r) of 0.9658 and a root mean squared error (RMSE) of 0.0786.squared is a statistical indicator usually used in multiple regression analysis to compare the reliability of the model with respect to reference points. Rsquared is defined as:y is the experimental value, y is the average of all the output values. Furthermore r is a measure of the linear correlation between experimental and predicted values in terms of direction, namely:RRMSE is defined as the root mean square of the summation of quadratic terms. These terms correspond to the difference between experimental and predicted data values:exp) and calculated  antiradical activity. Percent error is calculated as:Experimental and calculated antiradical activity, error and percent error are shown in The highest error value was 8.69% and the lowest one 0%. The average percent error was 3.77%. We propose the construction of a Back Propagation Neural Network (BPNN) with the most important descriptor Hy  in orderNeuroShell Predictor software  was usedThe BPNN model showed that in all the analyzed compounds Hy descriptor is the most important variable in the antiradical activity. The Hy descriptor indicates antiradical activity increases as we incorporate hydrophilic groups to the coumarin molecules. exp and log Ypred antiradical activity of coumarins was very successful. The graphic is showed in squered of 0.9196, r = 0.959 and RMSE = 0.0850.The linear correlation between log YGA.Experimental and calculated antiradical activity, error and percent error are shown in et al. [m2 must be >0.5 and ∆rm2 < 0.2, in this terms the QSAR model proposed here was good. In contrast m2 values for our GA model was of 0.9014 and 0.056 respectively, but we have to consider that in GA analysis 13 descriptors were used and in the ANN only one. These results show the importance to include ANN with the GA methodology. A previous QSAR study [2 and SH. Also in our study we validated our model with the statistical parameters Determination of reliability of our QSAR model was done by calculating the statistical parameters y et al. ,47. The AR study  made witand ∆rm2 ,45 that C1 and C2 compounds show the highest antiradical activities because both possess an -OH hydrophilic group. This functional group increases the Hy value in such a way that we could say that -OH group is crucial for antiradical activity of coumarins. It’s important to mention that the 1H and 13C-NMR spectra were recorded on a Varian Mercury 300  instrument in CDCl3 solutions or DMSO-d6, measured with SiMe4 as the internal reference, chemical shifts are in ppm and nJ(H-H) in Hertz.All chemicals and solvents were of reagent grade and used as received. Melting points were measured on an Electrothermal IA 9100 apparatus and were uncorrected. IR spectra were recorded neat using a Varian 3100 FT-IR with ATR system Excalibur Series spectrophotometer. Mass spectra were obtained in a Bruker Esquire 6000 spectrometer with an electron ionization mode. 1A–2D were synthesized by Knovenagel cyclization (The starting coumarins lization  between 3-Acetyl-2H-1-benzopyran-2-one (A1): Yellow solid. Yield 67.3%. m.p: 118–122 °C, IR (neat), ν (cm−1): 1719 (OC=O), 1680 (C=O), 1196 and 1161 (C-O). 1H-NMR : 8.63 , 7.92 , 7.72 , 7.43 , 7.39 , 2.56 . 13C-NMR : 195.1 (C-11), 158.5 (C-2), 154.6 (C-9), 147.1 (C-4), 134.5 (C-7), 130.8 (C-5), 124.9 (C-6), 124.4 (C-3), 118.2 (C-10), 116.1 (C-8), 30.1 (C-12). C11H8O3 188.05. m/z = 188.1 , 173.2 (100%), 145.3 (11.9%), 118.3 (11.1%).3-Acetyl-6-nitro-2H-1-benzopyran-2-one (B1): Yellow solid. Yield 54.6%. m.p: 200–203 °C; IR (neat), ν (cm−1): 1745 (OC=O), 1676 (C=O), 1530 and 1341 (C-NO2), 1275 y 1209 (C-O). 1H-NMR : 8.58 ; 8.55 ; 8.50 , 7.52 , 2.73 . 13C-NMR : 194.5 (C-11), 158.6 (C-2), 157.9 (C-9), 146.1 (C-4), 144.6 (C-6), 128.8 (C-7), 126.5 (C-3), 126.1 (C-5), 118.4 (C-10), 118.2 (C-8), 30.7 (C-12). C11H7NO5 233.03. m/z = 233.0 , 218.3 (100%), 172.3 (19.3%), 144.2 (5.0%).3-Acetyl-6-hydroxy-2H-1-benzopyran-2-one(C1): Yellow solid. Yield 90%. m.p: 247–248 °C; IR (neat), ν (cm−1): 3155 (O-H), 1736 (OC=O), 1643 (C=O). 1H-NMR: 9.89 , 8.53 , 7.20 ; 7.14 , 7.29 , 2.54 . 13C-NMR : 196.0 (C-11), 159.9 (C-2), 154.6 (C-6), 148.7 (C-9), 147.7 (C-4), 125.1 (C-3), 123.4 (C-7), 119.3 (C-10), 117.2 (C-5), 114.8 (C-8), 30.7 (C-12). C11H8O4 204.04. m/z = 204.1 , 189.1 (100%), 161.2 (19.0%), 134.2 (33.4%).3-Acetyl-6-methoxy-2H-1-benzopyran-2-one (D1): Yellow solid. Yield 89%. mp 180–183 °C. IR ν (cm−1): 1723 (OC=O), 1677 (C=O), 1226, 1197 (C-O).1H-NMR : 8.44 , 7.28 , 7.20 , 7.02 , 3.85 , 2.70 ; 13C-NMR : 195.9 (C11), 159.7 (C2), 156.6 (C6), 150.1 (C10), 147.6 (C4), 124.8 (C3), 123.2 (C7), 117.9 (C5), 118.7 (C9), 111.3 (C8), 56.1 (OCH3), 30.9 (CH3). C12H10O4 218.06 m/z = 218.0 , 203.0 (100%), 175.0 (16.6%), 148.0 (15.3%). EA (%) calculated for C12H10O4: 66.05 C, 4.62 H; found: 66.04 C, 4.61 H.Ethyl 2H-1-benzopyran-2-one-3-carboxyate (A2): White solid. Yield 90%. m.p. 91–92 °C, IR (neat), ν (cm−1): 1605 (OC=O), 11758 (C=O), 1196 y 1161 (C-O). 1H-NMR : 8.49 , 7.93 , 7.39 , 7.74 , 7.45 , 4.30 , 1.39 . 13C-NMR : 163.0 (C-11), 156.4 (C-2), 154.5 (C-9), 149.0 (C-4), 134.7 (C-7), 130.7 (C-5), 125.3 (C-6), 118.2 (C-3), 118.3 (C-10), 116.6 (C-8), 61.7 (O-CH2), 15.5 (-CH3). C12H10O4 218.06. m/z = 219.1 , 173.2 (76%), 146.2 (87.9%), 118.2 (27.4%).Ethyl 6-nitro-2H-1-benzopyran-2-one-3-carboxyate(B2): Yellow solid. Yield 89%. m.p = 191–192 °C, IR (neat), ν (cm−1): O-C=O (1716), C=O (1746). 1H-NMR: 8.90 ; 8.93 ; 8.50 , 7.65 , 4.32 , 1.33 . 13C-NMR : 162.5 (C-11), 158.5 (C-2), 155.2 (C-9), 147.1 (C-4), 148.8 (C-6), 128.8 (C-7), 118.0 (C-3), 125.4 (C-5), 120.7 (C-10), 118.3 (C-8), 62.7 (O-CH2), 14.3 (-CH3). C12H9NO6 263.04. m/z = 262.9 , 218.2 (91.9%), 191.0 (100%), 161.2 (98.0%).Ethyl 6-hydroxy-2H-1-benzopyran-2-one-3-carboxyate (C2): Beige solid, 91%, m.p. = 205–206 °C, IR  ν (cm−1): (O-H) 3324, (O-C=O) 1707, (C=O) 1722. 1H-NMR : 9.93 , 8.66 , 7.30 , 7.21 , 7.17 , 4.29 , 1.31 , 13C-RMN : 167.4 (C-11), 159.4 (C-2), 154.5 (C-9), 147.2 (C-4), 140.9 (C-6), 117.1 (C-7), 119.4 (C-3), 113.3 (C-5), 119.7 (C-10), 120.2 (C-8), 44.1 (O-CH2), 14.2 (-CH3). C12H10O5 234.05. m/z = 234.0 , 189.1 (36.1%), 161.8 (100.0%), 134.2 (31.3%)Ethyl 6-methoxy-2H-1-benzopyran-2-one-3-carboxyate (D2): Yellow solid, 92%, m.p. = 142–143 °C, IR (neat), ν (cm−1): (O-C=O)1733, (C=O)1740. 1H-NMR : 8.45 , 7.26 , 7.20 , 6.99 , 3.89  4.38 , 1.47 ; 13C-NMR : 163.3 (C-11), 157.1 (C-2), 156.4 (C-9), 148.6 (C-4), 149.9 (C-6), 118.0 (C-7), 118.3 (C-3), 110.8 (C-5), 118.6 (C-10), 122.8 (C-8), 62.1 (O-CH2), 56.1 (CH3O-), 14.4 (-CH3). C13H12O5 248.07. m/z = 248.2 , 203.1 (32.6%), 176.2 (100%), 148.2 (23.7%).A1–D2 was estimated according to a slight modification of the procedure reported by Morales and Jimenez-Perez [The antiradical activity of compounds ez-Perez . DilutioGA analysis we obtained an average percent error of 3.77% while in BPNN the average percent error was 7.18%. This result indicate that the combination of the two methodologies optimize the creation of QSAR models. The GA allows finding the most important descriptor for the development of the antiradical activity and ANN improves our model with the use of only one molecular descriptor to obtain accurate prediction values. The presence of hydroxyl groups on the ring structure of 3-carboxycoumarins is correlated with their DPPH• radical scavenging effects. The mixed QSAR model showed that Hy could indicate that antiradical activity would increase as we incorporate hydroxyl groups in the coumarin molecules. According to In


Eurotium amstelodami (015-2) inhibited nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells without cytotoxicity. By further bioassay-guided isolation, three compounds including asperflavin, neoechinulin A and preechinulin were successfully isolated from the BE of E. amstelodami. It was revealed that asperflavin showed no cytotoxicity up to 200 μM and significantly inhibited LPS-induced NO and PGE2 production in a dose-dependent manner. In the western blot results, asperflavin suppressed only inducible NOS (iNOS), but COX-2 were slightly down-regulated. Asperflavin was also observed to inhibit the production of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6. In conclusion, this study reports a potential use of asperflavin isolated from a marine fungus, E. amstelodami as an anti-inflammatory agent via suppression of iNOS and pro-inflammatory cytokines as well as no cytotoxicity.In the present study, 16 marine-derived fungi were isolated from four types of marine materials including float, algae, animals and drift woods along with the coast of Jeju Island, Korea and evaluated for anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW 24.7 cells. The broth and mycelium extracts from the 16 fungi were prepared and the broth extract (BE) of  Penicillium and Aspergillus. Representatives of other genera including Acremonium, Emericella, Epicoccum, Exophiala, Paraphaeospaeria, Phomopsis, and Halarosellinia were less common −. 1H-NMR : δ 6.78 , 6.54 , 6.42 , 3.84 , 2.95 , 2.85 , 2.79 , 1.26 . 13C NMR : δ 203.2 (C-1), 165.0 (C-9), 161.2 (C-8), 160.6 (C-6), 141.6 (C-10a), 137.8 (C-4a), 115.7 (C-10), 108.8 (C-8a), 108.1 (C-9a), 101.9 (C-5), 97.8 (C-7), 69.4 (C-3), 55.7 (CH3O-8), 51.5 (C-2), 42.7 (C-4), 28.9 (CH3-3).Asperflavin (1)) was isolated as greenish amorphous powder. The LR-ESIMS data give a molecular ion peak at m/z 287 [M − H]−. The molecular formula of the compound was determined as C16H16O5 base on the MS and NMR data. The 1H and 13C-NMR data showed the presence of a carbonyl, a methoxyl, a methyl, two aliphatic methylenes, three aromatic protons among which two were meta-coupled with each other, an aliphatic quaternary carbon bearing oxygen, seven aromatic quaternary carbons among which three were bearing oxygen in the compound. It was determined as asperflavin as comparison with the previous report [Asperflavin ): white powder. ESI-MS m/z: 324 [M + H]+, 1H-NMR : δ 1.38 , 1.47 , 4.16 , 5.01 , 5.05 , 6.07 , 6.89 , 7.01 , 7.08 , 7.19 , 7.42 , 8.32 , 13C-NMR : δ 166.4 (C-13), 159.9 (C-10), 145.1 (C-16), 143.9 (C-2), 135.1 (C-7a), 125.9 (C-3a), 124.9 (C-9), 120.7 (C-6), 119.3 (C-5), 118.8 (C-4), 111.6 (C-8), 111.5 (C-7), 110.1 (C-17), 103.4 (C-3), 50.5 (C-12), 38.9 (C-15), 27.5 (C-18/19), 19.6 (C-20).Neoechinulin A (2)), a white powder, has a molecular ion peak at m/z 322 [M − H]−. The molecular formula of the compound was determined as C19H21N3O2 base on the MS and NMR data. It was identified as neoechinulin A, which was previously isolated from fungal genera Eurotium and Apsergillus [Neoechinulin A (3)): white powder. ESI-MS m/z: 326 [M + H]+, 1H-NMR : δ 1.23 , 1.49 , 3.07 , 3.36 , 3.79 , 3.96 , 5.04 , 6.18 , 6.93 , 7.02 , 7.31 , 7.42 , 7.49 , 8.16 , 10.5 . 13C-NMR : δ 167.8 (C-13), 167.3 (C-10), 146.5 (C-16), 141.3 (C-2), 134.9 (C-7a), 128.9 (C-3a), 120.5 (C-6), 118.3 (C-5), 117.9 (C-4), 111.0 (C-17), 110.1 (C-7), 104.6 (C-3), 55.7 (C-9), 50.3 (C-12), 31.0 (C-8), 28.0 (C-19), 27.9 (C-18), 20.6 (C-20).Preechinulin (3)), a white powder, showed a molecular ion peak at m/z 326 [M + H]+. The molecular formula of the compound was determined as C19H23N3O2 base on the MS and NMR data. The structure of preechinulin was elucidated by analysis of its 1H, 13C-NMR and MS data, as well as by comparison of its spectral data to those reports in the literature  and kept on ice for 30 min. Cell lysates were washed by centrifugation, and protein concentrations were determined by using BCA™ protein assay kit. Aliquots of the lysates (30–50 μg of protein) were separated on a 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane  with a glycine transfer buffer . After blocking the nonspecific site with 1% bovine serum albumin (BSA), the membrane was then incubated with specific primary antibody at 4 °C for overnight. The membrane was further incubated for 60 min with a peroxidase-conjugated secondary antibody  at room temperature. The immune-active proteins were detected using an enhanced chemiluminescence (ECL) western blotting detection kit.RAW 264.7 macrophages were pre-incubated for 24 h, and then stimulated with LPS (1 μg/mL) in the presence of the active compound for the indicated time. After incubation, the cells were collected and washed twice with cold-PBS. The cells were lysed in a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVOIn order to determine the inhibitory effects of the active compound on production of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6, the RAW 264.7 macrophages were incubated with the active compound  in the presence or absence of LPS (1 μg/mL) for 24 h. The inhibitory effects of the compound on the production of pro-inflammatory cytokines in LPS-treated RAW 264.7 cells was determined as described in the Cho et al protocols . Supernap < 0.05, and ** p < 0.01, vs. LPS-stimulated group.All the data were presented as mean ± S.D. from three independent experiments unless stated otherwise. Statistical comparisons between different treatments were done by one-way ANOVA with Student Newman Keul’s post hoc tests using SPSS program (version 19.0). * E. amstelodami, inhibited the production of NO and PGE2 production through suppression of iNOS expression in LPS-stimulated RAW 264.7 macrophages. Asperflavin was also found to inhibit the pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 as well. Therefore, our study suggests that asperflavin might be considered a promising agent for the prevention and therapy of inflammatory disease.In conclusion, we have demonstrated that asperflavin isolated from the marine-derived fungus,


Miliusa mollis Pierre, three new compounds including -2,3-dihydro-2-(4-methoxyphenyl)-3-methyl-5-[1(E)-propenyl]benzofuran, - threo-Δ8′-4-methoxyneolignan and tyrosol-1-O-β-xylopyranosyl-(1→6)-O-β-gluco-pyranoside were isolated, along with seven known compounds. Their structures were elucidated through analysis of their spectroscopic data.From the twigs of  Miliusa Lesch. ex A.DC. (Annonaceae) comprises 30–40 species, which occur from India and South China to North Australia  benzofuran (1) and - threo-Δ8′-4-methoxyneolignan (3), and a new glycosidic phenylpropanoid, namely tyrosol-1-O-β-xylopyranosyl-(1→6)-O-β-glucopyranoside (10), together with seven known compounds: -2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-methyl-5-(E)-propenylbenzofuran (2)  was present at C-4′, as indicated from the HMBC correlations from the protons at δ 3.81 to C-4′ (δ 158.3), and from H-2′(6′)  to C-2 (δ 92.6) and C-4′. In addition, a 2-propenyl moiety  was located at C-5 (δ 131.2), as evidenced by the 3J-coupling from C-5 to H-9 (δ 6.09). These spectral data appeared to be superimposable on those reported for synthetic (±)-trans-2,3-dihydro-2-(4-methoxyphenyl)-3-methyl-5-(1(E)-propenyl)benzofuran benzofuran , conjugated unsaturation , and ether  functionalities. The 13C-NMR spectrum of 3  and 6.88 , and at δ 7.09  and 6.87  in the 1H-NMR spectrum. In the HMQC spectrum, two tertiary oxygenated carbon signals appearing at δ 77.7 (C-7) and 79.3 (C-8) showed direct coupling with protons at δ 4.62  and 4.34 , respectively. These two methine protons constituted an ABX coupling system with the Me protons at δ 1.07  in the COSY spectrum.Compound rum of 3  showed a3 were similar to those of previously reported 8-O-4′neolignans + ion peak at m/z 455.1619 in the HRESITOFMS. The compound showed UV absorptions at 223 and 273 nm, and IR bands at 3,366 (hydroxyl), 1,510 (conjugated unsaturation), and 1,071 and 1,043 (ether) cm−1. Compound 10 appeared to be a glycoside with tyrosol (4-hydroxyethylphenol)  , with its anomeric carbon linked to C-6′ of the glucose moiety through an ether bridge  , and itsr bridge ,33. ThisMiliusa, and this is the first time that neolignans were isolated from a plant of this genus.It should be noted that although neolignans are frequently identified from the Annonaceae, they were not previously found in the genus Optical rotations were measured on a Perkin-Elmer 341 polarimeter, and the CD spectra were recorded on a JASCO J-715 spectropolarimeter. UV spectra were obtained on a Shimadzu UV-160A UV/vis spectrometer and IR spectra on a Perkin-Elmer FT-IR 1760X spectrophotometer. Mass spectra were recorded on a Micromass LCT mass spectrometer or a Thermo-Finnigan Polaris Q mass spectrometer. NMR spectra were obtained with a Bruker Avance DPX-300 FT-NMR spectrometer (300 MHz) or a JEOL JMN-A 500 NMR spectrometer (500 MHz). Vacuum-liquid column chromatography (VLC), column chromatography (CC) and medium pressure liquid chromatography (MPLC) were performed with silica gel 60 , silica gel 60 , Diaion HP20SS  and Sephadex LH-20 . Preparative TLC was carried out with silica gel plate .Miliusa mollis Pierre were collected in Bangkok, Thailand by one of us (T.C.) and identified by R. W. J. M. van der Ham, as previously described benzofuran (1): colorless oil; c 0.42, MeOH); CD : [θ]300 −401, [θ]264 −2,296, [θ]226 +1,791; EI-MS m/z 281 [M+1]+, 280 [M]+, 265, 251, 157, 148, 135, 131, 115, 103, 91; HRESITOFMS m/z 303.1280 [M+Na]+ ; UV λmax (MeOH) nm (log ε) 228 (3.76), 274 (3.26); IR νmax (film): 1515, 1486, 1243 cm-1; 1H-NMR : δ 7.35 , 7.14 , 7.12 , 6.91 , 6.76 , 6.37 , 6.09 , 5.09 , 3.81 , 3.39 , 1.86 , 1.39 ; 13C-NMR : δ 159.6 (C-7a), 158.3 (C-4′), 132.7 (C-3a), 132.4 (C-1′), 131.2 (C-5), 130.8 (C-8), 127.6 , 126.3 (C-6), 122.9 (C-9), 120.7 (C-4), 114.0 , 109.2 (C-7), 92.6 (C-2), 45.2 (C-3), 55.3 (MeO), 18.3 (C-10), 17.8 (Me-3).-threo-8Δ′-4-methoxyneolignan (3): colorless oil; c 0.05, MeOH); CD : [θ]276 −1,932, [θ]233 +2,392; EI-MS m/z 298 [M]+, 281, 162, 161, 137, 133, 121, 115, 105, 91, 77; HRESITOFMS m/z 321.1375 [M+Na]+ ; UV λmax (MeOH) nm (log ε) 227 (4.18), 275 (3.48); IR νmax (film): 3448 (br), 1509, 1243 cm-1; 1H-NMR  and 13C-NMR : see Tyrosol-1-O-β-xylopyranosyl-(1→6)-O-β-glucopyranoside (10): colorless amorphous powder; c 0.08, MeOH); EI-MS m/z 414, 207, 167, 149, 138, 107, 77; HRESITOFMS m/z 455.1619 [M+Na]+ ; UV λmax (MeOH) nm (log ε) 223 (3.51), 273 (2.77); IR νmax (film): 3366 (br), 1510, 1071, 1043 cm-1; 1H-NMR  and 13C-NMR : see S,3S)-2,3-dihydro-2-(4-methoxyphenyl)-3-methyl-5-[1(E)-propenyl]benzofuran, - threo-Δ8′-4-methoxyneolignan and tyrosol-1-O-β-xylopyranosyl-(1→6)-O-β-glucopyranoside were isolated from the twigs of Miliusa mollis Pierre. The presence of neolignans in the genus Miliusa was reported for the first time in this study.Three new compounds including (2


Nicotinamide adenine dinucleotide (NAD), one of the most important coenzymes in the cells, is a substrate of the signaling enzyme CD38, by which NAD is converted to a second messenger, cyclic ADP-ribose, which releases calcium from intracellular calcium stores. Starting with 2′-deoxy-2′-fluoroarabinosyl-β-nicotinamide adenine dinucleotide (ara-F NAD), a series of NAD analogues were synthesized and their activities to inhibit CD38 NAD glycohydrolase (NADase) were evaluated. The adenosine-modified analogues showed potent inhibitory activities, among which 2′-deoxy-2′-fluoroarabinosyl-β-nicotinamideguanine dinucleotide (ara-F NGD) was the most effective one. The structure-activity relationship of NAD analogues was also discussed. It also catalyzes the synthesis of nicotinic acid adenine dinucleotide phosphate (NAADP) from nicotinamide adenine dinucleotide phosphate (NADP) with a base-exchange reaction in acidic condition. All the three molecules are calcium-mobilizing related , 680.1277; found, 680.1281.2′-Deoxy-2′-fluoro-5′-thio-phosphate-arabinofuranoside-β-nicotinamide adenine dinucleotide . Compound 9  , 682.0892; found, 682.0881.FA salt)  (0.10 mm1-(Adenosine)-P3-(2′-deoxy-2′-fluoro-β-nicotinamide arabinofuranoside) triphosphateP . Ara-F NMN  , 746.0784; found, 746.0788.FA salt)  . Ara-F NMN   and Na2·IMP  were reacted following the general procedure to yield 12   as a white cotton-shaped solid. 1H-NMR  δ 9.35 , 9.24–9.15 , 8.90 , 8.28–8.18 , 6.64 , 6.13 , 5.45 , 4.63–4.51 , 4.41–4.13 ; 31P-NMR  δ −11.47; 19F-NMR  δ −75.78, −199.16; HRMS (ESI-TOF+) calcd for C21H26FN6O14P2 [(M+H)], 667.0961; found, 667.0978.2′-Deoxy-2′-fluoro-arabinosyl-β-nicotinamide guanine dinucleotide . Ara-F NMN   and Na2·GMP  were reacted following the general procedure to yield 13   as a white cotton-shaped solid. 1H-NMR  δ 9.43 , 9.30 , 9.07–8.95 , 8.35–8.28 , 6.72 , 6.01 , 5.55 , 4.70–4.58 , 4.50–4.18 ; 31P-NMR  δ −11.45; 19F-NMR  δ −75.74, −199.21; HRMS (ESI-TOF+) calcd for C21H27FN7O14P2 [(M+H)], 682.1070; found, 682.1074.2′-Deoxy-2′-fluoro-arabinosyl-β-nicotinamide 6-O-methyl-hypoxanthine dinucleotide . Ara-F NMN  and 6-OMe-IMP , 681.1117; found, 681.1118.-OMe-IMP  (0.12 mmBis(2′-deoxy-2′-fluoro-β-nicotinamide-arabinosyl) pyrophophate (Bis(ara-F NMN), 20). Ara-F NMN  and another ara-F NMN  were added respectively following the general procedure to yield 20   as a white cotton-shaped solid. 1H-NMR  δ 9.41 , 9.28 , 8.97 , 8.29 , 6.71 , 5.52 , 4.61 , 4.41 , 4.34–4.27 ; 31P-NMR  δ −11.45; 19F-NMR  δ −75.68, −199.09; HRMS (ESI-TOF+) calcd for C22H28F2N4O13P2 [(M+H)], 655.1012; found, 655.1007.Bis(2′-deoxy-2′-fluoro-β-nicotinamide-arabinosyl)-methylenediphosphonate (Bis(ara-F NMN)[CH2], 21). Compound 8a  was dissolved in TMP (2.5 mL), (Cl2PO)2CH2  was added to the reaction mixture under ice bath cooling. The mixture was stirred for 5 h at 0 °C, then aqueous sodium hydroxide was added to the neutralize excess acid to pH = 7. The solution was reduced to dryness, and then the gummy residue was dissolved in water (10 mL) and extracted with ethyl acetate (3 × 10 mL). The aqueous layer was evaporated again, and dissolved in 1‰ TFA aqueous solution (10 mL). After purification by HPLC and lyophilization, 21   was generated as a white cotton-shaped solid. 1H-NMR  δ 9.43 , 9.25 , 8.96 , 8.26 , 6.71 , 5.51 , 4.59 , 4.41 , 4.34–4.26 , 2.50 ; 31P-NMR  δ 18.35; 19F-NMR  δ −75.69, −198.97; HRMS (ESI-TOF+) calcd for C23H30F2N4O12P2 [(M+H)], 653.1220; found, 653.1224.Bis(2′-deoxy-2′-fluoro-2′-methyl-β-nicotinamide)-methylenediphosphonate (Bis(2′-CH3-2′-F NMN)[CH2], 22). Compound 22 was synthesized from 5 using the same procedure as for 21. Yield 55% as a white cotton-shaped solid. 1H-NMR  δ 9.35 , 9.13 , 8.99 , 8.26 , 6.58 , 4.72 , 4.52–4.38 , 4.25–4.17 , 2.42 , 1.57 ; 31P-NMR  δ 18.59; 19F-NMR  δ −75.70, −173.53; HRMS (ESI-TOF+) calcd for C25H34F2N4O12P2 [(M+H)], 681.1533; found, 681.1517.50 values of inhibitors were calculated by the GraphPad Prism 5 software .To study the inhibition of the NAD-glycohydrolase (NADase) activity of CD38 by the compounds, an enzymatic assay was performed . Briefly12, 13 and 19) with a modified purine ring showed higher activities than the others, while the analogues with the purine ring replaced by nicotinamide showed obviously decreased activities, suggesting that the purine ring is important for the activity but subtle substituent changes at C-2 and C-6 of the purine ring did not significantly affect the activity. The charge, length and flexibility of the pyrophosphate bridge are important for retaining inhibitory activity; the replacement by phosphorothioate or triphosphate reduces the activity. A single fluoro substituent at C-2′ of the furan ring is critical for activity, and 2′-disubstitution, such as the introduction of fluoro and methyl leads to weak activity. The findings of this study present some insights into the structure-activity relationship of NAD analogues, which should be helpful for the discovery of more active probes to investigate NAD-related biological effects.NAD analogues with ribose, nucleobase, or pyrophosphate modifications were designed, prepared and their inhibitory activities against CD38 NADase were evaluated. SAR studies  revealed


To search for 5-HT2A receptor antagonists, a new indole alkaloid termed 6-bromo-N-propionyltryptamine (1), together with one known homologue 6-bromo-N-acetyltryptamine (2) were isolated and identified from a marine bacterium Pseudoalteromonas rubra QD1-2. Compound 1 with an N-propionyl side chain exhibited stronger 5-HT2A receptor antagonist activity than that of N-acetyl derivative (2), indicating that 6-bromotryptamine analogues with a longer chain acyl group perhaps displayed a more potent capacity to the target. Therefore, a series of new 6-bromotryptamine analogues (3–7) with different chain length of the acyl group (C4–C8) were prepared and evaluated activity against 5-HT2A receptor. Remarkably, 6-bromo-N-hexanoyltryptamine (5) displayed the most effective inhibitory activity, which was 5-fold stronger than that of the parent compound 1 and showed 70% efficacy of the positive control (ketanserin tartrate).5-Hydroxytryptamine type 2A (5-HT Among them, the 5-HT2 receptor subfamily contains three members, namely 5-HT2A, 5-HT2B, and 5-HT2C. Extensive studies have revealed that the 5-HT2A receptor played a critical role in the regulation of neuropsychiatric disorders associated with depression, Parkinson’s and Alzheimer’s disease +, suggesting 1 contained one bromine atom. The 13C-NMR spectrum of 1 showed signals for one amide carbonyl group at δC 177.0 (C-13), four sp2 quaternary carbons at δC 113.8 (C-3), 115.7 (C-6), 127.3 (C-9), 138.9 (C-8), four sp2 methines at δC 115.1 (C-7), 120.8 (C-4), 122.7 (C-5), 124.3 (C-2), three methylenes at δC 26.1 (C-10), 43.3 (C-11), and one methyl at δC 10.5 (C-15). The 1H-NMR signals at δH 7.07 , 7.10 , 7.47 , and 7.48  displayed a classic pattern for a 3,5- or 3,6-disubstituted indole moiety, which was supported by the HMBC correlations of H-2/C-3 and C-8, H-4/C-6 and C-8, H-5/C-7 and C-9, and H-7/C-5 and C-9 /H-11  for 1 disclosed the presence of a pair of coupled methylene groups, which was attached to the 6-bromoindole moiety at C-3 due to the HMBC correlations of H-10/C-2 and C-3 and H-11/C-3, thus forming a 6-bromotryptamine moiety /H-15  and HMBC correlations of H-14 and H-15/C-13  were evaluated at a concentration of 10 μM for their antagonist activity on 5-HT2A receptor detectable by calcium flux assay. The results (N-hexanoyltryptamine (5) thus exhibited an approximately 70% inhibition activity compared with the positive control, which was the most potency in the series. Notably, compound 5 displayed a roughly 4-fold higher inhibitory activity than the parent analogue 1. These observations might be explained by two mechanisms. A longer acyl side chain related to a higher lipophilicity, has the positive consequence in membrane permeation, probably eliciting more effective inhibitory potency for the target. However, the bulk chain (when > C6) caused an acyl group-dependent decrease in the binding affinity, and likely reduced the inhibitory potency for 5-HT2A receptor +.6-Bromo-N-pentoyltryptamine (4): Yield 81%, red brown solid; 1H-NMR  δ (ppm) 8.94 , 7.49 , 7.41 , 7.17 , 6.95 , 5.77 , 3.54 , 2.90 , 2.11 , 1.55 , 1.28 , 0.87 ; MS (ESI+) m/z 344.97, 346.96 [M + Na]+.6-Bromo-N-hexanoyltryptamine (5): Yield 83%, red brown solid; 1H-NMR  δ (ppm) 8.95 , 7.49 , 7.41 , 7.17 , 6.95 , 5.76 , 3.54 , 2.91 , 2.11 , 1.57 , 1.26 , 0.86 ; MS (ESI+) m/z 359.01, 360.99 [M + Na]+.6-Bromo-N-heptanoyltryptamine (6): Yield 80%, white solid; 1H-NMR  δ (ppm) 7.48 , 7.46 , 7.10 , 7.07 , 3.45 , 2.91 , 2.13 , 1.54 , 1.27 , 0.89 ; MS (ESI+) m/z 373.09, 375.08 [M + Na]+.6-Bromo-N-octanoyltryptamine (7): Yield 85%, white solid; 1H-NMR  δ (ppm) 7.48 , 7.46 , 7.10 , 7.06 , 3.45 , 2.91 , 2.12 , 1.53 , 1.26 , 0.89 ; MS (ESI+) m/z 387.11, 389.14 [M + Na]+.1–7 and the positive control were tested at 10 μM in HBSS buffer  with 0.1% DMSO. Ketanserin tartrate was employed as positive control. According to the previous method, the inhibitory activity was performed in CHO-K1 (Chinese hamster ovary) cells where recombinant human 5-HT2A receptor was expressed, measured by the calcium flux assay [All the tested compounds were prepared as follows and stored at −20 °C. Compounds ux assay ,17. The 2A receptor antagonist activity, including a new 6-bromo-N-propionyltryptamine (1) and a known 6-bromo-N-acetyltryptamine (2) were isolated and characterized from the culture broth of a marine-derived P. rubra QD1-2. Moreover, we synthesized a series of new 6-bromotryptamine congeners (3–7) with different chain lengths (C4–C8) and evaluated their antagonist activity for 5-HT2A receptor. Notably, the preliminary SAR studies indicated that the length of the acyl chain dramatically influenced the antagonist activity of 6-bromotryptamine derivatives. Their antagonist activity increased with elongation of the side chain acyl group (C2–C6), while the activity of compounds 6 and 7 with the side chain longer than that of 6-bromo-N-hexanoyltryptamine (C6) significantly declined or disappeared. As the most potent one in this series, 6-bromo-N-hexanoyltryptamine (5) displayed roughly 4-fold activity enhancement over the parent analogue 1, which possessed an approximately 70% inhibition activity comparable to the positive control. Overall, 6-bromo-N-hexanoyltryptamine (5) with a sound antagonist activity for 5-HT2A receptor was identified and our work provides an insight into the further structural optimization of 6-bromotryptamine derivatives for developing potential therapeutic agents to treat neurological disorders.In this paper, two 6-bromotryptamine derivatives with the 5-HT


Subsequent chemical modification by O-acylation led to a novel class of molecules, among which compound 25 demonstrated the most potent anti-HCV activity in the virus assay (EC50 = 0.19 μM), but low cytotoxicity and hERG cardiac toxicity. The following studies (a solution stability assay and a simple pharmacokinetic test together with a CypA enzyme inhibition assay) preliminarily indicated that 25 was a prodrug of 1. To the best of our knowledge, 25 is probably the most potent currently reported small molecule anti-HCV agent acting via the CypA inhibitory mechanism. Consequently, our study has provided a new potential small molecule for curing HCV infection.In this work, the relationship between cyclophilin A (CypA) and HCV prompted us to screen a series of small molecule CypA inhibitors which were previously reported by our group. Among them, compound  Infection with hepatitis C virus (HCV) is well recognized as a worldwide health issue that chronically affects more than 170 million people ,3. Therecis-trans isomerase (PPIase) −; HRMS (ESI) m/z calcd C23H18N2O5 [M − H]− 401.1137, found 401.1143.1-(2-Acetoxy-6-hydroxybenzoyl)-3-(9H-fluoren-9-yl)-urea (1-(2-Benzoyloxy-6-hydroxybenzoyl)-3-(9H-fluoren-9-yl)-urea (18). Prepared in the same manner as described for 14, but the acetic anhydride was replaced by benzoyl chloride: mp 211–214 °C; 1H-NMR (DMSO-d6) δ: 11.07 , 10.45 , 8.61 , 8.01 , 7.85 , 7.78 , 7.58 , 7.40 , 7.38–7.30 , 7.26 , 6.84 , 5.91 ; ESI-MS m/z 463.0 [M − H]−; HRMS (ESI) m/z calcd C28H20N2O5 [M − H]− 463.1294, found 463.1299.1-(2-Hydroxy-6-propionyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea (19). Prepared in the same manner as described for 14, but the acetic anhydride was replaced by propionic anhydride: mp 206–216 °C; 1H-NMR (acetone-d6) δ: 9.72 , 8.71 , 7.86 , 7.70 , 7.46 , 7.43–7.34 , 6.89 , 6.75 , 6.11 , 2.60 , 1.23–1.12 ; ESI-MS m/z 415.0 [M − H]−; HRMS (ESI) m/z calcd C24H20N2O5 [M − H]− 415.1294, found 415.1299.1-(2-Hydroxy-6-n-butyryloxybenzoyl)-3-(9H-fluoren-9-yl)-urea (20). Prepared in the same manner as described for 14, but the acetic anhydride was replaced by butyric anhydride: mp 204–210 °C; 1H-NMR (acetone-d6) δ: 8.71 , 7.87 , 7.71 , 7.47 , 7.44–7.36 , 6.91 , 6.75 , 6.12 , 2.56 , 1.76–1.64 , 0.96 ; ESI-MS m/z 429.1 [M − H]−; HRMS (ESI) m/z calcd C25H22N2O5 [M − H]− 429.1450, found 429.1456.1-(2-Cyclopropyl formyloxy-6-hydroxybenzoyl)-3-(9H-fluoren-9-yl)-urea (21). Prepared in the same manner as described for 14, but the acetic anhydride was replaced by cyclopropanecarboxylic acid chloride: mp 194–199 °C; 1H-NMR (DMSO-d6) δ: 10.85 , 10.36 , 8.71 , 7.90 , 7.61 , 7.47 , 7.38 , 7.25 , 6.79 , 6.63 , 6.00 , 1.75 , 0.99–0.83 ; EI-MS m/z 428.1(M+); 165.1 (100%); HRMS (EI) m/z calcd C25H20 N2O5 (M+) 428.1372, found 428.1373.1--3-(9H-fluoren-9-yl)-urea (22). Prepared in the same manner as described for 14, but acetic anhydride was replaced by valeric anhydride: mp 193–199 °C; 1H-NMR (acetone-d6) δ: 8.73 , 7.86 , 7.70 , 7.46 , 7.43–7.35 , 6.89 , 6.73 , 6.11 , 2.57 , 1.64 , 1.37 , 0.86 ; ESI-MS m/z 443.0 [M − H]−; HRMS (ESI) m/z calcd C26H24N2O5 [M − H]− 443.1607, found 443.1612.1-(2-Hydroxy-6-trimethylacetoxyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea (23). Prepared in the same manner as described for 14, but the acetic anhydride was replaced by trimethylacetic anhydride: mp 187–189 °C; 1H-NMR (acetone-d6) δ: 9.71 , 8.76 , 7.86 , 7.68 , 7.46 , 7.37 , 6.87 , 6.69 , 6.11 , 1.31 ; ESI-MS m/z 443.0 [M − H]−; HRMS (ESI) m/z calcd C26H24N2O5 [M − H]− 443.1607, found 443.1612.1-(2-Hexanoyloxy-6-hydroxybenzoyl)-3-(9H-fluoren-9-yl)-urea (24). Prepared in the same manner as described for 14, but the acetic anhydride was replaced by hexanoic anhydride: mp 200–222 °C; 1H-NMR (DMSO-d6) δ: 11.09 , 10.42 , 8.73 , 7.89 , 7.60 , 7.46 , 7.36 , 7.25 , 6.77 , 6.57 , 5.98 , 2.39 , 1.48 , 1.22–1.03 , 0.72 . ; ESI-MS m/z 457.0 [M − H]−; HRMS (ESI) m/z calcd C27H26N2O5 [M − H]− 457.1763, found 457.1769.1-(2-Hydroxy-6-n-Octanoyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea (25). Prepared in the same manner as described for 14, but the acetic anhydride was replaced by n-octanoic anhydride: mp 184–189 °C; 1H-NMR (CDCl3) δ: 11.56 , 9.45 , 8.48 , 7.73 , 7.65 , 7.43 , 7.34 , 6.89 , 6.69 , 6.16 , 2.81 , 1.85 , 1.53–1.28 , 0.91 ; EI-MS m/z 486.2 (M+); 165.1 (100%); HRMS (EI) m/z calcd C29H30N2O5 (M+) 486.2155, found 486.2157.1--3-(9H-fluoren-9-yl)-urea (15). To a solution of 1 (0.9 g) and triethylamine (1.38 mL) in anhydrous DMF (15 mL), was added acetic anhydride (0.93 mL) with stirring at room temperature for 12 h. The reaction mixture was poured into H2O (300 mL), and extracted with DCM (120 mL) three times. The organic layers were combined, dried with anhydrous sodium sulfate, then concentrated to dryness. The residue was purified by flash column chromatography on silica gel, eluted with a mixture of THF/DCM/PE , to afford 15  as a white solid: mp 191–194 °C; 1H-NMR (acetone-d6) δ: 8.64 , 7.86 , 7.69 , 7.56 , 7.46 , 7.38 , 7.18 , 6.10 , 2.24 ; ESI-MS m/z 445.0 [M + H]+; HRMS (ESI) m/z calcd C25H20N2O6 [M + Na]+ 467.1219, found 467.1214.3-(9H-fluoren-9-yl)-urea (26). Prepared in the same manner as described for 15, but the acetic anhydride was replaced by benzoyl chloride: mp 211–213 °C; 1H-NMR (acetone-d6) δ: 10.33 , 8.54 , 8.18 , 7.84–7.68 , 7.62 , 7.48 , 7.37 , 7.31 , 7.21 , 5.96 ; EI-MS m/z 568.2(M+); 105.0 (100%); HRMS (ESI) m/z calcd C35H24N2O6 [M + Na]+ 591.1532, found 591.1523.1--1--3-(9H-fluoren-9-yl)-urea (27). Prepared in the same manner as described for 15, but the acetic anhydride was replaced by propionic anhydride: mp 165–167 °C; 1H-NMR (acetone-d6) δ: 9.89 , 8.67 , 7.87 , 7.68 , 7.58 , 7.48 , 7.43–7.35 , 7.20 , 6.11 , 2.59 , 1.18 ; EI-MS m/z 472.2(M+); 165.1 (100%); HRMS (ESI) m/z calcd C27H24N2O6 [M + Na]+ 495.1532, found 495.1527.)-urea (28). Prepared in the same manner as described for 15, but the acetic anhydride was replaced by butyric anhydride: mp 186–188 °C; 1H-NMR (ad6) δ: 9.90 , 8.68 , 7.86 , 7.67 , 7.56 , 7.47 , 7.38 , 7.18 , 6.10 , 2.53 , 1.78–1.62 , 0.96 ; EI-MS m/z 500.2 (M+); 165.1 (100%); HRMS (ESI) m/z calcd C29H28N2O6 [M + Na]+ 523.1845, found 523.1840.1--3-(9H-fluoren-9-yl-9-yl)-urea (29). Prepared in the same manner as described for 15, but the acetic anhydride was replaced by cyclopropanecarboxylic acid chloride: mp 183–186 °C; 1H-NMR (CDCl3) δ: 8.50 , 7.92 , 7.73 , 7.65 , 7.49 , 7.43 , 7.33 , 7.08 , 6.17 , 1.84 , 1.21–1.13 , 1.08–0.99 ; EI-MS m/z 496.2(M+); 165.1 (100%); HRMS (ESI) m/z calcd C29H24N2O6 [M + Na]+519.1532, found 519.1523.1--3-(9H-fluoren30). Prepared in the same manner as described for 15, but the acetic anhydride was replaced by valeric anhydride: mp 172–174 °C; 1H-NMR (CDCl3) δ: 8.47 , 8.01 , 7.72 , 7.62 , 7.49 , 7.42 , 7.31 , 7.07 , 6.13 , 2.55 , 1.75–1.63 , 1.38 , 0.91 ; EI-MS m/z 528.2(M+); 165.1 (100%); HRMS (ESI) m/z calcd C31H32N2O6 [M + Na]+ 551.2158, found 551.2153.1--3-(9H-fluoren-9-yl)-urea (3-(9H-fluoren-9-yl)-urea (31). Prepared in the same manner as described for 15, but the acetic anhydride was replaced by trimethylacetic anhydride: mp 202–205 °C; 1H-NMR (CDCl3) δ: 8.55 , 7.71 , 7.50 , 7.49–7.37 , 7.28 , 7.04 , 6.13 , 1.32 ; EI-MS m/z 528.2(M+); 165.1 (100%); HRMS (ESI) m/z calcd C31H32N2O6 [M + Na]+ 551.2158, found 551.2153.1--3-(9H-fluoren-9-yl)-urea (32). Prepared in the same manner as described for 15, but the acetic anhydride was replaced by hexanoic anhydride: mp 162–165 °C; 1H-NMR (CDCl3) δ: 8.46 , 7.93 , 7.72 , 7.62 , 7.50 , 7.42 , 7.31 , 7.07 , 6.14 , 2.54 , 1.69 , 1.30 , 0.88 ; EI-MS m/z 556.3(M+); 165.1 (100%); HRMS (ESI) m/z calcd C33H36N2O6 [M + Na]+ 579.2471, found 579.2466.1--3-(9H-fluoren-9-yl)-urea (33). Prepared in the same manner as described for 15, but the acetic anhydride was replaced by n-octanoic anhydride: mp 152–153 °C; 1H-NMR (CDCl3) δ: 8.48 , 8.17 , 7.71 , 7.61 , 7.51–7.45 , 7.42 , 7.31 , 7.07 , 6.12 , 2.54 , 1.77–1.63 , 1.34–1.17 , 0.87 ; HRMS (ESI) m/z calcd C37H44N2O6 [M + H]+ 613.3278, found 613.3267.1--−1 for 5 min at RT. Pellets were added into 10 mL erythrocytes lysis buffer  followed by centrifugation to remove erythrocytes. After washing twice with complete medium , they were re-suspended in the complete medium and used for cell culture.Preparation of Splenocyte Suspension: The spleen was aseptically taken from female ICR mice strains , crushed gently and separated into single cells by squeezing in 20 mL RPMI 1640 medium . The cells obtained were passed through 70 μM nylon cell strainer and centrifuged at 1000 rpm∙min1 inhibition activity. 5 × 105 spleen cells were cultured in 96-well flat-bottom microplates  in RPMI 1640 complete medium containing 5 μg∙mL−1 of concanavalin A (Con A) in the presence of increasing concentration of compounds: CsA ; compound 1 . After 72 h of incubation, the cell growth was evaluated with MTT assay. The assay plates were added 10 μL of 5 mg∙mL−1 MTT per well and incubated for 4 h. The formazan generated was solubilized with 100 μL of solution  after incubated at 37 °C overnight. The absorption at 580 nm relative to 680 nm was measured by SpectraMax Plus 384 Reader .Proliferation Assay of Spleen Cells: During this assay, CsA was used as a control for evaluation of compound 4 cells per well at 37 °C overnight. All of the synthetic compounds were diluted with dimethyl sulfoxide (DMSO) to 10 mM of stock solution. The initial concentration of compounds was 20 μM and then diluted at a gradient of 1:4 to the concentrations ranging from 20 μM to 0.02 μM containing 0.5% DMSO. For the HCV virus assay, serial diluted compounds were mixed with a certain titer of JFH-1 virus, and the final concentration of JFH-1 virus titer was diluted to the numbers of relative luminescence units (RLU) ranging from 20,000 to 50,000 RLU and then added to the Huh 7.5.1 cells. For the HCV replicon system, serially diluted compounds was added to the Huh 7.5.1 replicon cells and the cells were cultured for 2 days at 37 °C. Then the cells were harvested and the luminescence was detected following the manufacturer’s protocol for the Renilla-Glo™ Luciferase Assay System. EC50 was the concentration of compound at which the HCV luminescence level in the Huh7.5.1 cells is reduced by 50%. Each data point represents the average of two replicates in cell culture. The values of EC50 were plotted by the GraphPad Prism 5 software.Viral Inhibition Assay: The HCV virus assay was constructed by using the method according to ,33 with 4 cells per well in 96-well plates overnight. The cells were respectively incubated with serial diluted compounds for 48 h, and the viability of Huh7.5.1 cells was determined in 96-well tissue culture plates using cell proliferation reagent WST-1 . The absorbance (OD450 /reference OD630) was measured to detect the cytotoxicity of compounds, according to the manufacturer’s protocol of Cell Proliferation Reagent WST-1.Cell Proliferation Assay: Huh 7.5.1 cells were seeded at a density of 2 × 104 per well in 96-well microtiter plates. After overnight culture, Huh 7.5.1 cells were infected with JFH-1 virus with luciferase reporter gene and treated with various concentrations of 25 alone, Sofosbuvir alone, or in combinations for 24 h. Antiviral activities were determined by measuring the reduction of luciferase activities in the cells. 3D-analysis of combinations on JFH1 infection was evaluated using the method of Prichard and Shipman [Huh 7.5.1 cells were seeded at 1 × 10 Shipman . CombinaN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide  as described in [−1 chymotrypsin (20 mg∙mL−1 in 1 mM HCl). The reaction was initiated by adding peptide substrate (2 mg∙mL−1 in 500 mM LiCl in tetrahydrofuran (THF)). After a delay from the onset of mixing , the cis-to-trans isomerization of the Ala-Pro peptide bond, coupled with the chymotryptic cleavage of the trans peptide, then the p-nitroanilide was quantified by the increase in absorbance at 380 nm for 60 s by a U-3010 spectrophotometer. Absorbance readings were collected every 0.1 s and the temperature was controlled at 4 °C during the assay. The progress curves were analyzed by nonlinear least-squares fit. The inhibition assays of compounds were performed in the same manner as above-mentioned. 10 µM of test compounds in DMSO, at various concentrations, was added to the CypA solution in the assay buffer. After being preincubated for 1 h at 4 °C, the assay was started by the addition of chymotrypsin and the substrate. To calculate the half-maximal inhibitory concentration (IC50), a percent of the remaining PPIase activity was plotted against the common logarithm of the compound concentration, and the data were fitted using the sigmoidal fitting model of Origin 7.0 software.The PPIase activity of CypA was conducted in a coupled assay with chymotrypsin using the synthetic tetrapeptide ribed in  with sma1 and its O-acetylated derivatives 14, 15 with potent anti-HCV activity were discovered. The subsequent assessment showed that compound 1 can be regarded as a non-immunosuppressive lead compound for further research, hence a series of acylated derivatives of the hydroxyl groups of compound 1 were prepared to give another 16 derivatives. Among these compounds, 25 exhibited the most potent antiviral activities in replicon and virus assays but low cytotoxicity (CC50 > 16 μM) and hERG (the human Ether-à-go-go-related gene) cardiac toxicity  25 was inactive at the enzyme level (CypA enzyme inhibition: IC50 > 5 μM), whereas it was active at the cellular level (virus assay: EC50 = 0.19 μM); (2) 25 showed sufficient stability in vitro (stable in PBS buffer at pH 7.4 after 12 h), but could rapidly release 1in vivo (AUC1/AUCA8 ≈ 60). However, further study is still necessary to explicitly demonstrate the hypothesized prodrug mechanism. To the best of our knowledge, compound 25 is probably the most potent small molecule anti-HCV agent in virus assay via CypA inhibitory mechanism. In conclusion, our present study indicated that compounds 1 and 25 could be used as a new class of potent anti-HCV agents and the further pre-clinical studies of the two compounds are currently underway in our laboratory.Considering the important role of CypA for HCV proliferation, we were enlightened and prompted to screen a series of compounds previously reported as CypA inhibitors and their derivatives. Fortunately, the small molecule CypA inhibitor


Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. Among the various medicinal plants, the alcohol extract of Pittosporum tobira, which significantly exhibited antibacterial effect for all tested strains, showed the highest activity in the antimicrobial assays. NMR analyses revealed that R1-barrigenol, a triterpene sapogenin, was the most effective compound in P. tobira. These results demonstrated that P. tobira possesses antimicrobial properties and would be beneficial for the prevention and treatment of periodontitis.The study of medicinal plants for treatment of periodontitis is of great value to establish their efficacy as sources of new antimicrobial drugs. Five hundred and fifty eight Korean local plant extracts were screened for antibacterial activity against representative periodontopathic bacteria such as  Porphyromonas gingivalis and Prevotella intermedia, representative periodontopathogens, are resistant to many antibiotics including penicillins, cephalosphorins, and tetracyclines   were obtP. gingivalis ATCC33277, P. intermedia ATCC 25611, and F. nucleatum subsp. nucleatum ATCC 23726 were grown in half-strength brain heart infusion (BHI) broth  supplemented with 5 mg/mL yeast extract, 5 µg/mL hemin, and 1 μg/mL vitamin K1 (BHI-HK). The bacteria grown at 37 °C anaerobically . The disc diffusion method ; flow rate 2 mL/min; UV detection, 210 nm], affording compound (The EtOAc fraction (3 g from 29 g) was fractionated by adsorption chromatography over silica gel eluting with EtOAc followed by increasing concentrations of methanol to yield five fractions (Fr.1–5). Fr.2 (442 mg) was firstly injected into a Sephadex LH-20 column eluting with 70% MeOH to yield four fractions (sFr.1–4). sFr.2 (38 mg) was purified by semi-preparative HPLC on a YMC silica  column : δ 0.98, 1.05, 1.11, 1.24, 1.35, 1.40, 1.88 , 3.78 , 3.79, 4.13 , 4.43 , 4.62 , 4.83 , 4.97 , and 5.54 ; 13C-NMR : δ 15.9 (C-25), 16.5 (C-24), 17.5 (C-26), 19.1 (C-6), 19.3 (C-30), 20.8 (C-27), 23.9 (C-11), 28.1 (C-2), 28.6 (C-23), 30.5 (C-29), 36.2 (C-20), 36.7 (C-7), 37.2 (C-10), 39.1 (C-1), 39.3 (C-4), 41.4 (C-8), 42.0 (C-18), 47.1 (C-9), 47.3 (C-14), 47.8 (C-19), 48.1 (C-17), 55.6 (C-5), 67.4 (C-15), 67.7 (C-28), 72.3 (C-16), 77.1 (C-22), 78.0 (C-3), 78.3 (C-21), 124.4 (C-12), 144.6 (C-13).P. gingivalis, P. intermedia, and F. nucleatum. Our findings provide evidence that R1-barrigenol, which is a triterpene sapogenin from P. tobira successfully inhibits periodontal bacteria strains. Although a detailed study of the action mechanism of this molecule remains to be elucidated, it could be more beneficial than traditional antibiotics as a potential agent for prevention and/or treatment of periodontitis.Periodontitis is a destructive inflammatory disease that leads to the loss of tooth support. It is initiated in the oral microbial biofilm constituted by Gram-negative anaerobic bacteria, including


Fusarium, Botrytis and Beauveria). Most of the selected microorganisms transformed these lactones by hydrolytic dehalogenation into the new compound cis-2-hydroxy-4,6-dimethyl-9-oxabicyclo[4.3.0]- nonan-8-one, mainly the (−)-isomer. When iodo-γ-lactone was used as the substrate, two products were observed: a hydroxy-γ-lactone and an unsaturated lactone. The structures of all substrates and products were established on the basis of their spectral data. The mechanism of dehalogenation of three halolactones was also studied.Bicyclic chloro-, bromo- and iodo-γ-lactones with dimethylcyclohexane rings were used as substrates for bioconversion by several fungal strains ( Halogenated organic compounds are often found in Nature. They are typically isolated from many marine organisms such as red algae, bacteria, sponges and corals. Among them there are found sesquiterpenoids ,2,3, ditThere are many different methods used for the biodegradation of halocompounds containing aromatic rings using algae ,23, bactFusarium, Beauveria and Botrytis are known for their hydrolytic properties in relation to miscellaneous compounds. Filamentous fungi of the genus Fusarium were often used for biohydroxylation of different substrates such as sesquiterpenoids nonan-8-one (3). A mixture of acid 2  and NCS  in THF (30 mL) was stirred at room temperature. After 24 h, water was added to the mixture and the product was diluted with diethyl ether. The separated organic layer was washed with a saturated NaHCO3 solution and brine, then dried with anhydrous magnesium sulfate. The crude product was purified on silica gel  and 2.1 g (yield 50%) of chlorolactone 3 was obtained. 1H-NMR (CDCl3): 0.93 , 1.06 , 1.39 , 1.53 , 1.62 , 1.92 , 2.05 , 2.26 and 2.48 , 4.29 , 4.50 ; 13C-NMR (CDCl3): 21.25 (C-9), 21.14 (C-4), 23.47 (C-10), 36.48 (C-3), 39.06 (C-6), 41.83 (C-5), 46.43 (C-7), 54.70 (C-2), 84.06 (C-1), 174.69 (C-8); IR : 2958 (s), 1788 (s), 1375 (m), 1207 (s), 1009 (m), 670 (m). Anal. Calcd. for C10H15ClO2 (202.68): C, 59.26; H, 7.46. Found: C, 59.18; H, 7.43.2-Bromo-4,6-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (4). Acid 2  was dissolved in THF (30 mL), then NBS  was added. The mixture was stirred for 24 h at room temperature and then water was added. The product was extracted with diethyl ether. The ether fractions were combined, washed with a saturated NaHCO3 solution and brine, then dried with anhydrous magnesium sulfate. The crude product was purified on silica gel  giving 2.3 g (yield 92%) of bromolactone 4. 1H-NMR (CDCl3): 0.95 , 1.07 , 1.45 , 1.56 , 1.70 , 1.97 , 2.09 , 2.27 and 2.48 , 4.43 , 4.57 ; 13C-NMR (CDCl3): 21.30 (C-9), 21.88 (C-4), 24.15 (C-10), 37.52 (C-3), 39.15 (C-6), 40.68 (C-2), 41.95 (C-5), 46.62 (C-7), 85.33 (C-1), 174.38 (C-8); IR : 2954 (s), 1789 (s), 1371 (m), 1204 (s), 1002 (s), 632 (m). Anal. Calcd. for C10H15BrO2 (247.13): C, 48.60; H, 6.12. Found: C, 48.57; H, 6.17.2-Iodo-4,6-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (5). 1H-NMR (CDCl3): 0.97 , 1.09 , 1.50 , 1.53 , 1.59 , 1.97 , 2.07 , 2.25 and 2.47 , 4.55 , 4.70 ; 13C-NMR (CDCl3): 21.34 (C-9), 33.41 (C-4), 23.77 (C-2), 25.39 (C-10), 38.69 (C-3), 39.62 (C-6), 42.02 (C-5), 46.72 (C-7), 85.79 (C-1), 173.98 (C-8); IR : 2924 (s), 1776 (s), 1453 (m), 1140 (s), 989 (s), 615 (s).2-Hydroxy-4,6-dimethyl-9-oxabicyclo[4.3.0]nonan-8-one (6). 1H-NMR (CDCl3): 0.94 , 1.02 , 1.19 , 1.22 , 1.41 , 1.60 , 1.83 , 2.00 , 2.31 and 2.46 , 3.80 , 4.24 ; 13C-NMR (CDCl3): 21.65 (C-10), 21.39 (C-9), 26.88 (C-4), 37.41 (C-3), 40.54 (C-6), 41.21 (C-5), 46.25 (C-7), 68.07 (C-2), 85.81 (C-1), 1753.56 (C-8); IR : 3340 (s), 2912 (s), 1788 (s), 1134 (s), 1052 (s). Anal. Calcd. for C10H15O3 (184.24): C, 65.20; H, 8.75. Found: C, 65.15; H, 8.71.4,6-Dimethyl-9-oxabicyclo[4.3.0]non-2-en-8-one (7). 1H-NMR (CDCl3): 1.04 , 1.15 , 1.22 , 1.52 , 2.24 , 2.33 and 2.52 , 4.31 , 5.83 , 5.94 ; IR : 1780 (s), 1651 (m), 1212 (s), 999 (s).Fusarium culmorum AM10, Fusarium avenaceum AM11, Fusarium oxysporum AM13, Fusarium tricinctum AM16, Fusarium semitectum AM20, Fusarium solani AM203, Botrytis cinerea AM235 and Beauveria bassiana AM278). They all are available in Department of Chemistry, University of Environmental and Life Sciences. These strains were cultivated on Sabouraud’s agar containing 5 g of aminobac, 5 g of peptone, 40 g of glucose and 15 g of agar dissolved in 1 L of distilled water at 28 °C and stored in refrigerator at 4 °C.The fungal strains, which were used in this study, were obtained from the collection of the Institute of Biology and Botany, Medical University, Wrocław . After 4 days, 10 mg of the substrate dissolved in 1 mL of acetone was added to the culture in each flask. Incubation of the shaken cultures with the substrate was continued for 14 days. After 5, 9 and 14 days of incubation, 30 mL of the reacting mixture including mycelium, unreacted substrate and expected product was taken from the two flasks and extracted with 30 mL of dichloromethane. After evaporated the solvent the residue was dissolved in 2 mL of acetone and analyzed by TLC  and GC (HP-5 column).3–5 (100 mg) were dissolved in 10 mL of acetone and distributed between 10 Erlenmeyer flasks with the 4-day cultures of fungal strains prepared as described in the screening procedure. The selected microorganisms were incubated with the substrates for 14 days, then the products were extracted with dichloromethane (3 × 50 mL). The organic solutions were dried (MgSO4) and the solvent was evaporated in vacuo. Mixtures containing the product, the unreacted substrate and metabolites of the fungi were separated by column chromatography  to obtain pure products.The halolactones 3, 4 and 5 showed very high regio- and stereoselectivity. The OH group was introduced at C-2 in the equatorial position. Since the halogen atom was in an axial position in all three substrates, this allowed us to conclude that the hydrolytic dehalogenation reaction proceeded analogously to the mechanism of SN2 nucleophilic substitution. Two substrates, chlorolactone 3 and bromolactone 4, were transformed by five and six fungal strains, respectively, in good yield (conversions over 50%). The iodolactone 5 was transformed by only three microorganisms in yields over 50%, and additionally this substrate gave an unsaturated lactone 7 in some cases. The best microorganisms for the transformation of halolactones 3, 4 and 5 in the best yields were F. solani, F. oxysporum and B. cinerea. Taking into account its ability to transform substrates with high enantiomeric excess, the best biocatalyst was F. solani. The filamentous fungal strains chosen for the conversion of halolactones


As the commonly applied solution phase synthesis of dendritic scaffolds is cumbersome and time-consuming, a synthesis strategy was developed that allows for the efficient assembly of acid amide bond-based highly modular dendrons on solid support via standard Fmoc solid phase peptide synthesis protocols. The obtained dendritic structures comprised up to 16 maleimide functionalities and were derivatized on solid support with the chelating agent DOTA. The functionalized dendrons furthermore could be efficiently reacted with structurally variable model thiol-bearing bioactive molecules via click chemistry and finally radiolabeled with 68Ga. Thus, this solid phase-assisted dendron synthesis approach enables the fast and straightforward assembly of bioactive multivalent constructs for example applicable as radiotracers for in vivo imaging with Positron Emission Tomography (PET).Dendritic structures, being highly homogeneous and symmetric, represent ideal scaffolds for the multimerization of bioactive molecules and thus enable the synthesis of compounds of high valency which are e.g., applicable in radiolabeled form as multivalent radiotracers for  Dendritic structures, exhibiting highly branched and ideally well-defined and homogeneous structures, have extensively been studied as carriers for a large variety of bioactive compounds over several decades ,2,3. The standard procedure for the preparation of dendrimers is still the solution-phase synthesis as by this technique, very large dendritic structures can be synthesized on a large scale. However, this synthesis approach is often time-consuming due to the long reaction times required for a complete conversion of all functional groups and the cumbersome purification procedures that have to be implemented after each reaction step. An alternative is the synthesis of dendrimers using a solid support, which exhibits the advantages of comparatively fast reaction kinetics  and very efficient purifications which are executed by simple washing of the resin. So far, several examples for the solid phase-assisted synthesis of dendrimers are available , includiSuch a modular dendrimer design can be of special interest if the dendrimers are to be used as drug carriers with a certain, optimal distance required between two drug moieties within the molecule. So was for example shown very recently that the distance between peptide moieties within a dendritic peptide multimer has a crucial influence on the achievable binding avidities of the multivalent peptides to their respective receptor . in vivo molecular imaging application, these dendrons also have to provide the opportunity to introduce a label such as a radionuclide. The optimal position for the introduction of this label is the focal point of the dendron to ensure a highly homogeneous and defined structure of the whole construct.Only very few examples of solid phase-supported syntheses of structurally flexible polylysine dendrimers have been described recently and in these systems, the structural flexibility was achieved by the introduction of different linkers ,24. To bin vivo imaging settings 3+ : 526.31 (526.31); [M+2H]2+ : 788.96 (788.96); [M+2Na]2+ : 810.94 (810.94). 2N-CO-Lys-PEG3-Mal4H2: Reaction times for conjugation of APG-1: 1 h, APG-2: 2 h, 6-maleimidohexanoic acid: 1 h; gradient: 10%–50% MeOH + 0.1% FA in 5 min, Rt = 3.5 min; ESI-MS (m/z) for [M+3H]3+ : 560.33 (560.33); [M+2H]2+ : 839.99 (839.99); [M+H+Na]2+ : 850.98 (850.98); [M+2Na]2+ : 861.97 (861.97); [M+Na+K]2+ : 869.96 (870.03). 2N-CO-Lys-PEG5-Mal4H3: Reaction times for conjugation of APG-1: 1 h, APG-2: 2 h, 6-maleimidohexanoic acid: 1 h; gradient: 10%–50% MeOH + 0.1% FA in 5 min, Rt = 3.7 min; MALDI-MS (m/z) for [M+H]+ : 1766.55 (1767.03); [M+Na]+ : 1788.98 (1789.01); [M+K]+ : 1805.58 (1805.12). 2N-CO-Lys-PEG7-Mal4H4: Reaction times for conjugation of APG-1: 1 h, APG-2: 2 h, 6-maleimidohexanoic acid: 1 h; gradient: 10%–50% MeOH + 0.1% FA in 5 min, Rt = 3.8 min; ESI-MS (m/z) for [M+3H]3+ : 619.04 (619.03); [M+2H]2+ : 928.05 (928.04); [M+H+Na]2+ : 939.04 (939.04); [M+2Na]2+ : 950.03 (950.03); [M+Na+K]2+ : 958.02 (958.08). 2N-CO-Lys-PEG11-Mal4H5: Reaction times for conjugation of APG-1: 1 h, APG-2: 2 h, 6-maleimidohexanoic acid: 1 h; gradient: 10%–50% MeOH + 0.1% FA in 5 min, Rt = 4.1 min; MALDI-MS (m/z) for [M+H]+ : 2031.36 (2031.19); [M+Na]+ : 2053.29 (2053.17). 2N-CO-Lys-(PEG1)3-Mal4H6: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, 6-maleimido-hexanoic acid: 1h; gradient: 20%–55% MeOH + 0.1% FA in 5 min, Rt = 2.9 min; ESI-MS (m/z) for [M+3H]3+ : 816.46 (816.46); [M+2H+Na]3+ : 823.78 (823.78). 2N-CO-Lys-(PEG3)3-Mal4H7: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, 6-maleimido-hexanoic acid: 1 h; gradient: 20%–55% MeOH + 0.1% FA in 5 min, Rt = 3.8 min; ESI-MS (m/z) for [M+4H]4+ : 791.21 (791.21); [M+Na+3H]4+ : 796.71 (796.71); [M+2Na+2H]4+ : 802.20 (802.20); [M+4Na]4+ : 813.20 (813.19); [M+3H]3+ : 1054.61 (1054.61); [M+Na+2H]3+ : 1061.94 (1061.94); [M+H+2Na]3+ : 1069.27 (1069.27); [M+3Na]3+ : 1076.60 (1076.60). 2N-CO-Lys-(PEG5)3-Mal4H8: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1h, 6-maleimido-hexanoic acid: 1 h; gradient: 20%–55% MeOH + 0.1% FA in 5 min, Rt = 4.3 min; MALDI-MS (m/z) for [M+H]+ : 3779.96 (3778.20). 2N-CO-Lys-(PEG7)3-Mal4H9: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, 6-maleimido-hexanoic acid: 1 h; gradient: 20%–55% MeOH + 0.1% FA in 5 min, Rt = 4.8 min; ESI-MS (m/z) for [M+5H+Na]6+ : 736.93 (736.93); [M+4H+2Na]6+ : 740.59 (740.59); [M+3H+3Na]6+ : 744.43 (744.26); [M+5H]5+ : 879.72 (879.72); [M+4H+Na]5+ : 884.12 (884.11); [M+3H+2Na]5+ : 888.91 (888.51). 2N-CO-Lys-(PEG11)3-Mal4H10: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, 6-maleimidohexanoic acid: 1 h; gradient: 0%–100% MeCN + 0.1% TFA in 5 min, Rt = 3.2 min; ESI-MS (m/z) for [M+7H]7+ : 804.76 (804.76); [M+6H+Na]7+ : 807.9 (807.9); [M+5H+2Na]7+ : 811.04 (811.04); [M+6H]6+ : 938.72 (938.72); [M+5H+Na]6+ : 942.39 (942.39); [M+4H+2Na]6+ : 946.05 (946.05). 2N-CO-Lys-PEG1-Mal8H11: Reaction times for conjugation of APG-1: 1 h, APG-2: 2 h, APG-3: overnight, 6-maleimido-hexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.0 min; MALDI-MS (m/z) for [M+H]+ : 3033.76 (3033.75). 2N-CO-Lys-PEG3-Mal8H12: Reaction times for conjugation of APG-1: 1 h, APG-2: 2 h, APG-3: overnight, 6-maleimido-hexanoic acid: 2h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.0 min; ESI-MS (m/z) for [M+5H]5+ : 627.97 (627.77); [M+4H]4+ : 784.46 (784.46); [M+3H]3+ : 1046.28 (1045.61). 2N-CO-Lys-PEG5-Mal8H13: Reaction times for conjugation of APG-1: 1 h, APG-2: 2 h, APG-3: overnight, 6-maleimido-hexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.0 min; MALDI-MS (m/z) for [M+H]+ : 3224.24 (3223.87). 2N-CO-Lys-PEG7-Mal8H14: Reaction times for conjugation of APG-1: 1 h, APG-2: 2 h, APG-3: overnight, 6-maleimido-hexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 1.9 min; MALDI-MS (m/z) for [M+H]+ : 3312.95 (3311.93). 2N-CO-Lys-PEG11-Mal8H15: Reaction times for conjugation of APG-1: 1h, APG-2: 2h, APG-3: overnight, 6-maleimidohexanoic acid: 2h; gradient: 20%–50% MeOH + 0.1% FA in 5 min, Rt = 3.5 min; MALDI-MS (m/z) for [M+H]+ : 3488.57 (3488.03). 2N-CO-Lys-(PEG1)4-Mal8H16: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.4 min; MALDI-MS (m/z) for [M+H]+ : 5071.36 (5064.79); [M+2H]2+ : 2533.63 (2532.90). 2N-CO-Lys-(PEG3)4-Mal8H17: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 3.5 min; ESI-MS (m/z) for [M+7H]7+ : 943.12 (943.12); [M+Na+6H]7+ : 946.41 (946.26). 2N-CO-Lys-(PEG5)4-Mal8H18: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 4.1 min; MALDI-MS (m/z) for [M+H]+ : 7946.57 (7938.58); [M+2H]2+ : 3962.96 (3958.80). 2N-CO-Lys-(PEG7)4-Mal8H19: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 4.7 min; MALDI-MS (m/z) for [M+H]+ : 9283.40 (9275.47); [M+2H]2+ : 4626.83 (4619.19). 2N-CO-Lys-(PEG3)3-Mal8H20: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 20%–50% MeOH + 0.1% FA in 5 min, Rt = 3.7 min; MALDI-MS (m/z) for [M+H]+ : 4623.72 (4618.67); [M+2H]2+ : 2310.27 (2309.84). 2N-CO-Lys-(PEG3)2-Mal8H21: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, APG-3: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 20%–50% MeOH + 0.1% FA in 5 min, Rt = 3.3 min; MALDI-MS (m/z) for [M+H]+ : 3631.08 (3630.11).2N-CO-Lys-PEG11-(PEG3)3-Mal8H22: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 3.3 min; MALDI-MS (m/z) for [M+Na]+ : 6970.43 (6970.00).2N-CO-Lys-PEG11-(PEG3)2-Mal8H23: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.4 min; MALDI-MS (m/z) for [M+H]1+ : 4975.39 (4970.88).2N-CO-Lys-PEG11-PEG3-PEG1-Mal8H24: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.3 min; MALDI-MS (m/z) for [M+H]+ : 4567.08 (4562.61).2N-CO-Lys-PEG5-PEG3-PEG1-Mal8H25: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 20%–50% MeOH + 0.1% FA in 5 min, Rt = 3.4 min; MALDI-MS (m/z) for [M+H]+ : 4303.96 (4298.46).2N-CO-Lys-PEG11-(PEG1)3-Mal8H26: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.4 min; MALDI-MS (m/z) for [M+H]+ : 5526.38 (5519.07), [M+2H]2+ : 2760.11 (2760.04).2N-CO-Lys-PEG11-(PEG1)2-Mal8H27: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–45% MeOH + 0.1% FA in 5 min, Rt = 2.8 min; MALDI-MS (m/z) for [M+H]+ : 4361.71 (4358.48).2N-CO-Lys-PEG11-(PEG1)3-Mal16H28: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, APG-4: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.3 min; MALDI-MS (m/z) for [M+K]+ : 8469.42 (8470.84).2N-CO-Lys-PEG11-PEG3-(PEG1)2-Mal16H29: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, APG-4: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.4 min; MALDI-MS (m/z) for [M+K]+ : 8673.61 (8674.98), [M+2H]2+ : 4324.72 (4318.95).2N-CO-Lys-PEG11-(PEG3)2-PEG1-Mal16H30: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, APG-4: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.5 min; MALDI-MS (m/z) for [M+K]+ : 9086.1 (9083.25), [M+2H]2+ : 4527.44 (4523.09).2N-CO-Lys-PEG11-(PEG3)3-Mal16H31: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, APG-4: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.8 min; MALDI-MS (m/z) for [M+K]+ : 9914.00 (9899.80), [M+2H]2+ : 4938.16 (4931.36).2N-CO-Lys-PEG11-PEG5-PEG3-PEG1-Mal16H32: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, APG-4: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% FA in 5 min, Rt = 2.6 min; MALDI-MS (m/z) for [M+K]+ : 9259.57 (9259.36), [M+2H]2+ : 4615.74 (4611.14).2N-CO-Lys(DOTA)-PEG11-Mal1H33: gradient: 35%–60% MeOH + 0.1% TFA in 8 min, Rt = 5.1 min; yield: 2.9%; MALDI-MS (m/z) for [M+H]+ : 1324.60 (1324.73), [M+Na]+ : 1347.37 (1346.72), [M+K]+ : 1362.67 (1362.82).2N-CO-Lys(DOTA)-PEG11-Mal2H34: Reaction times for conjugation of APG-1: 1 h, 6-maleimidohexanoic acid: 45 min; gradient: 35%–60% MeOH + 0.1% TFA in 8 min, Rt = 6.0 min; yield: 11.2%; MALDI-MS (m/z) for [M+H]+ : 1688.97 (1688.95), [M+Na]+ : 1711.39 (1710.93).2N-CO-Lys(DOTA)-PEG11-PEG1-Mal4H35: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, 6-maleimidohexanoic acid: 1 h; gradient: 45%–55% MeOH + 0.1% TFA in 8 min, Rt = 5.0 min; yield: 14.1%; MALDI-MS (m/z) for [M+H]+ : 2707.90 (2707.52).2N-CO-Cys-PEG36: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, 6-maleimidohexanoic acid: 1 h; gradient: 45%–65% MeOH + 0.1% TFA in 8 min, Rt = 6.9 min; yield: 6.4%; MALDI-MS (m/z) for [M+H]+ : 2384.42 (2384.28), [M+Na]+ : 2406.62 (2406.27), [M+K]+ : 2422.40 (2422.37).2N-CO-Lys-PEG11-PEG1-Mal4H37: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, 6-maleimidohexanoic acid: 1 h; gradient: 30%–55% MeOH + 0.1% TFA in 8 min, Rt = 7.7 min; yield: 17.0%; MALDI-MS (m/z) for [M+H]+ : 2321.25 (2321.34).2N-CO-Lys(DOTA)-PEG11-(PEG1)2-Mal8H38: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 30%–55% MeOH + 0.1% TFA in 8 min, Rt = 4.8 min; yield: 10.7%; MALDI-MS (m/z) for [M+H]+ : 4748.32 (4744.66), [M+2H]2+ : 2372.99 (2372.83).2N-CO-Cys(SH-11-(PEG1)2-Mal8tBu)-PEG39: Reaction times for conjugation of APG-1: 1 h, PEGX: 45 min, APG-2: 2 h, PEGY: 1 h, APG-3: overnight, PEGZ: 2 h, APG-4: overnight, 6-maleimidohexanoic acid: 2 h; gradient: 45%–65% MeOH + 0.1% TFA in 8 min, Rt = 6.7 min; yield: 13.0%; MALDI-MS (m/z) for [M+H]+ : 4424.95 (4421.42), [M+Na]+ : 4448.00 (4443.41), [M+K]+ : 4465.11 (4459.51). 35, 36, 38 or 39, 0.46–2.5 μmol) in MeCN (100–400 μL) was added a solution of the respective thiol-bearing synthon to be multimerized , c(RGDfC) or thiol-DOTA; at least 1.25 eq. per maleimide functionality) in phosphate buffered saline , resulting in a pH of 6.8–7.2. After 5 min reaction time, the products were analyzed/isolated via HPLC and obtained as white solids or colorless oils after purification and lyophilization. Gradients used for HPLC analysis/purification and analytical data for each compound are given below. Reaction on solid phase: The resin containing the respective multivalent maleimide (35 or 36) was rinsed after the final maleimide conjugation with first MeCN and subsequently phosphate buffered saline . The conjugation of thiol-bearing synthons was carried out by the addition of a solution of the respective thiol  in phosphate buffered saline  and incubation with the multivalent maleimide on solid support for 1h. The products were obtained by acidic cleavage from the resin and precipitation as described before and analyzed via HPLC. Gradients used for HPLC analysis/purification and analytical data for each compound are given below.Reaction in solution: To a solution of the respective maleimide-multimer  for [M+Na]+ : 6334.62 (6334.96), [M+2H]2+ : 3162.4 (3156.99).8-DOTAL-glutathione41: Gradient: 30%–55% MeOH + 0.1% TFA in 8 min, Rt = 4.8 min; yield: 45.2%; MALDI-MS (m/z) for [M+Na]+ : 7222.99 (7223.31), [M+2H]2+ : 3601.52 (3600.16).8-DOTAc(RGDfC)42: Gradient: 45%–65% MeOH + 0.1% TFA in 8 min, Rt = 4.3 min; yield: 19.8%; MALDI-MS (m/z) for [M+Na]+ : 9397.17 (9392.45), [M+2H]2+ : 4686.46 (4685.74).8-SS-tBuDOTA43: Gradient: 20%–30% MeCN + 0.1% TFA in 8 min, Rt = 5.5 min; yield: 18.1%; MALDI-MS (m/z) for [M+Na]+ : 8156.95 (8149.09), [M+2H]2+ : 4068.7 (4064.06).4-DOTAL-glutathione44: Gradient: 0%–100% MeCN + 0.1% TFA in 5 min, Rt = 1.8 min; MALDI-MS (m/z) for [M+H]+ : 3935.32 (3935.85).4-DOTAGalactose45: Gradient: 0%–100% MeCN + 0.1% TFA in 5 min, Rt = 1.8 min; MALDI-MS (m/z) for [M+H]+ : 3491.79 (3491.68), [M+Na]+ : 3514.30 (3513.66), [M+K]+ : 3529.59 (3529.77), [M+Na+K]+ : 3552.07 (3551.75), [M+2K]+ : 3567.82 (3567.86).4-SS-tBuDOTA46: Gradient: 0%–100% MeCN + 0.1% TFA in 5 min, Rt = 2.2 min; MALDI-MS (m/z) for [M+H]+ : 4239.60 (4237.12).2O was added to 244–277 MBq of 68Ga3+ in a solution obtained by fractioned elution of a 68Ge/68Ga generator  with HCl  and subsequent titration to pH 3.5–4.0 by addition of sodium acetate solution . After reaction for 10 min at 95 °C and cooling, the reaction mixtures were analyzed by analytical radio-HPLC. The radiolabeled products [68Ga]40, [68Ga]42 and [68Ga]43 were found to be ≥95% pure and obtained in specific activities of up to 147 GBq/µmol for 40, up to 55 GBq/µmol for 42 and up to 163 GBq/µmol for 43. A solution of the respective DOTA-derivatized multimer (1.5–5 nmol) in tracepur H68Ga after the multimerization reactions. This shows the applicability of the presented technique to the synthesis of multivalent radiotracers which can be used for molecular imaging purposes with PET.It was shown that the synthesis of even complex dendritic scaffold structures based on acid-amide bonds on solid supports is possible using standard solid phase peptide synthesis protocols. By systematically investigating optimal building blocks and reaction parameters, the desired homogeneous and symmetric dendrons could be obtained. In addition, molecular designs deviating from the optimized structure could also be synthesized, enabling a highly modular dendron assembly on solid support. The obtained dendritic structures comprised up to 16 maleimide functionalities and could subsequently be efficiently reacted with structurally variable thiol-bearing bioactive molecules via click chemistry. Furthermore, some dendritic scaffolds were derivatized on solid support with the chelator DOTA and finally successfully radiolabeled with