N6-benzyladenosine derivative (nL) provided the [Pt(ox)(nL)2]∙1.5H2O oxalato (ox) complexes 1–5, involving the nL molecules as monodentate coordinated N-donor ligands. The complexes were thoroughly characterized by elemental analysis, multinuclear  and two dimensional NMR, infrared and Raman spectroscopy, and mass spectrometry, proving their composition and purity as well as coordination of nL through the N7 atom of the purine moiety. Geometry of [Pt(ox)(4FL)2] (5) was optimized at the B3LYP/LANLTZ/6-311G** level of theory. The complexes were screened for their in vitro cytotoxicity against two human cancer cell lines (HOS osteosarcoma and MCF7 breast adenocarcinoma), but they did not show any effect up to the concentration of 50.0 µM  or 20.0 µM (compounds 3–5).A one-step synthetic procedure using the reaction of potassium bisplatinate(II) with the corresponding  N-donor ligands have been published to date. Cleare and Hoeschele reported the in vivo cytotoxicity of a platinum(II) dichlorido complex with the adenosine ligand against mice sarcoma 180 cells 2− decomposition after several hours), the preparation of the title compounds was quite time-consuming . The products (yields >80%) were simply collected by filtration and washed by distilled water and methanol with no further purification needed (as proved by 1H-NMR). The empirical formulas [Pt(ox)(nL)2]·1.5H2O platinate(II) as the starting salt allow us to perform the syntheses at higher temperature H2O 1–5; , followei.e., N6-benzyladenosine derivatives and oxalate dianion) involved in the structures of 1–5 were detected by IR and Raman spectroscopy . In connection with the fact that IR and Raman peaks characteristic for the corresponding vibrations were found within the same regions, the below given data represent the results obtained by both methods . The presence of the oxalate dianion is proved by the maxima of νas(C=O) at 1,669–1,714 cm−1 and νs(C–O) at 1,366–1,371 cm−1 + molecular peak was detected only in the ESI+ mass spectra of 1, the adducts whose masses correspond to [{Pt(ox)(nL)2}+Na]+ and [{Pt(ox)(nL)2}+K]+ were found in the spectra of all the studied complexes measured in the 200–1,300 m/z range (nL+H]+ fragments. Negative-mode electrospray mass spectrometry (ESI-) detected [{Pt(ox)(nL)2}–H]− molecular peaks of 1–5, and the peaks of deprotonated nL ligands, [nL–H]−, were also observed in the case of 3–5 (nL)(nL')}–H]− (nL' symbolizes the appropriate adenine derivative without ribose [Although the [{Pt(ox)(/z range . The ESIe of 3–5 . Next onL')}–H]− , where nt ribose .m/z contain the intensive peaks belonging to the [{Pt(nL)3}–H]+ species with several times higher intensity as compared with the above-discussed [{Pt(ox)(nL)2}+H]+, [{Pt(ox)(nL)2}+Na]+ and [{Pt(ox)(nL)2}+K]+ ones. The MS2 spectra of this species contained the series of peaks, which should be associated with the formation of the following species: [{Pt(nL)3]–H]}+, [{Pt(nL)2(nL')}–H]+, [{Pt(nL)(nL')2}–H]+, [{Pt(nL)2}–H]+, [{Pt(nL')3}–H]+, [{Pt(nL)(nL')}–H]+, [{Pt(nL')2}–H]+ and [{Pt(nL)}–H]+.The above-mentioned fragmentation of the studied complexes through the cleavage of the ribose substituent was also observed in the positive-ion spectra  Figure . Concret1H–1H gs-COSY, 1H–13C gs-HMQC, 1H–13C gs-HMBC and 1H–15N gs-HMBC experiments . The 1H, 13C and 15N signals of free nL molecules were shifted in the appropriate spectra of 1–5 due to their coordination to the Pt(II) atom (see Synthesis section). Focusing on the adenine moiety, we summarized the coordination shifts  in nL ligands through the N7 atom of the adenine moiety. Concretely, the 15N-NMR |Δδ| values calculated for the N7 atom equalled ca 113 ppm, while those of the other nitrogen atoms did not exceed 7 ppm  atom led to the considerable shifts (|Δδ| > 3 ppm) of the C5 and C8 atoms adjacent to the mentioned N7 atom.All the signals belonging to hydrogen, carbon and nitrogen atoms of both types of ligands involved in the structures of the studied complexes were found in appropriate NMR spectra and they were interpreted by means of ed 7 ppm ; Table 11H-NMR, the C8–H signal of 1–5 was strongly shifted downfield. However, the Δδ values of N6–H were found to be comparable or, in the case of 1, even higher as compared with those of the mentioned C8–H protons. This is most likely the consequence of the mutual steric hindrance of both the coordinated nL molecules combined with intramolecular interaction of the N6–H proton.In the case of 13C-NMR spectra of the studied complexes. The 195Pt chemical shifts equal ca −1,690 ppm (for the detailed information see the section Synthesis).The single signal, proving the symmetric bidentate coordination of the oxalate dianion to the Pt(II) atom, was detected at 165.53–165.81 ppm in the 1–5 suitable for a single crystal X-ray analysis were performed, but all of them were unsuccessful. That is why the structural aspects of the studied complexes, particularly the complex 5 as a representative one 2] (5) contains tetracoordinated central Pt(II) atom with two monodentate adenosine-based ligands coordinated through their N7 atoms and one bidentate oxalate dianion -9-isopropyladenine (L1) [5 differ from those crystallographically determined. The N7–Pt–N7A bond angle formed by both the 4FL molecules coordinated to the metal centre 2(ado)2](ClO4)2∙3.5H2O (see 4FL in the case of this work) on the N7–Pt–N7A bond angle value or, in other words, on the degree of square-planar geometry deformation. A dihedral angle formed by the purine rings is another relevant parameter describing the molecular structure of the studied complex. We compared the value of 70.6° calculated for [Pt(ox)(4FL)] (5) with the mentioned cis-[Pt(NH3)2(ado)2]2+ complex (82.9(3)°) and with the platinum(II) oxalato complex [Pt(ox)(L1)]∙2DMF with differently substituted adenine-based ligands (83.1(6)°).The structure of , K2(ox)·H2O and solvents were purchased from Sigma‑Aldrich Co.  and Acros Organics Co.  and they were used as received. K2[Pt(ox)2]∙2H2O was prepared according to the literature  in D2O (found at 0 ppm) and 1H–15N gs-HMBC experiments against the residual signals of DMF adjusted to 8.03 ppm (1H) and 104.7 ppm (15N). The splitting of proton resonances in the reported 1H spectra is defined as s = singlet, d = doublet, t = triplet, q = quartet, br = broad band, m = multiplet. Hydrolysis studies: the DMF-7d/H2O (4:1 v/v) solution of the representative complex 5 was carried out by means of 1H and 195Pt NMR after on the fresh solution, and after 24 and 48 h of standing at laboratory temperature.5, whose built model  was optimized by the ORCA software package . The cells were cultured according to the ECACC instructions and they were maintained at 37 °C and 5% CO2 in a humidified incubator. The cells were treated with nL, 1–5 and cisplatin (applied up to 50 μM) for 24 h, using multi-well culture plates of 96 wells. In parallel, the cells were treated with vehicle  and Triton X-100  to assess the minimal  and maximal  cell damage, respectively. The MTT assay was measured spectrophotometrically at 540 nm . The data were expressed as the percentage of viability, when 100% and 0% represent the treatments with DMF and Triton X-100, respectively. The cytotoxicity data from the cancer cell lines were acquired from three independent experiments (conducted in triplicate) using cells from different passages.The human breast adenocarcinoma MCF7 (ATCC HTB-22) and human osteosarcoma HOS (ATCC CRL-1543) cancer cell lines were supplied from European Collection of Cell Cultures. 5 and cisplatin were carried out on MCF7 human cancer cells. The cells were seeded in 100-mm tissue culture dishes  and incubated overnight (37 °C and 5% CO2 in a humidified incubator). After that the cells were treated with 5 , cisplatin  or with the pure medium (control) for 24 h. The cells were washed with PBS (2 × 2 mL), harvested by trypsin treatment, collected and centrifuged in PBS. The supernatants were discarded after centrifugation and pellets were digested in hydrochloric acid using a microwave system . The platinum content was determined by ICP-MS . The obtained values were corrected for adsorption effects.The cellular accumulation experiments (performed in triplicate) of 2[Pt(ox)2]∙2H2O (0.25 mmol) dissolved in a minimum volume of hot distilled water was added to 0.5 mmol of the appropriate N6-benzyladenosine derivative (nL) dissolved in a minimum volume of hot methanol. The reaction mixture was stirred at 60 °C for five days and another two days at laboratory temperature. Slow evaporation of methanol led to the formation of white to light yellow products, which were filtered off and washed by distilled water (5 mL) and methanol (5 mL). The obtained complexes 1–5 were dried at the temperature of 40 °C and stored in desiccator over silica gel.Ketc.) in combination with different techniques  did not lead to the formation of single-crystals suitable for single-crystal X-ray analysis.The recrystallization of the products from different organic solvents platinum(II) sesquihydrateBis{N6-(2-methoxybenzyl)adenosine)-κN7}. C38H42N10O14Pt∙1.5H2O; white solid; yield 75%; 1H-NMR (DMF-d7): δ = 9.24 , 8.90 , 8.35 , 7.24 , 7.23 , 7.03 , 6.79 , 6.12 , 5.82 , 5.46 , 5.38 , 4.92 , 4.78 , 4.38 , 4.15 , 3.92 , 3.85 , 3.74  ppm; 13C-NMR (DMF-d7): δ = 165.59 (C-ox), 157.17 (C-11), 153.90 (C-6), 152.98 (CH-2), 147.98 (C-4), 143.62 (CH-8), 128.09 (CH-13), 127.26 (CH-15), 126.54 (C-10), 120.41 (CH-14), 117.04 (C-5), 110.33 (CH-12), 90.37 (CH-16), 86.92 (CH-19), 74.70 (CH-17), 70.71 (CH-18), 61.68 (CH2-20), 55.30 (CH3-11'), 39.64 (CH2-9) ppm; 15N NMR (DMF-d7): δ = 90.64 (NH-6), 127.63 (N-7), 178.16 (N-9), 223.88 (N-3), 235.84 (N-1) ppm; 195Pt NMR (DMF-d7): δ = −1691.25 ppm; ESI MS (30 eV): m/z = 1096.6 (M + K), 1080.2 (M + Na), 1058.3 (M + H), 1056.2 (M − H), 388.3 (2OMeL + H); Anal. Calc.: C, 42.1; H, 4.2; N, 12.9. Found: C, 42.0; H, 4.0; N, 12.9%.2O,O')platinum(II) sesquihydrateBis{N6-(4-methoxybenzyl)adenosine)-κN7}. C38H42N10O14Pt∙1.5H2O; white solid; yield 70%; 1H-NMR (DMF-d7): δ = 9.19 , 8.93 , 8.35 , 7.32 , 6.82 , 6.09 , 5.80 , 5.37 , 4.84 , 4.74 , 4.35 , 4.13 , 3.86 , 3.83 , 3.77  ppm; 13C-NMR (DMF-d7): δ = 165.53 (C-ox), 158.70 (C-13), 153.76 (C-6), 152.66 (CH-2), 147.88 (C-4), 143.47 (CH-8), 131.01 (C-10), 128.53 , 116.89 (C-5), 113.70 , 90.23 (CH-16), 86.79 (CH-19), 74.66 (CH-17), 70.58 (CH-18), 61.56 (CH2-20), 54.87 (CH3-11´), 43.55 (CH2-9) ppm; 15N-NMR (DMF-d7): δ = 95.48 (NH-6), 127.80 (N-7), 178.15 (N-9), 223.74 (N-3), 236.18 (N-1) ppm; ESI MS (30 eV): m/z = 1096.2 (M + K), 1080.2 (M + Na), 1056.1 (M − H), 388.1 (4OMeL + H); Anal. Calc.: C, 42.1; H, 4.2; N, 12.9. Found: C, 42.0; H, 4.0; N, 12.9%.2O,O')platinum(II) sesquihydrateBis{N6-(4-methylbenzyl)adenosine)-κN7}. C38H42N10O12Pt∙1.5H2O; white solid; yield 80%; 1H-NMR (DMF-d7): δ = 9.22 , 8.98 , 8.35 , 7.27 , 7.07 , 6.10 , 5.60 , 5.40 , 5.30 , 4.87 , 4.76 , 4.37 , 4.14 , 3.86 , 3.75 , 2.28  ppm; 13C-NMR (DMF-d7): δ = 165.68 (C-ox), 153.88 (C-6), 152.86 (CH-2), 148.03 (C-4), 143.62 (CH-8), 136.26 (C-10), 136.18 (C-13), 129.08 , 127.24 , 117.04 (C-5), 90.39 (CH-16), 86.95 (CH-19), 74.77 (CH-17), 70.76 (CH-18), 61.72 (CH2-20), 43.95 (CH2-9), 20.44 (CH3-13') ppm; 15N-NMR (DMF-d7): δ = 94.91 (NH-6), 128.00 (N-7), 178.08 (N-9), 223.84 (N-3), 236.40 (N-1) ppm; 195Pt-NMR (DMF-d7): δ = −1688.92 ppm; ESI MS (30 eV): m/z = 1064.1 (M + K), 1048.2 (M + Na), 1024.3 (M − H), 372.1 (4MeL + H), 370.2 (4MeL − H); Anal. Calc.: C, 43.4; H, 4.3; N, 13.3. Found: C, 43.3; H, 4.0; N, 13.4%. 2O,O')platinum(II) sesquihydrateBis{N6-(2-chlorobenzyl)adenosine)-κN7}. C36H36N10Cl2O12Pt∙1.5H2O; white solid; yield 80%; 1H-NMR (DMF-d7): δ = 9.36 , 9.11 , 8.35 , 8.48 , 8.38 , 7.29 , 7.15 , 6.17 , 5.87 , 5.49 , 5.40 , 5.00 , 4.80 , 4.42 , 4.18 , 3.90 , 3.79  ppm; 13C-NMR (DMF-d7): δ = 165.81 (C-ox), 153.84 (CH-2), 152.82 (C-6), 148.13 (C-4), 143.63 (CH-8), 136.28 (C-10), 131.93 (C-11), 129.15 (CH-15), 128.39 (CH-12), 127.84 (CH-13), 127.32 (CH-14), 117.11 (C-5), 90.26 (CH-16), 86.82 (CH-19), 74.77 (CH-17), 70.61 (CH-18), 61.60 (CH2-20), 41.97 (CH2-9) ppm; 15N-NMR (DMF-d7): δ = 88.32 (NH-6), 127.63 (N-7), 178.18 (N-9), 224.86 (N-3), 235.91 (N-1) ppm; 195Pt-NMR (8DMF-d7): δ = −1697.63 ppm; ESI MS (30 eV): m/z = 1104.3 (M + K), 1088.3 (M + Na), 1064.1 (M − H), 392.2 (2ClL + H), 390.3 (2ClL − H); Anal. Calc.: C, 39.5; H, 3.6; N, 12.8. Found: C, 39.2; H, 3.1; N, 12.6%.2O,O')platinum(II) sesquihydrateBis{N6-(4-fluorobenzyl)adenosine)-κN7}. C36H36N10F2O12Pt∙1.5H2O; white solid; yield 65%; 1H-NMR (DMF-d7): δ = 9.24 , 9.06 , 8.36 , 7.49 , 7.11 , 6.12 , 5.87 , 5.47 , 5.14 , 4.93 , 4.76 , 4.38 , 4.15 , 3.84 , 3.75  ppm; 13C-NMR (DMF-d7): δ = 165.76 (C-ox), 163.02, 160.61 (C-13), 153.81 (CH-2), 152.48 (C-6), 148.00 (C-4), 143.61 (CH-8), 135.45 (C-10), 129.65, 129.57, 129.24, 129.16 , 116.97 (C-5), 115.11, 115.08, 114.90, 114.87 , 90.28 (CH-16), 86.84 (CH-19), 74.75 (CH-17), 70.62 (CH-18), 61.60 (CH2-20), 43.69 (CH2-9) ppm; 15N-NMR (DMF-d7): δ = 93.84 (NH-6), 127.52 (N-7), 178.12 (N-9), 224.03 (N-3), 235.88 (N-1) ppm; 195Pt-NMR (DMF-d7): δ = −1688.68 ppm; ESI MS (30 eV): m/z = 1071.9 (M + K), 1056.2 (M + Na), 1032.2 (M − H), 376.3 (4FL + H), 374.3 (4FL − H); Anal. Calc.: C, 40.8; H, 3.7; N, 13.2. Found: C, 40.3; H, 3.4; N, 13.1%.The IR and Raman spectroscopy results can be found in the Supplementary Materials.1–5 with the general formulae [Pt(ox)(nL)2]·1.5H2O was synthesized and characterized by relevant methods. The absence of single-crystals suitable for a crystallographic study was compensated by a detailed NMR study involving the multinuclear  and two-dimensional NMR spectroscopy, which clearly proved the monodentate N7-coordination mode (Δδ = ca −113 ppm for N7 in 15N-NMR spectra) of N6-benzyl-adenosine-based ligands and bidentate coordination of the oxalate dianion. One of the main goals of the work was to find whether the platination of N6-benzyladenosine derivatives affect in vitro cytotoxicity of the final [Pt(ox)(nL)2]·1.5H2O complexes 1–5 against HOS osteosarcoma and MCF7 breast adenocarcinoma human cancer cell lines. The results of the testing revealed that although usually very sensitive cell lines to the action of platinum complexes have been used, all the complexes have been found as inactive up to the concentrations of 50.0 µM (for compounds 1 and 2) and 20.0 µM , given by their limited solubility. The in vitro cytotoxic inactivity of the studied complexes corresponds with low cellular accumulation (ca 50-fold lower than cisplatin) as observed on the representative complex 5, which was moreover found  as resistant to the hydrolytic processes.A series of five platinum(II) oxalato complexes


H-1,5-benzodioxepin-2-one (5a) and its five derivatives with methoxy groups in different positions of A and B rings (5b–f), were synthesized from corresponding flavanones. Three of the obtained lactones  have not been previously described in the literature. Structures of all synthesized compounds were confirmed by complete spectroscopic analysis with the assignments of signals on 1H and 13C-NMR spectra to the corresponding atoms. In most cases, lactones 5a–f exerted an inhibitory effect on the growth of selected pathogenic bacteria , filamentous fungi , and yeast . The broadest spectrum of activity was observed for unsubstituted lactone 5a, which was particularly active against filamentous fungi and yeast. Lactones with methoxy groups in the 3′ (5c) and 4′ (5d) position of B ring were more active towards bacteria whereas lactone substituted in the 7 position of the A ring (5e) exhibited higher antifungal activity. In most cases, the introduction of lactone function increased the activity of the compound compared to its flavonoid precursors, chalcones 3a–e, and flavanones 4a–f.Six γ-oxa-ε-lactones, 4-phenyl-3,4-dihydro-2 Staphylococcus aureus strains are resistant to methicillin. A growing number of diseases, such as tuberculosis, pneumonia, or salmonellosis, are becoming difficult to treat as antibiotics become less effective, which leads to longer hospital stays, increasing medical costs and mortality. Another serious problem are fungal diseases, which affect over a billion people. For example, 3,000,000 cases of chronic pulmonary aspergillosis, about 700,000 cases of invasive candidiasis, over 10,000,000 cases of fungal asthma, and about 1,000,000 cases of fungal keratitis have been noticed annually. Early diagnosis allows the application of proper therapy; incorrect treatment may cause complications, including serious chronic illness or even death  × H2O in 10% H2SO4.Analytical thin layer chromatography was carried out on silica gel-coated aluminum plates (DC-Alufolien Kieselgel 60 FGas chromatography (GC) analysis was performed on an Agilent Technologies 6890N instrument equipped with an autosampler, split injection (5:1), and Flame Ionization Detector (FID) detector using a DB-5HT column (30 m × 0.32 mm × 0.10 µm) with hydrogen as the carrier gas. For the analysis of compounds, the following temperature program was applied: Injector 200 °C, detector (FID) 280 °C, column temperature: 140 °C, 140–340 °C (rate 30 °C /min), and 340 °C (hold 1 min).3 solutions on a Bruker Avance II 600 MHz Bruker spectrometer  with signals of residual solvent  as references for chemical shifts. Infrared spectroscopy (IR) spectra were determined using a Mattson IR 300 Thermo Nicolet spectrophotometer using KBr pellets or as neat. High-resolution mass spectra (HRMS) were recorded on a spectrometer Waters ESI-Q-TOF Premier XE  by the electron spray ionization (ESI) technique.Nuclear magnetic resonance (NMR) spectra were recorded in CDClThe melting points (uncorrected) were determined on a Boetius apparatus.3a–e are given below:A mixture of 2′-hydroxyacetophenone (1 equiv) and the corresponding benzaldehyde (1 equiv) was heated at reflux in 50 mL of 2% KOH methanolic solution and a drop of water. When the substrates reacted completely , the reaction mixture was transferred to the flask containing 50 mL of water, 80 mL of 1M HCl, and 50 g of crushed ice. The resulting solution was gently mixed with a glass rod until the precipitate was formed (2–3 min). The precipitate was then filtered under reduced pressure and washed with 30 to 40 mL of water and recrystallized from ethanol. The yields of the reaction and physical and spectral data of the obtained 2′-hydroxychalcones 2′-Hydroxychalcone (3a): Obtained from 2′-hydroxyacetophenone  and benzaldehyde ; yield 14.6 g (86%); yellow crystals; mp 77 °C (lit. 75–78 °C +: 293.0790, found: 293.0792.4-(3′-Methoxyphenyl)-3,4-dihydro-2H-1,5-benzodioxepin-2-one (5c): Obtained from 3′-methoxyflavanone 4c ; yield 2.68 g (67%); dense oil; 1H-NMR  δ: 3.09 , 3.15 , 3.82 , 5.69 , 6.91 , 6.94 , 6.95 , 7.07 , 7.15–7.19 , 7.31 ; 13C-NMR  δ: 38.66 (C-3), 55.45 (OCH3), 83.45 (C-4), 112.00 (C-2′), 114.55 (C-4′), 118.41 (C-6′), 120.57 and 125.84 (C-7 and C-8), 124.38 (C-9), 126.68 (C-6), 130.01 (C-5′), 140.28 (C-1′), 145.30 (C-5a), 145.73 (C-9a), 159.99 (C-3′), 167.56 (C-2); IR : 1773, 1490, 1437, 1268, 1211, 1035, 765. HRMS: calcd for C16H14O4[M + Na]+: 293.0790, found: 293.0794.4-(4′-Methoxyphenyl)-3,4-dihydro-2H-1,5-benzodioxepin-2-one (5d): Obtained from 4′-methoxyflavanone 4d ; yield 4.20 g (79%); white crystals; mp 108–109 °C  δ: 3.09 , 3.12 , 3.82 , 5.69 , 6.90–6.92 , 6.90–7.18 , 7.28–7.29 ; 13C-NMR  δ: 38.54 (C-3), 55.46 (-OCH3), 83.39 (C-4), 114.22 (C-3′ and C-5′), 120.46, 124.52, 125.77 and 126.60 , 127.77 (C-2′ and C-6′), 130.73 (C-1′), 145.13 (C-5a), 145.87 (C-9a), 160.21 (C-4′), 167.78 (C-2); IR : 1770, 1488, 1437, 1258, 1210, 1030, 766.–122 °C, ); 1H-NMR7-Methoxy-4-phenyl-3,4-dihydro-2H-1,5-benzodioxepin-2-one (5e); Obtained from 7-methoxyflavanone 4e ; yield 4.01 g (69%); white crystals, mp 71–73 °C  δ: 3.09–3.15 , 3.75 , 5.72 , 6.58 , 6.71 , 7.10 , 7.37–7.42 ; 13C-NMR  δ: 38.64 (C-3), 55.89 (-OCH3), 83.50 (C-4), 109.63 (C-6), 110.79 (C-8), 120.87 (C-9), 126.27 (C-2′ and C-6′), 128.96 (C-3′ and C-5′), 129.19 (C-4′), 138.82 (C-1′), 139.25 (C-9a), 145.92 (C-5a), 157.90 (C-7), 168.07 (C-2); IR : 1768, 1500, 1470, 1260, 1211, 1027, 703.72–74 °C ); 1H-NMR8-Methoxy-4-phenylo-3,4-dihydro-2H-1,5-benzodioxepin-2-one (5f): Obtained from 6-methoxyflavanone 4f ; yield 2.36 g (74%); white crystals; mp 122–123 °C; 1H-NMR  δ: 3.09 , 3.15 , 3.80 , 5.67 , 6.67 , 6.74 , 6.93 , 7.37–7.39 ; 13C-NMR  δ: 38.60 (C-3), 55.94 (-OCH3), 83.70 (C-4), 105.98 (C-9), 111.63 (C-7), 124.58 (C-6), 126.36 (C-2′ and C-6′), 128.90 (C-3′ and C-5′), 129.13 (C-4′), 138.64 and 138.71 (C-1′ and C-5a), 146.27 (C-9a), 157.32 (C-8), 167.65 (C-2); IR : 1766, 1502, 1468, 1445, 1260, 1236, 1028, 764, 704. HRMS: calcd for C16H14O4[M + Na]+: 293.0790, found: 293.0790.Escherichia coli PCM 2560, Bacillus subtilis B5, Staphylococcus aureus D1; filamentous fungi: Fusarium graminearum 109, Aspergillus niger XP, Alternaria sp., and yeast Candida albicans KL-1. All strains originated from the collection of the Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences.Antimicrobial tests were carried out using the bacteria strains: 6 cells mL−1. The bacteria were cultivated for 48 h in a liquid medium composed of nutrient broth (Biocorp) 15 g · L−1 and glucose 10 g · L−1. Yeast and fungi were cultured in YPG medium containing 10 g · L−1 of yeast extract, 10 g · L−1 of bacteriological peptone, and 10 g · L−1 of glucose for 48 and 96 h, respectively. Tested compounds were added to the cultures as 0.1% solutions in 10 μL of dimethyl sulfoxide (DMSO) (w/v). The cultures were carried out for 2 to 3 days at 30 °C  or 25 °C (filamentous fungi), under constant agitation. The optical density of the cell suspensions were measured automatically at the 560-nm wavelength at 30-min intervals. Each culture was performed in three replicates. The results . The working volume in the wells of the Bioscreen plate was adjusted to 200 μL, containing 180 μL of culture medium and 10 μL of cell or spore solution. The final density of the cultures was 1 × 10 results  were ana5a–f containing methoxy groups with different substitution patterns in the A and B ring inhibited the growth of selected bacteria, filamentous fungi, and yeast. The most sensitive strains were S. aureus, F. graminearum, and Alternaria sp. whereas the most resistant one turned out to be A. niger. The broadest spectrum of activity was observed for unsubstituted lactone 5a, which was a particularly strong inhibitor of fungi and yeast. The activity and selectivity of the methoxy-substituted lactones depended on the substitution pattern of the A and B rings. A-ring substitution with a methoxy group in position 7 improved the activity and selectivity against fungal strains and yeast whereas substitution at position 8 increased the antibacterial activity. Lactones possessing methoxy groups in the 3′ and 4′ position of the B ring were highly active against bacteria whereas the compound with the methoxy group at the 2′ position was a strong inhibitor of F. graminearum and Alternaria sp. In most cases, the activity of the compounds was improved after introduction of the lactone function into the molecule compared to their flavonoid precursors, chalcones 3a–e and/or flavanones 4a–f. The most spectacular example was 4′-methoxy-substituted lactone 5d, which exhibited significantly higher activity against all bacterial strains than both its flavonoid precursors. Unsubstituted lactone 5a was a much stronger inhibitor of filamentous fungi and yeast than the starting chalcone 3a whereas the activity of 7-methoxy lactone 5e was significantly higher than flavanone 4e towards these microorganisms. The γ-Oxa-ε-lactone scaffold derived from the flavanone skeleton is a good candidate for the screening of compounds with high antimicrobial activity. Our results indicate that future studies on the antifungal flavanone-derived lactones should focus on compounds substituted in the A ring whereas a candidate for an antibacterial drug will probably be a derivative substituted in the B ring.It was shown that γ-oxa-ε-lactones


Their structures were demonstrated by spectral analyses along with 1D and 2D nuclear magnetic resonance (NMR) techniques and mass spectrometry (MS). The cytotoxicity of the compounds was also tested by the 3--2,5-diphenyltetrazolium bromide (MTT) method on the cell lines Hela, MCF-7, BT-549 and MDA-MB-231. Vernoniamyoside A, vernoniamyoside B, and vernonioside B2 showed cytotoxicity towards BT-549 cell lines. Vernoniamyoside C, vernoniamyoside D and vernoamyoside D showed different levels of cytotoxic activities.In the present study, four new steroidal saponins, namely vernoniamyoside A–D ( Vernonia amygdalina Del. from the family Asteraceae is distributed throughout tropical Africa, especially in West Africa. It has received considerable scientific interest due to the observation that adult chimpanzees with malaria returned to normal activity after chewing the extract of the bitter juice of this species + , has 11 degrees of unsaturation. In the 1H NMR spectrum of compound 1, two olefinic proton signals at δH 5.40  and 5.62 , two angular methyl singlets at δH 0.52  and 0.87 , two methyl doublets at δH 0.83 , 0.91 , and one methyl singlet 2.15  were observed. The 13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra indicated that compound 1 contained 35 carbon signals, including five methyls (at δC 13.8 (C-18), 19.4 (C-19), 17.1 (C-26), 17.0 (C-27) and 29.2 (C-29)), eight methines , 133.1 (C-8), 143.9 (C-9), and 117.6 (C-11)), eight quaternary carbons  and 214.2 (C-16) and a lactone carbon signal at δC 177.3 (C-21)), and 14 methylenes. Particularly, an obvious carbonyl signal at δC 214.2, corresponding to δH 2.63 , 2.98 (H-20), 2.02 and 2.34 (H-15), in 1H-detected heteronuclear multiple-bond correlation (HMBC) was observed, suggesting a carbonyl group is connected to C-16. The 1H and 13C NMR analysis  stigmastane-type steroid derivative, with the same skeleton as vernonioside A3 + ; + ; + ; + ; [α3OH); UV (DMSO): λmax: 252.0, 208.8 nm; IR (KBr) vmax: 3735, 3421, 2933, 2361, 1750, 1716, 1558, 1507, 1024 cm−1; 1H and 13C NMR data: see White amorphous powder; HR-ESI-MS 1H and 13C NMR data comparable to published data [13H NMR : δH 5.33  and 5.51 ,0.44 , 0.83 , 0.84 , 0.91  and 2.16 . 13C NMR : δC 33.7 (C-1),29.3 (C-2), 76.3 (C-3), 34.2 (C-4), 38.7 (C-5), 29.8 (C-6), 121.9 (C-7), 135.1(C-8), 143.8 (C-9), 35.6 (C-10), 118.8 (C-11), 40.7 (C-12), 42.4 (C-13), 48.1 (C-14), 34.0 (C-15), 73.5 (C-16), 59.1 (C-17), 12.8 (C-18), 19.4 (C-19), 39.4 (C-20), 177.8 (C-21), 27.4 (C-22), 80.4 (C-23), 83.8 (C-24), 33.4 (C-25), 17.2 (C-26), 17.0 (C-27), 213.5 (C-28), 29.4 (C-29), 100.9 (C-1′), 73.5 (C-2′), 76.7 (C-3′), 70.1 (C-4′), 76.7 (C-5′), 61.2 (C-6′).White amorphous powder; hed data . 13H NMR1H and 13C NMR data: comparable to published data [13H NMR : δH 5.36  and 5.47 ,0.48 , 0.83 , 0.85 , 0.88 , 1.33  and 3.12 . 13C NMR : 35.2 (C-1), 29.7 (C-2), 77.1 (C-3), 34.9 (C-4), 38.9 (C-5), 29.9 (C-6), 121.8 (C-7), 135.7 (C-8), 143.7(C-9), 36.0 (C-10), 118.6 (C-11), 41.1 (C-12), 43.1 (C-13), 48.5 (C-14), 34.9 (C-15), 76.7 (C-16), 54.9 (C-17), 14.3 (C-18), 19.7 (C-19), 47.9 (C-20), 98.4 (C-21), 80.2 (C-22), 90.2 (C-23), 81.3 (C-24), 31.7 (C-25), 17.5 (C-26), 18.4 (C-27), 112.4 (C-28), 17.5 (C-29), 101.4 (C-1′), 75.5 (C-2′), 80.2 (C-3′), 72.9 (C-4′), 77.2 (C-5′), 61.6 (C-6′), 48.2 (OCH3).White amorphous powder; hed data . 13H NMR1–4 (1 mg each) were dissolved in 2 M HCl (5.0 mL) and stirred at 90°C for 4 h. The reaction solution was neutralized with NH4OH and partitioned between EtOAc and H2O. The residue was obtained from the water layer under vacuum distillation and dissolved in pyridine (1 mL), which was added to 0.1 M l-cysteine methyl ester hydrochloride in pyridine (1 mL). After being heated to 60 °C for 1 h, the mixture was added to phenyl isothiocynate in pyridine (1 mL) and stirred at 60 °C for an additional 1 h. After removal of the solvent, the residue was dissolved in MeOH and analyzed by HPLC, with the mobile phase CH3CN-H2O  containing 0.1% formic acid; the flow rate was 1 mL/min. The standard d-glucose derivative was prepared in the same way. We then compared the retention times of sugar derivatives obtained from compounds with standard d-glucose derivatives (d-glucose: 19.5 min).Compounds 1–6 was tested on cell lines MCF-7, BT-549, MDA-MB-231, and Hela via the MTT method. Cells were seeded into 96-well microplates at 100 μL per well  supplemented with 10% fetal calf serum and 1% penicillin-streptomycin solution, while BT-549 was cultured in Roswell Park Memorial Institute (RPMI) . After 48 h, we added MTT (0.5 mg/mL) dissolved in 100 μL of fetal calf serum per well. After 4 h, we removed the MTT reagent and added 150 μL DMSO to each well. Doxorubicin was used as the positive control. The sample without test compounds was used as the negative control. Absorbance was measured at 490 nm in an automated microplate reader. All experiments were performed in triplicate. Inhibition was calculated via the following equation:test sampleA is the test sample absorbance, blankA is the blank absorbance, negative controlA is the negative control absorbance.The cytotoxicity of compounds 1–4), together with the two known steroidal saponins vernoamyoside D (5) and vernonioside B2 (6) from V. amygdalina. Of these, 1, 2, and 6 showed an excellent cytotoxicity on BT-549 cell lines in the cytotoxicity activity assay (1 and 2 were selective for different tumor cell lines. Our results indicate that substances 1 and 2 were toxic to BT-549 cell lines. Further studies should consider the screening of more types of tumor cells. We obtained four new steroidal saponins, namely vernoniamyoside A–D (ty assay , as expe1–4 is represented by the highly oxidized groups in the side chain, which may be associated with their cytotoxic expression. Saponin 1 has a strong cytotoxicity to BT-549 cell lines, including two ketones and one ester group. It is assumed that the cytotoxicity of saponin 1 may be derived from the C=O group. This group, present in the highly oxidized side chains of saponins 2–4, may provide a basis for the cytotoxicity for BT-549. However, the different inhibitory effects may be due to the relationships between the different structures and spatial configurations. The specific mechanisms and verifications offer a new direction for further experiments.The novelty of the saponins V. amygdalina, are mainly hypoglycemic steroidal saponins. However, it is essential to separate polysaccharide steroidal saponins, which always exist in ethyl acetate and n-butanol extraction, from V. amygdalina. In addition to the steroidal saponins, V. amygdalina contains a series of active substances such as sesquiterpene lactones and flavonoids. Therefore, the separation and identification of these chemical components should be one of the most promising trends in the study of V. amygdalina.The separated steroidal saponins from the dichloromethane extraction of V. amygdalina in anti-tumor research, with a potential value in the treatment of cancer.Our results provide a scientific basis for the use of


Transition-metal-free synthesis of 4-pyrones via TfOH-promoted nucleophilic addition/cyclization of diynones and water has been developed. This transformation is simple, atom economical and environmentally benign, providing rapid and efficient access to substituted 4-pyrones. The gen2,6-Di-p-tolyl-4H-pyran-4-one (2b) ; found 277.1219.one (2b) . The gen2,6-Bis(4-methoxyphenyl)-4H-pyran-4-one (2c) ; found 309.1115.one (2c) . The gen2,6-Bis(4-(tert-butyl)phenyl)-4H-pyran-4-one (2d) ; found 361.2153.one (2d) . The gen2,6-Bis(4-fluorophenyl)-4H-pyran-4-one (2e) ; found 285.0716.one (2e) . The gen2,6-Bis(4-pentylphenyl)-4H-pyran-4-one (2f). The general procedure was used with 1,5-bis(4-pentylphenyl)penta-1,4-diyn-3-one  and water (1 mL). The crude obtained was purified by column chromatography (petroleum ether/ethyl acetate = 5:1 to 2:1) to afford the product as a yellow solid ; m.p. 66.7–67.9 °C; 1H-NMR  δ 7.76 , 7.32 , 6.77 , 2.70–2.66 , 1.69–1.61 , 1.36–1.33 , 0.90  ppm; 13C-NMR  δ 180.5, 163.5, 146.9, 129.2, 128.9, 125.9, 110.7, 35.8, 31.4, 30.8, 22.5, 13.9 ppm; IR (KBr): 3032, 2956, 2929, 2857, 1717, 1649, 1609, 1419, 1380, 1186, 944, 849, 649 cm−1; HRMS (m/z) (APCI): calcd. for C27H33O2 389.2482 [M + H+]; found 389.2466.2,6-Bis(4-ethylphenyl)-4H-pyran-4-one (2g) ; found 305.1532.one (2g) . The gen2,6-Di-m-tolyl-4H-pyran-4-one (2h) ; found 277.1219.one (2h) . The gen2,6-Dipropyl-4H-pyran-4-one (2i) ; found 181.1221.one (2i) . The gen2,6-Dicyclopropyl-4H-pyran-4-one (2j) ; found 177.0908.one (2j) . The gen2-Phenyl-6-propyl-4H-pyran-4-one (2k) ; found 215.1065.one (2k) . The gen2-Phenyl-6-(p-tolyl)-4H-pyran-4-one (2l) ; found 263.1061.one (2l) . The gen2-(4-Methoxyphenyl)-6-phenyl-4H-pyran-4-one (2m) ; found 279.1013.one (2m) . The gen2-(4-Fluorophenyl)-6-phenyl-4H-pyran-4-one (2n). The general procedure was used with 1-(4-fluorophenyl)-5-phenylpenta-1,4-diyn-3-one  and water (1 mL). The crude obtained was purified by column chromatography (petroleum ether/ethyl acetate = 5:1 to 2:1) to afford the product as a yellow solid ; m.p. 145.5–150.6 °C; 1H-NMR  δ 7.89–7.82 , 7.56–7.51 , 7.22 , 6.82 , 6.77  ppm; 13C-NMR  δ 180.1, 165.6, 163.6, 163.4, 162.5, 131.5, 131.3, 129.2, 128.1 , 127.6, 125.9, 116.4 , 111.3  ppm; IR (KBr): 3061, 2924, 1659, 1505, 1508, 1417, 1449, 1388, 1232, 1162 cm−1; HRMS (m/z) (APCI): calcd. for C17H12FO2 267.0823 [M + H+]; found 267.0813.2-Cyclopropyl-6-phenyl-4H-pyran-4-one (2o) ; found 213.0908.one (2o) . The gen2-(2-Chlorophenyl)-6-phenyl-4H-pyran-4-one (2p) ; found 283.0513.one (2p) . The gen2-(4-Fluorophenyl)-6-(4-methoxyphenyl)-4H-pyran-4-one (2q) ; found 297.0913.one (2q) . The gen2-Phenyl-4H-pyran-4-one (2r) ; found 173.0603.one (2r) . The gen1 (0.5 mmol), TfOH (1 equiv.), and D2O (1 mL) in a 15 mL test tube was stirred at 100 °C for 36 h, and monitored periodically by TLC. Upon completion, the reaction mixture was diluted with water (5 mL) and extracted with ethyl acetate (3 × 5 mL). The combined organic layers were washed with water and brine, dried over MgSO4 and filtered. The solvent was removed under vacuum. The residue was purified by flash column chromatography  to afford 4-pyrone 2a-d  as a yellow solid; m.p. 116.1–119.5 °C; 1H-NMR  δ 7.90–7.83 , 7.55–7.51 , 6.84   .1a (0.5 mmol), TfOH (1 equiv.), and H2O18 (1 mL) in a 15 mL test tube was stirred at 100 °C for 36 h, and monitored periodically by TLC. Upon completion, the reaction mixture was diluted with water (5 mL) and extracted with ethyl acetate (3 × 5 mL). The combined organic layers were washed with water and brine, dried over MgSO4 and filtered. The solvent was removed under vacuum. The residue was purified by flash column chromatography  to afford 4-pyrone 18-2aO (78%) (The reaction mixture of 2a (78%) .1a (5 mmol), TfOH (1 equiv.) and H2O (10 mL) in a 50 mL round-bottom flask was stirred at 100 °C for 36 h, and monitored periodically by TLC. Upon completion, the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with water and brine, dried over MgSO4 and filtered. The solvent was removed under vacuum. The residue was purified by flash column chromatography  to afford 4-pyrone 2a (53%) (The reaction mixture of 2a (53%) .2O. Water is a cheap, green and readily available staring material, which converted to the desired 4-pyrone products via a nucleophilic addition/cyclization/ dehydrogenation process. The operational simplicity, good yields, and environmentally benign nature of this method make it an attractive route to 4-pyrones. Further studies on the applications of 4-pyrones in drug design are currently ongoing in our laboratory.We have developed a simple and efficient transition-metal-free method for the synthesis of substituted 4-pyrones from diynones and H


Aconitum kusnezoffii Reichb. On the basis of spectral analysis, these three compounds were determined to be 1,15-dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline, benzoylaconine and aconitine.Three diterpenoid alkaloids, including an unreported compound, were isolated from the roots of  Aconitum kusnezoffii Reichb., belonging to the genus Aconitum L., is distributed in the Xinjiang, Sichuan and Jilin provinces of China. Although recently there were some reports on flowers +). The 1H-NMR spectroscopic data of compound 1 showed three groups of absorptions for a benzoyl moity at δH 7.47 , 7.61 , and 7.96 ; N-ethyl protons at δH 2.72 , 2.74 , and 1.14 ; and four O-methyl protons at δH 3.28  3.29 , 3.30 , and 3.82 . The 13C-NMR and DEPT spectra of 1 revealed the presence of 32 carbons, which were assignable to a tertiary methyl group at δC 12.2 (C-2), an oxygenated methylene group at δC 76.3 (C-18), an oxygenated quaternary carbon at δC 77.5 (C-8), two quaternary carbons at δC 43.6 (C-4) and 51.2 (C-11), four methylene groups at δC 31.8 (C-12), 32.6 (C-2), 48.5 (C-1ʹ) and 49.3 (C-19), two carbonyl groups at δC 166.0 (C-1ʺ) and 211.8 (C-16) and aromatic hydrocarbon signals at δC 128.7 (C-4ʺ), 129.1 (C-2ʺ), 129.7 (C-3ʺ) and 133.8 (C-5ʺ), respectively. After further analysis of the spectroscopic data, compound 1 was identified as a neoline-type diterpenoid alkaloid [Compound d 51.2 C-, four me1H–1H COSY, gHSQC and gHMBC spectra allowed the complete assignment of the chemical shifts of compound 1  respectively in compound 1, and δC 35.0, 38.7, 81.9 and δH 4.12  respectively in chasmanine, which suggested that C-3, C-15 and C-16 in compound 1 belong to an oxygen-containing group. In the gHMBC spectrum, the correlations between H-18 and C-3, and between H-15 and CH3O-15 were important to confirm the presence of oxygen groups at C-3 and C-15. Further analysis of the 13C NMR data suggested that C-16 in compound 1 could be a carbonyl group, and not methoxyl group. The gHMBC correlations from C-1ʺ to H-14 and H-3ʺ confirmed the position of a benzoyl group. In addition, it is noteworthy that the chemical shift of H-14 in compound 1 was more deshielded than the same proton in chasmanine as the result of the steric effect of the carbonyl group at C-16. On the basis of the above spectral data, compound 1 was determined to be 1,15-dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline  signal. DEPT, g1H-1H COSY, gHSQC, and gHMBC experiments were carried out with the pulse sequences given by Bruker. LC-MS were performed on a UPLC instrument  coupled with a ZQ 2000 mass spectrometer  using an XTerra MS C18 2.1 × 150 mm column  and a C18HCE 4.6 × 150 mm column (self-made). A Waters Auto-Purification Factory was used in this study and consisted of a sample inJector (Waters 2777 sample manager), a passive splitter, a compensation pump (515 HPLC pump), an eight-channel UV detector (MUX-UV 2488), a four-channel MS detector (Micromass ZQ2000), four Waters 2525 binary gradient modules, and four Waters 2757 sample managers using XTerra MS C18 19 × 150 mm  and C18HCE 10 × 150 mm (self-made) columns. Data were collected using a MassLynx workstation.IR spectra were recorded using a Thermo Nicolet Nexus spectrophotometer. 1D and 2D NMR spectra were obtained on Bruker AV400; Aconitum kusnezoffii Reichb. were collected in YiLi, Xinjiang Province, China, in July 2008. The herb was authenticated by Xi Rong He, Institute of Medication, Xiyuan Hospital of China Academy of Traditional Chinese Medicine. A voucher specimen (CAOWU 200807) was deposited at the School of Chemical Engineering, Dalian University of Technology.Roots of 2O followed in sequence by 30%, 50% and 100% EtOH . The EtOH fractions were separated by column chromatography on silica gel eluted with petroleum, petroleum/EtOAc , EtOAc and EtOH to obtain four fractions. Fraction 4 (15.0 g) was treated with a Waters Auto-Purification Factory using an XTerra 19 × 150 mm column. The injection volume was 1.2 mL. The flow rate was 17 mL/min. The mobile phases were 30 mM ammonium acetate buffer (pH = 8.0) and acetonitrile. Linear gradient elution was adopted starting from 10% Bp to 90% Bp: 0–40 min, 10–50% Bp; 40–45 min, 50–90% Bp and 45–60 min, 90–90% Bp. Fraction 4 was separated to afford 40 fractions of 1.5 min each. Fractions 16-17 were separated by Pre-HPLC with formic acid/water  and acetonitrile on C18HCE 10 × 150 mm. The injection volume was 1.0 mL. The flow rate was 3 mL/min. Linear gradient elution was as follows: 0–10 min, 5–20% Bp; 10–30 min, 20–25% Bp and 30–50 min, 25–50% Bp. Fractions of 1 min each were collected. This separation yielded 1,15-dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline (4.1 mg), benzoylaconine (5.2 mg) and aconitine (3.2 mg).Dried powdered roots of the plant (50.0 kg) were defatted with EtOH (500.0 L) over 12 h. After removing the solvent under vacuum at 45 ºC, the residue was applied to X-4 macroporous absorption resin and eluted with Hvmax 3480 (OH), 2935 (CH), 1780 , 1451, 1382, 1320, 1282, 1180, 1,097, 983, 711 cm-1; UV λmax (CAN/H2O) 232 nm; 1H- and 13C-NMR data, + .1,15-Dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline: amorphous solid; IR (KBr) Aconitum kusnezoffii Reichb. contain diterpenoid alkaloids, which possess diverse biological properties, as the principal secondary metabolites. A new diterpenoid alkaloid, 1,15-dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline, together with two known alkaloids, benzoylaconine and aconitine, were separated from roots of Aconitum kusnezoffii Reichb., and their structures identified by spectral analysis.Based on the results of our present study, roots of


N-cis-hibiscusamide (5) and (7′S)-N-cis-feruloylnormetanephrine (9), and eight known feruloyl amides were isolated from Portulaca oleracea L. and the geometric conversion of the ten isolated feruloyl amides by UV light was verified. The structures of the feruloyl amides were determined based on spectroscopic data and comparison with literature data. The NMR data revealed that the structures of the isolated compounds showed cis/trans-isomerization under normal laboratory light conditions. Therefore, cis and trans-isomers of feruloyl amides were evaluated for their convertibility and stability by UV light of a wavelength of 254 nm. After 96 h of UV light exposure, 23.2%–35.0% of the cis and trans-isomers were converted to trans-isomers. Long-term stability tests did not show any significant changes. Among all compounds and conversion mixtures collected, compound 6 exhibited the strongest inhibition of IL-6-induced STAT3 activation in Hep3B cells, with an IC50 value of 0.2 μM. This study is the first verification of the conversion rates and an equilibrium ratio of feruloyl amides. These results indicate that this natural material might provide useful information for the treatment of various diseases involving IL-6 and STAT3.Two new feruloyl amides,  P. oleracea is an annual plant widely distributed in the Middle East, Central Europe, America, Australia and Asia +. The UV spectrum displayed absorption peaks at 201, 223 and 314 nm. The IR spectrum suggested the presence in the molecule of OH and NH groups, as indicated by a band at 3332 (br) cm−1 and an amide carbonyl group band at 1650 cm−1. The obtained 13C-NMR data clearly revealed 20 resonance signals, which were classified as three methoxy, two methylene, seven methine, and eight quaternary carbons  and 5.78 ; ABX aromatic protons at δH 7.34 , 6.90 , and 6.69 ; meta-coupled aromatic protons at δH 6.45 ; two methylene protons at δH 2.70  and 3.42 ; and three methoxy groups at δH 3.75 , and 3.79 . These proton and carbon assignments were further confirmed by detailed analyses of the 1H-1H COSY, HMQC and HMBC spectra  and 5.80  were compared with those of 5 at δH 6.57  and 5.78 , the cis conformation was evident. Additionally, when comparing 5 with 6, which is a previously reported feruloyl amide that is a trans-isomer of hibiscusamide, the NMR signals for 5 were similar to those of 6 except for two olefinic protons in trans-geometry δH 7.41 , and 6.39 . This result indicates that 5 and 6 are cis and trans-isomers. Accordingly, compound 5 was determined to be N-cis-hibiscusamide. To the best of the authors’ knowledge, this is the first time a cis-isomer has been reported from this plant.Compound  carbons . The 1H- spectra B. In the9 was collected as a yellowish oil and has a molecular formula of C19H21NO6 as deduced from the molecular peak at m/z 382.1257 [M + Na]+ in the HRESI-MS spectrum. The UV spectrum revealed bands at 221, 283 and 314 nm. The OH and NH groups in the molecule were observed at 3320 (br) cm−1, and an amide carbonyl group was seen at 1651 cm−1 in the IR spectrum. The 1H and 13C-NMR spectra of 9 were similar to those of 3, except for the presence of a hydroxyl group at H-7′. In the 13C-NMR spectrum of 9, the presence of 19 resonance signals were classified as two methoxy, one methylene, nine methine, and seven quaternary carbons. The 1H-NMR spectrum revealed two olefinic protons at δH 6.59  and 5.79 ; ABX aromatic protons from the ferulic moiety at δH 7.38 , 6.91 , and 6.72 ; ABX aromatic protons from the normetanephrine moiety at δH 6.93 , 6.76 , and 6.69 ; methylene protons at 3.39  and δH 3.46 ; a methine proton at 4.66 ; and two methoxy groups at δH 3.79  and 3.80 . The 1H and 13C resonances of 9 were assigned with a combination of 1H-1H COSY, HMQC and HMBC experiments  and 12.8 Hz (H-8), respectively. The configuration of olefinic hydrogens was confirmed as the cis-conformation. The J values of H-7 and H-8 of 9 were calculated to be 12.6 Hz and 12.6 Hz compared to those of 3, respectively. Thus, compound 9 was identified as the cis-isomer of N-feruloylnormetanephrine. The configuration of C-7′ was determined based on a comparison of optical rotations and J-coupling constants with reported data for 8 + and 312.1 [M − H]. 1H-NMR : δH 7.35 , 7.00 , 6.93 , 6.74 , 6.69 , 6.61 , 5.81 , 3.83 , 3.40 , 2.69 . 13C-NMR : δC 157.2 (C-4′), 148.8 (C-4), 148.8 (C-3), 138.5 (C-7), 131.4 (C-1), 130.9 (C-2′ and C-6′), 128.8 (C-1′), 125.0 (C-6), 122.0 (C-8), 116.5 (C-3′ and C-5′), 116.1 (C-5), 114.2 (C-2), 56.6 (C-3-OCH3), 42.4 (C-8′), 35.7 (C-7′).N-trans-Feruloyltyramine (2): Dark brown oil. ESI-MS ion peaks at m/z 314.1 [M + H]+ and 312.1 [M − H]+. 1H-NMR : δH 7.45 , 7.10 , 7.05 , 7.01 , 6.80 , 6.72 , 6.42 , 3.86 , 3.47 , 2.75 . 13C-NMR : δC 157.1 (C-4′), 150.0 (C-4), 149.4 (C-3), 142.2 (C-7), 131.5 (C-1), 130.9 (C-2′ and C-6′), 128.5 (C-1′), 123.4 (C-6), 119.0 (C-8), 116.6 (C-5), 116.4 (C-3′ and C-5′), 111.7 (C-2), 56.5 (C-3-OCH3), 42.5 (C-8′), 35.8 (C-7′).N-cis-Feruloyl-3′-methoxytyramine (3): Dark green oil. ESI-MS ion peaks at m/z 344.0 [M + H]+ and 342.2 [M − H]+. 1H-NMR : δH 7.35 , 6.91 , 6.75 , 6.71 , 6.68 , 6.59 , 6.59 , 5.80 , 3.81 , 3.77 , 3.41 , 2.69 . 13C-NMR : δC 149.2 (C-3), 148.9 (C-3′), 148.8 (C-4), 146.3 (C-4′), 138.6 (C-7), 132.2 (C-1), 128.8 (C-1′), 125.1 (C-6), 122.4 (C-6′), 121.9 (C-8), 116.4 (C-5), 116.1 (C-5′), 114.2 (C-2′), 113.6 (C-2), 56.5 (C-3-OCH3), 56.5 (C-3′-OCH3), 42.4 (C-8′), 36.1 (C-7′).N-trans-Feruloyl-3′-methoxytyramine (4): Dark brown oil. ESI-MS ion peaks at m/z 344.0 [M + H]+ and 342.2 [M − H]+. 1H-NMR : δH 7.38 , 7.04 , 6.95 , 6.75 , 6.73 , 6.66 , 6.60 , 6.36 , 3.80 , 3.75 , 3.42 , 2.70 . 13C-NMR : δC 150.1 (C-4), 149.5 (C-3), 149.2 (C-3′), 146.2 (C-4′), 142.3 (C-7), 132.3 (C-1′), 128.5 (C-1), 123.4 (C-6), 122.5 (C-6′), 119.0 (C-8), 116.7 (C-5), 116.4 (C-5′), 113.7 (C-2), 111.7 (C-2′), 56.5 (C-3-OCH3), 56.5 (C-3′-OCH3), 42.6 (C-8′), 36.3 (C-7′).N-cis-Hibiscusamide (5): Dark brown oil. 3OH). HRESI-MS ion peak at m/z 396.1418 [M + Na]+ . UV (CH3OH) λmax (log ε): 201 (3.95), 223 (3.56) and 314 nm (3.33). IR (KBr) νmax: 3332 (br), 1650, 1580, 1517 and 1459 cm−1. 1H-NMR data in CD3OD (600 MHz) and 13C-NMR data CD3OD (150 MHz) are reported in (orted in .N-trans-Hibiscusamide (6): Dark brown oil. ESI-MS ion peaks at m/z 374.0 [M + H]+ and 372.2 [M − H]+. 1H-NMR data in CD3OD (600 MHz) and 13C-NMR data CD3OD (150 MHz) are reported in (orted in .7′S)-N-cis-Feruloyloctopamine (7): Yellowish oil. 3OH). ESI-MS ion peak at m/z 328.2 [M − H]+. 1H-NMR : δH 7.37 , 7.15 , 6.91 , 6.72 , 6.70 , 6.59 , 5.79 , 4.65 , 3.81 , 3.44 , 3.37 . 13C-NMR : δC 170.5 (C-9), 158.3 (C-4′), 149.6 (C-4), 148.8 (C-3), 139.1 (C-7), 134.5 (C-1), 128.4 (C-2′ and C-6′), 127.9 (C-1′), 125.2 (C-8), 120.9 (C-6), 116.2 (C-3′ and C-5′), 116.1 (C-5), 114.0 (C-2), 73.4 (C-7′), 56.4 (C-3-OCH3), 48.1 (C-8′).(7′S)-N-trans-Feruloyloctopamine (8): Yellowish oil. 3OH). ESI-MS ion peak at m/z 328.1 [M − H]+. 1H-NMR : δH 7.44 , 7.22 , 7.11 , 7.01 , 6.79 , 6.77 , 6.46 , 4.73 , 3.86  3.54 , 3.45 . 13C-NMR : δC 169.8 (C-9), 158.3 (C-4), 150.1 (C-4′), 149.5 (C-3), 142.5 (C-7), 135.0 (C-1), 128.7 (C-2′ and C-6′), 128.5 (C-1′), 123.5 (C-8), 118.9 (C-6), 116.7 (C-5), 116.3 (C-3′ and C-5′), 111.8 (C-2), 73.6 (C-7′), 56.5 (C-3-OCH3), 50.0 (C-8′).(7′S)-N-cis-Feruloylnormetanephrine (9): Yellowish oil. 3OH). HRESI-MS ion peak at . UV (CH3OH) λmax (log ε): 221 (3.24), 283 (3.06) and 314 nm (3.04). IR (KBr) νmax: 3320 (br), 1651, 1599, 1521 and 1458 cm−1. 1H-NMR data in CD3OD (600 MHz) and 13C-NMR data CD3OD (150 MHz) are reported in ((orted in .7′S)-N-trans-Feruloylnormetanephrine (10): Yellowish oil. 3OH). ESI-MS ion peak at m/z 358.1 [M − H]+. 1H-NMR data in CD3OD (600 MHz) and 13C-NMR data CD3OD (150 MHz) are reported in  were dissolved in MeOH to a concentration of 1 mg/mL, and the solutions were stored in the dark at room temperature for stability testing. For conversion tests, 0.1 mL of each solution was used to determine the initial ratio of cis and trans- by HPLC. Subsequently, the remainder of each solution was then exposed to a UV lamp  in a dark room for 96 h. A 0.1-mL aliquot of each solution was removed and analyzed by HPLC at 1, 2, 4, 8, 24, 48 and 96 h to determine the ratio of cis and trans- isomers. HPLC analysis was performed on a Phenomenex Luna 5 μ C18, 150 mm × 4.60 mm i.d column under isocratic solvent conditions . The flow rate was 1.0 mL/min, and the injection volume was 10 μL. The detection wavelength was 210 nm, and the column temperature was maintained at 40 °C. The wavelength for detection was set at 210 nm, where all compounds exhibited maximum absorption.All isolated pure compounds TM) were obtained from American Type Culture Collection  and were maintained in a DMEM medium, supplemented with 10% fetal bovine serum, 50 U/mL penicillin and 50 mg/mL streptomycin, at 37 °C in a 5% CO2 incubator. All of the cell culture reagents were purchased from GibcoBRL .Human hepatoma Hep3B cells (ATCC No. HB-8064) and myeloma U266 cells (ATCC No. TIB-196705) antibody was purchased from Calbiochem  and anti-total STAT3, anti-phospho JAK2 (Tyr1007/1008), anti-phospho ERK (Tyr202/204) were from Cell Signaling Technology . All reagents including genistein were obtained from Sigma-Aldrich Ltd .Recombinant human IL-6 was purchased from R & D systems . Anti-phospho STAT3 (TyrHep3B cells were cotransfected with pStat3-Luc encoding the Stat3 binding site and pcDNA3.1 (+) carrying a hygromycin selection marker  using Lipofectamine plus . Two days after transfection, cells that stably expressed luciferase were selected with hygromycin (100 μg/mL), and stable clones were expanded. The expression of luciferase in the clones stably expressing pStat3-Luc was confirmed by luciferase assays.4 cells/well. After 24 h, cells were subjected to starvation for 12 h, followed by treatment with IL-6 (10 ng/mL) with or without compounds for 12 h. Luciferase activity was measured according to the manufacturer’s protocol .Hep3B cells stably expressing pSTAT3-Luc were established as described previously . The HepHep3B cells were seeded and cultured for 24 h in 96-well culture plates. After 24 h, the culture medium was replaced with serum-free medium supplemented with a sample at the indicated dose. Following culture with the sample for 48 h, MTT (0.5 mg/mL) was added, and after 4 h of incubation at 37 °C, 200 μL of DMSO was added to each well. Cell viability was determined by MTT assay according to the manufacturer’s protocol .Hep3B or U266 cells were stimulated with IL-6 (10 ng/mL) for 20 min after pre-treatment with compound for 1 h. Whole-cell lysates were prepared using a cell lysis buffer  and subjected to western blot .P. oleracea. Their structures were determined by NMR and MS spectral data. Among the isolated compounds, with cis-configured double bonds, compounds 5 and 9 were reported for the first time. In experiments studying the UV-induced cis/trans-isomerization of the isolated feruloyl amides, we found that all isomeric pairs equilibrated to trans/cis-ratios of about 22%/77% to 35%/65%. As the number of methoxy groups in the tyramine moiety increased, the equilibrium ratio increased in favor of the trans-isomers, and the presence of the 7′-hydroxyl group also increased the trans-isomer proportion. Although all isomer compounds exhibited potent inhibitory activity in the STAT3-dependent luciferase assay, the trans-feruloyl amides had stronger activity than the cis-isomers, and the conversion mixtures maintained in the ranges of IC50 values on their pure cis and trans-isomers. Among the tested compounds, 5 and 6 exhibited the most potent inhibitory activity against IL-6/STAT3. Also, compound 5, 6 and their converted mixture (5 + 6) exhibited inhibition on the phosphorylation of p-STAT3, p-JAK2 and p-ERK. Based on these results, these isolated feruloyl amides from P. oleracea might be useful candidates for lead compounds against inflammatory diseases and their conversion study could be the basis for the design of IL-6 inhibitors.In this work, we evaluated the IL-6/STAT3 inhibitory activity of ten feruloyl amides isolated from


Their structures were elucidated by using high resolution mass spectrometry (HRMS) and NMR spectroscopy. In this study, the antimicrobial, cytotoxic, anti-biofilm, and nematicidal activities of the new compounds were evaluated. The distribution pattern of secondary metabolites in the species was also revealed in which more isolates of A. novoguineensis were encountered and their secondary metabolite profiles were examined using analytical HPLC with diode array and mass spectrometric detection (HPLC-DAD/MS). Remarkably, all isolated compounds are specifically produced by A. novoguineensis.Five new compounds including the glycosylated β-naphthol ( A. novoguineensis + . The sugar moiety signals were preserved in the NMR data of 2. In addition, the remaining 10 carbon signals were determined as follow: four douplets assigned to two sets of germinal methyls, two methines, and four sp2 quaternary carbons. COSY spectra showed correlations between the germinal methyls H3-8/H3-9 and the methine H-7 and between the germinal methyls H3-11/H3-12 and the methine H-10. Furthermore, HMBC correlations from the germinal methyls H3-8/H3-9 to C-3, from H3-11/H-12 to C-6, and from H-7 to C-2, together with the two nitrogen atoms, implied by the molecular formula, determined the agylcone as 3,6-diisopropylpyrazine-2-ol  suggested that the sugar in 2 possesses the same absolute d-configuration as 1. Consequently, Compound 2 was determined as a new natural product, 3,6-diisopropylpyrazine-2-ol bearing 4-O-methyl-β-d-glucopyranose and was named akanthozine.Akanthozine  with two unsaturation degrees. The relatively small molecular weight suggested the lack of the sugar moiety in 3. 13C- and HSQC NMR data revealed the presence of four methyls, three methines, and two carbonyls, which is required for the unsaturation degrees, so Compound 3 must be acyclic. COSY and HMBC data established two substructures 3-methylbutanamide and N-hydroxyisobutyramide as shown in H 4.60, δC 65.8) to the carbonyl carbon 1′-C established the linkage of the N-hydroxyisobutyramide at C-2 of the 3-methylbutanamide by a C–N bond. Furthermore, 1H-15N HSQC and 1H-15N HMBC experiments were performed in order to confirm the structure of 3, which showed correlations from 2-H/3-H to the tertiary amide nitrogen (δN 177.2) and from 2-H to the primary amide nitrogen (δN 106.4). Additionally, HR-ESI-MS spectra of 3 showed two characteristic fragments at m/z 186.1133  and at m/z 158.1178 . This further confirmed the structure of 3 -3-methylbutanamide. To the best of our knowledge, this is the first report on this compound.Compound ure of 3 . Consequ4 was obtained as a white amorphous powder; it showed the molecular formula of C9H18N2O4 calculated from the ion peak at m/z 219.1343 [M + H]+ . At first glance, Compound 4 differs from 3 by the presence of an additional oxygen atom. The 1H- and 13C-NMR data of 4 revealed a high similarity to those of 3; nevertheless, slight shifts were observed. Particularly, in the 13C-NMR spectrum, the carbonyl carbon C-1 was shifted from δC 175.2 in 3 to δC 168.9 in 4 suggested that the additional oxygen could be attached to the amide group (C-1). The 2D NMR data for 4 showed no further remarkable differences to those of 3. Nevertheless, the HRMS spectrum of 4 showed a different fragmentation pattern to that of 3. A fraction ion peak at m/z 186.1129 (C9H16NO3) was assigned to the cleavage of hydroxyamide (NHOH) and a fraction peak at m/z 158.1180 (C8H16NO2) was assigned to the cleavage of hydroxamic group (CONHOH) (−1 (secondary amine). Thus, the structure for 4 was determined as a new hydroxamic acid derivative, namely N-hydroxy-2-(N-hydroxyisobutyramido)-3-methylbutanamide. The newly released ACD labs’ Structure elucidator was used to confirm the suggested structure of 4; the structure showed the lowest ΔδC  dN13C = 1.34 and thus was assigned as the best structure among the 54 generated structures by the software + (17), 359 [M + Na]+ (73), 695 [2M + Na]+ (100), 161 [M + H-4-O-methyl-glucopyranose]+ (26), 435 [M − H]− (100), 159 [M − H-4-O-methyl-glucopyranose]− (84); HRESIMS m/z 359.1107 [M + Na]+ . Spectra see Akanthozine (2): brown gum; c 0.17, MeOH); 1H- and 13C-NMR see m/z 373 [M + H]+ (84), 359 [M + Na]+ (48), 197 [M + H-4-O-methyl-glucopyranose]+ (100), 371 [M − H]− (100), 743 [2M − H]− (5); HRESIMS m/z 373.1971 [M + H]+ . Spectra see Compound 3: white amorphous powder; c 0.99, MeOH); 1H-NMR : δH 4.61 , 3.22 , 2.38 , 1.13 , 1.11 , 1.02 , 0.96 . δC : 180.6 (C-1′), 175.1 (C-1), 65.8 (C-2), 31.3 (C-2′), 28.4 (C-3), 19.8 (C-4), 19.8 (C-5) 19.5 (C-3′), 19.1 (C-4′). 15N-NMR : δN 106.4 (NH2), 177.2 (ter. N). IR (KBr) υ 3346, 3319, 2943, 2831, 1448, 1366, 1113, 1022 cm−1, LCMS m/z 203 [M + H]+ (4), 225 [M + Na]+ (33), 201 [M − H]− (26); HRESIMS m/z 203.1389 [M + H]+ . Spectra see Compound 4: white amorphous powder; c 1.7, MeOH); 1H-NMR : δH 4.46 , 3.21 , 2.45 , 1.11 , 1.09 , 0.96 , 0.92 . δC : 180.4 (C-1′), 169.1 (C-1), 63.6 (C-2), 31.3 (C-2′), 28.0 (C-3), 19.9 (C-4′), 19.8 (C-4), 19.7 (C-5), 19.4 (C-3′). IR (KBr) υ 3466, 3326, 2944, 2831, 1738, 1448, 1366, 1217, 1022 cm−1, LCMS m/z 241 [M + Na]+ (20), 219 [M + H]+ (3), 217 [M − H]− (100); HRESIMS m/z 219.1343 [M + H]+ . Spectra see Compound 5: pale brown gum; c 0.3, MeOH); 1H-NMR : δH 4.39 , 3.47 , 2.26 , 2.13 , 1.08 , 1.05 , 0.98 , 0.97 . δC : 167.0 (C-1), 85.7 (C-1´), 72.6 (C-2), 29.3 (C-3), 29.2 (C-2´), 18.9 (C-4), 17.8 (C-5), 17.4 (C-4´), 15.9 (C-3´). LCMS m/z 203 [M + H]+ (7), 427 [2M + Na]+ (9), 201 [M − H]− (30), 403 [2M − H]−; HRESIMS m/z 203.1395 [M + H]+ . Spectra see 1 (1 mg) was hydrolyzed with 10% aqueous HCl (1 mL) at 90 °C for 12 h. The reaction mixture was then diluted with H2O (2 mL) and extracted with EtOAc (2 × 3 mL). The aqueous layer was concentrated under vacuum to yield 4-O-methyl-d-glycopyranose .Compound Bacillus subtilis DSM10, Escherichia coli DSM498, Candida tenuis MUCL29892, and Mucor plumbeus MUCL49355, and the cytotoxicity against the cervix carcinoma cell line KB-3-1 and the established mouse fibroblast cell line L929 using the protocols previously described by Richter et al. [All isolated compounds were determined for the minimum inhibitory concentration (MIC) against r et al. .Staphylococcus aureus DSM1104 (ATCC25923) and Pseudomonas aeruginosa PA14 [S. aureus and P. aeruginosa, respectively. After incubation at 37 °C under shaking condition, the overnight cultures were adjusted to equal the turbidity of 0.5 McFarland standard using medium. The assay was carried out in 96-well tissue cultured-treated microplates  for S. aureus and non-treated plates  for P. aeruginosa, in which 10 μL of each compound (0.5 mg/mL) in six replicates were mixed together with 140 μL of bacterial suspension. Methanol and CASO medium with and without 4% glucose were used as a negative control and tetracycline (100 μg/mL) as a positive control. Plates were covered with a sterile adhesive porous paper  and incubated at 37 °C for 24 h. After incubation, bacterial biofilms were stained with 0.1% crystal violet solution  following the protocol of O’Toole [The determination of the ability of new compounds to prevent biofilm formation of osa PA14  was perfosa PA14  with min O’Toole .Caenorhabditis elegans was tested in a microwell plate assay slightly modified from the method reported by Stadler et al. [C. elegans was monoxenically cultured on nematodes agar , 0.5 mL, 1 M CaCl2, 1 mL, 1 M MgSO4, 1 mL, 40 mM potassium phosphate buffer, 12.5 mL, pH 6.8) with living E. coli DSM498, at 20 °C in the dark for a week. After incubation, adult nematodes were suspended in sterile distilled water and transferred to a sterile tube. Finally, the nematodes were counted using a Malassez counting chamber and further adjusted to obtain a concentration of 500 nematodes/mL with sterile distilled water. Nematicidal activity screening was performed in a 24-microwell plate  and 4 concentrations of 100, 50, 20, and 10 μg/mL of each compound were tested. Standard nematicide, ivermectin, and 1% MeOH were used as positive inhibitory control and solvent control, respectively. The plate was incubated at 20 °C in the dark and nematicidal activity was recorded after 18 h of incubation.The nematicidal activity of the new compounds against r et al. . The comO-methyl-β-d-glucopyranose, akanthol (1), akanthozine (2), and three new amide derivatives including a hydroxamic acid derivative (3–5) were obtained from the spider-associated fungus Akanthomyces novoguineensis and proved to be the species-specific secondary metabolites in addition to the recent reported akanthopyrones A–D. The new metabolites showed no significant activities in antimicrobial, cytotoxic, anti-biofilm, and nematicidal assays. Two new natural products bearing 4-


N-6 of 4-amino-5-cyano-6-bromo-pyrrolopyrimidine, a Vorbrüggen glycosylation with 1,2,3-tri-O-acetyl-5-deoxy-β-D-ribofuranose afforded a completely regioselective N-9 glycosylation product, which is unambiguously confirmed by X-ray diffraction analysis. All of the involved intermediates were well characterized by various spectra.In present paper, an expeditious total synthesis of naturally occurring 5′-deoxytoyocamycin and 5′-deoxysangivamycin was accomplished. Because of the introduction of a benzoyl group at  The synthesis and biological evaluation of nucleoside/tide analogs as agents to treat cancers, viruses, and parasites, as well as fungal and bacterial infections, spans the past half century ,2,3. TheN-7 atom with a carbon atom results a more electron rich pyrrole ring more prone to cation–π or π–π interactions, which subsequently leads to increased nucleobase-pairing in DNA or RNA and possibly better binding to proteins . I. I13 and16 in 93% yield. Deprotection of the remaining four benzoyl groups proved troublesome. When heated with saturated ammonium in methanol, 5′-deoxytoyocamycin (3) and 5′-deoxysangivamycin (4) were obtained at the same time along with other side products.Subsequently, the bromide at C-8 was hydrogenated in a solvent mixture of methanol and THF using 10% Pd/C as catalyst to give nucleoside 12 and nucleoside 14, it was obvious that the carbonyl of the N-6 benzoyl was close and syn to the C-7 nitrile group  and 5′-deoxysangivamycin (4) were both targets, and they could be conveniently separated by chromatography. In the end, 5′-deoxytoyocamycin (3) and 5′-deoxysangivamycin (4) were obtained in 67% and 24% yield, respectively, after removing all the benzoyl protecting groups with ammonia in dioxane. All the spectra of 5′-deoxytoyocamycin (3) were in accordance with those of the naturally occurring product. Because different solvents were used in the original NMR determination of naturally occurring 5′-deoxytoyocamycin (3) [A variety of other conditions were tried with no improvement. Looking back the X-ray structures of nucleobase le group  37]. Th. Th12 anycin (3) , the cor3) and 5′-deoxysangivamycin (4) using a Vorbrüggen glycosylation between 1,2,3-tri-O-acetyl-5-deoxy-β-D-ribofuranose (13) and N4-benzoyl-5-cyano-6-bromo-7H-pyrrolopyrimidine (12) as the key step. The structure of 7-deazapurine nucleoside 14 was unambiguously confirmed by X-ray diffraction analysis, which showed an anti glycosylic bond conformation and an envelope C2′-endo (2E) sugar conformation. It was demonstrated that the regioselectivity during the Vorbrüggen glycosylation completely favored the N-9 glycosylation product after introducing a benzoyl group at the N-6 position of 4-amino-5-cyano-6-bromo-7H-pyrrolopyrimidine. Thus, this newly developed nucleobase 12 could be used to synthesize other toyocamycin analogues while avoiding the formation of N-3 glycosylation side products. In conclusion, we have accomplished an improved total synthesis of 5′-deoxytoyocamycin (d6 as solvent. Tetramethylsilane (TMS) was used as an internal reference. Specific rotations were acquired on an Autopol IV polarimeter . High resolution mass spectrometry (HRMS) data were measured with an AB Sciex TOF 4600 instrument . Melting points were measured on an X-4 digital melting point apparatus . X-ray diffraction analysis was performed on a Bruker Smart Apex II system . Unless otherwise specified, all the reagents were acquired from commercial sources and used directly. NMR spectra (400 MHz/100 MHz) were recorded on an Advance DPX spectrometer  at room temperature with DMSO-9, 35.6 g, 277.93 mmol) was dissolved in fresh dried acetone (200 mL) and ethyl acetate (420 mL). Under an Ar atmosphere, 33% HBr in acetic acid (200 mL) was added slowly while keeping the internal reaction temperature below 2 °C. After addition, the reaction mixture was stirred for another 1.5 h. The formed yellow solid was filtered and washed with cold ethyl acetate (100 mL). Subsequently, the crude product was suspended in ice cold water (100 mL) and 50% NaOH in H2O (60 mL) was added slowly until a homogenous solution was obtained. After filtration through Celite, the filtrate pH was adjusted carefully to 5 using acetic acid. The thus obtained solid was filtered and washed thoroughly with water until neutral. After drying under vacuum with P2O5, a grey solid of 10 was obtained . Rf = 0.6 ; mp: 224–226 °C; 1H-NMR δ: 12.25 , 6.45 ; 13C-NMR δ: 149.8, 115.1, 114.4, 103.0, 93.5, 71.4; HRMS: calcd. for C6H2BrN4 [M − H]− 208.9468; found: 208.9446.Tetracyanoethylene  was suspended in fresh dried CH3CN (1.5 L) and ethyl orthoformate . The suspension was heated to reflux until TLC showed the reaction was finished (about 3 h). The solvent was removed under vacuum to give a grey solid (82 g). Subsequently, the obtained solid was suspended in freshly prepared saturated ammonia in ethanol (1150 mL) in a closed stainless-steel container and heated at 105 °C for 6 h. Once cooled to room temperature, the reaction mixture was poured to a 5 L beaker with activated charcoal (22 g). After stirring for 30 min, the mixture was filtered through Celite and washed thoroughly with concentrated ammonia in ethanol . The filtration was adjusted to pH = 5 with acetic acid (about 160 mL). The obtained solid was filtered and washed with H2O. After dried under vacuum with P2O5, 6 was obtained as greenish solid . Rf = 0.6 ; mp: 279–281 °C; 1H-NMR δ: 8.21 , 7.20 ; 13C-NMR δ: 156.0(C-4), 150.1(C-7a), 149.6 (C-2), 124.9 (C-6), 115.9 (CN), 103.7 (C-4a), 84.4(C-5); HRMS: calcd. for C7H3BrN5 [M−H]− 235.9577; found: 235.9585.2-Amino-5-bromo-3,4-dicyano-16  was suspended in fresh dried CH3CN (450 mL) and Et3N . After the above suspension was stirred for 1 h at room temperature, benzoyl chloride  was added and the reaction mixture was stirred overnight. After TLC showed the 7-deaazapurine base had vanished, the reaction pH was adjusted by acetic acid to 6 and he reaction mixture was filtered. The filtrate was extracted with DCM (300 mL × 3). The combined extracts were washed with H2O, saturated NaCl, and dried with anhydrous MgSO4. After filtration, the solvent was evaporated under vacuum. The obtained crude product was stirred with concentrated ammonium and H2O  for 5 h to give light yellow protected deazapurine 12 . Rf = 0.3 ; mp 235–237 °C; 1H-NMR δ: 14.23 , 11.36 , 8.75 , 8.06 , 7.65 , 7.56 ; 13C-NMR δ: 167.4 (C=O), 153.3 (C-4), 152.7 (C-2), 150.6 (C-7a), 133.5, 132.9, 128.9, 128.8, 124.1 (C-6), 114.8 (CN), 111.9 (C-4a), 88.8 (C-5); HRMS: calcd. for C14H7BrN5O [M − H]− 339.9839; found: 339.9852.Deazapurine base N,O-Bis(trimethylsilyl)acetamide  was added to a suspension of N4-benzoyl-5-cyano-6-bromo-7H-pyrrolopyrimidine  in dry MeCN (60 mL) under an argon atmosphere. The reaction mixture was stirred at room temperature for 60 min. A solution of 1,2,3-tri-O-acetyl-5-deoxy-β-D-ribofuranose 13  in dry MeCN (50 mL) along with TMSOTf  were added to the above reaction mixture under ice-bath condition. The solution was stirred for 30 min at room temperature before heating to 80 °C for 3.5 h. Then the mixture was cooled and poured into sat. NaHCO3 solution (150 mL). It was extracted with CH2Cl2 (150 mL × 3). The combined organic layer was washed with sat. NaHCO3 (100 mL × 2), brine (100 mL × 2), and dried over anhydrous MgSO4. After filtration, the filtrate was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography  to afford 14 as yellow solid . Rf = 0.4 ; m.p. 230–232 °C; 2Cl2); 1H-NMR δ 11.58 , 8.89 , 8.06 , 7.67 , 7.57 , 6.23 , 6.18–6.14 , 5.50 , 4.33 – 4.25 , 2.13 , 2.08 , 1.39 ; 13C-NMR δ 170.1 , 170.0 , 167.5 , 153.1 (C-2), 152.0 (C-4), 151.4 (C-7a), 133.1, 129.0, 128.9, 126.6 (C-6), 114.2 (CN), 111.8 (C-4a), 91.4 (C-5), 89.1 (C-1′), 77.8 (C-4′), 74.0 (C-3′), 72.5 (C-2′), 20.8(OAc), 20.7 (OAc), 18.2 (C-5′); HRMS: calcd for C23H21BrN5O6 [M+H]+ 542.0670; found 542.0679.3N  were added to a solution of nucleoside 14  in the mixture of THF (65 mL) and MeOH (35 mL). After stirring at room temperature for 2.25 h under H2 atmosphere (1 atm), the reaction mixture was filtered and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography  to afford 16 as light yellow solid . Rf = 0.3 ; m.p. 92–94 °C; 2Cl2); 1H-NMR δ 11.50 , 8.88 , 8.07 , 7.66 , 7.57 , 6.40 , 5.90 , 5.32 , 4.32–4.26 , 2.13 , 2.04 , 1.44 ; 13C-NMR δ 170.0 , 169.8 , 167.5 , 153.2 (C-2), 152.5 (C-4), 152.1(C-7a), 137.1 (C-6), 133.3, 133.0, 128.9, 128.8, 114.9 (CN), 111.4 (C-4a), 87.5 (C-5), 86.6 (C-1′), 78.6 (C-4′), 74.3 , 73.2 (C-2′), 20.8 (OAc), 20.6 (OAc), 18.8 (C-5′); HRMS: calcd for C23H22N5O6 [M + H]+ 464.1565; found 464.1579.10% Pd/C  and Et2Cl2/MeOH 20:1, v:v) to afford compounds 3  and 4 , respectively.Ammonium hydroxide (45 mL) was added to a solution of 5  in 1,4-dioxane (18 mL). The reaction mixture was sealed and heated to 45 °C for 21 h. Then the reaction mixture was evaporated to dryness under reduced pressure and the residue was purified by silica gel column chromatography ; m.p. 190–192 °C , 8.23 , 6.90 , 6.04 , 5.48 , 5.20 , 4.44–4.37 , 4.00–3.91 , 3.91–3.84 , 1.30 ; 13C-NMR δ 157.5 (C-6), 154.1 (C-2), 150.8 (C-4), 132.9 (C-8), 115.8 (CN), 101.7 (C-5), 88.4 (C-1′), 83.8 (C-7), 80.2 (C-4′), 75.0 (C-3′), 74.2 (C-2′), 19.4 (CH3); HRMS: calcd for C12H14N5O3 [M + H]+ 276.1091; found 276.1099.R7–188 °C ); αD25 8f = 0.15 ; m.p. 257–259 °C , 7.99 , 7.39 , 6.08 , 5.50 , 5.20 , 4.24–4.19 , 4.00–3.91 , 3.91–3.84 , 1.32 ; 13C-NMR δ 166.8 (C=O), 158.5 (C-6), 153.4 (C-2), 151.4 (C-4), 125.4 (C-8), 111.7 (C-7), 101.3 (C-5), 87.7 (C-1′), 79.5 (C-4′), 75.2 (C-3′), 74.8 (C-2′), 19.4 (CH3); HRMS: calcd for C12H16N5O4 [M + H]+ 294.1197; found 294.1208.R0–262 °C ); αD25 −


Compounds 1a–e, 2a, 3a, 4, 7–9, were tested for their antibacterial activity against Gram-positive and Gram-negative reference and multidrug-resistant strains isolated from the environment. Only 8 exhibited an in vitro growth inhibition of all Gram-positive bacteria whereas 9 showed growth inhibition of methicillin-resistant Staphyllococus aureus (MRSA). None of the compounds were active against Gram-negative bacteria tested.A previously unreported chromene derivative, 1-hydroxy-12-methoxycitromycin ( Penicillium (Family Aspergillaceae) are the most common fungi occurring in a diverse range of habitats from soil to vegetation to various food products, air, indoor environments, and marine environments. They have a worldwide distribution and a large economic impact on human life -4H-chromene-5-carboxylic acid (5), and a pyranochromone dimer (6) (1a) +, , indicating nine degrees of unsaturation. The IR spectrum showed absorption bands for the hydroxyl (3420 cm−1), conjugated ketone carbonyl (1662 cm−1), aromatic , and ether (1270 cm−1) groups. The 13C NMR spectrum of 1c (C 174.8), seven quaternary sp2 , three methine sp2 , two oxymethylene sp3 (δC 62.5 and 59.5), and one methoxyl (δC 56.4) carbon. The 1H NMR spectrum  and 4.41 (2H), and a singlet of methoxyl protons at δH 3.80 (3H). The general features of the 1H and 13C NMR spectra of 1c resembled those of 12-methoxycitromycin (1b), which was previously isolated from the Australian marine-derived and terrestrial Penicillium spp. +, , the molecular formula C14H14O6 was attributed to 3b. The 1H and 13C NMR spectra of 3b (a minor compound) resembled those of penialidin F (3a) (13C NMR spectrum of 3b (C 173.2), six quaternary sp2 , two methine sp2 (δC 107.4 and 102.7), one ketal (δC 97.7), one oxymethylene sp3 (δC 57.1), one methylene sp3 (δC 37.1), one methyl (δC 22.4), and one methoxyl (δC 48.3) carbon. The 1H NMR spectrum (H 7.26 (H-13) and 6.83 (H-10), two pairs of geminally coupled methylene protons at δH 4.52, dd /4.22, dt,  and 2.63, dd /2.96, dd , a methyl singlet at δH 1.44 and a methoxyl singlet at δH 3.21. Comparison of the 1H and 13C data of 3b with those of 3a  . The 13Cum of 3b  exhibiteum of 3b , can be spectrum  displayese of 3a  led to trelation  of the s2b) and 3b did not exhibit any Cotton effects. Consequently, we concluded that 3b is a mixture of both enantiomers.Surprisingly, the ECD spectrum of the mixture of myxotrichin C  , in 3a (2R). Dehydration of the hemiketal in 3a furnished myxotrichin C (2b), which underwent a nucleophilic addition of methanol (chromatographic solvent) at C-2 to form an enantiomeric mixture of 3b. Therefore, 3b can be an artifact and not a natural product. The co-occurrence of 2b and 3b can be a concrete proof of this hypothesis.The biogenesis of iate (i) . Enzyme-4 was isolated as white crystals (mp 150–152 °C), and its molecular formula was established as C13H10O6 on the basis of its (+)-HRESIMS m/z 263.0569 [M + H]+, , indicating nine degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl , conjugated ketone carbonyls , olefin (1648 cm−1), aromatic , and ether (1276 cm−1) groups. The 13C NMR spectrum (C 198.3 and 181.6), four oxyquaternary sp2 , one quaternary sp2 (δC 111.1), three methine sp2 , one quaternary sp3 (δC 86.3), one oxymethylene sp3 (δC 69.8) and one methyl (δC 16.4) carbons. The 1H NMR spectrum , a pair of doublets of the oxymethylene protons at δH 4.49 (J = 12.4 Hz)/4.63 (J = 12.4 Hz), and a methyl singlet at δH 2.31, in addition to a broad signal of the hydroxyl protons at δH 10.01. The presence of the 6,7-dihydroxy-2,3-dihydro-4H-1-benzopyran-4-one moiety was corroborated by the HMBC correlations  to C-4 (δC 181.6), C-6 (δC 141.9), C-7 (δC 155.9) and C-8a (δC 156.9); H-8  to C-4, C-4a (δC 111.1), C-6, C-7 and C-8a, and from H2-2  to C-4 and C-8a. That another portion of the molecule was a 5-methylfuran-3(2H)-one ring was substantiated by the COSY correlation , as well as the HMBC correlations (C 86.3), C-2′ (δC 191.4), C-4′ (δC 198.3), and from the methyl singlet at δH 2.31 to C-2′ and C-3′ (δC 103.8). Finally, the 5-methylfuran-3(2H)-one moiety and the 6,7-dihydroxy-2,3-dihydro-4H-1-benzopyran-4-one were connected through C-3 since the HMBC spectrum exhibited correlations from H-2  to C-3 and C-4′, and from H-3′ to C-3. Therefore, the planar structure of 4 corresponds to 5′-methyl-2H,3′H,4H-spiro -3′,4-dione. Since 4 was obtained as a suitable crystal, an X-ray analysis was carried out to determine the absolute configuration of the stereogenic carbon (C-3).Compound spectrum  displayespectrum , as two spectrum  showed telations  from H-5relation  from theelations  from H-34 but also determined unequivocally the absolute configuration of C-3 as 3S. Therefore, the absolute structure of 4 is (3S)-6,7-dihydroxy-5′-methyl-3′H,4H-spiro-3′,4-dione, which was named erubescenschromone A.The ORTEP view, shown in 5 was isolated as a white solid (mp 276–277 °C), and displayed its (+)-HRESIMS m/z at 305.0667 [M + H]+, . Therefore, its molecular formula was established as C15H12O7, indicating ten degrees of unsaturation. The IR spectrum exhibited absorption bands for hydroxyl (3446 cm−1), conjugated ketone carbonyls , aromatic , olefin (1618 cm−1) and ether (1276 cm−1) groups. However, its 13C NMR spectrum (C 198.2 and 173.4), one conjugated carboxyl carbonyl (δC 167.1), three oxyquaternary sp2 , two quaternary sp2 (δC 117.4 and 112.0), one oxymethine sp2 (δC 158.9), three methine sp2 , one methoxyl (δC 61.0) and one methyl (δC 27.5) carbons. The 1H NMR spectrum (H 8.73 (1H), 7.35 (2H), 7.03 (1H), one methoxyl singlet at δH 3.75 and one methyl singlet at δH 2.29. That 5 consists of a 7-hydroxy-6-methoxy-4-oxo-4H-chromene-5-carboxylic acid nucleus, with a substituent on C-3, was supported by the HMBC correlations (H 8.73) to C-4 (δC 173.4), C-8a (δC 152.8) and C-3 (δC 117.4); H-8 (δH 7.03) to C-4a (δC 112.0), C-6 (δC 143.2), C-7 (δC 157.1), and C-8a, from OCH3-6 (δH 3.75) to C-6, as well as the carbon chemical shift value of OCH3-6 (δC 61.0), characteristic of the methoxyl group flanked by one oxygenated substituent and one carboxyl group. Like many other quaternary sp2 carbon linked to the carboxyl substituent, the intensity of the signal of C-5 was not strong enough to be observed in the 13C NMR spectrum. Moreover, since there is no proton two or three bonds away from C-5, it was not possible to localize the C-5 signal in the HMBC spectrum. The existence of a 3-oxobut-1-en-1-yl substituent was supported by the presence of a singlet of two protons at δH 7.35 (H-10 and H-11) which, through the HSQC spectrum, connected to the two methine sp2 carbons at δC 134.9 (C-10) and δC 128.7 (C-11), as well as the HMBC correlations from H-10/H-11 to the ketone carbonyl carbon at δC 198.2 (C-12), and from the methyl singlet at δH 2.29 (H3-13) to C-12 and C-11. That the 3-oxobut-1-en-1-yl substituent was on C-3 was also supported by the HMBC correlations . The only difference between PI-4 and 5 is the substituent on C-6 which is a hydroxyl group in the former and a methoxyl group in the latter. Therefore, 5 is identified as 7-hydroxy-6-methoxy-4-oxo-3-[(1E)-3-oxobut-1-en-1-yl]-4H-chromene-5-carboxylic acid.Compound spectrum  displayespectrum , can be spectrum  exhibiteelations  from H-2elations  from H-1ls 1719, 46 cm−1, 5 and the trans double bond between C-10 and C-11 are confirmed by X-ray analysis, as shown in the ORTEP view in The structure of 6 was isolated as a pale yellow viscous oil, and its molecular formula C26H20O11 was established based on its (+)-HRESIMS m/z 509.1085 [M + H]+, , indicating twelve degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3443 cm−1), ketone carbonyls , conjugated ketone carbonyls , aromatic , and ether (1261 cm−1) groups. The 13C NMR spectrum , two conjugated ketone carbonyls , ten quaternary sp2 , four methine sp2 , two oxyquatermary sp3 (δC 61.9 and 78.2), two methine sp3 (δC 71.4 and 69.8), two methylene sp3 (δC 67.2 and 33.4), and two tertiary methyl (δC 32.7 and 29.3) carbons. The 1H NMR spectrum  and 3.59 (J = 12.8 Hz), two doublets of the magnetically inequivalent methylene protons at δH 3.47 (J = 19.2 Hz) and 2.89 (J = 19.2 Hz), in addition to two methyl singlets at δH 2.16 and 1.51. The presence of the 7,8-dihydroxy-3-methyl-3,4-dihydro-1H,10H-pyranochromen-10-one moiety was substantiated by the HMBC correlations  to C-4 (δC 172.2), C-8a (δC 152.5), C-7 (δC 150.2), and C-6 (144.7); H-8  to C-4, C-8a, C-7, C-6, C-4a (δC 115.1); H-12  to C-4, C-2 (δC 161.3), C-3 (δC 112.3), C-10 (δC 78.2), and from Me-13  to C-2, C-10, and C-9 (δC 33.4). Another portion of the molecule was identified as 3,3-disubstituted 6,7-dihydroxy-2,3-dihydro-4H-1-benzopyran-4-one, based on the HMBC correlations from H-5′  to C- 4′ (δC 185.3), C-8′a (δC 156.0), C-7′ (δC 155.4), C-6′ (δC 141.4); H-8′  to C-4′, C-8′a, C-6′ and C-4′a (δC 109.8), as well as from H2-2′  to C-4′, C-8′a and C-3′ (δC 61.9). That the disubstituted 6,7-dihydroxy-2,3-dihydro-4H-1-benzopyran-4-one was connected to the 7,8-dihydroxy-3-methyl-3,4-dihydro-1H,10H-pyranochromen-10-one moiety, through C-3′ of the former and C-12 of the latter, was confirmed by the HMBC correlations from H-12 to C-3′ and H2-2′ to C-12. Moreover, since the HMBC spectrum also exhibited correlations from H-12 and H2-2′ to the ketone carbonyl carbon at δC 200.9 (C-14), from H-15  to C-9, C-10 (δC 78.2), C-14, and from Me-13 to C-15, the 7,8-dihydroxy-3-methyl-3,4-dihydro-1H,10H-pyranochromen-10-one moiety was connected through C-10 and C-15 of the oxan-4-one ring. The acetyl group on C-15 was corroborated by the HMBC correlations from Me-17  to C-15 and the carbonyl carbon at δC 204.6 (C-16), as well as from H-15 to C-16. Taking together the molecular formula, the NMR data, and the HMBC correlations, the planar structure of 6 was unambiguously established. In order to determine the relative configurations of the stereogenic carbons C-10, C-12, C-15 and C-3′, the ROESY spectrum was obtained. The ROESY spectrum  to H-15  and the methylene proton at δH 2.89, d (J = 19.2 Hz), implying that these three protons are on the same face. Additionally, H-15 also shows a correlation with Me-17 . Since the pyran ring and the oxan-4-one ring of the 9-oxabicyclo[3.3.1]nonan-3-one ring system are in a rigid half-chair conformation, Me-13 must be in a pseudoequatorial position while the methylene proton at δH 2.89, d (J = 19.2 Hz) and H-15 are in a pseudoaxial position. Therefore, the acetyl group on C-15 must be in a pseudoequatorial position. This was confirmed by the higher chemical shift value  of the pseudoequatorial H-9 as it is in the deshielding zone of the carbonyl (C-16) of the acetyl group. On the other hand, H-12  showed a weak correlation to one of H-2′ at δH 3.59, d (J = 12.9 Hz). Therefore both of these protons should be in the pseudoequatorial position since the pseudoaxial H-2′  is under the anisotropic effect (deshielding) of the carbonyl at C-14 of ring D. With these ROESY correlations, the relative configurations of C-10, 12, 15, and 3′ were proposed as 10S*, 12S*, 15S*, and 3′S*. However, it is necessary to determine the absolute configurations of these stereogenic carbons.Compound spectrum  exhibitespectrum  as two kspectrum , in combelations  from H-5spectrum  exhibite6 could not be obtained as a suitable crystal for an X-ray analysis, the determination of its stereogenic carbons had to be carried out by comparison of the calculated and experimental ECD spectra. Although the ROESY correlations pointed to the relative configuration of C-10 and C-15 as 10S, 15S, it is possible that it can be 10R, 15R, thus reducing its number of possible configurations from 16 (eight pairs of diastereoisomers) to eight (four pairs of diastereoisomers). Hence, four computational models were constructed by combining the two configurations of C-3′ with the two of C-12. The conformational analysis of 6 by molecular mechanics (MM2 and MMFF95 force fields) focused on combinations of hydroxyl 120° rotations and rings conformations. Most diastereoisomers did not show ring conformational freedom which limited the number of models to compute. The most stable APFD/6-31G conformation of 6 whose absolute configurations of C-10, C-12, C-15, and C-3′ are 10S, 12S, 15S, 3′S, as deduced from ROESY correlations, is shown in Since 6 diastereoisomers. The fitting between the experimental and calculated spectra is presented in 6 is the C-10S, C-12S, C-3′S, C-15S enantiomer.All conformations were energetically minimized and ranked using a DFT model. The lowest energy ones, representing at least 95% of the model Boltzmann population, were used to calculate the expected Boltzmann-averaged ECD spectra of the four 6 has never been previously reported and therefore it is a new compound which was named erubescenschromone B.The literature search revealed that 7 was isolated as a pale yellow oil and the (+)-HRESIMS showed the [M + H]+ peak at m/z 509.1085 . Therefore, its molecular formula is C26H20O11, indicating eighteen degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl , conjugated ketone carbonyls , olefin (1648 cm−1), aromatic , and ether (1206 cm−1) groups. The 13C NMR spectrum , fifteen quaternary sp2 , five methine sp2 , one methylene sp3 (δC 66.2) and two methyl (δC 32.4 and 16.6) carbons. The 1H NMR spectrum  and two methyl singlets at δH 2.71 and 2.32. The presence of the 6,7-dihydroxy-2,3-dihydro-4H-chromen-4-one moiety was corroborated by the HMBC correlations  to C-4′ (δC 183.6), C-6′(δC 155.9), C-8′a (δC 154.9) and C-7′ (δC 141.6); H-8′  to C-4′a (δC 111.9), C-6′, C-7′, C-8′a, and H2-2′  to C-3′ (δC 103.9) and C-4′ (δC 183.6). That the substituent on C-3′ was an enolic exocyclic double bond was substantiated by the HMBC correlations from H2-2′ to C-3′ (δC 103.9) and the enolic carbon . Another moiety was established as 7-substituted 5-acetyl-2,3-dihydroxy-6-methyl-9H-xanthen-9-one since the HMBC spectrum showed correlations from H-5  to C-7 (δC 143.3), C-8a (δC 113.5), C-10a (δC 154.2); H-8  to C-6 (δC 150.7), C-7, C-9 (δC 173.5), C-10a; H-1  to C-3 (δC 138.0) and C-4a (δC 152.1); Me-11  to C-2 (δC 129.5), C-3, C-4 (δC 132.2). Since H-1 also showed the HMBC correlation to C-9′, the 5-acetyl-2,3-dihydroxy-6-methyl-9H-xanthen-9-one was linked to the 6,7-dihydroxy-2,3-dihydro-4H-chromen-4-one moiety through C-9′. An extensive literature search revealed that the structure of 7 is the same as that of the enol tautomer of the compound, named SPF-3059-30, isolated from the acetone extract of the mycelium of Penicillium sp. SPF-3050 (FERM BB-7663), cultured in the liquid medium + .White solid, mp 232–233 °C (CHCl3/MeOH); + .White crystal, mp 150–152 °C (CHCl3/MeOH); IR (KBr) νmax 3446, 2922, 1719, 1646, 1618, 1560, 1541, 1521, 1276 cm−1; For 1H and 13C spectroscopic data , see m/z 305.0667 [M + H]+ .White crystal, mp 276–277 °C ; IR (KBr) νmax 3443 (br), 2922, 1731, 1715, 1697, 1648, 1634, 1556, 1540, 1506, 1261 cm−1; For 1H and 13C spectroscopic data , see m/z 509.1085 [M + H]+ .Yellowish oil; + .Yellowish oil; IR (KBr) ν3a and 6 (1.5 mM in methanol) were obtained in a Jasco J-815 CD spectropolarimeter  with a 0.01 mm cell (40 accumulations for 3a). The dihedral driver and MMFF95 minimizations were done in Chem3D Ultra . All DFT minimizations with model chemistries APFD/6-31G and APFD/6-311+G  for Gram-positive strains. When it was possible to determine a MIC, four concentrations of compound were tested, i.e., 2 × MIC, MIC, ½ MIC and ¼ MIC. A control with appropriate concentration of DMSO, as well as a negative control , was included. Sterile 96-well flat-bottomed untreated polystyrene microtiter plates were used. After a 24 h incubation at 37 °C, the biofilms were heat-fixed for 1 h at 60 °C and stained with 0.5% (v/v) crystal violet  for 5 min. The stain was solubilized with 33% (v/v) acetic acid  and the biofilm biomass was quantified by measuring the absorbance of each sample at 570 nm in a microplate reader  [The effect of all compounds on biofilm formation was evaluated using the crystal violet method, as follows: the highest concentration of the tested compound in the MIC assay was added to bacterial suspensions of 1 × 10MA, USA) ,34. ThisE. coli SA/2, vancomycin  for E. faecalis B3/101 and E. faecium 1/6/63, and oxacillin  for S. aureus 66/1. Antibiotic discs impregnated with 15 µg of each compound were placed on seeded MH plates. The controls used included antibiotic discs alone, blank paper discs impregnated with 15 µg of each compound alone and blank discs impregnated with DMSO. Plates with CTX were incubated for 18–20 h and plates with VAN and OXA were incubated for 24 h at 37 °C [1e, the concentration used was 32 mg/L while it was 64 mg/L for the other compounds. The antibiotic tested was serially diluted whereas the concentration of each compound was kept fixed. Antibiotic MICs were determined as described above. For 7, it was possible to determine the MIC for E. faecalis B3/101 and E. faecium 1/6/63, so the checkerboard method was used instead, as previously described [The potential synergy between the compounds and clinically relevant antimicrobial drugs was screened using the Kirby-Bauer method, as previously described . A set ocin VAN,  µg for Eescribed . The fraescribed .Penicillium. In the past ten years, the Penicillium species from the marine environment received more attention than other fungal genera since compounds isolated from members of the Penicillium genus accounted for more than 25% of compounds of marine fungal origin. Although polyketides are the major secondary metabolites isolated from marine-derived Penicillium species, other structural classes of secondary metabolites such as alkaloids, terpenoids, and sterols are also isolated. In this work, we have described isolation and structure elucidation of two common fungal sterol derivatives: β-sitostenone and ergosterol 5,8-endoperoxide, fifteen polyketides, five of which have not been previously described, and a macrocyclic ether containing 1,4-disubstituted phenyl and succinamide moiety called GKK1032B, from the culture of the fungus P. erubescens strain KUFA 0220, which was isolated from the marine sponge Neopetrosia sp., collected from the Gulf of Thailand. From the compounds evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria of reference strains and multidrug-resistant isolates, their capacity to inhibit biofilm formation and synergistic effect, only GKK1032B displayed significant activities in all assays. Although the rest of the compounds, including those which have not been previously described, did not show significant antibacterial activity, it does not mean that they are void of bioactivities. Therefore, it is necessary to test these compounds in other bioassay platforms to explore their potential. Finally, it is worth mentioning that this is the first report of the chemical study of the marine-derived P. erubescens.Marine-derived fungi have proved to be important sources of bioactive secondary metabolites, many of which exhibit cytotoxic and antibiotic activities. One of the most studied marine-derived fungi is of the genus


The synthesis, characterization and crystal structure of the title compounds are reported and the crystal supramolecularity is discussed. E)-2-cyano-3-(3-methylthiophen-2-yl)acrylate (1), C11H11NO2S, and ethyl (E)-2-cyano-3-(thiophen-2-yl)acrylate (2), C10H9NO2S, are reported. Derivative 1 crystallized with two independent molecules in the asymmetric unit, and derivative 2 represents a new monoclinic (C2/m) polymorph. The molecular conformations of 1 and the two polymorphs of 2 are very similar, as all non-H atoms are planar except for the methyl of the ethyl groups. The intermolecular interactions and crystal packing of 1 and 2 are described and compared with that of the reported monoclinic (C2/m) polymorph of derivative 2 .The synthesis, crystal structure and structural motif of two thiophene-based cyanoacrylate derivatives, namely, ethyl ( al. 2017. Acta Cr Derivative 2 is a polymorph of a reported structure , C14—C15—C16—C17 = 179.71 (15)° and C14—O15—C26—C27 = −88.66 (2)° (molecule B) in 1 and C1—C2—C3—C4 = −178.77 (11) and C1—O2—C11—C12 = −83.41 (13)° in 2] indicate an out-of-plane deviation of the methyl group. The planarity of the molecules allows intramolecular hydrogen bonds to occur   ring motif with the carbonyl O and cyano N atoms consequently exhibiting an anti-configuration to each other. The conformation of the ethene bond is always E .The molecular structures of the title compounds are depicted in Fig. 12] Fig. 1 and 2 ▸,2 is a polymorph of ethyl (E)-2-cyano-3-(thiophen-2-yl)acrylate  are further connected through C—H⋯N interactions, leading to infinite chains propagating along the c-axis direction. The resulting chains interact via van der Waals forces to form sheets parallel to the ac plane . The sheets are connected through van der Waals forces and/or weak dipolar interactions, thus consolidating the three-dimensional framework structure. Compounds 1, 2 and the polymorph of 2 acrylate -2-cyano-3-(1-methyl-1H-pyrrol-2-yl)prop-2-enoate -ethyl-2-cyano-3-(1H-pyrrol-2-yl)acrylate -ethyl-2-cyano-3-(furan-2-yl)acrylate .In a 250 ml round-bottom flask connected with a condenser, a mixture of the corresponding thiophene-2-carboxaldehyde (1 mmol), ethylcyanoacetate (1.1 mmol) and ammonium acetate (8 mmol) in absolute ethanol was refluxed for 6 h. The reaction was monitored using thin layer chromatography (TLC plates coated with silica gel). After completion, the reaction mixture was cooled to room temperature, and the obtained yellowish-brown precipitate was filtered off, washed with cooled water, dried and recrystallized from ethanol solution to give the final products as pale-yellow crystals (90% yield for both E)-2-cyano-3-(3-methylthiophen-2-yl)acrylate (1): m.p. 381–382 K, 1H NMR : δ (ppm) = 1.39 , 2.48 , 4.36 , 7.07 , 7.74 , 8.46 . 13C NMR  δ (ppm) = 14.4 (CH2CH3), 14.9 (CH3-3′), 62.7 (CH2CH3), 98.0 (C-2), 116.4 (CN), 131.2 (C-2′), 131.4 (C-4′), 134.3 (C-5′), 145.0 (C-3), 149.9 (C-3′), 163.4 (C-1). (+)-ESIMS m/z = 244 , 465 .Ethyl (E)-2-cyano-3-(thiophen-2-yl)acrylate (2): mp. 371–372 K, 1H NMR : δ (ppm) = 1.39 , 4.37 , 7.27 , 7.85 , 8.38 . 13C NMR  δ (ppm) = 14.4 (CH2CH3), 62.9 (CH2CH3), 99.8 (C-2), 116.1 (CN), 129.0 (C-4′), 135.5 (C-5′), 136.5 (C-2′), 137.8 (C-3′), 146.9 (C-3), 162.9 (C-1). (+)-ESIMS m/z = 230 , 237 .Ethyl  and refined using a riding model with Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl). Methyl groups were allowed to rotate to fit best the electron density. All hydrogen atoms in 2 were located in difference-Fourier maps and refined isotropically.Detailed crystal data and structure refinement for the title compounds are listed in Table 310.1107/S2056989019011435/tx2012sup1.cifCrystal structure: contains datablock(s) 1, 2, New_Global_Publ_Block. DOI: 10.1107/S2056989019011435/tx20121sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989019011435/tx20122sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989019011435/tx20121sup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019011435/tx20122sup5.cmlSupporting information file. DOI: 10.1107/S2056989019011435/tx2012sup6.pdfSupporting information file. DOI: 1947083, 1947082CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: