Here we report for the first time the NMR characterization of this rare lipid from marine organisms along with a synthetic strategy for the preparation of a PGDG analog (2). PGDG 1 exhibited immunostimulatory activity in human dendritic cells (DCs) and the synthetic PGDG 2 was prepared to explore its mechanism of action. A Toll-like receptor-4 (TLR-4) agonistic activity was evidenced in human and murine DCs underlying the antigen-specific T-cell activation of this class of molecules.An unprecedented phosphatidylmonogalactosyldiacylglycerol pool (PGDG,  Thalassiosira weissflogii (CCMP 1336) was selected due to its immunostimulatory properties, measured as production of IL-6, a cytokine with pleiotropic activity in immune regulation, hematopoiesis, inflammation and oncogenesis . . 2), who1H and 13C NMR resonances  and a monogalactosyldiacylglycerol (MGDG) ,23. Inde1  matches In the literature, there are very few examples of bacterial galactose-based phosphoglycolipids  and a unT. weissflogii compared to other related galactolipids like MGDG but also with respect to other lipid classes, such as sulfoquinovosides, phospholipids and triacylglycerols -1,2-distearoyl-sn-glycerol (X), subsequently methylated at phosphate (XI). Deprotection of acetonide and condensation with palmitic acid gave 3-O--1,2-distearoyl-sn-glycerol (XIII) that was finally de-O-methylated by trimethyl-silyl-chloride and deacetylated by hydrazine monohydrate [O-[6′-O--1′-β-d-galactopyranosyl]-1,2-distearoyl-sn-glycerol (2). NMR data of the final synthetic product were superimposable to those of 1  ,41. Amon1 and 2 activity were TLR-2/TLR-4 mediated, we tested their effects in a cell-based assay with HEK-293 cells stably co-transfected to express full-length human TLR-2 or TLR-4 and the secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of a NF-kB response element + .6′-d-1′,2′,3′,4′-Tetracetyl-galactopyranose (II): 6′-O-trityl-d-1′,2′,3′,4′-tetracetyl-galactopyranose (I)  was dissolved in acetic acid (0.64 mL) at 10 °C and bromidric acid  was added; after one minute of stirring the mixture was filtered and washed with water (0.4 mL); the filtrate was diluted with 2 mL of water and extracted three times with dichloromethane (1.3 mL). The organic phase was purified by silica gel chromatography using a light petroleum ether/diethyl ether gradient to give d-1′,2′,3′,4′-tetracetyl-galactopyranose (II) ; Rf (light petroleum ether/diethyl ether 3:7) = 0.20; 1H-NMR  (mix of α- and β-anomers): δ 6.31 , 5.68 , 5.41 , 5.31 , 5.12 , 3.69 , 3.49 , 2.16-1.91 ; HRESIMS m/z 371.0957 [M + Na]+ .O-dimethylphosphoryl-d-1′,2′,3′,4′-Tetracetyl-galactopyranose (III): d-1′,2′,3′,4′-tetracetyl-galactopyranose (II)  was dissolved in pyridine (0.28 mL); trimethylphosphite  and carbon tetrabromide  were added at 0°C. The mixture was stirred in argon atmosphere for 2 h at room temperature. The solvent was evaporated and the residue was purified by by silica gel chromatography using a chloroform/methanol gradient to give 6′-O-dimethylphosphoryl-d-1′,2′,3′,4′-tetracetyl-galactopyranose (III) ; Rf (diethyl ether) = 0.15; 1H-NMR  (mix of α- and β-anomers): δ 6.34 , 5.68 , 5.4 , 5.29 , 5.08 , 4.15 , 4.10-4.02 , 3.86-3.70 , 2.19-1.95 ; HRESIMS m/z 479.0926 [M + Na]+ .6′-O-dimethylphosphoryl-d-2′,3′,4′-triacetyl-galactopyranose (IV): 6′-O-dimethylphosphoryl-d-1′,2′,3′,4′-tetracetyl-galactopyranose (III)  was dissolved in tetrahydrofurane (0.36 mL); benzylamine  was added; the mixture was stirred overnight at room temperature and after evaporation of solvent was purified by silica gel chromatography using a chloroform/methanol gradient to give 6′-O-dimethylphosphoryl-d-2′,3′,4′-triacetyl-galactopyranose (IV) ; Rf (chloroform:methanol 95:5) = 0.49; 1H-NMR  (mix of α- and β-anomers): δ 5.49 , 5.43 , 5.13 , 5.29 , 5.08 , 4.95 , 4.51 , 4.12-4.01 , 3.88–3.69 , 2.20-1.95 ; HRESIMS m/z 437.0832 [M + Na]+ .6′-O-dimethylphosphoryl-1′-α-d-2′,3′,4′-triacetyl-galactopyranosyl-trichloroacetimidate (V): 6′-O-dimethylphosphoryl-d-2′,3′,4′-triacetyl-galactopyranose (IV)  was dissolved in dichloromethane (0.38 mL); 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)  and trichloroacetonitrile  were added and the mixture was stirred on molecular sieves for 2 h at 0 °C; after evaporation of solvent the mixture was purified by silica gel chromatography using a dichloromethane/methanol gradient to give 6′-O-dimethylphosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-trichloroacetimidate (V) ; Rf (chloroform:methanol 95:5) = 0.70; 1H-NMR : δ 8.65 , 6.59 , 5.58 , 5.40 , 5.33 , 4.42 , 4.12–3.96 , 3.77-3.68 , 2.21–1.96 ; HRESIMS m/z 579.9928 [M + Na]+ .6′-O-dimethylphosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-sn-isopropylidene-glycerol (VI): 6′-O-dimethylphosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-trichloroacetimidate (V)  was dissolved in anhydrous dichloromethane (0.44 mL) and 1.5 equivalents of 1,2-(S)-O-isopropylidene glycerol was added; the reaction mixture was kept at −20 °C on activated 3 Å molecular sieves under argon; boron trifluoride etherate  was added and stirring was maintained for 2 h; after another addition of boron trifluoride etherate , the temperature was raised to −10 °C and the reaction mixture was stirred overnight at that temperature. After neutralization with triethylamine (4 μL) and filtration on celite, the mixture was evaporated under reduced pressure and purified by silica gel chromatography using a dichloromethane/methanol gradient to give 6′-O-dimethylphosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-sn-isopropylidene-glycerol (VI) ; Rf (chloroform:methanol 95:5) = 0.65; 1H-NMR : δ 5.38 , 5.14 , 4.97 , 4.55 , 4.34 , 4.14–3.78 , 3.75–3.68 , 2.11 , 2.02 , 1.93 , 1.36 , 1.29 ; HRESIMS m/z 551.1512 [M + Na]+ .6′-O-dimethylphosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-sn-glycerol (VII): 6′-O-dimethylphosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-sn-isopropylidene-glycerol (VI)  was dissolved in methanol-water solution  and Dowex 50WX8-H+ resin (0.4 g) was added; after 3 h under stirring, the reaction mixture was filtered, evaporated and purified by silica gel chromatography using a gradient of dichloromethane/methanol to give 6′-O-dimethylphosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-sn-glycerol (VII) ; Rf (chloroform:methanol 95:5) = 0.30; 1H-NMR : δ 5.43 , 5.20 , 5.03 , 4.53 , 4.17–4.10 , 4.08–4.01 , 3.97 , 3.88–3.84 , 3.79–3.75 , 3.70 , 3.59 , 2.16 , 2.07 , 1.98 ; HRESIMS m/z 511.1197 [M + Na]+ .6′-O-dimethyl-phosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-1,2-distearoyl-sn-glycerol (VIII): 6′-O-dimethylphosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-sn-glycerol (VII)  was dissolved in anhydrous dichloromethane (0.4 mL); stearic acid , dicyclohexylcarbodiimide  and DMAP  were added under argon; the reaction mixture was stirred overnight at room temperature; after evaporation under reduced pressure, the mixture was purified by silica gel chromatography using a gradient of dichloromethane/methanol to give 6′-O-dimethyl-phosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-1,2-distearoyl-sn-glycerol (VIII) ; Rf (chloroform:methanol 95:5) = 0.80; 1H-NMR : δ 5.44 , 5.19–5.14 , 5.02 , 4.48 , 4.13 , 4.16–4.07 , 3.95 , 3.75 , 3.65 , 2.30 , 2.16 , 2.06 , 1.98 , 1.61 , 1.37–1.20 , 0.88 ; HRESIMS m/z 1043.6418 [M + Na]+ .6′-O-phosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-1,2-distearoyl-sn-glycerol (IX): 6′-O-dimethyl-phosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-1,2-distearoyl-sn-glycerol (VIII)  was dissolved in acetonitrile (1.36 mL); TMS-Cl  and sodium iodide  were added and after stirring in the dark at room temperature overnight, the mixture was filtered and washed with dichlorometane; the filtrate was evaporated to get 6′-O-phosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl-1,2-distearoyl-sn-glycerol (IX) ; Rf (chloroform:methanol 85:15) = 0.10; 1H-NMR : δ 5.44 , 5.22–5.15 , 5.02 , 4.50 , 4.13–3.92 , 3.68 , 2.30 , 2.16 , 2.05 , 1.98 , 1.66–1.54 , 1.36–1.18 , 0.88 ; HRESIMS m/z 991.6127 [M − H]− .6′-O--1,2-distearoyl-sn-glycerol (X): 6′-O-phosphoryl-1′-β-d-2′,3′,4′-triacetyl-galactopyranosyl]-1,2-distearoyl-sn-glycerol (IX)  and 1,2-(S)-O-isopropylidene glycerol  were dissolved in small quantities of pyridine and evaporated different times by nitrogen flow and finally were dissolved in anhydrous pyridine (0.3 mL) and trichloroacetonitrile  was added; after stirring at 75 °C under argon atmosphere overnight, the mixture was evaporated and purified by silica gel chromatography using a gradient of dichloromethane/methanol to give 3-O--1,2-distearoyl-sn-glycerol (X) ; Rf (chloroform:methanol 85:15) = 0.25; 1H-NMR : δ 5.49 , 5.24 , 5.16-5.09 , 4.70 , 4.38-4.23 , 4.15 , 4.15–4.10 , 4.02 , 3.96–3.89 , 3.84 , 3.75 , 2.39–2.31 , 2.19 , 2.09 , 1.99 , 1.68–1.60 , 1.43 , 1.37 , 1.34–1.15 , 0.93 ; HRESIMS m/z 1105.6798 [M−H]− .3-O--1,2-distearoyl-sn-glycerol (XI): 3-O--1,2-distearoyl-sn-glycerol (X)  was dissolved in anhydrous acetonitrile (0.54 mL); silver (I) oxide  and iodomethane  were added; after stirring at 40°C overnight, the mixture was evaporated and purified by silica gel chromatography using a gradient of dichloromethane/methanol to give 3-O--1,2-distearoyl-sn-glycerol (XI) ; Rf (chloroform:methanol 85:15) = 0.85; 1H-NMR : δ 5.43 , 5.22-5.16 , 5.01 , 4.48 , 4.35-4.28 , 4.16–4.02 , 3.99–3.92 , 3.82-3.72 , 2.30 , 2.16 , 2.06 , 1.98 , 1.64–1.56 , 1.43 , 1.36 , 1.33–1.21 , 0.88 ; HRESIMS m/z 1143.6910 [M + Na]+ .3-O--1,2-distearoyl-sn-glycerol (XII): 3-O--1,2-distearoyl-sn-glycerol (XI)  was dissolved in methanol-water-chloroform suspension  and Dowex 50WX8-H+ resin (0.17 g) was added; after 7 h under stirring at 60 °C, the reaction mixture was filtered, evaporated and purified by silica gel chromatography using a gradient of dichloromethane/methanol to give 3-O--1,2-distearoyl-sn-glycerol (XII) ; Rf (chloroform:methanol 95:5) = 0.45; 1H-NMR : δ 5.45 , 5.24–5.14 , 5.02 , 4.50 , 4.31 , 4.17-4.09 , 4.06 , 3.99–3.91 , 3.84–3.74 , 3.71–3.63 , 2.30 , 2.18 , 2.06 , 1.99 , 1.68–1.53 , 1.36–1.27 , 0.88 ; HRESIMS m/z 1103.6619 [M + Na]+ .3-O--1,2-distearoyl-sn-glycerol (XIII): 3-O--1,2-distearoyl-sn-glycerol (XII)  was dissolved in anhydrous dichloromethane (0.3 mL); palmitic acid , dicyclohexylcarbodiimide  and DMAP  were added; the reaction mixture was stirred overnight at 45 °C; after evaporation of solvent under reduced pressure, the mixture was purified by silica gel chromatography using a gradient of chloroform/methanol to give 3-O--1,2-distearoyl-sn-glycerol (XIII) ; Rf (chloroform:methanol 95:5) = 0.90; 1H-NMR : δ 5.43 , 5.25–5.14 , 5.02 , 4.49 , 4.36–4.25 , 4.20–4.08 , 4.04 , 3.98–3.93 , 3.75 , 3.65 , 2.36–2.25 , 2.16 , 2.06 , 1.98 , 1.64–1.55 , 1.34–1.17 , 0.88 ; HRESIMS m/z 1580.1205 [M+Na]+ .3-O--1,2-distearoyl-sn-glycerol (XIV): 3-O--1,2-distearoyl-sn-glycerol (XIII)  was dissolved in acetonitrile (0.4 mL); TMS-Cl  and sodium iodide  were added and after stirring in the dark at room temperature overnight, the mixture was filtered and washed with dichlorometane; the filtrate was evaporated to get 3-O--1,2-distearoyl-sn-glycerol (XIV) ; Rf (chloroform:methanol 95:5) = 0.30; 1H-NMR : δ 5.42 , 5.24–5.11 , 5.00 , 4.53 , 4.35-4.24 , 4.15–4.24 , 4.02–3.87 , 3.68 , 2.35–2.22 , 2.14 , 2.04 , 1.95 , 1.63–1.51 , 1.32–1.15 , 0.86 ; HRESIMS m/z 1542.1059 [M−H]− .3-O-[6′-O--1′-β-d-galactopyranosyl]-1,2-distearoyl-sn-glycerol (2): 3-O--1,2-distearoyl-sn-glycerol (XIV)  was dissolved in aq. ethanol (85%) (0.4 mL), hydrazine monohydrate  was added, and the reaction mixture was stirred for 7 h at 48°C. After evaporation of solvent under a stream of nitrogen, the residue was purified by silica gel chromatography using a gradient of chloroform/methanol to give 3-O-[6′-O--1′-β-d-galactopyranosyl]-1,2-distearoyl-sn-glycerol (2) ; Rf (chloroform/methanol 8:2) = 0.40; See m/z 1416.0792 .3-5/mL in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% l-glutamine 2 mM, 1% penicillin and streptomycin, human IL-4 (5 ng/mL) and human GM-CSF (100 ng/mL) for five days.For each assay, human peripheral blood mononuclear cells (PBMC) were isolated from two healthy donors by routine Ficoll density gradient centrifugation. Monocytes were purified from human peripheral blood mononuclear cells using MACS CD14 microbeads  according to the manufacturer’s instructions. Purity was checked by staining with a FITC-conjugated anti-CD14 antibody  and FACS analysis. Immature DCs were obtained by incubating monocytes at 8 × 105 cell/mL. Stimulation with PAM2CSK4 1 µg/mL  was used as positive control. Cells treated only with vehicle (DMSO) were used as the control. For the experiment with the anti TLR-4 neutralizing antibody, cells were incubated 30 min with only the antibody (1 µg/mL) at 37 °C before addition of PGDG. After 24 h, expression of all surface markers was estimated again by staining with fluorochrome-conjugated antibodies.After five days in culture, surface staining was performed on monocyte-derived dendritic cells (MoDCs) for flow cytometry analysis with mouse anti-human HLA-DR FITC, CD83 PE, CD86 APC, CD54 PeVio770, CD14 FITC, CD3 PercP  and analyzed by flow-cytometer LSRFortessa™ X-20 cell analyzer  according to standard protocol. MoDC were then incubated with natural and synthetic compound in 12-well plates at concentration of 8 × 10Total RNA was isolated using Trizol Reagent, according to the manufacturer’s protocol. RNA quantity and purity were measured with a NanoDrop 2000 spectrophotometer . Sample purity was checked by A260/A280 ratios between 1.80 and 2.00. Extracted RNAs from all preparations were in this range. Cytokines mRNA expression was measured by quantitative Real Time-PCR. Results are expressed as means ± SD. 5 cells/well. After 16 h TLR4 cells for functional assays were transiently transfected with MD2/CD14 plasmid vector  for 24 h. Cells were then stimulated with compounds for 24 h. LPS for TLR-4 cell line and PAM2CSK4 for TLR-2 cell line were used as positive control under the same conditions. SEAP  was analyzed using the SEAPorter Assay  in according to manufacturer’s instructions. Quantitative data (ng/mL) were obtained by a standard curve for the SEAP protein.Human TLR-4/NF-kB/SEAP and TLR-2/NF-kB/SEAP (SEAPorter™) HEK 293 reporter cells  were plated in 24-well plates at 2 × 10Eight/nine-week-old female C57BL/6 mice were purchased from Charles River  and housed in IGB “A. Buzzati-Traverso” Animal House Facility under standard pathogen-free conditions abiding institutional guidelines. Bone marrow-derived dendritic cells (BM-DCs) were produced from precursors isolated from the bone marrow of C57BL/6 mice by culturing them with 200 U/mL recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF)  in RPMI 1640  medium supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate and 50 µM 2-mercaptoethanol. Immature DCs were collected at day 7 of culture and were assayed for dendritic cell phenotype by staining with the monoclonal antibody anti-CD11c-PE-Cy7 .6/mL immature BM-DCs were incubated for 3 h with different concentrations of OVA257–264 synthetic peptide (from 0.0004 to 4 µM) in presence of 1 µg/mL of LPS, MPLA, or PGDG 2. As control, DCs incubated only with medium were used. After the incubation, cells were washed and co-cultured  with the OTI hybridoma cell line B3Z  for 40 h. The IL-2 released into cell co-culture supernatants was measured by ELISA. Supernatants (0.1 mL/well) were assayed in duplicate using mouse IL-2 ELISA MAX™ Standard , according to the manufacturer’s instructions. B3Z OTI hybridoma cell line, recognizing the OVA257–264 SIINFEKL determinant, was growth in complete RPMI 1640  and was a kind gift of Dr. Bénédicte Manoury . 1 × 10Comparative analysis was performed using the analysis of variance (ANOVA) followed by the Bonferroni post hoc comparisons or the Tukey test for multiple comparison test. A p-value less than 0.05 was considered as statistically significant. All analyses were performed using the GraphPad Prism 5.00 for Windows software . 1), was isolated and characterized for the first time from the marine diatom T. weissflogii. Phosphogalactolipid 1 is a tetra-acyl derivative featured by (poly)unsaturated fatty acid chains on a 3-O-[6′-(sn-glycero-3″-phosphoryl)-β-d-galactopyranosyl]-sn-glycerol. Its molecular core structure was defined by spectroscopic methods and confirmed by total synthesis of the analog 2, performed according to a versatile and general approach that gives access to a series of regio- and stereo-pure phosphatidyl-β-glycosyl-diacylglycerol derivatives for biological and pharmacological development. PGDG lipids gave immunostimolatory activity on DCs in the range 1–10 µg/mL and TLR-based assays evidenced a TLR-4 agonistic activity: this represents the first report of an immunomodulant activity for these uncommon lipids. Functional studies with hybridoma T cells revealed an adjuvant potential of synthetic PGDG 2, significantly higher than standard lipid adjuvants such as LPS and MPLA at 1 µg/mL in presence of OVA257–264 antigen. These results encourage further studies to define the specific intracellular signaling pathway mediated by TLR-4, to assess structure-activity relationship through preparation of molecular analogs and to explore the full pharmacological potential of this class of bioactive lipids. A rare pool of lipids, namely phosphatidylmonogalactosyl diacylglycerols PGDG (


The previous discoveries of butyl fenbufen amide analogs with antitumor effects were further examined. The amide analogs with 1, 3, 4 and 8 carbons chains were prepared in 70–80% yield. Fenbufen had no cytotoxic effects at concentrations ranging from 10 to 100 μM. Methyl fenbufen amide had significant cytotoxic effects at a concentration of 100 μM. As the length of the alkyl amide side chain increased, the cytotoxic effects increased, and the octyl fenbufen amide had the greatest cytotoxic effect. After treatment with 30 μM octyl fenbufen amide, nearly seventy percent of the cells lost their viability. At the concentration of 10 μM, fenbufen amide analogs did not show cytotoxicity according to the MTT assay results. The NO scavenging activities of the fenbufen amide analogs were not significantly different from those of fenbufen. Flash chromatography was performed using Geduran Si 60 silica gel (230-400 mesh).DMF was dried and distilled over CaHThe chemical synthesis for the amide products followed a similar procedure to that described before .N-methyl-4-(biphenyl-4-yl)-4-oxobutanamide (1). Anal. C17H17NO2, M  = 267.2 (m/z), ESI+Q-TOF: M = 267.2 (m/z), [M+H]+ = 268.2 (99%), 269.2 (18%), [M+Na]+ = 290.2 (100%), 291.2 (15%), [2M+H]+ = 535.3 (6%), [2M+Na]+ = 557.2 (53%), 558.2 (19%), 559.3 (3%), equivalent to the calculated isotopic ratio 100:38:8 for [2M+Na]+; 1H-NMR (CD6D6): δ 1.11 , 2.26 , 3.08 , 4.52 , 7.16-7.22 , 7.33-7.37 , 7.89-7.92 .N-propyl-4-(biphenyl-4-yl)-4-oxobutanamide (2). Anal. C19H21NO2, M  = 295.2 (m/z), ESI+Q-TOF: M = 295.2 (m/z), [M+H]+ = 296.3 (100%), 269.2 (19%), [M+Na]+ = 318.2 (74%), 319.2 (10%), [2M+H]+ = 591.3 (3%), [2M+Na]+ = 613.3 (67%), 614.3 (28%), 615.3 (6%), equivalent to the calculated isotopic ratio 100:42:9 for [2M+Na]+; 1H-NMR (CD3OD): δ 0.93 , 1.52 , 2.61 , 3.14 , 3.36 , 7.37-7.40 , 7.45-7.48 , 7.66-7.68 , 7.74-7.75 , 8.06-8.08 .N-butyl-4-(biphenyl-4-yl)-4-oxobutanamide (3). A solution of fenbufen , HBTU , DIEA  and DMF (15 mL) was stirred. The corresponding carboxylic acid analog was added. TLC (acetone/n-hexane = 3/7) indicated the consumption of starting material (Rf = 0.39) and the formation of the product (Rf = 0.78). After stirring for 1 h, the mixture was concentrated under high vacuum with oil pump at 60 °C. The residue was purified by flash chromatography using acetone/n-hexane 3:7 as eluents to provide a snow-white solid in 77% yield (269 mg). Anal. C20H23NO2, M  = 309.2 (m/z), ESI+Q-TOF: M = 309.2 (m/z), [M+H]+ = 310.3 (68%), 311.3 (13%), [M+Na]+ = 332.2 (65%), 333.2 (12%), [2M+Na]+ = 641.3 (100%), 642.3 (42%), 643.3 (10%), equivalent to the calculated isotopic ratio 100:44:10 for [2M+Na]+; 1H-NMR (CD3OD): δ 0.78 , 1.28-1.40 , 1.46-1.52 , 2.60 , 3.18 , 3.35 , 7.37-7.42 , 7.46-7.48 , 7.67-7.68 , 7.74-7.76 , 8.07-8.08 ; 1H-NMR (C6D6): δ 0.78 , 1.07-1.16 , 1.16-1.23 , 2.33 , 3.07 CH2, n-butyl], 3.11 , 4.83 , 7.10-7.22 , 7.32-7.39 , 7.88-7.93 ; 13C-NMR (C6D6): δ 13.9 (CH3), 20.2 (CH2), 30.4 (CH2), 32.0 (CH2), 34.3 (CH2), 39.3 (CH2), 127.4 (CH), 127.5 (CH), 128.2 (CH), 129.0 (CH), 129.1 (CH), 136.0 (C), 140.4 (C), 145.7 (C), 171.1 (C), 198.1 (C).N-octyl-4-(biphenyl-4-yl)-4-oxobutanamide (4). Anal. C24H31NO2, M  = 365.2 (m/z), ESI+Q-TOF: M = 365.3 (m/z), [M+H]+ = 366.3 (30%), 367.3 (7%), [M+Na]+ = 388.3 (15%), 389.3 (4%), [2M+H]+ = 731.5 (8%), 732.5 (4%), [2M+Na]+ = 753.4 (13%),754.4 (8%), equivalent to the calculated isotopic ratio 100:26 for [M+H]+; 1H-NMR (C6D6): δ 0.89 , 1.15-1.22 , 1.25-1.30 , 2.36 , 3.10-3.14 , 7.10-7.22 , 7.33-7.38 , 7.90-7.92 .Escherichia coli serotype 055:B5), sulfanilamide, N-(1-naphthyl)ethylenediamine, phosphoric acid, sodium nitrite, α,α-diphenyl-2-picrylhydrazyl, 3--2,5-diphenyltetrazolium bromide (MTT), and fenbufen were purchased from Sigma-Aldrich Chemical Co. . All samples were dissolved in dimethylsulfoxide (DMSO) and further diluted in culture medium. The final DMSO concentration in the medium was 0.1% and did not affect cellular function or the assay systems used in this study.Fetal bovine serum, dimethyl sulfoxide, lipopolysaccharide . RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics , and 10% heat-inactivated fetal bovine serum (FBS). The cells were incubated in a humidified incubator at 37 °C in 5% CO4/mL on 96-well plates and cultured overnight as described above. The medium was then replaced with fresh medium containing fenbufen derivatives at various concentrations. After incubation for 24 h at 37 °C in 5% CO2/air, MTT  was added, and the cells were then incubated at 37 °C for 2 h. Finally, the cells were lysed and absorbance was detected at 550 nm. For cell number determination, a standard correlation between the known cell numbers and the absorbance density values was constructed for measuring the cell number from various detected absorbance density values.Cell viability was determined using MTT assay . Briefly5 cell/mL) and treated with 100 ng/mL of LPS in the presence or absence of fenbufen derivatives for 24 h. Nitrite, a soluble oxidation product of NO, in the culture supernatanr, was determined using the Griess reaction [2- concentration.In order to measure NO production, macrophage RAW 264.7 cells were plated into 96-well plate (5 × 10reaction . The supt-test. P values less than 0.05 were considered significant.Data are expressed as mean ± S.D. of the indicated number of separate experiments. A one-way analysis of variance was performed for multiple comparisons, and if there was significant variation between treatment groups, the mean values were compared with the respective control using Student’s The methyl, propyl, butyl and octyl analogs of fenbufen were successfully synthesized in good yield. As the length of the alkyl amide side chain increased, the cytotoxic effects increased. The NO scavenging activities of the fenbufen amide analogs were not significantly different from those of fenbufen itself.


Fusarium species, Syncephalastrum racemosum and Botrytis cinerea) were tested on their ability to convert these lactones into other products. Some of the tested fungal strains transformed chloro-, bromo- and iodolactone with a methyl group at C-5 into 2-hydroxy-5-methyl-9-oxabicyclo[4.3.0]nonan-8-one during hydrolytic dehalogenation. When the same lactones had the methyl group at C-3, no structural modifications of halolactones were observed. In most cases, the optical purity of the product was low or medium, with the highest rate for chlorolactone (45.4%) and iodolactone (45.2% and 47.6%). All of the obtained compounds were tested with reference to their smell. Seven halolactones and the hydroxylactone obtained via biotransformation of halolactones with 5-methylcyclohexane ring were examined for their antimicrobial activity. These compounds were capable of inhibiting growth of some bacteria, yeasts and fungi.Eight new lactones , each with a methylcyclohexane ring, were obtained by chemical means from (4-methylcyclohex-2-en-1-yl) acetic acid or (6-methylcyclohex-2-en-1-yl) acetic acid. Whole cells of ten fungal strains ( Modern life sciences are focused on the search for new bioactive compounds of natural origin. It is reported that many plants used in traditional medicine systems contain a variety of compounds responsible for their healing properties. A large part of these compounds are sesquiterpenoid hydroxylactones ,3,4,5,6,However, the isolation of such compounds from plants is often complicated, because their content is usually not impressive. Therefore, it is necessary to look for alternative methods of the preparation of more potentially effective bioactive molecules. One such method is the use of microorganisms capable of introducing a hydroxy group into a molecule of, for example, a lactone. Halogenolactones or saturated lactones are useful compounds for this purpose ,27,28,29As part of our ongoing research on the biotransformation of halolactones with a methyl-substituted cyclohexane rings ,33,34,351a and 1b—were obtained during earlier syntheses +, 108 (11), 94 (100), 79 (81), 67 (46), 43 (34), 39 (45). EA for C9H14O2 (154.21) calculated 70.10% C, 9.15% H, found 70.19% C, 9.10% H.Basic hydrolysis of ester rocedure  yielded cis-trans mixture of acid 2a , according to the procedure of Grabarczyk and Białońska , 126 (17), 112 (68), 84 (68), 69 (100), 56 (18), 43 (50), 39 (33). EA for C9H13ClO2 (188.66), 57.30% C, 6.95% H, found 57.20% C, 6.90% H.Chlorolactonization of a diastereoisomeric 82:18 iałońska  gave 1.62a , according to the known method , 111 (39), 95 (22), 85 (100), 67 (33), 55 (42), 41 (26), 39 (46). EA for C9H13BrO2 (233.11) calculated 46.37% C, 5.62% H, found 46.29% C, 5.66% H.After bromolactonization acid n method , 1.6 g (2a (82:18 diastereoisomeric cis-trans mixture), according to the known procedure , 112 (26), 84 (25), 69 (37), 43 (21), 39 (13). EA for C9H13IO2 (280.11) calculated 38.59% C, 4.68% H, found 38.48% C, 4.57% H. 6a: m.p. = 48–49 °C, 1H-NMR (CDCl3): 0.98 , 1.22 , 1.47 , 1.95 , 2.25 , 2.33 , 2.38 , 2.70 , 3.80 , 4.70 , 13C-NMR (CDCl3): 19.49 (C-9), 27.17 (C-2), 27.40 (C-7), 30.48 (C-4), 31.01 (C-5), 36.59 (C-3), 42.80 (C-6), 86.24 (C-1), 175.13 (C-8), IR : 2936 (s), 1784 (s), 1454 (s), 1177 (s), 1016 (s). EI-MS m/z (%): 153 (100) [M−HI], 135 (26), 127 (13), 107 (20), 93 (28), 81 (36), 67 (21), 55 (34), 39 (31). EA for C9H13IO2 (280.11) calculated 38.59% C, 4.68% H, found 38.51% C, 4.60% H.Iodolactonization of 1.8 g (0.012 mol) of acid rocedure  gave iod1b , according to the known procedure +, 136 (5), 108 (9), 94 (100), 79 (61), 67 (39), 53 (8), 39 (31). EA for C9H14O2 (154.21) calculated 70.10% C, 9.15% H. Found: 70.04% C, 9.10% H.After basic hydrolysis of ester rocedure , acid 2bcis-trans mixture of acid 2b  according to the known procedure +, 167 (12), 159 (22), 153 (68), 109 (45), 93 (89), 81 (43), 67 (62), 55 (53), 39 (100). EA for C9H13ClO2 (188.66), 57.30% C, 6.95% H, found 57.24% C, 6.88% H.Chlorolactonization of a 79:21 diastereoisomeric rocedure  yielded 4b was obtained from 0.3 g (0.0019 mol) of 79:21 diastereoisomeric cis-trans mixture of acid 2b. The physical and spectral data of the product are as follows: nD = 1.5181, 1H-NMR (CDCl3): 1.03 , 1.21 , 1.37 , 1.52 , 1.80 , 2.23 , 2.65 , 2.75 , 4.53 , 4.71 , 13C-NMR (CDCl3): 20.26 (C-9), 25.83 (C-5), 27.37 (C-4), 30.66 (C-3), 31.00 (C-6), 38.23 (C-7), 57.08 (C-2), 81.88 (C-1), 176.13 (C-8), IR : 2930 (s), 1788 (s), 1423 (s), 1138 (s), 950 (s). EI-MS m/z (%): 153 (100) [M−HBr], 135 (23), 109 (43), 93 (37), 81 (16), 67 (39), 55 (17), 39 (35). EA for C9H13BrO2 (233.11) calculated 46.37% C, 5.62% H, found 46.42% C, 5.70% H.Using the previously described method  0.28 g , according to a known procedure , 135 (27), 107 (28), 93 (50), 79 (10), 67 (9), 55 (11), 39 (25). EA for C9H13IO2 (280.11) calculated 38.59% C, 4.68% H, found 38.71% C, 4.61% H. 5b: oil, 1H-NMR (CDCl3): 1.12 , 1.72–1.78 , 2.31 , 2.45 , 2.67 , 3.70 , 4.78 , 13C-NMR (CDCl3): 23.77 (C-9), 27.78 (C-5), 27.90 (C-4), 31.59 (C-7), 35.58 (C-6), 38.02 (C-3), 39.44 (C-2), 86.25 (C-1), 175.32 (C-8), IR : 2954 (s), 1772 (s), 1464 (s), 1178 (s), 952 (s). EA for C9H13IO2 (280.11) calculated 38.59% C, 4.68% H, found 38.53% C, 4.63% H.Iodolactonization of acid rocedure  gave iodFusarium culmorum AM10, Fusarium avenaceum AM11, Fusarium oxysporum AM13, Fusarium tricinctum AM16, Fusarium semitectum AM20, Fusarium equiseti AM22, Fusarium scirpi AM1199, Fusarium solani AM203, Syncephalastrum racemosum AM105 and Botrytis cinerea AM235). These strains were cultivated on Sabouraud’s agar containing 0.5% of aminobac, 0.5% of peptone, 4% of glucose and 1.5% of agar dissolved in distilled water at 28 °C and stored in refrigerator at 4 °C. Screening and preparative biotransformations were performed as described before [2-hydroxy-5-methyl-9-oxabicyclo[4.3.0]nonan-8-one (7a) are as follows: m.p. = 89–90 °C, 1H-NMR (CDCl3): 0.93 , 1.06 , 1.26 , 1.53 , 1.73 , 1.88 , 2.00 , 2.10 , 2.40 , 2.68 , 3.72 , 4.57 , 13C-NMR (CDCl3): 19.60 (C-9), 28.86 (C-3), 30.78 (C-4), 31.88 (C-5), 37.02 (C-7), 42.98 (C-6), 70.08 (C-1), 81.83 (C-2), 176.33 (C-8), IR : 3305 (sb), 2927 (s), 1769 (s), 1160 (s). EI-MS m/z (%):170 (6) [M]+, 170, 153 (11), 123 (8), 111 (39), 85 (100), 67 (33), 39 (42). EA for C9H14O3 (170.21) calculated 63.51% C, 8.29% H, found 63.43% C, 8.18% HThe fungal strains used in all biotransformations came from the collection of the Institute of Biology and Botany, Medical University, Wrocław, Poland  and 10 g of glucose dissolved in 1 L of distilled water. Yeast and fungi cultures were growing in YPG medium which was composed of 10 g of yeast extract, 10 g of bacteriological peptone and 10 g of glucose dissolved in 1 L of distilled water for 48 and 96 h, respectively. The test were performed in the automated Bioscreen C system .The tests were conducted on certain bacteria strains: Working volume in the wells of the Bioscreen plate was 300 µL, comprised of 280 µL of culture medium, 10 µL of cells or spore solution . Tested lactones were dissolved in DMSO and used at the final concentration of 0.1% (w/v). Temperature was controlled at 30 °C  and 25 °C (filamentous fungi). The optical density of the cell suspensions was measured automatically at 560 nm in regular intervals of 30 min, for 3–4 days. The cell cultures were continuously shaken. Each culture was performed in three replicates. Data was analyzed by using spreadsheet software (Excel 97) and were calculated as the averages from the tree replicate analysis for each type of culture medium. The averages were used to generate the growth curves for each strain studied, constructed as a function of the incubation time and the absorbancy of the culture medium. The resulting microbial growth curves were compared to control cultures in medium supplemented with DMSO.The odour evaluation was performed for ethanolic solutions (10%) of samples with the use of a strip blotter.N2 mechanism. The tested strains were also characterized by substrate specificity—iodolactone 5a was transformed at the greatest rate (eight strains) and chlorolactone 3a at the lowest rate (only three strains). The best results were obtained when F. equiseti AM22, F. avenaceum AM11, F. oxysporum AM13 and S. racemosum AM105 were used as biocatalysts. Degrees of conversion of the substrates 3a–5a were over 80%. Most of the microorganisms formed the (‒)-isomer of the hydroxylactone and the (+)-isomer was produced only by two microrganisms. The halolactones 3a–6a and 3b–5b and hydroxylactone 7a were tested for their ability to inhibit the growth of some bacteria, yeast and fungal strains. The biological tests indicated the ability of these compounds to inhibit or limit growth of most microorganisms. Odor evaluation of all the described compounds revealed that most of them were characterized by uninteresting smells, except for acid 2a, chlorolactone 3b and iodolactone 5b. The smell of acid 2a  was particularly interesting. Chlorolactone 3b and iodolactone 5b were characterized by a slight dried fruit scent and a slight dusty scent, respectively.Two-step chemical synthesis yielded eight halolactones with methylcyclohexane rings. Six of them were subjected to a screening biotransformation by ten fungal strains. Most of these strains were capable of transforming the halolactones with the methyl group at the C-4 position into the corresponding hydroxylactone. The selected microorganisms showed very high regioselectivity, because the hydroxy group was introduced at C-2 in the equatorial position due to the operative S


Additionally, recent studies showed an anti-inflammatory efficacy in vivo. Because of its interference with many targets, YS-121 is a promising drug candidate for the treatment of inflammatory diseases. Ongoing preclinical studies will thus necessitate huge amounts of YS-121. To cope with those requirements, we have optimized the synthesis of YS-121. Surprisingly, we isolated and characterized byproducts during the resulting from nucleophilic aromatic substitution reactions by different tertiary alkylamines at a heteroaromatic halide. These amines should actually serve as assisting bases, because of their low nucleophilicity. This astonishing fact was not described in former publications concerning that type of reaction and, therefore, might be useful for further reaction improvement in general. Furthermore, we could develop a proposal for the mechanism of that byproduct formation.YS-121 [2-pyrimidin-2-ylthio)octanoic acid] is the result of target-oriented structural derivatization of pirinixic acid. It is a potent dual PPAR POCl3  was added through a dripping funnel at 0 °C to the thioether derivative 4  resulting from step (i), followed by N,N-diethylaniline  at 0 °C. That mixture was stirred at 90 °C for 45 h (TLC control). Then, excess POCl3 was distilled off at atmospheric pressure, and the resulting oil was poured onto crushed ice. The aqueous solution was extracted with EtOAc, and the combined organic layers were washed with diluted HCl, saturated NaHCO3 solution and brine. Then, the organic layer was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was purified by column chromatography  to give 5  as a red oil. Rf = 0.6 ; 1H-NMR : δ = 0.86 , 1.20 , 1.24–1.34 , 1.36–1.43 , 1.79–1.97 , 4.15 , 4.37 , 7.75 ; MS (ESI+): m/e = 351.1 [M + H]+.Step (iii a). A solution of the chlorinated pyrimidine derivative 5  obtained from step (ii), triethylamine  and 2,3-dimethylaniline  in EtOH (62.5 mL) was refluxed under stirring for 65 h (TLC control). Then, EtOH and excess triethylamine were evaporated under reduced pressure. The residue was dissolved in EtOAc, and the organic layer was extracted with diluted HCl, saturated NaHCO3 solution and brine. Then, the organic layer was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was roughly purified by column chromatography  to give a mixture of the desired compound 6 together with one byproduct as a yellow oil (14.0 g). MS (ESI+): m/e = 436.2 [M + H]+, byproduct: MS (ESI+): m/e = 473.3 [M + H]+. That mixture was used for step (iv) without further purification.6 (210 mg) as a yellow oil. Therefore, purification by RP-18 silica gel is already useful and applicable at that stage of synthesis. By extrapolating the result of RP-18 column chromatography of 500 mg up to 14.0 g, a yield of 5.88 g, 25% of 6 could have been achievable. Rf = 0.2 ; 1H-NMR 2SO): δ = 0.83 , 1.15 , 1.16–1.34 , 1.63–1.84 , 2.05 , 2.26 , 4.08 , 4.25 , 6.20 , 7.06-7.13 , 9.51 ; MS (ESI+): m/e = 436.2 [M + H]+.After completion of step (iv), further purification of 500 mg of that mixture by column chromatography  gave the desired compound Step (iv). The mixture of 6 and one byproduct (13.5 g) obtained in step (iii a) was dissolved in EtOH (420 mL) at room temperature. An approximate excess of LiOH  was added, and the resulting mixture was stirred at room temperature for 120 h (TLC control). Then, EtOH was removed under reduced pressure, and the obtained residue was dissolved in water under heating, while low amounts of MeOH were added. The solution was acidified with diluted HCl, and the precipitate was filtered, washed to neutrality with water and then with n-hexane. A solution of the precipitate in EtOAc was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was purified by column chromatography . Finally a solution of the purified product in EtOAc was extracted with diluted HCl, washed to neutrality with water and was dried over MgSO4. After filtration, the solvent was evaporated under reduced pressure and the product was precipitated from EtOAc/n-hexane. After two weeks at 26 °C, the solid product was filtered and was washed with n-hexane. In the end 2.00 g of the carboxylic acid derivative 2 were obtained as a pale yellow solid. By including the extrapolated content of 6 in the mixture used in that step, a yield of 38% was achieved. Rf = 0.4 ; 1H-NMR : δ = 0.85 , 1.10–1.37 , 1.59–1.91 , 2.08 , 2.28 , 4.23 , 6.21 , 7.08–7.16 , 9.51 , 12.82 ; 13C-NMR : δ = 13.85 (CH3-Hex), 13.99 (Ph-2-CH3), 20.06 (Ph-3-CH3), 21.90 (CH2-Hex), 26.38 (CH2-Hex), 28.08 (CH2-Hex), 30.94 (CH2-Hex), 31.95 (CH2-Hex), 46.93 (SCH), 98.90 (Pyr-5-C), 123.99 (Ph-6-C), 125.67 (Ph-5-C), 127.83 (Ph-4-C), 132.33 (Ph-2-C), 135.61 (Ph-3-C), 137.59 (Ph-1-C), 157.73 (Pyr-4-C), 162.16 (Pyr-2-C), 169.63 (Pyr-6-C), 172.47 (COOH); MS (ESI+): m/e = 408.1 [M + H]+; HRMS (MALDI): calcd. for C20H27ClN3O2S+ [M + H]+ = 408.1507, found 408.1509; Anal. Calcd. C20H26ClN3O2S C, H, N: Calc. C 58.88, H 6.42, N 10.30, found C 59.12, H 6.52, N 10.15.8 could be isolated as well as a pale, yellow solid. Rf = 0.6 ; 1H-NMR : δ = 0.85 , 1.05 2, J = 7.0 Hz), 1.19–1.41 , 1.69–1.88 , 2.08 , 2.26 , 3.27–3.43 2), 4.25 , 5.19 , 6.98–7.15 , 8.38 , 12.74 ; 13C- NMR : δ = 12.79 (N(CH2CH3)2), 13.84 (CH3-Hex), 14.01 (Ph-2-CH3), 20.16 (Ph-3-CH3), 21.92 (CH2-Hex), 26.80 (CH2-Hex), 28.30 (CH2-Hex), 31.07 (CH2-Hex), 32.42 (CH2-Hex), 41.49 (N(CH2CH3)2), 46.96 (SCH), 77.39 (Pyr-5-C), 123.43 (Ph-6-C), 125.32 (Ph-5-C), 126.38 (Ph-4-C), 131.59 (Ph-2-C), 137.13 (Ph-3-C), 137.52 (Ph-1-C), 160.42 (Pyr-4-C), 161.80 (Pyr-2-C), 167.63 (Pyr-6-C), 173.29 (COOH); MS (ESI+): m/e = 445.3 [M + H]+; HRMS (MALDI): calcd. for C24H37N4O2S+ [M + H]+ = 445.2632, found 445.2635.During purification procedure of step (iv), 5  obtained from step (ii), triethylamine  and 2,3-dimethylaniline  in EtOH (1.33 mL) was refluxed under stirring for 14 h (TLC control). Then, EtOH and excessive triethylamine were evaporated under reduced pressure. The residue was dissolved in EtOAc, and the organic layer was extracted with diluted HCl, saturated NaHCO3 solution and brine. Then, the organic layer was dried over MgSO4, filtered and evaporated under reduced pressure. Compound 9 was precipitated from EtOAc/n-hexane at 26 °C to give 9  as a colorless solid. 1H-NMR : δ = 0.86 , 1.11–1.39 3, OCH2CH3, CH2-Hex), 1.71–1.89 , 2.10 , 2.30 , 3.65–3.82 3), 4.04–4.16 , 4.30 , 6.91–7.24 , 10.22 ; 13C-NMR : δ = 7.68 (N(CH2CH3)3), 13.82 (CH3-Hex), 13.92 (OCH2CH3), 14.18 (Ph-2-CH3), 20.08 (Ph-3-CH3), 21.85 (CH2-Hex), 26.39 (CH2-Hex), 28.08 (CH2-Hex), 30.87 (CH2-Hex), 30.95 (CH2-Hex), 47.07 (SCH), 53.41 (N(CH2CH3)3), 61.05 (COOCH2), 95.39 (Pyr-5-C), 123.57 (Ph-6-C), 125.63 (Ph-5-C), 127.94 (Ph-4-C), 131.92 (Ph-2-C), 135.42 (Ph-3-C), 137.59 (Ph-1-C), 159.10 (Pyr-4-C), 162.76 (Pyr-2-C), 170.26 (Pyr-6-C), 171.21 (COOCH2CH3); MS (ESI+): m/e = 502.3.A solution of the chlorinated pyrimidine derivative 5  obtained from step (ii), N-methylpyrrolidine  and 2,3-dimethylaniline  in EtOH (0.667 mL) was refluxed under stirring for 21 h (TLC control). Then, EtOH and excessive N-methylpyrrolidine were evaporated under reduced pressure. The residue was dissolved in EtOAc, and the organic layer was extracted with diluted HCl, saturated NaHCO3 solution and brine. Then, the organic layer was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was roughly purified by column chromatography  to give a mixture of compound 11 and compound 12. To separate both compounds a second column chromatography step followed using silica gel and an eluent gradient of n-hexane-EtOAc from 50:1 to 20:1 to give 11  as a colorless oil and 12  as a light yellow oil.A solution of the chlorinated pyrimidine derivative Compound11. Rf = 0.4 ; 1H-NMR : δ = 0.86 , 1.17 , 1.25–1.43 , 1.75–1.99 , 3.24–3.42 , 4.06–4.16 , 4.21–4.26 , 4.35 , 5.42 ; 13C-NMR : δ = 13.80 (CH3-Hex), 13.93 (COOCH2CH3), 14.43 (ArOCH2CH3), 21.89 (CH2-Hex), 24.62 , 26.62 (CH2-Hex), 28.11 (CH2-Hex), 30.93 (CH2-Hex), 31.37 (CH2-Hex), 46.07 , 46.88 (SCH), 60.70 (COOCH2),61.43 (ArOCH2), 81.23 (Pyr-5-C), 161.02 (Pyr-2-C), 167.38 (Pyr-4-C), 168.39 (Pyr-6-C), 171.72 (COOCH2CH3); MS (ESI+): m/e =396.2.Compound12. Rf = 0.3 ; 1H-NMR : δ = 0.86 , 1.17 , 1.25–1.43 , 1.59–1.99 , 2.95 , 3.43–3.59 , 4.06–4.16 , 4.21–4.26 , 4.34 , 5.60 ; 13C-NMR : δ = 13.80 (CH3-Hex), 13.93 (COOCH2CH3), 14.43 (ArOCH2CH3), 21.89 (CH2-Hex), 24.13 (NCH2CH2CH2CH2Cl), 26.62 (CH2-Hex), 28.09 (CH2-Hex), 29.36 (NCH2CH2CH2CH2Cl), 30.93 (CH2-Hex), 31.32 (CH2-Hex), 35.13 (NCH3), 45.09 (NCH2CH2CH2CH2Cl), 46.79 (SCH), 47.48 (NCH2CH2CH2CH2Cl), 60.74 (COOCH2), 61.51 (ArOCH2), 80.63 (Pyr-5-C), 162.83 (Pyr-2-C), 167.31 (Pyr-4-C), 168.98 (Pyr-6-C), 171.71 (COOCH2CH3); MS (ESI+): m/e = 446.2.Step (iii b). A solution of the chlorinated pyrimidine derivative 5  obtained from step (ii), sodium carbonate  and 2,3-dimethylaniline  in EtOH (1.33 mL) was refluxed under stirring for 22 h (TLC control). Then, excessive sodium carbonate was filtrated and EtOH was evaporated under reduced pressure. The residue was dissolved in EtOAc, and the organic layer was extracted with diluted HCl, saturated NaHCO3 solution and brine. Then, the organic layer was dried over MgSO4, filtered and evaporated under reduced pressure. The crude product was purified by column chromatography  to give compound 6  as a yellow oil. For analytical data of 6, see 3.4.2) as a pure compound for use in ongoing preclinical studies. By a change of the assisting base triethylamine to sodium carbonate in step (iii b), we succeeded in optimizing that step. Byproducts were formed to a lesser extent and the purification procedure was dramatically simplified.Here, we have demonstrated that syntheses conducted in a larger scale can generate problems and byproducts that did not appear at these syntheses at the original small scale, but we also show that it is possible to resolve these problems by a change in the purification procedure or reagents used as well as in the way of synthesis and thus obtained clean compounds. By conducting the described synthesis, it was possible to generate up to 2.00 g of YS-121 (7 during step (iii a) that was isolated later, in step (iv), after saponification, as 8.The reaction times are probably reducible, but our aim was first and foremost to get the highest yield possible during each respective step. After having conducted several reactions with two different assisting bases in step (iii a) and after having isolated and characterized different byproducts formed during those reactions, we could develop a proposal for the mechanism of byproduct formation. Therefore, we could clearly identify triethylamine as the reason for the formation of byproduct N,N-dialkylaminyl substituent at the heteroaromatic core was not described before during the used conditions. That unexpected fact can be very useful for reaction improvement of other nucleophilic aromatic substitution reactions at heteroaromatic halides in general.Additionally, we could show that also other tertiary alkylamines could act as nucleophiles during a nucleophilic aromatic substitution reaction at a heteroaromatic halide. Such a formation of byproducts resulting from an initial nucleophilic attack of a tertiary alkylamine and a following elimination of one alkyl residue leading to a


E)-3,7-dimethylocta-2,6-dienoate-6-O-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside (1), -3,7-dimethyl-3,6-octadiene-1,2,8-triol (2) and (6E)-7-methyl-3-methylene-6-octene-1,2,8-triol (3) were isolated from Acanthopanax sessiliflorus fruits, along with three known monoterpenoid compounds. The structures of the new compounds were determined by means of extensive spectroscopic analysis  and chemical methods.Three new acyclic monoterpenoids named (2 Acanthopanax species  are shrubs found mainly in China, Korea, and Japan. Acanthopanax sessiliflorus (Rupr. et Maxim) Seem. is known to be one of the most abundant species, and the pharmacology research has demonstrated that the fruits of Acanthopanax have many biological functions, including antitumor -β-d-glucopyranose (6) , [δ 1.62 ], [δ 2.18 ], two trisubstituted alkenes , and two anomeric protons [δ 5.47 ], [δ 4.27 ]. 1H-1H correlations were observed between the following protons pairs: δ 2.20 (H-4) and δ 2.21 (H-5); δ 2.21 (H-5) and δ 5.10 (H-6), suggesting a –CH2CH2CH– fragment. Two anomeric proton signals were assigned to two anomeric carbon signals at δ 95.2 (C-1') and 104.8 (C-1'') respectively, in the HMQC experiment. By comparing coupling constants and the chemical shifts of the sugar signals with those reported , two methylenes bearing an oxygen function , a methylene  and a methine [δ 4.61 ].Compound tra of 2  showed t1H-1H COSY spectrum showed correlations between δ 3.46 (H-1a), 3.55 (H-1b) and δ 4.61 (H-2), suggesting a –CHOHCH2OH fragment. 1H-1H correlations were observed between the following proton pairs: δ 5.29 (H-4) and δ 2.87 (H-5a); δ 2.87 (H-5a) and δ 5.38 (H-6), suggesting a –CHCH2CH– fragment. Moreover, in the HMBC spectrum, long-range correlations between the methyl proton signals at δ 1.68 (H-10) and the carbon signals at δ 71.6 (C-2), 136.3 (C-3) and 127.6 (C-4) could be identified. Correlations were also observed between the methyl proton signals at δ 1.67 (H-9) and the carbon signals at δ 125.2 (C-6), 136.2 (C-7) and 68.9 (C-8) (The .9 (C-8) .δ 1.68 (H-10) and δ 5.29 (H-4); δ 5.38 (H-6) and δ 3.91 (H-8), which indicated a trisubstituted alkene with the cis-configuration in the 3 position and another alkene with the trans-configuration in the 6 position. Thus, the structure of compound 2 could be elucidated as -3,7-dimethyl-3,6-octadiene-1,2,8-triol.In the NOESY spectrum, NOE correlations were observed between the following proton pairs: 3 was isolated as a yellow oil. c 0.3, MeOH). The molecular formula C10H18O3 was defined by HRESIMS . The NMR data , 5.11], a methyl [δ 1.66 ] and a methine [δ 5.43 ]. The structure of 3 was subsequently assigned by HMQC, DQF COSY and HMBC. In the HMBC spectrum (δ 4.93 (H-10a) and δ 76.6 (C-2), 150.3 (C-3), 33.1 (C-4); δ 1.66 (H-9) and δ 126.3 (C-6), 136.3 (C-7), 68.9 (C-8). Finally, the configuration of the trisubstituted alkene was clarified by a difference NOE experiment. The irradiation of δ 3.91 (H-8) resulted in an enhancement at δ 5.43 (H-6), suggesting the trans-configuration at the 6 position, Therefore, compound 3 was identified as (6E)-7-methyl-3-methylene-6-octene-1,2,8-triol.Compound NMR data  showed aspectrum , the lon1H, 13C and 2D  NMR spectra were recorded on a JNM-ECA600 spectrometer  using standard pulse sequence. Chemical shifts were reported in ppm (δ), and scalar coupling were reported in Hz. GC analyses were carried out using a Fuli 9790 instrument, DM-5 column .Open column chromatography was carried out using silica gel  or octadecyl silica gel  as stationary phase. TLC employed precoated silica gel plates . Preparative HPLC was carried out on a Waters 600 instrument equipped with a Waters RID-2414 detector. A Waters Sunfire prep C18 OBD  column was used for preparative purpose. The IR spectra were recorded as KBr pellets on a Jasco 302-A spectrometer . The UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer . Optical rotation was recorded on a Jasco P-2000 polarimeter. HRESIMS were measured on a FTMS-7 instrument . The A. sessiliflorus (Rupr. et Maxim.) Seem. were collected from WuJia Agriculture Sci-Tech Co. Ltd. of Dandong, Liaoning Province, China in October, 2009 and identified by Professor Jincai Lu of School of Traditional Chinese Materia Medica of Shenyang Pharmaceutical University, China.The fruits of A. sessiliflorus (Rupr. et Maxim.) Seem. were extracted with 70% EtOH (3 × 32 L) under reflux (1 h). The combined extract was concentrated under vacuum yielding a residue (2.2 kg) which was dissolved in water, loaded on a D101 macroporous adsorption resin column and eluted successively with H2O, 30% EtOH, 60% EtOH and 95% EtOH.The dried and powdered fruits (12 kg) of 3–MeOH , and finally with MeOH alone, to give five fractions 1–6. Fraction 4 (9.7 g) was subjected to reversed-phase silica gel column chromatography  to afford six fractions . Fr. 4.2 (506 mg) was separated by HPLC [MeCN–H2O ] to give 2 (10.6 mg) and 3 (11.2 mg). Fraction 4.3 (755 mg) was separated by HPLC [MeOH–H2O ] to give 6 (15.8 mg). Fraction 5 (15.1 g) was subjected to reversed-phase silica gel column chromatography  to afford ten fractions . Fraction 5.2 (533 mg) was separated by HPLC [MeOH–H2O ] to give 1 (20.1 mg). Fraction 5.3 (197 mg) was separated by HPLC [MeOH–H2O ] to give 4 (15.3 mg). Fraction 5.4 (426 mg) was separated by HPLC [MeOH–H2O ] to give 5 (8.5 mg). The known compounds kenposide A (4) [5) [O-[(S)-oleuropeyl]-β-d-glucopyranose (6) + ; UV max (MeOH) 221 nm; IR (KBr) 3410, 1715, 1645, 1423, 1385, and 1073 cm−1; 1H- and 13C-NMR (CD3OD) data, see -3,7-Dimethyl-3,6-octadiene-1,2,8-triol (2). c 0.3, MeOH). HR-ESI-MS m/z 209.1153 [M+Na]+ ; UV max (MeOH): 205 nm; IR (KBr) 3355, 2971, 1717 and 1160 cm−1; 1H- and 13C-NMR (CD3OD) data, see (6E)-7-Methyl-3-methylene-6-octene-1,2,8-triol (3). c 0.3, MeOH). HR-ESI-MS m/z 209.1150 [M+Na]+ ; UV max (MeOH): 202 nm; IR(KBr) 3903, 2965, 1668 and 1055 cm−1; 1H- and 13C-NMR (CD3OD) data, see 1 (3.03 mg) was hydrolyzed with 1 M HCl (1.0 mL) for 2 h at 85 °C. The reaction mixture was cooled and partitioned between CHCl3 (2.0 mL) and H2O (2.0 mL). The aqueous layer was washed with CHCl3 (3.0 mL × 3), neutralized with Ba(OH)2, filtered, and evaporated under reduced pressure. The residue was dissolved in pyridine (1.0 mL) and 0.1 M L-cysteine methyl ester hydrochloride in pyridine (2.0 mL) was added. The mixture was heated at 60 °C for 1 h. An equal volume of Ac2O was added with heating continued 1 h. The acetylated thiazolidine derivatives were analyzed by GC using a DM-5 Column . Temperatures of injector and detector were 280 °C for both. A temperature gradient system was used for the oven; starting at 160 °C and increasing up to 195 °C at a rate of 5 °C/min. Peaks of the hydrolysate were detected by comparison with retention time of authentic samples of d-glucose (10.08 min) and l-arabinose (6.55 min) after treatment with l-cysteine methyl ester hydrochloride in pyridine.Compound E)-3,7-dimethylocta-2,6-dienoate-6-O-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside (1), -3,7-dimethyl-3,6-octadiene-1,2,8-triol (2) and (6E)-7-methyl-3-methylene-6-octene-1,2,8-triol (3) were isolated from the EtOH extract of the dried fruits of Acanthopanax sessiliflorus together with three known monoterpenoids. To the best of our knowledge, this is the first scientific report of acyclic monoterpenoids from Acanthopanax plants.In this paper, three new acyclic monoterpenoids, (2


S-epoxycycloneran-3,15-diol, 7,10R-epoxycycloneran-3,15-diol, and (10Z)-15-acetoxy-10-cycloneren-3,7-diol, were isolated in addition to the known (10Z)-cyclonerotriol, (10E)-cyclonerotriol, catenioblin C, and chokol E from the culture of Trichoderma asperellum A-YMD-9-2, an endophytic fungus obtained from the marine red alga Gracilaria verrucosa. The structures of previously unreported compounds were established by spectroscopic techniques, including 1D/2D NMR, MS, and IR. The isolation of these new cyclonerane derivatives greatly adds to the structural diversity of unusual cyclonerane sesquiterpenes, and several isolates exhibit potent inhibition against some marine phytoplankton species.Seven previously unreported cyclonerane derivatives, namely, 3,7,11-trihydroxycycloneran-10-one, cycloneran-3,7,10,11-tetraol, cycloneran-3,7,11-triol, 11,12,15-trinorcycloneran-3,7,10-triol, 7,10 Trichoderma species have proven to be prolific sources of diterpenes and sesquiterpenes, especially some unusual terpene skeletons, such as harziane, proharziane, wickerane, citrinovirin, trichaspin, and cyclonerane + , requiring two degrees of unsaturation. Its IR absorption bands at 3440 and 1713 cm−1 suggested the presence of hydroxy and carbonyl groups. In combination with HSQC data, the 1H NMR spectrum  to C-10 (δC 215.1), C-11 (δC 79.4), and C-15 (δC 28.0) and from H3-15 (δH 1.29) to C-10, C-11, and C-12 (δC 27.8) established the connectivity between C-10 and C-11, which then extended to C-8 (δC 31.5) as corroborated by the HMBC correlation from H2-9 (δH 2.51) to C-10 as well as by the COSY correlation from H2-8 (δH 2.12 and 1.89) to H2-9. The connectivity around ring A was deduced to be the same as those of cyclonerodiol based on their identical NMR data, and this was confirmed by the COSY correlations from H3-1 (δH 1.03)/H-2 (δH 1.56)/H-6 (δH 1.97)/H2-5 (δH 1.88 and 1.63)/H2-4 (δH 1.67 and 1.56) and HMBC correlations from H3-1 to C-2 (δC 44.9), C-3 (δC 81.4), and C-6 (δC 55.1), from H3-13 (δH 1.25) to C-2, C-3, and C-4 (δC 40.4), and from H3-14 (δH 1.19) to C-6, C-7 (δC 76.0), and C-8 (δC 31.5)  and from H3-13 to H-5b (δH 1.63) suggested Me-1 to be opposite to Me-13. The above information evidenced 1 to be 3,7,11-trihydroxycycloneran-10-one.Compound spectrum  showed ospectrum  were assnerodiol . HMBC coδC 31.5) . The NOE2 was isolated as a colorless oil, and its molecular formula was established as C15H30O4, which has two more hydrogen atoms than that of 1, based on the (+)-HREIMS m/z 297.2037 [M + Na]+ . A detailed comparison of its NMR signals  at δH 3.38 in the 1H NMR spectrum. Combined with the HSQC correlation of this proton signal to the carbon signal at δC 79.0, this signal was attributed to the oxymethine proton on C-10. This was supported by the HMBC correlations from oxymethine sp3 carbon at δC 79.0 (C-10) to H3-12 (δH 1.16) and H3-15 (δH 1.21) as well as a COSY correlation from H-10 to H2-9 (δH 1.61 and 1.40)  . Thus, 2NMR data . To conf-15 δH 1. as well 3 was purified as a colorless oil, and its HREIMS m/z 258.2198 [M]+  produced the molecular formula C15H30O3, which has one oxygen atom less than that of 2. Its 13C NMR spectrum , which was supported by its HMBC correlations to H3-12 (δH 1.22) and/or H3-15 (δH 1.22). Other HMBC and COSY correlations, as shown in 3 to be cycloneran-3,7,11-triol, a 10-deoxy derivative of 2.Compound spectrum  showed a4 was also isolated as a colorless oil. A molecular formula C12H24O3 was assigned by HREIMS m/z 216.1724 [M]+ , implying one degree of unsaturation. Its 1H and 13C NMR data  and the presence of signals for a hydroxymethylene group (δH 3.68 and δC 63.6). This group was deduced to be situated at the side chain terminus on the basis of HMBC correlations from H3-14 (δH 1.17) to C-6 (δC 54.9), C-7 (δC 74.8), and C-8 (δC 36.8) and COSY correlations from H2-10 (δH 3.68) to H2-9 (δH 1.68) and from H2-9 to H2-8 (δH 1.57). HMBC correlations (3-1 (δH 1.05) to C-2 (δC 44.5), C-3 (δC 81.4), and C-6, and from H3-13 (δH 1.26) to C-2, C-3, and C-4 (δC 40.5), and COSY correlations from H3-1 to H2-4 (δH 1.69 and 1.56) via H-2 (δH 1.60), H-6 (δH 1.87), and H2-5 (δH 1.88 and 1.55), further verified the connectivity of ring A. Thus, 4 was proposed to be 11,12,15-trinorcycloneran-3,7,10-triol, possibly a degradation product of 1 or 2. To ascertain the relative configuration at C-7, the energy-minimized conformers, regardless of the rotation of the methyl and hydroxy groups as well as the side chain, of 7α- and 7β-methyl isomers optimized at the B3LYP/6-31G(d) level in chloroform, were subjected to the 13C NMR calculations using the gauge-independent atomic orbital (GIAO) method at the B3LYP/6-31+G level via Gaussian 09 software + . Its 1H and 13C NMR data . Thus, 6 was deduced to be a C-10 epimer of 5, as supported by the NOESY correlation from H3-12 (δH 0.90) to H3-14 (δH 1.17). The similar chemical shifts of C-7 (δC 86.0) and C-10 (δC 84.2) to those of epicyclonerodiol oxide further evidenced the opposite orientation of H-10 and C-14 20D − 24 ; IR (KBr) vmax 3440, 2960, 2932, 1713, 1460, 1663, 1374, 1239, 1125, 1013, 921 cm−1; 1H and 13C NMR data, m/z (%) 272 [M]+ (<1), 196 (12), 178 (45), 141 (95), 136 (44), 121 (39), 95 (39), 71 (40), 56 (40), 44(100); HREIMS m/z 272.1991 [M]+   .2): Colorless oil; [α]20D − 30 ; IR (KBr) vmax 3416, 2967, 1654, 1460, 1378, 1266, 1162, 1074, 1032, 921, 885 cm−1; 1H and 13C NMR data, +MS m/z 297 [M + Na]+; HRESI+MS m/z 297.2037 [M + Na]+   .3): Colorless oil; [α]20D − 18 ; IR (KBr) vmax 3406, 2965, 1654, 1460, 1377, 1164, 920, 885 cm−1; 1H and 13C NMR data, m/z (%) 258 [M]+ (<1), 207 (25), 157 (28), 139 (81), 127 (70), 109 (81), 95 (38), 81(45), 44(100); HREIMS m/z 258.2198 [M]+   .4): Colorless oil; [α]20D − 39 ; IR (KBr) vmax 3426, 2928, 1720, 1635, 1460, 1383, 1054, 920 cm−1; 1H and 13C NMR data, m/z (%) 216 [M]+ (<1), 165 (11), 157 (11), 139 (79), 103 (31), 96 (40), 85 (98), 81(58), 44(100); HREIMS m/z 216.1724 [M]+   .5): Colorless oil; [α]20D +11 ; IR (KBr) vmax 3426, 2965, 2931, 2876, 1654, 1460, 1376, 1205, 1092, 1024, 919 cm−1; 1H and 13C NMR data, m/z (%) 256 [M]+ (<1), 183 (20), 143 (100), 139 (32), 125 (35), 113 (30), 59 (42), 44(100); HREIMS m/z 256.2034 [M]+   .6): Colorless oil; [α]20D +6.5 ; IR (KBr) vmax 3426, 2965, 2931, 2868, 1635, 1460, 1376, 1206, 1152, 1093, 1024, 919 cm−1; 1H and 13C NMR data, m/z (%) 256 [M]+ (<1), 237 (8), 179 (10), 143 (90), 125 (32), 95 (28), 56 (41), 44(100); HREIMS m/z 256.2028 [M]+   .7): Colorless oil; [α]20D − 11 ; IR (KBr) vmax 3452, 2963, 2932, 1735, 1654, 1460, 1370, 1240, 1025, 920, 885 cm−1; 1H and 13C NMR data, m/z (%) 256 [M]+ (<1), 139 (33), 125 (98), 107 (28), 81 (27), 44(100); HREIMS m/z 298.2138 [M]+  ((10Z)-15-Acetoxy-10-cycloneren-3,7-diol (98.2144) .Trichoderma asperellum A-YMD-9-2 led to the isolation and identification of seven new (1–7) and four known (8–11) cyclonerane derivatives, including an unprecedented trinorcyclonerane (4). The discovery of these new isolates greatly diversifies the unusual cyclonerane sesquiterpenes. Among the isolates, 10 displays significant inhibition of the four phytoplankton species and features no toxicity to any of the zooplankton tested.Chemical examination of the marine-alga-endophytic fungus


Subsequent study on its active components led to the isolation and identification of two new dibenzylbutyrolactone lignans named 4-demethyltraxillaside (1) and nortrachelogenin 4-O-β-d-glucopyranoside (2), together with six known compounds. The lignan compounds 1–4 together with other lignan compounds isolated in previous study were tested the activities on IFN-γ/STAT1 and IL-6/STAT3 pathways. The following result showed that the main components trachelogenin and arctigenin had corresponding activities on IFN-γ/STAT1 pathway with IC50 values of 3.14 μM and 9.46 μM as well as trachelogenin, arctigenin and matairesinol strongly inhibiting IL-6/STAT3 pathway with IC50 values of 3.63 μM, 6.47 μM and 2.92 μM, respectively.Initial investigation for new active herbal extract with inhibiting activity on JAK/STAT signaling pathway revealed that the extract of Caulis Trachelospermi, which was separated by 80% alcohol extraction and subsequent HP-20 macroporous resin column chromatography, was founded to strongly inhibit IFN-γ-induced STAT1-responsive luciferase activity (IFN-γ/STAT1) with IC Trachelospermum jasminoides (Lindl.) Lem, is mainly distributed in Henan, Anhui, Hubei, Shandong and Guangxi provinces in China. It has been used in Traditional Chinese Medicine for the treatment of rheumatic arthralgia, aching of the loins and knees, traumatic injuries + ion at m/z 559.1788 . 1H-NMR and 13C-NMR spectral data  in 1H-NMR spectrum suggested compound 2 was a 8'-hydroxydibenzylbutyrolactone lignan. 1H-NMR spectrum also displayed signals of two sets of ABX systems of the phenyl protons to present two 3,4-disubstituted phenyl groups. The presence of two methoxy groups at δ 3.70 and 3.73 which had respective HMBC correlation with the carbon at δ 148.8 (C-3) and carbon at δ 147.2 (C-3') as well as a phenolic hydroxy at δ 8.85  suggested the aglycone of 2 was nortrachelogenin which was proved by HMBC correlations as shown in δ 4.82 (H-1'') with coupling constant of 7.2 Hz in 1H-NMR spectrum and its corresponding spectral data in 13C-NMR spectrum indicated the presence of a β-d-glucopyranosyl group. HMBC correlation between the anomeric proton (H-1'') and carbon at δ 145.1 (C-4) or δ 145.4 (C-4') implied that the glucopyranosyl group was connected to C-4 or C-4'. According to compound 2 having evident different NMR spectral data from that of nortracheloside which had been isolated from Caulis Trachelospermi in our previous research, it was determined that the location of β-d-glucopyranoside was at C-4. Concerned the β effect of 8'-OH, optical isomers of 8'-hydroxydibenzylbutyrolactone lignan produced different chemical shifts at C-7' and C-8. Trans isomers were at about δ 42.0 and δ 43.7, but cis isomers were at about δ 38.3 and δ 48.1. Moreover, 8S-configuration produced negative optical rotation in contrast with positive optical rotation of its epimer  to give compound 8 (38.5 mg). Fr-A-5was fractionated by ODS CC eluted with MeOH–H2O (25:75) and then purified by preparative RP-HPLC [MeOH–H2O (17:83)] to yield compound 4 (45.0 mg). Fr-A-6 was further isolated by ODS CC eluted with MeOH–H2O (3:7) then purified by Sephadex LH-20 (MeOH) and semi-preparative RP-HPLC [MeOH–H2O (2:8)] to afford compound 7 (1.9 mg). Fr-A-8 was chromatographed with identical method of Fr-A-6 to yield compound 2 (8.0 mg). Fr-C was fractionated by ODS CC eluted with MeOH–H2O (4:6) and seven fractions were obtained. Fr-C-4 was chromatographed over ODS CC [MeOH–H2O (25:75)] and purified by semi-preparative RP-HPLC [MeOH–H2O (28:72)] to afford compounds 1 (15.7 mg) and 3 (7.8 mg).Guided by HPLC detection, the product (50 g) was applied on column chromatography over ODS eluted with MeOH–H4-Demethyltraxillaside (1). White amorphous powder.  −44.1 . CD (MeOH) λmax nm (∆ε): 233 (−2.72), 276 (−0.58). ESIMS: m/z 573 [M+Na]+ (Pos.), 549 [M−H]− (Neg.). HRESIMS: m/z: 573.1945 [M+Na]+ . 1H-NMR and 13C-NMR data see d-glucopyranosideNortrachelogenin 4-O-β- (2). White amorphous powder.  −48.5 . CD (MeOH) λmax nm (∆ε): 233 (−1.94), 276 (−0.28). ESIMS: m/z 559 [M+Na]+ (Pos.), 535 [M−H]−(Neg.). HRESIMS: m/z: 559.1788 [M+Na]+ . 1H-NMR and 13C-NMR data see d-glucopyranosideMatairesinol 4-O-β- (3). White amorphous powder.  −34.0 . CD (MeOH) λmax nm (∆ε): 233 (−2.23), 276 (−0.39). ESIMS: m/z 543 [M+Na]+ (Pos.), 519 [M−H]−(Neg.). HRESIMS: m/z: 543.1838 [M+Na]+ . 1H-NMR (DMSO-d6): δH 6.67 , 6.96 , 6.57 , 2.44–2.48 , 4.05 , 3.87 , 6.76 , 6.69 , 6.60 , 2.83 , 2.73 , 2.69 , 8.84 , 3.72 , 4.82 . 13C-NMR (DMSO-d6): δc 132.6 (C-1), 112.9 (C-2), 148.8 (C-3), 145.1 (C-4), 115.3 (C-5), 120.5 (C-6), 36.9 (C-7), 40.9 (C-8), 70.8 (C-9), 129.0 (C-1'), 113.5 (C-2'), 147.5 (C-3'), 145.1 (C-4'), 115.4 (C-5'), 121.6 (C-6'), 33.8 (C-7'), 45.7 (C-8'), 178.6 (C-9'), 100.2 (C-1''), 73.3 (C-2''), 77.1 (C-3''), 69.7 (C-4''), 76.9 (C-5''), 60.7 (C-6''), 55.6 .d-glucopyranosideNortrachelogenin 4,4'-di-O-β- (4). White amorphous powder. ESIMS: m/z 716 [M+NH4]+, 721 [M+Na]+ (Pos.), 697 [M−H]− (Neg.). 1H-NMR (DMSO-d6): δH 6.72 , 6.99 , 6.74 , 2.65 , 2.46 , 2.40 , 3.97 , 6.83 , 6.97 , 6.62 , 3.02 , 2.88 , 6.30 , 3.70, 3.73 , 4.85, 4.82 . 13C-NMR (DMSO-d6): δc 132.9 (C-1), 129.2 (C-1'), 113.0 (C-2), 114.8 (C-2'), 148.4, 148.8 , 145.1, 145.5 , 115.3 , 120.5 (C-6), 122.5 (C-6'), 30.9 (C-7), 42.8 (C-8), 70.0 (C-9), 75.4 (C-8'), 178.0 (C-9'), 100.2, 100.0 , 73.2 , 77.0 , 69.7 , 76.9 , 60.6, 60.7 , 55.6, 55.7 .HepG2/STAT1 (HepG2/STAT3) cells, a gift from Prof. Xinyuan Fu , were a human hepatoma derived cell line cells stably transfected with a STAT1 (STAT3)-responsive firefly luciferase reporter plasmid. Luciferase assay kit was purchased from Promega Corp., Madison, WI, USA. Recombinant human IFN-γ was purchased from Shanghai Clone High Biological Technology Co Ltd., Shanghai, China. Recombinant human IL-6 was purchased from Pepprotech Inc., Rocky Hill, NJ, USA. Pyridone 6 and AG490 were purchased from Merck Biosciences, Darmstadt, Germany and Calbiochem, San Diego, CA, USA, respectively.4 per well) maintained in a DMEM medium were seeded into 96-well cell culture microplates and allowed to grow for 24 h at 37 °C in a 5% CO2 incubator, and then treated with test samples for 1h followed by stimulation with 50 IU/mL IFN-γ (10 ng/mL IL-6) for 6 h. Luciferase activity was determined using the Promega luciferase kits according to the manufacturer’s instruction. The cell number was counted at seeding and controlled by equal seeding. All luciferase assays were repeated at least twice to minimize the variance caused by cell number. IFN-γ (IL-6)-induced STAT1 (STAT3)-responsive luciferase activity (% of control) was calculated as {[Fluorescence intensity with sample and IFN-γ (IL-6) treatment − Fluorescence intensity without IFN-γ (IL-6) treatment]/[Fluorescence intensity with IFN-γ (IL-6) treatment − Fluorescence intensity without IFN-γ (IL-6) treatment]} × 100.Luciferase assay was performed as previous described . HepG2/S1) and nortrachelogenin 4-O-β-d-glucopyranoside (2), together with six known compounds were isolated from Caulis Trachelospermi. Their structures and stereochemistries were established by the analysis of 1D, 2D-NMR, MS, OR and CD spectra. The extract of Caulis Trachelospermi and its main dibenzylbutyrolactone lignan components had strong inhibiting activity on JAK/STAT pathway, and it may be an important mechanism for the reported anticancer and anti-inflammation function of Caulis Trachelospermi. However, it is still unclear whether the inhibition of IFN-γ/STAT1 and IL-6/STAT3 signaling pathways by the active components is direct on these pathways or whether it occurs via the upregulation of the expression of other factors that then inhibit these signaling pathways. Further research is needed for the determination of their precise target.In summary, two new dibenzylbutyrolactone lignans, 4-demethyltraxillaside (


E)-5′-vinylphosphonate (5′-VP) is a metabolically-stable phosphate mimic for siRNA and demonstrated that 5′-VP improves the potency of the fully modified siRNAs in vivo. Here, we report an alternative synthesis of 5′-VP modified guide strand using S-pivaloyl-2-thioethyl (tBu-SATE) protecting group. The tBu-SATE group is readily removed during the final cleavage of the oligonucleotide from the solid support and providing a more convenient route for the synthesis of siRNA guide strand carrying a 5′-vinylphosphonate.We recently reported that ( RNA interference (RNAi) is a powerful tool for gene regulation and modulation of disease causing genes ,2. PotenE)-5′-vinylphosphonate as a phosphate mimic for guide strand -5′-vinyl (dimethylphosphonate)-2′-O-(2-methoxyethyl)-thymidine-3′-phosphoramidite 1  group has been successfully utilized in the synthesis of nucleotide prodrugs and charge neutral oligonucleotides −m/z = 983.3, found 982.1.Compound 6: To a solution of 5  in CH2Cl2 (35 mL) were added DIEA  and benzoylchloride  and stirred at room temperature overnight. Reaction mixture was concentrated by rotary evaporation under reduced pressure and the residue was purified by silica gel column chromatography by a gradient of hexanes:ethyl acetate (1:1) to ethyl acetate to afford 6 . 1H-NMR : δ 7.83–7.95 , 7.58–7.77 , 7.32–7.55 , 6.94 , 6.56–6.77 , 5.79–5.98 , 4.65 , 3.98 , 3.67–3.71 , 3.32–3.61 , 3.29 , 1.9 , 1.01 ; 31P-NMR : δ 19.44; LR MS (ESI) calcd for C39H47N2O10PSi [M − H]−m/z = 761.2, found 761.3.Compound 7: To a solution of 6  in CH2Cl2 (15 mL) were added pyridine  and TMSI  and stirred at room temperature. Reaction was completed after 30 min as determined by LC-MS . The reaction mixture was concentrated under reduced pressure and the residue was suspended in ethyl acetate and filtered. The filtrate was washed with 0.1 N HCl, dried over MgSO4, filtered and concentrated to afford 7 . 1H-NMR : δ 7.81–7.94 , 7.56–7.77 , 7.30–7.53 , 6.97 , 6.49–6.72 , 5.89–6.01 , 4.62 , 4.01 , 3.32–3.61 , 3.28 , 1.84 , 1.09 ; 31P-NMR : δ 10.2; LR MS (ESI) calcd for C37H43N2O10PSi [M + H]+m/z = 735.2, found 735.1.Compound 10: To a solution of 7 (2.5 g 3. 41 mmol) in CH2Cl2 (8.4 mL) were added 2,6-di-tert-butyl-4-methylpyridine , oxalyl chloride  and DMF . The reaction mixture was stirred at room temperature for 1 h to afford 8 in situ. The reaction mixture was concentrated under reduced pressure and residue dissolved in CH2Cl2. tBu-SATE alcohol 9  was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and the residue was partitioned between ethyl acetate and H2O. The organic layer was separated, dried (MgSO4), filtered and concentrated. The crude product was purified by silica gel column chromatography  to yield 10 . 1H-NMR : δ 7.84–7.95 , 7.59–7.77 , 7.33–7.55 , 6.96 , 6.57–6.78 , 5.80–6.01 , 4.65 , 3.93–4.16 , 3.31–3.65 , 3.29 , 3.10 , 1.90 , 1.23 , 1.10 ; 31P-NMR : δ 17.21; LR MS (ESI) calcd for C51H67N2O12PS2Si [M − H]−m/z = 1021.3, found 1021.4.Compound 11: To a solution of 10  in THF (3.0 mL) was added TREAT-HF  and stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and the residue obtained was purified by silica gel column chromatography using a gradient of ethyl acetate to 50% acetone/CH2Cl2 to afford 11 . 1H-NMR : δ 7.92–8.04 , 7.75–7.86 , 7.70 , 7.56–7.66 , 6.73–6.95 , 6.00–6.20 , 5.85 , 5.45 , 4.40 , 4.12–4.26 , 3.93–4.10 , 3.60–3.82 , 3.48 , 3.24 , 3.11 , 1.89 , 1.19 ; 31P-NMR : δ 22.24; LR MS (ESI) calcd for C35H49N2O12PS2 [M − H]−m/z = 783.2, found 783.3.Compound 13: The synthesis of modified oligonucleotides was performed on an ABI 394 synthesizer  on a 2 µmol scale. A 0.1 M solution of 2′-F, 2′-O-Me, and 2′-O-MOE, modified nucleoside phosphoramidites in anhydrous acetonitrile (CH3CN) and were used for the synthesis. Phosphoramidite 2 was dissolved in 30% dichloromethane in anhydrous CH3CN (0.1 M) and used for the oligonucleotide synthesis. The modified oligonucleotides were synthesized on VIMAD UnyLinkerTM  solid support. Dichloroacetic acid (6%) in toluene was used as detritylating reagent. 4,5-Dicyanoimidazole in the presence of N-methylimidazole in CH3CN was used as activator during the coupling step. During the coupling step, nine equivalents of phosphoramidite solutions were delivered and the coupling was carry out for 10 min. Phosphorothioate linkages were introduced by sulfurization with a 0.05 M solution of DDTT (3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione) in 1:1 pyridine/CH3CN for a contact time of 3 min. Phosphate diester linkages were incorporated via oxidation of phosphite triesters using a solution of tert-butyl hydroperoxide/CH3CN/water (10:87:3) for a contact time of 12 min. All other steps in the protocol supplied by the manufacturer were used without modification. After the desired sequence was assembled, the solid-support bound oligonucleotide was washed with 1:1 TEA/CH3CN for 30 min. The solid-support bound oligonucleotide was suspended in aqueous ammonia (28–30 wt%) containing 1M 2-mercaptoethanol (used 1 mL/µmol of solid support) and either heated at 55 °C . Fractions containing full-length oligonucleotides were pooled together  and the tributylammonium counter ion was exchanged to sodium by HPLC on a strong anion exchange column  Source 30Q, 30 µm, 2.54 × 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH3CN, B = 1.5 M NaBr in A, 0–40% of B in 60 min, flow 14 mL min−1). The residue was desalted by HPLC on reverse phase column to yield the oligonucleotide in an isolated yield of 15–20% based on solid-support loading. The oligonucleotides were characterized by ion-pair-HPLC-MS analysis with Agilent 1100 MSD system .Compound 5 °C 13,  for 6 h  13, 13,  to complE)-5′-vinyl-phosphonate)-2′-O-(2-methoxyethyl)-thymidine-3′-phosphoramidite with a base-labile tBu-SATE protected vinylphospho- nate in good yield. To examine the compatibility of the tBu-SATE-protecting group under solid-phase oligonucleotide synthesis conditions, we synthesized a guide strand of fully modified siRNA containing (E)-5′-vinylphosphonate using this phosphoramidite. The tBu-SATE-protected phosphor- amidite was successfully incorporated at the 5′-terminus of a 2′-F/2′-O-Me guide strand of siRNA using solid-phase DNA synthesis reagents and methods. Moreover, the tBu-SATE group was conveniently removed under aqueous ammonia mediated deprotection conditions. The yield and purity of resulting oligonucleotides were identical to the one obtained using previously reported phosphoramidite containing vinylphosphonate methyl esters [E)-5′-vinylphosphonate and eliminates the need for any additional steps or reagents.In this study, we designed and synthesized 5′-deoxy-(l esters . The str


Among the synthesized analogues, the tetrafluorophthalimide LASSBio-1439 (2e) stands out showing an in vitro anti-TNF-α effect similar to the standard thalidomide. The relevance of tetrafluorination of the phthalimide nucleus was also confirmed by the anti-inflammatory profile of 2e, through oral administration, in a murine model of pulmonary inflammation. The corresponding tetrafluorocarboxyamide metabolite LASSBio-1454 (15), generated from partial hydrolysis of the derivative 2e, presented a significant in vitro effect and a pronounced anti-inflammatory activity in vivo.In this paper we report the design, synthesis and pharmacological evaluation of a new series of phenyl sulfonamide derivatives  Inflammation is the body’s response to insults, including trauma, infections and hypersensitivity. In the lung, this response is usually induced by pathogens, toxins, pollutants, irritants and allergens . NormallSince the lung is a vital organ for gas exchange, excessive inflammation can be life threatening . Thus, tKnowing that inflammation is an important feature of many pulmonary diseases, several strategies have been adopted to interfere with the lung immune response, including the modulation of pro-inflammatory cytokines and the regulation of cellular signal transduction .Tumor necrosis factor alpha (TNF-α) is a crucial cytokine in immunity and inflammation. On the other hand, despite its physiological relevance, it is well-known that the increased biosynthesis and release of TNF-α lead to exacerbation of inflammatory and oxidative responses, which are related to the pathogenesis of a wide range of diseases ,7. Among1), a thalidomide analogue with pronounced anti-inflammatory activity in a murine model of acute lung inflammation  ..14 and 114) and LASSBio-1454 (15) confirmed that they corresponded to the metabolites formed by chemical (pH 7.4) or enzymatic (plasma) hydrolysis of the phthalimides LASSBio-468 (1) and LASSBio-1439 (2e), respectively.The HPLC and UV analyses of the synthesized compounds LASSBio-596 , comparing the results to those obtained for the phthalimide precursors 1 and 2e (The carboxyamide metabolites 1 and 2e .15) showed an anti-TNF-α effect in vitro  similar to that observed for the tetrafluorophthalimide 2e , indicating that 15 could be partly responsible for the anti-TNF-α activity observed for 2e.The tetrafluorinated carboxyamide LASSBio-1454 (14), probably generated in vivo as a metabolite of the prototype 1, showed significant anti-TNF-α effect . This fact can justify why the prototype 1 did not demonstrate any statistically significant TNF-α inhibition in vitro in the screening assay, although it had shown a pronounced anti-TNF-α effect in vivo after intraperitoneal administration acetamide . Thiomorpholine  was added to a solution of the sulfonylchloride derivative 12  in methylene chloride (50 mL). The reaction mixture was stirred for 30 min at room temperature and the end of the reaction was checked by TLC. The sulfonamide 7 was isolated by addition of 50 mL of methylene chloride and extraction, washing the organic layer with 10% aq HCl and brine. The organic layer was then dried over anhydrous Na2SO4, filtered and concentrated at reduced pressure to give the title compound as a yellow powder, 65% yield, mp 207–209 °C. The melting point, 1H-NMR, 13C-NMR and IR data for compound 7 are in agreement with previous reports pyridine-5,7(6H)-dione . The title compound was obtained by condensation of 8 with pyridine 2,3-dicarboxylic anhydride as yellow crystals, 50% yield after recrystallization from ethanol, mp > 250 °C. 1H-NMR  δ (ppm): 2.74 , 3.42 ; 7.76 ; 7.91 , 8.32 ; 9.10 . 13C-NMR  δ (ppm): 27.5 (C2'' and C6''); 48.0 (C3'' and C5''); 126.6 (C-3' and C-5'); 127.0 (C-7a); 128.3 (C-6); 128.5 (C-2' and C-6'); 132.1 (C-7); 135.5 (C-4'); 136.7 (C-1'); 151.0 (C-3a); 156.5 (C-5); 164.62 (C-3); 164.65 (C-1). IR  υ (cm−1): 3073, 2931, 2847, 1753, 1730, 1375, 1333, 1172, 838, 806, 700. Anal. Calcd. for C17H15N3O4S2: C, 52.43; H, 3.88; N, 10.79. Found: C, 52.50; H, 3.76; N, 10.87.1-(4-(Thiomorpholinosulfonyl)phenyl)pyrrolidine-2,5-dione . The title compound was obtained by condensation of 8 with succinic anhydride as a white shiny powder, 85% yield after purification in a silica gel chromatographic column using CH2Cl2 100% as eluent, mp > 250 °C. 1H-NMR  δ (ppm): 2.68 ; 2.81 ; 3.25 ; 7.58 ; 7.89 . 13C-NMR  δ (ppm): 26.4 (C2'' and C6''); 28.6 (C-4 and C-5); 47.8 (C3'' and C5''); 127.8 (C-3' and C-5'); 127.9 (C-2' and C-6'); 135.5 (C-4'); 136.8 (C-1'); 176.6 (C-1 and C-3). IR  υ (cm−1): 2916, 2858, 1781, 1706, 1393, 1160, 826. Anal. Calcd. for C14H16N2O4S2: C, 49.40; H, 4.74; N, 8.23. Found: C, 49.27; H, 4.68; N, 8.17.® followed by washing with methylene chloride. Subsequently, the organic extract obtained was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated at reduced pressure. The residue was purified in a silica gel chromatographic column using MeOH:CH2Cl2 1% as eluent.In a 50 mL flask equipped with a reflux condenser, a reaction mixture containing the nitrophthalimide derivative (0.46 mmol), metallic iron dust , ammonium chloride , ethanol (12 mL) and distilled water (6 mL) was stirred and heated at 78 °C for 2 h. The end of the reaction was checked by TLC. The product was isolated by filtration on Celite4-Amino-2-(4-(thiomorpholinosulfonyl)phenyl)isoindoline-1,3-dione . The title compound was obtained from the nitrophthalimide precursor LASSBio-1437 (2a) as yellow crystals, 61% yield after purification by chromatographic column, mp 195–197 °C. 1H-NMR  δ (ppm): 2.69 ; 3.26 ; 6.62 ; 7.08 ; 7.10 ; 7.52 ; 7.75 ; 7.89 . 13C-NMR  δ (ppm): 26.5 (C2'' and C6''); 47.8 (C3'' and C5''); 108.6 (C-7); 111.3 (C-5); 121.8 (C-3a); 127.3 (C-3' and C-5'); 127.9 (C-2' and C-6'); 132.0 (C-7a); 134.7 (C-6); 135.7 (C-4'); 136.3 (C-1'); 147.1 (C-4); 166.6 (C-3); 167.8 (C-1). IR  υ (cm−1): 3464, 3365, 2927, 2849, 1768, 1715, 1637, 1370, 1164, 834, 814, 719. Anal. Calcd. for C18H17N3O4S2: C, 53.58; H, 4.25; N, 10.41. Found: C, 53.47; H, 4.28; N, 10.39.5-Amino-2-(4-(thiomorpholinosulfonyl)phenyl)isoindoline-1,3-dione . The title compound was obtained from the nitrophthalimide precursor 2c as yellow crystals, 57% yield after purification by chromatographic column, mp 230°C. 1H-NMR  δ (ppm): 2.68 ; 3.25 ; 6.64 ; 6.89 ; 7.02 ; 7.62 ; 7.73 ; 7.87 . 13C-NMR  δ (ppm): 26.5 (C3'' and C5''); 47.8 (C2'' and C6''); 107.2 (C-4); 116.1 (C-6); 117.4 (C-7a); 125.6 (C-7); 127.1 (C-3' and C-5'); 127.8 (C-2' and C-6'); 134.2 (C-4'); 134.5 (C-1'); 136.6 (C-3a); 155.5 (C-5); 166.3 (C-3); 166.8 (C-1). IR  υ (cm−1): 3464, 3365, 2918, 2856, 1768, 1710, 1637, 1379, 1164, 834. Anal. Calcd. for C18H17N3O4S2: C, 53.58; H, 4.25; N, 10.41. Found: C, 53.49; H, 4.19; N, 10.32.2-(4-(Thiomorpholinosulfonyl)phenyl)isoindolin-1-one . In a 25 mL flask equipped with a reflux condenser, the reaction mixture containing the phthalimide derivative 1 , zinc dust  and 20 mL of acetic acid was stirred and heated at 140 °C for 72 h. The end of the reaction was observed by TLC and the warm reaction medium was then filtered, followed by addition of distilled water to the filtrate until precipitation. Subsequently, the obtained precipitate was filtered through a Büchner funnel, giving the title compound as a white solid, 71% yield, mp > 250 °C. 1H-NMR  δ (ppm): 2.68 ; 3.22 ; 5.10 ; 7.55 ; 7.69 ; 7.81 ; 8.18 . 13C-NMR  δ (ppm): 26.3 (C2'' and C6''); 47.7 (C3'' and C5''); 50.3 (C-3); 118.6 (C-3' and C-5'); 123.4 (C-7); 123.5 (C-6); 128.3 (C-4); 128.4 (C-2' and C-6'); 130.2 (C-7a); 131.7 (C-4'); 132.8 (C-5); 141.0 (C-1'); 143.4 (C-3a); 167.2 (C-1). IR  υ (cm−1): 2927, 2852, 1692, 1384, 1163, 843, 729. Anal. Calcd. for C18H18N2O3S2: C, 57.73; H, 4.84; N, 7.48. Found: C, 57.68; H, 4.81; N, 7.38.N-(4-(Thiomorpholinosulfonyl)phenyl)benzamide . Benzoyl chloride  was slowly added to a solution of the key intermediate 8  in 20 mL of methylene chloride. The reaction mixture was stirred for 20 min at room temperature and the end of the reaction was checked by TLC. The benzamide 6 was isolated by addition of 50 mL of methylene chloride and extraction, washing the organic layer with 10% aq HCl and brine. The organic layer was then dried over anhydrous Na2SO4, filtered and concentrated at reduced pressure to give the title compound as a white shiny powder, 76% yield, mp 204 °C. 1H-NMR  δ (ppm): 2.67 ; 3.20 ; 7.55 ; 7.74 ; 7.97 ; 8.07 ; 10.67 . 13C-NMR  δ (ppm): 26.4 (C2'' and C6''); 47.8 (C3'' and C5''); 120.0 (C-3 and C-5); 127.8 (C-3' and C-5'); 128.3 (C-2' and C-6'); 128.5 (C-2 and C-6); 130.2 (C-1); 132.0 (C-4); 134.4 (C-1'); 143.5 (C-4'); 166.2 (C=O). IR  υ (cm−1): 3381, 2916, 2858, 1683, 1336, 1160, 833, 733, 712. Anal. Calcd. for C17H18N2O3S2: C, 56.33; H, 5.01; N, 7.73. Found: C, 56.22; H, 4.88; N, 7.68.The reaction mixture containing the phthalimide precursors (0.43 mmol), potassium hydroxide  and ethanol (5 mL) was vigorously stirred at room temperature for 1 hour and the end of the reaction was checked by TLC. The reaction medium was then diluted with 10 mL of methylene chloride followed by addition of 5 mL of distilled water. Subsequently, the extraction was performed in a separatory funnel. The aqueous layer was acidified with 10% aq HCl until pH 3 and the obtained precipitate was, then, filtered through a Büchner funnel to provide the carboxyamide derivatives in good yields.2-((4-(Thiomorpholinosulfonyl)phenyl)carbamoyl)benzoic acid . The title compound was obtained from alkaline hydrolysis of the phthalimide precursor 1 as a white shiny powder, 86% yield, mp 187–189 °C. The melting point, 1H-NMR and IR data for compound 14 are in agreement with previous reports [1H-NMR  δ (ppm): 2.67 ; 3.18 ; 3.61 ; 7.56 ; 7.60 ; 7.65 ; 7.72 ; 7. 90 ; 7.94 ; 10.82 . IR  υ (cm−1): 3495, 3325, 2929, 2857, 1731, 1693, 1330, 1159, 837, 724. Anal. Calcd. for C18H18N2O5S2.H2O: C, 50.93; H, 4.75; N, 6.60. Found: C, 51.08; H, 4.78; N, 6.59. 2,3,4,5-Tetrafluoro-6-((4-(thiomorpholinosulfonyl)phenyl) carbamoyl) benzoic acid . The title compound was obtained from alkaline hydrolysis of the phthalimide precursor 2e as a white powder, 84% yield, mp > 250 °C. 1H-NMR  δ (ppm): 2.67 ; 3.19 ; 3.91 ; 7.72 ; 7.85 ; 11.25 . IR  υ (cm−1): 3497, 3323, 2919, 2855, 1730, 1694, 1402, 1375, 1161, 838. Anal. Calcd. for C18H14F4N2O5S2: C, 45.19; H, 2.95; N, 5.86. Found: C, 45.23; H, 3.01; N, 5.81.1) and LASSBio-1439 (2e) and the appearance of the corresponding metabolites LASSBio-596 (14) and LASSBio-1454 (15), respectively.The compounds (5 µM) were incubated at 37 °C under stirring in 250 µL of PBS (pH 7.4) or rat plasma (50% in PBS) for different times . The reaction was stopped by addition of 750 µL of acetonitrile (0.4% trifluoroacetic acid). The samples were centrifuged and the supernatants were filtered and analyzed by HPLC. The HPLC analyses were conducted in a Shimadzu LC-20AD apparatus equipped with a SHIM-PACK CLC-ODS analytical column (4.6 mm × 250 mm) or a Kromasil 100-5C18 (4.6 mm × 250 mm) and with a Shimadzu SPD-M20A diode array detector. The mobile phase was consisted of acetonitrile-water (60:40% v/v), both acidified with 0.1% trifluoroacetic acid, and the flow rate was 1 mL/min. This method allowed monitoring the consumption of the phthalimide precursors LASSBio-468 (A/J and Swiss-Webster mice (18–20 g) were obtained from the Oswaldo Cruz Foundation breeding colonies . They were kept in the animal-housing facilities at a controlled room temperature (22–25 °C) and a 12h/12h light:dark cycle (6 a.m. to 6 p.m.). All the procedures related to care and use of laboratory animals in this study were examined and approved by the Animal Ethics Committee of the Oswaldo Cruz Foundation .E.coli serotype 0127:B8; SIGMA, St. Louis, MO, USA) (1 mg/mL) or saline (control group). The testing compounds were dissolved in saline solution containing 0.5% Tween 80 and administered orally or intraperitoneally (50 or 100 mg/kg) 1 h before LPS instillation. All solutions were prepared immediately before use. The evaluation was conducted 24 h after LPS instillation.Lung inflammation was induced in anesthetized A/J mice by an intranasal instillation of LPS (from L) (cmH2O/mL/s) and dynamic lung elastance (mL/cmH2O) in each breath cycle were calculated. Lung elastance was calculated as the inverse of compliance values. Analogical signals from the computer were digitized using a Buxco Analog/Digital Converter (Buxco Electronics). Increasing concentrations of methacholine  were aerosolized for 5 min each and basal lung resistance and lung function were assessed under condition of phosphate buffered saline (PBS) aerosolization.Airway responsiveness was assessed as a change in airway function after challenge with aerosolized methacholine  in a Buxco System (Finepoint). Mice were anesthetized with nembutal (60 mg/kg) and neuromuscular activity was blocked with bromide pancuronium (1 mg/kg). Tracheostomized mice were mechanically ventilated and the lung function was assessed. The trachea was cannulated and the cannula connected to a pneumotachograph. The air flow and the transpulmonary pressure were recorded using a Buxco pulmonary mechanics processing system (Finepoint). The parameters of resistance . The right lung lobes were quick-frozen by immersion in liquid nitrogen.o-dianisidine dihydrochloride (SIGMA) and 0.0005% hydrogen peroxide. The absorbance was read at 460 nm using a spectrophotometer and the results were expressed as optical density.Lung samples were homogenized in 5 μM EDTA buffer solution containing 0.5% hexadecyl-trimethylammonium bromide  using a tissue homogenizer. Samples were centrifuged at 1,700 × g for 15 min at 4 °C. The supernatants were collected, centrifuged at 15,295 × g for 15 min at 4 °C and assayed spectrophotometrically for MPO determination. 50 μL of sample were mixed with 50 μL of a EDTA/HTBA solution containing 0.68 mg/mL of 2 asphyxiation and peritoneal macrophages obtained by washing the peritoneal cavity with 10 μM EDTA solution. The cells were pelleted  and ressuspended in RPMI 1640 Medium . Total mononuclear cells counts were performed in Neubauer chamber after sample dilution in Turk’s solution (acetic acid 0.2%). The cells were added (0.5 × 106 cells/mL) to a 24-well plate and incubated (37 °C and 5% CO2) in RPMI 1640 medium with bovine fetal serum (BSA) (2%)  supplemented with 200 U/mL penicillin and 200 mg/mL streptomycin for adhesion (1 h). After the adhesion step, the medium was removed and the macrophages were incubated in RPMI 1640 medium without BSA and supplemented with 200 U/mL penicillin and 200 mg/mL streptomycin. The cells were incubated with the testing compounds (100 μM) at 37 °C and 5% CO2 for 1 h and then stimulated with LPS (0.1 μg/mL). Twenty four hours later, the cells were centrifuged  and the supernatants were recovered and frozen at -80°C for further cytokine quantification by ELISA.Swiss-Webster mice were killed by COLevels of TNF-α in macrophage culture supernatants and lung tissue samples were measured by commercial ELISA kits  according to manufacturer’s protocols. The statistical analysis was performed with ANOVA followed by the Newman-Keuls Student test or with Student’s t-test.2a–h and 3–8, designed by structural modification on the anti-inflammatory lead-compound LASSBio-468 (1), the tetrafluorophthalimide analogue LASSBio-1439 (2e) stands out showing an anti-TNF-α effect in vitro similar to the standard thalidomide (9). These results confirm the data previously described in the literature [Among the synthesized phenyl sulfonamide derivatives terature ,13,25,262e after oral administration in a murine model of pulmonary inflammation. Moreover, this functionalization increased the chemical and enzymatic lability of the phthalimide subunit, indicating that the anti-inflammatory and anti-TNF-α effects observed for 2ein vivo are probably due to the corresponding carboxamide metabolite LASSBio-1454 (15).The relevance of tetrafluorination in the phthalimide nucleus was also demonstrated by the anti-inflammatory profile of 15 has shown an anti-TNF-α effect in vitro similar to that observed for the phthalimide precursor 2e. Additionally, the tetrafluorometabolite 15 presented pronounced anti-inflammatory effects in vivo after intraperitoneal administration; and a higher inhibitory effect in the control of neutrophils infiltration into the lung tissue when compared with the non-fluorinated analogue 14, showing once again the relevance of this functionalization.The tetrafluorinated carboxamide metabolite