Their structures were elucidated using extensive MS, 1D, and 2D NMR spectroscopic analyses as well as by comparing with data in the literature. The absolute configurations of 1 and 2 were determined using TDDFT-ECD calculations. Antimicrobial activity was evaluated. Compound 5 displayed significant activity against methicillin-resistant Staphylococcus aureus strain ATCC 700699 and moderate activity against S. aureus strain ATCC 29213.In this study, we report the isolation of two new meroterpenoids, miniolutelide D ( Nature has granted mankind plenty of drug leads since the beginning of history. Many of these are widely used now in clinical settings and in everyday use to ameliorate various diseases . OriginaFungal endophytes represent outstanding sources of new secondary metabolites spanning different classes of secondary metabolites, including polyketides, steroids, alkaloids, peptides, terpenoids, and quinones, among others, displaying potential biological activities. Although it has been estimated that approximately one million species of endophytic fungi exist, only 5% have been investigated. There are still many fungi yet to be explored that hold many undiscovered treasures ,4,5.Talaromyces purpureogenus, formerly known as Penicillium purpurogenum −  4.07 , 4.23 , and 5.97 , where the former two methines are oxygenated, while the latter is di-oxygenated. Also, three methylene protons were found at δH (ppm) 3.34, 2.93 , 2.89, 2.26 , and 2.39, 2.20 , in addition to six methyl protons at δH (ppm) 1.42 , 1.90 , 1.62 , 1.49 , 1.45 , and 1.38 . The APT spectrum  to δC 168.71 (C-1), and δC 128.97 (C-3), δH 2.89, 2.26 (H2-6) to δC 131.65 (C-4), δC 133.8 (C-5), δC 46.53 (C-7), δC 173.36 (C-8), δC 52.02 (C-12), δC 80.68 (C-22), and δC 18.64 (C-24), δH 4.07 (H-9) to δC 13.31 (C-21), and δC 98.53 (C-23), δH 4.23 (H-13) to δC 133.8 (C-5), and δC 27.91 (C-19), δH 2.39, 2.20 (H2-14) to δC 128.97 (C-3), δC 52.02 (C-12), δC 92.91 (C-13), δC 130.88 (C-15), and δC 85.05 (C-16), δH 1.49 (H3-17) to δC 130.88 (C-15), δC 85.05 (C-16), and δC 27.79 (C-18), δH 1.62 (H3-18) to δC 130.88 (C-15), δC 85.05 (C-16), and δC 27.69 (C-17), δH 1.45 (H3-19) to δC 133.8 (C-5), δC 72.38 (C-11), δC 52.02 (C-12), and δC 92.91 (C-13), δH 1.42 (H3-21) to δC 70.33 (C-9), and δC 99.10 (C-10), δH 5.97 (H-23) to δC 173.36 (C-8), and δC 72.38 (C-11), δH 1.38 (H3-24) to δC 32.51 (C-6), δC 46.53 (C-7), δC 173.36 (C-8), and δC 80.68 (C-22), δH 1.90 (H3-25) to δC 128.97 (C-3), δC 131.65 (C-4), and δC 133.8 (C-5) were observed.By inspection of the COSY spectrum , it was elations  confirme1 showed a close similarity with the previously reported data of miniolutelide C +  4.62 , 4.16 , 2.85 , and 5.43 , where the H-9 and H-13 are oxygenated, while H-23 is di-oxygenated. Three methylene protons were found at δH (ppm) 3.27, 2.83 , 2.94, 2.26 , and 2.66, 2.47 , in addition to seven methyl protons at δH (ppm) 1.24 , 1.71 , 1.56 , 1.48 , 1.07 , 1.39 , and 3.68 . APT spectrum  to δC 169.11 (C-1), and δC 128.57 (C-3), δH 2.94, 2.26 (H2-6) to δC 129.57 (C-4), δC 137.35 (C-5), δC 41.2 (C-7), δC 173.36 (C-8), δC 51.12 (C-12), δC 48.78 (C-22), and δC 21.53 (C-24), δH 4.62 (H-9) to δC 109.34 (C-10), δC 13.26 (C-21), and δC 90.90 (C-23), δH 4.16 (H-13) to δC 137.35 (C-5), δC 109.34 (C-10), and δC 27.12 (C-19), δH 2.66, 2.47 (H2-14) to δC 128.57 (C-3), δC 51.12 (C-12), δC 94.47 (C-13), δC 131.25 (C-15), and δC 85.2 (C-16), δH 1.48 (H3-17) to δC 131.25 (C-15), δC 85.2 (C-16), and δC 27.47 (C-18), δH 1.56 (H3-18) to δC 131.25 (C-15), δC 85.2 (C-16), and δC 27.65 (C-17), δH 1.07 (H3-19) to δC 137.35 (C-5), δC 57.14 (C-11), δC 51.12 (C-12), and δC 94.47 (C-13), δH 1.24 (H3-21) to δC 67.23 (C-9), and δC 109.34 (C-10), δH 2.85 (H-22) to δC 41.2 (C-7), δC 173.36 (C-8), δC 109.34 (C-10), δC 57.14 (C-11), δC 90.90 (C-23), and δC 21.53 (C-24), δH 5.43 (H-23) to δC 41.2 (C-7), δC 67.23 (C-9), δC 57.14 (C-11), and δC 48.78 (C-22), δH 1.39 (H3-24) to δC 42.54 (C-6), δC 41.2 (C-7), δC 173.36 (C-8), and δC 48.78 (C-22), δH 1.71 (H3-25) to δC 128.57 (C-3), δC 129.57 (C-4), and δC 137.35 (C-5), δH 3.68 (H3-26) to δC 169.82 (C-20).The COSY spectrum  revealedelations  confirme2 was found to be the same as that of miniolutelide C [δc 94.47 in 2, while it was located at δc 79.3 in miniolutelide C. The large difference between the chemical shift values suggests that the relative configuration might be different.Hence, the planar structure of telide C . However3 (1H NMR and NOESY spectra were measured in dimethylsulfoxide (DMSO)  instead of (R*). Therefore, the relative configuration was determined as . Thus, 2 is a new metabolite for which the trivial name miniolutelide E (13-epi-miniolutelide C) was given. Key COSY, HMBC, and NOESY correlations are shown in After being measured in CDCl3 , the 1H e (DMSO)  to confi2 was also confirmed by TDDFT-ECD calculations computing for -2. DFT re-optimization of the initial 9 MMFF conformers resulted in a single major conformer with 99.3% Boltzmann population. ECD spectra computed at various levels for this conformer gave acceptable to good agreement with the experimental ECD spectrum -2.The absolute configuration of spectrum , allowinS. aureus ATCC 29213, which causes food poisoning, toxic shock syndrome, and scalded skin syndrome, among other diseases, as well as the Gram-negative bacterial strain E. coli ATCC 25922 known to cause food-borne illnesses [S. aureus ATCC 29213, with an MIC value of 6.3 µg/mL. Subsequently, all isolated compounds 1–6 were tested for antimicrobial activity against S. aureus ATCC 29213, as well as the methicillin-resistant S. aureus (MRSA) strain ATCC 700699. Only compound 5 exhibited considerable activity against S. aureus ATCC 700699 with an MIC of 25 µM, and modest activity against S. aureus ATCC 29213 with an MIC of 100 µM. None of the other compounds showed significant activity at a dose of 100 µM.The emergence and prevalence of antimicrobial resistance are currently depicted as a worldwide public health threat. Most pathogens develop resistance to common antimicrobials, which risk treatment regimens’ failure. Therefore, the urgent need for new antimicrobial agents to combat this serious problem is steadily increasing ,27. Inhillnesses . The extM. tuberculosis, is the major cause of death by an infectious bacterium, accounting for more than a million deaths every year. Although huge efforts are being made by health communities, the morbidity and mortality rates are still high, especially in developing countries, along with the problem of emerging resistant strains, which put great importance in finding new anti-tubercular agents [M. tuberculosis. However, none of them showed any significant activity at 100 µM.Tuberculosis, an infectious disease caused by r agents ,30. Henc254 plates , followed by UV detection at 254 and 365 nm and spraying with vanillin sulphuric acid reagent. All solvents used were priorly distilled, and spectroscopic grade solvents were used for spectroscopic measurements.NMR spectra were recorded on a Bruker Avance III HD 400 MHz NMR spectrometer . LC-MS Shimadzu 8045 spectrometer coupled with photodiode array (PDA) detector (LC-2030/2040) was used to record the mass spectra. HRESIMS spectra were acquired on FTHRMS-Orbitrap  mass spectrometer. Semi-preparative HPLC  was utilized for purification, with Kromasil C-18 RP semi-preparative column (10 mm × 250 mm), with 5 mL/min flow rate, and UV detection at 254 nm with λ max absorption at 220–400 nm. Medium-pressure liquid chromatography was performed using the Puriflash 4125 system, Montlucon, France  coupled with a PDA detector. HiMedia silica gel GRM7484 (0.037–0.063 mm) was utilized for column chromatography. TLC analyses were carried out on precoated silica gel 60 FP. granatum, family Lythraceae. The fruit was cleaned and cut longitudinally. Inside the aseptic area, different fruit parts were cut by sterile blades, washed with distilled water, followed by treatment with 70% ethanol to exclude any contaminants and epiphytes that might arise. The sterile dissected parts were inoculated aseptically on malt extract agar medium (MEA). Plates were grown at room temperature (25 °C). After successive purification steps, the pure fungal strain was successfully isolated from the tissues of the P. granatum fruit. The same fungal strain was simultaneously isolated from the mesocarp, calyx, and exocarp, which confirms its indigeneity in P. granatum fruits. The fungus was identified as T. purpureogenus according to a molecular biological protocol using DNA amplification and sequencing of the ITS region [Pomegranate fruit collected in Cairo, Egypt, in the summer of 2019 was identified and authenticated as S region . The obtSolid rice medium was prepared in 22 1-L Erlenmeyer flasks, each containing 100 g of commercial rice and 110 mL of water. The solid rice medium was autoclaved at 121 °C for 20 min prior to fungal fermentation. The fungal strain was cultivated on the rice medium for 7 weeks at room temperature. Afterwards, fungal biomass was extracted using 700 mL ethyl acetate (EtOAc) for each flask. The flasks were left in static mode overnight. The following day, the flasks were put on electric shakers at 150 rpm for 8 h. Filtration and evaporation steps were followed to obtain a dried extract. The extraction step was run four successive times, yielding 75 g of crude extract.n-hexane and 90% aqueous methanol (aq. MeOH) yielding 40 g of methanolic extract. The obtained methanolic extract was fractionated by vacuum liquid chromatography (VLC) with silica gel 230–400 mesh-packed column. Gradient elution was performed using n-hexane–EtOAc 100:0 to 0:100 and CH2Cl2–MeOH 100:0 to 0:100, collecting 23 fractions (F1–F23). All fractions were evaporated and weighed.The crude extract was partitioned between n-hexane–EtOAc 1:1, was subjected to further chromatographic fractionation by VLC (230–400 mesh), applying the same described technique. This resulted in 12 sub-fractions (F4-1 to F4-12). Sub-fractions F4-5 and F4-6, obtained from 60% and 40% n-hexane, respectively, were combined together based on high similarity in their thin layer chromatography (TLC) pattern and thus named F-4-6c (850 mg). Semi-preparative HPLC was performed on sub-fraction F-4-6c, which led to the isolation of four compounds, 1 (9.2 mg), 3 (17.2 mg), 4 (20.1 mg), and 5 (10.4 mg).Fraction F4 (2.14 g), eluted from n-hexane-EtOAc 8:2, 9:1 and 10:0, respectively, were combined (2.0 g), and subjected to another VLC subfractionation, from which 27 sub-fractions were obtained. Sub-fractions collected from n-hexane-EtOAc 4:6 and 3:7 (twice) F7-9, F7-10, and F7-11 were combined obtaining “F7-9+”, which was subjected to flash chromatography yielding compound 2 (20.0 mg).Fractions 7, 8, and 9 eluted from n-hexane solvent system, which was collected separately from the original fraction F-3. F-3′ (590 mg) was subjected to semi-preparative HPLC to yield 6 (7.4 mg).While running the VLC on the total extract, an orange band F-3′ was observed in a 60% All fractionation and isolation steps were continually tracked by TLCs of isolated fractions, sub-fractions, and compounds in different mobile phase systems.Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 700699, Escherichia coli ATCC 25922, and the minimum inhibitory concentration (MIC) values were measured according to the Clinical and Laboratory Standards Institute (CLSI) recommendations [Mycobacterium tuberculosis H37Rv was determined in 96-well microtiter plates employing the resazurin reduction assay, as described previously [S. aureus and E. coli, whereas rifampicin was used as a positive control for M. tuberculosis. DMSO was used as the solvent control.The broth micro-dilution method was applied to evaluate the antimicrobial activity against ndations . The MICeviously . Moxiflo3 [−1 width at half-height, using dipole-velocity-computed rotational strength values [Mixed torsional/low-frequency mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the MMFF with an implicit solvent model for CHCl3 . Geometr3  with the3 . ECD speh values . Boltzmah values .Talaromyces purpureogenus, derived from Punica granatum fruits, led to the isolation of two new meroterpenoids together with two other known meroterpenoids and two known phenolic compounds. The compounds were identified based on their spectral data and by comparison with previously reported data in the literature. The absolute configuration of newly isolated compounds was confirmed by TDDFT-ECD calculations. Additionally, antimicrobial activity was evaluated. Compound 5 displayed significant activity against the MRSA strain S. aureus ATCC 700699, as well as moderate activity against the drug-susceptible reference strain S. aureus ATCC 29213. Further biological investigations of the isolated compounds from T. purpureogenus are suggested for future research.Chemical investigation of the endophytic fungus


N-thiocyanatosaccharin and the subsequentthiocyanation of a wide range of activated arenes. The procedure wasapplicable for the thiocyanation of biologically active compoundssuch as metaxalone and an estradiol derivative and was used as partof a one-pot tandem iron-catalytic process for the regioselective,dual functionalization of an arene building block.Aryl thiocyanates are flexible synthetic intermediatesthat canbe used in the preparation of a diverse range of arene building blocksfor medicinal chemistry. Here, we report a fast and efficient Lewisacid-catalyzed method for the regioselective thiocyanation of arenes.Iron(III) chloride was found to be an effective Lewis acid for theactivation of  In 1995, Still and co-workers reported N-thiocyanatosuccinimide  preparedfrom N-bromosuccinimide and sodium thiocyanate forthe thiocyanation of (hetero)arenes .8 As well as demonstratingthis as an effective reagent for the thiocyanation of β-ketocarbonyl compounds and oxindoles, N-thiocyanatosaccharin(2) was also shown to functionalize activated aromaticcompounds arenes in moderate to high yields, using triflic acid activationarenes 1a.7 Usinompounds 1b. Phenotivation1c.N-halosuccinimides and the subsequentregioselective halogenation of arenes.11 More recently, we have shown that iron(III) triflimide can activate N-thioaryl succinimides for the preparation of unsymmetricalbiaryl sulfides.12 Based on this previouswork, we proposed that an iron(III) salt may function as an activatorof an N-thiocyanating reagent, allowing efficientthiocyanation of a wide range of arenes, without the requirement ofacidic conditions or long reaction times. Here, we report the useof iron(III) chloride as a Lewis acid catalyst for the activationof N-thiocyanatosaccharin (2) and thesubsequent thiocyanation of various arenes. We also demonstrate theapplication of this method for the late-stage thiocyanation of biologicallyactive compounds and as the key step in a tandem iron-catalytic processfor the one-pot dual functionalization of an arene building block.We have shown that iron(III) salts can act asLewis acids for theactivation of N-Thiocyanatosaccharin(2) was chosenas the electrophilic reagent for this study due to its straightforwardsynthesis and higher reactivity compared to succinimide and phthalimidereagents.8 Initially, 2 was screened forthe thiocyanation of anisole (4a) (2 in thepresence of iron(III) triflimide (2.5 mol %) at a reaction temperatureof 20 °C required a reaction time of 96 h and gave 4-thiocyanatoanisole(5a) in 74% yield (entry 1).13 On increasing the temperature to 40 °C, the reaction was completeafter 2 h and gave 5a in an improved yield of 95% (entry2). Other Lewis acids known to effect electrophilic aromatic substitutionreactions were also screened (entries 3–6). The fastest reactionwas observed using iron(III) chloride (entry 3). With a catalyst loadingof 2.5 mol %, the transformation was complete after 0.5 h and gave 5a in 93% yield. Interestingly, at the same catalyst loading,aluminum(III) chloride gave no product (entry 4), while both silver(I)triflimide and indium(III) triflate gave excellent yields of 5a, although after much longer reaction times (entries 5 and6).14 It should be noted that in the absence ofany Lewis acid, minimal conversion was observed even after an extendedreaction time .ole (4a) 1. Using 15 Standard electron-rich arenes, such as anisoles, phenols,anilines, and indoles, were found to undergo fast transformationswith typical reaction times of 5–30 min. In all cases, onlythe para-substituted products were observed in highyields. Deactivated arenes such as bromobenzene were not tolerated;however, aromatic compounds with electron-deficient substituents butcontaining at least one activating group were good substrates forthe thiocyanation reaction. For example, thiocyanation of anilineswith deactivating groups such as 2-fluoroaniline (4m)and 2-aminobenzophenone (4n) was complete in 15 min,while 2-trifluoromethylaniline (4l) and 2-aminobenzonitrile(4o) required marginally longer reaction times . Under the standard conditions, two compounds,methyl salicylate (4i) and N-Cbz-protectedaniline 4p, were shown to require longer reaction timesof 20 h to achieve complete conversion. Lewis bases such as diarylselenides have been shown to accelerate challenging Lewis acid-catalyzedthioarylations by forming a more reactive cationic intermediate -catalyzed thiocyanation of 4i and 4p significantly improved the reaction times. The thiocyanated salicylate 5i was formed in 70% yield after a 15 min reaction time, while 5p was isolated in 87% yield following a 40 min reaction.It should be noted that for all anilines bearing electron-deficientsubstituents in this study, N-thiocyanated productswere never observed. The extent of the scope of this transformationwas demonstrated by the thiocyanation of minimally activated arenes.Under standard conditions, the reaction with mesitylene (4u) was complete after 0.5 h to give mono-thiocyanated product 5u in 93% yield. With the same catalyst loading, m-xylene (4v) reached 62% conversion after 24 h. In thiscase, an increase of catalyst loading to 10 mol % allowed completeconversion after 2 h and an 88% yield of 5v. While effectivereactions were possible with mesitylene and m-xylene,no reaction was observed with toluene. Using a catalyst loading of10 mol % and standard reaction conditions, no conversion was observedafter 72 h. During the substrate screening process, the scalabilityof the transformation was also investigated. Thiocyanation of anisole(4a) on a one-gram scale using the optimized conditionsproceeded in a similar manner, with completion after 0.5 h and a 95%yield of 4-thiocyanatoanisole (5a).Having identified iron(III) chloride as the most effectiveLewisacid in performing both rapid and efficient thiocyanation reactions,this was used to examine the scope of the transformation 2.15 Staniate see 3.12c,16 6), a drug used for pain relief and as amuscle relaxant.17 Using iron(III) chloride(2.5 mol %) and standard reaction conditions, thiocyanation was completeafter 0.5 h. Analysis of the reaction mixture by 1H NMRspectroscopy revealed a 6:1 ratio of the para- and ortho-products. Separation by column chromatography gavethe major para-product 7 in 72% yield.The study then focused on demonstrating the use ofiron(III) chloride-catalyzedthiocyanations for the preparation or derivatization of biologicallyactive compounds 3. Initiaortho-thiocyanation of phenols.Thiocyanation of para-cresol gave a mixture of inseparableproducts. We propose that as well as the ortho-thiocyanatedproduct, 5-membered oxathioimines, generated by intramolecular cyclizationof the hydroxyl group with the thiocyanate moiety and observed byBhat and co-workers during their oxidative ortho-thiocyanationof phenols, are also formed.18 To preventsimilar side reactions with estradiol, studies focused on the thiocyanationof dibenzyl derivative 8 . In this case,the reaction was complete after 10 min and gave an improved 10:1 ratioof regioisomers in favor of 2-thiocyanated product 9.Purification by column chromatography gave 9 in 86% yield.Here, the in situ-generated thiocyanated-diphenyl selenide cation(see insert) is more sterically hindered than the iron-activated N-thiocyanatosaccharin and thus leads to selective thiocyanationat the most accessible ortho-position.Iron(III)-catalyzed thiocyanation of estradiol was next investigated.During screening of the reaction scope, a limitation was observedduring attempted vative 8 3. An ini12, whichis a key intermediate for the synthesis of toltrazuril (13), used to treat coccidiosis in livestock and poultry.19 We proposed that an iron(III)-catalyzed thiocyanationcould be used for a two-step synthesis of 12 from 2-methyl-4-nitro-1-phenoxybenzene(10)  chloride was requiredfor good conversion. Although this resulted in selective para-thiocyanation of the electron-rich phenyl ether, a reaction timeof 18 h was required and gave 11 in moderate yield (43%).To improve the reaction rate and yield, the transformation was repeatedusing diphenyl selenide as a Lewis base catalyst. This gave 11 after a 1 h reaction time in 91% yield. Conversion of thethiocyanate group to the trifluomethyl thioether was achieved usinga Langlois-type nucleophilic substitution, reported by Goossen andco-workers.20 Reaction of 11 withthe Ruppert-Prakash reagent22 TMS-CF3 underbasic conditions gave trifluoromethyl thioether 12 in77% yield, thereby completing the formal synthesis of toltrazuril.Theiron(III)-catalyzed thiocyanation reaction was also investigatedfor the efficient synthesis of biaryl ether ene(10) 3. Initia4a), a one-pot, dual iron(III)-catalyzed para-thiocyanationand ortho-bromination was attempted (para-thiocyanation proceeded rapidly usingiron(III) chloride (2.5 mol %) and N-thiocyanatosaccharin(2), the more challenging second-stage ortho-bromination with N-bromosuccinimide (NBS) requiredthe use of the stronger Lewis acid, iron(III) triflimide, and an increasedcatalyst loading.11b Therefore, iron(III)triflimide (10 mol %) was added at the start of the one-pot processand resulted in fast para-thiocyanation (1 h), followedby slower ortho-bromination (18 h). This gave 2-bromo-4-thiocyanatoanisole(14) as the sole product in 87% yield. Scale-up (3.3mmol) demonstrated the compatibility of both transformations as partof a one-pot process, with the isolation of the dual functionalizedproduct 14 in similarly high yields. The use of 14 as a synthetic building block was then established. A Langlois-typereaction with the Ruppert-Prakash reagent was used to introduce themedicinally relevant trifluoromethyl thioether group and gave 15 in 71% yield.23 Then, standard palladium-catalyzedcross-coupling reactions were used to diversify the ortho-position, with the introduction of aryl, alkenyl, and alkynyl substituentsin good to high yields. Thus, diverse aryl synthetic building blockscan be readily accessed using the iron(III)-catalyzed thiocyanationreaction as part of a one-pot dual functionalization process, followedby selective transformation of each functional group.The project next investigated the use of iron(III)-catalyzed thiocyanationfor the rapid preparation of multifunctional synthetic building blocksfor medicinal chemistry. Due to the mild nature of iron(III)-catalyzedarene substitution reactions, we proposed that one-pot tandem iron-catalyzedprocesses involving dual functionalization could be used to preparearene building blocks for diversification. Using anisole  chloride.12a This is in agreement with otherapplications of metal triflimide salts in organic reactions, in whichthe high electronegativity, large volume, and low charge density ofthe triflimide counterion allows the metal cation to act as a superLewis acid.24 In contrast, this study hasshown that iron(III) chloride catalyzed faster thiocyanation of anisole(4a) compared to iron(III) triflimide (4i) and m-xylene (4v).15 Previousstudies using N-thiosaccharin reagents have suggestedthat activation with Bronsted or Lewis acids occurs via coordinationwith the amide oxygen atom.25 We proposethat the iron salts bind in a similar manner during the thiocyanationof arenes using N-thiocyanatosaccharin (2) (N-thiocyanatosaccharin(2) than with the smaller, weaker Lewis acid, iron(III)chloride. This rational also explains why faster electrophilic substitutionreactions are observed using iron(III) triflimide with smaller succinimide-basedreagents.12aPrevious work in our group has shown that iron(III)triflimideactivation of iflimide 1. Similaarin (2) 1. Althoupara-thiocyanationof activated arenes with N-thiocyanatosaccharin (2). Excellent scope and fast reaction times of 5–30min were observed for anisoles, phenols, anilines, and indoles. Areneswith weak activating groups, such as mesitylene and m-xylene, were also found to undergo a fast thiocyanation reaction.Many activated arenes bearing electron-deficient substituents werealso substrates for this transformation, with diphenyl selenide usedas a Lewis base catalyst to maintain short reaction times for someof the compounds. The synthetic utility of the transformation forthe thiocyanation of more complex arenes was demonstrated with thehigh-yielding and selective functionalization of biologically activecompounds such as metaxalone and a protected estradiol derivative.The thiocyanation reaction was also used as part of a one-pot iron-catalyzedprocess for the dual functionalization of anisole. Application ofthe resulting product as a synthetic building block was realized withthe selective introduction of a trifluoromethyl thioether substituentusing a Langlois-type reaction of the thiocyanate group, followedby various palladium-catalyzed cross-coupling reactions at the ortho-bromide position. Investigation of other arene functionalizationreactions using iron(III) salts are currently underway.In summary, iron(III) chloride has beenshown as an effective Lewisacid catalyst for the regioselective N-Thiocyanatosaccharin(2),8 β-estradiol dibenzylether (8),26 and 2-methyl-4-nitro-1-phenoxybenzene(10)27 were prepared accordingto the literature. Reactions were performed open to air unless otherwisementioned. All reactions performed at elevated temperatures were heatedusing an oil bath. Brine refers to a saturated aqueous solution ofsodium chloride. Flash column chromatography was performed using silicagel 60 (40–63 μm). Aluminum-backed plates precoated withsilica gel 60F254 were used for thin-layer chromatographyand were visualized with a UV lamp or by staining with potassium permanganate. 1H NMR spectra were recorded on a NMR spectrometer at either400 or 500 MHz, and data are reported as follows: chemical shift inppm relative to tetramethylsilane or the solvent as an internal standard, multiplicity. The abbreviationbr s refers to broad singlet. 13C NMR spectra were recordedon a NMR spectrometer at either 101 or 126 MHz, and data are reportedas follows: chemical shift in ppm relative to tetramethylsilane orthe solvent as an internal standard , multiplicity with respectto hydrogen . Infrared spectra were recorded on a FTIR spectrometer;wavenumbers are indicated in cm–1. Mass spectrawere recorded using an electrospray technique. HRMS spectra were recordedusing a dual-focusing magnetic analyzer mass spectrometer. Meltingpoints are uncorrected. Optical rotations were determined as solutionsirradiating with the sodium D line (λ = 589 nm) using a polarimeter.[α]D values are given in units of 10–1 deg cm–1 g–1.All reagents and starting materialswere obtained from commercialsources and used as received. N-thiocyanatosaccharin (2)  andiron(III) chloride  in dry dichloromethane(2 mL) under argon was added anisole (4a) . The reaction mixture was stirred at 40 °C in theabsence of light for 0.5 h. After cooling to room temperature, thereaction mixture was diluted with dichloromethane (10 mL) and washedwith water (10 mL). The aqueous layer was extracted with dichloromethane(2 × 10 mL), and the combined organic layers were washed withbrine (20 mL). The organic phase was dried (MgSO4), filtered,and concentrated in vacuo. Purification by flashcolumn chromatography (15% diethyl ether in hexane) gave 4-thiocyanatoanisole(5a)  as a colorless oil. Spectroscopicdata were consistent with the literature.4e1H NMR : δ 7.53–7.48, 6.97–6.93 , 3.83 ; 13C{1H} NMR : δ 161.5 (C), 134.0(2 × CH), 116.0 (2 × CH), 114.0 (C), 111.8 (C), 55.7 (CH3); MS (ESI) m/z 166 .To a solution of N-thiocyanatosaccharin (2)  and iron(III)chloride  in dry dichloromethane(60 mL) under argon was added anisole (4a) . The reaction mixture was stirred at 40 °C in theabsence of light for 0.5 h. After cooling to room temperature, thereaction mixture was diluted with dichloromethane (50 mL) and washedwith water (50 mL). The aqueous layer was extracted with dichloromethane(2 × 50 mL), and the combined organic layers were washed withbrine (100 mL). The organic phase was dried (MgSO4), filtered,and concentrated in vacuo. Purification by flashcolumn chromatography (15% diethyl ether in hexane) gave 4-thiocyanatoanisole(5a)  as a colorless oil. Spectroscopicdata as described above.To a solution of 4b)  and N-thiocyanatosaccharin(2) . The reaction mixture was stirredat 40 °C for 0.25 h. Purification by flash column chromatography(30% ethyl acetate in hexane) gave 2,4-dimethoxy-1-thiocyanatobenzene(5b)  as a colorless oil. Spectroscopicdata were consistent with the literature.281H NMR : δ 7.46 , 6.54 , 6.51 , 3.91, 3.83 ; 13C{1H} NMR : δ 163.2 (C), 159.2 (C), 134.0 (CH), 111.4(C), 106.3 (CH), 102.7 (C), 99.8 (CH), 56.3 (CH3), 55.8(CH3); MS (ESI) m/z 218.The reaction was performed accordingto the general procedure using 1,3-dimethoxybenzene (4c)  and N-thiocyanatosaccharin(2) . The reaction mixture was stirredat 0 °C for 0.25 h. Purification by flash column chromatography(30% ethyl acetate in hexane) gave 1,3,5-trimethoxy-2-thiocyanatobenzene(5c)  as a white solid. Mp 151–153°C (lit.29 151–152 °C); 1H NMR : δ 6.15 , 3.91, 3.84 ; 13C{1H} NMR : δ 164.4 (C), 161.5 (2 × C), 112.0 (C),91.5 (2 × CH), 89.9 (C), 56.5 (2 × CH3), 55.7(CH3); MS (ESI) m/z 226.The reaction was performed accordingto the general procedure using 1,3,5-trimethoxybenzene (4d)  and N-thiocyanatosaccharin (2). The reaction mixture was stirred at 40 °Cfor 0.5 h. Purification by flash column chromatography (10% ethylacetate in hexane) gave 2-methyl-4-thiocyanatoanisole (5d)  as a colorless oil. Spectroscopic data were consistentwith the literature.81H NMR: δ 7.37 , 7.35–7.32 , 6.84 , 3.84 , 2.22 ; 13C{1H} NMR : δ 159.6 (C), 134.3 (CH),131.4 (CH), 129.5 (C), 113.1 (C), 112.0 (C), 111.4 (CH), 55.6 (CH3), 16.2 (CH3); MS (ESI) m/z 180 .The reaction was performed according to the generalprocedure using 2-methylanisole (4e)  and N-thiocyanatosaccharin(2) . The reaction mixture was stirredat 40 °C for 1.5 h. Purification by flash column chromatography(30% diethyl ether in hexane) gave 2,3-dihydro-5-thiocyanatobenzofuran(5e)  as a colorless oil. Spectroscopicdata were consistent with the literature.301H NMR : δ 7.43–7.40, 7.32 , 6.80 , 4.63 , 3.24 ; 13C{1H} NMR : δ 162.4 (C), 133.3(CH), 130.1 (C), 129.6 (CH), 113.1 (C), 112.1 (C), 111.1 (CH), 72.1(CH2), 29.5 (CH2); MS (ESI) m/z 178 .The reaction was performed accordingto the general procedure using 2,3-dihydrobenzofuran (4f)  and N-thiocyanatosaccharin(2) . The reaction mixture was stirredat 40 °C for 0.25 h. Purification by flash column chromatography(10% ethyl acetate in hexane) gave 1-methoxy-4-thiocyanatonaphthalene(5f)  as a white solid. Mp 97–100°C (lit.31 104–106 °C); 1H NMR : δ 8.35 , 8.29 ,7.85 , 7.75–7.70 ,7.63–7.57 , 6.81 ,4.03 ; 13C{1H} NMR : δ 158.8 (C), 135.4 (CH), 133.8 (C), 128.8 (CH), 126.9(C), 126.5 (CH), 124.7 (CH), 123.2 (CH), 111.6 (C), 110.5 (C), 104.2(CH), 56.0 (CH3); MS (ESI) m/z 216 .The reaction was performed accordingto the general procedure using 1-methoxynaphthalene (4g)  and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °C for 0.25 h.Purification by flash column chromatography (40% diethyl ether inhexane) gave 4-thiocyanatophenol (5g) as a yellow solid. Mp 53–55 °C (lit.18 51–53 °C); 1H NMR : δ 7.46–7.41 , 6.90–6.86 , 4.94 ; 13C{1H} NMR : δ 158.2 (C), 134.4 (2 × CH), 117.6(2 × CH), 113.3 (C), 112.4 (C); MS (ESI) m/z 152 .The reaction was performed according to the general procedureusing phenol (4h)  and N-thiocyanatosaccharin(2) . The reaction mixture was stirredat 40 °C for 0.25 h. Purification by flash column chromatography(30% ethyl acetate in hexane) gave 3,5-dimethyl-4-thiocyanatophenol(5h)  as a yellow solid. Mp 115–117°C. Spectroscopic data were consistent with the literature.181H NMR :δ 6.66 , 5.25 , 2.54 ; 13C{1H} NMR : δ 158.0 (C), 145.3(2 × C), 116.3 (2 × CH), 113.1 (C), 111.5 (C), 22.3 (2 ×CH3); MS (ESI) m/z 180.The reaction was performed accordingto the general procedure using 3,5-dimethylphenol (4i) , iron(III)chloride , diphenyl selenide , and N-thiocyanatosaccharin(2) . The reaction mixture was stirredat 40 °C for 0.25 h. Purification by flash column chromatography(10% ethyl acetate in hexane) gave methyl 2-hydroxy-5-thiocyanatobenzoate(5i)  as a white solid. Mp 75–77°C; IR (neat) 3166, 2955, 2154, 1674, 1571, 1388, 1333, 1185,750 cm–1; 1H NMR : δ 11.02 , 8.10 , 7.65 , 7.07 , 4.00 ; 13C{1H}NMR : δ 169.4 (C), 163.3 (C), 139.3(CH), 134.6 (CH), 120.3 (CH), 114.1 (C), 113.0 (C), 111.1 (C), 53.0(CH3); MS (ESI) m/z 210; HRMS (ESI) m/z: [M + H]+ calcd for C9H7NO3SH 210.0219; found 210.0220.The reaction was performed according to the general procedure usingmethyl salicylate (4j)  and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °C for 5 min. Purificationby flash column chromatography (30% ethyl acetate in hexane) gave4-thiocyanatoaniline (5j)  as a yellowsolid. Mp 54–55 °C (lit.32 50–52°C); 1H NMR : δ 7.37–7.33, 6.68–6.65 , 3.97 ; 13C{1H} NMR : δ 148.9 (C), 134.6(2 × CH), 116.2 (2 × CH), 112.5 (C), 109.6 (C); MS (ESI) m/z 151 .The reaction was performed according to the general procedureusing aniline (4k) and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 0 °C for5 min. Purification by flash column chromatography (25% ethyl acetatein hexane) gave 2-methoxy-4-thiocyanato-5-methylaniline (5k)  as a brown solid. Mp 40–42 °C; IR (neat)3297, 2916, 2148, 1618, 1575, 1503, 1260, 1217, 1031, 883 cm–1; 1H NMR : δ 6.96 , 6.60 , 4.03 , 3.85 , 2.40 ; 13C{1H} NMR : δ145.9 (C), 139.4 (C), 135.2 (C), 116.7 (CH), 116.2 (CH), 112.1 (C),108.0 (C), 56.0 (CH3), 20.2 (CH3); MS (ESI) m/z 195 ; HRMS(ESI) m/z: [M + Na]+ calcdfor C9H10N2OSH 195.0587; found 195.0587.The reaction was performed according to the general procedure using2-methoxy-5-methylaniline (4l)  and N-thiocyanatosaccharin(2) . The reaction mixture was stirredat 40 °C for 1 h. Purification by flash column chromatography(25% ethyl acetate in hexane) gave 4-thiocyanato-2-(trifluoromethyl)aniline(5l)  as an orange oil. Spectroscopicdata were consistent with the literature.331H NMR : δ 7.66 , 7.51 , 6.78 , 4.51; 13C{1H} NMR : δ 146.7 (C), 137.6 (CH), 132.0 , 124.0 , 118.7 (CH), 114.8 , 111.6 (C), 109.6(C); MS (ESI) m/z 219 .The reaction was performed accordingto the general procedure using 2-(trifluoromethyl)aniline (4m)  and N-thiocyanatosaccharin (2). The reaction mixture was stirred at 40 °Cfor 0.25 h. Purification by flash column chromatography (30% ethylacetate in hexane) gave 2-fluoro-4-thiocyanatoaniline (5m)  as a brown solid. Mp 32–34 °C (lit.34 33–34 °C); 1H NMR : δ 7.24 , 7.17 , 6.78, 4.04 ; 13C{1H} NMR : δ151.1 ,137.5 ,129.9 ,119.9 ,117.4 ,111.7 (C), 109.7 ; MS (ESI) m/z 169 .The reaction was performed according to the generalprocedure using 2-fluoroaniline (4n)  and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °C for 0.25 h.Purification by flash column chromatography (10–25% ethyl acetatein hexane) gave 2-amino-5-thiocyanatobenzophenone (5n)  as a yellow solid. Mp 84–86 °C. IR (neat)3332, 3029, 2152, 1635, 1572, 1249, 943, 829 cm–1; 1H NMR : δ 7.71 , 7.65–7.56 , 7.53–7.47, 6.79 , 6.45 ; 13C{1H} NMR : δ197.9 (C), 152.6 (C), 140.1 (CH), 139.0 (C), 138.5 (CH), 132.0 (CH),129.2 (2 × CH), 128.6 (2 × CH), 119.1 (CH), 118.8 (C), 112.0(C), 107.0 (C); MS (ESI) m/z 253; HRMS (ESI) m/z: [M – H]− calcd forC14H9N2OS 253.0440; found 253.0441.The reaction was performed according to the general procedure using2-aminobenzophenone (4o)  and N-thiocyanatosaccharin (2). The reaction mixture was stirred at 40 °Cfor 0.5 h. Purification by flash column chromatography (30% ethylacetate in hexane) gave 2-cyano-4-thiocyanatoaniline (5o)  as a white solid. Mp 119–121 °C (lit.34 126–127 °C); 1H NMR : δ 7.63 , 7.53 , 6.80 , 4.78 ; 13C{1H} NMR : δ 151.3 (C), 138.4 (CH),137.2 (CH), 116.9 (CH), 115.9 (C), 111.0 (C), 110.5 (C), 97.5 (C);MS (ESI) m/z 174 .The reaction was performed according to the generalprocedure using 2-cyanoaniline (4p) , diphenyl selenide , and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °Cfor 0.75 h. Purification by flash column chromatography (20% ethylacetate in hexane) gave benzyl (4-thiocyanatobenzene)carbamate (5p)  as a white solid. Mp 75–77 °C(lit.35 83–85 °C); 1H NMR : δ 7.48 , 7.43–7.32, 6.85 , 5.21 ; 13C{1H} NMR : δ 153.0 (C), 139.9 (C),135.7 (C), 132.6 (2 × CH), 128.8 (2 × CH), 128.7 (CH), 128.5(2 × CH), 120.0 (2 × CH), 117.3 (C), 111.2 (C), 67.6 (CH2); MS (ESI) m/z 307 .The reaction was performed accordingto the general procedure using benzyl benzenecarbamate (4q)  and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °C for 5 min. Purificationby flash column chromatography (30% ethyl acetate in hexane) gave3-thiocyanatoindole (5q)  as a brown solid.Mp 65–67 °C (lit.8 72–74°C); 1H NMR : δ 8.65, 7.84–7.79 , 7.51 , 7.47–7.40 , 7.36–7.28 ; 13C{1H} NMR : δ136.1 (C), 131.1 (CH), 127.8 (C), 124.1 (CH), 122.1 (CH), 118.9 (CH),112.2 (CH), 112.0 (C), 92.5 (C); MS (ESI) m/z 173 .The reaction was performed according to the general procedureusing indole (4r) and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °C for10 min. Purification by flash column chromatography (40% ethyl acetatein hexane) gave 3-thiocyanato-5-nitroindole (5r)  as a pale yellow solid. Mp 210–212 °C (lit.29 207–209 °C); 1H NMR : δ 8.55 , 8.29 , 8.15 , 7.73 ; 13C{1H} NMR : δ142.2 (C), 139.5 (C), 137.1 (CH), 126.9 (C), 118.2 (CH), 114.4 (CH),113.7 (CH), 111.9 (C), 93.2 (C); MS (ESI) m/z 218 .The reaction was performed according to the generalprocedure using 5-nitroindole (4s) and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °C for5 min. Purification by flash column chromatography (40% ethyl acetatein hexane) gave 3-thiocyanato-7-azaindole (5s)  as a white solid. Mp 203–206 °C (lit.8 197–199 °C); 1H NMR : δ 12.60 , 8.39, 8.17 , 8.12 , 7.30; 13C{1H} NMR : δ 148.4(C), 144.5 (CH), 134.0 (CH), 126.5 (CH), 119.8 (C), 117.4 (CH), 112.1(C), 89.0 (C); MS (ESI) m/z 176.The reaction was performed according to the generalprocedure using 7-azaindole (4t)  and N-thiocyanatosaccharin (2) . The reaction mixture was at 40 °Cfor 10 min. Purification by flash column chromatography (10% ethylacetate in petroleum ether) gave N-phenylsulfonyl-3-thiocyanatoindole(5t)  as a white solid. Mp 133–135°C; IR (neat) 3032, 2150, 1582, 1447, 1370, 1269, 1173, 1130,999 cm–1; 1H NMR : δ 8.02 , 7.97–7.93, 7.74 , 7.64–7.58, 7.54–7.38 ; 13C{1H} NMR: δ 137.5 (C), 134.9 (CH), 134.8(C), 131.2 (CH), 129.8 (2 × CH), 129.3 (C), 127.2 (2 × CH),126.5 (CH), 124.7 (CH), 119.8 (CH), 113.9 (CH), 109.6 (C), 101.2 (C);MS (ESI) m/z 315 ; HRMS (ESI) m/z: [M + H]+ calcd for C15H10N2O2S2H 315.0256; found 315.0259.The reaction was performed according to the generalprocedure using 1-(phenylsulfonyl)indole (4u)  and N-thiocyanatosaccharin (2) . The reaction mixture was stirredfor at 40 °C for 0.5 h. Purification by flash column chromatography(10% ethyl acetate in petroleum ether) gave 2,4,6-trimethylthiocyanatobenzene(5u)  as a white solid. Mp 58–62°C (lit.5c 62 °C); 1H NMR : δ 7.01 , 2.56 , 2.30 ; 13C{1H} NMR : δ 142.8 (2 × C), 141.6 (C), 130.2 (2 ×CH), 119.2 (C), 111.0 (C), 22.0 (2 × CH3), 21.2 (CH3); MS (ESI) m/z 178 .The reaction was performed accordingto the general procedure using mesitylene (m-xylene (4v) , iron(III) chloride , and N-thiocyanatosaccharin (2) . The reaction mixture was stirredat 40 °C for 2 h. Purification by flash column chromatography(30% dichloromethane in hexane) gave 1,3-dimethyl-4-thiocyanatobenzene(5v)  as a colorless oil. Spectroscopicdata were consistent with the literature.4e1H NMR : δ 7.50 , 7.14–7.12 , 7.09–7.04, 2.46 , 2.34 ; 13C{1H}NMR : δ 141.1 (C), 139.9 (C), 132.9(CH), 132.4 (CH), 128.6 (CH), 119.9 (C), 111.1 (C), 21.2 (CH3), 20.6 (CH3); MS (ESI) m/z 164 .The reaction was performed accordingto the general procedure using 6)  and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °Cfor 0.5 h. Purification by flash column chromatography (80–90%ethyl acetate in hexane) gave 5-[methyl]-1,3-oxazolidin-2-one(7)  as a white solid. Mp 174–176°C; IR (neat) 3246, 2959, 2148, 1747, 1583, 1309, 1244, 1168,1074, 856 cm–1; 1H NMR : δ 6.74 , 5.67 , 5.01–4.93, 4.15 , 3.78 , 3.60 , 2.57 ; 13C{1H} NMR : δ 160.0 (C), 159.2 (C), 145.1 (2 × C), 115.4(2 × CH), 114.4 (C), 111.0 (C), 74.0 (CH), 68.1 (CH2), 42.7 (CH2), 22.5 (2 × CH3); MS (ESI) m/z 279 ; HRMS(ESI) m/z: [M + H]+ calcdfor C13H14N2O3SH 279.0798;found 279.0802.The reaction was performed according tothe general procedure using metaxalone (8) , diphenyl selenide ,and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 0 °C for10 min. Purification by flash column chromatography (60% dichloromethanein hexane) gave 2-thiocyanato-β-estradiol dibenzyl ether (9)  as a white solid. Mp 123–125 °C;IR (neat) 2926, 2153, 1596, 1496, 1307, 1258, 1078, 738 cm–1; [α]D23 +63.3 ; 1H NMR :δ 7.49–7.25 , 6.71 , 5.13 , 4.58, 3.51 , 2.90–2.78, 2.35–2.26 , 2.22–2.00 , 1.92–1.84, 1.73–1.14 , 0.88 ; 13C{1H} NMR : δ 154.0 (C), 140.4(C), 139.4 (C), 136.2 (C), 135.1 (C), 128.8 (2 × CH), 128.4 (2× CH), 128.33 (CH), 128.32 (CH), 127.5 (3 × CH), 127.3 (2× CH), 113.5 (CH), 111.3 (C), 109.9 (C), 88.3 (CH), 71.9 (CH2), 71.1 (CH2), 50.3 (CH), 44.1 (CH), 43.5 (C),38.4 (CH), 37.9 (CH2), 29.9 (CH2), 28.2 (CH2), 27.0 (CH2), 26.5 (CH2), 23.3 (CH2), 11.9 (CH3); MS (ESI) m/z 510 ; HRMS (ESI) m/z: [M + H]+ calcd for C33H35NO2SH 510.2461; found 510.2464.The reaction was performed according to the general procedureusing β-estradiol dibenzyl ether (10) , iron(III) chloride , diphenyl selenide , and N-thiocyanatosaccharin (2) . The reaction mixture was stirred at 40 °Cfor 0.25 h. Purification by flash column chromatography (5% diethylether in hexane) gave 1-(2′-methyl-4′-nitrophenoxy)-4-thiocyanatobenzene(11)  as a white solid. Mp 62–64°C; IR (neat) 2925, 2156, 1581, 1486, 1340, 1244, 1091, 843 cm–1; 1H NMR : δ8.18 , 8.05 , 7.61–7.56 , 7.09–7.04 ,6.90 , 2.37 ; 13C{1H} NMR : δ 159.6 (C),157.6 (C), 143.8 (C), 133.4 (2 × CH), 130.7 (C), 127.2 (CH),123.4 (CH), 120.7 (2 × CH), 119.1 (C), 117.9 (CH), 110.8 (C),16.5 (CH3); MS (ESI) m/z 287 ; HRMS (ESI) m/z: [M + H]+ calcd for C14H10N2O3SH 287.0485; found 287.0484.The reaction was performed according tothe general procedure using 2-methyl-4-nitro-1-phenoxybenzene (11)  and cesium carbonate  in dry acetonitrile (0.7 mL) under argon was addedtrimethyl(trifluoromethyl)silane , and the reactionmixture was stirred at room temperature for 3 h. The reaction mixturewas diluted with ethyl acetate (10 mL) and washed with water (10 mL).The aqueous layer was extracted with ethyl acetate (2 × 10 mL),and the combined organic layers were washed with brine (30 ml). Theorganic phase was dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash column chromatography (3%ethyl acetate in hexane) gave 1-(2′-methyl-4′-nitrophenoxy)-4-(trifluoromethylsulfanyl)benzene(12)  as a colorless oil. Spectroscopicdata were consistent with the literature.371H NMR : δ 8.19 , 8.06 , 7.70–7.65 , 7.06–7.01 ,6.93 , 2.38 ; 13C{1H} NMR : δ 159.6 (C),158.5 (C), 143.9 (C), 138.8 (2 × CH), 130.9 (C), 129.6 , 127.2 (CH), 123.4(CH), 119.7 (2 × CH), 119.6 , 118.2 (CH), 16.5 (CH3); MS (ESI) m/z 330 .To a suspensionof 1-(2′-methyl-4′-nitrophenoxy)-4-thiocyanatobenzene(N-thiocyanatosaccharin (2) in dry dichloromethane (20 mL) under argon. Anisole (4a)  was added, and the reaction mixture was stirredin the dark at 40 °C for 1 h. The reaction mixture was then cooledto room temperature, and N-bromosuccinimide  was added. The reaction mixture was stirred at 40 °Cfor 18 h. After cooling to room temperature, the reaction mixturewas diluted with dichloromethane (20 mL) and washed with water (20mL). The aqueous layer was extracted with dichloromethane (2 ×20 mL), and the combined organic layers were washed with brine (50mL). The organic phase was dried (MgSO4), filtered, andconcentrated in vacuo. Purification by flash columnchromatography (7.5–10% ethyl acetate in hexane) gave 2-bromo-4-thiocyanatoanisole(14)  as a white solid. Mp 61–62°C; IR (neat) 2944, 2152, 1577, 1483, 1438, 1256, 1010, 810 cm–1; 1H NMR : δ7.77 , 7.51 , 6.94 , 3.93; 13C{1H} NMR : δ 157.9 (C), 136.6 (CH), 132.7 (CH), 115.2 (C), 113.3(C), 113.1 (CH), 111.0 (C), 56.7 (CH3); MS (ESI) m/z 244 ; HRMS(ESI) m/z: [M + H]+ calcdfor C8H679BrNOSH 243.9426; found243.9427.Iron(III)chloride  was dissolved in 1-butyl-3-methylimidazoliumbis(trifluoromethanesulfonyl)imide  and stirredfor 0.5 h at room temperature and then added to a solution of 14)  and cesium carbonate  in dry acetonitrile (5 mL) under argon was added trimethyl(trifluoromethyl)silane, and the reaction mixture was stirred at roomtemperature for 4 h. The reaction mixture was diluted with ethyl acetate(30 mL) and washed with water (30 mL). The aqueous layer was extractedwith ethyl acetate (2 × 30 mL), and the combined organic layerswere washed with brine (50 mL). The organic phase was dried (MgSO4), filtered, and concentrated in vacuo. Purificationby flash column chromatography (7.5% ethyl acetate in hexane) gave2-bromo-4-(trifluoromethylsulfanyl)anisole (15)  as a colorless oil. Spectroscopic data were consistent withthe literature.381H NMR : δ 7.84 , 7.58 , 6.92 , 3.93 ; 13C{1H}NMR : δ 158.4 (C), 141.1 (CH), 137.4(CH), 129.5 , 116.2 , 112.39 (C), 112.36 (C), 56.6 (CH3); MS (ESI) m/z 286 .To a suspension of 2-bromo-4-thiocyanatoanisole(15) , 4-nitrophenylboronic acid and potassium phosphate tribasic  in degassed tetrahydrofuran (0.7 mL) and water (1.3 mL)was added XPhos Pd G2 , and the reactionmixture was stirred at 40 °C for 4 h. After cooling to ambienttemperature, the reaction mixture was filtered through a short padof Celite and washed with ethyl acetate (10 mL). The filtrate waswashed with water (10 mL), and the aqueous layer was extracted withethyl acetate (2 × 10 mL). The combined organic layers were washedwith brine (20 mL), dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash column chromatography (5%ethyl acetate in hexane) gave 2-(4′-nitrophenyl)-4-(trifluoromethylsulfanyl)anisole(16) as a brown oil . IR (neat) 2950,1595, 1509, 1345, 1267, 1099, 857 cm–1; 1H NMR : δ 8.30–8.26 ,7.72–7.65 , 7.62 ,7.06 , 3.88 ; 13C{1H} NMR : δ 158.8 (C), 147.2 (C), 143.8 (C), 138.9 (CH), 138.8 (CH), 130.5 (2× CH), 129.8 (C), 129.6 , 123.5 (2 × CH), 115.8 , 112.4 (CH), 56.0(CH3); MS (ESI) m/z 330; HRMS (ESI) m/z: [M + H]+ calcd for C14H10F3NO3SH 330.0406; found 330.0405.To a solution of 2-bromo-4-(trifluoromethylsulfanyl)anisole(15)  in degassed dimethylformamide(4 mL) was added methyl acrylate  and N,N-diisopropylethylamine , followedby bis(triphenylphosphine)palladium(II) dichloride , and the reaction mixture was stirred under argon at 100 °Cfor 18 h and at then 120 °C for 24 h. After cooling to ambienttemperature, the reaction mixture was diluted with ethyl acetate (25mL) and washed with water (3 × 25 mL) and brine (25 mL). Theorganic layer was dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash column chromatography (10%ethyl acetate in hexane) gave methyl (2E)-3-(2′-methoxy-5′-trifluoromethylsulfanylbenzene)acrylate(17) as a white solid . Mp 67–68°C; IR (neat) 2945, 1711, 1586, 1484, 1438, 1253, 1093, 814 cm–1; 1H NMR : δ7.92 , 7.77 , 7.62 , 6.95, 6.55 , 3.93 , 3.81 ; 13C{1H} NMR : δ 167.6 (C), 160.3 (C),139.7 (CH), 138.8 (CH), 137.3 (CH), 129.6 , 125.0 (C), 120.2 (CH), 115.6, 112.3(CH), 56.0 (CH3), 51.9 (CH3); MS (ESI) m/z 293 ; HRMS(ESI) m/z: [M + H]+ calcdfor C12H11F3O3SH 293.0454;found 293.0459.To a solution of 2-bromo-4-(trifluoromethylsulfanyl)anisole(15) , tetrakis(triphenylphosphine)palladium(0), and copper(I) bromide . Degassed tetrahydrofuran (0.7 mL) was added, and the solutionwas stirred for 0.1 h. Triethylamine  and phenylacetylene were added, and the reaction mixture was stirredat 75 °C under argon for 3 h. The reaction mixture was cooledto ambient temperature, diluted with water (10 mL), and extractedwith ethyl acetate (3 × 10 mL). The combined organic layers werewashed with brine (20 mL), dried (MgSO4), filtered, andconcentrated in vacuo. Purification by flash columnchromatography (5% ethyl acetate in hexane) gave 2-(phenylethynyl)-4-(trifluoromethylsulfanyl)anisole(18) as a white solid . Mp 69–71°C; IR (neat) 2919, 1585, 1479, 1250, 1091, 1015, 750 cm–1; 1H NMR : δ7.79 , 7.61–7.54 ,7.39–7.34 , 6.93 ,3.95 ; 13C{1H} NMR : δ 162.1 (C), 141.7 (CH), 138.4 (CH), 131.9 (2 ×CH), 129.6 , 128.7 (CH), 128.5 (2 × CH), 123.1 (C), 115.1 , 114.4 (C), 111.7(CH), 94.9 (C), 84.3 (C), 56.3 (CH3); MS (ESI) m/z 309 ; HRMS (ESI) m/z: [M + H]+ calcd for C16H11F3OSH 309.0555; found 309.0556.A reaction vial was charged with 2-bromo-4-(trifluoromethylsulfanyl)anisole(


Enantiomerically highly enriched unsaturated β-ketoesters bearing a quaternary stereocenter can be utilized as building blocks for the synthesis of natural occurring terpenes, i. a., trisporic acid and its derivatives. An advanced building block has been synthesized in a short reaction sequence, which involves an oxidative allylic rearrangement initiated by pyridinium dichromate (PDC) as the key step. To. To1c isd workup  12,14].,14.1c is1c was monitored during the reaction, and after completion of the reaction the enantiomeric excess was determined to be >99% by GLC using a cyclodextrin modified stationary phase (LIPODEX E). The R-configuration at the stereocenter was established by chemical correlation -sigmatropic rearrangement is as shown in The first step towards the synthesis of cyclohexenone (+)-r (−)-1c . This waR-(+)-7, which can be utilized as an enantiomerically pure starting material in Suzuki’s elegant protocol +, 175 (30), 147 (18), 131 (40), 107 (85), 91 (28), 79 (100); Anal. Calcd for C12H16O3 (208.3): C, 69.21; H, 7.74. Found: C, 69.05; H, 7.91.Methyl (+)-(1R)-2-ethynyl-1,3-dimethyl-4-oxocyclohex-2-ene-1-carboxylate (7). PDC  was added to a solution of 6  in abs CH2Cl2 (5 mL) in the presence of a catalytic amount of hydroquinone. The resulting suspension was heated under reflux in an argon atmosphere for 7 h. At the end of the reaction, ethyl acetate (1 mL) was added and the product separated from the chromium salts by passage through a short column of silica gel (petroleum/ethyl acetate 9:1) to give 7  as a colorless oil. The product required storage in a freezer to prevent polymerization. Rf = 0.1 (petroleum/ethyl acetate 9:1);  +48.4 ; IR (film):  3262, 2984, 2955, 2100, 1736, 1674, 1593 cm−1; 1H NMR (CDCl3): δ 1.57 , 1.89–2.00 , 2.03 , 2.39–2.57 , 2.50 , 3.76 ; 13C NMR (CDCl3): δ 14.8 (q), 24.4 (q), 33.9, 34.7 (2 t), 47.5 (s), 53.0 (q), 80.6 , 92.5 , 138.2, 141.5 , 174.7 , 197.4 ; GC–MS (70 eV) m/z (%): 207 (100) [M + H]+, 191 (50), 178 (23), 150 (21), 119 (65), 91 (50); Anal. Calcd for C12H14O3 (206.2): C, 69.89; H, 6.84. Found: C, 69.72; H, 7.03.


The structures of 1–7 were determined by spectroscopic techniques. Among the isolates, endiandric acid M (4) exhibited moderate iNOS inhibitory activity, with an IC50 value of 31.70 μM.Four new endiandric acid analogues, tsangibeilin C ( B. tsangii Merr. (Lauraceae) has shown potent inhibition of NO production, with no cytotoxicity against RAW 264.7 cells. B. tsangii is a medium-sized evergreen tree, distributed throughout Tonkin, Vietnam, southern and western China and southern Taiwan. About 200 Beilschmiedia species are found in these tropical regions, including two species in Taiwan  in beilschmiedic acid D. The length of the alkyl side chain at C-11 of 1 was two methylenes less than beilschmiedic acid D, as supported by the molecular formula of 1 (C20H26O3). The rigid tetracyclic skeleton was indicated by HMBC correlations, including: H-5 to C-3, C-6, C-7 and C-14, H-3 to C-4, and C-7, H-13 to C-8 and C-10, H-8 to C-6 and C-10, H-9 to C-7, H-2 to C-3, C-4, C-11 and C-13, H-1 to C-3 and C-13, H-12 to C-3, C-9 and C-11 and H-11 to C-9. The relative configuration of 1, rel-, was supported on the basis of NOESY , 1 should be racemic, as are many endiandric acid analogues  at C-6 and C-7 in 1, as supported by HRESIMS, IR and DEPT spectra. The NOESY spectrum  was supported on the basis of NOESY and structural similarity with beilschmiedic acid D + ion at m/z 341, in agreement with the molecular formula of C20H30O3, with six degrees of unsaturation as confirmed by HRESIMS. The 13C NMR  connected the fragment C1-C2-C3-C4-C5-C6 with the cyclohexane ring, as supported by HMBC correlations, H-6 to C-4, C-5 and C-7, H-5 to C-3 and C-7 and H-9 to C-7. According to the above evidence, the carboxyl group, the fragment C1-C2-C3-C4-C5-C6, the cyclohexane ring and the cyclobutane ring become a cyclododecane ring, which is also supported by six degrees of unsaturation. The HMBC spectrum showed correlations between H-1′ and C-1, C-11, C-2′ and C-3′, thus establishing the presence of an alkyl chain (C-1′~C-6′) to be connected at C-12. Finally, the HRESIMS, IR, and DEPT spectra indicated the presence of a hydroxy group located at C-10. The NOESY spectrum , was supported on the basis of NOESY and its structural similarity with know endiandric acid analogues. Based on further spectrographic evidence, the structure of 3 was elucidated and designated as tricyclotsangibeilin.Compound  13C NMR  and DEPTand COSY  spectra and COSY  correlatspectrum  showed c4 indicated the molecular formula C22H22O4, four methylenes less than endiandric acid L  in 4 replaced the pentamethylene group in endiandric acid L. The relative configuration of 4 was supported by NOESY spectrum and its structural similarity with endiandric acid L  were similar to those of beilschminol A [3-3′ [δ 3.88 ] and OCH3-4′ [δ 3.83 ] in 5 replaced a methylenedioxy group [δ 5.96 ] in beilschminol A. Thus, the planar structure of 5 was proposed to be 3-hydroxy-4,5,3′,4′,5′-pentamethoxy-7,7′-epoxylignan. The relative configuration of 5 was based on the NOESY spectrum and its structural similarity with -3-hydroxy-3′,4′-methylenedioxy-4,5,5′-trimethoxy-7,7′-epoxylign 25D + 123.8 . The HRESIMS analysis of 6 gave a quasi-molecular ion peak at m/z 257.1516 [M+Na]+ , consistent with a molecular formula of C15H22O2 with five degrees of unsaturation. The IR, 1H, and 13C NMR ] in C-5 (δc 71.2) of 6 replaced a methylene  in C-5 (δc 41.9) of (+)-barbatenal, resulting in the downshifting of the olefinic proton  in 6 from δH 5.87 (H-4) in (+)-barbatenal. The relative configuration of 6 was determined by NOESY spectrum, where Ha-1 showed correlations with H-2, H-12, H-13 and H-14, but no correlation with H-5. It has been suggested that Ha-1, H-2, H-12, H-13 and H-14 are on the same side of the molecule and that Hb-1 and H-5 are on the opposite side of the molecule. Furthermore, H-5 showed a correlation with H-14 due to a spatial distance between these two protons of <4 Å, as supported by simulating the ChemDraw 3D Ultra (version 10.0). Based on the NOESY spectrum, the optical rotation and comparison to values in the literature, the relative configuration of 6 was determined to be rel-. Hence, the structure of 6 was elucidated and designated as (+)-5-hydroxybarbatenal, which was further confirmed by DEPT, HSQC, NOESY . IR absorption bands at 3396 cm−1 (OH) and 1637 cm−1 (C=C) were observed. From the HRESIMS data, the molecular formula was determined to be C15H26O2 . The 13C NMR -4,5-dihydroxycaryophyll-8(13)-ene and identified with its synthetic compound 23D ±0 ; IR (neat) νmax cm−1: 3432 (hydroxy group of carboxylic acid), 1696 (C=O); for 1H and 13C NMR spectroscopic data, see m/z 337 [M + Na]+; HRESIMS: m/z 337.1782 [M + Na]+ .Tsangibeilin C (2): Light yellowish oil; [α]24D ±0 ; IR (neat) νmax cm−1: 3397 (OH), 1692 (C=O); for 1H and 13C NMR spectroscopic data, see m/z 325 [M + Na]+; HREIMS: m/z 302.1879 [M]+ .Tsangibeilin D (3): Light yellowish oil; [α]24D + 36.3 ; IR (neat) νmax cm−1: 3422 (OH), 1729 (C=O); for 1H and 13C NMR spectroscopic data, see m/z 341 [M + Na]+; HRESIMS: m/z 341.2096 [M + Na]+ .Tricyclotsangibeilin (4): Light yellowish oil; [α]25D ±0 ; IR (neat) νmax cm−1: 3461 (hydroxy group of carboxylic acid), 1694 (C=O), 1040, 937 (OCH2O); for 1H and 13C NMR spectroscopic data, see m/z 373 [M + Na]+; HRESIMS: m/z 373.1418 [M + Na]+ .Endiandric acid M (5): Light yellowish oil; [α]25D –18.6 ; UV (MeOH) λmax (log ɛ): 209 (4.63), 240 sh (3.92), 270 (3.12) nm; UV (MeOH+KOH) λmax (log ɛ): 210 (4.71), 242 sh (3.97), 280 (3.25) nm; IR (neat) νmax cm−1: 3415 (OH), 1593, 1508, 1461 (benzene ring); 1H NMR  δ 1.07 , 1.08 , 1.78 , 3.83 , 3.88 , 4.61 , 4.63 , 5.78 , 6.55 , 6.61 , 6.64 ; 13C NMR  δ 13.9 (C-9′), 14.0 (C-9), 50.9 (C-8′), 51.0 (C-8), 55.9 (OCH3-5), 56.1 , 60.8 (OCH3-4′), 60.9 (OCH3-4), 88.2 (C-7), 88.5 (C-7′), 101.6 (C-6), 102.9 , 105.8 (C-2), 134.7 (C-4), 137.2 (C-4′), 138.0 (C-1′), 138.7 (C-1), 149.1 (C-3), 152.4 (C-5), 153.2 ; ESIMS: m/z 441 [M + Na]+; HRESIMS: m/z 441.1887 [M + Na]+ .Beilschminol B (6): Light yellowish oil; [α]25D +123.8 ; IR (neat) νmax cm−1: 3408 (OH), 1681 (C=O); for 1H and 13C NMR spectroscopic data, see m/z 257 [M + Na]+; HRESIMS: m/z 257.1516 [M + Na]+ .(+)-5-Hydroxybarbatenal -4,5-Dihydroxycaryophyll-8(13)-ene (7): Light yellowish oil; [α]25D –23.9 ; IR (neat) νmax cm−1: 3396 (OH), 1637 (C=C); for 1H and 13C NMR spectroscopic data, see m/z 261 [M + Na]+; HRESIMS: m/z 261.1828 [M + Na]+ .(4Beilschmiedia species in the world have revealed endiandric acids [B. tsangii, together with the roots [B. erythrophloia. Both species possess endiandric acids, 7,7′-epoxylignans, 7′,8′-seco-7,7′-epoxylignans, benzopyrans, arylpropanoids, amides, sesquiterpenoids and steroids. In this study, endiandric acids, 7,7′-epoxylignans, benzenoid, sesquiterpenoids and triterpenoid were also reported. The constituents of the roots of the two Formosan Beilschmiedia species showed 19 new endiandric acid analogues as key secondary metabolites. The roots of other Beilschmiedia species found in other regions have not yet been studied. However, several isolates from two Formosan Beilschmiedia species have shown cytotoxic [There are about 200 Beilschmiedia species in the tropical regions mentioned earlier, including two species in Taiwan . Except ic acids ,18,19, fic acids , alkaloiic acids –24, benzic acids  and sesqic acids  and antiic acids  activitiic acids  and the ytotoxic ,9 or ant


This strain was an associated actinomycete isolated from the marine sponge Hymeniacidon perlevis collected from the coast of Dalian (China). Their structures were elucidated by detailed NMR and MS spectroscopic analysis as well as comparison with literature data.One new nucleoside derivative, named 3-acetyl-5-methyl-2′-deoxyuridine ( The ava1), along with two known nucleoside derivatives , were isolated from Streptomyces microflavus strain No. HVG29 belonging to actinomycetes. To date, there have been no reports of deoxyuridine structures isolated from this species. We report herein the isolation and structure elucidation of 1–3.A number of reports have been published on the isolation of actinobacteria from marine organisms . Screeni2.2O followed by diluting the aqueous layer to 3:2 MeOH–H2O and solvent partitioning with EtOAc. The EtOAc fraction was subjected to further purification by gel permeation on Sephadex LH-20 to yield compounds 1–3 . The IR spectrum possessed absorptions for hydroxyl groups (3394 cm−1) and amide carbonyl groups (1693 cm−1). The EIMS gave a molecular ion peak at m/z 284 [M]+, and the molecular formula C12H16N2O6 was determined by HRESIMS at m/z 307.0905 [M + Na]+ .Compound δH 6.28 , 2.22 , 3.90 , 4.40 , 3.79 , and 3.83  and carbon resonances at δC 86.3 , 41.2 , 72.2 , 88.9 , and 62.9  supported the presence of a pentose moiety in the molecule. The HMBC spectrum showed correlations from H-6′ to C-4′, from H-5′ to C-2′ and C-3′, from H-4′ to C-2′, from H-3′ to C-5′and C-2′, and from H-2′ to C-5′ and C-4′  and 1.88  and carbon resonances at δC 152.4 (C-2), 166.4 (C-4), 111.5(C-5), 138.2 (C-6), and 12.4 (C-7) evidenced the presence of a thymine moiety in the molecule. An acetyl group was supported by proton signal at δH 1.88  and carbon signals at δC 23.4 (C-9) and 179.2 (C-8). In combination with the chemical shifts of the corresponding carbons and the elemental composition, the acetyl group was assigned to N-3 of the thymine moiety, because only N-3 had a vacancy in the molecule. Meanwhile, HMBC long-range correlations from H-2′ to C-2 and C-6  and Sephadex LH-20 (Pharmacia). TLC was carried out with precoated silica gel plates . All solvents were of analytical grade.NMR spectra were recorded at 500 and 125 MHz for 3.2.Streptomyces microflavus was isolated from the inner tissue of the marine sponge Hymeniacidon perlevis collected from the inter-tidal beach of the Yellow Sea at Dalian, China  in March 2003. Sponge specimens were placed in plastic bags containing seawater and immediately transported to the laboratory. Actinobacteria identification was carried out by the method reported by Zhang et al. 20D +5.4° ; UV (MeOH) λmax 264 (log ε) (3.52), λmax 256 (log ε) (3.46); 1H NMR  δ 7.81 , 6.28 , 4.40 , 3.90 , 3.83 , 3.79 , 2.22 , 1.88 , 1.88 ; 13C NMR  δ 179.2 (C-8), 166.4 (C-4), 152.4 (C-2), 138.2 (C-6), 111.5 (C-5), 88.9 (C-5′), 86.3 (C-2′), 72.2 (C-4′), 62.9 (C-6′), 41.2 (C-3′), 23.4 (C-9), 12.4 (C-7). EIMS m/z (relative intensity): m/z 284 (5), 256 (9), 241 (5), 239 (9), 213 (15), 206 (9), 163 (11), 98 (43), 96 (80), 95 (74), 60 (100); HRESIMS m/z: 307.0905 [M + Na]+, calcd. for C12H16N2O6Na, 307.0906.3-Acetyl-5-methyl-2′-deoxyuridine  were originally isolated from a marine sponge [Geodia barretti, and now, for the first time, from sponge-associated microorganisms. This finding strengthens the hypothesis that marine microbial symbionts are possibly the true producers or take part in the biosynthesis of some bioactive marine natural products isolated from the marine organism hosts.The known compounds  , herpes


Tilia cordata), two colorless nonfluorescent chlorophyll catabolites (NCCs) were identified, named Tc-NCC-1 and Tc-NCC-2, as well as a polar yellow chlorophyll catabolite (YCC), named Tc-YCC. The constitution of the two NCCs was determined by spectroscopic means. In addition, a tentative structure was derived for Tc-YCC. The three chlorophyll degradation products exhibited tetrapyrrolic structures, as are typical of NCCs or YCCs, and turned out to be rather polar, due to a glucopyranosyl group at their 82-position. At their 3-positions, the more polar Tc-NCC-1 carried a 1,2-dihydroxyethyl group and the less polar Tc-NCC-2 a vinyl group. Tc-YCC was identified as the product of an oxidation of Tc-NCC-1.In cold extracts of senescent leaves of the Lime tree ( Evidence for another type of further oxidative transformation of NCCs was also provided more recently by the yellow chlorophyll catabolites (YCCs) and pink chlorophyll catabolites (PiCCs) detected in senescent leaves of the Katsura tree (Cercidiphyllum japonicum), which were identified as dehydrogenation products of the tetrapyrrolic NCC 3b (Cj-NCC-1) About ten years ago, two ‘urobilinogenoidic’ chlorophyll catabolites were discovered in extracts of senescent primary leaves of barley (Acer platanoides), classified as dioxobilanes, and indicating a different path to these tetrapyrroles (Musa acuminata) fruits , a tropical evergreen However, several recent observations suggested the existence of divergent pathways of chlorophyll breakdown in higher plants. A strikingly contrasting stereo-chemical variant of the ‘urogenobilinoidic’ catabolites from barley was found in naturally de-greened leaves of Norway Maple (Tilia cordata), a first representative of the genus Tilia  to be investigated in this respect. Lime trees are well-known deciduous trees native to the forests of the northern hemisphere in Europe, Asia, and Eastern North America and Central America Tilia sp. in central Europe can be found in the Middle High German ‘Nibelungenlied', where a lime tree leaf ('linden leaf) covered Siegfried's back during his bath in the blood of a wounded dragon and gave rise to his vulnerable spot1). Beside a special medicinal relevance of lime trees , two polar colorless and nonfluorescent chlorophyll catabolites (NCCs) and a yellow chlorophyll catabolite (YCC) were provisionally identified by analytical HPLC, on the basis of their characteristic UV-absorbance properties ) 31.8 min, R (Tc-YCCt (13)) 33.8 min, and Rt (Tc-NCC-2 (4b)) 47.8 min) reflected the different polarities of the three catabolites.In a cold MeOH extract of yellow (senescent) fall leaves of a lime tree ties see  24 durin4b and 5 matched those of the chlorophyll catabolite Hv-NCC-1 (1a). An absorbance maximum near 314 nm indicated the presence of an α-formyl-pyrrole moiety at ring BUV/VIS and CD spectra of the NCCs Exper. Part) to give 5.1 mg (6.1 mmol) of Tc-NCC-1 (5), 1 mg (1.2 mmol) of the less polar Tc-NCC-2 (4b), and 0.2 mg of the yellow catabolite Tc-YCC (13) (determined by UV/VIS spectroscopy).For further structural analysis, 100 g (wet weight) of senescent lime tree leaves were extracted according to a three-stage purification procedure based on a cold extraction, followed by separation by MPLC and by preparative HPLC  of Tc-NCC-1 (5). In the mass spectrum, characteristic fragment-ion peaks at m/z 809.5, 684.4, and 679.3 were also detected, which corresponded to loss (from [M+H] + ) of MeOH, as is typical of the methyl ester functionality, to loss of ring A, and to loss of the sugar moiety (as [C6H10O5]), respectively.Mass spectrometry was utilized to derive a tentative molecular formula of Tc-NCC-1 (5) as C4O15 see , a. ESI-1H-NMR spectrum of a solution of Tc-NCC-1 (5) in CD3OH revealed signals of 44 of the 52 H-atoms: a singlet (at low field) of the formyl H-atom, four singlets (at high field) of the four Me groups attached at the b-pyrrole positions, and a singlet at 3.74 ppm (due to a methyl ester function). In addition, the signals of the H-atoms H-N(21), H-N(24), and H-N(23) were detected between 8.0 and 11.3 ppm, and could be assigned with the help of COSY, ROESY, and HMBC data. However, in contrast to the 1H-NMR spectrum of Tc-NCC-2 (4b), the typical signals for a peripheral vinyl group in the intermediate field range were absent. 1H,13C-Hetero-nuclear NMR correlations (from HSQC and HMBC spectra Tc-NCC-1 (5) in CD3OH) allowed assignment of the signals of a 1,2-dihydroxyethyl side chain. 1H,1H-Homonuclear correlations from ROESY spectra and 1H,13C-heteronuclear correlations from HMBC spectra 2) as the site of the attachment of the sugar moiety. The shifts of the 1H and 13C signals for the CH2(82) group were consistent with an O-bridge to the peripheral sugar substituent. The latter was identified as a b-glucopyranoside unit by comparing the 1H and 13C chemical shifts with the spectra of methyl b-d-glucopyranoside Tc-NCC-1 (5) with those of Bn-NCC-2, Nr-NCC-2, and Zm-NCC-1, where a peripheral b-glucopyranosyl group at C(82) had also been found doublet  = 2.5 Hz, in CD3OH) due to coupling with H-C(132). Therefore, a relative trans-configuration of H-C(132) and H-C(15), which is typical for stable isomers of NCCs Tc-NCC-1 was derived using the Karplus relation A 500-MHz with H-C1. TherefoTc-NCC-2 (4b) as C41H50N4O13 . The basic substitution pattern of the tetrapyrrolic core of Tc-NCC-2 could again be determined by the analysis of characteristic fragment-ion peaks at m/z 775.3, 684.3, and 645.3 due to loss of MeOH, of ring A, and of the sugar moiety (as [C6H10O5]), respectively.ESI-MS in the positive-ion mode was also used to deduce the tentative molecular formula of 4O13 see , b. The 1H-NMR-spectrum of 4b (in CD3OD) exhibited signals for 40 of the 50 H-atoms: a singlet (at low field) for the formyl H-atom, four singlets (at high field) for the four Me groups in the b-pyrrole positions, and a singlet at 3.74 ppm (due to the methyl ester function). In addition, the typical signal pattern for a vinyl group was detected around 6 ppm. 1H,13C-Heteronuclear correlations . A sugar moiety was identified as a b-glucopyranosyl group at C(82) from 1H,1H-ROESY correlations, as well as 1H,13C-heternuclear correlations from HMBC spectra 2)) via a bridging O-atom, O(83), was consistent with the typical downfield shifts of the 1H and 13C signals of CH2(82). The lack of the signal for H-C(132) in the 1H-NMR spectra (in CD3OD) of Tc-NCC-2 (4b) indicated the exchange-labile a-position of the b-keto ester functionality at ring E to have undergone H/D exchange. HPLC Co-injection of Tc-NCC-1 (5) and of the constitutionally identical analog from maize, Zm-NCC-1 R of ca. 30 min, suggesting the two NCCs to be identical .A 600-MHz Tc-NCCs 5 and 4b were consistent with the suggested common configuration of C(15) in naturally occurring NCCs from higher plants 2), with H-C(15) cis to the COOMe group at C(132) in (the prevailing epimer of) 5 and 4b is a likely result of a nonenzymatic equilibration reaction at the acidic b-keto ester position C(132), which adjusts its configuration to that at C(15). The latter process would occur in the vacuoles of senescent plant cells The CD spectra of the roup at C2 in (theTc-YCC (13) could be deduced as C41H50N4O15 by LC/ESI-MS in the positive-ion mode . The mass spectrum of 13 showed characteristic fragment-ion peaks at m/z 821.1,789.3, and 659.2, due to loss of H2O , loss of MeOH, and combined loss of the sugar moiety and H2O , respectively. The loss of ring A, a typical fragmentation pathway of NCCs , could not be observed in the mass spectrum of Tc-YCC (13), compatible with the C=C bond at ring A. For further characterization of 13 as oxidation product of Tc-NCC-1 (5), a nearly colorless spot of analytically pure 5 on a silica-gel TLC plate was irradiated for 10 min with a 365-nm UV lamp Exper. Part). A major yellow product fraction formed, which had a UV/VIS spectrum typical for a YCC with (Z)-configuration of the C(l)=C(20) bond Tc-YCC (13).A tentative molecular formula of mode see , c, whicTilia cor data) leaves, a first representative of the genus Tilia to be investigated in this respect. Two colorless nonfluorescent chlorophyll catabolites (NCCs) were identified in the senescent leaves, Tc-NCC-1 (5) and Tc-NCC-2 (4b), and their structures were characterized. The basic build-up of 4b and 5 was found to be the same as known for NCCs from higher plants. The available NMR data revealed the functionalization at C(82) of the Tc-NCCs to imply a common β-glucopyranosyl moiety, as in Bn-NCC-2 (12) from oilseed rape (Brassica napus), in Nr-NCC-2 (4b) from tobacco, and in Zm-NCCs 4b and 5 from senescent maize leaves (Zea mays). Furthermore, the spectroscopic data indicated the tetrapyrrolic cores of the two Tc-NCCs 5 and 4b to have a common relative configuration at the stereogenic centers C(1), C(132), and C(15). The absolute configuration at C(1) was deduced to be the same in the Tc-NCCs 5 and 4b as in the glucosylated Nr-NCCs 4b and 6, and Zm-NCCs 4b and 5. Therefore, the absolute configuration at C(1) was indicated to be opposite to that in Bn-NCC-2 (12). NCCs (and FCCs) fall into two classes with respect to the configuration at C(1) due to the evolution of two types of RCC-reductases (RCCR) in higher plants Tc-NCC-1 and Tc-NCC-2, in senescent lime tree leaves parallels the occurrence of NCCs in other senescent plants, where a range of peripheral groups was observed Tc-NCCs provide further support for the view that the main constitutional variations of the chlorophyll catabolites in various higher plants involve enzyme catalyzed, peripheral refunctionalization reactions. The peripheral adaptions with polar functions have been suggested to be of relevance for the transport and for the final deposition of chlorophyll catabolites in the vacuoles In the present work, non-green chlorophyll catabolites were analyzed in fresh extracts of naturally de-greened lime tree ( C(1), C1, and C(1Tc-YCC (13) was identified in fresh extracts of lime tree leaves, and it was suggested to be a product of a naturally occurring oxidation reaction of 5 during senescence. Our study, therefore, provides (further) evidence for the notion that NCCs may not generally be the ‘final products’ of chlorophyll in senescent plants A yellow chlorophyll catabolite C. Kreutz for acquiring the NMR-spectra, and Hans-Jçrg and Hildegard Patscheider for giving us access to the Hotel Linde lime tree. Financial support by the Bundesministerium fur Wissenschaft und Forschung  and by the Austrian Science Foundation  is gratefully acknowledged.We would like to thank General. Commercial solvents (reagent-grade) were redistilled before use for extractions. HPLC-Grade MeOH and Et2O were purchased from Merck (DE-Darmstadt) and Acros Organics (B-Geel). Potassium dihydrogen phosphate,puriss. p.a., potassium phosphate dibasic-anhydrous,puriss. p.a., and ammonium acetate,puriss. p.a., were from Fluka (CH-Buchs).Sep-Pak-C18 cartridges were from Waters Associates. The pH values were measured with a WTW Sentix 21 electrode connected to a 535WTW pH digital pH-meter.Five-g and 1-g HPLC: Dionex Summit HPLC system with manual sampler, P680 pump, online degasser and diode array detector, 1-ml or 20-μl injection loop. Data were collected and processed with Chromeleon V6.50.Phenomenex HyperClone ODS5 μm 250 x 4.6-mm i.d. column at 20° protected with a Phenomenex ODS 4 mm x 3-mm i.d. pre-column was used with a flow rate of 0.5 ml min−1. Solvent A:50 mm aq. potassium phosphate (pH 7.0), solvent B: MeOH; sovent composition (A/B) as function of time (0-75 min): 0-5,80:20; 5-55,80:20 to 30:70; 55-60,30:70 to 0:100; 60-70,0:100;70-75,0:100 to 80:20.a) Anal. HPLC: Phenomenex HyperClone ODS5 μm 250 x 21.2-mm i.d. column at 20° protected with a Phenomenex ODS 10 mm x 5-mm i.d. pre-column was used with a flow rate of 5 and 7 ml min−1. Solvent A: 50 mm aq. potassium phosphate (pH 7.0), solvent B: MeOH.b) Prep. HPLC: MPLC: Buchi MPLC system equipped with two C-605 pumps, a C-615 pump manager, and a C-630 UV-detector at 254 nm. A 460 x 55-mm i.d. column at 208 filled with Phenomenex Spepra-C18-E, 50 mm, 65A was used with a flow rate of 10 ml min−1. Solvent A: 50 mm aq. potassium phosphate (pH7.0), solvent B: MeOH.LC/MS: a) HPLC: Dionex Ultimate HPLC system with manual sampler, He degasser, and diode array detector; 20 μl injection loop; Phenomenex HyperClone ODS5 mm 250 x 4.6-mm i.d. column at 20° protected with a Phenomenex ODS 4 mm x 3-mm i.d. pre-column was used with a flow rate of 0.5 ml min−1. Solvent A: 10 mm ammonium acetate (pH 7.0), solvent B: MeOH; solvent composition (A/B) as function of time (in 75 min): 0-5, 80:20; 5-55, 80:20 to 30:70; 55-60, 30:70 to 0:100; 60-70, 0:100; 70-75, 0:100 to 80:20. Data were collected and processed with Chromeleon V6.50.b) Mass Spectrometry: Finnigan MAT 95, electrospray ionization (ESI) source, positive-ion mode, 1.4 kV spray voltage. MS and MS/MS: Finnigan LCQ Classic, ESI source, positive-ion mode, 4.5-kV spray voltage (rel. abundance).Spectroscopy. UV/VIS Spectra: Hitachi U-3000 spectrophotometer; λmax [nm] (log e/rel. E). CD Spectra: JASCO J715; λmax and λ [nm], 1H-De.  and 13C-NMR: Varian Unity Inova 500MHz spectrometer; Bruker 600 MHz Avance II+ (d(C1HD2OH) 3.31 ppm, and d(13CD3OD) 49.0 ppm) Analysis of Chlorophyll Catabolites in Senescent Leaves by Anal. HPLC. Freshly picked lime tree leaves were collected from a lime tree grown near the Hotel Linde  and immediately stored in liquid N2. A leaf (with the area of ca. 20 cm2) was grounded in a mortar and extracted with 2 ml of MeOH. The resulting suspension was centrifuged 5 min at 13,000 rpm. The methanolic supernatant was diluted with 50 mm aq. potassium phosphate (pH 7.0) 80:20 (v/v). After centrifugation for 5 min at 13,000 rpm, 1 ml of the extract was injected into the anal. HPLC system, to be analyzed with parallel detection at 320 and 420 nm (see 0 nm see .Determination of Chlorophyll in Green and Senescent (yellow) Lime Tree Leaves by UV/VIS Spectroscopy, of Nonfluorescent and of Yellow Chlorophyll Catabolites  in Senescent (yellow) Leaves by Anal. HPLC. Chlorophyll a and b in Green Leaves. A total area of 9 cm2 was cut out of a green lime tree leaf. The leaf was frozen in liquid N2, pulverized in a mortar, and extracted with MeOH. The slurry was filtered through a sintered glass filter, and the residue was grounded in a mortar and extracted with MeOH. The procedure was repeated, until the residue was colorless. The MeOH extracts were combined and diluted with MeOH to 100.00 ml in a volumetric flask. The extracts were analyzed by UV/VIS spectrometry. In green Tilia cordata leaves, 72.09 ± 3.93 mg·cm”2 (80.37 ± 4.38 nmol · cm−2) of chlorophyll a and b were found (n = 4), the data analysis was based on Chlorophyll a and b in Senescent (Yellow) Leaves. A total area of 99 cm2 was cut out of eleven senescent lime tree leaves. The extraction and the UV/VIS analysis were performed as described above. Yellow Tilia cordata leaves were found to contain 0.55±0.10 μig·cm”2 (0.61 ±0.11 nmol·cm−2) of residual chlorophyll a and b.NCCs in Senescent (Yellow) Leaves. The quantification of the Tc-NCCs was accomplished by anal. HPLC. An anal. pure sample of Cj-NCC-1 from Cercidiphyllum japonicum was used to prepare a standard soln.  of Tc-NCC-1, 3.7±1.1 u,g-cm-2 (4.6± 1.4nmol-cm-2) of Tc-NCC-2, and 1.6±0.2 ug-cm-2 (1.9±0.3 nmol-cm-2) of Tc-YCC  leaves (100 g (wet weight)) were frozen in liquid N2, crushed into small pieces, and freeze-dried in vacuo for 3 d, resulting in a dry-weight of ca. 40 g. The dried leaves were pulverized using liquid N2 and a 300-W blender, and extracted with 70 ml of MeOH. The suspension was filtered with suction over a Buchner funnel. The extraction was repeated using another 70 ml of MeOH. The combined 140 ml of MeOH extracts were added portionwise to 350 ml of Et2O to precipitate a raw product that was enriched in chlorophyll catabolites. The raw product was cooled in an ice-bath for 15 min. After filtration with suction the white precipitate was dried in vacuo (dry weight: 1.77 g) and stored at -80° for further purification by MPLC: The crude product was dissolved in 48 ml of aq. potassium phosphate buffer soln.  using an ultrasonic bath. After filtration with a Sartorius filter two aliquots of the filtrate (24 ml of clear red-brownish solutions) were injected into the MPLC system (flow rate: 10 ml min−1); solvent A: 50 mm aq. potassium phosphate (pH 7.0); solvent B: MeOH; solvent composition (A/B) as function of time (0-120 min): 0-90, 70:30; 90-120, 70:30 to 0:100.Tc-NCC-1 and Tc-NCC-2 were collected after 50 and 105 min, resp. The collected fractions were concentrated using a rotary evaporator, freeze-dried in vacuo, and stored at - 808 for further purification.The fractions containing Tc-NCC-1 (5) was dissolved in 0.2 ml MeOH and 2.5 ml of H2O using an ultrasonic bath. After filtration of the suspension through a Sartorius filter the sample was divided in three aliquots and applied to prep. HPLC; injection vol., 1 ml; flow rate, 7 ml min−1; solvent A: 50 mm aq. potassium phosphate (pH 7.0); solvent B: MeOH; solvent composition (A/B) as function of time (0-198 min): 0-180, 90:10 to 65:35; 180-185, 65:35 to 0:100; 185-195, 0:100; 195-198 0:100 to 70:30. Three consecutive prep. HPLC runs were performed, and fractions containing Tc-NCC-1 were collected between 62.5 and 68 min. For de-salting, the aq. soln. was then applied to a pre-conditioned Sepak cartridge, washed with 20 ml of H2O, and eluted with a minimum amount of MeOH. After the sample was dried under high vacuum, 5.1 mg of anal. pure Tc-NCC-1 (5) were obtained.The (more-polar) fraction containing Tc-NCC-2 (4b) fraction was likewise dissolved in 0.6 ml of MeOH and 2.4 ml of H2O using an ultrasonic bath. After centrifugation for 3 min at 13,000 rpm, prep. HPLC  was applied for three aliquots; solvent A: 50 mm aq. potassium phosphate (pH 7.0); solvent B: MeOH; solvent composition (A/B) as function of time (0-180 min): 0-5, 80:20; 5-120,80:20 to 35:65; 120-180: 35:65 to 0:100. Three consecutive prep. HPLC runs were performed, and fractions containing Tc-NCC-2 were collected at 79 min. For de-salting, the aq. soln. was then applied to a pre-conditioned Sepak cartridge, washed with 20 ml of H2O, and eluted with a minimum amount of MeOH. The solvents were removed using a rotary evaporator. To obtain 1.1 mg of an anal. pure Tc-NCC-2, one more prep. HPLC run (the sample was dissolved 0.2 ml of MeOH and 0.6 ml of H2O), followed by Sep-Pak desalting (see above) had to be performed.The (less-polar) Data ofTc-NCC-1 (5). Rt 31.8 min . UV/VIS : 245 (4.16), 314 (4.23). CD ; 226 (18), 258sh (-6), 281 (-17), 315 (3). 1H-NMR : 1.91 ); 2.04 ); 2.10 ); 2.23 ); 2.25-2.36 ); 2.44 ); 2.57-2.67 ,H CH2(81)); 2.68-2.77 ); 2.90 ); 3.16 ); 3.23-3.29 , H-C(5')); 3.32-3.40 , H-C(3')); 3.60-3.73 , Ha-C(6'), Hb-C(82)); 3.74 ); 3.78 ); 3.84 ); 3.94-4.09 , CH2(10)); 4.16 ); 4.56 ); 4.87 ); 8.04 ); 9.30 ); 9.42 ); 11.25 ). 13C-NMR : 8.2 (C(71); 9.1 (C(181)); 9.1 (C(121)); 12.3 (C(21)); 21.8 (C(171)); 23.7 (C(10)); 24.9 (C(81)); 29.4 (C(20)); 37.1 (C(15)); 39.3 (C(172)); 52.6 (C(135)); 62.4 (C(6')); 62.6 (C(1)); 65.9 (C(32)); 67.9 (C(132)) 68.4 (C(31)); 70.3 (C(82));71.2 (C(4')); 75.1 (C(2')); 77.5 (C(5'));77.9 (C(3')); 104.0 (C(1')); 112.3 (C(12)); 115.0 (C(18)); 120.4 (C(17)); 120.5 (C(8)); 124.4 (C(16)); 124.4 (C(19)); 125.9 (C(13)); 129.0 (C(6)); 131.4 (C(3)); 134.2 (C(11)); 135.3 (C(7)); 139.9 (C(9)); 158.7 (C(2)); 161.5 (C(14)); 171.4 (C(133)); 175.1 (C(4)); 176.8 (C(5)); 181.4 (C(173)); 191.6 (C(131)). ESI-MS: 879.43 , 863.49 , 841.49 , 809.47 , 684.35 , 679.33 .Tc-NCC-2Data of (4b). Rt 47.8 min . UV/VIS (50 mm aq. potassium phosphate (pH7.0)/MeOH 65:35, c = 2.6-10-s M): 240 (1.0), 312 (0.83). CD (50 mm aq. potassium phosphate (pH7.0)/MeOH 65:35, c = 2.6-10-s M): 225 (26), 262sh (9), 281 (-19), 318 (3). 1H-NMR : 1.92 ); 1.99 ); 2.14 ); 2.21 ); 2.29-2.38 , CH2(172)); 2.54-2.68 , Ha-C(171)); 2.74–2.81 ); 2.89 ); 3.17 ); 3.24-3.28 , H-C(5')); 3.33-3.36 ); 3.42 ); 3.64 ) superimposed by 3.67 ); 3.74 ); 3.84 ); 3.92-4.13 , CH2(10)); 4.16 ); 4.88 ); 5.35 ); 6.12 ); 6.46 ); 9.27 ). 13C-NMR : 8.4 (C(71)); 9.1 (C(181)); 9.2 (C(121)); 12.2 (C(21)); 22.5 (C(171)); 23.8 (C(10)); 24.9 (C(81)); 30.3 (C(20)); 37.0 (C(15)); 40.3 (C(172)); 52.4 (C(135)); 61.9 (C(1)); 62.3 (C(6')); 67.5 (C(132)); 70.3 (C(82)); 71.2 (C(4')); 75.1 (C(2')); 77.6 (C(3')); 77.6 (C(5')); 103.9 (C(1')); 112.1 (C(12)); 115.2 (C(18)); 118.6 (C(32)); 120.1 (C(8)); 120.3 (C(17)); 124.2 (C(16)); 124.4 (C(19)); 125.4 (C(13)); 126.9 (C(31)); 128.1 (C(3)); 128.7 (C(6)); 133.7 (C(11)); 135.4 (C(7)); 139.5 (C(9)); 156.5 (C(2)); 161.8 (C(14)); 171.3 (C(133)); 174.2 (C(4)); 182.7 (C(173)). ESI-MS: 845.32 , 829.38 , 807.37 , 775.34 , 684.24 , 645.29 .Co-injection Experiments Using Anal. HPLC. Anal. HPLC was used to identify NCCs from freshly prepared MeOH extracts of senescent leaves of the lime tree and of maize. A yellow lime tree leaf was grounded in a mortar with 0.2 g of sea sand, frozen with liquid N2, and extracted with 2.5 ml of MeOH. The suspension was centrifuged at 13,000 rpm for 5 min. The clear supernatant (200 ml) was diluted with 1.3 ml of phosphate buffer (pH 7) and again centrifuged at 13,000 rpm for 4 min. Fractions of Tc-NCC-1 or of Zm-NCC-1 eluted (both) at a Rt of ca. 30 min . For co-injection experiments, separated samples of Tc-NCC-1 and of Zm-NCC-1, as well as a 1:2 mixture of Tc-NCC-1 and Zm-NCC-1 were analyzed by anal. HPLC .Tc-YCCFormation of (13) Tc-NCC-1 (5), and Data ofTc-YCCby Oxidation of  (13). For further characterization of Tc-YCC (13) as oxidation product of Tc-NCC-1 (5), a MeOH soln. of anal. pure 5 was applied to a silica-gel TLC plate. The nearly colorless ‘spot’ acquired a brownish-red color after 10 min of irradiation with a 365-nm UV lamp . HPLC Analysis and a co-injection experiment with isolated Tc-YCC (13) showed 13 to co-elute with the yellow oxidation product of Tc-NCC-1 (5), which had an UV/VIS spectrum typical for YCCs.RData of 13. t 33.8 min . UV/VIS /MeOH 50:50): 247 (0.7), 313 (1.0), 413 (1.3) nm. LC/ESI-MS: 899.20 , 883.20, 877.27 , 861.27 , 839.07 , 821.13 , 789.27 , 659.20 , 627.27, 518.87 . ESI-MS : 821.13 , 807.20 , 659.13 . ESI-MS : 789.07 , 659.13 , 627.07 .Verse 902: “When from the wounded dragon / reeking flowed the blood, And therein did bathe him / the valiant knight and good, Fell down between his shoulders / full broad a linden leaf. There may he be smitten; / ‘tis cause to me of mickle grief”, in ‘Project Gutenberg's The Nibelungenlied', translated into rhymed english verse in the metre of the original, translation by George Henry Needler, January, 2005 .


Structures of 2–6 were confirmed by 1D- and 2D-NMR and HR-ESI-MS spectra. In addition, antifeedant activities of these compounds (1–6) were tested against the 3rd-instar larvae of Mythimna separata. Antifeedant effects of compounds 2 and 4 were greater than the parent compound 1 whereas other compounds exhibited low to no feeding deterrent effects against third instar M. separata larvae in lab bioassays. Therefore, our results suggest that acylated and reduced derivatives at C-8 and C-2, respectively, of 1 may improve the antifeeding effect. This preliminary information will be useful in designing new insect control agents against M. separata and other important pests.Five new derivatives ( During the last three decades, both synthetic and botanical pesticides from a variety of sources have been identified and developed, with most of the plant defensive chemicals discouraging insect herbivory, either by deterring feeding or by impairing larval growth, rather than by killing insects outright ,2. One oParnassia wightiana Wall. (Family: Saxifragaceae) is a widely occurring perennial rhizomatous herb in East Asia, North American and Europe + ion at m/z 615.2219 in the HR-ESI-MS, corresponding to the molecular formula C33H36O10. In the 1H-NMR spectrum of 2, the signal of H-8 for 1 at 4.45 ppm had moved to 5.39 ppm. Further assignments were made based on DEPT, HSQC, and HMBC experiments spectra. Structure, numbering and key NOESY correlations of 2 are shown in Compound cylation  of 1 in 3 was obtained through mild Dess-Martin oxidation at the 8-OH using 1,1,1-tris(acetoxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (DMP). Signal of the C-8 for 3 appears at δ 202.2, stating existence of a carbonyl group. Reduction with NaBH4 at the C-2 carbonyl of 1 at room temperature yielded mainly compound 4 with α-OH configuration at position C-2. This result was supported by the presence of coupling constant (J = 3.0 Hz) of Hax-1 through Hex-2 being located at chemical shift δ 5.56 ppm. This is due to that coupling constant of Hax and Hex is 2~3 Hz, which is smaller than that of Hax and Hax (J = 6~8 Hz).Compound 5 and 6 were obtained using esterification of 1 followed by amination. It is well-known that morpholine is one of the commonly used chemical groups because it plays an important role in medicinal chemistry and agrochemicals, such as morpholine derivatives Amorolfine +, 615.94 [M + Na]+; HR-ESI-MS m/z: 615.2219 [M + Na]+ .Compound 3 : To a stirred solution of compound 1  in DCM (3 mL) was added DMP  at room temperature, and then the mixture was further stirred for 3 h, with monitoring by TLC analysis. The reaction mixture was poured into ice water (10 mL) and extracted with AcOEt . The combined organic layer was washed with NaHCO3 solution and dried over anhydrous Na2SO4. After evaporation in vacuum, the resulting solid was subjected to a silica-gel column chromatography with AcOEt/petroleum ether (1/6) as the eluent to give compound 3 in 90% yields. White solid; AcO-1, BzO-6, BzO-9 was certified by 1H NMR δ: 1.67 , 8.06 , 7.52 , 7.65 , 8.00 , 7.43 , 7.58 ;13C NMR δ: 169.3 (1C), 20.2 (1C), 164.5 (1C), 133.9 (1C), 130.0 (2C), 128.6 (2C), 128.9 (1C), 128.3 (1C), 164.8 (1C), 134.1 (1C), 129.7 (1C), 128.8 (2C), 128.9 (1C), other data see m/z: 565.83 [M + NH4]+, 571.22 [M + Na]+; HR-ESI-MS m/z: 571.1962 [M + Na]+ .Compound 4 : To a stirred solution of compound 1  in anhydrous methanol (10 mL) was added NaBH4  in 10 min at room temperature, and then the mixture was further stirred for 1 h with monitoring by TLC analysis. The reaction mixture was extracted with AcOEt (3 × 10 mL). The combined organic layer was washed with HCl (1 mol/L) solution to pH = 7 and dried over anhydrous Na2SO4. After evaporation in vacuum, the resulting solid was subjected to a silica-gel column chromatography with AcOEt/petroleum ether (1:3) as the eluent to give compound 4 in almost 85% yield. White solid; AcO-1, BzO-6, BzO-9 was certified by 1H NMR δ: 1.73 , 8.01 , 7.53 , 7.65 , 8.10 , 7.48 , 7.60 ; 13C NMR δ: 170.0 (1C), 20.8 (1C), 165.5 (1C), 133.3 (1C), 129.7 (1C), 128.8 (1C), 130.2 (1C), 128.7 (1C), 129.6 (1C), 165.6 (1C), 133.3 (1C), 130.0 (1C), 128.6 (1C), 128.4 (1C), 128.6 (1C), 130.1 (1C), other data see m/z: 552.77 [M + H]+, 575.12 [M + Na]+; HR-ESI-MS m/z: 575.2273 [M + Na]+ .Compound 5 : To a solution of chloroactic acid  in toluene (0.2 mL) at room temperature were added Et3N  and 2,4,6-trichlorobrnzoyl chloride . The resulting mixture was stirred for 1 h before a solution of compound 1  and DMAP  in toluene (0.15 mL) added. The resulting mixture was heated to 80 ºC and stirred for 24 h, then it was quenched with a NaHCO3 (1 mL). The layers were separated and the aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (3 mL), dried (Na2SO4) and concertrated in vacuum. The resulting solid was subjected to a silica-gel column chromatography with AcOEt/petroleum ether (1:10) as the eluent to give compound 5 in almost 85% yield. White solid; AcO-1, BzO-6, BzO-9 was certified by 1H NMR δ: 1.72 , 8.02 , 7.50 , 7.63 , 8.04 , 7.47 , 7.60 ; 13C NMR δ: 169.3 (1C), 20.0 (1C), 164.6 (1C), 133.8 (1C), 129.6 (2C), 128.7 (2C), 129.9 (1C), 166.0 (1C), 133.1 (1C), 128.8 (2C), 128.7 (2C), 128.2 (1C), other data see m/z: 626.71 [M + H]+, 649.01 [M + Na]+; HR-ESI-MS m/z: 649.1829 [M + Na]+ .Compound 6 : To a stirred solution of compound 5  in DCM (10 mL) was added morpholine (0.05 mL) at room temperature, and then the mixture was further stirred for 20 h, with monitoring by TLC analysis. The reaction mixture was poured into ice water (10 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic layer was washed with NaHCO3 solution and dried over anhydrous Na2SO4. After evaporation in vacuum, the resulting solid was subjected to a silica-gel column chromatography with methanol/chloroform (1:50) as the eluent to give compound 6 in almost 50% yield. Yellow solid; AcO-1, BzO-6, BzO-9 was certified by 1H NMR δ: 1.71 , 8.02 , 7.49 , 7.62 , 8.02 , 7.47 , 7.60 ; 13C NMR δ: 169.3 (1C), 20.0 (1C), 164.6 (1C), 133.9 (1C), 129.5 (2C), 128.8 (2C), 129.9 (1C), 168.9 (1C), 133.8 (1C), 129.4 (2C), 128.7 (2C), 128.4 (1C), other data see m/z: 678.2915 [M + H]+ .Compound 3.3.M. separata Walker (armyworm), provided by the Institute of Pesticides, Northwest A & F University, were used as test insect for evaluation of antifeedant activity using a leaf disk choice bioassay  × 100, where C and T are areas consumed of the control and treated leaf disks, respectively. Experiment was repeated twice.Third instar larvae of bioassay . M. sepa4.1) from P. wightiana as a starting material. Some sesquiterpenes showed certain antifeedant activity, and structure-activity relationships suggested that the antifeedant effects of the derivatives depend on the position of hydroxyl and acetoxyl substituents at C-8 and C-2 positions (compounds 2 and 4). This will be helpful in designing new dihydro-β-agarofuran insect control agents against M. separata and other important pests.In conclusion, five new dihydro-β-agarofuran sesquiterpenes were semisynthesized using a dihydro-β-agarofuran with C-2 carbonyl (compound


Rubus chingii was studied in vitro. Ethanolic extract, ethyl acetate and n-butanol fractions from dried R. chingii fruits revealed strong DPPH free radical scavenging activity with IC50 values of 17.9, 3.4 and 4.0 μg/mL, respectively. The ethyl acetate and n-butanol fractions were further purified by a combination of silica gel chromatography, Lobar RP-8 chromatography, and high-pressure liquid chromatography (HPLC). Nine compounds were isolated, where methyl -acetate (2), vanillic acid (5), kaempferol (7), and tiliroside (9) showed stronger DPPH free radical scavenging activity than that of ascorbic acid (131.8 μM) with IC50 values of 45.2, 34.9, 78.5, and 13.7 μM, respectively. In addition, rubusine (1) is a new compound discovered in the present study and methyl -acetate (2), methyl dioxindole-3-acetate (3), and 2-oxo-1,2-dihydroquinoline-4-carboxylic acid (4) were isolated from the fruits for the first time.The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of the fruits of  Fruits and vegetables are known to contain different antioxidant compounds, and high consumption of these products has long been associated with a lower incidence of degenerative diseases. This beneficial association is considered to be partially due to the various antioxidant compounds present in these foods; these antioxidants scavenge free radicals and, thereby, reduce the manifestation of degenerative pathologies –3. ColorR. chingii Hu (Rosaceae), referred to as “fu-pen-zi” in Chinese, have been used as a food and a tonic in traditional Chinese medicine  at m/z 189.0420, corresponding to the molecular formula C10H7NO3. The IR spectrum of 1 revealed absorption bands at νmax 3450–3200 (OH), 2978, 2895 (NH), and 1650 (C = O) cm−1 indicating hydroxyl, imide, and ketone groups, respectively. The 1H NMR spectrum of 1 exhibited an aromatic ring at δ 7.22 , 7.36 , 7.54 , and 8.13 . The 13C NMR spectrum was δ 115.8, 122.3, 126.1, 130.9, 139.4, and 141.2. The 13C NMR chemical shifts for two ketone groups at δ 161.1 were in the B ring. The B ring has a double bond as indicated by the 1H NMR peak of δ 6.86. The 13C NMR peaks of δ 123.4 and 166.8 suggest that the double bond possesses one hydroxyl group. The results of correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond connectivity (HMBC), and NOESY of the 1H and 13C NMR signals are shown in 1) (Rubusine (wn in 1) .2) was a pale tinted powder and exhibited [α]D +10° (MeOH). HR-EIMS revealed an [M+] at m/z 221.0683, corresponding to the molecular formula C10H7NO3. The UV, IR, 1H and 13C NMR spectral data of 2 were similar to those of methyl dioxindole-3-acetate (3), except that the methylene proton peaks were recorded at δ 3.05 and 3.08 , and shifted downfield. In addition, C-2 (δ 180.9) and C-7a (δ 143.7) were shifted downfield by δ 6.1 and δ 5.3, respectively. Comparing with the 1H and 13C NMR data of compound 3, indicated that compound 2 may be an enantiomer of compound 3. The 1H NMR spectrum of 2 also exhibited an ABCD system of aromatic rings at δ 6.88 , 7.01 , 7.25 , and 7.35  and a methoxyl group of esters at δ 3.46. HMBC spectrum of compound 2 showed H-8  correlation to C-2 (δ 180.9), C-3 (δ 42.3), C-3a (δ 131.8), and C-9 (δ 171.1). H-4  was correlated with C-3 (δ 42.3), C-3a (δ 131.8), and C-7a (δ 143.7). Taken together, the structural data point to 2 being methyl -acetate (2). The 1H- and 13C-NMR signals of 2 were completely assigned using contemporary 2-D NMR techniques  and are listed in Methyl -acetate , vanillic acid (5), kaempferol (7), and tiliroside (9) showed significant free radical scavenging activity with IC50 values of 45.2, 34.9, 78.5, and 13.7 μM, respectively. Compound 9 appeared to 9.62-fold as potent as ascorbic acid, whereas 2, 5, and 7 were about 2.92, 3.78, and 1.68-fold, respectively, as potent. Compound 8 had the same antioxidant activity as that of ascorbic acid , the DPPH scavenging activity of kaempferol  was 1.66-fold higher. However, tiliroside  exhibited 5.73-fold higher activity than that of kaempferol (7). Many of the benefits associated with consumption of phenolic-rich foods are correlated with their antioxidant activities +, Measured: 189.0420; EI-MS m/z : 189 , 161 (12), 144 (31), 117 (34); 1H and 13C NMR data are presented in Rubusine (2): white powder; mp 131–132 °C; [α]D +10° ; UV λmax nm: 254.3, 285.5; IR νmax cm−1: 3550–3280, 2982, 2894, 1646; HREIMS m/z 221.0688 [M]+ ; EI-MS m/z : 221 , 161 (81), 148 (100), 133 (20), 120 (38); 1H and 13C NMR data are presented in Methyl -acetate (3): Pale yellow oil; [α]D −3.1°; UV λmax (MeOH) nm: 252.8; IR vmax (KBr) cm−1: 3590–3125, 2975, 1649; EI-MS m/z : 221 , 204 (100), 162 (90); 1H-NMR: δ 2.81, 3.07 , 3.73 , 6.92 , 7.05 , 7.28 ,7.37; 13C NMR : δ 43.1 (C-8), 53.5 (OCH3), 75.2 (C-3), 117.2 (C-7), 124.5 (C-5), 125.9 (C-6), 127.3 (C-4), 131.1 (C-3a), 138.4 (C-7a), 170.8 (C-9), 174.8 (C-2).Methyl dioxindole-3-acetate  free radical scavenging activity was determined by the spectro-photometric method previously described . A methan-butanol, and water fractions of fupenzi exhibit potent DPPH free radical scavenging activity. Nine compounds were isolated, including rubusine (1), methyl -acetate (2), methyl dioxindole-3-acetate (3), 2-oxo-1,2-dihydroquinoline-4-carboxylic acid (4), vanillic acid (5), p-hydroxybenzoic acid (6), kaempferol (7), nicotiflorin (8), and tiliroside (9). Compound 1 is a new alkaloid compound. Compounds 2, 5, 7, 8, and 9 showed potent DPPH free radical scavenging activity. These results suggest that fupenzi may be used as a natural antioxidant to improve the quality, stability, and safety of cosmetics, foods, and medicines.This study demonstrated that the ethanolic extract, ethyl acetate,