Ki values ranged from 0.10 to 2.19 nM. Inclusion of an N-bromobenzyl substituent at the ε-amino group of lysine enhanced PSMA+ PIP tumor uptake, as did hydrophilic substituents within the linker. The presence of a polyethylene glycol chain within the linker markedly decreased renal uptake. In particular, DyLight800-10 demonstrated high specific uptake relative to background signal within kidney, confirmed by immunohistochemistry. These compounds may be useful for FGS in prostate, renal or other PSMA-expressing cancers.We have synthesized a series of 10 new, PSMA-targeted, near-infrared imaging agents intended for use in vivo for fluorescence-guided surgery (FGS). Compounds were synthesized from the commercially available amine-reactive active NHS ester of DyLight800. We altered the linker between the PSMA-targeting urea moiety and the fluorophore with a view to improve the pharmacokinetics. Chemical yields for the conjugates ranged from 51% to 86%. The  As surgeons use robot-assisted laparoscopy in more operations and for an increasing number of indications, they are less likely to palpate the tumor and surgical bed to determine the extent of their resection. Accordingly, they must increasingly rely on visual inspection, which real-time fluorescence imaging can enhance by helping to avoid leaving behind a positive margin . This isOthers and we have developed targeted NIR agents to enable specific evaluation of nerves, vascular structures within tumors, and tumor epithelium ,11,12,13We have previously tested our initial PSMA-targeted NIR agent, YC-27, which bears the IRDye 800CW fluorophore, in xenograft-bearing mice and showed that it was useful in preventing recurrence in the surgical margin . We havet-Boc-N-amido-PEG8-acid was purchased from BroadPharm, Inc. . The N-hydroxysuccinimide (NHS) ester of DyLight800 was purchased from Thermo Fisher Scientific . Care was taken to limit the exposure of DyLight800-NHS and DyLight800-urea conjugates 1–10 from light. 1H NMR spectra were recorded on a Bruker Ultrashield 500 MHz spectrometer. ESI mass spectra were obtained on a Bruker Esquire 3000 plus system. . High-performance liquid chromatography (HPLC) purifications were performed on a Varian Prostar System .General Methods. All chemicals and solvents were purchased from either Sigma-Aldrich  or Fisher Scientific . Boc-15-amino-4,7,10,13-tetraoxapentadecanoic acid and H-Lys(Boc)-Ot-Bu.HCl were purchased from Chem-Impex International . 1: To the trifluoroacetate salt of (S)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentan-1-amine, 1 2− 603.7 found 603.6.DyLight800-amine, 1  -N-(4-bromobenzyl)-5-carboxy-5-(3-((S)-1,3-dicarboxypropyl)ureido)pentan-1-amine 2 (20)  in DMSO (0.1 mL), N,N-diisopropylethylamine  was added, followed by the NHS ester of DyLight800 . After stirring overnight at room temperature, the reaction mixture was purified by HPLC  to afford 0.2 mg (51%) of DyLight800-2. ESI-Mass calculated for C64H74BrN5O18S32− [M-2H]2− 687.7 found 687.5.DyLight800-3: To the trifluoroacetate salt of -1,3,7,22-tetracarboxy-5,13,20-trioxo-4,6,12,21-tetraazahexacosan-26-amine 3 (9),  in DMSO (0.1 mL), N,N-diisopropylethylamine  was added, followed by the NHS ester of DyLight800 . After 1 h at room temperature, the stirred reaction mixture was purified by HPLC  to afford 0.3 mg (70%) of DyLight800-3. ESI-Mass calculated for C71H94N7O22S3− [M-H]− 1492.6 found 1492.4.DyLight800-S,7S)-12-(4-Bromobenzyl)-1,3,7-tricarboxy-5,13,20-trioxo-4,6,12,21-tetraazahexacosan-26-aminium trifluoroacetate, 4. To a solution of (S)-N-(4-bromobenzyl)-6-(tert-butoxy)-5-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-6-oxohexan-1-amine, 12 + 772.3 found 772.2. in DMSO (0.1 mL), N,N-diisopropylethylamine  was added, followed by the NHS ester of DyLight800 . After stirring for 1 h at room temperature, the reaction mixture was purified by HPLC  to afford 0.5 mg (63%) of DyLight800-4. ESI-Mass calculated for C78H98BrN7O22S32− [M-2H]2− 829.9 found 830.5.DyLight800-S,7S)-1,3,7-Tricarboxy-5,13,20-trioxo-4,6,12,21-tetraazahexacosan-26-aminium trifluoroacetate 5: To the formate of (S)-6-(tert-butoxy)-5-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-6-oxohexan-1-amine, 15  + 828.6 found 828.6. To this, a solution of TFA/H2O  was added. The mixture was stirred at ambient temperature for 2 h, then purified on a Sep-Pak Vac 12 cc syringe column (Waters) using a gradient of water to acetonitrile/water 5:5 (v:v) to give compound 5 . 1H NMR  δ 3.97–4.02 , 3.05 , 2.83–2.86 , 2.31 , 2.09 , 1.79 , 1.66 , 1.40–1.56 , 1.19–1.28 . ESI-Mass calculated for C25H46N5O9+ [M + H]+ 560.3 found 560.3.(305 mmol)  in CH2Cl5: To a solution of compound 5  in DMSO (0.1 mL), N,N-diisopropylethylamine  was added, followed by the NHS ester of DyLight800 . After stirring 2 h at room temperature, the reaction mixture was purified by HPLC  to afford 0.3 mg (72%) of DyLight800-5. ESI-Mass calculated for C70H93N7O20S32− [M-2H]2−, 723.8, found 723.6.DyLight800-S,7S)-12-(4-Bromobenzyl)-1,3,7-tricarboxy-5,13,20-trioxo-4,6,12,21-tetraazahexacosan-26-aminium trifluoroacetate, 6: To a solution of compound 12  in CH2Cl2 (2 mL), triethylamine  was added, followed by 13 . After stirring at room temperature for 2 h, the solvent was evaporated and the residual was purified by HPLC  to give 0.013 g (54%) of the protected intermediate. ESI-Mass calculated for C49H83BrN5O11 [M + H]+ 996.5 found 996.4. To the protected intermediate, a solution of TFA/H2O  was added. The mixture was kept at ambient temperature for 2 h, then purified on a Sep-Pak Vac 12 cc syringe column (Waters) using a gradient of water to acetonitrile/water of 5:5 (v:v) to give compound 6 . 1H NMR  δ 7.43–7.49 , 7.08 , 4.43–4.49 , 4.02–4.08 , 3.18–3.26 , 3.04–3.08 , 2.82–2.85 , 2.23–2.36 , 2.08–2.11 , 1.79–1.82 , 1.66 ,1.49–1.58 , 1.13–1.26 . ESI-Mass calculated for C32H51BrN5O9+ [M + H]+ 728.3 found 728.2. in DMSO (0.1 mL), N,N-diisopropylethylamine  was added, followed by the NHS ester of DyLight800 . After stirring 2 h at room temperature, the reaction mixture was purified by HPLC  to afford 0.4 mg (86%) of DyLight800-6. ESI-Mass calculated for C77H98BrN7O20S32− [M-2H]2−, 807.8, found 808.6.DyLight800-S,25S)-21,25,27-Tricarboxy-15,23-dioxo-3,6,9,12-tetraoxa-16,22,24-triazaheptacosan-1-aminium trifluoroacetate, 7: To a solution of 15  in CH2Cl2 (2 mL), triethylamine  was added, followed by 2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oate, 17 . After stirring at room temperature for 2 h, the solvent was evaporated and the residue was purified by a silica gel column (5% MeOH in CH2Cl2) to give the protected intermediate . ESI-Mass calculated for C40H74N4O14Na [M + Na]+ 857.5 found 857.5. To the protected intermediate, a solution of TFA/H2O  was added. The mixture was stirred at ambient temperature for 2 h, then purified using a Sep-Pak Vac 12 cc syringe column (Waters) using a gradient of 100% water to acetonitrile/water 3:7 (v:v). Product fractions were collected and lyophilized to give compound 7 . 1H NMR  δ 4.20 , 4.12 , 3.76 , 3.65–3.70 , 3.17–3.20 , 2.46–2.50 , 2.12–2.15 , 1.92–1.98 , 1.78–1.83 , 1.64–1.72 , 1.49–1.55 , 1.37–1.41 . ESI-Mass calculated for C23H43N4O12+ [M + H]+ 567.3 found 567.2. in DMSO (0.1 mL), N,N-diisopropylethylamine  was added, followed by the NHS ester of DyLight800 . After stirring for 2 h at room temperature, the reaction mixture was purified by HPLC  to afford 0.3 mg (72%) of DyLight800-7. ESI-Mass calculated for C68H90N6O23S32− [M-2H]2− 727.3 found 727.1.DyLight800-S,25S)-16-(4-Bromobenzyl)-21,25,27-tricarboxy-15,23-dioxo-3,6,9,12-tetraoxa-16,22,24-triazaheptacosan-1-aminium trifluoroacetate,8: To a solution of compound 12  in CH2Cl2 (2 mL), triethylamine  was added, followed by 2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oate, 17 . After stirring for 2 h at room temperature, the solvent was evaporated, and the residue was purified by a silica gel column (5% MeOH in CH2Cl2) to give the protected intermediate  ESI-Mass calculated for C47H79BrN4O14Na+ [M + Na]+ 1025.5 found 1025.3. To the protected intermediate, a solution of TFA/H2O  was added. The mixture was kept at an ambient temperature for 2 h, and the solvent was evaporated and the residue purified on a Sep-Pak Vac 12 cc syringe column (Waters) using a gradient of 100% water to acetonitrile/water 1:1 (v:v). The fractions were collected and lyophilized to give compound 8 . 1H NMR  δ 7.49–7.55 , 7.12–7.15 , 4.52–4.61 , 4.10 , 4.03 , 3.68–3.80 , 3.54–3.64 , 3.32–3.38 , 3.13–3.15 , 2.62–2.76 , 2.37–2.40 , 2.04–2.08 , 1.86–1.89 , 1.67–1.73 , 1.51–1.61 , 1.26–1.30 . ESI-Mass calculated for C30H48BrN4O12+ [M + H]+ 735.2 found 735.2. in DMSO (0.1 mL), N,N-diisopropylethylamine  was added, followed by the NHS ester of DyLight800 . After stirring for 2 h at room temperature, the reaction mixture was purified by HPLC  to afford 0.3 mg (65%) of DyLight800-8. ESI-Mass calculated for C75H95BrN6O23S32− [M-2H]2− 811.2 found 811.0.DyLight800-20: To a solution of t-Boc-N-amido-PEG8-acid  and N-hydroxysuccinimide  in CH2Cl2 (4 mL), dicyclohexylcarbodiimide  was added. After stirring at room temperature overnight, the mixture was filtered, and the filtrate was evaporated to give compound 20. ESI-Mass calculated for C28H50N2O14Na [M + Na]+ 661.3 found 661.3.2,5-Dioxopyrrolidin-1-yl 2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nonaoxa-5-azadotriacontan-32-oate, S,37S)-33,37,39-Tricarboxy-27,35-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,34,36-triazanonatriacontan-1-aminium trifluoroacetate 9: To a solution of the formate salt of 15  in CH2Cl2 (2 mL), triethylamine  was added, followed by 2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nonaoxa-5-azadotriacontan-32-oate, 20 . After 2 h of stirring at room temperature, the solvent was evaporated, and the residual was purified by a silica gel column (5% MeOH in CH2Cl2) to give the protected intermediate . A solution of TFA/H2O  was added to the protected intermediate . The mixture was kept at an ambient temperature for 2 h, then purified by Sep-Pak Vac 12 cc (Waters) using a gradient of water to acetonitrile/water of 3:7 (v:v). The fractions were collected and lyophilized to give compound 9 . 1H NMR  δ 4.12 , 4.07 , 3.76 , 3.65–3.70 , 3.17–3.20 , 2.48–2.50 , 2.41–2.44 , 2.06–2.10 , 1.88–1.92 , 1.76–1.79 , 1.65–1.69 , 1.49–1.53 , 1.35–1.38 . ESI-Mass calculated for C31H59N4O16+ [M + H]+ 743.4 found 743.3. in DMSO (0.1 mL), N,N-diisopropylethylamine  was added, followed by the NHS ester of DyLight800 . After stirring for 2 h at room temperature, the reaction mixture was purified by HPLC  to afford 0.4 mg (86%) of DyLight800-9. ESI-Mass calculated for C76H106N6O27S32− [M-2H]2− 815.3 found 815.1.DyLight800-S)-28-(4-Bromobenzyl)-33,37,39-tricarboxy-27,35-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,34,36-triazanonatriacontan-1-aminium trifluoroacetate 10: To a solution of compound 12  in CH2Cl2 (3 mL), triethylamine  was added, followed by 2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nonaoxa-5-azadotriacontan-32-oate, 20, . After stirring overnight at room temperature, the solvent was evaporated, and the residue was purified on a silica gel column (5% MeOH in CH2Cl2) to give the protected intermediate . To the protected intermediate , a solution of TFA/H2O  was added. The mixture was kept at ambient temperature for 2 h, then purified by HPLC  to give compound 10 . 1H NMR  δ 7.49–7.53 , 7.12–7.16 , 4.52–4.61 , 4.21 , 4.10 , 3.71–3.80 , 3.54–3.66 , 3.32–3.38 , 3.15–3.16 , 2.63–2.78 , 2.44–2.47 , 2.09–2.15 , 1.88–1.96 , 1.71–1.77 , 1.50–1.64 , 1.28–1.30 . ESI-Mass calculated for C38H64BrN4O16+ [M + H]+ 912.9 found 913.3. in DMSO (0.1 mL), N,N-diisopropylethylamine , followed by the NHS ester of DyLight800  was added. After stirring for 2 h at room temperature, the reaction mixture was purified by HPLC  to afford 0.3 mg (58%) of DyLight800-10. ESI-Mass calculated for C83H111BrN6O27S32− [M-2H]2− 899.3 found 899.1.DyLight800-4–10 are presented in the Synthetic details for the linkers used in compounds 50 values were determined as the concentration at which enzymatic activity was inhibited by 50%. Assays were performed in triplicate, with the entire inhibition study being repeated at least once to confirm the affinity and mode of inhibition. Enzyme inhibitory constants  were generated using the Cheng–Prusoff conversion [Cell lysates of LNCaP cell extracts were incubated with PSMA-targeted imaging agents (0.01 nM–100 μM) in the presence of 4 μM NAAG at 37 °C for 2 h. The amount of released glutamate from NAAG was measured by incubating with a working solution of the Amplex Red glutamic acid kit  at 37 °C for 60 min. Fluorescence was determined by reading with the Cytation 5 Cell Imaging Multi-Mode Reader  with excitation at 545 nm and emission at 590 nm . Inhibitnversion . Data an+ PC3 PIP and PSMA− PC3 flu cell lines were originally obtained from Dr. Warren Heston (Cleveland Clinic). Cells were grown to 80–90% confluence in a single passage before trypsinization and formulation in Hank’s balanced salt solution  for implantation into mice. PC3-ML-PSMA [6 PSMA+ PC3 PIP and PSMA− PC3 flu cells in opposite flanks. Mice were imaged when the tumor xenografts reached 3–5 mm in diameter. For the metastatic model of a PSMA-expressing prostate tumor, 1 × 106 PC3-ML-PSMA cells were injected intravenously into 4 to 6-week-old NSG (NOD/Shi-scid/IL-2Rγnull) mice . Four weeks post-injection of the cells, animals were used for imaging.PSMA-ML-PSMA  cells ma1 to DyLight800-10, 3–4 mice per compound), and images were acquired at 1 h, 2 h, 4 h and 24 h time points using a Pearl Impulse Imager . Following the 24 h image, each mouse was sacrificed by cervical dislocation, and tumor, muscle, liver, spleen, kidneys and stomach were collected and assembled on a petri dish for image acquisition. All images were scaled to the same intensity for direct comparison.After image acquisition at baseline (pre-injection), each mouse was injected intravenously with 1 nmol of DyLight800-urea conjugate  with 1:2 dilution in the presence of background reducing components . Slides were then stained with anti-mouse HRP linked antibody from EnVision plus system-HRP (DAB) . Processed slides were scanned by Johns Hopkins Oncology Tissue Services, and the images were analyzed using Aperio Image Scope software .t-test was performed to determine whether there were differences in PSMA+ PC3 PIP tumor uptake and the kidney and corresponding PSMA− PC3 flu tumor for DyLight800-1–10. Statistical significance was determined to be present at p < 0.01. Calculations were performed using GraphPad Prism 9 .An unpaired 1 and -2 (no linker) are presented in 3 through -10 (with linker) are shown in General syntheses of DyLight800-1–10 to produce the dye-PSMA inhibitors shown in Commercially available amine-reactive active NHS ester of DyLight800 with principal excitation/emission wavelength at 777/794 nm was conjugated with amines Ki values were determined using a modification of the Amplex Red glutamic acid assay [Ki values range from 0.10 to 2.19 nM, which is similar to other compounds of this class [The id assay  and are is class .1 to DyLight800-10 in mice with PSMA+ PC3 PIP and PSMA- PC3 flu tumors. All ten compounds demonstrated robust PSMA+ PC3 PIP tumor uptake and little uptake in PSMA- PC3 flu tumors, indicating target selectivity in vivo. Fluorescence intensities of PIP, flu, kidney and liver are shown in 3, DyLight800-4, DyLight800-5 and DyLight800-6 demonstrated the highest PSMA+ PIP tumor uptake. Comparing DyLight800-7 and DyLight800-9 with DyLight800-8 and DyLight800-10, it was demonstrated that the N-bromobenzyl substituent significantly increased the PSMA+ PIP tumor uptake. DyLight800-1 and DyLight800-2, which have no linker between the dye and Lys-Glu urea, had much lower PSMA+ PIP tumor uptake than other compounds with linkers, confirming the importance of the linker moiety for modifying pharmacokinetics. DyLight800-7, DyLight800-8, DyLight800-9 and DyLight800-10, which have PEG4 or PEG8 linker had significantly less kidney uptake compared to DyLight800-3, DyLight800-4, DyLight800-5 and DyLight800-6. While Dylight800-1, Dylight800-9 and DyLight800-10 showed the most favorable tumor/background, their absolute uptake levels were relatively low. Nevertheless, for renal surgery, one of them, particularly DyLight-10, may have the best properties  chain in the linker, improve pharmacokinetics. The presence of the N-bromobenzyl group increased PSMA+ PIP tumor uptake. We hope that inclusion of the N-bromobenzyl substituent will also promote a decrease in salivary gland uptake, as it has for our corresponding radiotheranostics [Although these agents are suggested for use in enhancing surgery, which means that in the context of prostate cancer, only local images in the tumor margin or of local-regional lymph nodes would be obtained, since PSMA is expressed in the neovasculature of many other tumors, it is important to minimize uptake in other organs, such as the kidney. For example, we have previously shown that we can target clear cell renal cell carcinoma (ccRCC) to good effect with a positron-emitting version of our PSMA-targeting ureas, DCFPyL (piflufolostat F 18) ,33,34, atic foci . That coanostics .One strategy to improve the performance of PSMA-targeting agents for FGS is to use a fluorescent molecular rotor , which p3 to YC-27 did not reveal a substantial difference in tumor uptake, although the latter had significantly lower renal uptake. The literature also provides little evidence for the superiority of DyLight800 over IRDye 800CW, and actually suggests the contrary in terms of quantum yield and brightness in certain in vitro assays [We undertook this study to optimize a PSMA-targeting NIR agent with respect to fluorophore and pharmacokinetics. Our qualitative, head-to-head comparison of DyLight800-o assays ,40. RemaN-bromobenzyl substituent promoted increased PSMA+ PIP tumor uptake, while a hydrophilic linker, particularly with the inclusion of PEG, may decrease nonspecific binding, particularly to the kidney, in this new series of PSMA-targeted NIR agents. Such modifications, perhaps coupled with what could be a truly superior flurophore [In conclusion, inclusion of an urophore , may ren


Some oxime ethers, namely thiazole and benzothiophene derivatives (24–27), also reduced HeLa cell viability at similar concentrations but with lower efficiency. Further cytotoxicity evaluation confirmed the specific toxicity of (E)-acetophenone O-2-morpholinoethyl oxime (22) against A-549, Caco-2, and HeLa cancer cells, with an EC50 around 7 μg/mL (30 μM). The most potent and specific compound was (E)-1-(benzothiophene-2-yl)ethanone O-4-methoxybenzyl oxime (27), which was selective for Caco-2 (with EC50 116 μg/mL) and HeLa (with EC50 28 μg/mL) cells. Considering the bioavailability parameters, the tested derivatives meet the criteria for good absorption and permeation. The presented results allow us to conclude that oxime ethers deserve more scientific attention and further research on their chemotherapeutic activity.Chemotherapy is one of the most commonly used methods of cancer disease treatment. Due to the acquisition of drug resistance and the possibility of cancer recurrence, there is an urgent need to search for new molecules that would be more effective in destroying cancer cells. In this study, 1-(benzofuran-2-yl)ethan-1-one oxime and 26 oxime ethers containing heterocyclic, alicyclic or aromatic moiety were screened for their cytotoxicity against HeLa cancer cell line. The most promising derivatives with potential antitumor activity were 2-(cyclohexylideneaminoxy)acetic acid ( The cancer diseases are a very serious health problem. Currently, about 18 million cases of various types of cancer are diagnosed each year. According to WHO predictions, this number is expected to reach 29.4 million in 2040 +) in positive ESI mode. The measurement results were processed with MassLynx 4.1 software (Waters).Solvents: chloroform, dimethylsulfoxide, ethyl acetate, ethyl alcohol, hexane, triethylamine—POCh, Poland .Reagents for synthesis: 2-acetylhiophene, 2,4-dichlorobenzyl chloride, 4-methoxybenzyl chloride, 2-chloroethylamine hydrochloride 99%, 3-chloropropylamine hydrochloride 98%, 2-chloro-N,N-diethylethylamine hydrochloride 99%, Sigma Aldrich Poznań Poland. 2-Acetylbenzothiophene 98% and 2-acetylthiazole 98% AmBeed .Auxiliary reagents: hydroxylamine hydrochloride, magnesium sulfate, potassium iodide, potassium hydroxide—POCh Poland .TLC: 5 cm × 10 cm TLC plates coated with silica gel with F-254 .13–15,  and DMSO (6 mL), an appropriate chloride (5.0 mmol), KI (0.10 g), and strongly pulverized KOH (20 mmol) were added + . Rf : 0.05.NMR and MS spectra of the tested compounds are available in E)-1-(thiophen-2-yl)ethanone O-2-(diethylamino)ethyl oxime (14)—Yield: 78%, orange oil. 1H-NMR , δ ppm, J Hz): 7.26 , 7.21 , 7.02 , 4.27 , 2.85 , 2.65 , 2.26 , 1.08 . 13C-NMR : 150.41 (CC=N), 140.62 (C), 126.89 (CH), 125.62 (CH), 125,91 (CH), 72.70 (OCH2), 51.31 (CH2), 47.86 , 12.98 (CH3), 11.87 (2 × CH3). HR-MS m/z 241.1373 [M + H]+ . Rf : 0.2.(E)-1-(thiophen-2-yl)ethanone O-3-aminopropyl oxime (15)—Yield: 76%, pale yellow oil. 1H-NMR , δ ppm, J Hz): 7.27 , 7.22 , 7.02 , 4.26 , 2.87 , 2.26 , 1.90–2.10 , 1.90 . 13C-NMR : 150.33 (CC=N), 140.63 (C), 126.89 (CH), 126.59 (CH), 125.90 (CH), 72.03 (OCH2), 39.20 (NCH2), 33.15, 12.85 (CH3). HR-MS m/z 199.0909 [M + H]+ . Rf : 0.05.(E)-1-(thiazol-2-yl)ethanone O-4-methoxybenzyl oxime (24)—Yield: 84%, yellow oil. 1H NMR: : δ (ppm) 7.84 , 7.38 , 7.30 , 6.93 , 5,21 , 3.83 , 2.41 . 13C NMR: : δ (ppm) 165.44 (C=N), 159.58 (C), 151.88 (C), 142.79 (CH), 130.21 (2 × CH), 129.35 (C), 120.07 (CH), 113.84 (2 × CH), 76.79 (CH2), 55.28 (OCH3), 12.14 (CH3). HR-MS m/z 263.0848 [M + H]+ . Rf : 0.85.(E)-1-(thiazol-2-yl)ethanone O-4-bromobenzyl oxime (25)—Yield: 69%, white solid, mp. 67–70 °C (ethanol). 1H NMR: : δ (ppm) 7.85 , 7.52 , 7.29–7.32 , 5.22 , 2.41 . 13C NMR: : δ (ppm) 165.05 (C=N), 152.39 (C), 142.87 (CH), 136.36 (C), 131.59 (2 × CH), 130.03 (2 × CH), 122.10 (C), 120.24 (CH), 76.12 (CH2), 12.18 (CH3). HR-MS m/z 310.9856 [M + H]+ . Rf : 0.85.(E)-1-(benzothiophene-2-yl)ethanone O-2,4-dichlorobenzyl oxime (26)—Yield: 51%, pale yellow solid, mp. 113–117 °C (ethanol). 1H-NMR: : δ (ppm) 7.79 , 7.77 , 7.48 , 7.44 , 7.35 , 7.29 , 5.34 , 2.38 . 13C NMR: : δ (ppm) 151.90 (C=N), 140.39 (C), 140.06 (C), 139.33 (C), 134.18 (CH), 134.11 (C), 130.82 (CH), 129.25 (CH), 127.05 (CH), 125.56 (CH), 124.39 (C), 123.92 (CH), 123.52 (CH), 122.26 (CH), 72.96 (CH2), 12.65 (CH3). HR-MS m/z 350.0172 [M + H]+ . Rf : 0.8.(E)-1-(benzothiophene-2-yl)ethanone O-4-methoxybenzyl oxime (27)—Yield: 51%, white solid, mp. 132–134 °C (ethanol). 1H NMR: : δ (ppm) 7.79 , 7.74 , 7.44 , 7.41 , 7.35 , 6.94 , 5.20 , 3.84 , 2.32 . 13C NMR: : δ (ppm) 159.41 (C=N), 150.99 (C), 140.97 (C), 140.01 (C), 139.44 (C), 130.27 (2 × CH), 129.66 (C), 125.38 (CH), 124.32 (CH), 123.83 (CH), 123.06 (CH), 122.23 (CH), 113.80 (2 × CH), 76.40 (CH2), 55.28 (OCH3), 12.61 (CH3). HR-MS m/z 312.1054 [M + H]+ . Rf : 0.7. and 1% penicillin/streptomycin. HDF cells were grown in DMEM  medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Caco-2 cells were grown in EMEM medium containing 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA) and 1% penicillin/streptomycin. HeLa cells were grown in DMEM  medium containing 10% fetal bovine serum (FBS) and 1% gentamycin. After thawing, cells were cultured until they reached sub-confluent state. The cells were then detached using 0.25% trypsin solution and seeded into 24-well or 96-well plates (3 × 103 cells per well) for further experiments. After 24 h growth, the tested compounds were added to the cell cultures in concentrations of 1, 10, 50, 100 and 250 μg mL−1, respectively, and incubated for the next 24 h or 72 h. The MTT test, based on the ability to reduce 3--2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenases, was performed in triplicate to assess cell metabolic activity and viability. The plates were then read spectrophotometrically at a wavelength of 570 nm. The dose-dependence curves were charted directly on x-/y-axes, with the drug dosages on the x-axis and the cell viability (percentage of viable cells) on the y-axis.2-Acetylbenzofuran, 2-acetylbenzothiophene, 2-acetylthiophene, 2-acetylthiazole, cyclohexanone and acetophenone derivatives were used for in vitro cytotoxicity assays with cervical adenocarcinoma (HeLa), human dermal fibroblasts (HDF), lung epithelial carcinoma (A549), and intestinal epithelial carcinoma (Caco-2) cell lines. A549, Caco-2, and HeLa cells were obtained from Sigma-Aldrich (Germany), and HDF from Biokom (Poland). All cell lines were cultured according to the manufacturer’s protocol under sterile conditions in the presence of 4.9% COAdditionally, the lactate dehydrogenase (LDH) activity test reflecting cell-membrane damage was performed for cells exposed to selected tested compounds. LDH activity was determined in the culture medium by measuring the decrease in NADH . The decrease in the amount of NADH was directly correlated to the increase in the number of damaged cells. To measure LDH activity, the culture medium was collected from the wells with cells exposed to concentrations equal to the EC50 values of tested compounds after short- (24 h), and long-time (72 h) exposure. The LDH activity test was performed as follows: 25 μL of NADH (2.5 mg/mL) and 25 μL of sodium pyruvate (2.5 mg/mL) were added to 150 μL of the collected culture medium. Absorbance at 340 nm was spectrophotometrically measured. The number of damaged cells was compared to the untreated control sample, also considering the positive control treated with 1% Triton X-100 as 100% damaged cells.18) and (E)-acetophenone O-2-morpholinoethyl oxime (22), which reduced the viability of HeLa cells to below 20% of control at concentrations over 100 μg/mL. However, in further evaluations of cytotoxic influence, compound (18) showed higher toxicity against normal cells than cancerous cells, a common feature of many molecules that results from the lower sensitivity of cancer cells. We confirmed the specific toxicity of acetophenone derivative (22) against the cancer cell lines . From another group of oxime ether derivatives, compound 27 exhibited specific toxicity against Caco-2 and HeLa cells with EC50 values of 370 µM and 90 µM, respectively. Comparison of 22 and 27 modes of action revealed that they initiate different processes, leading to reduced proliferation and cell death. Considering the bioavailability parameters, the tested derivatives met the criteria for good absorption and permeation, and can be further investigated.In conclusion, 1-(benzofuran-2-yl)ethan-1-one oxime and 26 oxime ethers containing benzofuran, benzothiophene, thiophene, thiazole, cyclohexyl or phenyl moiety were screened for cytotoxicity against the human cervical adenocarcinoma (HeLa cell line) representative cancer cell model. The most promising derivatives were 2-(cyclohexylideneaminoxy)acetic acid (


Olyra latifolia L. leaves. This plant, known to have several therapeutic properties, belongs to the family of Poaceae and is found in the neotropics and in tropical Africa and Madagascar. Among the purified compounds, feddeiketone B, 2,3-dihydroxy-1--l-propanone, and syringylglycerol were shown to present structural analogy with DANA, and their effects on membrane NEU-1 sialidase activity were evaluated. Our results show that they possess inhibitory effects against NEU-1-mediated sialidase activity at the plasma membrane. In conclusion, we identified new natural bioactive molecules extracted from Olyra latifolia as inhibitors of human NEU-1 of strong interest to elucidate the biological functions of this sialidase and to target this protein involved in several pathophysiological contexts.Sialidases, also called neuraminidases, are involved in several human pathologies such as neurodegenerative disorders, cancers, as well as infectious and cardiovascular diseases. Several studies have shown that neuraminidases, such as neuraminidase 1 (NEU-1), may be promising pharmacological targets. Therefore, the discovery of new selective inhibitors of NEU-1 are necessary to better understand the biological functions of this sialidase. In the present study, we describe the isolation and characterization of nine known compounds from  Neuraminidase 1 (NEU-1) is an exoglycosidase which removes terminal sialic acid residues from glycoproteins, glycolipids, and oligosaccharides. Sialidases are widely distributed among species . For exaAt present, there are no commercially available inhibitors that are selective for NEU-1, especially due to the lack of structural data. Indeed, only the crystallographic structure of NEU-2 was determined . NEUs arOlyra latifolia L. is a cane-like grass, thin woody culms which can be up to 4 m erected, more or less scandent, from a brief rhizome, and belongs to the family of Poaceae. O. latifolia is distributed in the neotropics, from the Southeast of the United States, passing through Central America to the Caribbean in South America and is also found in tropical Africa and Madagascar +; 1H-NMR : 7.05 , 6.82 , 3.44 , 3.43 , 3.74 , 3.70 ; 13C-NMR : 130.4 (C-1), 111.4 (C-2), 149.0 (C-3), 138.4 (C-4), 152.7 (C-5), 106.0 (C-6), 198.1 (C-7), 43.8 (C-8), 58.6 (C-9), 55.7 (3-OCH3), 60.7 (4-O CH3). The structure was determined from above data according to literature +; 1H-NMR : 7.38 , 5.15 , 3.99 , 3.70 ; 3.95 ; 13C-NMR : 124.0 (C-1), 106.0 , 148.0 , 142.0 (C-4), 198.0 (C-7), 74.0 (C-8), 65.0 (C-9), 55.5 . The structure was determined from the above data according to literature +; 1H-NMR : 6.58 , 4.95 , 3.70 , 3.40 , 3.55 , 3.74 ; 13C-NMR : 134.1 (C-1), 104.1 , 147.4 , 133.4 (C-4), 72.9 (C-7), 75.8 (C-8), 62.5 (C-9), 55.8 . The structure was determined from the above data according to literature +; 1H-NMR : 6.70 , 8.00 , 6.95 , 6-C-glc: 4.88 , 3.70 , 3.60 , 3.63 , 3.51 , 3.84 , 3.80 , 8-C-glc: 4.75 , 4.15 , 3.51 , 3.70 , 3.35 , 3.91 , 3.78 ; 13C-NMR : 165.6 (C-2), 102.2 (C-3), 183.0 (C-4), 161.8 (C-5), 109.6 (C-6), 163.3 (C-7), 102.9 (C-8), 156.2 (C-9), 105.6 (C-10), 122.0 (C-1′), 128.0 , 115.8 , 161.0 (C-6′), 6-C-glc: 73.0 (C-1), 72.2 (C-2), 77.6 (C-3), 69.8 (C-4), 81.0 (C-5), 60.0 (C-6), 8-C-glc: 74.0 (C-1), 72.2 (C-2), 78.2 (C-3), 71.0 (C-4), 81.6 (C-5), 61.5 (C-6). The structure was determined from the above data according to literature +; 1H-NMR : 7.9 , 6.8 ; 13C-NMR : 121.5 (C-1), 131.6 , 114.6 , 161.9 (C-4), 168.9 (C-7). The structure was determined from the above data according to literature +; 1H-NMR : 7.90 , 6.85 , 9.80 ; 13C-NMR : 128.0 (C-1), 132.0 , 115.0 , 163.5 (C-4), 191.0 (C-7). The structure was determined from the above data according to literature +; 1H-NMR : 2.48 , 1.75 , 4.23 , 2.10 , 1.5 , 5.77 , 1.76 , 1.49 , 1.3 ; 13C-NMR : 184.3 (C-1), 87.6 (C-2), 45.0 (C-3), 65.8 (C-4), 46.6 (C-5), 35.8 (C-6), 111.9 (C-7), 173.0 (C-8), 26.0 (2-Me), 25.6 (6a-CH3), 29.6 (6-CH3). The structure was determined from the above data according to literature +; 1H-NMR : 6.04 , 7.40 , 7.18 , 7.27 , 3.66 , 4.86 , 3.94 ; 13C-NMR : 158.9 (C-2), 90.4 (C-3), 164.8 (C-4), 119.9 (C-4a), 113.4 (C-5), 150.0 (C-6), 114.9 (C-7), 125.9 (C-8), 129.9 (C-8a), 39.9 (N-CH3), 55.8 (4-OCH3), 55.6 (6-OCH3). The structure was determined from the above data according to literature +; 1H-NMR : 5.90 , 2.55 , 2.18 , 5.88 , 5.86 , 4.42 , 1.32 , 1.94 , 1.20 , 1.26 , Glc: 4.45 , 3.43 , 3.47 , 3.22 , 3.25 , 3.87 , 3.63 , Rha: 5.26 , 3.92 , 3.67 , 3.38 , 4.10 , 1.27 ; 13C-NMR : 199.8 (C-1), 125.8 (C-2), 165.8 (C-3), 78.1 (C-4), 41.1 (C-5), 49.3 (C-6), 130.2 (C-7), 133.9 (C-8), 75.8 (C-9), 19.8 (C-10), 18.2 (C-11), 23.3 (12-CH3), 22.0 (13-CH3), Glc: 99.8 (C-1), 77.3 (C-2), 78.0 (C-3), 70.5 (C-4), 76.6 (C-5), 61.5 (C-6), Rha: 100.6 (C-1), 70.8 (C-2), 70.9 (C-3), 72.6 (C-4), 68.3 (C-5), 16.7 (C-6). The structure was determined from the above data according to literature [1), 2,3-dihydroxy-1--1-propanone (compound 2), syringylglycerol (compound 3), and the reference DANA molecule were evaluated in COS-7 and THP-1 cell lines. For all these compounds, no cell toxicity was noticed both in COS-7   b. A trea control b. Simila control c. Given  control d. Moreov control e. Altogek-elastin stimulation of THP-1-derived macrophages is dependent of NEU-1 [k-elastin stimulation (50 µg/mL), NEU-1 sialidase activity of macrophages increases by 54.1 ± 9.0% compared to non-stimulated cells for an incubation period of 15 min and by 51.5 ± 26.9% for an incubation period of 30 min (not shown). k-elastin stimulated sialidase activity decreases in presence of DANA and the vegetal compounds after 15 min of incubation at 0.1 µM. Indeed, in presence of compounds 1–3 and DANA, decreases of k-elastin induced NEU-1 sialidase activity of 32.3 ± 7.3, 28.9 ± 6.9, 25.2 ± 7.7, and 27.7 ± 6.4% are observed, respectively  were shown to possess structural analogy with DANA: the feddeiketone B, the 2,3-dihydroxy-1--1-propanone, and the syringylglycerol. Two cell models have been used in the present work, COS-7 cells which overexpressed NEU-1 and THP-1-derived macrophages which endogenously express this target protein. For all these compounds and DANA, no significant effect on cell toxicity was observed in the two cell lines after 2 or 3 h of incubation at 0.1 and 1 µM. Therefore, we studied the effects of these compounds on NEU-1 sialidase activity in COS-7 cells and in THP-1-derived macrophages at this concentration range. In all experiments, final percentages of DMSO used were under 0.03% in order to avoid cell toxicity. Our results suggest that compounds 1–3 are able to decrease significantly NEU-1 mediated sialidase activity in both cell models, but the inhibitory effects were much greater in THP-1 cells than in COS-7 for all the tested concentrations (0.1 and 1 µM). These results might be explained by the fact that in COS-7 cells, NEU-1 is overexpressed and, therefore, the ratio between the NEU-1 expression level and the compound concentrations is higher in these cells compared to THP-1 cells. Kawecki et al. have previously shown that incubation with k-elastin significantly increased sialidase activity at the plasma membrane and that silencing NEU-1 by siRNA completely blocked the effects of k-elastin: these data definitely demonstrated involvement of NEU1 in k-elastin-induced sialidase activity at the plasma membrane of THP-1 cells [k-elastin: these experiments suggest that these 3 compounds are able to directly inhibit human NEU-1, the ERC catalytic subunit, after activation of its sialidase activity by k-elastin. In addition to these results, we observed that the inhibitory effects of the three tested compounds extracted from Olyra latifolia are very similar to those obtained with DANA in the same experimental conditions which indicate that these natural molecules are potent inhibitor of NEU-1 as well as DANA. The glycerol lateral chain in syringylglycerol (3) and DANA seems to be important for the activity or at least a ketone and a primary alcohol group on a C3-substituant. In this preliminary study, we tested the effects of these three compounds at very low concentrations (0.1 and 1 µM). Indeed, these compounds are solubilized in DMSO which cannot be used at higher concentrations due to its toxicity. However, the effects of these molecules obtained at low concentrations are very promising and suggest that at higher concentrations (which requires less toxic solvents for solubilization), similar to those classically used for DANA, these inhibitory effects could be more important. Consequently, these vegetal compounds could be used as natural putative inhibitors of NEU-1 and could serve as models to design more potent synthetic inhibitors. Furthermore, as these compounds appear to be effective in inhibiting membrane NEU-1 in vitro, they could be used in in vivo models to inhibit this protein involved in several diseases such as atherosclerosis, thrombosis, insulin resistance, non-alcoholic steatohepatitis, and cancer.The key role of NEU-1 in the degradation of sialyloconjugates in lysosomes has been known for many years , but itsoenzymes . In factoenzymes . Furtheroenzymes . The aimic wound  and this-1 cells . Our res1–9) isolated for the first time from O. latifolia leaves. Among them, compounds 1–3—having structural analogy with DANA—showed inhibitory effects against human NEU-1-mediated sialidase activity at the plasma membrane. The effects of these molecules obtained at low concentrations are very promising and suggest that at higher concentrations, similar to those classically used for DANA, these inhibitory effects could be more important. Thus, these compounds are considered as new natural bioactive inhibitors of human NEU-1 which may be further exploited as potential leads to elucidate the biological functions of this sialidase and to target this protein involved in several pathophysiological contexts. Consequently, this preliminary study predicted an inhibitory potential of these vegetal compounds that could be a starting point for the development of new natural putative inhibitors of NEU-1 and for the design of more potent synthetic inhibitors . Furthermore, in order to better characterize compounds 1–3 and their possible use as tools for a better understanding of sialic acid biology, it could be interesting to evaluate their effect on another plasma membrane associated sialidase such as NEU-3.but also on two other members of sialidase family, NEU-2 and NEU-4. Last, as these compounds appear to be effective in inhibiting membrane NEU-1 in vitro, they could be used in in vivo models to down-regulate this protein involved in several diseases such as atherosclerosis, thrombosis, insulin resistance, non-alcoholic steatohepatitis, and cancer.In this preliminary study, we report the isolation and characterization of nine compounds (


Saccharomonospora sp. CNQ-490 has yielded three novel compounds: saccharobisindole (1), neoasterric methyl ester (2), and 7-chloro-4(1H)-quinolone (3), in addition to acremonidine E (4), pinselin (5), penicitrinon A (6), and penicitrinon E (7). The chemical structures of the three novel compounds were elucidated by the interpretation of 1D, 2D nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) data. Compound 2 generated weak inhibition activity against Bacillus subtilis KCTC2441 and Staphylococcus aureus KCTC1927 at concentrations of 32 μg/mL and 64 μg/mL, respectively, whereas compounds 1 and 3 did not have any observable effects. In addition, compound 2 displayed weak anti-quorum sensing (QS) effects against S. aureus KCTC1927 and Micrococcus luteus SCO560.Analysis of the chemical components from the culture broth of the marine bacterium  Streptomyces, the largest genus of actinomycetes +  indicating 14 degrees of unsaturation. The infrared (IR) spectrum of this compound indicated the presence of a hydroxy group at 3431 cm−1, a carbonyl group at 1676 cm−1, and a double bond at 1638 cm−1. Saccharobisindole , H-8 , H-9 , H-6′ , H-8′ , and H-9′ , four methyl singlets H-13 , H-14 , H-13′ , and H-14′ , two triplet olefinic proton signals H-11  and H-11′, two doublet protons H-10  and H-10′ , and one singlet proton H-3 . Furthermore, three exchangeable protons at NH-1 (δH 10.08), NH-1′ (δH 10.22), and 3′-OH (δH 6.40) were also observed in the 1H NMR spectrum. The analysis of 13C NMR and heteronuclear single quantum coherence (HSQC) spectroscopic data indicated two carbonyl C-2 (δC 174.6) and C-2′ (δC 177.3), eight sp2 quaternary C-4 (δC 123.2), C-5 (δC 143.4), C-7 (δC 141.8), C-12 (δC 131.8), C-4′ (δC 125.8), C-5′ (δC 142.9), C-7′ (δC 143.1), and C-12′ (δC 131.9), six aromatic methine C-6 (δC 108.8), C-8 (δC 120.9), C-9 (δC 126.2), C-6′ (δC 109.4), C-8′ (δC 120.8), and C-9′ (δC 123.5), two olefinic C-11 (δC 123.2) and C-11′ (δC 122.9), two methylene C-10 (δC 33.8) and C-10′ (δC 33.7), and four methyl singlet carbons C-13 (δC 25.5), C-14 (δC 17.7), C-13′ (δC 25.4), and C-14′ (δC 17.6), along with one sp3 methine group C-3 (δC 51.1) and one sp3 quaternary carbon C-3′ (δC 75.4) (The δC 75.4) .1. Two sets of indolin-2-one moieties were identified by the interpretation of COSY (COrrelated SpectroscopY) and HMBC spectral data. The COSY cross peaks H-6/H-8/H-9 and H-6′/H-8′/H-9′ and the long-range HMBC correlations H-8 (δH 6.80)/ C-4 (δC 123.2), C-6 (δC 108.8); H-9 (δH 7.35)/ C-5 (δC 143.4), C-7 (δC 141.8); singlet aromatic proton H-6 (δH 6.55)/ C-4, C-8 (δC 120.9); H-8′ (δH 6.45)/ C-4′ (δC 125.8), C-6′ (δC 109.4); H-9′ (δH 6.09)/ C-5′ (δC 142.9), C-7′(δC 143.1); and H-6′ (δH 6.53)/ C-4′, C-8′(δC 120.8) confirmed the presence of two sets of 1,2,4-trisubstituted benzene moieties. Furthermore, two sets of 3-methylbut-2-enyl moieties were also identified from the interpretation of COSY and log-range HMBC analyses. COSY crosspeaks H-10/H-11 and H-10′/H-11′ and long-range HMBC correlations H-13 (δH 1.72) and H-14 (δH 1.69)/C-11 (δC 123.2), C-12 (δC 131.8); H-10 (δH 3.30)/ C-11 (δC 123.2), C-12 (δC 131.8); H-13′ (δH 1.68) and H-14′ (δH 1.64)/ C-11′ (δC 122.9), C-12′(δC 131.9); and H-10′ (δH 3.19)/ C-11′ (δC 122.9), C-12′ (δC 131.9) allowed for the assignment of two sets of 3-methylbut-2-enyl units.Further interpretation of 2D NMR spectroscopic data including two-bond HMBC (heteronuclear multiple bond correlation) analysis allowed for the structural construction of δC 141.8), and from H-6 (δH 6.55) and H-8 (δH 6.80) to C-10 (δC 33.8) allowed for the attachment of a 3-methylbut-2-enyl group at C-7 of the benzene ring system. Further, HMBC correlations from the exchangeable NH-1 proton (δH 10.08) to C-5 (δC 143.4) and the carbonyl carbon C-2 (δC 174.6), and from the methine proton H-3 (δH 3.91) to C-2, C-4 (δC 123.2), and C-5, allowed for the assignment of an indolin-2-one moiety with a 3-methylbut-2-enyl group. Similarly, the other indolin-2-one moiety with a 3-methylbut-2-enyl group was also assigned by interpretation of HMBC correlations. Briefly, HMBC correlations of H-10′ (δH 3.19)/ C-7′ (δC 143.1); H-6′ (δH 6.53) and H-8′ (δH 6.45)/ C-10′ (δC 33.7); and NH-1′/ C-3′ (δC 75.4), C-4′ (δC 125.8), C-5′ (δC 142.9) provided further evidence for the assignment of an indolin-2-one moiety with a 3-methylbut-2-enyl group. A hydroxy group at δH 6.40 was attached to C-3′ based on HMBC correlations from this exchangeable proton to C-3′ and C-4′, as well as the consideration of the carbon chemical shift of C-3′ (δC 75.4). Lastly, the connection between C-3 and C-3′ of two indolin-2-one moieties was secured from the observation of HMBC correlations from H-3 to C-3′, thus completing the assignment of 1 (1 were deduced from the interpretation of nuclear Overhauser effect spectroscopy (NOESY) data. The NOESY correlation of 1 between H-3 and the 3′-OH protons indicated that the proton and hydroxy group should be placed at the same side of the rings  modeling. The possible enantiomers of compound 1 were selected based on NOESY NMR data. The observed ECD spectrum of compound 1 showed a positive Cotton Effect around 291 nm. Comparison of the calculated ECD data of the two enantiomers with that measured, allowed the absolute configurations of compound 1 to be assigned as R, S  was isolated as a yellowish oil and its molecular formula was assigned as C17H16O8 as determined by high-resolution fast atom bombardment mass spectrometry which illustrated a [M+H]+m/z value of 348.0849 , (HRFABMS). The IR absorptions at 3320 cm−1 and 1660 cm−1 indicated hydroxy and carbonyl functionalities, respectively. The 1H NMR spectrum showed bands for a 1,2,3,5-tetrasubstituted phenyl unit with two protons at H-2  and H-4 , a 1,2,3,4-tetrasubstituted phenyl group at H-2′  and H-3′ , two methoxy groups H-8  and H-8′ , and one methyl singlet H-9 . The 1H, 13C and HSQC spectroscopic data revealed one methyl C-9 (δC 21.4), two methoxys C-8 (δC 51.8) and C-8′ (δC 51.8), two carbonyls C-7 (δC 166.9) and C-7′ (δC 165.5), four aromatic methines C-2 (δC 105.1), C-4 (δC 109.7), C-2′ (δC 113.3), and C-3′ (δC 118.9), and eight quaternary carbons C-1 (δC 156.3), C-3 (δC 141.1), C-5 (δC 156.0), C-6 (δC 107.2), C-1′ (δC 142.2), C-4′ (δC 138.0), C-5′ (δC 147.7), and C-6′ (δC 116.6) (Neoasterric methyl ester (C 116.6) .2 to be assigned (δH 5.77) to C-1 (δC 156.3), C-4 (δC 109.7), and C-6 (δC 107.2) and from H-4 (δH 6.32) to C-1 and C-2 (δC 105.1) supported the presence of a 1,2,3,5 substituted benzene moiety. Furthermore, the observation of HMBC correlations from the exchangeable proton 5-OH (δH 10.06) to C-4, C-5 (δC 156.0), and C-6 permitted the C-5/5-OH connectivity. HMBC correlations from the methyl singlet protons H-9 (δH 2.08) to C-2, C-3 (δC 141.1) and C-4 indicated that a methyl group was positioned at C-3. A methoxy group was then assigned as part of a methyl ester located at C-7 (δC 166.9) according to analysis of the three-bond HMBC correlation from H-8 (δH 3.75) to C-7. The long-range HMBC correlation from H-4 to C-6 (δC 107.2) and C-7 (δC 166.9) with the typical carbon chemical shift of C-6 (δC 107.2) suggested the attachment of C-6 to C-7. The ortho-coupled protons  displayed the COSY cross peaks H-2′/H-3′ and the HMBC correlations with C-1′ (δC 142.2), C-5′ (δC 147.7), and C-6′ (δC 116.0) and C-1′, C-4′ (δC 138.1), and C-5′, respectively. Based on these correlations, a 1,2,3,4 substituted aromatic moiety was established. The two exchangeable protons of 1′-OH (δH 9.56) and 4′-OH (δH 9.70) were placed at C-1′ and C-4′, respectively, based on the observation of HMBC correlations from 1′-OH to C-1′, C-2′ (δC 113.3), and C-6′ and from the 4′-OH to C-3′ (δC 118.9), C-4′, and C-5′. The HMBC correlation from the methoxy protons H-8′ to C-7′ (δC 165.5) suggested the presence of a methyl ester moiety and this functional group was placed at C-6′ (δC 116.6) based on the identification of a long-range HMBC correlation from H-2′ (δH 6.64) to C-6′. Lastly, the linkage of two aromatic rings through an oxygen atom (C-5′/O/C-1) was deduced from the typical oxygenated carbon chemical shifts of C-1 (δC 156.3) and C-5′ (δC 147.7). Compound 2 is an analog of asterric acid. Methyl asterrate, an asterric acid derivative, is most structurally similar to 2 except for the hydroxy group at C-4′ and the ortho-coupled protons  +  in the HRFAB mass spectrum. The 1H NMR spectrum of 3 displayed two deshielded olefinic protons H-2  and H-3 , and a 1,3,4-trisubstituted benzene ring with protons at H-5  and H-6 , and H-8 . The 13C NMR spectrum exhibited nine carbons C-2 (δC 142.2), C-3 (δC 110.3), C-4 (δC 179.8), C-4a (δC 125.1), C-5 (δC 128.3), C-6 (δC 126.1), C-7 (δC 142.2), C-8 (δC 118.9), C-8a (δC 139.8). A quinolone moiety was identified based on the interpretation of COSY and HMBC spectroscopic data. The COSY correlation of H-2 (δH 8.01) with H-3 (δH 6.37) and long-range HMBC correlations from H-2 to C-4 (δC 179.8) indicated the presence of an α,β-unsaturated carbonyl group. Furthermore, three-bond HMBC correlations from H-2 to C-8a and from H-3 to C-4a, along with the carbon chemical shifts of C-2 (δC 142.2) and C-8a (δC 142.2), and correlations from H-6 to C-4a (δC 125.1) and C-8 (δC 118.9), and from H-5 to C-4, C-7 (δC 142.2) and C-8a (δC 139.8) allowed for the construction of the quinolone moiety. The carbon chemical shift at δC 142.2 and the isotope ratio (3:1) of two pseudomolecular ion peaks [M + H]+ and [M + H + 2]+ in the LR-ESI-MS spectroscopic data (3 was assigned as 7-chloro-4(1H)-quinolone  due to its capacity to damage DNA and block DNA synthesis in tumor cells + .Saccharobisindole (2): Yellowish oil, UV (MeOH) λmax (log ε) 216 (2.42), 262 (1.93), 397 (1.65) nm; IR (KBr) νmax 3320, 1660, 1616, 1439, 1314, 1201, 1077 cm−1, 1H and 13C NMR data: m/z 348.0849 [M+H]+ .Neoasterric methyl ester (3): White amorphous powder, UV (MeOH) λmax (log ε) 218 (2.58), 278 (1.87), 279 (1.77) nm; IR (KBr) νmax 3326, 1729, 1457, 802 cm−1, 1H and 13C NMR data: + .7-Chloro-4(1H)-quinolone (4): 1H NMR: ; δH 11.36 , 11.36 , 9.70 , 9.18 , 6.94 , 6.80 , 6.10 , 6.10 , 3.54 , 2.16 , 13C ; δC 180.2 (C-8), 167.9 (C-7′), 161.5 (C-2), 161.5 (C-6), 151.1 (C-1′). 147.7 (C-4), 145.6(C-4′), 131.2 (C-3′), 122.1 (C-5′), 117.4 (C-6′), 112.3 (C-2′), 108.9 (C-1), 107,4 (C-3), 107.4 (C-5), 51.9 (C-8′), 21.6 (C-7), LR-ESI-MS m/z 319.1 [M+H]+.Acremonidine E (5): 1H NMR:; δH 12.18 , 10.47 , 7.45 , 7.60 , 6.89 , 6.65 , 3.84 , 2.39 ; 13C ; δC 180.2 (C-8), 166.8 (C-7′), 160.4 (C-2), 155.4 (C-6), 150.7 (C-1′), 149.3 (C-4), 148.8 (C-4′), 125.3 (C-5′), 120.1 (C-6′), 117.2 (C-3′), 117.0 (C-2′), 110.7 (C-3), 107.4 (C-5), 106.0 (C-1), 52.2 (C-8′), 22.0 (C-7), LR-ESI-MS m/z 301.1 [M+H]+.Pinselin (6): 1H NMR: ; δH 8.36 , 6.68 , 5.38 , 4.71 , 3.48 , 3.36 , 2.30 , 2.22 , 1.45 , 1.42 , 1.35 , 1.34 , 13C ; δC 177.8 (C-6), 167.5 (C-1), 160.3 (C-8), 150.7(C-6′), 145.6 (C-4′), 138.5 (C-7′a), 137.4 (C-5), 128.8 (C-4a), 120.1 (C-5′), 105.7 (C-7′), 102.7 (C-8a), 101.6 (C-7), 89.0 (C-2′), 86.3 (C-3), 46.3 (C-3′), 45.8 (C-4), 21.2 (2′-CH3), 19.5 (3′-CH3), 18.9 (3-CH3), 18.8 (4-CH3), 12.1(5′-CH3), 10.7 (5-CH3), LR-ESI-MS m/z 381.1 [M+H]+.Penicitrinon A (7): 1H NMR: ; δH 5.17 , 4.77 , 3.29 , 3.25 , 2.28 , 2.22 , 1.49 , 1.47 , 1.38 , 1.36 , LR-ESI-MS m/z 425.1 [M+H]+.Penicitrinon E  and three Gram-negative bacteria  following the recommendations of previous studies. The bacterial inoculum was prepared in Muller-Hinton broth at 37 °C and 225 rpm for 24 h. The stock solution of compounds 1–7 and the positive control were dissolved at a 10 mg/mL concentration in DMSO and diluted with Muller-Hinton broth to obtain a 0.25–256 µg/mL concentration range. Bacterial inoculum (5 × 105 CFU/mL concentration) was then dispensed into each well. The resulting mixtures consisting of varying concentrations of compounds 1–7 with a 1 × 106 CFU/mL concentration of bacterial inoculum were transferred to 96 well microtiter plates and incubated at 37 °C for 24 h.For the antibacterial susceptibility assays, compounds C. marina JEA023, M. luteus SCO560, S. aureus KCTC1927, P. aeruginosa SNC165, P. fluorescens SNA239, and A. tumefaciens SND195). S. aureus KCTC1927 and A. tumefaciens SND195 were cultured in tryptic soy broth (TSB), whereas P. aeruginosa SNC165 and P. fluorescens SND204 were cultured in King’s broth (KB). C. marina JEA023 and M. luteus SCO560 were cultured in marine broth maintaining a 0.5 McFarland standard turbidity (1.0 × 108 CFU/mL). Dimethyl sulfoxide (100% DMSO) was used as a negative control, and kanamycin and rifampin (10 mg/mL each) were used as a positive control. Next, 50 µL of inoculum was inoculated into each well and 10 mg/mL of compound 2 and 4–7 dissolved in DMSO was diluted with marine broth media to produce concentrations ranging from 0.25 to 256 µg/mL. The mixtures of compounds and broth were dispensed into each inoculated well. The 96-well plates were then incubated for 16–18 h at 37 °C depending on the bacterial strains.The 96-well plate method was employed to detect the anti-quorum sensing activity of different compounds against six bacterial strains (1) was carried out by MacroModel with the Merck molecular force field (gas phase), a 10 kJ/mol upper energy limit, and a 0.001 kJ (mol Å)−1 convergence threshold on the rms gradient to minimize computational complexity and expense. The possible enantiomers of 1 were selected based on NOESY NMR data and the energy-minimized enantiomer structures were generated by Avogadro 1.2.0. Energy minimization of the two structures was performed by Turbomole X 4.3.2. The calculated ECD spectra corresponding to two enantiomer models were calculated using DFT at the functional B3LYP/DFT level and the def-SV(P) basis set. The ECD spectra were simulated by overlapping each transition, where σ is the width of the band at 1/e height. ∆Ei is the excitation energies and Ri is rotatory strengths for transition i. In this calculation, the σ value was at 0.10 eV. The observed ECD spectrum of compound 1 showed positive cotton effect around 291 nm. Comparing the calculated spectra of the two enantiomers with the measured ECD spectrum, the absolute configurations of compound 1 were deduced as R, S.A conformational analysis of saccharobisindole (1), neoasterric methyl ester (2), and 7-chloro-4(1H)-quinolone (3), in addition to four known compounds. Compound 1 is an isatin derivative and 2 contains two substituted benzene methyl esters with an ether linkage. Compound 3 possesses a chloroquinolone moiety and our study was the first to isolate this compound from a natural source. Additionally, our study was the first to isolate compounds 4–7, reported as fungal metabolites, from a bacterial strain. Compound 2 exhibited antibacterial activity against B. subtilis KCTC 1021 and S. aureus KCTC 1927, as well as a weak QS inhibitory activity against S. aureus KCTC 1927.The identification of the chemical components produced by strain CNQ-490 has led to the discovery of the new compounds saccharobisindole (


All structures were unambiguously elucidated based on 1D and 2D NMR spectra as well as by HRESIMS data. The absolute configuration of sesquiterpenoids was determined by comparison of the experimental and computed ECD spectra. In vitro anti-inflammatory assessment showed that compound 9 exhibited inhibition of NO production in LPS-stimulated RAW264.7 cells with an IC50 value of 62.22 ± 1.27 μM.Nine new sesquiterpenoids ( Aquilaria and Gyrinops trees as a self-treatment mechanism to suppress various forms of injury, such as chopping, holing, nailing, microbial infection, etc. octane core and acetal group at C-4. The relative configuration of 3 was deduced by NOEs between H-11/Me-15, H-11/H-12a (δH 4.02), H-11/H-8, H-6/H-10b (δH 1.63), H-6/Me-14, H-2/H-3b (δH 1.59), H-4/H-3b, and H-3a (δH 2.25)/H-10a (δH 2.14) as shown .6 The existence of 12-OH was confirmed by COSY correlations between H2-12/H-2 (δH 2.32)/H2-3 (δH 2.40 and 2.10) together with HMBC correlations from H2-12 to C-1 (δC 52.0), C-2 (δC 42.1) and C-3 (δC 43.8)  . The rem spectra .5 had the molecular formula C15H22O3 according to HRESIMS, indicating that the addition of one further oxygen compared to 4. The NMR data of 5 . The attachment of hydroxy group at C-11 was elucidated by key HMBC correlations from H-11 to C-9 (δC 23.1) and C-10 (δC 27.7). The NOE cross-peaks from H-11 to H-2 (δH 2.44), H-8 (δH 1.83) and Me-14 (δH 1.23) indicated a cis relationship between H-8 and H-11. The remaining structure of 5 was shown to be identical to that of 4 by 2D NMR analysis.Compound ata of 5  were ide6 was isolated as a white amorphous powder. The molecular formula of C15H24O2 was deduced from its HRESIMS, differing from 5 by the loss of one oxygen and the addition of two extra protons. Comparison of its 1H and 13C NMR data  and the disappearance of keto group in 5, which was confirmed by COSY correlations between H2-12 (δH 3.63 and 3.45)/H-2 (δH 2.12)/H2-3 (δH 1.75 and 1.30)/H2-4. The remaining structure of 6 was shown to be identical to that of 5 by detailed analysis of the 2D NMR spectra of 6.Compound NMR data  with tho7 was obtained as a colorless oil. It possessed the molecular formula C16H26O2 as determined by HRESIMS. Its 1H and 13C NMR spectra were similar to those of 4. The appearance of a methoxy group  in 7 and the absence of keto group at C-4 in 4 suggested the methoxy group located at C-4, which was confirmed by COSY correlations between H2-12 (δH 3.76 and 3.62)/H-2 (δH 2.25)/H2-3 (δH 2.09 and 1.34)/H-4 (δH 4.12) together with the HMBC correlations from the methoxy singlet to C-4 (δC 79.7). Detailed analysis of the 2D NMR spectra of 7 determined the compound to be identical to 4 except for the methoxy group at C-4. The coupling constant of H-4  was comparable to that of (4S)-albaflavenol , but differed from that in (4R)-albaflavenol , suggesting the trans configuration between H-2 and H-4 .,14.13,141–9) were tested for α-glucosidase inhibition and anti-inflammatory activities in vitro. Acarbose was used as a positive control for α-glucosidase inhibition with an IC50 value of 743.4 ± 3.3 μM. Quercetin and indomethacin were used as positive controls for anti-inflammatory activity with IC50 values of 8.22 ± 0.80 μM and 35.40 ± 1.77 μM, respectively. However, none of them exhibited α-glucosidase inhibition activity. Only compound 9 exhibited weak inhibition of NO production in LPS-stimulated RAW264.7 cells with an IC50 value of 62.22 ± 1.27 μM , their effects on cell viability also had been measured using the MTT method. None of them (up to 100 μM) showed cytotoxicity with LPS treatment.All isolated sesquiterpenes   using a G1311C 1260 Quat Pump VL and detected with a G1315D 1260 DAD VL detector (190–500 nm) . Column chromatography was performed with silica gel , ODS gel , and Sephadex LH-20 . TLC was carried out on NUSCRIPT silica gel GF254 , and peaks were detected by spraying with 5% H2SO4 in EtOH followed by heating.Optical rotations were measured with a Rudolph Autopol I polarimeter . UV spectra were recorded on a Shimadzu UV-2550 spectrometer . ECD spectra were measured on a JASCO J-715 spectrophotometer . IR absorptions were obtained on a Nicolet 380 FT-IR instrument  using KBr pellets. HRESIMS were determined by an API QSTAR Pulsar mass spectrometer  or 6200 series TOF/6500 series . The NMR spectra were recorded on Bruker Avance 500 NMR spectrometers , using TMS as an internal standard. HPLC purifications were performed on an analytical reversed-phase column . A voucher specimen (201408SLLK) has been deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.The plant material was collected in NANA International Agarwood Market of Thailand, in August of 2014, and identified as agarwood originated from 2O  to provide 16 fractions (Fr.1–Fr.16). Fr.6 (0.6 g) was subjected to Sephadex LH-20 gel CC eluded with petroleum ether–CHCl3–MeOH  to give six fractions (Fr.6-1–Fr.6-7). Fr.6-3 (140.4 mg) was separated on silica gel CC with petroleum ether–acetone , then purified by silica gel CC using CHCl3–MeOH  to afford compound 5 (2.3 mg). Fr.7 (1.3 g) was separated to Sephadex LH-20 gel CC eluded with CHCl3–MeOH  and then fractionated by silica gel CC eluted with petroleum ether–EtOAc  to yield 13 fractions (Fr.7-1–Fr.7-13). Fr.7-7 and Fr.7-8 were merged together (547.7 mg) to be subjected on silica gel CC with petroleum ether–EtOAc–MeOH  and silica gel CC using CHCl3–MeOH , then purified by semi-preparative HPLC  to afford compounds 2 (32.2 mg) and 1 (1.2 mg). Fr.8 (799.1 g) was subjected to silica gel CC eluted with petroleum ether–EtOAc  to give 12 fractions (Fr.8-1–Fr.8-12). Fr.8-6 (36.8 mg) was separated on silica gel CC with petroleum ether–CHCl3–MeOH , then purified by semi-preparative HPLC  to afford compound 8 (4.4 mg). Fr.8-11 (48.0 mg) was applied to silica gel CC with petroleum ether–EtOAc , then purified by semi-preparative HPLC  to afford compound 7 (8.3 mg). Fr.9 (1.8 g) was divided to Sephadex LH-20 gel CC eluting with petroleum ether–CHCl3–MeOH  to obtain 11 fractions (Fr.9-1–Fr.9-11). Fr.9-3 (472.4 mg) was separated on silica gel CC with petroleum ether–EtOAc  and recrystallization to get compound 4 (30.5 mg). Fr.10 (2.0 g) was chromatographed on Sephadex LH-20 gel CC eluting with petroleum ether–CHCl3–MeOH  to yield Fr.10-1–Fr.10-5. Fr.10-2 (944.8 mg) was subjected to silica gel CC with CHCl3–MeOH  and silica gel CC with petroleum ether–EtOAc , then further purified by HPLC  to get compound 6 (15.0 mg), 9 (12.0 mg), 3 (36.4 mg).Air-dried of agarwood (384.0 g) was extracted with ethyl ether (1.5 L × 3). The extract was filtered and concentrated to get the ethyl ether extract (27.6 g). The ethyl ether extract (23.4 g) was applied to ODS gel CC eluting with MeOH–HR,6S,8R,11R)-10(1→5)abeo-11-hydroxy-ziza-1(2)-en-3-one (1): colorless bulk crystals (MeOH); c 0.11, MeOH); UV (MeOH) λmax: 246 nm; ECD  λmax (Δε): 315 (+1.39), 251 (+2.05), 216 (−5.69) nm; IR (KBr) νmax: 3441, 2964, 2914, 2875, 1682, 1650, 1442, 1406, 1287, 1093, 1050, 961 cm−1; 1H and 13C NMR data see m/z 257.1513 [M + Na]+ .-11(1→5)abeo-ziza-5(6)-en-1,12-diol (2): colorless bulk crystals (MeOH); c 0.12, MeOH); UV (MeOH) λmax: 216 nm. IR (KBr) νmax: 3425, 2956, 1617, 1450, 1397, 1090, 1024, 803 cm−1; 1H and 13C NMR data see m/z: 259.1672 [M + Na]+ .-agarozizone (3): colorless oil; c 0.05, CH3OH); UV (MeOH) λmax: 204 nm. ECD  λmax (Δε): 296 (−2.00), 207 (−0.29) nm; IR (KBr) νmax: 2954, 2924, 1701, 1635, 1399, 1185, 1104, 1027, 799 cm−1; 1H and 13C NMR data see m/z: 273.1471 [M + Na]+ .-12-hydroxy-ziza-5(6)-en-4-one (4): colorless bulk crystals (MeOH); c 0.16, CH3OH); UV (MeOH) λmax: 261 nm; ECD  λmax (Δε): 348 (+1.37), 260 (−3.15), 214sh (+4.51) nm; IR (KBr) νmax: 3423, 2960, 1701, 1615, 1406, 1202, 1030, 794 cm−1; 1H and 13C NMR data see m/z 257.1512 [M + Na]+ .-11,12-dihydroxy-ziza-5(6)-en-4-one (5): colorless oil; c 0.04, CH3OH). UV (MeOH) λmax: 259 nm; ECD  λmax (Δε): 349 (+1.67), 234 (−0.66) nm; IR (KBr) νmax: 3414, 3210, 2957, 1626, 1398, 1099, 1029, 800 cm−1; 1H and 13C NMR data see m/z 273.1459 [M + Na]+ .-ziza-5(6)-en-11,12-diol (6): white amorphous powder; c 0.27, CH3OH); UV (MeOH) λmax: 205 nm; IR (KBr) νmax: 3203, 2938, 1638, 1400, 1033, 794 cm−1; 1H and 13C NMR data see m/z 259.1674 [M + Na]+ .-4-methoxy-ziza-5(6)-en-12-ol (7): colorless oil; c 0.18, CH3OH); UV (MeOH) λmax: 210, 260 nm; IR (KBr) νmax: 3421, 2957, 1702, 1617, 1453, 1373, 1200, 1071, 1028, 805 cm−1; 1H and 13C NMR data see m/z 273.1817 [M+Na]+ .-4-methoxy-ziza-5(6)-en-12-ol (8): colorless oil; c 0.11, CH3OH); UV (MeOH) λmax: 209, 260 nm; IR (KBr) νmax: 3411, 2955, 1615, 1394, 1087, 1028, 800 cm−1; 1H and 13C NMR data see m/z 273.1829 [M + Na]+ .-ziza-5(6)-en-13,15-diol (9): colorless oil; c 0.15, CH3OH); UV (MeOH) λmax: 215 nm; IR (KBr) νmax: 3375, 2948, 1641, 1458, 1400, 1027, 799 cm−1; 1H and 13C NMR data see m/z 259.1669 [M + Na]+ . macrophages using the Griess assay as described before . The RAW1–3), together with six zizaane-type sesquiterpenoids (4–9) were identified from ethyl ether extract of agarwood originated from Aquilaria sp., which could be the characteristic chemicals of this kind of agarwood. Their structures were unambiguously elucidated on the basis of HRESIMS data, 1D and 2D NMR and comparison of the experimental and computed ECD spectra. In vitro anti-inflammatory assessment showed that only compound 9 exhibited weak anti-inflammatory activity with an IC50 value of 62.22 ± 1.27 μM.Three novel zizaane derivatives (


Sarcophyton contains the enzymatic machinery to synthesize a multitude of cembrene-type diterpenes. Herein, highly oxygenated cembrenoids, sarcoconvolutum A–E (1–5) were purified and characterized from an ethyl acetate extract of the red sea soft coral, Sarcophyton convolutum. Compounds were assemblies according to spectroscopic methods including FTIR, 1D- and 2D-NMR as well as HRMS. Metabolite cytotoxicity was tested against lung adenocarcinoma, cervical cancer, and oral-cavity carcinoma . The most cytotoxic compound, (4) was observed to be active against cell lines A549 and HSC-2 with IC50 values of 49.70 and 53.17 μM, respectively.The soft coral genus  Sarcophyton soft coral, isolated metabolites include cembrenoids + was assigned predicting a molecular formula of C20H30O6  with six degrees of unsaturation suggesting a bicyclic skeleton. FTIR spectrum exhibited bands at 3465 cm−1 and 1765 cm−1 corresponding to hydroxyls and carbonyl groups. From 1H NMR spectrum (δH 3.26 br d (J = 10.8 Hz) and 5.53 d (J = 10.4 Hz), three olefinic protons at δH 4.84 br d (J = 10.4 Hz), 5.48 br d (J = 16.3 Hz), and 5.50 m, in addition to four methyls were at δH 1.28 s, 1.37 s, 1.83 s, and 1.87 s were characterized. Twenty carbon resonances were predicted from 13C NMR , five methines , five methylenes, four methyls  depending upon DEPT and HSQC experiments. The above analyses of 1D NMR of 1 revealed the cembrene-based diterpenoid [S. glaucum [1H 1H COSY and HMBC spectral analysis. H-2 [δH 5.53 d (J = 10.4 Hz)]/C-4 , and 1H 1H COSY of H-2/H-3 [δH 4.84 br d (J = 10.4 Hz)] indicated the location of Δ3(4). The HMBC correlation of H3-18 (δH 1.87 s) and CH2-5  followed by 1H 1H COSY correlations of H2-5 [δH 2.18 br d (J = 12.7 Hz)]/ H-6 (δH 1.77 br d (J = 13.4 Hz)], H-6/H-7 (δH 3.26 br d (J = 10.8 Hz) confirmed a hydroxyl group at C-7. HMBC correlation of H3-19 (δH 1.28 s)/C-7 , H3-19/C-8 , and H-7/C-8 (J2) established hydroxylation of the C-8 quaternary carbon. Subsequently, HMBC correlation of H3-19/CH2-9 , H3-20 (δH 1.37 s)/CH2-11 , in addition to 1H 1H COSY of H2-9 [δH 2.34 m]/H-10 (δH 5.50 m), H-10/H-11 [(δH 5.48 br d (J = 16.3 Hz)] established Δ10(11). The down field shift of C-12 by ca. 7 ppm / H3-18 [δH 1.87 s], H3-18/ H-5α [δH 2.18 br d (J = 12.7 Hz)], H3-18/ H-6α [δH 1.77 br d (J = 13.4)], H-5α/ H-6α, and H-6α/ H-7 [δH 3.26 br d (J = 10.8 Hz)] established an α orientation for H-7. NOESY correlations of H-6β [δH 1.41 ddd ]/ H3-19 (δH 1.28 s), H-9β (δH 2.34 m)/ H3-19, H-9β/ H-11 [δH 5.48 br d (J = 16.3 Hz)], H-11/ H-13β [δH 1.72 br t (J = 13.2 Hz)], H-13β/ H3-20 [δH 1.37 s], H-11/ H3-20 indicated β configurations for both Me-19 and Me-20. Based on the described spectral analysis, 1 was identified as 7β,8α-dihydroxy-12α-hydroperoxide-16-keto-cembra-1E,3E,10E-triene (sarcoconvolutum A).The relative configuration of 1 is based on coupling constants and NOESY data . From thablished ,24,25. S2) was isolated as yellow oil with a positive optical rotation (+43.1) in MeOH. From the HRCIMS molecular ion peak at m/z 367.2122 [M + H]+, the molecular formula of C20H30O6  a bicylic skeleton with six degrees of unsaturation was predicted. The FTIR broad bands corresponding to hydroxyls and carbonyl groups were detected at 3462 cm−1 and 1768 cm−1. The 1H NMR spectrum (δH 3.32 d (J = 10.9 Hz) and 5.45 d (J = 10.2 Hz), three olefinic protons at δH 4.88 d (J = 10.1 Hz), 5.50 d (J = 16.1 Hz), and 5.57 dt , along with four methyls were at δH 1.28 s, 1.42 s, 1.82 s, 1.85 s. From the observed twenty signals in the 13C NMR spectrum , 5 methenes , 5 methylenes, 4 methyls  were identified based on DEPT and HSQC signals. A down field shift of δC 84.0 by ca. 7 ppm suggested the presence of a hydroperoxyl unit at C-12 [13C NMR comparison of the two isolated compounds indicated a down field shift of Me-20 for 2. The 2D NMR comparisons showed otherwise similar 1H 1H COSY and HMBC signals ]/ H3-18 [δH 1.82 s], H-18/ H-5 [δH 2.37 td ], H-18/ H-6 [δH 1.49 td ], H-5α/ H-6, H-6/ H-7 [δH 3.32 br d (J = 10.9 Hz)], H-7/ H-19 [δH 1.28 s] and H-13β [δH 1.95 td ]/ H3-19 indicated a H-7α Me-19 and Me-20orientation. Based upon these spectral data, 2 was identified as 7β,8α-dihydroxy-12β-hydroperoxide-16-keto-cembra-1E,3E,10E-triene (sarcoconvolutum B).Relative stereochemistry was determined based on coupling constants and NOESY data . The coun of H-2 ,24,25. T3) was isolated as yellow oil with a positive optical rotation . The molecular formula was deduced as C21H32O6  based on a HREIMS molecular ion peak at m/z 364.2258 [M-OH + H]+ indicating a bicylic skeleton with six degrees of unsaturation. FTIR broad bands corresponded to hydroxyls and carbonyl groups were detected at 3457 cm−1 and 1761 cm−1. From 1H NMR spectrum (δH 3.27 br d (J = 10.9 Hz) and δH 5.49 d (J= 9.9 Hz), three olefinic protons at δH 4.86 br d (J = 9.9 Hz), δH 5.44 br d (J = 16.1 Hz) and δH 5.56 dt , four methyls as well as one methyl of methoxy group at δH 3.23 s were assigned. Based on the 13C NMR , 5 methines , 5 methylenes, 5 methyls . As with 1 and 2, down field shift at δC 84.4 ppm of ca. 7 ppm along with a mass shift of [M-H2O], suggested the addition of a hydroperoxy group + that predicted a molecular formula of C20H28O5  with a bicylic skeleton and seven degrees of unsaturation. The corresponding FTIR broad bands to hydroxyls and carbonyl groups at 3456 cm−1 and 1763 cm−1 were assigned. The 1H NMR , and 5.45 d (J = 9.4 Hz), three olefineic protons at δH 5.00 d (J = 9.4 Hz), 5.42 m, and 5.47 m, in addition to four methyls at δH 1.31 s, 1.41 s, 1.86 s, and 1.87 s. From 13C NMR, twenty carbon resonances were characterized and categorized to six quartenary , five methylenes, four methyls based on DEPT-135 and HSQC analysis. The compound is very similar to 1 except for an up-field shift of δH 2.65 dd  by 0.67 ppm and an up-field shift of δC 57.0 by 14.6 ppm both associated with C-7 and an up-field shift of δC 60 by 14.5 ppm associated with C-8. These chemical shift changes were deduced to be a result of a C-7/C-8 epoxide ring that was consistent with a molecular ion peak at m/z 348.1939 (C20H28O5). The localization of this group was derived via the HMBC correlations of Me-19 (δH 1.31 s)/C-7 (J3), Me-19/C-8 (J2), H-6 (δH 1.60 m)/C-7 (J2), and H-6/C-8 (J3)  .4 exhibited almost same NOESY correlations as 1 except the correlations of Me-19/H-7, and H-6β/H-7 that elucidated the β orientation of H-7. These data confirmed 4 as 12α-hydroperoxide-16-keto-cembra-1E,3E,10E-triene-7α, 8α-epoxide (sarcoconvolutum D).The relative stereochemistry was identified via coupling constants ,24,25 an5) was obtained as yellow oil exhibiting a positive optical rotation . The mass spectroscopy exhibited a HREIMS molecular ion peak at m/z 367.2130 [M + H]+ that was identified as C20H30O6  with a bicylic skeleton and six degrees of unsaturation. The FTIR broad bands corresponding to hydroxyls and carbonyl groups were detected at 3464 cm−1 and 1759 cm−1. 1H NMR (δH 3.45 br d (J = 10.5 Hz), 4.43 dd , and 5.47 d (J = 10.1 Hz), one olefineic proton at δH 4.92 br d (J = 10.1 Hz), one exomethylene proton at δH 5.07 s, and 5.13 s, and three methyles at δH 1.26 s, 1.86 s (6H). The 13C NMR showed twenty carbon resonances. Six quaternary , four methenes , 7 methylenes (comprising exomethylene carbon at δC 113.4), three methyls  that were characterized via DEPT-135 and HSQC experiments. NMR spectral data of 5 were similar to 1 and 2 except for the following signals: (i) a down-field shift of δC 75.0, by ca. 3.5 ppm associated with C-7; (ii) an downfield shift of δH 3.45 brd (J = 10.5 Hz) by 0.2 ppm associated with H-7; and (iii) an up-field shift of δH 4.43 dd  by 1 ppm and a shift of ca. 45 ppm of δC 134.0 to 89.0 associated with C-11 indicating the oxygenation of this carbon; (iv) the appearance of δH 1.32  associated with H-10 and C-10 (at δC 25.0) indicating a disappearance of Δ10(11); and (v) the presence of δC 146.0 associated with C-12 and signals δH 5.07 s, 5.13 s and δC 113.4 associated with an exomethylene carbon at C-20 indicating the presence of Δ12(20). These new functional group assignments based on spectral data were consistent with 2D HMBC spectral signals including H3-19 [δH 1.26 s]/C-7 , H3-19/C-7  confirmed the hydroxylation of both C-7 and C-8. The HMBC correlations of H2-20 /C-12 , H2-20/C-11 , and H2-20/C-13 , H-11 [at δH 4.43 dd ]/C-12 [J2], H-11/C-13 [J3] confirmed the Δ12(20) and the oxygenation of C-11 (Sarcoconvolutum E (. 1H NMR  displaye of C-11 . Consistα and cis orientation of H-2 [δH 5.47 d (J = 10.1 Hz)]/ H3-18 [δH 1.86 s], H-3α [δH 4.92 br d (J = 10.1 Hz)]/ H-5α [δH 2.35 dt ], and H-5α/ H-6 [δH 1.75 m], H3-18/ H-6, H-5/ H-7 [δH 3.45 br d (J= 10.5 Hz)], H-6/ H-7, H-7/ H3-19 [δH 1.26 s], H3-19β/ H-9β [δH 1.51 m], H-9β/ H-11 [δH 4.43 dd ], H-13β [δH 2.25 m]/ H-identified a β orientation for H-7, H-11 and Me-19. Therefore, 5 was assigned as 7α,8α-dihydroxy-11α-hydroperoxide-16-keto-cembra-1E,3E,12(20)-triene (sarcoconvolutum E). 1D, 2D-NMR, HRMS and FTIR for all isolated secondary metabolites (1–5) were deposited in the The relative stereochemistry assignment was based the coupling constant of H-2 (10.1 Hz) and the vicinal coupling of H-3 (10.1 Hz) the indicated a n of H-2 ,24,25. N1–5  equipped with a Jasco PU-980 pump, a Jasco UV-970 intelligent UV/VIS detector at 210 nm and a semi preparative reversed-phase column .A JASCO P-2300 polarimeter  and shimazu FTIR-8400S instrument  was operated for optical rotation and IR spectra, respectively. Then, 1D and 2D NMR spectra were recorded on a Bruker 600 or 500 Hz NMR spectrometer . HR-MS spectra were obtained on a JEOL JMS-700 instrument . For Chromatography: column chromatography (CC) [silica gel 60 ]; TLC analysis: 3OH); FT-IR (KBr) νmax: 3465, 2961, 1765, 1435, and 987 cm−1; 1H and 13C NMR data , see m/z 367.2122 ; .7,8-Dihydroxy-12-hydroxy-16-keto-cembra-1E,3E,10E-triene : yellow oil; [α]3OH); FT-IR (KBr) νmax: 3461, 2957, 1759, 1439, and 991 cm−1; 1H and 13C NMR data , see m/z 367.2122 ; .7,8-Dihydroxy-12-hydroxy-16-keto-cembra-1E,3E,10E-triene : yellow oil; [α]3OH); FT-IR (KBr) νmax: 3456, 2951, 1763, 1446, and 993 cm−1; 1H and 13C NMR data , see m/z 364.2258, [M-OH+H]+]; .7β-Hydroxy-8α-methoxy-12α-hydroperoxide-16-keto-cembra-1E,3E,10E-triene : yellow oil; [α]3OH); FT-IR (KBr) νmax: 3456, 2958, 1763, 1442, and 996 cm−1; 1H and 13C NMR data , see m/z 348.1939 [M]+; .12α-Hydroperoxide-16-keto-cembra-1E,3E,10E-triene-7α,8α-epoxide : yellow oil; [α]3OH); FT-IR (KBr) νmax: 3464, 2955, 1759, 1433, and 997 cm−1; 1H and 13C NMR data , see m/z 367.2130 ; .7α,8α-Dihydroxy-11α-hydroperoxide-16-keto-cembra-1E,3E,12(20)-triene  and were maintained as monolayer culture in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 4 mM l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. Monolayers were passaged at 70–90% confluence using a trypsin-EDTA solution. All cell incubations were maintained in a humidified CO2 incubator with 5% CO2 at 37 °C. All materials and reagents for the cell cultures were purchased from Lonza .Human cancer cell lines of non-small cell lung adenocarcinoma (A549), squamous cell carcinoma of the oral cavity (HSC-2), and human cervical cancer cell (HeLa). All cell lines were purchased from American Type Culture Collection (ATCC2 at 37 °C. The prepared tested compounds (1–5) were screened and the dose-dependent toxicity with concentrations range (0.001–100 μg/mL) were investigated according to our protocol [A modified MTT  assay was used for cytotoxicity investigation were performed using based on a previously published method . A549, Hprotocol . Doxorub® v6.0 software  were used to calculate the concentration-response curve fit to the non-linear regression for IC50 values.GraphPad PrismSarcophyton convolutum tissue. Chemical structures were elucidated based upon spectroscopic analyses. The cytotoxic activity of the isolated compounds was screened against three cancer cell lines  and 4 was found to be the most active compound against cell lines A549 and HSC-2 with IC50 values of 49.70 and 53.17 μM, respectively.Five compounds, sarcoconvolutum A–E were isolated from an organic extract of


TW58-16. The planar structures of these compounds were elucidated by extensive 1D and 2D NMR, which was supported by HR-ESI-MS data. The absolute configurations of these compounds were determined by experimental and calculated electronic circular dichroism (ECD), and their optical rotations compared with those reported. Evaluation of the anti-inflammatory activity of compounds 1–18 revealed that compound 5 significantly inhibited the release of nitric oxide (NO) induced by lipopolysaccharide (LPS) in RAW264.7 cells, correlating with the inhibition of expression of inducible nitric oxide synthase (iNOS). In addition, we revealed that compounds 1, 3–6, 14, 16, and 18 showed strong α-glucosidase inhibitory effects with inhibition rates of 35.4%, 73.2%, 55.6%, 74.4%, 32.0%, 36.9%, 88.0%, and 91.1%, respectively, which were comparable with or even better than that of the positive control, acarbose. Together, our results illustrate the potential of discovering new marine-based therapeutic agents against inflammation and diabetes mellitus.Marine fungi-derived natural products represent an excellent reservoir for the discovery of novel lead compounds with biological activities. Here, we report the identification of two new drimane sesquiterpenes ( Marine-derived natural products (MNPs) represent a new and promising source of therapeutic agents . As the Penicillium sp. TW58-16. Eighteen compounds in total were isolated and identified from the ethyl acetate (EtOAc) extract of the fungus Penicillium sp. TW58-16, including two new drimane sesquiterpenes (1 and 2), six new polyketides (3–8), and 10 known compounds (9–18). Additionally, we evaluated the anti-inflammatory and hypoglycemic activities of these compounds in cell cultures and in vitro, respectively.In this study, we report the discovery of biologically active secondary metabolites from the marine fungal strain Penicillium sp. TW58-16. In summary, two new drimane sesquiterpenes 1 and 2, six new polyketides 3–8, and 10 known compounds (9–18) were obtained (9) +  in the HR-ESI-Q-TOF spectrum, implying four degrees of unsaturation. The IR spectrum showed the presence of hydroxyl (3388 cm−1), alkyl (2976 cm−1), and conjugated carbonyl (1649 cm−1) groups , an olefin carbon signal at δC 162.2 (C-8) and two others at δC 39.1 (C-4) and δC 43.5 (C-10); three methines including an olefin signal at δH 5.80 /δC 129.0 (C-7) and two others at δH 2.44 /δC 65.2 (C-5) and δH 2.39 /δC 59.7 (C-9); five methylenes including two oxygenated methylene signals at δH 3.91  and δH 3.76 /δC 59.9 (C-11) and δH 4.13  and δH 3.65 /δC 63.8 (C-13), and three others at δH 1.48  and δH 2.06 /δC 40.1 (C-1), δH 1.48  and δH 1.60 /δC 18.9 (C-2), and δH 1.94  and δH 0.96 /δC 37.2 (C-3), and three methyls at δH 2.08 /δC 22.3 (C-12), δH 1.15 /δC 27.4 (C-14), and δH 0.90 /δC 17.0 (C-15). Comparison of the 1H and 13C NMR data of 1 with a reported drimane sesquiterpene, fudecadione A + , accounting for five degrees of unsaturation. Comparison of the 1H and 13C NMR data of 2 with 1 indicated that 2 was also a drimane sesquiterpene. Replacement of signals for a hydroxymethyl [δH 4.13  and δH 3.65 /δC 63.8 (C-13)] in 1 with a carboxyl [δC 179.7 (C-13)] in 2 indicated 2 is an oxidation derivative of 1. The planar structure of 2 was confirmed by 1H-1H COSY and HMBC correlations -11-hydroxy-13-carboxy-drim-7-en-6-one.The relative configuration of periment . In the ated ECD . Thus, t3 was isolated as a brown solid powder. It has the molecular formula of C13H16O3 determined by the HR-ESI-MS data at m/z 221.1178 [M + H]+ , implying six degrees of unsaturation. The IR spectrum showed the presence of hydroxyl (3359 cm−1), alkyl (2971 cm−1), and aromatic ring . Analyses of the 1H and 13C NMR data of 3 aided by HSQC indicated the presence of a 1,3,5-trisubstituted aromatic ring fragment with phenolic hydroxyl groups at C-1 and C-3 ; four olefinic signals , and an oxymethine [δH 3.66 /δC 65.9 (C-12)]; a methylene [δH 2.17 /δC 42.6 (C-11)], and a methyl [δH 1.04 /δC 23.2 (C-13)]. Considering the spin-coupling system of H-7–H-8–H-9–H-10–H2-11–H-12–H3-13 in the 1H–1H COSY spectrum and HMBC correlation from H-9 to C-7, a 4,6-heptadien-2-ol fragment was ascertained. Furthermore, according to the HMBC correlations of H-7 with C-4 and C-5 and of H-6/H-4 with C-7, the 4, 6-heptadien-2-ol fragment was attached at C-5 of the 1,3,5-trisubstituted aromatic ring -7-phenyl-4,6-heptadien-2-ol, a known compound, (4)) . The HMBC correlations of H-8 with C-5, and of H-6 with C-7 linked the 4,6-heptadien-2-ol fragment to C-5 of aromatic ring fragment. Further, according to the molecular formula and degrees of unsaturation of 4, the carboxyl group was attached at C-4 of aromatic ring fragment, which was confirmed by the HMBC correlation of H-2 with C-14 (-COOH). Thus, the planar structure of compound 4 was deduced.Compound elations . However7,8 and Δ9,10 were ascertained as Z and E, respectively, by the coupling constants of 11.0 Hz between H-7 and H-8, and 14.9 Hz between H-9 and H-10. In addition, the positive optical rotation value of compound 4 ) indicated the R configuration of C-12 + , indicating a molecular formula of C14H16O4, one oxygen atom less than 4, and accounting for seven degrees of unsaturation. The 1H and 13C NMR spectra of 5 showed great similarities with those of 4 and the main difference laid in the 1H and 13C resonances of C-12. The upfield shift of 1H and 13C resonances of C-12 from δH 3.77 /δC 68.6 (C-12) in 4 to δH 1.41 /δC 23.6 (C-12) in 5 indicated that the oxymethine at C-12 in 4 was replaced by a methylene in 5. The planar structure of 5 was confirmed by the 1H-1H COSY and HMBC correlations -1,3-heptadien-1-yl)-1,3-benzenediol. Compound elations . In addi6 was obtained as a brown solid powder. It has a molecular formula of C11H10O4 as determined by the HR-ESI-MS at m/z 207.0661 [M + H]+ , implying seven degrees of unsaturation. The 1H and 13C NMR spectra combined with the HSQC spectrum displayed a 1,3,5-trisubstituted aromatic ring fragment with phenolic hydroxyl groups at C-1 and C-3 ; four olefinic signals , and a carbonyl carbon signal [δC 168.7 (C-11)]. The spin-coupling system of H-7–H-8–H-9–H-10 in the 1H–1H COSY spectrum, together with the HMBC correlations of H-9 and H-10 with C-11, indicated the presence of a 2,4-pentadienoic acid fragment. In addition, the HMBC correlations of H-7 with C-5 and C-6, and of H-4 with C-7, indicated that the 2,4-pentadienoic acid fragment was attached to C-5 of the aromatic ring. Based on the coupling constants of 11.4 Hz between H-7 and H-8, and 14.8 Hz between H-9 and H-10, the Z and E configurations of double bonds of Δ7,8 and Δ9,10 were determined. Therefore, the structure of 6 was identified as 5--4-carboxy-1,3-butadienyl-1-yl)-1,3-benzenediol.Compound 7 was obtained as a white solid powder and has a molecular formula of C12H10O6 determined by the HR-ESI-MS at m/z 251.0556 [M + H]+ , implying eight degrees of unsaturation. The IR spectrum showed the presence of hydroxyl groups (3451 cm−1) and an aromatic ring . The 1H NMR spectrum of 7 exhibited two meta-aromatic protons at δH 6.26  and δH 6.19 , indicating the presence of a tetrasubstituted aromatic ring. Additionally, a pair of olefinic protons at δH 6.68  and δH 6.00  indicated the presence of a double bond with an E configuration. The 13C NMR spectrum exhibited a total of 12 carbon signals, including two carbonyl carbon signals at δC 168.6 (C-1) and δC 167.6 (C-11). Comparison of the 1H and 13C NMR data of 7 with 6,8-dihydroxy-3--penta-1,3-dien-1-yl) isochroman-1-one -2-propenoic acid.Compound elations . The CD n of C-3 ,21. Thus8 was obtained as a white solid powder. Its molecular formula was elucidated as C12H12O6 based on the HR-ESI-MS  and 13C NMR data, implying seven degrees of unsaturation. Comparison of the 1H and 13C NMR data of 8 with that of 7 suggested that they had significant similarities and the main difference between them falls in the side chain. The NMR data of C-9 and C-10 shifted from δH 6.68 /δC 140.1 (C-9) and δH 6.00 /δC 126.6 (C-10) (in 7) to δH 1.92 /δC 29.6 (C-9) and δH 2.35 /δC 29.6 (C-10) (in 8), indicating that the double bond in 7 was reduced to two methylene groups. In addition, compound 8 had an S configuration at C-3 according to the ECD result (8 was determined and named as 3--propanoic acid.Compound D result  and posi1-18 on NO production induced by LPS in murine macrophage RAW264.7 cells were evaluated. Dexamethasone (DXM) and curcumin (Cur) were used as positive controls. Our results show that compounds 5–7 and 16, especially 5, significantly inhibited NO production induced by LPS , from the marine-derived fungus Penicillium sp. TW58-16. We first presented data to show that these compounds inhibited inflammation as they suppressed LPS-stimulated NO production in macrophages. Consistently, compounds 5–7 and 16, and 5 in particular, greatly inhibited the expression of iNOS, the enzyme that produces NO. We expect to perform structure modification and further explore the anti-inflammatory effects of these compounds. Polyketides are a large family of natural products that are derived from acetate building blocks . Due to 3–6, 14, 16, and 18 exhibited potent α-glucosidase inhibitory activities at levels that were comparable with or better than acarbose, a known α-glucosidase inhibitor. Among them, the new polyketides 3–6 share structural similarities, but display distinct α-glucosidase inhibitory activities. Structure-activity relationship (SAR) analysis revealed that the length of the side chain or the introduction of carboxylic acid in the side chain may have a crucial effect on their activities, as compounds 3–5 exhibited significantly stronger α-glucosidase inhibitory effects than compound 6. To our surprise, compound 16 baring 3,4-dihydroxyl groups in the aromatic ring and an acetic acid side chain also exhibited significant α-glucosidase inhibitory activity, suggesting the favour of ortho-dihydroxyl groups for the α-glucosidase inhibitory activity. These were the first report to show a potent α-glucosidase inhibitory activity of the polyketide ε-caprolactone derivative 18.In addition, scattered reports showed α-glucosidase inhibitory activities of polyketides ,27,28,291 and the known analogue 3 demonstrated strong anti-inflammation and α-glucosidase inhibitory activities. SAR analysis indicated that carboxy substituent in C-13 and lactone in C-15 and C-11 unfavour for the α-glucosidase inhibitory activity of these compounds.Drimane sesquiterpenes are widely distributed in metabolites of higher plants and terrestrial and marine fungi. They also demonstrate a broad range of bioactivities, including antifungi and antibacteria, cytotoxicity, piscicidal and molluscicidal activity, etc. . Here weα-glucosidase inhibitors may promote the study and development of new derivatives of this compound class for the treatment of inflammation and diabetes mellitus.To sum up, these discoveries of new 3OD (δH 3.31/δC 49.0) and DMSO-6d (δH 2.50/δC 39.5) as an internal reference. HR-ESI-Q-TOF-MS spectra were acquired on a Waters Synapt G2 mass spectrometer . A Chirascan plus  was used to acquire the CD spectra. HPLC analyses were conducted on a Shimadzu LC-20AB Liquid Chromatography with SPD-M20A DAD detector . The column for analytical HPLC was a YMC-Triart C18 column . The semi-preparative HPLC was conducted on a Shimadzu LC-20AT Liquid Chromatography with SPD-20A UV/Vis detector . The column was a YMC-Pack ODS-A column . Silica gel for column chromatography (200–300 mesh) was purchased from Qingdao Marine Chemical Ltd. . Pre-coated silica gel plates  for TLC analysis were from Yantai Chemical Industry Research Institute . Octadecylsilanized (ODS)  for column chromatography was from YMC Co. Ltd. .Optical rotations were taken on a P-1020 digital polarimeter . The UV/vis and IR spectra were measured by a JASCO V-550 UV/Vis spectrometer and a JASCO FT/IR-480 plus spectrometer , respectively. NMR data were taken by a Bruker AV 600  with signals of CDα-Glucosidase was purchased from Sigma-Aldrich Chemical Co. Ltd. . Antibodies of iNOS (#13120), COX-2 (#12282), and HRP conjugated secondary anti-rabbit (#7076) antibodies were from Cell Signaling Technology ; β-Actin (# AP0060) was from Bioworld .Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco . Methyl thiazolyl tetrazolium (MTT) and acarbose (#A129816) were purchased from Aladdin Reagent Co., Ltd. . Curcumin (#T1516) was from TargetMol . Dexamethasone was purchased from MYM Biological Technology Company Limited. Cell lysis buffer (#P0013J), phenylmethylsulfonyl fluoride (#ST505) and NO assay kit (#S0021) were purchased from Beyotime Inst Biotech . BCA protein assay kit (#23225) was obtained from Pierce . Polyvinylidenedifluoride (PVDF) membrane (#IPVH00010) was purchased from Millipore . Penicillium sp. according to the morphological characteristics and the internal transcribed spacer (ITS) sequence (MZ558028), which is 100.00% similar to Penicillium citrioviride isolate D5 (GU388431.1). The strain was deposited at Ocean College, Zhejiang University, Zhejiang, China. The fungus strain TW58-16 was isolated from hydrothermal vent sediment, collected from Kueishantao, Taiwan, and identified as Penicillium sp. TW58-16 was inoculated on a PDA agar plate, which was composed of 200 g potatoes, 20 g glucose, and 20 g agar in 1 L ddH2O. The spores from the agar plate were transformed to a triangular flask containing 100 mL PDA liquid medium and placed in a constant temperature shaking incubator for 6 days  to obtain 2000 mL of seed culture solution (100 mL × 20). Then 20 mL of seed culture solution was inoculated to solid rice medium that was composed of 100 g rice in 150 mL ddH2O. A total of 7.0 kg of largescale fermentation was conducted in solid rice medium and incubated at room temperature for 30 days. The fermentation product was extracted with EtOAc and the filtrate was concentrated to yield extracts (50.7 g) under vacuum.Strain The extracts were chromatographed by silica gel column  eluted with gradient Petroleum ether-EtOAc  and EtOAc-MeOH  to obtain 14 fractions (Fr. 1–14) according to TLC analyses.2O (20–100%) to obtain 15 subfractions (Fr. 8-1–Fr. 8-15). Fr. 8-6 (9.7 mg) was applied to preparative RP HPLC eluted with 43% MeOH-H2O (0.1% HCOOH) to get compound 15 (1.3 mg) and Fr. 8-8 (38.0 mg) was applied to preparative RP HPLC eluted with 35% MeOH-H2O (0.1% HCOOH) to get compound 5 (2.0 mg). Fr. 9 (1.8 g) was isolated by ODS CC eluted with gradient MeOH-H2O to get 15 subfractions (Fr. 9-1–Fr. 9-15). Fr. 9-5 (19.6 mg) was applied to preparative RP HPLC (35% MeOH-H2O with 0.1% HCOOH) to obtain compound 6 (4.6 mg). Fr. 9-9 (101.7 mg) was applied to preparative RP HPLC (55% MeOH-H2O with 0.1% HCOOH) to produce compounds 11 (26.8 mg), 12 (3.4 mg), and 13 (7.4 mg). Fr. 9-12 (290.6 mg) was subjected to silica gel CC, eluted with gradient Petroleum-EtOAc to produce 4 subfractions (Fr. 9-12-1–Fr. 9-12-4). Fr. 9–12-2 (155.5 mg) was applied to preparative RP HPLC (70–85% MeOH-H2O with 0.1% HCOOH) to get compound 18 (6.5 mg). Fr. 10 (1.2 g) was subjected to ODS CC, eluted with gradient MeOH-H2O to produce 9 subfractions (Fr. 10-1–Fr. 10-9). Fr. 10-1 (205.6 mg) was isolated by silica gel CC eluted with gradient CH2Cl2-MeOH to get 3 subfractions (Fr. 10-1-1–Fr. 10-1-3). Fr. 10-1-2 (53.0 mg) was applied to preparative RP HPLC (20% MeOH-H2O with 0.1% HCOOH) to get compound 16 (1.8 mg). Fr. 10-2 (43.1 mg) was applied to preparative RP HPLC with elution of 20% MeOH-H2O (0.1% HCOOH) to get compounds 7 (1.1 mg) and 8 (1.3 mg). Fr. 10-3 (56.8 mg) was applied to preparative RP HPLC eluted with 18% CH3CN-H2O (0.1% HCOOH) to get compounds 1 (2.1 mg), 2 (2.0 mg), 3 (1.1 mg), 4 (3.5 mg), and 14 (27.3 mg). Fr. 11 (6.4 g) was subjected to ODS CC eluted with gradient MeOH-H2O to get 19 subfractions (Fr. 11-1–Fr. 11-19). Fr. 11-5 (201.9 mg) was purified by preparative RP HPLC (10% CH3CN-H2O with 0.1% HCOOH) to obtain compound 9 (9.3 mg). Fr. 11-8 (128.2 mg) was chromatographed by silica gel CC eluted with gradient CH2Cl2-MeOH to get 6 subfractions (Fr. 11-8-1–Fr. 11-8-6). Fr. 11-8-5 (19.3 mg) was applied to preparative RP HPLC (35% MeOH-H2O with 0.1% HCOOH) to obtain compounds 10 (6.5 mg) and 17 (7.4 mg).Fr. 8  was chromatographed by ODS CC (ϕ 27.0 × 150.0 mm) eluted with MeOH-HS,5R,9S,10R)-11,13-dihydroxy-drim-7-en-6-one (1): White solid; c 0.55, in MeOH); UV (MeOH) λmax (log ε): 204 (3.7), 240 (3.9) nm; IR (KBr) νmax: 3388, 3316, 2976, 2933, 2852, 1649, 1383, 1031 cm−1; CD (MeOH) λmax (Δε): 204 (−5.46), 228 (−1.51), 242 (−2.04), 328 (+0.88); HR-ESI-MS: m/z 253.1808 [M + H]+ ; 1H and 13C NMR spectral data -11-hydroxy-13-carboxy-drim-7-en-6-one (2): White solid; c 1.4, in MeOH); UV (MeOH) λmax (log ε): 201 (3.1), 241 (3.5) nm; IR (KBr) νmax: 3449, 2939, 1710, 1623, 1414, 1383, 1215, 1078 cm−1; CD (MeOH) λmax (Δε): 212 (−2.34), 228 (−1.48), 248 (−2.18), 316 (+1.0); HR-ESI-MS: m/z 267.1591 [M + H]+ ; 1H and 13C NMR spectral data -6-hydroxy-1,3-heptadien-1-yl)-1,3-benzenediol (3): Brown solid; c 0.2, in MeOH); UV (MeOH) λmax (log ε): 205 (4.3), 227 (4.1), 279 (4.0) nm; IR (KBr) νmax: 3359, 2971, 2924, 1602, 1452, 1386, 1156, 1003 cm−1; HR-ESI-MS: m/z 221.1178 [M + H]+ ; 1H and 13C NMR spectral data -6-hydroxy-1,3-heptadien-1-yl)-1,3-benzenediol (4): Brown solid; c 0.4, in MeOH), c 0.5, in CHCl3); UV (MeOH) λmax (log ε): 206 (4.3), 219 (4.2), 282 (4.0) nm; IR (KBr) νmax: 3451, 2976, 2930, 1600, 1484, 1374, 1161, 1070 cm−1; HR-ESI-MS: m/z 265.1075 [M + H]+ ; 1H and 13C NMR spectral data -1,3-heptadien-1-yl)-1,3-benzenediol (5): Brown solid; UV (MeOH) λmax (log ε): 204 (3.3), 240 (3.1), 283 (2.9) nm; IR (KBr) νmax: 3369, 2958, 2930, 2868, 1719, 1611, 1580, 1462, 1371, 1269, 1167, 1023 cm−1; HR-ESI-MS: m/z 249.1133 [M + H]+ ; 1H and 13C NMR spectral data -4-carboxy-1,3-butadienyl-1-yl)-1,3-benzenediol (6): Brown solid; UV (MeOH) λmax (log ε): 209 (4.0), 307 (3.7) nm; IR (KBr) νmax: 3420, 1628, 1600, 1507, 1429, 1380, 1313, 1165, 1011 cm−1; HR-ESI-MS: m/z 207.0661 [M + H]+ ; 1H and 13C NMR spectral data -3--2-propenoic acid (7): White solid; c 0.3, in MeOH); UV (MeOH) λmax (log ε): 210 (4.1), 269 (3.7), 301 (3.3) nm; IR (KBr) νmax: 3451, 2924, 2854, 1662, 1634, 1496, 1467, 1379, 1246, 1172, 1105 cm−1; CD (MeOH) λmax (Δε): 214 (−2.13), 233 (+1.55), 254 (−0.21), 268 (+0.01); HR-ESI-MS: m/z 251.0556 [M + H]+ ; 1H and 13C NMR spectral data -3,4-dihydro-6,8-dihydroxy-1-oxo-1H-2-benzopyran-3-yl]-propanoic acid (8): White solid; c 0.3, in MeOH); UV (MeOH) λmax (log ε): 216 (3.9), 269 (3.7), 304 (3.4) nm; IR (KBr) νmax: 3445, 2924, 2854, 1725, 1648, 1379, 1254, 1172, 1114 cm−1; CD (MeOH) λmax (Δε): 208 (+0.15), 228 (−0.03), 246 (−0.06), 269 (+0.02), 307 (−0.04); HR-ESI-MS: m/z 253.0721 [M + H]+ ; 1H and 13C NMR spectral data -1, -1, -2, -2, (3R)-7, (3S)-7, (3R)-8, and (3S)-8 were afforded before their initial 3D structures were generated using CORINA version 3.4. Subsequently, CONFLEX version 7.0 was used to acquire the conformer databases based on the MMFF94s force-field. In that process, an energy window of 5 kcal mol−1 above the ground state, a maximum number of conformations per molecule (maxconfs) of 100 for acceptable conformers (ewindow), and an RMSD cutoff (rmsd) of 0.5 Å were limited. Then, all the acceptable conformers were optimized with the HF/6-31G(d) method in Gaussian 09 ) .2. The cells in the logarithmic phase were used for the following experiments.The murine macrophage RAW 264.7 cells were from the American Type Culture Collection  and cultured in DMEM supplemented with 10% FBS at 37 °C in a 98% humidified incubator with 5% CO5 cells/mL were seeded in each well of a 96-well culture plate (100 μL) and cultured overnight. Cell-free wells were set as blank controls. After attachment, the cells were co-treated with tested compounds (50 μM) and LPS (100 ng/mL) for 24 h. Then, the culture medium was replaced with DMEM full media containing 0.5 mg/mL MTT (100 μL) and incubated for another 2 h. After aspiration of the culture medium, DMSO (150 μL) was added to dissolve the formazan. Finally, the optical densities (OD) were measured at 490 nm.Cell viability was assessed by the MTT assay. In brief, cells with a density of 1.5 × 105 cells/well) were co-treated with tested compounds and LPS (100 ng/mL) for 24 h. Finally, the nitrite concentration in the culture supernatants was taken by the NO assay kit.The NO concentrations were measured by the Griess method. After being cultured in 96-well plates overnight, cells  membranes, and blocked with 5% skim milk in TBST solution for 1 h at room temperature. Finally, the membranes were sequentially incubated with primary and secondary antibodies, followed by chemiluminescence detection.α-glucosidase inhibitory assay was conducted as per a previous report  × 100.The s report . Acarbosp < 0.05 was considered statistically significant.GraphPad Prism software version 5  were used to perform the statistical analyses. Each experiment was conducted in triplicate, and the final data were expressed as mean ± standard error of mean (SEM). Multiple comparisons were carried out by one-way ANOVA, followed by Tukey’s test.