Z10 is the most active against Pseudomonas aeruginosa (32 mm) and least active against Candida stellatoidea (27 mm) and Vancomycin Resistant Enterococci (VRE) (27 mm), while Z7 shows the least zone of inhibition (22 mm) against Methicillin Resistant Staphylococcus aureus (MRSA). The minimum inhibition concentration (MIC) determination reveals that Z10 inhibits the growth of tested microbes at a low concentration of 6.25 μg/mL, while Z9 and Z12 inhibits the growth of most microbes at a concentration of 12.5 μg/mL, recording the least MIC. The Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) results revealed that Z10 has the highest bactericidal/fungicidal effect on the test microbes, at a concentration of 12.5 μg/mL, with the exception of Candida stellatoidea and Vancomycin Resistant Enterococci (VRE) with MBC/MFC of 25 μg/mL. The result of this investigation reveals the potential of the target compounds  in the search for new antimicrobial agents. Our search for new antibiotics led to the syntheses and biological evaluation of new classes of dicarboxylic acid analogues. The syntheses involve nucleophilic addition of different substituted benzylamine, aniline, alkylamine, and 4-hydroxyl-L-proline with carbamoylbenzoic acid. The results of the antimicrobial activity as indicated by the zone of inhibition (ZOI) showed that Development of novel bioactive drugs in chemical warfare against bacteria, fungi, and other infectious diseases has become an important and challenging task for the synthetic and medicinal chemists. Many research programs are tailored towards the design and synthesis of new drugs, for their chemotherapeutic application. The emergence of antimicrobial resistance threatens the effective prevention and treatment of an ever increasing range of infections caused by bacteria, parasites, virus, and fungi. New resistance mechanisms are emerging and spreading globally; the appearance and widespread use of fake and substandard drugs have further compounded the problem . The HIV3, CH3, NO2, and F) and explored their potentials as antibacterial and antifungal drugs.Currently, there are more than 450 clinically approved drugs containing a free carboxylic acid group . To the 254) from MERCK (Germany). Spots on the TLC plates were visualized under UV light (254 nm and 366 nm) and by heating with 10% sulphuric acid in MeOH. The melting point was recorded using a Gallenkamp melting point apparatus. The UV-VIS analysis was carried out on a Perkin Elmer Lambda 35 UV/VIS Spectrometer, scanning from 200 to 400 nm. Absorbance was measured with a 1 cm quartz cell. The infrared spectra of the solid products were recorded on a Perkin Elmer Spectrum 100 FTIR spectrometer using ATR sampling accessory. 1H and 13C-NMR spectra were recorded using Bruker AvanceIII 400 MHz Spectrometer at room temperature (400 MHz for 1H and 100 MHz for 13C), using TMS as reference. Chemical shift values (δ) were reported in parts per million (ppm) relative to TMS and coupling constants are given in Hz. The solvent used for these measurements was deuterated DMSO. Multiplicities are given as follows: singlet (s), doublet (d), doublet of doublets (dd), triplet (t), and multiplet (m).All chemicals and solvents were purchased from Sigma-Aldrich (Germany) and used as purchased without further purification. Thin-layer chromatography was performed using precoated silica gel 60 (Faq (5%) and then stirred for 30 min; the solid product  formed was then filtered under suction. The products were pure enough for further reaction without further purification; this acid  was further transferred to a 10 mL round bottom flask containing dichloromethane (7 mL) and a magnetic stirrer and 1.5 equiv. of different classes of amine were each added individually to the respective flasks and the mixture refluxed for at least two hours. The heat was then removed and the reaction stirred for a further 30 min. The reaction mixtures were allowed to stand at room temperature for a further 30 min; the solid precipitates formed were then filtered under suction and washed thoroughly with dichloromethane to remove any excess amines  was weighed into a round bottom flask containing 100 mL of KOHs amines . After ds amines .4-[(4-Fluorobenzyl)carbamoyl]benzene-1,5-dicarboxylic Acid ( Z1). Bone white solid powder (99% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , 4-fluorobenzylamine , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 173–175°C, UV analysis λmax(logε), 245 (4.39), 300 (3.92). IR (cm−1); 2872 (COOH), 1603 and 1536 (CONH), 896 (=CH) and 828–808 (C=C). 1H-NMR  δH 8.72 , 8.18 , 7.96 , 7.54 , 7.26 , 4.03 . 13C-NMR ; 168.33 (C-7), 167.88 (C-9), 167.81 (C-8), 161.94 , 137.21 (C-2), 136.82 (C-1), 134.43 (C-5), 133.57 (C-6), 132.39 (C-4), 131.25 , 131.02 , 130.46 (C-3), 115.32 , 41.64 (C-10). GC-MS  317.1 [M]+ (74), 299.1 (100), 244.1 (24), 122.1 (23), LRMS 313.3 [M − Na]+ for C16H12FNO5, calculated mass 317.3 .4-[(3-Fluorobenzyl)carbamoyl]benzene-1,5-dicarboxylic Acid ( Z2). Bone white solid powder (55% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , 3-fluorobenzylamine , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 200-201°C, UV analysis (λmax(logε)ethanol), 250 (4.26), 300 (3.48). IR (cm−1); 2868 (COOH), 1271 and 1246 (O-C), 1691 and 1501 (CONH), 1691 and 1574 (C=C) and 913 (=CH). 1H-NMR  δH 8.72 , 8.18 , 7.96 , 7.46–7.40 , 7.33–7.27 , 7.33–7.27 , 7.18–7.14 , 4.00 . 13C-NMR ; 168.21 (C-7), 167.74 (C-9), 167.69 (C-8), 162.04 , 139.36 , 136.99 (C-1), 134.51 (C-2), 133.58 (C-3), 133.58 (C-5), 132.42 (C-6), 130.43 , 130.42 (C-4), 124.44 , 115.12 , 114.59 , 42.35 (C-10). GC-MS  317.1 [M]+ (72), 299.1 (100), 226.1 (24), 122.1 (21), LRMS 317.1 [M − Na]+ for C16H12FNO5 calculated 317.3 (Appendixes  8–15).2-[(2-Fluorobenzyl)carbamoyl]benzene-1,5-dicarboxylic Acid ( Z3). Bone white solid powder (35% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , 2-fluorobenzylamine , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 198–200°C, UV analysis λmax(logε), 245 (4.24), 300 (3.43). IR (cm−1); 2839 (COOH), 1272 and 1248 (O-C), 1684 and 1574 (CONH), 1613 and 1500 (C=C) and 907 (=CH).t6 1H-NMR  δH 8.71 , 8.15 , 7.94 , 7.56–7.53 , 7.39–7.34 , 7.24–7.19 , 3.98 . 13C-NMR ; 168.85 (C-7), 168.03 (C-9), 167.95 (C-8), 160.15 , 138.53 (C-2), 136.36 (C-1), 134.31 (C-5), 133.53 (C-6), 132.22 (C-3), 130.42 (C-4), 130.42 , 129.76 , 124.78 , 124.44 , 115.15 , 36.77 . GC-MS  317.1 [M]+ (91), 299.1 (100), 244.1 (32), 122.1 (24), LRMS 313.1 [M − Na]+ for C16H12FNO5, calculated mass 317.3 (Appendixes  16–23).2-{[-4-Carboxy-2-hydroxypyrrolidin-1-yl]carbonyl}benzene-1,5-dicarboxylic Acid ( Z5). White solid powder (78% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , 4-hydroxy-L-proline , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 187–180°C, UV analysis λmax(logε), 245 (4.02), 300 (3.18). IR (cm−1); 2855 (COOH), 1285 and 1250 (O-C), 1689 and 1574 (CONH), 1639 and 1502 (C=C) and 916 (=CH). 1H-NMR  δH 8.43 , 8.05 , 7.96 , 4.41 , 4.31 , 3.32 , 2.21 . 13C-NMR ; 170.41 (C-7), 167.92 (C-10), 167.34 (C-9), 166.21 (C-8), 137.87 (C-2), 133.22 (C-1), 132.29 (C-5), 131.25 (C-4), 131.03 (C-6), 130.28 (C-3), 68.75 (C-2′), 57.98 (C-4′), 53.23 (C-5′), 37.31 (C-3′). GC-MS  323.1 [M − 4]+ (100), 226.1 (29), 282.1 (18), 122.1 (13), LRMS 324.3 [M − Na]+ for C14H13NO8, calculated mass 324.3 (Appendixes  24–31). 2-[carbamoyl]benzene-1,5-dicarboxylic Acid ( Z7). Pale yellow solid powder (90% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , 2,4-difluorobenzylamine  and toluene (7 mL) as solvent and purified by recrystallization with DCM. Melting point 152–154°C, UV analysis λmax(logε), 245 (3.86), 300 (2.95). IR (cm−1); 2878, 2631 (COOH), 1273 and 1297 (O-Cacid), 1687 and 1561 (CONH), 1603 and 1507 (C=C) and 847 (=CH). 1H-NMR  δH 8.74 , 8.24 , 8.01 , 7.64 , 7.35 , 7.20 , 4.07 . 13C-NMR ; 167.46 (C-7), 167.46 (C-9), 167.16 (C-8), 161.34 , 160.55 , 138.05 (C-2), 134.73 (C-5), 133.70 (C-1), 133.63 (C-6), 132.77 (C-4), 132.69 , 117.89 , 111.77 , 104.04 , 35.41 . GC-MS  331.1 [M + 4]+ (100), 244.1 (32), 300.1 (16), 103.1 (10), LRMS 333.1 [M − Na]+ for C16H11F2NO5, calculated mass 335.3 (Appendixes  32–39).2-(Butylcarbamoyl)benzene-1,5-dicarboxylic Acid ( Z8). Bone white solid powder (54% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , butylamine , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 118–120°C, UV analysis λmax(logε), 245 (4.08), 300 (3.26). IR (cm−1); 3061, 2959 (COOH), 1239 and 1267 (O-Cacid), 1654 and 1562 (CONH), 1622 and 1516 (C=C) and 780 (=CH). 1H-NMR  δH 8.71 , 8.19 , 7.97 , 2.81 , 1.53 , 1.34 , 0.88 . 13C-NMR ; 167.99 (C-7), 167.84 (C-9), 167.79 (C-8), 137.03 (C-2), 136.52 (C-1), 134.45 (C-5), 133.55 (C-6), 132.47 (C-3), 130.48 (C-4), 38.44 (C-1′), 29.12 (C-2′), 19.07 (C-3′), 13.43 (C-4′). GC-MS  261.1 [M + 4]+ (41), 218.1 (100), 185.1 (74), 261.1 (41), LRMS 261.1 [M − Na]+ for C13H15NO5, calculated mass 265.3 (Appendixes  40–46). 2-[(3-Methoxypropyl)carbamoyl]benzene-1,5-dicarboxylic Acid ( Z9). White solid powder (24% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , 3-methoxypropylamine , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 135-136°C, UV analysis λmax(logε), 245 (3.68), 300 (2.48). IR (cm−1); 3136, 2932 (COOH), 1356 and 1297 (O-Cacid), 1622 and 1529 (CONH), 1477 (C=C) and 752 (=CH). 1H-NMR  δH 8.67 , 8.12 , 7.91 , 3.39 , 3.22 , 2.86 , 1.82 . 13C-NMR ; 169.04 (C-7), 168.19 (C-9), 168.08 (C-8), 139.91 (C-2), 135.83 (C-1), 134.15 (C-5), 133.45 (C-6), 132.05 (C-4), 130.34 (C-3), 68.92 (C-3′), 57.87 (C-4′), 36.52 (C-1′), 27.44 (C-2′). GC-MS  281.1 [M]+ (28), 207.1 (100), 32.1 (93), 45 (77), LRMS 277.1 [M − Na]+ for C13H15NO6, calculated mass 281.3 (Appendixes  47–54). 2-(Benzylcarbamoyl)benzene-1,5-dicarboxylic Acid ( Z10). Cream coarse powder (65% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , benzylamine , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 163–165°C, UV analysis λmax(logε), 245 (3.78), 300 (2.78). IR (cm−1); 3396, 3034 (COOH), 1361 and 1288 (O-Cacid), 1622 and 1551 (CONH), 1499 (C=C) and 693 (=CH). 1H-NMR  δH 8.72 , 8.15 , 7.94 , 7.46–7.31 , 4.00 . 13C-NMR ; 169.03 (C-7), 168.15 (C-9), 168.04 (C-8), 139.38 (C-1′), 136.05 , 134.23 (C-1), 133.52 (C-6), 132.14 (C-3), 130.40 (C-4), 128.46 , 128.45 , 127.91 (C-4′). GC-MS  295.1 [M + 4]+ (100), 278 (34), 208.1 (25), 104.1 (24), LRMS 295.1 [M − Na]+ for C16H13NO5, calculated mass 299.3 (Appendixes  55–62). 2-[carbamoyl]benzene-1,5-dicarboxylic Acid ( Z11). Bone white powder (79% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , 2-amino-4,6-dimethylpyridine , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 189-190°C, UV analysis λmax(logε), 245 (4.31), 300 (3.88). IR (cm−1); 3331, 3097 (COOH), 1343 and 1296 (O-Cacid), 1675 and 1534 (CONH), 1604 and 1477 (C=C) and 752 (=CH). 1H-NMR  δH 8.58 , 8.09 , 8.04 , 6.47 , 2.33 . 13C-NMR ; 167.93 (C-7), 167.63 (C-9), 166.79 (C-8), 154.86 (C-2′), 154.81 (C-6′), 147.41 (C-4′), 138.32 (C-2), 134.24 (C-1), 132.57 (C-5), 132.28 (C-6), 131.49 (C-4), 130.80 (C-3), 113.24 (C-5′), 108.20 (C-3′), 21.12 (6′-CH3), 19.14 (4′-CH3). GC-MS  314.2 [M]+ (5), 32.1 (100), 43 (65), 207.1 (20), 77.1 (18), LRMS 304.1 [M + Na]+ for C16H14N2O5, calculated mass 314.3 (Appendixes  63–70). 2-[(2-Nitrophenyl)carbamoyl]benzene-1,5-dicarboxylic Acid ( Z12). Pale brown powder (36% yield) was prepared according to the general procedure from 1,3-dioxo-2-benzofuran-5-carboxylic acid 2 , 2-nitrobenzylamine , and DCM (7 mL) as solvent and purified by recrystallization with DCM. Melting point 204-205°C, UV analysis λmax(logε), 245 (4.26), 300 (3.48). IR (cm−1); 3317, 3062 (COOH), 1290 and 1249 (O-Cacid), 1679 and 1574 (CONH), 1500 (C=C) and 752 (=CH). 1H-NMR  δH 8.21 , 8.12 , 7.75 . 13C-NMR ; 168.41 (C-7), 167.47 (C-9), 165.95 (C-8), 137.34 (C-2), 132.42 (C-1), 131.76 , 129.29 , 128.58 . GC-MS  331.1 [M − H]+ (94), 32.1 (100), 44 (71), 207.1 (67), 244.1 (39), LRMS 333.2 [M − 3H]+ for C15H10N2O7, calculated mass 330.3 (Appendixes  71–78).Z1–3,5,7–12) were evaluated on the following isolates, obtained from the Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital Zaria, Nigeria (ABUTH): Candida stellatoidea, Candida tropicalis, Candida krusei, Candida albicans, Shigella dysenteriae, Salmonella typhi, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, Corynebacterium ulcerans, Streptococcus pyogenes, Staphylococcus aureus, Vancomycin Resistant Enterococci (VRE), and Methicillin Resistant Staphylococcus aureus (MRSA).The test compounds  tubes that showed no growth, on sterile nutrient Agar (OXOID) plates incubated at 38°C for bacteria and 34°C for fungi for 48 h. The MBC/MFC was recorded as the lowest concentration of compounds showing no bacterial/fungal growth at all.Our synthetic approach involved two steps . In the 1H and 13C NMR with application of 2D NMR where necessary.The structures of the compounds were confirmed by the use of  Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococci (VRE), and four fungi. The test compounds had significant zones of inhibitions against all tested organism as compared to the standard drug. Compound Z10 had the best activity among the compounds tested, with zone of inhibition ranging from 32 to 27 mm on the test microbes, but was not able to inhibit five bacteria  .The synthesised compounds were tested against eleven bacteria including two resistance bacteria, Methicillin Resistantyogenes) . This waμg/mL of the test compounds  inhibited the growth of the resistance bacteria (MRSA); the only exception was Z1 which inhibited at 12.5 μg/mL. Three of the compounds  inhibited the resistance VRE at a concentration of 6.25 μg/mL while Z8, Z9, and Z12 inhibited VRE at a higher concentration of 12.5 μg/mL. Generally, other test microbes have shown MIC ranging between 6.25 and 50 μg/mL. These test compounds were also found to be both bactericidal and fungicidal at a concentration of 12.5 μg/mL, as recorded by the minimum bactericidal/fungicidal concentration (MBC/MFC) analysis (Minimum inhibitory concentration (MIC) results  reveal tanalysis . 2-(benzylcarbamoyl)benzene-1,5-dicarboxylic acid (Z10) showed a better activity against resistance bacteria MRSA and VRE and other microbes. Although the test compounds were not as active as the standard drugs, sparfloxacin and fluconazole, the compounds may be employed in situations where there is resistance to antimicrobial drugs. Compound Z10 is therefore a lead candidate in the search for an antimicrobial agent.The different substituted test compounds were successfully synthesised and their structures were confirmed by NMR analysis. Generally,1 for example has its spectral data in Appendix 1-7, where Appendix 1 is the UV Spectrum, Appendix 2 is the 1H NMR spectrum, Appendix 3 is the 13C NMR spectrum, Appendix 4 is the DEPT-90, DEPT-135 and decouple 13C NMR Spectra combined. Appendix 5 shows the COSY spectrum, Appendix 6 is the HSQC spectrum and Appendix 7 is the NOESY spectrum. A similar arrangement is obtainable for the other compounds.The supplementary materials Appendix 1-78 shows the spectroscopy results for the synthesized compounds. Compound Z


Thiosulfonate derivatives based on quinones were synthesized for studying “structure-activity relationship” compounds with an acylated and a free amino-group. Anti-platelet activity of the synthesized compounds was determined and the influence of substituents on the activity of the derivatives was assessed. It is widely recognized that platelet activation and aggregation plays a crucial role in the initiation and progression of ischemic diseases –3. Given2-CH2-S-S-R fragment are also known as the agents that possess antithrombotic behavior [In paper , it was behavior . An inteHerein we report the synthesis of new thiosulfonate derivatives of quinones by the interaction of the corresponding salts of thiosulfoacids that have properties of S,N-binucleophiles with quinone derivatives. Selected salts of thiosulfoacids, as bifunctional molecules, can react with the halogen derivatives of quinones by two alternative ways: both by the thiosulfonate fragment and also by the amino group. The choice of the solvent and reaction conditions predetermine the direction of the substitution.In optimizing parameters of the reaction, we based it on our previous works in the synthesis of thiosulfonate derivatives –20.2a were obtained while carrying out the reaction in THF at room temperature. The general scheme of transformations is shown below:The target of this work was to synthesize derivatives by the S-nucleophilic center. Experimentally, it was found that the highest yields of the S-substituted products using the sodium salt of 4-aminobenzenethiosulfonic acid The formation of the products was monitored by TLC and LC-MS analysis. The NMR spectrum of quinoid derivatives of thiosulfonates showed signals of the amino group in diapason from 6.09–6.34 ppm.4a–d were synthesized. For excluding the possibility of the obtained products of N-alkylation, we directly used the sodium salt of 4-(acetylamino)benzenethiosulfonic acid 2b, which allowed us to carry out the reaction in DMF, and due to its high polarity, caused rapid dissociation of the S-Na bond with the formation of the thiosulfonate anion and -CH2-Hal bond with the formation of the corresponding carbocation.By studying the influence of an acetyl group on the biological activity, the derivatives Platelet function can be regulated by various agonists. A major signaling molecule causing platelet aggregation is adenosine-5’-diphospate (ADP), which activates platelets and is known to play an important role in hemostasis and thrombosis. Moreover, ADP receptor antagonists are widely in clinical use. That is why the activities of new thiosulfonate derivatives of quinones as inhibitors of ADP-induced platelet aggregation were assayed.in vivo, has two types of G protein-coupled receptors in the platelet plasma membrane: P2Y1 and P2Y12 [Adenosine diphosphate (ADP), an important platelet agonist nd P2Y12 . The endnd P2Y12 , 22, 23.3c had the best inhibitory activity against platelet aggregation and the degree of inhibition was proportional to its concentration. Compounds 3d and 4d inhibited ADP-induced platelet aggregation only at high concentration (100 μmol/L). In contrast, the closely structurally related analogs, compounds 4b and 4c, were not effective in inhibiting platelet aggregation in the same assay at both concentrations , the maximal changes in light transmission observed at the ADP (5 μmol/L) was 46±4% (control). The data suggested that the vehicle 1% DMSO had no effect on platelet aggregation induced by ADP . Our restrations . These r50 of derivative 3c, samples of citrated PRP were pre-incubated with vehicle 1% DMSO or with increasing concentrations of the studied compound  for 2 minutes. The aggregation was induced by adding ADP  and we monitored the change of light transmission for 8 minutes, measuring the maximal increase after the addition of the inducer. The concentration at which the test compound showed 50% inhibition was taken as the IC50.To investigate the IC3c (50) – 25 μM.Our results suggest that the degree of inhibition was proportional to the concentration of compound 3c . As show3c on ADP-induced platelet aggregation, samples of PRP were pre-incubated with 50 μM of the studied compound for 0, 2, 3, 5, 20, 40, and 60 minutes at 37°C with continuous stirring. The aggregation was induced by adding 5 μM ADP and monitored for the maximal changes in light transmission.To study the time-dependent inhibitory effect of derivative 3c for 0, 2, 3, or 5 minutes did not differ from each other. On the other hand, the inhibitory effect of the test compound was significantly reduced after more than twenty minutes of incubation. Moreover, after pre-incubation of PRP with 50 μM of the derivative for 60 minutes, the level of aggregation was identical to that in untreated PRP  had considerable activity as an inhibitor of ADP-induced platelet aggregation. We have established that this compound had less activity compared with that of the clinically used one. But, unlike the thienopyridine derivatives, which irreversibly bind to P2Y12 receptors, thereby prolonging bleeding time, compound 3c is a reversible ADP receptor antagonist and perhaps may be safer.In this report, we describe the synthesis and evaluation of new thiosulfonate derivatives of quinones, that were obtained by the interaction of the sodium salt of 4-amino or 4-(acetylamino)benzenethiosulfonic acid 2- fragment predetermined the anti-platelet activity of the investigated products, compared with the results given in [The research of the structure-activity relationships of the synthesized compounds allowed the establishment of certain interesting facts about the nature of active groups that provide anti-platelet activity. Herein, it was revealed that the free amino group and -S-SOgiven in , 15. The1H- and 13C-NMR spectra were determined on a Bruker AC 200 spectrometer. The 1H the 13C chemical shifts are reported from CDCl3 peaks: 1H (7.26 ppm) and 13C (77.16 ppm) and from DMSO peaks: 1H (2.50 ppm) and 13C (39.52 ppm).Melting points were determined on a Büchi capillary melting point apparatus and remain uncorrected. Element analyses were performed by the Centre of Microanalyse of the Aix-Marseille University. Both The following adsorbents were used for column chromatography: silica gel 60 . TLC was performed on 5 cm × 10 cm aluminum plates coated with silica gel 60 F254 (Merck) in an appropriate solvent.1a–d in THF (or DMF) and dissolved in a portion of THF (or DMF) sodium salt of 4-aminobenzenethiosulfonic acid 2a (or 4-(acetylamino)benzenethiosulfonic acid 2b). The solution was stirred and maintained at room temperature for 5 hours. After this time, TLC analysis showed that compound 2a (or 2b) was totally consumed. The reaction mixture was treated with ice water and extracted three times with dichloromethane. The organic phase was washed with water, and then dried over anhydrous sodium sulfate. After evaporation, the product was purified by silica gel chromatography and recrystallized from ethanol and gave corresponding 4-amino-benzenethiosulfonic acid S-(R-methyl) esters 3a–d (or 4-(acetylamino)benzenethiosulfonic acid S-(R-methyl) esters 4a–d).Into a two-necked flask equipped with a nitrogen inlet was added solution of the appropriate methyl halogenated derivative 3a–d and 4a–d were synthesized:By these procedure compounds 1H-NMR  δ, ppm: 3.89 , 4.46 , 6.34 , 6,52 , 6.58 , 7.50 , 7.77–7.81 , 7.99–8.07 . 13C-NMR  δ: 28.21 (CH2), 33.87 (N-CH3), 104.02 (CH), 114.31 (2CH), 126.28 (C), 127.63 (CH), 127.89 (CH), 129.80 (2CH), 131.21 (C), 131.93 (C), 133.47 (C), 134.72 (C), 134.90 (CH), 135.21 (CH), 135.74 (C), 152.38 (C), 174.61 (CO), 181.23 (CO). Calculated for (C20H16N2O4S2), %: C 58.24; H 3.91; N 6.79; O 15.52; S 15.55. Found, %: C 58.37; H 4.02; N 6.73; S 15.72.Eluent: dichloromethane–diethyl ether: 97.5:2.5. Yellow crystals, mp 183–185°C, yield 70%. 1H-NMR  δ, ppm: 3.85 , 4.30 , 6.55 , 7.00 , 7.44 , 7.71–7.78 , 7.91–7.97 . 13C-NMR  δ: 30.49 (CH2), 36.42 (CH3), 112.51 (2CH), 118.29 (C), 123.83 (C), 126.02 (CH), 126.10 (CH), 129.38 (2CH), 130.21 (C), 132.69 (C), 133.23 (CH), 133.30 (CH), 133.51 (CH), 133.59 (C), 154.22 (C), 175.01 (CO), 180.68 (CO). Calculated for (C20H16N2O4S2), %: C 58.24; H 3.91; N 6.79; O 15.52; S 15.55. Found, %: C 58.32; H 3.97; N 6.72; S 15.69.Eluent: dichloromethane–diethyl ether: 90:10. Grey-yellow crystals, mp 204–207°C, yield 32%. 1H-NMR  δ, ppm: 3.70 , 3.83 , 4.33 , 6.22 , 6.60 , 7.39 , 7.55 , 7.79–7.84 , 7.97–8.06 . 13C-NMR  δ: 34.54 (CH2), 56.82 (OCH3), 62.71 (OCH3), 114.03 (2CH), 120.37 (CH), 125.42 (C), 126.44 (CH), 126.61 (CH), 127.13 (C), 129.59 (CH), 133.02 (C), 133.41 (CH), 133.63 (C), 133.84 (2CH), 134.21 (C), 138.29 (C), 151.10 (C), 152.32 (C), 156.17 (C), 182.73 (CO), 183.03 (CO). Calculated for (C23H19NO6S2), %: C 58.84; H 4.08; N 2.98; O 20.45; S 13.66. Found, %: C 58.02; H 4.41; N 2.76; S 12.85.Eluent: dichloromethane–diethyl ether: 90:10. Yellow crystals, mp 215–217°C, yield 30%. 1H-NMR  δ, ppm: 3.64 , 4.32 , 6.12 , 6.62–6.69 , 7.72–7.78 , 7.84–7.88 , 8.04–8.07 . 13C-NMR  δ: 27.31 (2CH2), 61.78 (2CH3), 113.86 (4CH), 122.83 (2C), 126.71 (2CH), 129.40 (2CH), 134.94 (2CH), 135.62 (2C), 135.91 (2C), 135.83 (2C), 152.61 (2C), 154.83 (2C), 182.30 (2CO). Calculated for (C30H26N2O8S4), %: C 53.72; H 3.91; N 4.18; O 19.08; S 19.12. Found, %: C 53.57; H 3.84; N 4.24; S 18.89.Eluent: dichloromethane-diethyl ether: 85:15. Yellow crystals, mp 121–124°C, yield 40%. 1H-NMR  δ: 1.82 , 3.87 , 4.57 , 6.40 , 7.56 , 7.67 , 7.76–7.80 , 7.96–8.02 , 10.13 . 13C-NMR  δ: 24.09 (CH3), 31.03 (CH2), 33.10 (N-CH3), 109.51 (CH), 118.04 (CH), 118.23 (CH), 126.11 (CH), 126.32 (CH), 126.76 (C), 128.09 (2CH), 130.92 (C), 132.94 (C), 133.62 (2CH), 133.71 (C), 135.70 (C), 137.32 (C), 144.31 (C), 168.93 (C(O)N), 175.04 (CO), 179.70 (CO). Calculated for (C22H18N2O5S2), %: C 58.14; H 3.99; N 6.16; O 17.60; S 14.11. Found, %: C 58.37; H 3.78; N 6.26; S 14.23.Eluent: dichloromethane–diethyl ether: 95:5. Yellow crystals, mp 208–211°C, yield 55%. 1H-NMR  δ: 2.07 , 3.87 , 4.44 , 7.15 , 7.68 , 7.78–7.82 , 7.97–8.04 , 10.33 . 13C-NMR  δ: 24.41 (CH3), 31.04 (CH2), 36.40 (N-CH3), 117.56 (C), 118.32 (2CH), 123.78 (C),126.04 (CH), 126.15 (CH), 128.44 (CH), 130.30 (C), 133.01 (CH), 133.19 (C), 133.42 (C), 133.74 (CH), 133.81 (CH), 137.70 (C), 144.21 (C), 169.30 (C(O)N), 175.12 (CO), 180.74 (CO). Calculated for (C22H18N2O5S2), %: C 58.14; H 3.99; N 6.16; O 17.60; S 14.11. Found, %: C 58.43; H 3.82; N 6.37; S 14.03.Eluent: dichloromethane-diethyl ether: 85:15. Yellow crystals, mp 224–227°C, yield 70%. 1H-NMR  δ: 1,97 , 3.70 , 3.82 , 4.43 , 7.40 , 7.68 , 7.77–7.88 , 7.98–8.06 , 10,10 . 13C-NMR  δ: 24.34 (CH3), 34.60 (CH2), 56.67 (OCH3), 62.21 (OCH3), 118.53 (2CH), 121.30 (CH), 126.14 (CH), 126.22 (2CH), 126.61 (C), 128.49 (2CH), 133.40 (C), 133.67 (CH), 134.01 (CH), 134.13 (C), 137.32 (C), 137.96 (C), 144.54 (C), 151.69 (C),155.50 (C), 169.22 (C), 181.44 (CO), 182.31 (CO). Calculated for (C25H21NO7S2), %: C 58.70; H 4.14; N 2.74; O 21.89; S 12.54. Found, %: C 58.83; H 4.02; N 2.67; S 12.62.Eluent: dichloromethane–diethyl ether: 80:20. Orange crystals, mp 207–212°C, yield 30%. 1H-NMR  δ: 2.00 , 3.63 , 4.41 , 7.72–7.85 , 8.03 , 10.37 . 13C-NMR  δ: 24.63 (2CH3), 31.72 (2CH2), 63.01 (2OCH3), 119.04 (4CH), 126.56 (2CH), 128.68 (4CH), 133.75 (2C), 134.50 (2CH), 136.32 (2C), 137.31 (2C), 144.90 (2C), 155.21 (2C), 169.59 (2CO), 181.72 (2CO). Calculated for (C34H30N2O10S4), %: C 54.10; H 4.01; N 3.71; O 21.19; S 16.99. Found, %: C 54.19; H 4.23; N 3.64; S 16.87.Eluent: dichloromethane-diethyl ether: 80:20. Orange crystals, mp 214–217°C, yield 22%. All procedures were conducted at room temperature. Blood was collected from the auricular artery of healthy rabbits into 3.8% citrate in a ratio of 9:1 and then centrifuged at 150 g for 15 min in order to obtain platelet-rich plasma (PRP). The PRP was carefully removed and placed into a plastic tube. Platelet-poor plasma (PPP) was prepared by further centrifugation of the remaining plasma at 1500 g for 40 minutes. Throughout all the experiments, the platelet number was adjusted to 250 x 10-9/L by diluting PRP with PPP.Platelet aggregation in PRP was recorded under constant stirring conditions (500 rpm) at 37°C for 10 min by the aggregometer . The baseline value was set using PRP while PPP served as a full transmittance control. The PRP suspension was incubated with the studied compounds  or with dimethylsulphoxide (DMSO) alone for 2 minutes. The aggregation was induced by adding ADP  and the change of light transmission was monitored for 8 minutes, measuring the maximal increase after the addition of the inducer. Concentrations of ADP leading to maximal aggregation were determined in preliminary experiments. To minimize the effect of DMSO, the solvent, on the aggregation, the final concentration of DMSO was fixed at 1% (v/v). In each experiment, all the samples were tested in triplicate.Results were expressed as the mean±SEM. The difference between groups was analyzed by the standard Student’s t-test. P-values less than 0.05 were considered statistically significant.The authors declare no conflict of interest.


The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6β,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6β,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11β,16β-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15β,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6β, -11β, -14α, -15β, -16β positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC50= 30.3 μg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC50 values between <0.2–10.4 μg/mL. Compound 7 was found to be the most potent inhibitor (IC50 < 0.2 μg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC50 = 14.6–20.0 μg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa , or against PC3 (prostate cancer) cancer cell lines.Microbial transformation of the anti-inflammatory steroid medrysone ( Microbial transformation is an effective tool for structural derivatizations that are difficult to achieve by conventional chemical methods. Microbial systems are also extensively employed in the study of drug metabolism and bioremediation , 2.Steroids are among the most widely marketed pharmaceutical products. Several steroids are used as anabolic, contraceptive, anti-androgenic, anti-inflammatory, and anti-cancer agents. Microbial hydroxylation of steroids is an efficient method for the synthesis of new hydroxysteroids with high stereo- and regio-selectivity, and for study of the steroidal metabolism , 3–5. Th1) by biotransformation. Medrysone  is an anti-inflammatory agent used in ophthalmic treatments. This is the first report of the microbial transformation of medrysone (1) with the filamentous fungi, C. blakesleeana (ATCC 8688a), N. crassa (ATCC 18419), and R. stolonifer (TSY 0471). Fermentation of medrysone with these fungi yielded seven new metabolites 2–8. Various cellular assays, such as phagocyte oxidative burst, T-cell proliferation, and cytokines analysis, were performed on the substrate 1, and metabolites 2–8 to evaluate their anti-inflammatory potential.During the last two decades, we have been focusing on the structural modifications of various classes of steroids in search of their new bioactive analogues –22. The 1) was obtained from Sigma-Aldrich. Precoated TLC plates  were used for thin layer chromatography, ceric sulfate solution was used as staining reagent. Column chromatography was performed on silica gel . Recycling preparative HPLC separation was performed on a JAI LC-908W instrument, equipped with YMC L-80  using MeOH-H2O as the mobile phase, with UV detection at 254 nm. Electron impact mass spectra (EI-MS) and high resolution electron impact mass spectra (HREI-MS) were recorded on JEOL JMS600H mass spectrometer . Electrospray ionization mass spectra (ESI-MS) and high resolution electrospray ionization mass spectra (HRESI-MS) were measured on QSTAR XL mass spectrometer . 1H- and 13C-NMR spectra were recorded on a Bruker Avance 300 and 600 MHz NMR spectrometers  in CDCl3, CD3OD and DMSO-d6. Melting points (m.p.) were determined on Buchi-560  apparatus. Hitachi U-3200  spectrophotometer was used to collect UV spectra (nm). FT-IR-8900  and Bruker VECTOR 22  spectrophotometers were employed to obtain infrared (IR) spectra (cm-1). Digital polarimeter JASCO P-2000  was used to measure optical rotations in chloroform or methanol. Bruker SMART APEX II single-crystal X-ray diffractometer, fitted with CCD detector, was used to collect X-ray data  (84.3), 340 (69), 269 (37), 177 (100), 161 (63), 136 (73), 43 (62); HREI-MS m/z : 358.2128 ; Single-crystal X-ray diffraction data: Empirical formula = C22H30O4, Mr = 358.46, Crystal system: Orthorhombic, space group: P212121, Unit cell dimensions: a = 6.1067(8) Å, b = 13.813(2) Å, c = 23.065(3) Å, Volume: 1945.6(5) Å3, Z = 4, ρcalc = 1.224 Mg/m3, F(000) 776, Crystal size: 0.32 × 0.10 × 0.10 mm, θ range for data collection: 1.72 to 25.50°, Total 11,616 reflections were collected, out of which 3,620 reflections were judged observed (Rint = 0.0509). Final R indices were, R1 = 0.0587 for [I>2σ (I)], wR2 = 0.1333, R indices were R1 = 0.0982, wR2 = 0.1581 for all data, largest difference peak and hole: 0.471 and -0.185 e. Å-3. Crystallographic data for compound 2 was deposited in the Cambridge Crystallographic Data Center (CCDC 1053672).Colorless crystalline solid: m.p.: 209–210°C; c = 0.1, CHCl3); UV (MeOH) λmax nm (log ⊰): 248 (6.0); IR (CHCl3) νmax cm-1: 3479 (OH), 1707 (C = O), 1678 (C = C = O); 1H-NMR : 13C-NMR : m/z : 358 [M+] (4), 315 (100), 175 (5), 149 (7), 123 (6), 85 (8), 43 (39); HREI-MS m/z : 358.2115 .Colorless amorphous solid; c = 0.14, CHCl3); UV (MeOH) λmax nm (log ⊰): 248 (5.9); IR (CHCl3) νmax cm-1: 3423 (OH), 1703 (C = O), 1658 (C = C = O); 1H-NMR : 13C-NMR : m/z  358 [M+] (63), 343 (44), 259 (24), 161 (30), 136 (100), 121 (25), 43 (62); HREI-MS m/z : 358.2126 ; Single-crystal X-ray diffraction data: Empirical formula = C22H30O4, Mr = 358.46, Crystal system: Triclinic, space group: P1, Unit cell dimensions: a = 6.5208(6) Å, α = 97.416(2)°, b = 7.4210(6) Å, β = 101.439(2)°, c = 10.9061(9) Å, γ = 107.064(2)°, Volume: 484.52(7) Å3, Z = 1, ρcalc = 1.229 mg/m3, F(000): 194, Crystal size: 0.55 × 0.38 × 0.30 mm, θ range for data collection: 1.94 to 28.29°. Total 6,591 reflections were collected, out of which 4,741 reflections were judged observed (Rint = 0.0118). Final R indices were, R1 = 0.0461 for [I>2sσ (I)], wR2 = 0.1169, R indices were R1 = 0.0503, wR2 = 0.1213 for all data, largest difference peak and hole: 0.295 and -0.187 e. Å-3. Crystallographic data for compound 4 was deposited in the Cambridge Crystallographic Data Center (CCDC 1402957).Colorless crystalline solid; m.p.: 185–186°C; c = 0.05, CHCl3); UV (MeOH) λmax nm (log ⊰): 248 (6.3); IR (CHCl3) νmax cm-1: 3450 (OH), 1699 (C = C = O); 1H-NMR : 13C-NMR : m/z : 374 [M+] (58), 356 (68), 303 (54), 270 (62), 255 (63), 220 (45), 175 (42), 43 (100); HREI-MS m/z : 374.2068 .Colorless crystalline solid; m.p.: 289–291°C; c = 0.1, CHCl3); UV (MeOH) λmax nm (log ⊰): 247 (5.7); IR (CHCl3) νmax cm-1: 3445 (OH), 1742 (C = O), 1662 (C = C = O); 1H-NMR : 13C-NMR : +m/z: 361.2382 (C22H32O4+H requires 361.2378); Single-crystal X-ray diffraction data: Empirical formula = C22H32O4, Mr = 360.48, Crystal system: Orthorhombic, space group: P212121, Unit cell dimensions: a = 6.0392(8) Å, α = 90°, b = 13.5095(16) Å, β = 90°, c = 23.795(3) Å, γ = 90°, Volume 1941.3(4) Å 3, Z = 4, ρcalc = 1.233 mg/m3, F(000): 784, Crystal size: 0.50 × 0.24 × 0.13 mm, θ range for data collection: 1.71 to 25.49°. Total 11,559 reflections were collected, out of which 2,108 reflections were judged observed (Rint = 0.1006). Final R indices were R1 = 0.0548 for for [I>2siσ (I)], wR2 = 0.0933, R indices were R1 = 0.1153, wR2 = 0.1163 for all data largest difference peak and hole: 0.142 and -0.123 e. Å-3. Crystallographic data for compound 6 was deposited in the Cambridge Crystallographic Data Center (CCDC 1053673).Colorless crystalline solid; m.p.: 229–230°C; c = 0.1, CHCl3); UV (MeOH) λmax nm (log ⊰): 247 (6.0); IR (CHCl3) νmax cm-1: 3402 (OH), 1703 (C = O), 1662 (C = C = O); 1H-NMR : 13C-NMR : +m/z: 361.2374 (C22H32O4+H requires 360.2378).Colorless crystalline solid; c = 0.1, CHCl3); UV (MeOH) λmax nm (log ⊰): 247 (6.0); IR (CHCl3) νmax cm-1: 3431 (OH), 1703 (C = O), 1662 (C = C = O); 1H-NMR : 13C-NMR : m/z : 360 [M+] (17), 342 (9), 225 (9), 177 (16), 136 (55), 105 (33), 91 (61), 55 (100); HREI-MS m/z : 360.2295 ; Single-crystal X-ray diffraction data: Empirical formula = C22 H34 O5, Mr = 378.49, Crystal system: Monoclinic, space group: P21, Unit cell dimensions: a = 9.3482(14) Å, α = 90°, b = 9.4131(14) Å, β = 102.433 Å, γ = 90°, Volume 1035.5(3) Å3, Z = 2, ρcalc = 1.214 mg/m3, F(000): 412, Crystal size: 0.45 × 0.38 × 0.16 mm, θ range for data collection: 1.73 to 25.49°. Total 6,011 reflections were collected, out of which 3,508 were judged observed (Rint = 0.0152). Final R indices were R1 = 0.0378 for [I>2sσ (I)], wR2 = 0.0899, R indices were R1 = 0.0434, wR2 = 0.0936 for all data, largest difference peak and hole: 0.205 and -0.144 e. Å-3. Crystallographic data for compound 8 was deposited in the Cambridge Crystallographic Data Center (CCDC 1053674).Colorless solid; m.p.: 274–275°C; 1 (600 mg/ 20 mL of acetone) was evenly transferred among 40 flasks containing 4 days old culture of R. stolonifer TSY (0471). After 14 days of fermentation, the biomass was separated from the medium by filtration and then washed with dichloromethane. The medium was then extracted with the same solvent i.e. dichloromethane, dried with anhydrous sodium sulfate (Na2SO4), and evaporated to obtain a brown gum (1.4 g). The gum was then subjected to silica gel column chromatography with the mobile phase consisting of petroleum ether and acetone. Three main fractions (1–3) were obtained and purified to acquire compounds 2, 3, and 5 by recycling RPHPLC  (Compound  mL/min) .1 (900 mg/ 60 mL of acetone) was distributed among 60 flasks, containing the fungal culture of N. crassa (ATCC 18419). These flasks were then kept on a shaker at 27°C for 15 days. After fermentation, the medium was filtered and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate (Na2SO4), and evaporated under reduced pressure to obtain brown a gummy material. The crude mass was then subjected to column chromatography using petroleum ether and acetone as the mobile phase. Three main fractions were thus purified to yield compounds 4, 6, and 8 by recycling RPHPLC  (Compound  mL/min) .et al. method with slight modifications  (1), was carried out for the first time. Fermentation of 1 with C. blakesleeana (ATCC 8688a), R. stolonifer (TSY 0471) and N. crassa (ATCC 18419) yielded seven new metabolites 2–8. Previously we have reported the biotransformation of several steroids for the synthesis of new anti-inflammatory compounds  with eight degrees of unsaturation. The IR spectrum showed the presence of hydroxyl (3479 cm-1), ketonic carbonyl (1707 cm-1), and α,β-unsaturated ketonic carbonyl groups (1678 cm-1). The 1H-NMR spectrum  and 2.45  which indicated a ketonic carbonyl at C-11. Furthermore, the appearance of CH3-22 as a singlet (instead of a doublet) indicated the hydroxylation at vicinal C-6. The 13C-NMR spectrum (2J HMBC correlations of H-9 (δ 2.70) and H2-12  with δ 207.9. This supported the presence of a ketonic functionality at C-11. Similarly, 3J HMBC of H-4 (δ 6.00), and 2J correlations of H2-7  with C-6 (δ 71.0) indicated the presence of an OH hydroxyl group at C-6 (d6) between OH (δ 4.88) and H-19 (δ 1.26)  . The str4 supported the molecular formula C22H30O4 [M+ = m/z 358.2126 ] with eight degrees of unsaturation. The IR absorptions at 3423, 1703, and 1658 cm-1 were due to the hydroxyl, ketonic carbonyl, and α,β-unsaturated ketonic carbonyl moieties, respectively. The 1H-NMR spectrum  and 2.48 , showed oxidation of the C-11 OH into a ketonic carbonyl. The 13C-NMR (2J HMBC correlations of H-9 (δ 1.97) with C-11 (δ 207.6) and C-10 (δ 38.6) supported a ketonic group at C-11. Furthermore, the 3J HMBC correlations of H-15 (δ 4.40) with C-13 (δ 46.6) and C-17 (δ 62.0) supported an OH at C-15  , B (C-5—C-10), C (C-8—C-9/C-11—C-14) and D (C-13—C-17). Ring A exists in a half-chair conformation. Trans fused rings B, C and D exist in chair, chair and envelope conformations, respectively. The acetyl substituent attached at C17 adopts a pseudo axial orientation, whereas the OH at C-15 is β   . Single-o-axial) . Compoun5 showed the M+ peak at m/z 374.2068, in agreement with the formula C22H30O5  with eight degrees of unsaturation. The IR spectrum showed absorptions at 3450 and 1699 cm-1 for hydroxyl and α,β-unsaturated ketonic carbonyl moieties, respectively. The 1H-NMR spectrum , and 2.12 , supported the presence of a ketonic carbonyl at C-11. Secondly, the appearance of CH3-22 as a singlet indicated a hydroxylation at the C-6 position. The 13C-NMR spectrum (2J HMBC correlations of H-9 (δ 2.07) and H2-12  with C-11 (δ 212.3) supported the presence of a ketonic carbonyl at C-11. The position of the hydroxyl group at C-6 was inferred from 3J and 2J HMBC correlations of H-4 (δ 5.98) and H2-7  with C-6 (δ 71.2). An OH at C-17 was deduced on the basis of 3J HMBC correlations of H-18 , and H-21 (δ 2.17) with C-17 (δ 90.0) (d6) between OH (δ 4.88) and H-19 (δ 1.25). Similarly, the stereochemistry of the OH at C-17 was deduced as α on the basis of NOESY correlation (DMSO-d6) between OH (δ 5.64) and H-14 (δ 2.07) .The HREI-MS of metabolite spectrum  showed aspectrum  showed t(δ 90.0) . The ste(δ 2.07) . Compoun6 exhibited the [M+H]+ at m/z 361.2382 , 16 a.m.u. greater than that of substrate 1, suggesting the multiple oxidation of the substrate. The IR spectrum showed absorptions at 3445, 1742, and 1662 cm-1 for hydroxyl, ketonic carbonyl, and α,β-unsaturated ketonic carbonyl moieties, respectively. The downfield shift of H-9 (δ 1.85) and the appearance of the C-12 methylene as AB doublets at δ 2.49 , and 2.22 (J12β. 12α = 12.5 Hz) indicated oxidation at C-11  and C-6 (δ 71.1). The presence of new methine OH-containing C-20 (δ 69.4) was deduced from upfield chemical shift of vicinal C-21 (δ 23.4). In the HMBC spectrum, 2J correlations of H-9 (δ 1.85) and H2-12  with C-11 (δ 209.3) supported a ketonic group at C-11. Furthermore, 3J correlation of H-19 (δ 1.60) with C-9 (δ 62.3) also supported C-11 C = O. 3J HMBC correlation of H-4 (δ 6.0), and 2J correlation of H2-7  with C-6 (δ 71.1), were used to place an OH at C-6. 2J and 3J HMBC correlations of H-21 (δ 1.20) with C-20 (δ 69.4) and C-17 (δ 57.0), respectively, were used to deduce the position of an OH-bearing methine at C-20 (6 indicated that the molecule consists of four fused rings i.e. rings A (C-1—C-5/C-10), B (C-5—C-10), C (C-8—C-9/C-11—C-14). and D (C-13—C-17). The stereochemistry at C-20 appeared as S, and the hydroxyl group at C-6 was β oriented , 16 a.m.u. greater than the substrate 1. The IR absorptions appeared at 3402, 1703, and 1662 cm-1 were for the hydroxyl, ketonic carbonyl, and α,β-unsaturated ketonic carbonyl moieties, respectively. The 1H-NMR spectrum  due to the hydroxylation of the substrate 1. The 13C-NMR spectrum (1. An OH group at C-16 was placed on the basis of 3J HMBC correlations between H-16 (δ 4.72) and C-14 (δ 56.4) and C-20 (δ 210.5)  orientation of OH group at C-16 was deduced on the basis of NOESY correlation between geminal H-16 (δ 4.72) and H-17 (δ 2.49). The stereochemistry of the newly generated OH at C-11 was also deduced to be β  on the basis of NOESY correlation between H-11 (δ 4.34) and H-9 (δ 1.10)  . The β ((δ 1.10) . Metabol8 exhibited the M+ at m/z 360.2295 which supported the formula as C22H32O4 , with seven degrees of unsaturation. The IR absorptions at 3431, 1703, and 1662 cm-1 were due to hydroxyl, ketonic carbonyl, α,β-unsaturated ketonic carbonyl moieties, respectively. The 1H-NMR spectrum , and 3.66 (m), due to the hydroxylation at the two sites. The appearance of an additional doublet for H3-21 at δ 1.11  indicated the reduction of the vicinal C-20 ketonic carbonyl into a CH-OH. The 13C-NMR spectrum (3J HMBC correlations of H-15 (δ 4.32) with C-8 (δ 33.8) and C-17 (δ 58.2) further indicated the presence of an OH at C-15. The presence of an OH at C-20 (δ 70.2) was deduced on the basis of 2J and 3J HMBC correlations of H-20 (δ 3.66) with C-17 (δ 58.2) and C-16 (δ 40.8), respectively. 3J HMBC correlation of H-18 with C-12 (δ 58.8) further supported a carbonyl at C-11  based on NOESY correlations between H-15 (δ 4.32) and H-14 (δ 1.62) (i.e. ring A (C-1—C-5/C-10), B (C-5—C-10), C (C-8—C-9/C-11—C-14) and D (C-13—C-17). The stereochemistry at C-20 appeared as R and the hydroxyl group at C-15 was β oriented, respectively  . Single-ectively . The str1), and its metabolites 2–8, different assays, such as phagocyte oxidative burst, T-cell proliferation, and cytokine inhibition assays were employed.To explore the anti-inflammatory effects of medrysone  by using luminol as a probe and zymosan as an activator [6 (IC50= 30.3 ± 8.8 μg/mL), which showed moderate activity against the zymosan-induced oxidative burst in PMNs (The production of reactive oxygen species (ROS) plays a key role in the development of many inflammatory disorders. This includes endothelial dysfunction and tissue injury by an enhanced ROS generation by polymorphonuclear neutrophils (PMNs) at the site of inflammation. In our study the luminol-enhanced chemiluminescence assay was used for the measurement of the production of ROS. Metabolites ctivator –36. The  in PMNs .1), and its metabolites 2–8 were evaluated for the inhibition of T-cell proliferation by using PBMCs, activated with PHA. The results indicated that compounds 1, 4, 5, 7, and 8 possess strong inhibitory activity with an IC50 values between < 0.2 to 10.4 μg/mL. Among them compound 7 was the most potent inhibitor (IC50 < 0.2 μg/mL) of T-cell proliferation. Compounds 2, 3, and 6 were found to be moderately active with an IC50 ranges between 14.6–20.0 μg/mL as compared with the standard drug prednisolone (IC50 < 3.1 μg/mL)  .2–8 were evaluated against the production of TNF-α [1 (IC50= 30.54 ± 1.69 μg/mL), and its metabolite 7 (IC50= 28.6 ± 11.5 μg/mL) were found to be moderate inhibitors of TNF-α production  and cytokines, which perpetuate the inflammatory process and activate the adaptive immune responses. During the current study, compound 6 exhibited moderate inhibitory activity on ROS produced from professional phagocytes, activated with the serum opsonized zymosan (as an antigen) by myeloperoxidase dependent pathway. It also showed moderate inhibition of proliferation of PHA activated T-cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the PHA activated proliferation of T-cells, whereas compounds 2, 3, and 6 were found to be the moderate inhibitors. Compound 7 was found to be the most potent inhibitor of T-cells proliferation from this group. These results indicate that all compounds have the potential to inhibit cellular immune responses and might be useful in suppressing various chronic inflammatory and autoimmune disorders as well as for treatment of transplantation rejection. Among all compounds, the compounds 1, and 7 were also found to inhibit pro-inflammatory cytokine TNF-α produced from LPS activated macrophages. The blockade of TNF-α proved to be beneficial in many pathological conditions including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis. All compounds showed their suppressive effects on various parameters of innate and adaptive immune responses, and can provide valuable insight for the treatment of different chronic inflammatory and autoimmune illnesses.These results have helped us to identify compounds with potential anti-inflammatory activity. Compounds 1 and its metabolites 2–8 were evaluated for their cytotoxicity against HeLa , PC3 (prostate cancer), and 3T3 (mouse fibroblast) cells. All the compounds were found to be non-cytotoxic against above mentioned cell lines.Substrate 1) into several new derivatives 2–8 with C. blakesleeana (ATCC 8688a), N. crassa (ATCC 18419), and R. stolonifer (TSY 0471). Through this study we identified an efficient route towards the synthesis of C-6β, 11β, 14α, 15β, 16β, and 20β oxidation products. Single-crystal X-ray diffraction analyses were performed to unambiguously deduce the structures of metabolites 2, 4, 6, and 8. Among all the metabolites, compound 6 (IC50= 30.3 μg/mL) showed moderate inhibitory activity against the zymosan-induced oxidative burst in human whole blood cells whereas rest of the compounds were found to be inactive. When tested for their effects on proliferation of T-cells, compounds 1, 4, 5, 7, and 8 showed a strong inhibitory activity against these cells. Compound 7 (IC50 < 0.2 μg/mL) was the most potent inhibitor of T-cell proliferation. The compounds 2, 3, and 6 showed moderate levels of inhibition with an IC50 values ranges between 14.6 to 20.0 μg/mL. When tested for their effect on production of pro-inflammatory cytokine, TNF-α, compounds 1, and 7 showed moderate levels of inhibition while remaining compounds showed no inhibitory activity. All the compounds were found to be non-toxic when tested on 3T3 (mouse fibroblast) cells and showed no activity when tested against HeLa , and PC3 (prostate cancer) cell lines. The work presented here can be helpful for the study of in vivo metabolism of medrysone (1), as well as for the identification of new anti-inflammatory agents, based on bio-catalysed structural transformation of various steroids.In conclusion, this is the first report of the fungal transformation of steroidal anti-inflammatory drug medrysone (S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(PDF)Click here for additional data file.


Salvia Mirzayanii was extracted with methanol. Methanol extract was suspended in water and defatted with petroleum ether. The defatted part was then partitioned between ethyl acetate and water. The ethyl acetate partition was chromatographed on a silica gel column to afford several fractions. Lymphocyte proliferation inhibitory assay of the resulted fractions was compared in-vitro and the fraction with more immunosuppressive activity was subjected to more purification to yield three methoxylated flavones: 5,7-dihydroxy,6,4’– dimethoxyflavone(1), 5-hydroxy,6,7,3’,4’–tetramethoxyflavone(2) and 5,3’-dihydroxy,6,7,4’– trimethoxyflavone (3). Aerial parts of Compounds 2 and 3 potently suppressed the proliferation of human blood lymphocytes with IC50 values of 1.3 ± 0.04 μg/mL and 1.3 ± 0.21 μg/mL in comparison with prednisolone as one of the lymphocyte suppressor drugs (IC50=1.45 ± 0.6 μg/mL). In phagocyte chemiluminescence assay, compounds 1 and 3 in peripheral mononuclear cells (PMNCs) exerted suppressive moderate activity against ROS with IC50 of 55.3 ± 0.4 and 36.2 ± 0.7 μg/mL, respectively, while compound 2 showed weak activity with IC50 values more than 100 μg/mL. In conclusion, compounds 2 and 3 have a similar suppressive effect more than compound 1 on PHA-activated lymphocyte proliferation, which might be because of their C-3’ oxidation pattern of ring B. It is indicated that the presence of 3’-OH or 3’-OMe in flavone ring B, caused more anti-proliferation activity than 3’-H. Oxidative burst assay showed more activity for compound 1 which is less methoxylated than others. It also showed more activity for compounds 3 than 2, which differ only in 3’-OH instead of 3’-OMe.  Salvia species to have a wide range of biological activities like antimicrobial, cytotoxic, and radical scavenging activities and are used in the folk medicine of different countries ( S. Mirzayanii Rech. f. & Esfand (Labiateae family) with local name "Morr-e-Porzo" in the Iranian folk medicine is used as the antispasmodic or for the treatment of diabetes , MN-polyamide SC-6  and thin layer chromatography . The structure elucidations were done using different spectral methods, including 1H-NMR, 13C-NMR (BB and DEPT), and EI-MS. The NMR spectra were obtained with Bruker AV-500. The EI-MS spectra were taken with Varian MAT 312 spectrometer.Plant materialS. Mirzayanii was collected from Kerman, Iran. It was identified by plant taxonomist in the Kerman Faculty of Pharmacy, Kerman University of Medical, Sciences, Kerman, Iran and a herbarium voucher specimen was deposited there. Extraction and isolationin-vitro on peripheral blood lymphocytes  to yield compounds 1-3 as the pure compounds. The air dried plant material (2 Kg) was macerated in methanol (10 L) at room temperature for 3 days. The extraction was repeated three times. The extract was concentrated under reduced pressure to achieve a green gummy residue. Methanol extract was suspended in water and defatted with petroleum ether. The defatted part was then partitioned between ethyl acetate and water. The ethyl acetate fraction was chromatographed on normal phase silica gel column using gradient mixtures of dichloromethane: methanol (0 → 100%) to afford several fractions. Lymphocyte proliferation inhibitory assay of the resulted fractions was compared phocytes . The mosphocytes . Obtaine. Light yellow powder. 1H-NMR , δH: 3.72, 3.91 , 6.83  , 6.91 , 6.93 , 7.96 , 10.37 (7-OH), 12.91 (5-OH). 13C-NMR , δC: 56.4 (4'-OMe), 59.9 (6-OMe), 91.5 (C-8), 102.6 (C-3), 105.0 (C-10), 115.9 , 121.1 (C-4'), 128.4 , 131.9 (C-6), 152.0 (C-9), 152.5 (C-5), 158.5 (C-7), 161.2 (C-4'), 164.0 (C-2), 182.1 (C-4). EI-MS m/z (%): 314 (100), 299 (86), 285 (21), 271 (32), 268 (20), 254 (5), 181 (18), 153 (37), 135 (5), 119 (14), 69 (15). 5,7-dihydroxy,6,4'–dimethoxyflavone (1). Light yellow powder. 1H-NMR , δH:3.73, 3.85, 3.88, 3.93 , 6.97  , 7.04 , 7.13 , 7.59 , 7.72 , 12.89 (5-OH). 13C-NMR , δC:55.7 (7-OMe), 55.9 (3'-OMe), 56.4 (4'-OMe), 59.9 (6-OMe), 91.6 (C-8), 103.6 (C-3), 105.1 (C-10), 109.6 (C-2'), 111.7 (C-5'), 120.1 (C-6'), 122.8 (C-1'), 131.9 (C-6), 149.0 (C-3'), 152.0 (C-4'), 152.3 (C-9), 152.6 (C-5), 158.6 (C-7), 163.6 (C-2), 182.2 (C-4). EI-MS m/z (%): 358 (100), 357 (21), 343 (92), 329 (21), 315 (25), 312 (24), 181 (14), 163 (13), 153 (25), 148 (6), 136 (6), 69 (10). 5-hydroxy,6,7,3',4'–tetramethoxyflavone (2). Light yellow powder. 1H-NMR , δH:3.72, 3.86, 3.92 , 6.81  , 6.90 , 7.08 , 7.47 , 7.57 , 9.42 (3'-OH), 12.89 (5-OH). 13C-NMR ,δC:55.8 (7-OMe), 56.4 (4'-OMe), 59.9 (6-OMe), 91.5 (C-8), 103.3 (C-3), 105.1 (C-10), 112.1 (C-5'), 113.1 (C-2'), 118.7 (C-6'), 122.9 (C-1'), 131.9 (C-6), 146.8 (C-3'), 151.2 (C-4'), 151.9 (C-9), 152.6 (C-5), 158.6 (C-7), 163.8 (C-2), 182.1 (C-4). EI-MS m/z (%): 344 (100), 343 (36), 329 (100), 315 (32), 301 (34), 298 (30), 181 (17), 153 (29), 149 (14). 5,3'-dihydroxy,6,7,4'–trimethoxyflavone (3)Lymphocyte proliferation assay2 environment for about 72 hours. Incubation for more eighteen hours after the addition of [3H]-thymidine was done. Then cells were harvested with cell harvester and proliferation percent was determined by the radioactivity count as CPM reading by the Beta-scintillation counter  of the flavones 1-3 in triplicates in supplemented RPMI-1640 containing 5.0 µg/mL phytohemagglutinin (PHA) at 37 ºC in CO counter .Phagocyte chemiluminscence assayThe reactive oxidants (ROS formation) in whole blood during the phagocytosis oxidative burst processes were measured by the luminol-enhanced chemiluminescence assay procedure . BrieflySalvia Mirzayanii with positive reaction to natural product reagent. NMR data (BB and DEPT) in addition to EI-MS molecular ion peak [M]+ at m/z 314, indicated the molecular formula of C17H14O6 consistent with eleven degrees of unsaturation. EIMS spectrum showed m/z 314 as a base ion together with fragment ions of m/z 181 and 131, characteristic retro-Diels-Alder (RDA) fragments for a flavone-skeleton with two hydroxyl and one methoxyl groups on ring A and one methoxyl group on ring B , and 59.9 (6-OMe). The 1H-NMR spectrum showed AA'XX' spin pattern for H-2', 6' and H-3', 5' of ring B at δH 7.95 , 6.92 , two singlets, each one proton at δH:6.91 (H-3) and 6.83 (H-8), in addition to two singlet signals each three protons related to the aryl methoxyl groups at δH: 3.91, and 3.72 (s) and two deshielded protons at δH: 12.91 and 10.37 ppm, which could be assigned as phenolic hydroxyl groups on flavone structure. Comparing the above data with published NMR data in the literature suggested the structure of compound 1 as 5,7-dihydroxy,6,4'–dimethoxyflavone  and EI-MS molecular ion [M]+ at m/z 358. The 1H-NMR spectrum showed ABX spin pattern similar to H-2', 6' and H-5' of ring B in flavones at δH: 7.71 , 7.59 , 7.13 , two singlets at δH:7.04 (H-3) and 6.97 (H-8), in addition to four singlet signals each integrated of three protons related to the aryl methoxyl groups at δH: 3.93, 3.88, 3.85, and 3.73 (s) and one deshielded proton at δH: 12.89 ppm, as 5-OH group , 7.46 , 7.10 , two singlets, each one proton at δH:7.04 (H-3) and 6.97 (H-8), in addition to three methoxyl singlet signals at δH: 3.92, 3.86, and 3.72 (s) and two deshielded protons at δH 12.89, and 9.41 ppm as 5-OH, and 3'-OH groups. Therefore, based on the above data and similar NMR data in the literature, compound 3 was determined as 5,3'-dihydroxy, 6,7,4'–trimethoxyflavone . The results agreed with the immiunoinhibitory activity of other methoxlylated flavonoids like casticin from Vitex agnus-castus with potent suppressive effect on PHA stimulated T-cell  of compounds 1-3. As shown in d T-cell .Effect on phagocyte oxidative burst and ROS production50 values of 55.3 ± 0.4 and 36.2 ± 0.7 μg/mL, while compound 2 showed weak activity with IC50 values more than 100 μg/mL (Compounds 1 and 3 on PMNCs in human whole blood, employing luminol and zymosan for the intracellular oxidative burst studies, exerted suppressive activities against ROS with IC00 μg/mL .Lymphocyte proliferation assay showed that compounds 2 and 3 have similar suppressive effects, but more than compound 1 on PHA-activated lymphocyte proliferation, which might be because of the C-3' oxidation pattern of ring B. It is indicated that the presence of 3'-OH or 3'-OMe in the flavones ring B, caused more anti-proliferation activity than 3'-H. It is agreed with another study on the structure activity requirements for methoxylated flavone cytotoxicity on human tumor cell lines. Oxidative burst assay showed more activity for compound 1, which is less methoxylated than others. It also showed more activity for compound 3 than 2, which differs only in 3'-OH instead of 3'-OMe. It means that 3'-methoxylation reduced phagocyte oxidative activity.


The chemical structures were established based on the spectroscopic analysis. In addition, phellinulin A (1) was subjected to the examination of effects on activated rat hepatic stellate cells and exhibited significant inhibition of hepatic fibrosis.Three new γ-ionylideneacetic acid derivatives, phellinulins A–C ( Phellinus linteus (Hymenochaetaceae) was usually called as “Sangwhang” in traditional Chinese medicines, and it has been utilized extensively as healthy foods or medicines in Asia to prevent various diseases, including cancer, ulcer, bacterial or viral infections and diabetes. In the literature, the fruiting bodies of P. linteus had been reported to possess bioactive compounds and display broadband bioactivities, such as cytotoxic, antioxidant, anti-inflammatory, anti-platelet aggregation, anti-dementia, anti-diabetic, and anti-viral activities D25 −111.6. The positive-mode HR-ESI-MS of 1 showed a pseudomolecular ion peak at m/z 259.1676  constructing the molecular formula as C15H24O2. The IR absorption bands at 1782 and 1643 cm−1 indicated the presence of lactone carbonyl and carbon–carbon double bond functionalities, respectively. The 1H NMR spectrum of 1 displayed three methyls at δ 0.81 , 0.93 , and 1.10 . There were also five methylenes at δ 1.24–1.29 , 1.38–1.42 , 1.54 , 1.64 , 1.75 , 2.03 , 2.12–2.19 , 2.63 ; and three methines at δ 2.12–2.19 , 3.99 , respectively. In the downfield region, there was also two olefinic protons at δ 4.61  and 4.83 . The 13C NMR, DEPT-135 and HMQC spectral data displayed fifteen carbon signals including three methyl groups at δ 16.8, 25.7, 28.2, six methylene groups at δ 23.6, 31.1, 32.6, 36.4, 36.7, 110.0, three methines at δ 37.2, 49.8, 85.4, two quaternary carbons at δ 34.6, 148.0, and one carbonyl at δ 176.6, respectively. In the HMBC spectrum of 1 -methyl)-4-methyl-dihydrofuran-2(3H)-one and named trivially as phellinulin A.In addition, there were NOESY correlations between H-6 and H-7, H-13, CHectively . These N2 was obtained as optically active syrup and [α]D25 −70.4. In the HR-ESI-MS analytical data, 2 displayed a sodiated ion peak at m/z 255.1362  and its molecular formula was determined as C15H20O2 with six degrees of unsaturation which is two more than that of 1. The UV maximum at 260 nm and IR absorption bands at 1744 and 1647 cm−1 indicated the occurrence of an extended α, β-unsaturated carbonyl moiety. The 1H and 13C NMR spectral characteristics also suggested 2 to possess the ionone basic skeleton. The significant differences between 2 and 1 were only two methyl groups at δ 0.84  and 0.92 , and three more olefinic protons at δ 5.86 , 6.24 , and 6.40  could be found in 2. In the HMBC spectrum of 2, there were 2J, 3J-correlations from CH3-14 and CH3-15 to C-1, C-2, and C-6; from CH2-12 to C-9, C-10, and C-11; from H-4 to C-5 and C-13; from H-7 to C-8 and C-9; from H-8 to C-6, C-7, C-9, and C-12; from H-10 to C-11 and C-12; and from H-13 to C-4 and C-6 respectively. These spectral characteristics indicated that 2 is also an ionone derivative as 1. The 3J-HMBC correlation from H-12 to C-11 constructed the formation of a γ-lactone ring between C-11 and C-12. NOE correlations of H-6/CH3-15 and H-7/CH2-12 in the NOESY spectrum determine the relative stereochemistry of 2. The complete assignments of all the proton and carbon signals were furnished with the aid of other two-dimensional spectral analysis and therefore the chemical structure of 2 was constructed as (E)-4-vinyl)furan-2(5H)-one and named trivially as phellinulin B.Compound 3 was yielded as colorless optically active syrup with [α]D25 −84.0. The positive-mode HR-EI-MS of 3 showed a molecular ion peak at m/z 507.3086 corresponding to a molecular formula of C30H44O5. The mass spectrometric data implied the existence of an ionone dimer and it was supported by the 1H and 13C NMR in which showed spectral characteristics similar with the presence of two ionone units. The 1H NMR spectrum of 3 displayed six methyls, six aliphatic methylenes, and six olefinic protons; however, only one set of terminal olefinic methylene was found at δ 4.53  and 4.76 . This indicated that the terminal olefinic methylene in one of the ionone units was replaced by other functional group and it was proved by the occurrence of one oxygenated methylene at δ 3.79  and 3.97 . In the HMBC spectrum of 3, there were 2J, 3J-correlations from CH3-14 and CH3-15 to C-1, C-2, and C-6; from CH3-12 to C-8, C-9, and C-10; from H-4 to C-13; from H-6 to C-1, C-5, C-7, and C-8; from H-7 to C-9; from H-8 to C-6, C-10, and C-12; from H-10 to C-8 and C-12; and from H-13′b to C-4′, C-5′ and C-11, respectively. In addition, there were 2J, 3J-correlations from CH3-14′ and CH3-15′ to C-1′, C-2′, and C-6′; from CH3-12′ to C-8′, C-9′, and C-10′; from H-6′ to C-1′, C-7′, and C-8′; from H-7′ to C-9′; from H-8′ to C-6′, C-10′, and C-12′; from H-10′ to C-8′ and C-12′, respectively. The 3J-HMBC correlation from H-13′a and H-13′b to C-11 (δ 167.1) determined the linkage of dimer to be through oxygen atom between C-11 and C-13′. The 2J-HMBC correlation from H-13′ to oxygenated quaternary carbon C-5′ (δ 73.0) also proved the presence of hydroxy group at C-5′. All the protons and carbons assignment and relative stereochemistry were performed with other 2D spectral analysis and the chemical structure of 3 was unambiguously constructed as shown . Therefore, PLE and the purified compound phellinulin A (1) were further examined for their inhibitory activities on activated rat HSCs (1) exhibited the significant effects against the activated rat HSCs with the inhibition percentage of 67%  and transforming growth factor-1 (TGF-1) can activate the quiescent hepatic stellate cells and transform them to proliferating myofibroblast-like cells to afford hepatic accumulation of extracellular matrix and result in liver fibrosis ,34,35,36rat HSCs  and the e of 67% . Thus th1H and 13C NMR spectra were examined and recorded by a Bruker AV-500 NMR spectrometer . Chemical shifts are expressed as δ values (ppm) using tetramethylsilane as an internal standard. The ESI-MS and HR-ESI-MS were taken on a Bruker Daltonics APEX II 30e spectrometer (positive-ion mode) . The EI and HR-EI mass spectra were measured on a JEOL JMS-700 spectrometer . Column chromatography was conducted on 70–230 mesh and 230–400 mesh silica gels, and pTLC (preparative thin layer chromatography) was performed on Merck precoated Si gel 60 F254 plates , and the spots on TLC was visualized by UV light.Melting points were recorded on a Yanagimoto MP-S3 apparatus  without corrections. Optical rotations were determined by the JASCO DIP-370 digital polarimeter . UV spectra were measured at room temperature with a Hitachi UV-3210 spectrophotometer , respectively. IR spectra were obtained with a Shimadzu FT-IR DR-8011 spectrophotometer . P. linteus was provided and identified from Gene Ferm Biotechnology Co., Ltd. in Taiwan in August 2003. A voucher specimen (Wu 2003010001) has been deposited in the Herbarium of National Cheng Kung University, Tainan, Taiwan.The fermentation cultivated mycelium dried powder of P. linteus  was refluxed with ethanol (4 L × 5 × 4 h). The extract was then filtered with Whatman No. 1 filter paper, and the ethanol extracts were combined and concentrated at 40 °C under reduced pressure to obtain the ethanol extract . PLE was further dissolved in water and partition with chloroform to give chloroform  and water extracts (280 g). The PLEC extract was purified by silica gel column chromatography eluted with n-hexane: ethyl acetate (4:1) to afford six fractions (Frs. 1–6). Fr. 3 was chromatographed on a silica gel column (chloroform: methanol = 29:1) to obtain 1 (21 mg) and 2 (3 mg), respectively. Fr. 5 was isolated by silica gel column chromatography with mixing eluent of 90% chloroform in acetone and further purified by pTLC to result in 3 (1 mg).The dried mycelium powder of D25 −111.6; IR (neat) vmax 2932, 2870, 1782, 1643, 1215, 1150 cm−1; 1H NMR  δ 0.81 , 0.93 , 1.10 , 1.24–1.29 , 1.38–1.42 , 1.54 , 1.64 , 1.75 , 2.03 , 2.12–2.19 , 2.12–2.19 , 2.63 , 3.99 , 4.61 , 4.83 ; 13C NMR  δ 16.8 (C-12), 23.6 (C-3), 25.7 (C-14), 28.2 (C-15), 31.1 (C-7), 32.6 (C-4), 34.6 (C-1), 36.4 (C-2), 36.7 (C-10), 37.2 (C-9), 49.8 (C-6), 85.4 (C-8), 110.0 (C-13), 148.0 (C-5), 176.6 (C-11). ESI-MS m/z (rel. int.) 259 ; HR-ESI-MS m/z 259.1676 .Colorless powder, mp 60–63 °C; [α]D25 −70.4; UV (MeOH) λmax (log ε) 260 (4.71) nm; IR (neat) vmax 2936, 2866, 1744, 1647, 1451, 1150 cm−1; 1H NMR  δ 0.84 , 0.92 , 1.33–1.39 , 1.47–1.51 , 1.55–1.63 , 2.07 , 2.25 , 2.59 , 4.52 , 4.80 , 4.99 , 5.86 , 6.24 , 6.40 ; 13C NMR  δ 23.0 (C-3), 23.8 (C-14), 29.3 (C-15), 34.1 (C-4), 35.7 (C-1), 38.7 (C-2), 58.3 (C-6), 70.5 (C-12), 109.6 (C-13), 114.4 (C-10), 123.2 (C-8), 140.4 (C-7), 148.5 (C-5), 162.0 (C-9), 174.0 (C-11); ESI-MS m/z (rel. int.) 255 ; HR-ESI-MS m/z 255.1362 .Yellow syrup, [α]D25 −84.0; UV (MeOH) λmax (log ε) 264 (4.57) nm; IR (neat) vmax 3499, 2943, 2870, 1694, 1609, 1242, 1150 cm−1; 1H NMR  δ 0.82 , 0.83 , 0.89 , 1.06 , 1.19–1.21 , 1.26–1.37 , 1.49–1.51 , 1.56–1.60 , 1.71–1.78 , 1.79 , 1.97–2.06 , 2.17–2.26 , 2.30 , 2.31 , 2.54 , 3.79 , 3.97 , 4.53 , 4.76 , 5.71 , 5.74 , 6.08 , 6.11 , 6.28 , 6.35 ; 13C NMR  δ 14.2 (C-12′), 14.2 (C-12), 17.4 (C-3′), 22.3 (C-14′), 23.3 (C-3), 23.6 (C-15), 29.4 (C-14), 32.3 (C-15′), 34.4 (C-4), 34.4 (C-1′), 35.7 (C-4′), 35.7 (C-1), 39.0 (C-2), 41.2 (C-2′), 55.3 (C-6′), 58.1 (C-6), 71.4 (C-13′), 73.0 (C-5′), 108.9 (C-13), 117.1 (C-10′), 117.2 (C-10), 135.2 (C-8), 135.5 (C-7′), 136.9 (C-7 and -8′), 149.5 (C-5), 153.6 (C-9), 154.6 (C-9′), 167.1 (C-11), 170.4 (C-11′). EI-MS m/z (rel. int.) 507 ; HR-EI-MS m/z 507.3086 .Colorless syrup, [α]2. HepG2 cells were cultured with Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% FBS for 24 h. Cell viability was examined by MTT assay. Totally 1 × 105 cells were seeded in 24-well plates for 24 hours (h) and made quiescent by cultivation in medium containing 0.2% FBS overnight. After treating with 40 μM of phellinulin A (1) for 72 h, tetrazolium salt mixed isopropanol solution was added to the wells and the resulting mixture was more incubated at 37 °C for 4 h [The immortalized rat myofibroblast cell line HSC-T6 was a gift kindly provided by Dr. Scott L. Friedman . The HSC-T6 cells were cultured in Waymouth medium with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO for 4 h . The opt® 20 (TBST)  four times, the membranes were cultured with the 5% nonfat milk-TBST solution containing peroxidase-labeled anti-rabbit IgG  for 2 h. After washing in TBST five times, enhanced chemiluminescence substrate (ECL)  was used for protein detection. The intensity of each band was quantified using GeneTools Image Software , as GAPDH was used as the internal control. The Western blot experiments were conducted in triplicate.Proteins were isolated on 10% denatured gels and transferred to PVDF membranes and then incubated for 1 h with blocking solution of 5% nonfat milk-TBST solution. The membranes were further immersed in the same solution containing an antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and α-SMA  overnight. After washing with the mixture of tris-buffered saline and TweenThe experimental data were expressed as the mean ± standard deviation (SD) of three parallel measurements .


O-methyl-β-cyclodextrin (DIMEB) and randomly methylated-β-cyclodextrin (RAMEB) were conjugated to propargylated hydroxyethyl starch (HES) by Cu+-catalysed [2 + 3] cycloaddition. The resulting water soluble polymers showed lower critical solution temperatures (LCST) at 52.5 °C (DIMEB-HES) and 84.5 °C (RAMEB-HES), respectively. LCST phase separations could be completely avoided by the introduction of a small amount of carboxylate groups at the HES backbone. The methylated CDs conjugated to the HES backbone exhibited significantly lower cytotoxicities than the corresponding monomeric CD derivatives. Since the binding potentials of these CD conjugates were very high, they are promising candidates for new oral dosage forms of anaesthetic actives.The mono-6-deoxy-6-azides of 2,6-di- Cyclodextrins (CDs), α1→4) linked cyclic oligomers of anhydroglucose, are produced nowadays in industrial scale →4 linked. β-CD, tO-methyl derivative, abbreviated as DIMEB  cycloaddition of methylated derivatives of mono-6-azido-6-deoxy-β-CD 2·8H2O and BaO as the base following the procedure of Szejtli et al. published for the methylation of native β-CD  1364.63, C55H95N3O34, (m/z): [M + Na+ + CH3] 1378.02, C56H97N3O34, (m/z): [M + Na+ + 2CH3] 1392.64, C57H99N3O34, most intense peak (m/z): [M + Na+ + 3CH3] 1406.29, C58H101N3O34, (m/z): [M + Na+ + 4CH3] 1420.20, C59H103N3O34; IR  (cm−1): 3412 (OH), 2930 (CH), 2102 (N3), 1453 (CH), 1361 (OH).Yield: 2.5 g ; DS: 2.2; (3-Propargyloxy-2-hydroxypropyl)-hydroxyethyl starch (2): 3.412 g (18.98 mmol) HES was dissolved in 40 mL 0.1 M NaOH and 1.8 mL (16.70 mmol) glycidyl propargyl ether were added. The temperature was increased to 35 °C and the solution was stirred. After 24 h further 1.8 mL (16.70 mmol) glycidyl propargyl ether were added and stirred for another 16 h. The product was precipitated in 500 mL 2-propanol, filtered and washed with 200 mL 2-propanol. The product was purified by ultrafiltration with water against a polyethersulfone membrane (cut-off: 5 kDa) and freeze-dried.1H NMR  δ 5.68–5.38 , 4.27 , 4.03–3.45 ; IR  (cm−1): 3400.25 (OH), 2925.80 (CH), 2113.83 (-C≡C), 1456.15 (CH), 1365.50 (OH).Yield: 3.61 g ; DS: 0.65; Conjugate of DIMEB and HES 5a: Under an atmosphere of N2 1.6 g (1.19 mmol) 1a was dissolved in 40 mL of degassed DMSO/H2O 1:1 (v/v) and 600 mg (2.4 mmol) 2 and 334 µL (119 µmol) of a solution of sodium ascorbate in water (70 mg/mL) were added. After reaching 50 °C, 211 µL (59 µmol) of a solution of CuSO4·5H2O in water (70 mg/mL) was added. The solution was stirred for 48 h and purified by ultrafiltration with water against a polyethersulfone membrane (cut-off: 5 kDa) and freeze-dried.1H NMR:  δ 8.03 , 5.58 , 5.14 , 3.86–3.07 ; IR  (cm−1): 3400.25 (OH), 2925.80 (CH), 1456.15 (CH), 1365.50 (OH).Yield: 1.67 g ; DS: 0.4; File 11b, 3, 4, 5b and for the oxidation of HES.General methods and experimental procedures for compounds File 21a, 1b, 6, and IR spectra of HES, 2, 5a.NMR spectra of HES,


We aimed to perform microbiological analysis of Gram positive infections in patients infected with HIV (PIH).We performed a retrospective and compared study, between 01 January 2007 – 31 December 2010, in the Infectious Diseases Hospital of Craiova, on two groups of adults patients (Px): group A - 114 PIH and group B - 1442 Px non HIV, which had been diagnosed with non-tuberculosis bacterial infections (BI) with established etiology. For each group we analyzed the incidence, clinical spectrum, etiology and antibiotic susceptibility phenotype of Gram positive infections (GPI). For the statistical analysis we used the Fischer test; threshold values are statistically significant for p<0.05 and relative risk (RR) >2.S aureus – 31 PIH (27.19%) vs. 58 Px (4.92%) , S pneumoniae – 24 PIH (21.05%) vs. 234 Px (16.22%) , Enterococcus spp. – 2 PIH (1.75%) vs. 7 Px (0.49%) (p<0.05), S pyogenes – 2 PIH (1.75%) vs. 77 Px (5.33%) (p<0.05) and S viridans – 2 Px (0.14%) only in group B. The clinical spectrum was dominated by lower respiratory tract infections: 29 PIH (49.15%) vs. 237 Px (69.09%) (p=0.004). Sensitivity to antibiotics: S pneumoniae, group A vs. group B: teicoplanin, linezolid 24 (100%) vs. 234 (100%) (p<0.05), moxifloxacin, 24 (100%) vs. 228 (97.43%) (p<0.05), vancomycin 24 (100%) vs. 228 (97.43%) (p<0.05), erythromycin -18 (75%) vs. 166 (70.94%) (p<0.05), co-trimoxazole 9 (37.5%) vs. 119 (50.84%) (p<0.05), penicillin 10 (41.62%) vs. 92 (39.31%) (p <0.05), ceftriaxone, 21 (87.5%) vs. 198 (84.61%) (p <0.05), rifampicin 18 (75%) vs. 217 (92.73%) (p=0.000) antibiotic sensitivity of S aureus, group A vs. group B (41 strains tested): oxacillin 24 (77.41%) vs. 33 (80.48%) (p>0.05) erythromycin, 13 (41.93%) vs. 21 (51.21%) (p<0.05), co-trimoxazole 8 (25.80%) vs. 29 (70.73%) (p<0.05), amikacin 29 (93.54%) vs. 38 (92.68%) (p<0.05), vancomycin 31 (100%) vs. 39 (95.12%) (p<0.05), linezolid 31 (100%) vs. 41 (100%), clindamycin 29 (93.54%) vs. 38 (92.68%) (p<0.05).Incidence: group A vs. group B: BI-114 PIH (22.66%) vs. 1442 Px (8.7%) , GPI-59 (51.75%) vs. 378 (26.21%) (p<0.0001); S pneumoniae to co-trimoxazole, rifampicin and of S aureus to co-trimoxazole.Gram positive infections are more common in PIH versus Px non-HIV; the clinical spectrum is dominated by acute respiratory tract infections, regardless of HIV status. HIV infection was associated with high risk of resistance of


Structures of these compounds were identified by 1D and 2D NMR as well as MS. The absolute configurations of compounds 1, 2, and 4 were determined by comparing CD spectra and optical rotations. Among the isolated compounds, 1–3 were novel and subsequently named trogopterins A, B, and C, respectively. Likewise, compound 4 was isolated from nature for the first time. Cytotoxic activities of compounds 1–4 were evaluated. Compounds 1–3 exhibited moderate cytotoxic activities against HL-60 cells with IC50 values of 34.77–45.68 μM.Seven compounds, including three neolignans  Trogopterus were selected since organic extracts from this material were found to exhibit potent cytotoxic activities in our preliminary study. Trogopterus feces, called “Wulingzhi”, are dried excrements of Trogopterus (T.) xanthipes Milne-Edwars (Petauristidae) that are known as complex-toothed flying squirrels to eat branches, leaves, and fruits of pine trees D25 +20.2, which was similar to that of (S)-2,3-diphenyl-1-propanol (+107°). Thus, C-8′ was determined to have S configuration.Compound mpound 2  was simiskeleton . HMBC copropanol –19. Comp3 was obtained as colorless oil. Its molecular formula was found to be C18H20O3 based on HREIMS (m/z 300.1360), requiring nine degrees of unsaturation. The 1H NMR spectrum , 6.43 , and 6.51 ] indicated the presence of a 1,3,4-trisubstituted benzene ring. Extension of the spin systems was confirmed by HMBC correlations (3 methylene protons H2-7 (δ 2.76) with C-1 (δ 139.1), C-2 (δ 132.4), C-6 (δ 115.3), and C-7′ (δ 48.0) indicated that the first spin system was connected to C-1 (δ 139.1) of the 1,3,4-trisubstituted benzene ring while C-8 was connected to C-7′ of the second spin system. HMBC correlations of H-7′ with C-6 (δ 115.3) together with the unsaturation requirement allowed the connection of C-8′ with C-2 to form a cyclohexene moiety. Furthermore, splitting patterns of the protons in the other aromatic moiety  and downfield-shifted chemical shift of C-3′ (δ 158.5) indicated that the 3-hydroxy-1,3-disubstituted benzene ring was attached to the C-7′ carbon. In this way, the planar structure of compound 3 was elucidated as shown in Compound spectrum  indicateelations . HMBC co3 was deduced based on NOESY data and comparison with interproton distances calculated by MM2 with ChemDraw. A strong NOESY correlation between H-7′ and H-8′ indicated that the phenyl group at C-7′ and the hydroxymethylene group at C-8′ are on the same face of the molecule -6-hydroxy-2-(3-hydroxyphenyl)-1,2,3,4-tetrahydronaphthalene-1,3-diyl)dimethanol and named trogopterin C.A careful modeling study considering all the possible conformers of compound 4 was isolated as light brown oil. The 1H and 13C NMR spectra contained signals similar to those of compound 2 except for those corresponding to the methyl propanoate moiety in compound 2. Based on the 1H, 13C, HMQC, and HMBC data, compound 4 had an aromatic proton instead of the methyl propanoate found in compound 2. Thus, compound 4 was determined to be ((R)-3,3'-diphenol as previously reported D25 −15.4 similar to (R)-2,3-diphenyl-1-propanol D25 +33.62 ; CD (MeOH) 234 (∆ε −2.53), 255 (∆ε +0.92), 327 (∆ε +2.12); 1H and 13C NMR , see 3OD, H-#→C-#) H-2→C-4, C-6, C-7, and C-8'; H-6→C-2, C-4, C-5, and C-7; H-7→C-1, C-2, C-8, and C-9; H-8→C-1, C-7, and C-9; H-2'→C-1', C-3', C-4', C-6', and C-7'; H-5'→C-1', C-3', and C-4'; H-6'→C-2', C-4', and C-7'; H-7'→C-3, C-4, C-1', C-2', C-6', C-8', and C-9'; H-8'→C-3, C-4, C-1', C-7', and C-9'; H-9'a→C-3, C-7', and C-8'; H-9'b→C-3, C-7', and C-8'; 9-OCH3→C-9; 4'-OCH3→C-4'; 1H,1H COSY correlations  H-7↔H-8, H-5'↔H-6', H-7'↔H-8', H-8'↔H-9'a and H-9'b, H-9'a↔H-8' and H-9'b; NOESY correlations  H-2↔H-8' and H-9', H-6'↔H-5', H-7', and H-8'; H-7'↔H-2', H-8', and H-9'; H-8'↔H-2' and H-9'; HREIMS (m/z): calcd for C20H22O7, 374.1365; found, 374.1364.Trogopterin A (2): light brown oil; [α]D25 +20.2 ; 1H and 13C NMR  spectroscopic analysis, see d5, H-#→C-#) H-2→C-4, C-6, and C-7; H-4→C-2, C-6, and C-8'; H-6→C-2, C-4, and C-7; H-7→C-1, C-2, C-6, and C-9; H-8→C-1 and C-9, H-2'→C-4' and C-6', H-4'→C-2' and C-6', H-5'→C-1', C-3', C-4', and C-6'; H-6'→C-2' and C-4', H-7'a→C-5, C-1', C-2', and C-6'; H-7'b→C-5, C-1', C-2', and C-6'; H-8'→C-2, C-3, C-4, and C-1'; H-9'→C-3, C-7', and C-8'; 9-OCH3→C-9; 1H,1H COSY correlations  H-7↔H-8, H-4'↔H-5', H-5'↔H-6', H-7'a↔H-7'b and H-8', H-7'b↔H-8', H-8'↔H-9'; HREIMS (m/z): calcd for C19H22O5, 330.1467; found, 330.1469.Trogopterin B (3): light brown oil; [α]D25 −14.8 ; 1H and 13C NMR  spectroscopic analysis, see 3OD, H-#→C-#): H-1→C-5 and C-9, H-3→C-1 and C-5, H-4→C-2, C-7, and C-10; H-6→C-12, H-7→C-4, C-5, C-6, C-10, C-12, C-1', C-2', and C-6'; H-8→C-6 and C-7, H-9→C-7, C-8, and C-11; H-11a→C-7, C-8, and C-9; H-11b→C-7, C-8, and C-9; H-12a→C-6, C-7, and C-8; H-12b→C-6, C-7, and C-8; H-2'→C-6, C-3', C-4', and C-6'; H-4'→C-2' and C-6', H-5'→C-1' and C-3', H-6'→C-6, C-2', and C-4'; 1H-1H COSY correlations  H-6↔H-7, H-12a, and H-12b; H-8↔H-9, H-11a, and H-11b; H-4'↔H-5', H-5'↔H-6'; NOESY correlations : H-7↔H-8, H-8', and H-9'a; H-8↔ H-7', H-8', H-9'a, and H-9'b; H-5'↔H-4' and H-6'; H-6'↔H-5', H-7', and H-8'; H-7'↔H-8' and H-9'a; H-8'↔H-9'a and H-9'b; H-9'a↔H-9'b; HREIMS (m/z): calcd for C18H20O3, 300.1362; found, 300.1360.Trogopterin C (4): light brown oil; [α]D25 −15.4 ; 1H and 13C NMR  spectroscopic analysis, see 3OD, H-#→C-#) H-2→C-3, C-6, and C-7; H-4→C-2, C-3, and C-6; H-5→C-1 and C-3; H-6→C-2, C-4, and C-7; H-7→C-1, C-2, C-6, C-8, C-9, and C-1'; H-8→C-1, C-7, and C-9; H-9a→C-1, C-7, C-8, C-1', C-2', and C-6'; H-9b→C-1, C-7, C-8, C-1', C-2', and C-6'; H-2'→C-9, C-3', C-4', and C-6'; H-4'→C-2', and C-6'; H-5'→C-1' and C-3', H-6'→C-9, C-2', and C-4'; HREIMS (m/z): calcd for C15H16O3, 244.1099; found, 244.1105.3-(1-hydroxy-3-(3-hydroxyphenyl)propan-2-yl)phenol (2 incubator. Cytotoxic activity was measured using a modified MTT assay [4 cells per well) and incubated at 37 °C in a 5% CO2 incubator. The test sample was dissolved in DMSO for the final sample concentrations to be adjusted from 5.0 to 150 µM by diluting with the growth medium. Each sample was prepared in triplicate. The final DMSO concentration was adjusted to be below 0.1%. After standing for 24 h, 10 µL of the test sample was added to each well. The same volume of DMSO alone was added to the control wells. The medium was removed after 48 h of incubation with the test samples, and 10 µL of MTT were then added to the each well . After an additional 4 h of incubation, the resulting formazan crystals were dissolved in 150 mL of DMSO and the optical density (O.D.) was measured at 570 nm. The IC50 value was defined as the concentration of sample that reduced absorbance by 50% relative to the vehicle-treated control. Adriamycin was used as a positive control.Different types of cancer cells  were maintained in RPMI 1640 medium supplemented with L-glutamine, 10% fetal bovine serum (FBS), and 2% penicillin–streptomycin. The cells were cultured at 37 °C in a 5% COTT assay . Viable File 1NMR and MS spectra of compounds.