Trypanosoma brucei. The enzyme trypanothione reductase (TryR) is a potential drug target within these organisms. Herein we report the screening of a 62000 compound library against T. brucei TryR. Further work was undertaken to optimise potency and selectivity of two novel-compound series arising from the enzymatic and whole parasite screens and mammalian cell counterscreens. Both of these series, containing either a quinoline or pyrimidinopyrazine scaffold, yielded low micromolar inhibitors of the enzyme and growth of the parasite. The challenges of inhibiting TryR with druglike molecules is discussed.There is an urgent need for new drugs for the treatment of tropical parasitic diseases such as human African trypanosomiasis, which is caused by  Trypanosoma brucei gambiense and T. b. rhodesiense, which initially reside within the blood stream and then subsequently penetrate into the central nervous system giving rise to the classical symptoms of HAT. The current drugs to treat HAT are inadequate due to poor efficacy, side effects and the requirement for parenteral administration, which is not appropriate for a rural African setting.2Human African trypanosomiasis (HAT) is a serious health problem in sub-Saharan Africa, with an estimated 50000 new cases each year. HAT, a fatal disease unless treated, is caused by the protozoan parasites N1,N8-bis(glutathionyl)spermidine disulphide, T[S]2; Figure 2).2 serves as a substitute for many of the metabolic and antioxidant functions ascribed to glutathione (GSH) in mammalian cells.5Herein we report a target-based approach to the discovery of novel inhibitors of trypanothione reductase (TryR) as a potential therapy for HAT. TryR is an NADPH-dependent flavoprotein disulphide oxidoreductase unique to, and essential for growth of, trypanosomatid parasites, whose function is to convert trypanothione disulphide  =12.4±0.14 μm. Clomipramine was used as a standard throughout the screening process.The TryR screening assay was miniaturised to a 384-well plate format and optimised to the standard required to support a large-scale screening campaign. The assay was assessed for robustness in an automated environment yielding the following typical performance statistics: Z′=0.84±0.001; %CV (plate)=3.65±0.4; S:B=10±0.25; ICm and the percentage inhibition (PI) value calculated for each compound . A total of 767 compounds were confirmed as hits giving a confirmation rate (primary to re-test screen) of 84% and an overall confirmed hit rate of 1.24%.TryR was initially screened in single-point mode at 30 μd Figure . The dism to 1 nm. In all cases, residual material from the potency compound plates was subjected to LCMS analysis for structure and purity confirmation. Figure R2) value of 0.967 between replicate pIC50 determinations.A preliminary chemistry assessment of the confirmed hits identified a selection of 120 compounds for potency determinations. Each compound was subjected to a ten-point half-log titration in duplicate ranging from 30 μm. Key compounds were resynthesised or resupplied to further confirm their activity and identity. From the five series initially identified, two were deemed of sufficient interest to progress into formal hit validation. The aim of this phase was to rapidly assess the existence of defined structure–activity relationships (SAR), whilst securing a significant increase (>10-fold) in activity over the initial hit compound.Analysis of the potency data for the best hits revealed multiple putative hit series covering a range potencies from 0.7 to 30 μSeries 1 was based on a quinoline scaffold, which afforded a number of possible points of variation: the 5-methylfuran in the 2-position, the amide in the 4-position, and the 6-bromo substituent. To expand the SAR, we prepared or purchased 84 analogues. Table The general synthetic route is shown in Scheme 1 and 17, 20 and 22; 8 and 13; 12 and 18); in the 4-position, amides with a basic substituent, such as dimethylaminoethyl were more potent than neutral aryl or alkylaryl amides ; and the 6-bromo group could be moved around the ring without too much effect on the activity . Although replacing the 6-bromo substituent with a chloro atom led to a small effect on activity , replacement of the 6-bromo with hydrogen or fluorine led to loss in activity . Particularly with substituents on the 4-position, increases in MW and clogP led to small increases in potency, suggestive of a general surface contact between inhibitors and protein. There may be a similar effect with the 6-bromo group, as changes in location to the 7- or 8-position of the quinoline ring system, or even replacing it with a chlorine, led to only small effects on activity. It was difficult to deduce which changes, if any, would increase potency at these positions. It is possible that the 5-methylfuran at the 2-position is making a very specific hydrogen bonding and/or π-stacking interaction, which accounts for the requirement for this group at this position.A summary of the SAR is given in Figure 35) was then condensed with aldehyde and cyclisation was achieved by treatment with sodium nitrite followed by dehydration through microwave heating in DMF with molecular sieves to give 42.Series 2, containing the pyrimidopyridazines scaffold, gave five compounds with an inhibition of >62% in the initial screen. The general synthetic route is outlined in Scheme 1 (methyl) and R2 (methyl and ethyl) as replacement of R2 or both R1 and R2 with hydrogen led to a loss in activity (42 and 43). The methyl at R2 could be replaced by a small alkyl group; however, replacement with larger alkyl groups led to a decrease in activity. A number of different aromatic substituents were tolerated at R3, however, there was no clear SAR.Small alkyl groups were required in positions RKm allowing direct comparison of IC50 values for selectivity assessment. All of the compounds tested demonstrated significant selectivity for TryR over GR (Table The selectivity of these series with respect to nearest human homologue of TryR was assessed in glutathione reductase (GR) assays. Both TryR and GR assays were performed with substrate concentrations equal to GR Table . This is2. The mixed inhibition of compounds in series 1 indicates the compounds can either bind to the free enzyme or to the enzyme–substrate complex, implying that the compounds are not binding in an orientation that excludes binding of T[S]2. For series 2, the binding appears to be linear and uncompetitive, with compounds binding only to the enzyme–substrate complex. In enzymes such as TryR that obey a bi bi ping pong reaction mechanism, such an inhibition pattern can arise due to an inhibitor competing for the second independent substrate-binding site, NADPH. However, a similar uncompetitive inhibition pattern was observed when the concentration of the second substrate, NADPH, was varied in the presence of a fixed concentration of T[S]2. This finding indicates that compounds from series 2 bind at a site distinct from both substrate-binding sites. Since compounds of series2 have some structural similarity to the FAD co-factor of TryR, we propose that disruption of the orientation or displacement of FAD could account for the uncompetitive inhibition pattern. In theory, both series present significant advantages over competitive inhibitors where build up of substrate trypanothione during inhibition in vivo could reduce the efficacy of the inhibitor. Indeed, substrate build-up in the case of uncompetitive inhibition may even drive binding of the inhibitor.Mode of inhibition studies were carried out on key representatives of series 1 and 2. The data indicated that neither compound series acted as simple linear competitive inhibitors with respect to T[S]T. brucei and MRC-5  proliferation in vitro ; it is likely that what we are observing is hydrophobic interactions between the hydrophobic regions of our inhibitors and the active site and electrostatic interactions between the positive charge on the inhibitors and the negatively charged region of the active site. In order to get a significant increase in potency, it will be necessary to build in some additional specific interactions between the inhibitor and the enzyme. This process would be greatly aided by a co-crystal structure of an inhibitor bound in the active site. Some cellular activity was observed, which implies that the compounds are able to penetrate into cells. However we believe that to get a significant correlation between enzyme and cellular activity will require enzyme inhibitors that are significantly more potent.Data from the gene knockout studies indicated that it would be necessary to cause >90% loss in activity of TryR for cell death to occur.In summary, we have identified two novel series of TryR inhibitors from a HTS screen and have carried out SAR studies. Further work is needed to increase the potency of these compounds to a level where the activity is sufficient to achieve significant cellular activity. This would be greatly facilitated by structural information.T. brucei was initially screened for inhibitors in single-point mode. Each test compound (500 nL) was transferred into 384-well clear polystyrene plates at an appropriate stock concentration. Clomipramine and DMSO were added to the appropriate control wells. Then 37.5 μL of reaction mix  was added to each well and the reaction initiated by addition of 150 μm NADPH (4 μL). Low signal controls received no NADPH. Plates were incubated for 35 min at room temperature and absorbance measured at 405±8 nm using the Envision platereader (Perkin–Elmer).Recombinant TryR from m; subsequent ten-point screening was conducted between 30 μm and 1 nm or between 100 μm and 3 nm depending on stock concentrations. The final DMSO level was 1% in all cases.Initial single-point screening of compounds was conducted at 30 μm HEPES; 1 mm EDTA, pH 7.4. Assay mixture (180 μL) containing three different concentrations of test compound was added to three rows of a microtitre plate, a fourth row contained only the assay mixture. The test compound concentration ranged up to ∼1×IC50 value. T[S]2 was serially diluted across a fifth row of the plate to produce a 12-point range from 500 μm to 5.8 μm. The assay was initiated by transferring 20 μL of the T[S]2 row to each of the assay rows. The final 200 μL assay contained 150 μm NADPH; 50 μm DTNB and 20 mUmL−1 U TryR.An assay mixture consisting of TryR, NADPH and DTNB was made up in 40 mReaction rates were measured from the linear increase in absorbance at 412 nm using a Molecular Devices Thermomax plate reader. Individual data sets were plotted as 1/v versus 1/[S] using Grafit 5.0 (Erithacus software) and inspected for diagnostic inhibition patterns. Data sets were then globally fitted by nonlinear regression to the appropriate equation for competitive, mixed or uncompetitive inhibition.T. brucei brucei single marker) and human  cell growth was performed using a modification of the cell viability assay previously described by Raz et al.15Measurement of the ability of the compounds to inhibit trypanosome  were seeded in 200 μL DMEM, 10% FCS and allowed to adhere overnight. One microlitre of test compound was added to each well on the day of assay initiation.2 for 69 h, prior to the addition of 20 μL rezasurin . After a further 4 h, fluorescence was measured  using a BioTek flx800 plate reader.Trypanosome and MRC-5 plates were incubated at 37°C in an atmosphere of 5% COm and 0.5 nm or between 50 μm and 2 nm, depending on stock concentrations. The final DMSO level was 0.5% in all cases.For cell assays, ten-point screening was conducted between 15 μ1H NMR were recorded on either a Bruker Avance DPX 500 or on a Bruker Avance 300 spectrometer. Chemical shifts (δ) are expressed in ppm. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br), or combination thereof. Low resolution electrospray (ES) mass spectra were recorded on a Bruker MicroTof mass spectrometer, run in positive ion mode, using either CH3OH, CH3OH/H2O (95:5) or H2O/CH3CN (1:1) + 0.1% formic acid as the mobile phase. High resolution electrospray measurements were performed on a Bruker MicroTof mass spectrometer. LC-MS analysis was performed using an Agilent HPLC 1100 with a diode array detector in parallel with a Bruker MicroTof mass spectrometer. A Phenomenex Gemini C18 column 110 A 50×3.0 mm, 5 μm particle size was used. The mobile phase was H2O/CH3CN + 0.1% HCOOH .4 for visualisation. Column chromatography was performed using RediSep® 4 or 12 g silica prepacked columns with the specified eluent system.Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 plates using UV light and/or KMnO6, 7, 13, 16, 17, 19, 21, 23 and 24 were purchased from ChemBridge and compounds 18, 39, 40, 41, 44, 45, 46, 47 and 49 were purchased from ASINEX and submitted to the screening assay. They were checked for purity by LCMS and were found to be >90% pure.Compounds All reactions were carried out under dry and inert conditions unless otherwise stated.N-(2-(dimethylamino)ethyl)-2-(5-methylfuran-2-yl)quinoline-4-carboxamide (1)6-Bromo-: Compound 27  and carbonyldiimidazole  were stirred in CH2Cl2 (15 mL) at 35°C for 20 min. N,N-dimethylethane-1,2-diamine  was added, and the solution stirred at RT for 18 h. The solution was diluted with CH2Cl2 (30 mL) and washed with aq NaHCO3 (2×20 mL), H2O (1×30 mL) and brine (2×20 mL). The organic layer was dried (MgSO4), filtered and concentrated to afford a yellow/brown solid. The crude product was purified by flash column chromatography  to give 1 as pale yellow solid : 1H NMR : δ=2.28 , 2.44 , 2.51 , 3.46 , 6.39 , 7.38 , 7.91 , 7.92 , 7.96 , 8.4 , 8.85 ppm ; LCMS (ES+): m/z (%): 402 (100) [M+H]+ (79Br), 404 (100) [M+H]+ (81Br); tR=5.0 min.N-(2-(methylamino)ethyl)-2-(5-methylfuran-2-yl)quinoline-4-carboxamide (2)6-Bromo-: Prepared as for 1 from 27  and CDI  in CH2Cl2 (15 mL). The mixture was stirred at 35°C for 20 min, then treated with N-methylethane-1,2-diamine  and stirred at RT for 18 h. The resultant solid product was collected by filtration and further purified by trituration with hot Et2O (3×10 mL) to give 2 as a pale yellow solid : 1H NMR : δ=2.29 , 2.40 , 2.72 , 3.41 , 6.39 , 7.37 , 7.89 , 7.96 , 8.34 , 8.88 ppm ; LCMS (ES+): m/z (%): 388 (100) [M+H]+ (79Br), 390 (100) [M+H]+ (81Br); tR=4.2 min.N6-Bromo-,N,N-trimethyl-2-(2-(5-methylfuran-2-yl)quinoline-4-carboxamido)ethanaminium iodide (3): CH3I  was added to a solution of 1  in dry acetone (5 mL). The solution was stirred at RT for 48 h then concentrated. A solid was precipitated from the resulting gum on the addition of acetone (15 mL), which was isolated by filtration and washed with acetone (3×10 mL) to afford 3 as a beige solid : 1H NMR : δ=2.41 , 3.19 , 3.6 , 3.79 , 6.39 , 7.36 , 7.94 , 7.98 , 8.34 , 9.25 ppm ; LCMS (ES+): m/z (%): 418 (100) [M+H]+ (79Br), 420 (100) [M+H]+ (81Br); tR=4.4 min.N-(2-morpholinoethyl)quinoline-4-carboxamide (4)6-Bromo-2-(5-methylfuran-2-yl)-: Prepared as for 1 from 27 , CH2Cl2 (15 mL), CDI  and 2-morpholinoethanamine . The reaction was stirred for 18 h then concentrated. The crude solid was recrystallised from refluxing EtOAc to give 4 as a white crystalline solid : 1H NMR : δ=2.38 , 2.48 , 2.49 , 4.49 , 4.66 , 6.39 , 7.38 , 7.91 , 7.92 , 7.96 , 8.32 , 8.86 ppm ; LCMS (ES+): m/z (%): 444 (100) [M+H]+ (79Br), 446 (100) [M+H]+ (81Br); tR=4.5 min.N-(pyridin-2-ylmethyl)quinoline-4-carboxamide (5)6-Bromo-2-(5-methylfuran-2-yl)-: Prepared as for 1 from 27 , CDI  and pyrin-2-ylmethanamine . The reaction was stirred for 18 h then concentrated. The crude solid was purified by trituration with hot Et2O (3×10 mL) to give 5 as pale yellow solid : 1H NMR : δ=2.39 , 4.79 , 6.39 , 7.35 , 7.41 , 7.49 , 7.84 , 7.92 , 7.99 , 8.03 , 8.52 , 8.61 , 9.51 ppm ; LCMS (ES+): m/z (%): 422 (100) [M+H]+ (79Br), 424 (100) [M+H]+ (81Br); tR=4.7 min.N-(2-(dimethylamino)ethyl)-2-(5-methylfuran-2-yl)quinoline-4-carboxamide (8)6-Chloro-: Compound 29  and CDI  were stirred in CH2Cl2 (15 mL) for 10 min at RT. N,N-Dimethylethane-1,2-diamine  was added, and the resulting solution was stirred at RT for 18 h. The reaction was diluted with CH2Cl2 (20 mL) and washed with aq NaHCO3 (2×20 mL), H2O (2×30 mL) and brine (1×50 mL). The organic layer was dried (MgSO4), filtered and concentrated in vacuo. Purification by flash column chromatography  gave 8 as a white solid : 1H NMR : δ=2.26 , 2.41 , 2.48 , 3.47 , 6.39 , 7.34 , 7.77 , 7.90 , 7.96 , 8.1 , 8.84 ppm ; LCMS (ES+): m/z (%): 358 [M+H]+; tR=5.0 min.N-(2-(dimethylamino)ethyl)-2-(5-methylfuran-2-yl)quinoline-4-carboxamide (9)7-Bromo-: Prepared as for 1 from 31 , CH2Cl2 (15 mL), CDI  and N,N-dimethylethane-1,2-diamine . The reaction was stirred at RT for 18 h. Purification by flash column chromatography  gave 9 as a white solid : 1H NMR : δ=2.3 , 2.45 , 2.68 , 3.70 , 6.21 , 7.35 , 7.92 , 8.05 , 8.16 ppm ; LCMS (ES+): m/z (%): 402 (100) [M+H]+ (79Br), 404 (100) [M+H]+ (81Br); tR=5.1 min.N-(2-(dimethylamino)ethyl)-2-(5-methylfuran-2-yl)quinoline-4-carboxamide (10)8-Bromo-: Prepared as for 1 from 32 , CH2Cl2 (10 mL), CDI  and N,N-dimethylethane-1,2-diamine . The reaction was stirred for 18 h at RT. Purification by flash column chromatography  gave 10 as a white solid : 1H NMR : δ=2.24 , 2.43 , 2.8 , 3.46 , 6.40 , 7.38 , 7.47 , 7.91 , 8.12 , 8.17 , 8.84 ppm ; LCMS (ES+): m/z (%): 402 (100) [M+H]+ (79Br), 404 (100) [M+H]+ (81Br); tR=5.2 min.N-(pyridin-2-ylmethyl)quinoline-4-carboxamide (11)2-(5-Methylfuran-2-yl)-: Compound 34  and CDI  were dissolved in CH2Cl2 (4 mL). The solution was stirred for 10 min at RT, then pyrin-2-ylmethanamine  was added. The reaction was stirred at RT for 48 h and worked up as previously described (see details for 8). The product was recrystallised from from Et2O to give 11 as a white solid : 1H NMR : δ=2.44 , 4.68 , 6.39 , 7.31 , 7.35 , 7.48 , 7.6 , 7.80 , 7.85 , 7.97 , 8.05 , 8.16 , 8.58 , 9.47 ppm ; LCMS (ES+): m/z (%): 344 [M+H]+; tR=4.7 min.N-(2-(Dimethylamino)ethyl)-2-(5-methylfuran-2-yl)quinoline-4-carboxamide (12): Prepared as for 11 from 34 , 8 mL CH2Cl2, CDI  and N,N-dimethylaminoethylenediamine . The reaction was stirred for 18 h at RT. Column chromatography  gave 12 as a white solid : 1H NMR : δ=2.22 , 2.41 , 2.48 , 3.46 , 6.39 , 7.32 , 7.59 , 7.78 , 7.86 , 8.03 , 8.11 , 8.8 ppm ; LCMS (ES+): m/z (%): 324 [M+H]+; tR=4.3 min.N-(2-morpholinoethyl)quinoline-4-carboxamide (14)2-(5-Methylfuran-2-yl)-: Prepared as for 11 from 34 , 10 mL CH2Cl2, CDI  and 2-morpholinoethanamine . The reaction was stirred for 18 h at RT, concentrated and purified by triturating the crude solid with hot Et2O to give 14 : 1H NMR : δ=2.41 , 2.48 , 2.52 , 3.49 , 3.64 , 6.39 , 7.34 , 7.59 , 7.79 , 7.85 , 8.05 , 8.18 , 8.79 ppm ; LCMS (ES+): m/z (%): 366 [M+H]+; tR=4.1 min.N-Benzyl-6-bromo-2-(5-methylfuran-2-yl)quinoline-4-carboxamide (15): Prepared as for 1 from 27 , CH2Cl2 (10 mL), CDI  and benzylamine . The reaction was stirred for 18 h at RT. Flash column chromatography  gave 15 as a beige solid : 1H NMR : δ=2.36 , 4.70 , 6.09 , 6.59 , 6.95 , 7.31 , 7.39 , 7.58 , 7.66 , 7.74 , 8.2 ppm ; LCMS (ES+): m/z (%): 421 (100) [M+H]+ (79Br), 423 (100) [M+H]+ (81Br); tR=5.7 min.N-(2-(dimethylamino)ethyl)-2-(furan-2-yl)quinoline-4-carboxamide (20)6-Bromo-: Prepared as for 1 from 33 , CH2Cl2 (15 mL), CDI  and N,N-dimethylethane-1,2-diamine . The reaction was stirred at RT for 18 h. Flash column chromatography  gave 20 as a pale yellow solid : 1H NMR : δ=2.29 , 2.51 , 3.48 , 6.78 , 7.47 , 7.95 , 7.99 , 8.44 , 8.9 ppm ; LCMS (ES+): m/z (%): 388 (100) [M+H]+ (79Br), 390 (100) [M+H]+ (81Br); tR=4.9 min.N-(2-(dimethylamino)ethyl)-2-(5-methylthiophen-2-yl)quinoline-4-carboxamide (22)6-Bromo-: Prepared as for 11 from 30 , CH2Cl2 (15 mL), CDI  and N,N-dimethylethane-1,2-diamine . The reaction was stirred for 18 h at RT. Flash column chromatography  gave 22 as a beige solid : 1H NMR : δ=2.28 , 2.44 , 2.60 , 3.50 , 6.92 , 7.76 , 7.89 , 8.11 , 8.40 , 8.9 ppm ; LCMS (ES+): m/z (%): 419 (100) [M+H]+ (79Br), 421 (100) [M+H]+ (81Br); tR=5.1 min.N-phenethylquinoline-4-carboxamide (25)6-bromo-2-(5-methylfuran-2-yl)-: Prepared as for 1 from 27 , CH2Cl2 (10 mL), CDI  and phenethylamine . The reaction was stirred for 18 h at RT. Purification by flash column chromatography  gave 25 as a cream solid : 1H NMR : δ=2.43 , 4.70 , 5.23 , 6.11 , 6.58 , 6.97 , 7.31 , 7.61 , 7.61 , 7.66 , 7.74 , 8.21 ppm ; LCMS (ES+): m/z (%): 434 (100) [M+H]+ (79Br), 436 (100) [M+H]+ (81Br); tR=5.2 min.N-(2-(dimethylamino)ethyl)-6-fluoro-2-(5-methylfuran-2-yl)quinoline-4-carboxamide (26): Prepared as for 1 from 28 , CH2Cl2 (10 mL), CDI  and N,N-dimethylethane-1,2-diamine . The reaction was stirred for 18 h at RT. Purification by flash column chromatography  gave 26 as a white solid : 1H NMR : δ=2.29 , 2.46 , 2.53 , 3.46 , 6.37 , 7.34 , 7.72 , 7.92 , 7.95 , 8.1 , 8.85 ppm ; LCMS (ES+): m/z (%): 341 [M+H]+; tR=4.0 min.6-Bromo-2-(5-methylfuran-2-yl)quinoline-4-carboxylic acid (27): A solution of 5-bromoindoline-2,3-dione  in EtOH/H2O  in a 30 mL microwave reactor tube was treated with 1-(5-methylfuran-2-yl)ethanone . This tube was transferred to the microwave reactor and, following a 1 min pre-stirring, was heated to 110°C for 13 min. The reaction mixture was acidified to pH 3 using AcOH, diluted with H2O (60 mL) and extracted with EtOAc (2×80 mL). The organic layer was washed with H2O (2×80 mL) and brine (80 mL), dried (MgSO4), filtered and concentrated to give a yellow/brown solid. The crude product was redissolved in a minimum amount of hot EtOAc (∼40 mL) and the pure product was allowed to crystallise out yielding 27 : RF=0.1 ; 1H NMR : δ=2.42 , 6.40 , 7.39 , 7.83 , 8.07 , 8.31 , 8.77 , 14.16 ppm ; LCMS (ES+): m/z (%): 332 (100) [M+H]+ (79Br), 334 (100) [M+H]+ (81Br); tR=0.5–0.6 min.6-Fluoro-2-(5-methylfuran-2-yl)quinoline-4-carboxylic acid (28): Prepared as for 27 from 5-fluoroindoline-2,3-dione , 1-(5-methylfuran-2-yl)ethanone  and KOH  in EtOH/H2O  to give 28 as a pale cream solid : 1H NMR : δ=2.45 , 6.40 , 7.40 , 7.94 , 8.00 , 8.31 , 8.92 , 14.10 ppm ; LCMS (ES+): m/z (%): 271 [M+H]+; tR=0.5–0.6 min.6-Chloro-2-(5-methylfuran-2-yl)quinoline-4-carboxylic acid (29): Prepared as for 27 from 5-chloroindoline-2,3-dione , 1-(5-methylfuran-2-yl)ethanone  and KOH  in EtOH/H2O  to give 29 as a pale cream solid : 1H NMR and LCMS confirm purity at >95%; 1H NMR : δ=2.42 , 6.40 , 7.39 , 7.94 , 8.00 , 8.3 , 8.92 , 14.15 ppm ; LCMS (ES+): m/z (%): 288 [M+H]+; tR=0.5–0.6 min.6-Bromo-2-(5-methylthiophen-2-yl)quinoline-4-carboxylic acid (30): Prepared as for 27 from 2-acetyl-5-methylthiophene  to give carboxylic acid 30 as a yellow solid : 1H NMR and LCMS confirm purity at >95%; 1H NMR : δ=2.47 , 6.92 , 7.92 , 8.42 , 8.88 , 14.15 ppm ; LCMS (ES+): m/z (%): 348 (100) [M+H]+ (79Br), 350 (100) [M+H]+ (81Br); tR=0.5–0.6 min.7-Bromo-2-(5-methylfuran-2-yl)quinoline-4-carboxylic acid (31): Prepared as for 27 from 6-bromoindoline-2,3-dione . The crude product was triturated with hot Et2O to isolate purified carboxylic acid 31 as a pale brown solid : 1H NMR and LCMS confirm purity at >95%; 1H NMR : δ=2.42 , 6.40 , 7.42 , 7.50 , 8.18 , 8.22 , 8.54 , 14.2 ppm ; LCMS (ES+): m/z (%): 332 (100) [M+H]+ (79Br), 334 (100) [M+H]+ (81Br); tR=0.5–0.6 min.8-Bromo-2-(5-methylfuran-2-yl)quinoline-4-carboxylic acid (32): Prepared as for 27 from 7-bromoindoline-2,3-dione . The crude product was triturated with hot Et2O to isolate purified carboxylic acid 32 as a pale brown solid : 1H NMR and LCMS confirm purity at >95%; 1H NMR : δ=2.42 , 6.42 , 7.39 , 7.53 , 8.2 , 8.28 , 8.62 , 14.2 ppm ; LCMS (ES+): m/z (%): 332 (100) [M+H]+ (79Br), 334 (100) [M+H]+ (81Br); tR=0.5–0.6 min.6-Bromo-2-(furan-2-yl)quinoline-4-carboxylic acid (33): 5-Bromoindoline-2,3-dione  and KOH flakes  were added to a 30 mL microwave reactor tube containing EtOH/H2O . The tube was transferred to the microwave reactor and, following a 1 min prestirring, was heated to 110°C for 3 min. The tube was then removed from the reactor and 2-acetylfuran  was added. Heating in the microwave was recommenced (110°C for 12 min) to achieve complete reaction. The reaction mixture was then acidified to pH 3 using AcOH, diluted with H2O (60 mL) and extracted with EtOAc (2×80 mL). The organic layer was washed with H2O (2×80 mL) and brine (80 mL), dried (MgSO4), filtered and concentrated to give a pale yellow solid. The crude solid was heated to reflux in EtOAc (20 mL) and the remaining insoluble cream solid was filtered off to give 33 : Product purity was determined by 1H NMR and LCMS as >98%; RF=0.3 ; 1H NMR : δ=6.77 , 7.50 , 7.97 , 8.01 , 8.38 , 8.91 , 14.17 ppm ; LCMS (ES+): m/z (%): 318 [M+H]+; tR=0.5–0.6 min.2-(5-Methylfuran-2-yl)quinoline-4-carboxylic acid (34): Prepared as for 23 from indoline-2,3-dione , KOH flakes  and 2-acetyl-5-methylfuran . The crude solid was redissolved in refluxing EtOAc (30 mL) and the purified product was precipitated out and filtered off, washing with cold EtOAc (20 mL) to give compound 34 : Product purity was determined by 1H NMR and LCMS as >98%; RF=0.15 ; 1H NMR : δ=2.42 , 6.38 , 7.36 , 7.62 , 7.79 , 7.87 , 8.02 , 8.11 ppm ; LCMS (ES+): m/z (%): 254 [M+H]+; tR=0.5–0.6 min.H,3H)dione (35)1-Methyl-6-(1-methylhydrazinyl)pyrimidine-2,4- and methyl-hydrazine  were refluxed in EtOH (20 mL) for 1.5 h. The reaction was cooled to RT and concentrated in vacuo to afford pyrimidine 35 as a pale yellow solid : 1H NMR : δ=2.67 , 3.09 , 3.11 , 3.41 , 4.23 ppm ; LCMS (ES+): m/z (%): 288 [M+H]+ (100); tR=2.7–2.9 min.H,3H)dione (36)6-(2-(5-Bromo-2-fluorobenzylidene)-1-methylhydrazinyl)-1-methylpyrimidine-2,4 and 5-bromo-2-fluoro-benzaldehyde  were refluxed in EtOH (40 mL) for 2 h. The reaction was cooled to RT, filtered and washed with Et2O to afford benzylidene 36 as a cream solid : 1H NMR : δ=3.13 , 3.39 , 5.31 , 7.28 , 7.60 , 7.88 , 8.74 , 11.05 ppm ; LCMS (ES+): m/z (%): 381 (100) [M+H]+ (79Br), 383 (100) [M+H]+ (81Br); tR=3.9 min.H,4H)dione (37)3-(4-Bromo-2-fluorophenyl)-4-hydroxy-8-methyl-6,8-dihydropyrimidotriazine-5,7 was dissolved in AcOH/H20 (3.45/0.17 mL) and the solution was cooled to 0°C. NaNO2  was added portionwise and the reaction was warmed to RT over 16 h. Et2O (10 mL) was added and the reaction was stirred at RT for 1 h. The resultant yellow precipitate was filtered and washed with Et2O to afford 37 : 1H NMR : δ=3.25 , 7.39 , 7.72 , 8.11 , 12.30 ppm .e,1,2,4]triazine-5,7dione (42)3-(5-Bromo-2-fluorophenyl)-6-methylpyrimido+ (79Br), 353 (100) [M+H]+ (81Br); tR=3.9 min.e,1,2,4]triazine-5,7dione (43)3-(5-Bromo-2-fluorophenyl)pyrimidotriazine-5,7-dione  was heated in anhyd DMF (5 mL) at 90°C for 3 h in a sealed tube with 4 Å molecular sieves. The reaction was cooled to RT and iced H2O was added. The resultant brown precipitate was filtered and dried in vacuo to afford pyrimido triazine 43 : 1H NMR : δ=7.29 , 7.86 , 8.01 , 11.97 , 12.49 ppm .H,6H)dione (48)3-(5-Bromo-2-fluorophenyl)-1-((2-dimethylamino)ethyl)-6-methylpyrimidotriazine-5,7, Cs2CO3 , 2-dimethylaminoethylchloride  and anhyd DMF (2 mL) were heated at 80°C for 3 h. The reaction mixture was then filtered and diluted with EtOAc, the organic layer was washed with H2O (3×10 mL), dried (MgSO4), filtered and concentrated in vacuo. The crude was purified by column chromatography  to give pyrdimido triazine 48 as a yellow solid : 1H NMR : δ=2.11 , 2.80 , 3.48 , 4.13 , 7.04 , 7.49 , 8.13 ppm ; LCMS (ES+): m/z (%): 395 (100) [M+H]+ (79Br), 397 (100) [M+H]+ (81Br); tR=0.9–1.1 min.H,6H)dione (50)3-(5-Bromo-2-fluorophenyl)-1-isopropyl-6-methylpyrimidotriazine-5,7, Cs2CO3 , isopropylbromide  and anhyd DMF (2 mL) were heated at 150°C for 10 min in a Biotage microwave reactor. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The crude was purified by column chromatography  to give pyrdimido triazine 50 as a yellow solid : 1H NMR : δ=8.35 , 7.65 , 7.18 , 5.90 , 3.57, 1.72 ppm ; LCMS (ES+): m/z (%): 399 (100) [M+H]+ (79Br), 401 (100) [M+H]+ (81Br); tR=4.8 min.e,1,2,4]triazine-5,7dione (51)3-(5-Bromo-2-fluorophenyl)-1-butyl-6-methylpyrimidotriazine-5,7dione (52)3-(5-Bromo-2-fluorophenyl)-1-cyclopentyl-6-methylpyrimido[5,4-: Compound 42 , K2CO3 , cyclopentylbromide  and anhyd DMF (2 mL) were heated at 150°C for 10 min in a Biotage microwave reactor. The reaction mixture was filtered and the filtrate concentrated in vacuo. The crude was purified by column chromatography (CH2Cl2/MeOH/NH3 95:5:0.1) to give pyrdimido triazine 52 as a yellow solid : 1H NMR : δ=8.19 , 7.54 , 7.11 , 4.96 , 3.51 , 2.36–2.31 , 2.09–2.03  and 1.73–1.65 ppm .


O-acetyl-D-glucal with a variety of nucleophiles, viz. alcohols, phenols, thiols, thiophenols, and allyl trimethyl silane (TMS), in the presence of 5 mol% of lanthanum(III) nitrate hexahydrate under solvent-free conditions yielded the corresponding 2,3-unsaturated glycopyranosides  in excellent yields.A mild and highly efficient stereoselective reaction of 3,4,6-tri- The direct and straightforward method for the synthesis of this class of compounds is an allylic rearrangement of glycols by the anomeric glycosidation with different nucleophiles in the presence of Lewis acids. This is well known as the Ferrier rearrangement, which includes BF3OEt2,[14] SnCl4,[16] InCl3, montmorillonite K-10, 2,3-dichloro-5,6-dicyano-p-benzoqui-none (DDQ), N-iodosuccinimide (NIS), I2, FeCl3, and some metal triflates such as Dy(OTf)3 and Yb(OTf)3. However, the use of strongly acidic conditions frequently leads to the formation of undesirable side products competing with the main reactions. Thus, a mild and efficient catalyst for the synthesis of 2,3-unsaturated glycopyranosides is highly desirable. In view of current interest in catalytic processes, there is a merit in developing the synthesis of pseudoglycals using an inexpensive, mild, and nonpolluting reagent.The anomeric products, uronic ahydrates,,5 nucleoibiotics,,7 and olccarides,–10 and tibiotics. The direangement, which in BF3OEt2,–14 SnCl44] SnCl4,,16 InCl36] InCl3, montmoriite K-10, 2,3-dichne (DDQ), N-iodosude (NIS), I2,21]  C-glycol FeCl3, and some Dy(OTf)3 and Yb(OYb(OTf)3. However, chemoselec-tive deprotection of acetonides, synthesis of quinazolinones, mild and efficient acetylation of phenols and amines, synthesis of α-amino nitriles, synthesis of benzodiazepines, and N-tert-butoxycarbonylation and N-benzyloxycarbonylation of amines.[32] In studying these transform-ations, it has been observed that the substrates containing other acid labile functional groups, such as TBDMS ethers, some isopropylidene protected diols, and N-tert-Boc-protected amines, were intact in the presence of La(NO3)3 · 6H2O. In continued efforts for utilizing La(NO3)3 · 6H2O, we found that it is an efficient and mild Lewis acid catalyst for the synthesis of 2,3-unsaturated glycopyranosides.Organic reactions using mild and water-tolerant catalysts received much attention in recent years. They can be conveniently handled and removed from the reaction mixture, making the experimental procedure simple and eco-friendly. Lanthanum(III) nitrate hexahydrate is relatively nontoxic, inexpen-sive, insensitive to air, and used in various organic transformations, such as chemoselective tetrahydropyranylation of primary alcohols, chemoseletonides, synthesiolinones, mild andd amines, synthesinitriles, synthesiazepines, and N-tef amines.,32 In st23)3 · 6H2O is highly oxophilic, forms a labile bond with carbonyl oxygen and initiates the formation of a C−X  bond with nucleophiles  nitrate hexahydrate under solvent-free conditions at room temperature proceeds efficiently and smoothly, and the reaction conditions are very mild. No by-products were observed. Furthermore, other functionalities such as N-tert-butylcarbamates are compatible under reaction conditions 3 · 6H2O (5 mol%) under solvent-free conditions 3 · 6H2O under solvent-free conditions.In conclusion, we have described a mild and efficient method for the synthesis of 2,3-unsaturated glycopyranosides using La(NO44.13)3 · 6H2O (5 mol%) was added to a mixture of 3,4,6-tri-O-acetyl-D-glucal (1 mmol), alcohols/phenols/thiols/allyl TMS (1.1 mmol), and the reaction mixture was stirred at room temperature under solvent-free conditions for the appropriate time ; 1H NMR : δ 2.02 , 3.30 , 3.46–3.50 , 3.55–3.62 , 3.77–3.92 , 3.97–4.17 , 4.95 , 5.20 , 5.77 ; IR (KBr) υmax: 3374, 2926, 1746, 1544, 1451 cm−1; LCMSD: m/z 289 (M+ + 1). Entry 4: solid, mp 49–52°C; [α]D25 80.9 ; 1H NMR : δ 2.07 , 2.35 , 3.51–3.58 , 3.75–3.83 , 3.99–4.01 , 4.14–4.20 , 4.97 , 5.02–5.12 , 5.24 , 5.81 ; LCMSD: m/z 285 (M+ + 1).Entry 3: Solid, mp 44–46°C; [α]D25 64.0 ; 1H NMR : δ 0.90 , 1.22 , 1.38–1.40 , 2.09 , 2.10 , 3.52 , 3.71 , 3.89 , 4.30 , 4.44 , 4.65 , 5.28 , 6.37 ; LCMSD: m/z 388 (M+ + 1).Entry 5: [α]D25 96.4 ; 1H NMR : δ 1.78 , 2.00 , 2.86 , 3.2–3.50 , 3.60  3.71–3.88 , 3.94–4.16 , 4.90 , 5.20 , 5.75 ; IR (KBr) υmax: 3414, 2927, 1742, 1664, 1660, 1542, 1456, 1444, 726 cm−1; FAB mass: m/z 360 (M+ + 1).Entry 6: Solid, mp 50–52°C; [α]


Oestrogen receptor protein (ER) was detected in 9 of 11 samples of malignant breast tissue and 8 of 9 samples of normal breast tissue. Levels of cytosolic ER (ERc) in malignant breast were 21-1102 fmol mg-1 soluble protein (Kd 1.8 X 10(-9)-3.1 X 10(-8) mol l-1) and those of nucleosolic ER (ERn), 13-526 fmol mg-1 soluble protein (Kd 2.1 X 10(-9)-1.4 X 10(-8) mol l-1). In normal breast tissue ERc levels were 33-640 fmol mg-1 soluble protein (Kd 1.3 X 10(-10)-3.2 X 10(-9) mol l-1), ERn was detected in only 2 samples, 8 and 87 fmol mg-1 soluble protein with Kd 3.2 X 10(-9) and 1.4 X 10(-9) l mol-1 respectively. 17 alpha-ethinyl-13 beta-ethyl-17 beta-hydroxy-4,15-gonadiene-3-one (gestodene), a new synthetic progestogen displaced 3H-oestradiol (3H-E2) from both ERc and ERn in malignant tissue but not in normal breast, or these receptors from endometrial tissue. In competition studies gestodene was approximately 3 times more effective in displacing 3H-E2 from ERc and ERn in malignant breast tissue than the natural ligand. Quantitation of ER by gestodene were ERc, 12-1134 fmol gestodene bound mg-1 soluble protein (Kd 1 X 10(-9)-8.1 X 10(-9) mol l-1); ERn, 17-531 fmol gestodene bound mg-1 soluble protein (Kd 1.6 X 10(-9)-1.1 X 10(-8) mol l-1). L-13-ethyl-17 alpha-ethinyl, 17 beta-hydroxy-gonen-3-one (levonorgestrel) showed no binding to ER in malignant breast, normal breast or endometrial tissue. In circulation both gestodene and levonorgestrel displaced E2 from sex hormone binding globulin more than any of the androgens tested. These results suggest that gestodene is a progestogen with oestrogenic and/or antioestrogenic properties and provide strong evidence for differences in ER from malignant and normal breast tissue.


A two step synthesis of 2-alkynyl-4-arylquinolines has been accomplished via Pd/C-mediated regioselective C-2 alkynylation of 2,4-dichloroquinoline in water followed by Suzuki coupling at C-4 of the resulting 4-chloro derivative. A, 2-Alkynyl pyridine and its benzo (i.e. quinoline) derivative possessing an aryl group at the C-4 position A,  have attA. Considering the possible C–C bond forming reactions on a pyridine/quinoline ring (A can be carried out following two main strategies e.g. (a) arylation at C-4 followed by alkynylation at C-2 or (b) alkynylation at C-2 followed by arylation at C-4. Methodologies based on strategy ‘a’ have been reported earlier. For example, Sonogashira coupling of a terminal alkyne with 2-chloro-4-aryl substituted quinoline [3)2PdCl2-CuI or treatment of 4-aryl pyridine-N-oxide with alkynyl Grignard [A following the strategy ‘b’ has not been explored. In our effort towards the synthesis of quinoline derivatives of potential biological significance we have reported Pd/C-mediated regioselective C-2 alkynylation of 2,4-dichloroquinoline in water [3) followed by successful Suzuki coupling at C-4 of compound 3 leading to the corresponding 4-arylated derivatives (5) in good yields  were prepared via coupling of 2,4-dichloroquinoline (1) in the presence of 10% Pd/C (10 mol%), PPh3 (20 mol%) and CuI (5 mol%) as a catalyst system in water. The results are presented in 3a. Irradiation of protons of the benzene ring attached to the alkynyl group resulted in enhancement of the singlet at δ 8.03 assigned to the C-3 hydrogen of the quinoline ring. If the alkyne was at C-4, NOE enhancement at C-5 is expected in addition to C-3. It is note worthy that the use of the Sonogashira coupling or its modified form has been employed for the preparation of 2-alkynylquinolines or related derivatives earlier [A number of 2-alkynyl-4-chloroquinolines ( earlier –17.5) we planned to exploit the reactivity of chloro group of 3 towards Suzuki arylation reaction. Accordingly, a variety of arylboronic acids were coupled with 3 and results of this study are summarized in 3)2PdCl2 as a catalyst, CsCO3 as a base, tricyclohexyl phophine, (PCy3) as a ligand in dioxane–water at 80 °C. The arylboronic acids used in this reaction include phenylboronic acid  were prepared in good to excellent yields without affecting the alkynyl substituents present in compound 3.Next, in order to prepare 2-alkynyl-4-arylquinolines (1) with terminal alkynes (2) in water proceeds via normal Sonogashira pathway [3 thus formed then undergo Suzuki reaction in the next step. Oxidative addition of Pd0 generated in situ to compound 3 followed by trans-organometallation of the resultant aryl-palladium complex formed with arylboronic acids provides the desired compound 5.The reaction mechanism of the present stepwise C–C bond forming reactions consisting of alkynylation followed by arylation is shown in  pathway . Due to  pathway –19 contrIn conclusion, a two-step method consisting of alkynylation followed by arylation has been developed for the synthesis of 2-alkynyl-4-arylquinolines. The alkynylation step involved Pd/C–Cu mediated regioselective C-2 alkynylation of 2,4-dichloroquinoline in water to afford 2-alkynyl-4-chloroquinoline. The arylation step is a Pd-mediated (Suzuki) coupling of 2-alkynyl-4-chloro derivative with arylboronic acids in aqueous media to give the target compounds. The process is amenable to the diversity-oriented synthesis of quinoline derivatives of potential pharmacological significance and may therefore find wide usage in organic/medicinal chemistry.5: A mixture of alkyne 3 (1.0 mmol) and (PPh3)2PdCl2 (0.05 mmol) in dioxane (5.0 mL) was stirred for 10 min under nitrogen at room temperature and then heated to 80 °C. To this mixture was added a solution of PCy3 (0.05 mmol) and CsCO3 (3.5 mmol) dissolved in water (3.0 mL) and arylboronic acid (1.5 mmol) dissolved in dioxane (3.0 mL) at the same temperature. The mixture was stirred at 80 °C according to the time indicated in 2SO4 and concentrated under vacuum. The crude product was purified by column chromatography on silica gel, using light petroleum ether (60–80 °C)-ethyl acetate to afford the desired product. Spectral data for selected compounds; Compound 5a; light brown gum, Rf (20% ethyl acetate/n-hexane) 0.21; 1H NMR  δ 8.14 , 7.85 , 7.69 , 7.50–7.41 , 2.49 , 1.69–1.21 , 0.91–0.8 ; IR  2927, 2225, 1587, 1543, 1357; m/z (ES Mass) 314 ; 13C NMR  148.5, 143.6, 137.5 (2C), 129.6 (3C), 128.4 (2C), 126.7 (2C), 125.5 (3C), 124.3, 92.2, 81.0, 76.3, 31.3, 29.6, 28.0, 24.6, 22.5; HRMS (ESI): calcd for C23H23N (M+H)+ 314.1909, found 314.1896. Compound 5b, low melting solid, Rf (20% ethyl acetate/n-hexane) 0.28; 1H NMR  δ 8.20 , 7.87 , 7.75–7.71 , 7.56–7.47 , 7.25–7.17 , 2.38 ; IR  2924, 2216, 1583, 1541; m/z (ES Mass) 320 ; 13C NMR  148.6, 148.5, 143.2 (2C), 139.3 (2C), 137.3 (2C), 134, 132, 129.5 (3C), 129.3 (2C), 126.9 (2C), 125.6 (2C), 124.4, 118.9, 90.2, 88.8, 29.0; HRMS (ESI): calcd for C24H17N (M+H)+ 320.1439, found 320.1454.General Procedure for the preparation of compound File 15c–h.Spectral data of 2-alkynyl-4-arylquinolines


Pyrrolopyridazine derivatives have various biological applications –8, and tb]pyridazine derivatives were reviewed in 1977 by Kuhla and Lombardino [b]pyridazinium perchlorates with malononitrile, ethyl cyanoacetate and ethyl malonate in the presence of sodium ethoxide pyridazine derivatives can also be synthesized from 1-aminopyrrole and its derivatives. This original method was reported by Flitsch and Krämer (in 1968–9) pyridazines through a hitherto unprecedented approach from a BOC-protected 1-aminopyrrole derivative and α,β-unsaturated ketones.As a part of our continued interest in the development of new synthetic methods for highly substituted pyrrole and indole derivatives –28, we h1 was reacted with tertiary butyl carbazate 2 in toluene and cyclohexane in the presence of p-toluenesulfonic acid (p-TSA) at reflux and the resulting [2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-phenylcarbamoyl-pyrrol-1-yl]-carbamic acid tert-butyl ester 3 was further condensed with 3-(4-fluorophenyl)-1-phenyl-propenone 4a pyridazine derivatives, i.e. 4,7-bis-(4-fluorophenyl)-4a-isopropyl-2,6-diphenyl-4a,7-dihydropyrrolopyridazine-5-carboxylic acid phenylamide 5a, were expected as products in this synthetic sequence -5-isopropyl-2,6-diphenyl-3,4-dihydropyrrolopyridazine 6a (2-[2-(4-Fluorophenyl)-2-oxo-1-phenylethyl]-4-methyl-3-oxo-pentanoic acid phenylamide enone 4a  in the psequence . Howeverazine 6a .m/z 502 (M+), instead of m/z 621 (M+); HRMS data also confirmed the m/z 502 (M+) and molecular formula as C34H28F2N2, in accord with structure 6a and not with 5a. The IR spectrum lacked any –C=O absorption. The 1H NMR spectrum of 6a exhibited signals due to two methyl groups and the –CH of an isopropyl group at δ 0.85 , δ 1.15 , and δ 2.84–2.91 , respectively; –CH2 and –CH of pyridazine ring at δ 3.1–3.26  and δ 4.71–4.74 , respectively; and aromatic protons at δ 6.88–7.63 . In the 13C NMR, the DEPT spectrum was characterized by the presence of signals due to 2 × CH3 and 1 × CH of isopropyl group; CH2 and CH of pyridazine ring at δ 22.6, 23.8, and 25.3; 31.5 and 34.7 ppm, respectively. Product 5a should exhibit a –C=O signal and no CH2 peak. In the DQCOSY spectrum, 1H–1H coupling between 2 × CH3 groups and –CH of the isopropyl group and also between the –CH2 and –CH groups of pyridazine ring was seen, but no coupling between the isopropyl group and the –CH2 and –CH of the pyridazine ring was observed. This indicates that the newly formed –CH2 and –CH are connected to each other, which is not possible in 5a. The HSQC spectrum exhibited 13C–1H coupling of two methyl groups at δ 23.0, 1.2 ; δ 24.0, 0.90  and –CH of isopropyl at δ 25.5, 2.85 , and also the –CH2 and –CH groups of pyridazine ring at δ 31.5, 3.1–3.3 ; δ 35.0, 4.75 . The newly formed –CH2 is linked to a -CH group. It is reported pyridazine structure 6a  in refluxing ethyl alcohol to provide alkenyl imine 9, which was characterized on the basis of its mass, 1H and 13C NMR, DEPT, and IR spectral data. Compound 9 was heated in toluene in the presence of p-TSA for 10–12 h and the resulting compound was found to be identical to product 6a. Hydrolysis of the amide group and subsequent decarboxylation was carried out on pyrrole derivative 15 to afford the 2,3-diaryl pyrrole derivative 16  the olefinic carbon is electron deficient and therefore cyclization is favorable.With a view to extending this protocol to aliphatic systems such as α,β-unsaturated ketones, carbamate 6a was heated in the presence of p-TSA in toluene at 110 °C for 25.0 h and the resulting compound to yield 4,7-bis-(4-fluorophenyl)-5-isopropyl-2,6-diphenylpyrrolopyridazine  were expressed in ppm with tetramethylsilane  as internal standard. Spin multiplicities are given as s (singlet), d (doublet), t (triplet), and m (multiplet). FT-IR spectra were recorded in KBr dispersion with a Perkin-Elmer 1650 FT-IR spectrometer. Mass spectra (70 eV) were recorded on HP-5989 A LC-MS spectrometer. The high resolution mass spectroscopy (HRMS) analysis was performed on the Micromass LCT Premier XE mass spectrometer equipped with an ESI Lack spray source for accurate mass values . Melting points were determined by the capillary method with a POLMON (Model MP-96) melting point apparatus. Solvent removal was accomplished by a Buchi rotary evaporator at house vacuum (30–40 Torr). Solvents and reagents were used without further purification. The purity of compounds was checked on silica gel coated aluminium plates (Merck).The (a) Procedure for compound 3: A mixture of 2-[2-(4-fluorophenyl)-2-oxo-1-phenylethyl]-4-methyl-3-oxo-pentanoic acid phenylamide , tertiary butyl carbazate , and p-TSA (0.006 mol) in toluene (20.0 mL) and cyclohexane (75.0 mL) was maintained at reflux until no more water collected (reaction monitored by TLC). The reaction mixture was cooled to 30 °C, allowed to stand for 3 h, filtered and washed with cyclohexane (10.0 mL). The compound was washed again with cyclohexane (30.0 mL) and dried to give a white solid. mp 191–193 °C; 1H NMR : 1.3 , 3.1 , 6.97–7.54 , 9.9 , 10.3 , both –NH groups were D2O exchangeable; IR KBr (cm−1): 3422, 3258, 1712, 1671; Anal. Calcd for C31H32FN3O3: C, 72.49; H, 6.28; N, 8.18. Found: C, 72.33; H, 6.42; N, 8.35.(b) A typical procedure for compound 6a: A mixture of [2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-phenylcarbamoyl-pyrrol-1-yl]-carbamic acid tert-butyl ester , 3-(4-fluorophenyl)-1-phenyl-propenone , and p-TSA  in toluene (150.0 mL) and cyclohexane (100.0 mL) was maintained at reflux (azeotropic) for 19.0 h (reaction monitored by TLC). The reaction mixture was cooled to ambient temperature; ethyl acetate (40.0 mL) was added and washed first with water (25.0 mL) and then with 10% sodium bicarbonate solution (25.0 mL). The resulting organic layer was concentrated under vacuum and the crude product recrystallized from ethyl acetate (25.0 mL) to remove unreacted 9 (~5.0%). The resulting filtrate was concentrated under vacuum and further recrystallized from isopropyl alcohol (20.0 mL) to give a cream solid (m/z 502 (M+); HRMS data: m/z 502 (M+) and mol. formula: C34H28F2N2; 1H NMR : 0.85 , 1.15 , 2.84–2.91 , 3.1–3.26 , 4.71–4.74 , 6.88–7.63 ; 13C NMR : 22.6, 23.8, 25.3, 31.5, 34.7, 114.0, 114.5, 115.2, 115.7, 118.4, 122.2, 124.0, 126.0, 126.2, 127.0, 127.7, 127.9, 128.3, 128.5, 129.7, 131.2, 132.50, 132.65, 136.1, 136.7, 139.5, 152.3, 159.0, 159.2, 163.8, 164.0; DEPT : CH3 carbons at δ 22.6, 23.8; aliphatic-CH carbons at δ 25.3, 34.7 and aromatic-CH carbons at δ 114.0–132.6; CH2 carbon at δ 31.5 ppm; DQCOSY : δ 0.8 and 1.2 coupled with δ 2.8 and δ 3.2 coupled with δ 4.8 ppm; HSQC : δ 23.0, 1.2 ; δ 24.0, 0.90 ; and δ 25.5, 2.85 ; δ 31.5, 3.1–3.3 ; δ 35.0, 4.75 ; and δ 113–134, 6.9–7.95 ; the HMBC spectrum: 13C–1H correlations of 2 × CH3 ; iPr-CH ; CH of pyridazine ring , C-2, C-9, and C-10); CH2 , C-13 (less), C-9, and C-2); IR KBr (cm−1): 3064, 1600, 1506, 1155; Anal. Calcd for C34H28F2N2: C, 81.25; H, 5.62; N, 5.57. Found: C, 81.41; H, 5.51; N, 5.74.am solid . mp 203–


Citrus sinensis flavedo var. Pineapple resulted in the isolation of six compounds characterized as tetracosane, ethyl pentacosanoate, tetratriacontanoic acid, tangertin, β-sitosteryl-β-D-glucoside and 3,5,4'-trihydroxy-7,3'-dimethoxy flavanone 3-O-β-glucoside. Of these 3,5,4'-trihydroxy-7,3'-dimethoxy flavanone 3-O-β-glucoside is a hitherto unreported compound.Phytochemical examination of  Citrus sinensis (L.) Osbeck var. Pineapple  belongs[mosambi. Its fruit is strengthening[) belongs to Rutacngthening, cardiotngthening antiinfl1H NMR spectra were recorded on Bruker AC-400F MHz NMR Spectrometer in CDCI3 using TMS as internal standard. IR spectra were obtained on Perkin Elmer Infrared Spectrophotometer. Mass spectra were recorded on VG-70S 11-250J GC-MS-DS Mass Spectrometer.Melting points were determined on Ganson Electrical Melting Point Apparatus. Fruits (15 kg) of this plant were collected from Department of Horticulture, CCSHAU, Hisar. Flavedos (outer part of peel) were chopped into small pieces and then extracted with hot methanol. The solvent was removed to obtain extractives. The extractives were concentrated on a water bath under reduced pressure to obtain a viscous mass. The viscous mass thus obtained was mixed with silica gel (60-120 mesh), dried on water bath and subjected to silica gel (60-120 mesh) column chromatography and six compounds were isolated which were characterized as tetracosane (A), ethyl pentacosanoate (B), tetratriacontanoic acid (C), tangertin (D), β-sitosteryl-β-D-glucoside (E), 3,5,4'-trihydroxy-7,3'-dimethoxy flavanone 3-O-β-glucoside . Of thes3 gave brown colour. Found 56.07, H 5.26; C23H26O12 requires C 55.87, H 5.26 %. UV λmax (nm) 250, 287 (MeOH); 250, 287 (MeOH+NaOAc), 250, 311 (MeOH+AlCl3); 250, 311 (MeOH+AlCl3+HCl). IR νmax : 815, 975, 1070, 1095, 1131, 1202, 1278, 1358, 1443, 1518, 1607, 1647, 2918, 3471. MS (m/z): 494 , 464 (8), 463 (27), 446 (12), 418 (33), 386(36), 374 (41), 330 (100), 214 (38), 159 (5), 158 (16), 134 (3), 132 (10), 102 (6). Molish test: positive. Kiliani hydrolysis: glucose, confirmed by direct comparison using paper chromatography. Aglycone: UV λmax (nm) 250, 288 (MeOH); 250, 288 (MeOH+NaOAc); 250, 310 (MeOH+AlCl3); 250, 310 (MeOH+AlCl3+HCl). Acetate of glycoside: Ac2O/Py, m.p. 144°. 1H NMR of glycoside acetate : 2.04, 2.06, 2.07, 2.09 ; 2.32, 2.38 ; 2.70-5.50 ; 3.87 ; 6.31 ; 6.46 ; 7.01 ; 7.15 ; 7.26 .Compound F  obtained on elution with methanol-ethyl acetate (1:9) and it crystallized out from ethyl alcohol, 85 mg, m.p. 210°. It responded to Mg/HCl test. Alcoholic FeCl−1) and OH (3471 cm−1) groups. Elemental analysis indicated its molecular formula: C23H26O12 and molecular mass: 494. Its acetate was prepared by using acetic anhydride and pyridine. The 1H NMR of the acetate in CDCl3 showed a double doublet  at δ 7.26, integrating to one proton, which could be H-6' of a flavanone. A doublet (J 2.5 Hz) was observed at δ 7.15, integrating to one proton, which could be H-2'. Another doublet (J 7.5 Hz.) was observed at δ 7.01, integrating to one proton, indicating it to be H-5'. There was a doublet (J 2.5 Hz.) at δ 6.46, integrating to one proton, which could be H-8. A doublet (J 2.5 Hz) at δ 6.31, for one proton, could be due to H-6. There was a singlet at δ 3.87, integrating to six protons, indicating two methoxy groups in the compound. There was a multiplet for nine protons in the range δ 2.70 - 5.50 and these could be seven protons of glucose and two protons of the C - ring of a flavanone. A singlet at δ 2.38, integrating to three protons, indicated a phenolic acetoxyl. Another singlet for second phenolic acetoxyl group was observed at δ 2.32. Four alcoholic acetoxyls at δ 2.09 (3H), 2.07(3H), 2.06 (3H), 2.04 (3H) indicated the presence of glucose in the molecule. This data suggested compound F to be 3,5,7,3',4'-pentaoxygenated flavanone-O-glucoside.The brown colour with alcoholic ferric chloride indicated it to be a phenolic compound. The positive Mg/HCl colour reaction suggested it to be a flavonoid. IR depicted the presence of CO  3,5,3'-trihydroxy-7,4'-dimethoxy (ii) 3,5,4'-trihydroxy-7,3'-dimethoxy (iii) 5,3',4'-trihydroxy-3,7-dimethoxy. The aglycone, obtained after Kiliani hydrolysis, had properties identical to (ii). The aglycone could therefore be settled as 3,5,4'-trihydroxy-7,3'-dimethoxy flavanone (1-a)[The original compound showed bathochromic shift with AlClThe compound F could be one of the following two (i) 3,5,4'-trihydroxy-7,3'-dimethoxy flavanone 4'-O-glucoside (ii) 3,5,4'-trihydroxy-7,3'-dimethoxy flavanone-3-O-glucoside. The positions of H-5' in the acetates of compound F and that in 3,5,4'-trihydroxy-7,3'-dimethoxy flavanone are comparable; δ 7.01 in the former and δ 7.07 in the latter[


The molecular structures were identifed by IR, 1H-NMR, 13C-NMR and MS analysis. The crystal structure of the title compound 2a, C38H36O8, is reported. Its crystal data are: monoclinic, space group P 2(1)/n with cell dimensions of a=9.0395(12) Å, b=12.6114(17) Å, c=13.8166(18) Å, β=95.875(3), V=1566.8(4) Å3, F.W.=620.67, ρcalc=1.316 gcm3 for Z=2, μ=0.092 mm−1Tetraketone and tetraaldehyde derivatives  Salmonella typhimurium , 135.05 (C=C), 196.24 (C=O); MS: m/z 621.34 (M+1)+1.4-Hydroxyacetophenone (0.04 mol) and potassium hydroxide (0.04 mol) were refluxed in absolute ethanol for 2 hours. 1,1,2,2-Tetra-yl-tetramethylene tetrabromide (0.01) was added to the reaction mixture which was refluxed for 20 hours. The mixture was filtered and the solvent was evaporated. The solid residue was recrystallized from chloroform-acetone (1:1) to give compound 2b . Analysis  for C58H44O8 (Mw 869.0) C: 80.17/80.26, H: 5.10/5.14; IR (KBr) , 3065 (Ar-CH), 2970 (-CH), 1652 (C=O); 1H-NMR (DMSO-d6) δ (ppm) 5.07 , 7.13–7.57 , 7.62–7.65 , 7.69–7.71 , 7.75 ; 13C-NMR (DMSO-d6) δ (ppm) 64.96 (O-CH2), Ar-C: , 135.12 (C=C), 194.30 (C=O); MS: m/z 869.31 (M+1)+1.4-Hydroxybenzophenone (0.04 mol) and potassium hydroxide (0.04 mol) refluxed in absolute ethanol for 2 hours. 1,1,2,2-Tetra-yl-tetramethylene tetrabromide (0.01) was added to the reaction mixture, which was then refluxed for 20 hours. The mixture was filtered and the solvent was evaporated. The solid residue was recrystallized from DMF-ethyl alcohol (2:1) to give compound 2c . Analysis  for C34H20Cl8O8 (Mw 840.1) C: 48.61/48.53, H: 2.40/2.38; IR (KBr) , 3067 (Ar-CH), 2972 (-CH), 2883–2894 (CHO), 1696 (C=O); 1H-NMR (DMSO-d6) δ (ppm) 5.07 , 7.63–7.64 , 7.98–7.99 , 10.05 ; 13C-NMR (DMSO-d6) δ (ppm) 55.99 (O-CH2), Ar-C: , 135.05 (C=C), 188.17 (C=O); MS: m/z 882.50 (M+Na+H2O)+1.3,5-Dichlorosalicylaldehyde (0.04 mol) and potassium hydroxide (0.04 mol) were refluxed in absolute ethanol for 2 hours. 1,1,2,2-Tetra-yl-tetramethylene tetrabromide (0.01) was added to the reaction mixture, which was refluxed for 20 hours. The mixture was filtered and the solvent was evoporated. The solid residue was recrystallized from ethyl alcohol to give compound 2d . Analysis  for C34H20Br4Cl4O8 (Mw 1017.9) C: 40.12/40.17, H: 1.98/2.01; IR (KBr) , 3075 (Ar-CH), 2977 (-CH), 2796–2884 (CHO), 1698 (C=O); 1H-NMR (DMSO-d6) δ (ppm) 5.01 , 7.44 , 7.83 , 10.18 ; 13C-NMR (DMSO-d6) δ (ppm) 55.93 (O-CH2), Ar-C: , 135.45 (C=C), 188.26 (C=O).3-Chloro-5-bromosalicylaldehyde (0.04 mol) and potassium hydroxide (0.04 mol) were refluxed in absolute ethanol for 2 hours. 1,1,2,2-Tetra-yl-tetramethylene tetrabromide (0.01) was added to the reaction mixture, which was refluxed for 20 hours. The mixture was filtered and the solvent was evaporated. The solid residue was recrystallized from DMF-ethyl alcohol (1:1) to give compound A summary of the key crystallographic information is given in 1) in absolute ethanol media with the corresponding potassium salts of phenolic ketones and aldehydes (obtained by potassium hydroxide solution) gave the corresponding tetraketones 2a,b or tetraaldehydes 2c,d in a good yield  methylene groups integrating for eight protons were seen between 4.99–5.07 ppm. Aldehyde protons (CHO) of compounds 2c,d were observed around 10.05–10.18 ppm, integrating for four protons. In the 13C-NMR of compounds 2a–d, OCH2 group was observed at 56–65 ppm. In addition, C=O and C=C functions of compounds 2a–d appeared at 188–196 ppm and 134–135 ppm, respectively. 1H-NMR and 13C-NMR spectral data of compounds 2a–d are presented in the Experimental section in this study.In the 2a has a centre of symmetry located at the mid-point of the C=C double bond. The centre of symmetry C=C bond length [1.332(6) Å] agrees with the values reported in the literature .The molecule of the title compound C50H36O8 , 1.316. So, we successfully prepared C=O functionalized chelates having ether bridges as a key intermediate for the synthesis of novel macromolecules containing a donor group. The compounds were prepared and identified by elemental analysis, IR, 1H-NMR, 13C-NMR and Mass spectroscopy. In addition, the crystal structure of the compound 2a, C38H36O8, was determined by single crystal X-ray diffraction technique, In order to investigate the influence of the flexibility of the ligand molecule, four new carbonyl compounds have been synthesized and the crystal structure of compound


N-(1-oxohex-5-ynyl)neuraminic acid (Neu5Hex) into the cell-surface glycocalyx of a human larynx carcinoma cell line (HEp-2) and its fluorescence labelling by click chemistry.Sialic acids are located at the termini of mammalian cell-surface glycostructures, which participate in essential interaction processes including adhesion of pathogens prior to infection and immunogenicity. Here we present the synthesis and bioorthogonal metabolic incorporation of the sialic acid analogue  N-acetylneuraminic acid , are believed to permeate the cell membrane by diffusion processes  cycloaddition reaction between N-tert-butyl nitrone 9 and 8 and subsequent base-catalyzed ring-opening and hydrolysis afforded N-(1-oxohex-5-ynyl)neuraminic acid  in 38% yield . The internalization of this acetylated monosaccharide was described previously as a diffusion process through the membrane of eukaryotic cells  triazole cycloaddition amine, TBTA) were applied in dimethylsulfoxide (DMSO)  with 10% fetal calf serum (FCS). At 80% confluence they were split into 6-well plates with DMEM containing the functionalized carbohydrates (Acaddition –16. The e (DMSO) . After oe (DMSO) . In both4GlcNAc 16 as well as the new substrate Neu5Hex 3 was successful. The copper-catalyzed [3+2] triazole formation (“click reaction”) proved very useful for the cell surface labelling because of its bioorthogonality. The incubation of HEp-2 cells with the sialic acid analogue Neu5Hex 3 guarantees its direct incorporation into the cell surface glycan patterns bypassing metabolic bottlenecks. Furthermore, the described genetic feedback inhibition by sialic acid leading to an accumulation of the fed Neu5Hex 3 ensures an efficient integration into the cell surface glycocalyx. A drawback of the reaction parameters and compounds used for the click reaction is the cytotoxicity of DMSO and copper. But this problem for in vivo labelling can be overcome by different reaction conditions and different detection molecules. For example, the strain-promoted click reaction with difluorinated cyclooctyne (DIFO) and cell-surface azido-glycans introduced recently has been proven to be suitable for in vivo labelling D20 : +19.8 ; 1H NMR  δ = 6.05 , 5.27 , 5.22 , 4.56 , 3.84–3.50 , 2.50 , 2.30–2.20 , 1.90–1.70 ; 13C NMR:  δ = 175.29 (CO), 137.04 (C-1”), 116.65 (C-2”), 84.12 (C-5’), 72.46, 72.11, 71.55 , 70.34 (C-6’), 64.94 (C-5), 55.11 (C-1), 35.89 (C-2’), 25.77 (C-3’), 18.58 (C-4’); MS (ESI): m/z [M+Na]+ calculated for C13H20NO5[Na]+, 294.14, found 294.1.A solution of D-arabinose  and nitrone  in dioxane (100 mL) were stirred at 30 °C for 14 d. After complete conversion of the starting material as monitored by TLC, the solvent was removed at reduced pressure. The residue was purified by normal silica gel chromatography  to afford the ester as colourless oil  in 82% yield. Rf = 0.42 ; [α]D20 = +7.2 ; 1H NMR  δ = 4.66–4.61 , 4.14–4.08 , 3.91–3.87 , 3.86–3.47 , 3.32–3.28 , 3.23–3.20 , 2.65–2.57 , 2.40–2.34 , 2.18–2.06 , 1.79–1.71 , 1.20–1.15 , 1.05 3); 13C NMR  δ = 177.07 (CONH), 174.22 (COO), 84.19 (C-5’), 77.34 (C-5), 72.15, 71.42, 70.63 , 70.35 (C-6’), 65.27 (C-5’’), 62.50 (OCH2CH3), 62.23 (C-3), 61.21 (C(CH3)3, 53.71 (C-1’’), 39.07 (C-4), 35.92 (C-2’), 25.91 (C-4’), 25.91 (C(CH3)3), 25.85 (C-3’), 14.45 (OCH2CH3); MS (ESI): m/z [M+Na]+ calculated for C21H36N2O8[Na]+ 467.2, found 467.2.Polyhydroxy olefin  in 46% yield. Rf = 0.29 ; [α]D20 = -19.04 ; 1H NMR , β-anomer: δ = ppm 4.09–4.02 , 4.03–4.00 , 3.87–3.81 , 3.81–3.79 , 3.74–3.89 , 3.64–3.60 , 3.52–3.49 , 3.23–3.20 , 2.42–2.38 , 2.26–2.20 , 2.17–2.11 , 1.86–1.80 ; 13C NMR , δ = 177.00 (2 × CONH), 173.49 (COOH), 96.49 (C-1), 84.05 (C-5’), 72.03 (C-8), 71.55 (C-6), 70.08 (C-7), 70.03 (C-6’), 67.63 (C-4), 64.68 (C-9), 53.94 (C-5), 40.94 (C-3), 35.67 (C-2’), 25.64 (C-3’), 18.49 (C-4’); MS (ESI): m/z [M-H]- calculated for C14H21NO9[H]- 360.13, found 360.2.Isoxazoline  and NaOMe (0.74 mL of 5.4 M solution in MeOH) in anhydrous MeOH (100 mL) were stirred at room temperature overnight. Water (100 mL) was added and the solution stirred for further 24 h. The mixture was then neutralized with acidic ion exchange resin containing formate ions (Amberlyte). The solvent was removed under reduced pressure and the crude product subjected for size exclusion chromatography with Biogel P2 (Bio-Rad) to afford pure Rf = 0.34 (EtOAc), 1H NMR , δ = 8.18 , 7.65 , 7.25 , 6.90 , 6.82 , 6.78 , 6.68 , 6.47 , 6.38 , 4.10 , 4.01 , 3.47 , 2.99 , 2.03, 1.55 . 13C NMR , δ = 185.56 (C-3’’’), 165.23 (C-1’), 163.10 (C-6’’’), 158.70 (C-4a’’’), 154.12 (C-5a’’’), 149.67 (C-9a’’’), 134.15 (C-2), 132.78, 131.31, 130.49, 130.11, 130.08, 129.72, 128.90 , 130.27 (C-9’’’), 117.71, 114.91 , 113.55 (C-5’’’), 105.88 (C-2’’’), 100.93 (C-4’’’), 65.38 (C-1’’’’), 62.37 (C-1’’), 47.92 (C-3’’’’), 47.77 (C-3’’), 28.45 (C-2’’’’), 27.76 (C-2’’).Iodopropyl azide  was added to a solution of fluorescein  in a mixture of distilled THF/MeOH  and the reaction mixture stirred overnight. The crude mixture was concentrated under reduced pressure, diluted with water and extracted with EtOAc (3 × 25 mL). The combined organic layers were dried, concentrated followed and purified by flash chromatography to afford the pure required product  in 84% yield. 2 atmosphere. At 80% confluence the medium was discarded and the cells washed with PBS buffer (Gibco). After the addition of 1.5 ml of a trypsin/EDTA mixture, the cells were detached for 5 min at 37 °C. They were supplied with 8.5 ml of fresh medium and split in a ratio of 1:10.Human larynx carcinoma (HEp-2) cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) at 37 °C under a 5% CO4GlcNAz 16 or Neu5Hex 3). The incubation time was 48 hours. The cells were detached using a cell scraper in order to retain the glycocalyx. 150 μL from each well was transferred into an 8-well microscopy cultivation slide and filled with 150 μL of the fresh medium. The cells were cultivated at the described growth conditions until reattachment. The medium was discarded and the cells were washed several times with PBS buffer (Gibco). The labelling reaction was performed in the dark with 2 mM of the complementary labelling molecule 9-[2-carboxy-4-[(2-propyn-1-ylamino)carbonyl]phenyl]-3,6-bis(dimethylamino)xanthylium, alkynylated TAMRA or azido-fluorescein 14) with 2 mM CuSO4, 10 mM sodium ascorbate and 2 mM Tris-[ methyl]amine (TBTA) in DMSO. After 1 h each well was washed several times with DMSO/water (1:1) and subsequently examined by fluorescence microscopy.For the metabolic labelling, HEp-2 cells were cultivated as described above. Subsequently, at 80% confluence they were seeded into 6-well dishes and incubated in 2 ml of the medium described above. The medium contained 25 μM of the modified carbohydrate to be incorporated (Ac


The structures of two products (R = C2H5O and n-C3H7O) were established by single crystal X-ray diffraction. The corresponding oxetane carrying the bulky t-C4H9O group has different reactivity towards BF3·OEt2, slowly producing a mixture of two acyclic, unsaturated products.Treatment of 2,2-bis(trifluoromethyl)-4-R-oxetanes (R = C3)2C(OH)CH2CH(OR)OR′.Clean and spontaneous reaction with alcohols is another interesting transformation of oxetanes described in this paper. The reaction leads to high yield formation of the corresponding acetals  have different reactivity towards electrophiles. They are totally inert to the action of BF3·OEt2 and rapidly react with a protic acid (H2SO4) forming the same product, 3,3,7,7-tetrakis(trifluoromethyl)-9-oxa-2,6-dithia-bicyclo[3.3.1]nonane in 35–50% yield. The structure of this product was established by single crystal X-ray diffraction.Structurally related 2,2-bis(trifluoromethyl)-4-R-thietanes (R =  Based on a combination of 1H, 19F NMR, mass spectrometry and elemental analysis data, the structure of 4--2,2-bis(trifluoromethyl)thietane 5a was proposed for that product nonane (5).Protonation of the thietane sulfur of 2H5, n-C3H7, n-C4H9) with BF3·OEt2 results in the fast formation of the corresponding 2,2,6,6-tetrakis(trifluoromethyl)-1,5-dioxocanes 2a–c, the corresponding thietanes 4b, c  are inert towards this Lewis acid, but rapidly react with concentrated H2SO4 with the formation of bicyclic 3,3,7,7-tetrakis(trifluoromethyl)-9-oxa-2,6-dithia-bicyclo[3.3.1]nonane 5. Reinvestigation of a previously reported reaction of 4c, (R = C2H5) with H2SO4, led us to the conclusion, that the product formed in this process has the structure 3,3,7,7-tetrakis(trifluoromethyl)-9-oxa-2,6-dithia-bicyclo[3.3.1]nonane (5), rather than 4--2,2-bis(trifluoromethyl)-thietane (5a) as proposed earlier [Despite the structural similarity, 2,2-bis(trifluoromethyl)-4-alkoxy-oxetanes and thietanes have very different reactivity towards Lewis and protic acids. While the reaction of 2,2-bis(trifluoromethyl)-4-R-oxetanes (R = CFile 12a–2e, 3a–3e and 5.Experimental details and analytical data for compounds File 22a, 2b, 2d and 5.X-ray data for compounds


The synthesized compounds were screened for their in vitro growth inhibiting activity against different strains of bacteria and fungi viz., Staphylococcus aureus, Bacillus subtilis, Bacillus megaterium, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Candida albicans, Aspergillus niger and Aspergillus flavus were compared with the standard antibiotics such as chloramphenicol (50 μg/ml) and griseofulvin (50 μg/ml) using well agar diffusion technique. Compounds 3e, 3g, 3h and 3m exhibits highest antibacterial activity and compounds 3d, 3g and 3h showed better antifungal activity. The synthesized compounds (3a-o) were screened for their in vitro antiinflammatory activity against carrageenan-induced rat paw oedema. Compounds 3f and 3i were found to be most active compound of this series, which shows 46.42% and 50% inflammation inhibitory activity, whereas standard drug phenylbutazone exhibit 53.57% antiinflammatory activity at a dose of 50 mg/kg po.In the present study, 2,5-disubstituted-1,3,4-oxadiazoles (3a-o) have been synthesized by the condensation of 4-methoxybenzohydrazide (1) with different aromatic acids (2a-o) in presence of phosphoryl chloride. The structural assignment of this compound (3a-o) has been made on the basis of elemental analysis, UV, IR,  In order to reduce or mask the side effects of carboxylic moiety we planned to synthesize various 2,5-disubstituted-1,3,4-oxadiazole via the condensation of 4-methoxybenzohydrazide with various aromatic acids in presence of phosphoryl chloride respectively in the hope of getting potent biodynamic agents and evaluate their antimicrobial and antiinflammatory activity.254) with Ethyl acetate: acetone (9:1) using iodine vapours and UV light as detecting agents and the Rf value were given below. Melting points were measured on open capillaries in a liquid paraffin bath and are uncorrected. The absorbance maxima (λmax) were determined on a Systronics UV-Vis double beam spectrophotometer (2201) in ethanol. IR Spectra were taken on a Perkin Elmer Spectrum RX I, FTIR Spectrophotometer using potassium bromide pellets. 1H NMR spectra were recorded in DMSO-d6 on AMX-400, NMR spectrometer using TMS as an internal standard . FAB mass spectra were taken out on a JEOL SX102/DA-6600 mass spectrometer using Argon/Xenon  as the FAB gas. Elemental analysis was obtained on a Carlo Erba 1108 Heraeus elemental analyzer. All the chemicals used were of synthetic and AR grade and were procured from Alfa Aesar (4-methoxybenzohydrazide), USA, S.D. Fine Chem. Ltd and Merck, Mumbai, India.The identification and purity of the products were checked by TLC (Merck Silica-60F254) using ethyl acetate: acetone (9:1) as eluent. The reaction mixture was cooled and poured on to crushed ice (∼200 g) with continuous stirring. The solid mass separated was neutralized with sodium bicarbonate solution (10% w/v). The resulting solid thus obtained was collected by filtration, washed well with cold water, dried in vacuum and recrystallized from absolute ethanol (95%) and analyzed. Adopting the above procedure fifteen different 2,5-disubstituted-1,3,4-oxadiazoles (3a-o) were synthesized and their characterization data are presented in A mixture of different aromatic acid(s) (0.01 mol) with 4-methoxybenzohydrazide  in phosphoryl chloride (15 ml) was refluxed over a steam bath for 5-6 h. The progress of the reaction was monitored by TLC (Merck Silica-60F−1): 3027 (aromatic C-H), 1602, 1493, 1456 (aromatic C = C), 1650 (C = N), 1210 (asymmetric C-O-C), 1028 (symmetric C-O-C), 2962 , 2872 ; 1H NMR : 6.87-7.0 , 7.21-7.38 , 3.74 ; MS (FAB) m/z: 252 (M+), 253  for C15H12N2O2.Yield: 69.37% (1.75 g); mp: 221°; Rf value: 0.69; UV : 348.3; IR : 3040 (aromatic C-H), 1595, 1499, 1472 (aromatic C = C), 1640 (C = N), 1250 (asymmetric C-O-C), 1032 (symmetric C-O-C), 2950 , 2859 , 750 (out-of-plane aromatic C-H bend); 1H NMR : 6.87-7.10 , 7.27-7.48 , 2.37 , 3.76 ; MS (FAB) m/z: 266 (M+), 267  for C16H14N2O2.Yield: 78.86% (2.1 g); mp: 229°; Rf value: 0.84; UV : 276.2; IR : 3055 (aromatic C-H), 1604, 1493, 1464 (aromatic C = C), 1668 (C = N), 2972 , 2840 , 1249 (asymmetric C-O-C), 1040 (symmetric C-O-C), 3048 , 1640 , 991 ; 1H NMR : 6.82-7.12 , 7.21-7.38 , 3.9 , 4.82-5.92 ; MS (FAB) m/z: 278 (M+), 279  for C17H14N2O2.Yield: 70.09% (1.95 g); mp: 231°; Rf value: 0.78; UV : 267.2; IR : 3048 (aromatic C-H), 1598, 1494, 1474 (aromatic C = C), 1654 (C = N), 1252 (asymmetric C-O-C), 1047 (symmetric C-O-C), 3510 , 3421 , 1292 , 2965 , 2874 ; 1H NMR : 6.85-7.07 , 7.29-7.48 , 3.82 , 4.48 ; MS (FAB) m/z: 267 (M+), 268  for C15H13N3O2.Yield: 80.44% (2.15 g); mp: 202°; Rf value: 0.82; UV : 278.2; IR : 3054 (aromatic C-H), 1603, 1498, 1470 (aromatic C = C), 1646 (C = N), 1247 (asymmetric C-O-C), 1046 (symmetric C-O-C), 1523 , 1347 , 852 , 2958 , 2843 ; 1H NMR : 6.83-7.0 , 7.44-7.48 , 3.81 ; MS (FAB) m/z: 297  for C15H11N3O4.Yield: 65.60% (1.95 g); mp: 235°; Rf value: 0.69; UV : 273.2; IR : 3048 (aromatic C-H), 1609, 1492, 1462 (aromatic C = C ring), 1648 (C = N), 1258 (asymmetric C-O-C), 1052 (symmetric C-O-C), 1538 , 1350 , 856 , 2963 , 2874 ; 1H NMR : 6.85-7.17 , 7.31-7.52 , 3.78 ; MS (FAB) m/z: 342  for C15H10N4O6.Yield: 82.39% (2.82 g); mp: 258°; Rf value: 0.78; UV : 282.0; IR : 3045 (aromatic C-H), 1596, 1496, 1472 (aromatic C = C), 1672 (C = N), 1260 (asymmetric C-O-C), 1050 (symmetric C-O-C), 1530 , 1348 , 854 , 3332 , 1310 , 2950 , 2836 ; 1H NMR : 6.86-7.0 , 7.21-7.48 , 3.80 , 2.18 ; MS (FAB) m/z: 433 (M+), 434  for C21H15N5O6.Yield: 87.68% (3.8 g); mp: 249°; Rf value: 0.69; UV : 268.4; IR : 3038 (aromatic C-H), 1596, 1492, 1458 (aromatic C = C), 1636 (C = N), 1246 (asymmetric C-O-C), 1046 (symmetric C-O-C), 1520 , 1342 , 854 , 3332 , 1298 , 2964 , 2870 ; 1H NMR : 6.89-7.14 , 7.20-7.52 , 3.78 , 2.21 ; MS (FAB) m/z: 388  for C21H16N4O4.Yield: 75.18% (2.92 g); mp: 224°; Rf value: 0.75; UV : 285.2; IR : 3065 (aromatic C-H), 1607, 1496, 1465 (aromatic C = C), 1654 (C = N), 1245 (asymmetric C-O-C), 1040 (symmetric C-O-C), 3430 , 1644 , 2968 , 2875 ; 1H NMR : 6.87-7.10 , 7.18-7.48 , 3.86 , 8.51 ; MS (FAB) m/z: 371 (M+), 372  for C22H17N3O3.Yield: 71.35% (2.65 g); mp: 242°; Rf value: 0.68; UV : 288.0; IR : 3030 (aromatic C-H), 3598 (O-H), 1224 (C-O), 1605, 1495, 1465 (aromatic C = C), 1645 (C = N), 1252 (asymmetric C-O-C), 1049 (symmetric C-O-C), 2960 , 2869 , 750 (out-of-plane aromatic C-H bend), 1361 (in-plane O-H bend); 1H NMR : 6.87-7.13 , 3.52 , 7.17-7.42 , 10.44 ; MS (FAB) m/z: 268 (M+), 269  for C15H12N2O3.Yield: 75.27% (2.125 g); mp: 279°; Rf value: 0.79; UV : 270.0; IR : 3045 (aromatic C-H), 1219 (C-O), 1608, 1492, 1459 (aromatic C = C), 1634 (C = N), 1242 (asymmetric C-O-C), 1021 (symmetric C-O-C), 2963 , 2872 ; 1H NMR : 6.37-6.92 , 7.18-7.42 , 3.9 ; MS (FAB) m/z: 342 (M+), 343  for C18H18N2O5.Yield: 77.48% (2.65 g); mp: 252°; Rf value: 0.81; UV : 310.2; IR : 3040 (aromatic C-H), 1601, 1492, 1473 (aromatic C = C), 1642 (C = N), 1251 (asymmetric C-O-C), 1045 (symmetric C-O-C), 2960 , 2869 , 748 (out-of-plane aromatic C-H bend), 690 (out-of-plane ring C = C bend); 1H NMR : 6.92-7.12 , 7.70-7.75 , 3.85 ; MS (FAB) m/z: 239 , 240 (M + 1)+ for C14H11N3O2.Yield: 73.05% (1.85 g); mp: 240°; Rf value: 0.79; UV : 285.4; IR : 3038 (aromatic C-H), 3602 (O-H), 1224 (C-O), 1606, 1446, 1466 (aromatic C = C), 1645 (C = N), 1249 (asymmetric C-O-C), 1035 (symmetric C-O-C), 2965 , 2876 , 1224 (C-O), 780 (out-of-plane aromatic C-H bend), 689 (out-of-plane ring C = C bend); 1H NMR : 6.87-7.11 , 7.17-7.38 , 10.7 , 3.86 ; MS (FAB) m/z: 284 (M+), 285  for C15H12N2O4.Yield: 68.59% (1.95 g); mp: 287°; Rf value: 0.80; UV : 281.6; IR : 3045 (aromatic C-H), 1602, 1497, 1470 (aromatic C = C), 1647 (C = N), 1250 (asymmetric C-O-C), 1041 (symmetric C-O-C), 3522 , 3416 , 2968 , 2877 , 1294 ; 1H NMR : 6.92-7.21 , 7.27-7.52 , 3.81 , 4.41 ; MS (FAB) m/z: 267 (M+), 268  for C15H13N3O2.Yield: 71.08% (1.90 g); mp: 217°; Rf value: 0.81; UV : 292.0; IR : 3050 (aromatic C-H), 3606 (O-H), 1226 (C-O), 1595, 1499, 1470 (aromatic C = C), 1652 (C = N), 1247 (asymmetric C-O-C), 1040 (symmetric C-O-C), 2962 , 2872 , 1378 , 1362 (in-plane O-H bend), 692 (out-of-plane ring C = C bend); 1H NMR : 6.42-6.92 , 6.97-7.21 , 10.28 , 2.42  3.82 ; MS (FAB) m/z: 282 (M+), 283  for C16H14N2O3.Yield: 74.39% (2.1 g); mp: 261°; Rf value: 0.72; UV : 291.0; IR , Staphylococcus aureus (ATCC 25923) and Bacillus megaterium (ATCC 1327) for gram positive and Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Shigella dysenteriae (ATCC 13313) for gram negative and for fungal strain viz., Candida albicans (ATCC 10231), Aspergillus niger (ATCC 16404) and Aspergillus flavus (ATCC 22547).The antimicrobial activity of all the newly synthesized compounds were determined by well plate methodThe compounds were tested at a concentration of 100 μg/ml (or) 0.00001 nanomoles were prepared in dimethylformamide (DMF). The petridishes used for antibacterial screening were incubated at 37 ± 1° for 24 h, while those used for antifungal activity were incubated at 28° for 48-72 h. The diameters of zone of inhibition (mm) surrounding each of the wells were recorded.The results were compared to chloramphenicol (50 μg/ml (or) 0.000005 nanomoles) and griseofulvin (50 μg/ml (or) 0.000005 nanomoles) for antibacterial and antifungal activity. The antibacterial and antifungal screening results were presented in 50 of the drug was found to be 500 mg/kg body wt. One tenth of this dose was selected as the therapeutic dose for evaluation (i.e. 50 mg/kg).Acute toxicity study was carried out by “Stair case” methodet al.ad libitum. The animals were acclimatized for one week under laboratory conditions before performing the test. They were housed in polypropylene cages under standard conditions . The standard groups received phenylbutazone 50 mg/kg body weight po, suspended in 1% w/v of carboxymethylcellulose (CMC) in distilled water. The test group received synthesized compounds (3a-o) . The control group received corresponding amount of vehicle (1% w/v of CMC). All the test compounds and standard drug were administered 30 min prior to carrageenan injection. The antiinflammatory activity of synthesized compounds (3a-o) was carried out in Periyar College of Pharmaceutical Sciences for Girls, Tiruchirappalli 21, Tamilnadu. Before performing these experiments, ethical clearance was obtained from Institutional Animal Ethics Committee and conducted according to the Indian National Science Academy guidelines for the use and care of experimental animals (CPCSEA Reg No. 418).Antiinflammatory activity was determined by carrageenan-induced rat paw method of winter Acute edema was induced in the right hind paw of rats by injecting 0.1 ml of freshly prepared 1% w/v of aqueous solution of carrageenan  in the subplanter region of right hind paw. After the carrageenan injection the paw volume was measured before and after 1, 2 and 3 h by plethysmometer . The difference between the left and right paw was taken as a measure of oedema. Any significant reduction in the volume of the paw compared to the control group was considered as anti-inflammatory response1H NMR and MS) and analytical data are in good agreement with their structures.2,5-Disubstituted-1,3,4-oxadiazole (3a-o) was synthesized by the condensation of 4-methoxybenzohydrazide with various aromatic acids in presence of phosphoryl chloride . The phy50 of the drug was found to be 500 mg/kg body wt. The therapeutic dose of the drug is considered as 1/10th of the LD50 value. Screening results of antimicrobial activity reveal  with the standard. Moreover, compounds 3d, 3g, 3j and 3o show less significant anti-inflammatory activity (p < 0.01). Carrageenan-induced paw edema was taken as a prototype of exudative phase of inflammation. The development of edema has been described as biphasic. The initial phase is due to the release of histamine, serotonins, 5-hydroxy tryptamine and kinins in the first hour after injection of carrageenan. More pronounced second phase is related to the release of prostaglandinThe results in


Their structures were elucidated by combined spectroscopic methods, and the structure of 1 was further confirmed by single-crystal X-ray data.Three new phenolic bisabolane-type sesquiterpenoids: (+)-methyl sydowate ( Aspergillus has been known to be a major contributor to the secondary metabolites of marine fungal origin, for example, four sesquiterpenoids with a unique nitrobenzoyl ester from Aspergillus versicolor + m/z of 278.1500 . The IR absorption bands indicated the existence of hydroxyl  and ester  groups. The 1H-NMR spectrum of 1 showed one exchangeable proton signal at δH 9.26 (s), one ABX spin system assignable to a 1,3,4-trisubstitued benzene ring at δH 7.10 , 7.48  and 7.50  (13C-NMR spectrum (δC 166.9) and six aromatic carbons. With six degrees of unsaturation accounted for by the molecular formula, the structure of 1 was suggested to contain another ring, in association with a benzene ring and a carboxylic group. The NMR data of 1 were closely related to those of sydowic acid (4), a bisabolane-type sesquiterpenoid previously isolated from a terrestrial fungus Aspergillus sydowi.(+)-Methyl sydowate ( 1.8 Hz) . The 13Cspectrum  displaye1 was the methyl ester of 4. The correlations from H2-8 through H2-9 to H2-10 in the COSY spectrum revealed the CH2-CH2-CH2 subunit in 1. The C-12 and C-13 methyl singlets (δH 0.94 and 1.28) were determined to be germinal and attached to C-11 based on mutual HMBC correlations to each other and correlations from two methyl protons to C-10 and C-11. The connection between C-6 and C7 was established based on the HMBC correlations from H3-14 to C-6 and C-7, and H-5 to C-7. Crystals of 1 suitable for X-ray diffraction were obtained by slow evaporation of solution of 1 in methanol-DMF (1:1). The structure of 1 was further confirmed by a single-crystal X-ray analysis, and its ORTEP plot is depicted in 1 exhibited a positive optical rotation similar to that of (+)-sydowic acid (4) , four methylenes , and one methine proton signal [H-11 (δH 1.51)]. From the 13C-NMR spectrum, one carboxylic carbon (δC 171.2), and eight sp2 carbons were observed. The NMR data of 2 closely resembled those of sydonic acid (5) previously isolated from a terrestrial strain of A. sydowi 25D + 24.7 ; UV (MeOH) λmax 204.0, 238.0, 293.5 nm; IR (KBr) νmax 3230, 2972, 2932, 1719, 1573, 1281, 1201 cm−1; 1H-NMR and 13C-NMR see m/z 278 [M]+ (59), 263 (37), 260 (46), 245 (42), 231 (29), 217 (100), 203 (52), 195 (62), 192 (51), 189 (32), 179 (37), 173 (29), 161 (23), 145 (17), 131 (14), 69 (32); HREIMS m/z [M]+ 278.1500 . Crystallizes in triclinic, space group P-1 with a = 7.0260(13) Å, b = 8.1016(15) Å, c = 13.939(3) Å, α = 87.266(2), β = 77.823(2), γ = 76.502(2)°, C16H22O4, Mr = 278.34, V = 754.2(2) Å3, Z = 2, Dc = 1.226 g/cm3, F(000) = 300, μ = 0.087 mm−1, the final R = 0.0461 and wR = 0.1095 for 5811 observed reflections (I > 2σ (I)). The crystallographic data for 1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC No.738932).7-Deoxy-7,14-didehydrosydonic acid (2): white powder; UV (MeOH) λmax 209.8, 247.4, 300.6 nm; IR (KBr) νmax 3071, 2946, 2860, 1692, 1640, 1533, 1507, 1407, 1288, 1215, 764 cm−1; 1H-NMR and 13C-NMR see m/z [M – H]− 247; HRESIMS m/z [M – H]− 247.1331 .nic acid : white p7-Deoxy-7,8-didehydrosydonic acid (3): white powder; UV (MeOH) λmax 206.3, 253.3, 304.2 nm; IR (KBr) νmax 3104, 3065, 3005, 1699, 1540, 1514, 1447, 1215, 758 cm−1; 1H-NMR and 13C-NMR see m/z [M – H] − 247; HRESIMS m/z [M – H] − 247.1346 .S. aureus and methicillin resistant S. aureus for their inhibitory activity. Antibacterial assays were performed using a modified version of the 2-fold serial dilutions method as Fromtling et al. [The compounds were tested against g et al. .


The other didemnid ascidian Diplosoma sp. contained didemnenones 1, 2 and 5, and five known metabolites 8–12. The structures of 1–7 were elucidated by spectroscopic analyses. Cytotoxicity of the isolated compounds was evaluated against three human cancer cell lines .A series of new C The specimens were extracted with acetone and the extract was partitioned between EtOAc and H2O. The EtOAc extract was suspended in aqueous MeOH and then successively extracted with hexane, CHCl3 and 1-BuOH. The CHCl3-soluble material was subjected to RP-OCC and eluted with H2O/MeOH, MeOH and MeOH/EtOAc. Further separation of the H2O/MeOH fraction by RP-HPLC eluted with a combination of H2O, MeOH and MeCN, led to the isolation of 1 (0.0021%), 2 (0.00066%), 5 (0.00047%), 8 (0.018%), 9/10 , 11 (0.00098%) and 12 (0.00033%).The colonial ascidian 1 by NMR (m/z 233.0786 (M + Na)+, calcd. for C11H14O4Na, 233.0784] provided a molecular formula of C11H14O4. The carbon resonating at δC 197.1 (s) suggested the presence of a carbonyl carbon in 1 and the IR absorption band at vmax 1675 cm−1 further supported the presence of the carbonyl group. Extensive analysis of 1H- and 13C-NMR data (1H-1H COSY data indicated the presence of a cis double bond , a trans double bond , a tetrasubstituted double bond , a methyl group , two oxygenated methylenes  and an oxygenated quaternary carbon [δC 80.5 (s)] in 1. Degrees of unsaturation for these partial structures amount to four. Thus, 1 must be monocyclic to account for the five degrees of unsaturation required by the molecular formula. The connectivity of the aforementioned partial structures was established from the HMBC correlations of H2-1/C-2, H-3/C-6, H-4/C-5, H-4/C-6, H-8/C-11, H3-10/C-7, H3-10/C-8, H3-10/C-9, H2-11/C-6 and H2-11/C-7, as shown in 1. Geometric configuration of two olefins in 1 at C-6/C-7 and C-8/C-9 were assigned to be E by NOEDS experiments +, calcd. for C11H14O4Na, 233.0784]. The IR absorption bands at vmax 1695 and 3360 cm−1 indicated the presence of carbonyl and hydroxyl groups. The NMR data  was at higher field than in 1 (δH 7.73) due to the magnetic anisotropy effect of the carbonyl group, suggesting a Z configuration for the C-6 olefin in 2. This was confirmed by NOEDS experiments +, calcd for C11H12O4Na, 231.0628] for 3 provided a molecular formula C11H12O4, which suggested six degrees of unsaturation. The carbon resonating at δC 203.8 (s) suggested the presence of a carbonyl carbon in 3 and the IR absorption band at vmax 1710 cm−1 further supported the presence of the carbonyl group. The 1H- and 13C-NMR analyses , 132.8 (d), 165.1 (d); δH 6.28 (d), 7.52 (d)], a conjugated diene moiety , an acetal , a methine , an oxygenated methylene  and an oxygenated quaternary carbon [δC 79.7 (s)] in 3. Extensive analysis of 1H-1H COSY demonstrated two isolated spin systems, C-3–C-4 and C-8–C-10. The connectivity of the aforementioned partial structures was established from the HMBC correlations of H2-1/C-2, H2-1/C-3, H2-1/C-6, H2-1/C-11, H-3/C-2, H-3/C-4, H-3/C-5, H-3/C-6, H-6/C-2, H-6/C-4, H-6/C-5, H-6/C-7, H-6/C-11, H-8/C-6, H-8/C-11, H-9/C-7, H-11/C-1, H-11/C-6 and H-11/C-7, to describe the entire carbon framework of 3. Geometric configuration of the olefins in 3 at C-7/C-8 was assigned to be E by NOEDS experiments, in which irradiation of H-6 caused enhancement of H-9 and irradiation of H-8 caused enhancement of H-11 and H-10. Therefore, the planar structure of 3 was concluded to be a class of didemnenone, as depicted in 3. The NOE observed between a hydroxyl proton (OH-2) and H-6 revealed a cis fusion of two rings. The NOEs; OH-2/H-11; H-1b/H-11 allowed the assignment of the H-11 as α.Analysis of the analyses  and 2 co4 was deduced to be C12H14O4 based on HR-ESIMS . The 1H- and 13C-NMR spectral data  and a carbon signal at δC 54.9 (q) in 4. Geometric configuration of the double bond at C-7/C-8 in 4 was assigned to be E by NOEDS experiments, in which irradiation of H-6 caused enhancement of H-9 and irradiation of H-8 caused enhancement of H-11 and H-10. Therefore, the planar structure of 4 was elucidated to be a methylacetal of 3. The NOE between a hydroxyl proton (OH-2) and H-6 also indicated the ring junction to have the cis-geometry. The NOEs; OH-2/ H-1b; H-1a /H-11 allowed the assignment of the H-11 as β. We cannot affirm that 4 is a natural product, because it is conceivable it arises from 3 in the isolation process.The molecular formula of ral data  and 2 of5 was determined to be C11H10O4, based on HR-ESIMS . The carbons resonating at δC 202.9 (s) and 166.9 (s) suggested the presence of a carbonyl carbon and an ester carbonyl carbon, respectively in 5  and H-6 allowed the ring junction to be assigned as cis.The molecular formula of ely in 5  and the  13C-NMR  and 2 an13C-NMR and HR-ESIMS  for 6 provided a molecular formula C11H14O5. The carbon resonating at δC 206.1 (s) suggested the presence of a carbonyl carbon in 6 , 135.3 (d); δH 7.27 , 6.31 ], a tetrasubstituted double bond , an oxygenated methylene , two oxygenated methines , an oxygenated quaternary carbon [δC 73.0 (s)], a methylene  and a methyl  in 6. These functionalities accounted for three of the five degrees of unsaturation, therefore 6 is bicyclic. The connectivity of the aforementioned partial structures was established from the HMBC correlations of H-1/C-2, H-1/C-6, H-1/C-11, H-4/C-5, H-4/C-6, H-8/C-11 and H-11/C-6, to describe the entire carbon framework of 6. The NOE between H-9 and H-11 also indicated the double bond at C-8 to have the trans-geometry, and the NOEs; OH-2/H-1a; OH-2/H-3; H-1a/H-11 allowed the assignment of the 3-OH and the 11-OH both as β.Analysis of the bon in 6  and the NMR data  and 2 su7 by 13C-NMR (m/z 401.1602 (M + H)+, calcd for C22H25O7, 401.1595] provided a molecular formula of C22H24O7. Extensive analysis of 1H- , 132.8 (d), 165.1 (d); δH 6.31 (d), 7.55 (d) and δC 196.7 (s), 134.9 (d), 161.6 (d); δH 6.28 (d), 7.37(d)], a conjugated diene moiety , a trans double bond , a tetrasubstituted double bond , an acetal , three oxygenated methylenes , two oxygenated quaternary carbons , a methine  and a methyl  in 7. The structure of 7 was elucidated to be a dimeric didemnenone composed of 1 and 3, from the molecular formula, the NMR data and the HMBC correlations of H-11/C-11′ and H2-11′/C-11. Geometric configuration of three olefins in 7 at C-7/C-8, C-6′/C-7′ and C-8′/C-9′ was assigned to be E by NOEDS experiments, in which irradiation of H-6 caused enhancement of H-9, irradiation of H-8 caused enhancement of H-11 and H-10, irradiation of H-9′ caused enhancement of H-11′ and irradiation of H-3′ resulted in enhancement of the H-1′ proton signal. Therefore, the structure of 7 was established as a didemnenone dimer as shown in 7. We could not determine the C-2/C-6 ring junction stereochemistry owing to decomposition of 7.Analysis of  13C-NMR  and HR-Es of 1H-  and 13C-8 was determined to be 4-amino-7-(5’-deoxy-β-d-xylofuranosyl) -5-iodopyrrolopyrimidine by 1D and 2D NMR spectra for 8 and 23, and by CD spectra of compounds 8, 24 and 25 (1–7 based on the assumption that there is a similar biogenetic relationship between these compounds and (+)-didemnenone A. (+)Didemnenones 9–12 and inosine (13) were unambiguously identified by comparison of their spectral data with those described in the literature .,20.11 co8 and 19  from unr11 compounds are derived from polyketides (six acetates-C1) or polyketides (five acetates + C1) . Inversed-detected heteronuclear correlations were measured using HMQC and HMBC pulse sequences with a pulse field gradient. HR-ESIMS data were obtained on a LTQ ORBITRAP , and HR-FABMS data were obtained on a JEOL JMS-700 mass spectrometer. LR-ESIMS data were measured on a Waters Quattro micro API triple quadruple mass analyzer. RP-OCC and RP-MPLC were performed on COSMOSIL® 140C18-OPN. Preparative RP-HPLC was run on a Waters 600 multi solvent system using ODS columns . All solvents used were reagent grade.Optical rotations were measured on either a JASCO P-1020 or JASCO DIP-1000 polarimeter. Ultraviolet-visible spectra were obtained in methanol on a JASCO V-550 spectrophotometer. Infrared spectra were recorded as dry films on either JASCO FT/IR-300 or Spectrum 2000 Explorer (PERKIN ELMER). CD spectra were recorded on a JASCO J-720W Circular Dichroism Spectrometer. 3.2.°C until extraction. The brown ascidian and the green ascidian were identified as Lissoclinum sp. and Diplosoma sp., respectively, by Euichi Hirose, University of the Ryukyus, Japan. The voucher specimens were deposited at the University of the Ryukyus (Specimen no. URKU-801 for the brown ascidian and URKU-802).The colonial brown ascidian was collected by hand at the tidal zone of Tarama island, Okinawa, Japan, and the colonial green ascidian was collected by hand from the coast of Hateruma island, Okinawa, Japan. The ascidians were stored at −15 3.3.Lissoclinum sp.  was initially extracted with acetone (18 L) and filtered to remove debris. The filtrate was concentrated in vacuo to remove acetone and the resultant mixture was partitioned between EtOAc (3.5 L) and H2O (3.5 L). The H2O layer was extracted with 1-BuOH to give the 1-BuOH extract (18.3 g) and the H2O extract (141.2 g). An aliquot of 27.8 g of the H2O-soluble material was subject to RP-MPLC on ODS  with H2O (500 mL), H2O/MeOH  and MeOH (500 mL) to give seven fractions. The second fraction (860 mg) was subjected to HPLC on ODS  to give 18 fractions. The 18th fraction contained pure 1 (66.0 mg). The 14th fraction (84.0 mg) was subjected to HPLC on ODS  to give 13 (7.6 mg). The EtOAc layer was concentrated in vacuo to give a brown material (29.3 g). The material was subjected to a modified Kupchan’s partitioning procedure as follows. The material was suspended in a mixture of H2O/MeOH (1:1) and then successively extracted with hexane and CHCl3. The resultant aqueous phase was concentrated to remove MeOH and then extracted with 1-BuOH. An aliquot of 0.58 g of the BuOH-soluble material (3.93 g) was separated by RP-MPLC  using H2O (500 mL), H2O/MeOH  and MeOH (500 mL) to give seven fractions (fr. 1–7). The first fraction  was purified by RP-HPLC  to afford 3 (165 mg) and 4 (3.2 mg). The second fraction  was separated by RP-HPLC  to give 18 fractions (fr. 2-1–2-18). Purification of the second fraction  by RP-HPLC  using H2O/MeOH (9:1) led to the isolation of 6 (24.6 mg), 2 (92.3 mg), 1 (92.7 mg) and an inseparable mixture of didemnenones A/B . HPLC separation  of seventh fraction  led to the isolation of 12 (0.8 mg). An aliquot of 3.35 g of the 1-BuOH extract (3.93 g) was chromatographed on ODS  with H2O/MeOH  and MeOH (100 mL) to give nine fractions. The second fraction (1.1 g) was purified by HPLC on ODS , to give 1 (186.8 mg), 5 (14.6 mg), 4 (29.7 mg) and 7 (0.9 mg).The ascidian Diplosoma sp.  was initially extracted with acetone (2.2 L). After filtration, the extracts were concentrated in vacuo to give an acetone extract. The acetone extract was partitioned between EtOAc and H2O. The H2O layer was further extracted with 1-BuOH. The 1-BuOH layer was concentrated in vacuo to give a BuOH-soluble material (2.14 g). The EtOAc layer was concentrated in vacuo to give an EtOAc extract (7 g). The EtOAc extract was suspended in H2O/MeOH  and then successively extracted with hexane and CHCl3 to give a hexane-soluble material (4.0 g), a CHCl3-soluble material (2.0 g) and an aqueous material (2.1 g). The aqueous material and the BuOH-soluble material (2.14 g) were combined. The combined polar fraction (4.24 g) was chromatographed on ODS (COSMOSIL® 140C18-OPN) with H2O/MeOH , MeOH and MeOH/EtOAc (9:1) as eluent. The first fraction (2.0 g) contained more than 95% of 1 from its 1H NMR data. The CHCl3-soluble material (2.0 g) was subjected to OCC on ODS . The first fraction  was further separated by OCC on ODS (COSMOSIL® 140C18-OPN) using H2O/MeOH , MeOH and MeOH/EtOAc (9:1). The first aqueous methanol fraction  was subjected to HPLC on ODS  using H2O/MeOH (1:1) to give six fractions (fr. 1-1-1-fr. 1-1-6). The first fraction  was separated by HPLC on ODS  first, and then by RP-HPLC  to yield 1 (18.7 mg) and 2 (5.9 mg). Further separation of the second fraction  by repeated HPLC  to give 5 (4.2 mg). An inseparable mixture (6.2 mg) of didemnenones A and B (9 and 10) was obtained along with their methylacetals 11 (8.8 mg) and 12 (3.0 mg) from the fourth fraction  by repeated ODS HPLC . The fifth fraction  was purified by RP-HPLC  with H2O/MeOH/MeCN (50:31:19) to afford 8 (9.9 mg). The sixth fraction  was purified by HPLC on ODS  using MeOH/EtOAc (19:1) to yield 8 (164.4 mg).The ascidian 3.3.1.27D +21.0° ; UV (MeOH) λmax (log ɛ) 246 (3.39), 308 (3.69) nm; FT/IR (film) vmax 3355, 1675, 1035 cm−1; 1H-NMR and 13C-NMR (DMSO-d6) see m/z 211 (M + H)+, 209 (M − H)− HR-ESIMS m/z (M + Na)+ 233.0786 .Pale yellowish oil; [α]3.3.2.27D +22.5° ; UV (MeOH) λmax (log ɛ) 245 (3.38), 307 (3.74) nm; FT/IR (film) vmax 3359, 1712, 1379 cm−1; 1H-NMR and 13C-NMR (DMSO-d6) see m/z 233 (M + Na)+, 211 (M + H)+, 209 (M − H)−; HR-ESIMS m/z (M + Na)+ 233.0786 .Pale yellowish oil; [α]3.3.3.24D +147° ; UV (H2O) λmax (log ɛ) 246 (3.51), 274 (3.54); FT/IR (film) vmax 3400, 1710, 1050 cm−1; 1H-NMR and 13C-NMR (DMSO-d6) see m/z 231 (M + Na)+, 207 (M − H)−; HR-ESIMS m/z (M + Na)+ 231.0632 .Pale yellowish oil; [α]3.3.4.24D +459° ; UV (MeOH) λmax (log ɛ) 249 (3.65); FT/IR (film) vmax 3410, 1700, 1045 cm−1; 1H-NMR and 13C-NMR (DMSO-d6) see m/z 223 (M + H)+; HR-ESIMS m/z (M + Na)+ 245.0794 .Pale yellowish oil; [α]3.3.5.27D + 112° ; UV (MeOH) λmax (log ɛ) 273 (3.83); FT/IR (film) vmax 3419, 1717, 1049 cm−1; 1H-NMR and 13C-NMR (DMSO-d6) see m/z 207 (M + H)+, 205 (M − H)−; HR-ESIMS m/z (M + H)+ 207.0654 .Pale yellowish oil; [α]3.3.6.24D +16.3° ; UV (MeOH) λmax (log ɛ) 294 (3.91); FT/IR (film) vmax 3390, 1720, 1050 cm−1; 1H-NMR and 13C-NMR (DMSO-d6) see m/z 249 (M + H)+, 223 (M + H)+; HR-ESIMS m/z (M + Na)+ 249.0742 .Pale yellowish oil; [α]3.3.7.24D +11.7° ; UV (MeOH) λmax (log ɛ) 248 (3.78), 308 (3.83); FT/IR (film) vmax 3400, 1580, 1050 cm−1; 1H-NMR and 13C-NMR (DMSO-d6) see m/z 423 (M + Na)+, 401 (M + H)+, 399 (M − H)−; HR-ESIMS m/z (M + Na)+ 423.1423 , (M + H)+ 401.1602 .Pale yellowish oil; [λ]3.3.8.26D −69° ; UV (MeOH) λmax (log ɛ) 283 nm (3.59); FT/IR (film) vmax 3461, 3317, 3132, 1633, 1584, 1474, 1084, 755 cm−1; NMR data were described in the previous paper 3.3.9.8,  in 2,2-dimethoxypropane (1 mL) and acetone (2 mL) was added a catalytic amount of camphorsulfonic acid. The mixture was stirred at rt for 24 h and at 45 °C for 4 h. The reaction mixture was diluted with ether, washed with saturated aqueous Na2CO3 and brine. The organic phase was dried (MgSO4) and concentrated in vacuo. The residual oil was purified by preparative TLC [CHCl3-MeOH (3:0.2)] to give the acetal as colorless oil . HR-FABMS and 1H NMR data for compound 23 were described in the earlier paper  to give dibenzoate 25 . Spectral data for compound 25 were described in the earlier paper  dye reduction assay. Test compounds were dissolved in DMSO (4.0 mg/mL) and diluted with H4.1–7 together with four known metabolites 9/10, 12 and 13 from Lissoclinum sp. We have also isolated didemnenones 1, 2 and 5 and five known metabolites 8–12 from the other didemnid ascidian Diplosoma sp. Compounds 1–4, 7–10 and 12 were significantly cytotoxic against the HCT116, A431 and/or A549 cancer cell lines. Didemnenone-related compounds 1–7, 9–12, 14–17 and 20–22, which have been ever isolated from marine organisms [1H-NMR spectra of the extracts of the separated Prochloron spp. from the body of the ascidians Lissoclinum sp. and Diplosoma spp.showed the presence of the same peaks as present in those of didemnenones, which suggested that Prochloron spp. are the actual producers of didemnenones.We have isolated seven new didemnenone derivatives rganisms –22,35,36