O-acetyl and 2,3,4,6-tetra-O-(3-bromo)benzoyl methyl α-D-glucopyranosides have been studied. In addition, a computational study on the deacylation of β-D-glucose pentaacetate has been carried out with density functional theory (B3LYP/6-31G*). The anomeric effect during deacetylation and dealkylation has been clearly demonstrated in both experimental and computational results.The deacylation of glucose, galactose and mannose pentaacetates, galactose and mannose penta(3-bromo)benzoates, as well as the dealkylation of 2,3,4,6-tetra- A. plantanifolium, which contains the glycoside 2-(1-hydroxy-6-oxo-cyclohex-2-ene-1-carboxymethyl)-phenyl-4,6-O--β-D-glucopyranose is used for the treatment of rheumatalgia, paralysis and cardianeuria [Eriobotrya japonica (Thunb.), containing a biphenyl glucoside, have been widely applied as a folk medicine (known as “Pipaye”) to treat stomachache and to promote antitussive, anti-inflammatory and diuretic effects in China [Cucurbita species (commonly known as pumpkin), being traditionally used for a deworming medicine (C. moschata) [C. pepo) [P. sieboldii, have shown allergy preventive effects [in vivo antitumor-promoting effects [2 or p-toluenesulfonic acid [O-benzoyl-α-D-glucopyranose from D-glucose pentabenzoate and phenol in dichloromethane in the presence of BF3·Et2O [O-acetyl-α-D-mannopyranoside through the fusion of 1,2,3,4,6-penta-O-acetyl-β-D-mannopyranose and 4-nitrophenol in the presence of ZnCl2 [2or AlCl3(either in anhydrous Et2O or in the absence of solvent), we only isolated the product deacetylated at the anomeric position, i.e., 2,3,4,6-tetra-O-acetyl-α-D-glucose. On the basis of this unexpected result, we extended our condition to different monosaccharide peresters as well as 2,3,4,6-tetra-O-acyl methyl glucosides. Reported here are the results for deacylation of glucose, mannose and galactose peresters, as well as the dealkylation of acylated methyl α-D-glucosides. Along with the experimental studies, a parallel computational study on the deacetylation of glucose pentaacetate has been performed using density functional theory (B3LYP/6-31G*).A variety of biological activities of plants have been found to be associated with the presence of phenolic glycosides ,5,6,7,8.ianeuria , and is ianeuria . Also, tin China . In addioschata) , and theC. pepo) , are fouC. pepo) . Moreove effects , and cyp effects . Therefonic acid . Althougnic acid . Other dBF3·Et2O , and theof ZnCl2 . However2, AlCl3 or p-toluenesulfonic acid by heating the reaction mixtures around 120–130 °C, no glycosylation product was observed. For example, in the presence of p-TsOH·H2O, the reaction mixture developed into a tar-like mass within one hour at a temperature of 120–130 °C. This substance had a very low solubility in EtOAc. Furthermore, the reaction mixture in the presence of anhydrous ZnCl2 melted at 107 °C and turned into a dark mixture, from which no glycosylation product was isolated by column chromatography except for a small amount of 4-acetylphenyl acetate. In order to promote the expected glycosylation, an even stronger Lewis acid (AlCl3) was applied. After heating the mixture of β-D-glucose pentaacetate and 4-hydroxyacetophenone in the presence of AlCl3 at 87 °C under vacuum for 4.5 hours, the mixture was fractionated by column chromatography to give two major products, i.e., 4-acetylphenyl acetate  and a sugar derivative of a higher polarity than β-D-glucose pentaacetate. This preliminary result indicates that at least one acyl group  has been removed under the thermolytic condition with Lewis acid  since the polarity of the resulting product is increased.Initially, we planned to prepare phenyl glycosides in order to synthesize carbohydrate-containing styrenes for our glycopolymer project. When we attempted the reactions between β-D-glucose pentaacetate and 4-hydroxyacetophenone in the presence of ZnCl3 (2.25 equiv.) was carried out at 110 °C. No reaction was observed when the mixture was maintained at 94 °C or lower. Several hours later, the resulting black solid was pulverized and loaded directly to a short silica gel column and washed with hexane/EtOAc (5:1) to afford a pure compound, that was identified as 2,3,4,6-tetra-O-acetyl-α-D-glucopyranose . In addition, this same reaction proceeded smoothly when the reaction mixture was heated under vacuum, and completed in a short period of time. Furthermore, when β-D-galactose pentaacetate and 2,3,4,6-tetra-O-acetyl methyl α-D-glucopyranoside were subjected to similar conditions, the corresponding 2,3,4,6-tetra-O-acetyl α-D-galacto- and α-D-glucopyranoses were obtained. When this procedure was extended to β-D-lactose octaacetate, the compound 2,3,4,6-tetra-O-acetyl-α-D-galactopyranose was identified, indicating the breakage of the glycosidic bond. Therefore, a direct deacetylation of β-D-glucose pentaacetate in the presence of AlCl3 at 110 °C. It was found that all of the anomeric acyl groups  have been removed from the aforementioned per(3-bromo)benzoates. Moreover, all of the deacylation reactions have been repeated under the optimized condition, in which the respective carbohydrate peresters (either peracetate or per(3-bromo)benzoate) in 4–6 mL of anhydrous Et2O was heated with a small amount of AlCl3 at 110 °C (oven) in a Parr acid digestion bomb for 5 hours. Upon completion of the reaction, the mixture was loaded directly to a short column of silica gel and washed with hexane/EtOAc (8:1 for the reactions from (3-bromo)benzoates and 3:1 for reactions from acetates) to collect the product. However, when these reactions were carried out in a polar solvent, e.g., DMSO, DMF, two or more spots were identified by TLC and they were too close to be isolated in pure forms for structural characterization.For comparison, per(3-bromo)benzoates of α-D-lactose, α-D-glucose, α-D-galactose, α-D-mannose and β-D-mannose  were hea2O or in the absence of solvent, as shown in the deacylation of D-glucose  peresters. In addition, alkyl protecting groups at the anomeric position can also be removed smoothly without affecting other acyl protecting groups on various hydroxyl groups of alkyl glycosides, as indicated by the breakage of the glycosidic bond in both D-lactose and methyl α-D-glucoside derivatives. In addition, the anomeric configuration for β-D-glucose and β-D-galactose derivatives have been inverted in the respective products, e.g., from β-D-glucose pentaacetate to 2,3,4,6-tetra-O-acetyl-α-D-glucopyranose. While α-D-glucose and α-D-galactose per(3-bromo)benzoates [O-(3-bromo)benzoyl-β-D-mannopyranose, indicating the conversion of configuration at the anomeric position during the debenzoylation. The conversion of configuration at the anomeric site has been affirmed by the small NMR coupling constants between H-1 and H-2 in the resulting α-D-glucose (4.0– 4.1 Hz) and α-D-galactose (3.9 Hz) derivatives, which are consistent with the coupling constant of 3.6 Hz between H-1 and H-2 observed in either 2,3,4,6-tetra-O-acetyl methyl α-D-glucopyranoside or 2,3,4,6-tetra-O-(3-bromo)benzoyl methyl α-D-glucopyranoside prepared in this study, whereas the normal coupling constant between H-1 and H-2 in β-D-glucose derivatives was observed in the range of 7–8 Hz [O-(3-bromo)benzoyl D-mannose (1.2 Hz) strongly indicates the β-configuration at the anomeric site.It is found in this study that all of the deacylations occur only at the anomeric position in anhydrous Etenzoates  also undf 7–8 Hz . LikewisD-glucose pentaacetate in the presence of BF3. Furthermore, possible reaction pathways between an oxonium intermediate and water were studied at the same level (III-a and III-b), and the bond energies between β-D-glucose pentaacetate and BF3 were also computed at MP2/6-31G* level with the geometries optimized at the B3LYP/6-31G* level [3 and the oxygen atom on the ring of pyranoside or anomeric acetyl group. It is calculated that the molecular complex arising from the interaction between BF3 and the anomeric acetyl group (structure I-b) is slightly more stable than that formed by the pyranoside oxygen and BF3 (structure I-a). In addition, at the MP2/6-31G* level the bond energy between BF3 and the anomeric acetyl group is -5.4 kcal/mol, which is about 2.5 kcal/mol more exothermic than the interaction between BF3 and the pyranoside oxygen . On the other hand, the facial preference during the attack of water molecule on the oxonium intermediate will determine the final geometry of the product. To rationalize the inversion of configuration at the anomeric position, DFT computations at the B3LYP/6-31G* level have been performed to derive the optimal geometries as well as energy variations for the deacetylation of β-G* level , as showe oxygen . AssuminIII-a) or top side (structure III-b), will lead to the formation of the corresponding 2,3,4,6-tetra-O-acetyl α- or β-glucopyranose, respectively. At the B3LYP/6-31G* level, computations show that the energy for the attack from the top is about 9.6 kcal/mol higher than the total energy of oxonium and water, whereas the bottom attack is 3.5 kcal/mol lower than the total energy of the oxonium intermediate and water combined, indicating that the formation of the α-anomer for the case of D-glucose is favored in kinetics. Interestingly, the gas phase free energy of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranose is computed to be -1297.5029 a.u. , then the yield of the α-anomer is expected to be about 35 times that of the β-anomer. Such a preference for α-D-glucopyranose is consistent with our experimental result that the deacetylation of both α-D-glucose and β-D-glucose pentaacetate either in anhydrous Et2O or in the absence of solvent gives 2,3,4,6-tetra-O-acetyl-β-glucopyranose. It should be pointed out that the order of free energies for α- and β-anomers in a polar solvent such as DMSO is reversed, since the β-anomer has a higher polarity (4.49 Debyes) and a larger solvation energy . The higher solvation energy for the β-anomer over the α-anomer  could reduce the preference to 1/5.91 in the formation of α-anomer if the reaction is carried out in such a polar solvent. This result also partially parallels our experimental result that the deacetylation of β-D-glucose pentaacetate in either DMSO or DMF gave two or more than two products as identified by TLC which are almost inseparable by column chromatography. It is proposed that upon deacetylation, the oxonium intermediate is formed, and the attack of water molecule on the oxonium intermediate either from the bottom  at the Keck/IMD NMR center founded by the W. M. Keck Foundation and the University of Houston. Column chromatography was performed on silica gel using hexane/EtOAc mixtures as eluents. Thin-layer chromatography (TLC) was performed on silica gel  and detected both by UV and heating with 1.5% H2SO4 in EtOH. The high pressure acid digestion bomb (model No. 4746), equipped with a 23 mL PTFE cup (A255 AC), was purchased from the Parr Instrument Company. The high-speed shaker was a Wrist ActionTM Shaker (Model 75) purchased from Burrell Scientific . The preparations of α-D-glucose and α-D-galactose peracetate, as well as α-D-glucose, α-D-galactose, α-D-lactose per(3-bromo)benzoates have been reported previously [All chemicals and solvents were purchased from either Aldrich or Alfa Aesar and were used as received. eviously . In conteviously .D-glucose pentaacetate and BF3 were computed at MP2/6-31G* level with the geometries optimized at the B3LYP/6-31G* level [All the computational work has been performed at the Department of Chemistry, Western Michigan University at Kalamazuo, using the Gaussian 03 package. Among these calculations, the optimal geometries of all the structures have been performed using DFT method at the B3LYP/6-31G* level, and bond energies between β-G* level . In addiG* level . All theD-glucopyranoside  and pyridine (3.0 mL) was added acetic anhydride (3.0 mL). The mixture was stirred overnight and then transferred to a separation funnel with EtOAc (50 mL). The solution was washed with 2 N HCl, saturated NaHCO3 and brine, dried over Na2SO4 and concentrated to afford 2.263 g of 2,3,4,6-tetra-O-acetyl methyl-α-D-glucopyranoside, which was further purified by crystallization in EtOAc/hexane, in almost quantitative yield. 1H-NMR (CDCl3): δ 5.47 , 5.07 , 4.96 , 4.90 , 4.27 , 4.11 , 3.99 , 3.42 , 2.10 , 2.08 , 2.03 , 2.01 ; 13C-NMR (CDCl3): δ 170.50, 169.98, 169.91, 169.45, 96.64 (C-1), 70.64 (C-2), 69.97 (C-3), 68.40 (C-4), 67.00 (C-5), 61.79 (C-6), 55.31 (OMe).To a cold mixture of methyl α-O-(3-bromo)benzoyl methyl-α-D-glucopyranoside, in a yield of 91.2%. 1H-NMR (CDCl3): δ 8.15 , 8.09 , 8.04 , 8.01 , 7.97 , 7.90 , 7.85 , 7.81 , 7.68 , 7.64 , 7.63 , 7.56 , 7.31 , 7.27 , 7.24 , 7.19 , 6.14 , 5.64 , 5.29 , 5.26 , 4.59 , 4.52 , 4.42-4.45 , 3.51 ; 13C-NMR (CDCl3): δ 164.73, 164.44, 164.40, 163.91, 136.52, 136.43, 136.30, 136.14, 132.85, 132.69, 132.62, 131.37, 130.73, 130.48, 130.01, 128.37, 128.31, 128.19, 96.84 (C-1), 72.11 (C-2), 70.82 (C-3), 69.82 (C-4), 67.24 (C-5), 63.28 (C-6), 55.72 (OMe).To a 50 mL flask were added methyl α-D-glucopyranoside (0.529 g) and pyridine (4.0 mL). Then the flask was sealed by septum, vacuumed by a needle, and mounted to a high-speed shaker. After shaking for 10 minutes, the flask was wrapped with dry ice, then 3-bromobenzoyl chloride  was added, and the mixture was left at -78ºC for 1 hour. Then the flask was shaken until all dry ice disappeared, and the light brown residue was transferred to a separation funnel, and worked up accordingly to afford 2.768 g of 2,3,4,6-tetra-2O (5 mL) and AlCl3 . The reaction vessel was sealed and put in an oven at 110 ºC. After 4.5 hours, the ether solution was loaded directly to a silica gel column, and washed by hexane/EtOAc (3:1) to afford 99.9 mg of 2,3,4,6-tetra-O-acetyl-α-glucopyranose (63.4 % yield). fR = 0.17 (hexane/EtOAc = 3:1); 1H-NMR (CDCl3): δ 6.30 , 5.56 , 5.14 , 5.03 , 4.30-4.34 , 4.13 , 2.105 , 2.101 , 2.05 , 2.04 ; 13C-NMR (CDCl3): δ 170.39, 169.75 (2C), 169.35, 90.00 (C-1), 70.61 (C-2), 70.29 (C-5), 69.31 (C-3), 67.30 (C-4), 61.01 (C-6).To a high pressure acid digestion bomb were added β-D-glucose pentaacetate (176.6 mg), Et3 . The vial was swirled to mix the reactants well and left on a hot plate at 110 ºC. After one and a half hours, a weight of 14.04 mg was lost. The residue was transferred to a short silica gel column and washed by hexane/EtOAc (5:1 to 3:1) to afford 27.7 mg of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranose, in a yield of 37.5%, fR = 0.14 (hexane/EtOAc = 3:1); 1H-NNR (CDCl3): δ 6.37 , 5.52 , 5.42 , 5.25 , 4.52 , 4.17 , 4.11 , 2.15 , 2.11 , 2.06 , 2.01 ; 13C-NMR (CDCl3): δ 170.32, 170.12, 169.92, 1369.78, 91.17 (C-1), 69.35 (C-5), 67.85 (C-2), 67.20 (C-4), 67.09 (C-3), 61.00 (C-6).To a 5 mL scintillation vial were added α-D-lactose octaacetate (0.144 g) and AlClO-(3-bromo)benzoyl methyl α-D-glucopyranoside (177.1 mg), AlCl3  and Et2O (4 mL). The reaction vessel was sealed and put in an oven at 110 ºC for 5 h, and the solution was loaded directly to a short silica gel column and washed with hexane/EtOAc (8:1) to afford 97.7 mg of 2,3,4,6-tetra-O-(3-bromobenzoyl)-α-D-glucopyranose, in a yield of 56.0%. fR = 0.39 (hexane/EtOAc = 3:1); 1H-NMR (CDCl3): δ 8.16 , 8.10 , 8.05 , 8.01 , 7.98 , 7.91 , 7.87 , 7.81 , 7.68 , 7.66 , 7.64 , 7.58 , 7.32 , 7.28 , 7.25 , 7.19 , 6.56 , 6.21 , 5.75 , 5.49 , 4.77 , 4.64 , 4.55 ; 13C-NMR (CDCl3): δ 164.58, 164.25, 163.98, 163.74, 136.78, 136.69, 136.48, 136.23, 135.66, 132.89, 132.71, 132.64, 132.45, 131.14, 130.36, 130.10, 129.97, 129.82, 128.62, 128.43, 128.28, 128.20, 128.05, 122.61, 122.50, 89.97 (C-1), 71.72 (C-2), 70.52 (C-5), 70.24 (C-3), 68.53 (C-4), 62.32 (C-6).To a high pressure acid digestion bomb were added 2,3,4,6-tetra-3  and Et2O (4 mL) in a high pressure acid digestion bomb was left in an oven for 5 h (110 ºC). After cooling down to room temperature, the solution was loaded directly to a short silica gel column and washed with hexane/EtOAc (5:1) to afford 177.4 mg of 2,3,4,6-tetra-O-(3-bromo)benzoyl-α-D-galactopyranose (69.6% yield), fR = 0.46 (hexane/EtOAc = 3:1); 1H-NMR (CDCl3): δ 8.17 , 8.12 , 8.11 , 8.02 , 7.92 , 7.88 , 7.76 , 7.73 , 7.67 , 7.66 , 7.59 , 7.39 , 7.30 , 7.28 , 7.17 , 6.67 , 6.08 , 6.04 , 5.78 , 4.95 , 4.64 , 4.50 ; 13C-NMR (CDCl3): δ 164.49, 164.21, 164.19, 163.90, 136.89, 136.75, 136.44, 136.30, 132.79, 132.74, 132.66, 132.62, 130.96, 130.45, 130.41, 130.33, 130.27, 130.09, 129.97, 128.35, 128.24, 128.16, 122.91, 122.60, 122.49, 122.45, 90.98 (C-1), 69.69(C-5), 68.98 (C-2), 68.58 (C-4), 68.14 (C-3), 61.92 (C-6).The mixture of α-D-galactopyranose penta(3-bromo)benzoate (305.9 mg), AlCl3  and Et2O (5 mL). After reaction at 110ºC for 4 hours, the solution was loaded directly to a short silica gel column and washed with hexane/EtOAc (5:1) to give 103.0 mg of 2,3,4,6-tetra-O-(3-bromo)benzoyl-α-D-mannopyranose (87.0% yield), fR = 0.48 (hexane/EtOAc = 3:1); 1H-NMR (CDCl3): δ 8.17 , 8.15 , 8.08 , 7.98 , 7.94 , 7.92 , 7.90 , 7.75 , 7.73 , 7.70 , 7.66 , 7.60 , 7.303 , 7.298 , 7.27 , 7.18 , 6.27 , 6.13 , 6.03 , 5.84 , 4.74 , 4.70 , 4.56 ; 13C-NMR (CDCl3): δ 164.59, 163.98, 163.91, 163.87, 136.92, 136.74, 136.53, 136.24, 132.95, 132.72, 131.33, 130.37, 130.09, 130.03, 128.37, 128.15, 122.91, 122.67, 122.61, 122.53, 88.57 (C-1), 72.64 (C-2), 71.15 (C-5), 69.06 (C-3), 66.57 (C-4), 62.45 (C-6), 20.54, 20.49, 20.45 (2C).To a high pressure acid digestion bomb were added α-D-mannose penta(3-bromo)benzoate (142.1 mg), AlClD-glucose pentaacetate by the action of Lewis acid will give 2,3,4,6-tetra-O-acetyl-α-D-glucopyranose. In addition, experimental results also indicate that the deacylation of D-galactose peresters will give results similar to that of D-glucose perester, whereas the corresponding debenzoylation of α-D-mannose per(3-bromo)benzoate will afford 2,3,4,6-tetra-O-(3-bromo)benzoyl β-D-mannopyranose. Both experimental and theoretical studies show that the deacetylation of α- or β-


E-isomers. To extend the understanding of conformational and thermodynamic preferences of Z-isomeric super-statin analogues, this study focused on analyzing pitavastatin and its lactonized derivatives via NMR spectroscopy and ab initio calculations. Z-isomeric pitavastatin analogues exist in solution as a pair of interconverting rotamers, where the Gibbs free energies between the major and minor rotamers are within 0.12 and 0.25 kcal mol−1 and the rotational energy barriers are between 15.0 and 15.9 kcal mol−1. The analysis of long-range coupling constants and ab initio calculations revealed that rotation across the C5'–C7 single bond is essential for generating a pair of atropisomers. The overall comparison of the results between Z-isomeric pitavastatin and rosuvastatin analogues demonstrated that the former are to some extent more flexible to attain numerous conformations. Demonstrating how structural differences between super-statin analogues induce distinctive conformational preferences provides important insight into the super-statins’ conformational variability and may well improve future drug design.Super-statins are synthetic inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is the rate-limiting enzyme responsible for the biosynthesis of cholesterol. All of the super-statins with a C=C double bond spacer between the heterocyclic and the dihydroxycarboxylic moiety that are currently on the market exist as  E-oriented geometric isomers. In the early discovery stage of super-statins it was shown that some of their Z-isomeric analogues showed only weak  of 0° ± 30° and 180° ± 30°  and θ and –1) and global minimum at –1). Conformations exhibiting  minima at torsion angle θ = 90°, 150° and 270° all minimized to 164° ; an additional local minimum was found at θ = 321° . Frequency calculations verified that the optimized geometries at  minima were stable points on the potential energy surface.Initial structures were generated by Chem3D Pro 10.0 software and energy minimization at B3LYP/6-311+G level was performed using Gaussian 09 . Torsion-6-((Z)-2-(2-Cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)vinyl)-4-hydroxytetrahydro-2H-pyran-2-one (P-2). A solution of Bu4NF∙3H2O  and AcOH  in THF (2.5 mL) was cooled in ice bath. Then a solution of P-1  in THF (2.5 mL) was added. The solution was left to stir for 24 h at room temperature. The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc (5 mL). The organic layer was washed with water (5 mL), saturated solution of NaHCO3 (5 mL), brine (5 mL) and water (5 mL) respectively. Organic layer was dried over Na2SO4 and evaporated under reduced pressure. The residue was purified by column chromatography  to give 170 mg (73% yield) of P-2. 1H-NMR : 7.91 , 7.67 , 7.54–7.24 , 6.61 , 5.80 , 5.16 , 4.24 , 2.61 , 2.50 2), 2.41 , 1.98–1.48 , 1.29 2), 1.06 2). 13C-NMR : 169.8 (C-1), 163.3 , 161.4 (Ar), 148.2 (Ar), 145.8 (Ar), 134.0 , 133.4 (C-6), 132.7 , 130.1 (Ar), 129.9 (Ar), 129.7 (C-7), 128.7 (Ar), 126.53 (Ar), 126.49 (Ar), 126.4 (Ar), 116.3 , 116.1 , 73.4 (C-5), 63.0 (C-3), 39.2 (C-2), 35.9 (C-4), 15.9 (CH(CH2)2), 11.7 2). IR (KBr): 3424, 3063, 3007, 2954, 2922, 1708, 1513, 1488, 1255, 1222, 1159, 1034, 764, 668, cm−1. Mp: 190.8 °C (onset), 192.7 °C (peak). HRMS (ESI): MH+, found: 404.1656. C25H23FNO3 requires 404.1656. c 0.90, CH2Cl2]. -7-(2-Cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)-3,5-dihydroxyhept-6-enoate calcium (P-3). To a solution of P-2  in THF–water, 4:1 (5 mL) at 35 °C was added 8M aq NaOH . After 16 h the reaction was finished and THF was evaporated under reduced pressure. Sodium salt of Z-isomeric pitavastatin spontaneously precipitated from the aqueous solution. The suspension was cooled in an ice bath and THF (1 mL approx) was added drop by drop till all of the precipitate has dissolved again. To a concentrated solution of sodium salt of Z-isomeric pitavastatin was added a solution of CaCl2  in water (0.5 mL). Immediately occurred white precipitate was left to stir for 1 h. Then it was filtered off, washed with water (5 × 2 mL) and dried at 90 °C under vacuum to give 340 mg (77% yield) of P-3. 1H-NMR : 7.90 , 7.60 , 7.49–7.12 , 6.42 , 5.62 , 4.17 , 3.94 , 2.52 2), 2.28–1.94 , 1.80–0.72 2, 2∙OH). 13C-NMR : 181.8 (C-1), 163.9 , 162.3 (Ar), 148.5 (Ar), 146.4 , 137.3 , 134.4 , 133.1 , 130.1 (Ar), 129.8 (Ar), 129.4 (Ar), 127.7 (C-7), 127.0 (Ar), 126.8 (Ar), 126.6 (Ar), 116.4 , 116.3 , 69.0 (C-3), 68.7 (C-5), 44.9 (C-4), 43.8 (C-2), 16.3 (CH(CH2)2), 11.6 2). IR (KBr): 3397, 3067, 3008, 2919, 1561, 1513, 1489, 1417, 1222, 1158, 844, 765, 560 cm−1. Mp:/(amorphous). HRMS (ESI): MH+, found: 422.1764. C25H25FNO4 requires 422.1762. c 0.98, MeOH] for sodium salt of pitavastatin P-3. c 0.25, DMSO] for calcium salt of pitavastatin P-3.Z-isomeric pitavastatin analogues P-1, P-2, and P-3 were synthesized and characterized using various NMR spectroscopy techniques. Acquiring NMR spectra at lower temperature revealed that the resonance line broadening observed at room temperature originates from the dynamic exchange between two rotamers. Firstly, according to the 4JH5-H7 long-range coupling constants, the preferred orientation along the C5–C6 bond for pitavastatin analogues is anti. This orientation is in addition supported by ab initio calculations. Secondly, the determination of mole fractions of minor conformers for pitavastatin analogues, which were between 1.4 and 2.7 fold greater than for rosuvastatin analogues, enabled calculations of thermodynamic preferences. The values of Gibbs free energies for the equilibrium between the major and minor conformers of pitavastatin analogues at room temperature are consequently smaller than for rosuvastatin analogues. Finally, the experimentally established values of rotational energy barriers ΔG‡ for pitavastatin and rosuvastatin analogues are very comparable with each other and in good agreement with their calculated values. The presented results confirm that for Z-isomeric pitavastatin analogues the rotation across the C5'–C7 bond is crucial for generating a pair of atropisomers. Nevertheless, the orientation across the C=C double bond is obviously essential for generating a pair of conformers, for which atropisomerism was not detected in E-isomeric pitavastatin and rosuvastatin analogues.The


Tabernaemontana catharinensis (Apocynaceae) roots, which contains alkaloids with several biological activities, was separated on a preparative scale using high-speed counter-current chromatography. The optimum solvent system was found to be a mixture of CHCl3-MeOH-H2O [5:10:6 (v/v/v)] and led to a successful separation of two monoterpenic indole alkaloids, voachalotine (1) and 12-methoxy-Nb-methylvoachalotine (2) in approximately 4.0 hours. The alkaloids were all isolated at purities over 95%, and their structures were established on the basis of spectroscopic methods, including 1D and 2D NMR and EI/MS.The methanolic extract of  Tabernaemontana includes about 110 species widespread in the pantropical regions, and these species are rich in alkaloids . The spots on the TLC sheets were observed under a UV lamp (254 nm) and revealed with sulfuric vanilin and Dragendorff reagents. Fractions of similar retention factors (RF) were combined and subimetted from the analyses NMR.An aliquot of fraction 12 and the collected 214 fractions were analyzed using silica gel on aluminium TLC sheets  developed with a solvent mixture composed of CHThe spectral data of the two alkaloids are as given below:Voachalotine (1): 1H-NMR : δH 7.50 , 7.21 , 7.11 , 7.30 , 1.63 , 5.34 , 4.22 , 4.33 , 3.25 , 3.15 , 2.99 , 2.02 , 1.83 , 3.71 , 3.62 , 3.60 , 3.64 , 3.76 . 13C-NMR : δC 137.4 (C-2), 105.0 (C-7), 126.0 (C-8), 138.0 (C-13), 53.4 (C-16), 137.5 (C-20), 176.4 (C-22), 48.0 (CH-3), 53.8 (CH-5), 118.3 (CH-9), 119.0 (CH-10), 121.2 (CH-11), 108.9 (CH-12), 29.9 (CH-15), 116.5 (CH-19), 22.3 (CH2-6), 28.4 (CH2-14), 63.1 (CH2-17), 54.0 (CH2-21), 12.9 (CH3-18), 30.3 (CH3N-1), 52.3 (CH3O-22).b-methylvoachalotine12-Methoxy-N (2): 1H-NMR : δH 6.28 , 4.96 , 7.27 , 7.25 , 6.93 , 3.31 , 5.60 , 3.96 , 3.30 , 2.40 , 1.86 , 3.78 , 3.13 , 5.31 , 3.90 , 1.82 , 4.18 , 3.35 , 4.18 , 3.99 . 13C-NMR : δC 132.4 (C-2), 101.4 (C-7), 126.9 (C-8), 147.6 (C-12), 126.9 (C-13), 55.0 (C-16), 127.6 (C-20), 172.8 (C-22), 56.5 (CH-3), 64.2 (CH-5), 111.7 (CH-9), 120.9 (CH-10), 103.1 (CH-11), 29.4 (CH-15), 119.9 (CH-19), 19.0 (CH2-6), 27.7 (CH2-14), 62.2 (CH2-17), 64.0 (CH2-21), 12.1 (CH3-18), 32.9 (CH3N-1), 48.7 (CH3N-4), 55.3 (CH3O-12), 52.8 (CH3O-22).K` (partition coefficient) values of the target compounds in a proper range: the suitable K` values for HSCCC are 0.5 ≤ K ≤ 1.0. In the present study, the suitable solvent system for HSCCC separation was developed according this rule considering the properties of the target compounds . Thus CHCl3-MeOH-H2O with different volume ratios were tested for HSCCC separation of these compounds.The selection of the two-phase solvent system is the most important and critical step in performing HSCCC. One rule for HSCCC mentioned in the literature  was the v/v/v) mixture of CHCl3-MeOH-H2O gave the best results, with the alkaloids of interest being almost equally distributed between the two phases . A good chromatographic resolution was obtained with the lower phase of this solvent system, with RF values of 0.7 and 0.6 for compounds 1–2, respectively. The relatively high proportion of MeOH in the solvent mixture and the RF values of the compounds indicate the medium or high polarity of the T. catharinensis alkaloids. Thus the lower phase was chosen as mobile phase and the upper phase was used as stationary phase for the CCC separation of the MeOH fraction of the methanolic extract of T. catharinensis. Under the conditions used, the retention of the stationary phase in the HSCCC was 82%. After the HSCCC separation, the collected fractions were monitored by UV-detector (300 nm) and combined based on TLC. Spectroscopic analyses allowed us to identify two main compounds, identified by comparison with literature data .,21.v/v/vby CG/MS .T. catharinensis roots: voachalotine  and 12-methoxy-Nb-methylvoachalotine . TLC silica gel plates were eluted in CH2Cl2-MeOH (9:1 (v/v)) and the chemical detection was done by spraying sulfuric vanillin (a) and Dragendorff reagents (b).Nb-methylvoachalotine (2) was the first compound to be eluted (after 2.4 h), because of its lower polarity compared to voachalotine (1). Thus, the separation of these alkaloids seems to depend on the methoxyl group attached to the benzene ring and the methyl group attached to the nitrogen atom at position 4 in alkaloid 12-methoxy-Nb-methylvoachalotine (2). Our results showed that the conditions used provided a very efficient method for the separation of the alkaloids from T. catharinensis roots.This approach led to the isolation of the two alkaloids in approximately 4.0 h. The alkaloid 12-methoxy-Nb-methylvoachalotine (2) exhibited a significative anti acetylcholinesterase effect (results not shown). Since 1–2 were obtained in good amounts and purity, without the need of further steps of purification, we can conclude that the HSCCC conditions used in our work fits well with our objective of obtaining these compounds to perform future acetylcholinesterase inhibitors tests.Preliminary assays with alkaloid 12-methoxy-1) and 12-methoxy-Nb-methylvoachalotine (2) from the roots of T. catharinensis. In particular, preparative HSCCC with its rapid separation and minimum solvent consumption offers a very efficient method for the separation and purification of monoterpenic indole alkaloids.The results of our studies clearly demonstrate the potential of HSCCC for the preparative isolation of voachalotine (


Acanthamoeba genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause Acanthamoeba keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen Laurencia oxasqualenoid metabolites and semisynthetic derivatives were tested against Acanthamoeba castellanii Neff. The results obtained point out that iubol (3) and dehydrothyrsiferol (1) possess potent activities, with IC50 values of 5.30 and 12.83 µM, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 µM, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies. Acanthamoeba spp.  [Acanthamoeba spp. are opportunistic pathogens to humans and other animals [Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE) are the main infections caused by Acanthamoeba spp. Moreover, it is also a causative agent of nasopharyngeal and cutaneous infections [Acanthamoeba is able to invade the stroma and secrete collagenolytic factors, dissolving the stromal matrix, which results in an inflammatory response that leads to corneal cell death and keratitis [Herpes simplex keratitis [Acanthamoeba infections [oebidae)  are oppooebidae) ,3. Acant animals ,5. Acantfections ,7. AK iseratitis ,9. It caeratitis ,11. The fections . In the fections .Laurencia species are prolific producers of sesquiterpenes, diterpenes, triterpenes, and C15 acetogenins, and they have been tested for a wide range of biological activities—cytotoxic, antiviral, anti-inflammatory—and against parasites and their vectors [Marine organisms are the source of structurally diverse and biologically active secondary metabolites, which have inspired the development of several drugs to treat human diseases, despite none being in the therapeutic area of antiparasitics . Among m vectors ,15,16.Laurencia johnstonii, including the most active compound, 3α-bromojohnstane, a halogenated semisynthetic derivative that showed an IC50 of 41.51 µM against Acanthamoeba castellanii Neff [In a previous work, we reported on the amoebicidal activity of the natural sesquiterpenes, α-bromocuparane and α-isobromocuparane, isolated from nii Neff .1) [2) [3) [4) [5) [6 and 7) [8 and 9) [10) [11) [Laurencia viridis (12); 18-ketodehydrothyrsiferol (13); 28-iodosaiyacenols A and B (14 and 15); and 28-hydroxythyrsiferol (16)—were evaluated in vitro against Acanthamoeba castellanii Neff, and a structure–activity relationship was also established.In this work, oxasquealenoids—including dehydrothyrsiferol (DT) (1) ; thyrsif1) [2) ; iubol ( [2) [3) ; 22-hydr [3) [4) ; 1,2-deh [4) [5) ; saiyace6 and 7) ; 28-hydr8 and 9) ; nivario10) [11) —were iso viridis . The sub1–11) obtained from specimens of Laurencia viridis and semisynthetic DT derivatives (12–16) were selected to screen their in vitro activities against Acanthamoeba castellanii Neff. The results of the antiamoeboid effect are indicated in A. castellanii trophozoites. Compound 3, iubol, was the most active compound, with an IC50 of 5.30 ± 0.87 µM, comparable to that of the reference drug chlorhexidine, followed by DT (1) with a value of IC50 of 12.83 ± 1.38 µM. A similar range of activity was observed for thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4) with IC50 values of 13.97 ± 1.57 µM and 17.00 ± 4.57 µM, respectively. In fact, ANOVA showed a nonsignificant difference (p < 0.05) in the activity effect between compounds (2) and (4).A discrete collection of natural oxasquealenoids (3) and DT (1) on the growth of A. castellanii trophozoites at a concentration of 42.5 µM (25 µg/mL). Compared with the negative control, many of the treated cells were dead and others lost their characteristic cellular shape, highlighting their trophocidal activity.1–16 were determined in vitro against murine macrophages (J774A.1). Iubol (3), the most active compound, turned out to be the most cytotoxic with a CC50 of 7.72 ± 0.22 µM. On the other hand, DT (1) was less toxic than 3 with a CC50 of 28.77 ± 3.10 µM, as well as 18-ketodehydrothyrsiferol (13), 28-iodosaiyacenol A (14), and saiyacenol A (6) with CC50 of 23.37 ± 1.76, 29.45 ± 0.20, and 59.91 ± 8.50 µM, respectively. The rest of the molecules showed CC50 values higher than 1000 µM; therefore, due to its cytotoxicity levels, iubol (3) was excluded from further analyses.The toxicities of compounds A. castellanii Neff, with an IC50 of 39.27 ± 0.14 µM. DT was tested against the trophozoite stage of two clinical strains, Acanthamoeba griffini and Acanthamoeba polyphaga, showing IC50 values of 56.66 ± 2.09 and 73.44 ± 4.02 µM, respectively.As it could be observed in 1) was selected as the candidate molecule to continue the studies on the mode of action on A. castellanii Neff. Drugs that do not produce necrotic cell death is the main focus in the search for new therapies. Thus, programmed cell death or apoptosis-like processes in protozoan parasites are the subject of study and include several morphological events such as chromatin condensation, nuclear DNA fragmentation, or a decrease of cellular ATP level, among others [Consequently, considering the preliminary screening values, the major metabolite DT  levels in cubation .1), as a model molecule for a structure–activity relationship (SAR) discussion, the main structural modifications that have been analyzed are summarized in 11) (fragment C-1-C-14), and bromine losses as for 1,2-dehydropseudodehydrothyrsiferol (5) or nivariol A (10) lead to less active substances. In the same way, chemical modifications were carried out on DT (1), including oxidation and sulfonation at C-18 as well as the diol formation at C-15-C-28, which led to substances with very low activities, with 28-hydroxythyrsiferol (16) being the least active compound of this series. All molecules that introduced an additional tetrahydrofuran ring between C-15 and C-18 did not improve their activity with respect to DT (1).Assuming the major compound in the alga, dehydrothyrsiferol (3), which was also the most toxic against murine macrophage J774A.1. Undoubtedly, the two specific modifications that showed the best results were both the introduction of a hydroxyl group, at C-15 as in thyrsiferol (2) and at C-22 as in 22-hydroxydehydrothyrsiferol (4). Thus, 2 and 4 possess a relevant biological activity against A. castellanii Neff, with IC50 of 13.97 ± 1.57 and 17.00 ± 4.57 µM, respectively. This was combined with a very low toxicity against murine macrophage J774A.1 (CC50 < 50), and these were the most important chemical modifications on the leader molecule to improve both its antiparasitic potency and mitigate its lateral toxic effects.On the other hand, modification of the tetrahydrofuran terminal ring between C-19 and C-22 by a tetrahydropyran ring between C-19 and C-23 led us to the most active compound, iubol (n-hexane and ethyl acetate (EtOAc), in different proportions given in each case, as the mobile phase. Melting points were determined on a Büchi B-540 model. Optical rotations were determined on a PerkinElmer 343 polarimeter using a sodium lamp operating at 589 nm. IR spectra were measured on a Bruker IFS 66 spectrometer  using a methanolic solution over a NaCl disk or neat. NMR spectra were performed on Bruker Avance 400, 500, or 600 instruments  at 300 K, and coupling constants are given in Hz. COSY, 1D/2D TOCSY, HSQC, HMBC, and ROESY experiments were performed using standard pulse sequences. Phase-sensitive ROESY spectra were measured using a mixing time of 500 ms. Data were processed using Topspin or MestRe software . Mass spectra were recorded on an LCT Premier XE Micromass spectrometer using electrospray ionization. EnSpire® Multimode Reader  used absorbance values of Alamar Blue® reagent . Thin Layer Chormatography (TLC) was performed on AL Si gel. TLC plates were visualized by UV light (254 nm) and by adding a phosphomolybdic acid solution 10% (w/v) in MeOH or a vanillin solution , 40 mL of EtOAc, and 30 mL of H2SO4).All reagents were commercially available and used as received. All solvents were dried and distilled under argon immediately prior to use or stored appropriately. Flash chromatography was performed with silica gel (230−400 mesh) as the stationary phase and mixtures of Laurencia viridis at the Paraiso Floral, Tenerife, Canary Islands . Dried specimens from the sterile plants, sporophytes, and gametophytes were filed at TFC Phyc 7180 . In all cases, the individual biomasses of L. viridis were extracted with chloroform:methanol (CHCl3:MeOH) (1:1) at room temperature (rt), and a dark green, viscous oil was obtained after concentration under reduced pressure. The extracts were first chromatographed using Sephadex LH-20 (7 × 50 cm) using CH2Cl2:MeOH (1:1) as the mobile phase. The enriched polyether fractions were subsequently processed on a silica gel column (7 × 50 cm) using a linear gradient of n-hexane:EtOAc (80:20−20:80), and the oxasqualenoids fractions were monitored by NMR. Next, medium-pressure chromatography was done on Lobar LiChroprep Si-60 using CH2Cl2:acetone (8:2) at 1 mL/min. Final purification was done on an HPLC with a μ-Porasil column using n-hexane:EtOAc:MeOH, 18:15:5. Using this protocol, the following pure powdery compounds were obtained for the biological assays (% dry weight): dehydrothyrsiferol (DT) (1), (0.093%); thyrsiferol (2), (0.00017%); iubol (3), (0.00018%); 22-hydroxydehydrothyrsiferol (4), (0.000041%); 1,2 dehydropseudodehydrothyrsiferol (5), (0.00029%); saiyacenol A(6), (0.00018%); saiyacenol B (7), (0.00019%); 28-hydroxysaiyacenol A (8), (0.00018%); 28-hydroxysaiyacenol B (9), (0.00019%); nivariol A (10), (0.00013%); and the truncated metabolite adejene B (11), (0.00019%).All metabolites were isolated from biomass obtained from several annual harvestings between April 2013–April 2018 of the red alga 3-pyridine complex  was added to a solution of DT (1)  in pyridine (5 mL) at room temperature. After stirring at 80 °C for 3 h, the reaction mixture was quenched with MeOH. Removal of the solvent gave a residue, which was purified by Sephadex LH-20 column (CHCl3:MeOH 1:1) followed by HPLC chromatography  that afforded 18-sulphatedehydrothyrsiferol (12)  (yield: 62%). 1H NMR : δ 1.18 , 1.18 , 1.18 (s H3-30), 1.21 , 1.21 (s H3-27), 1.26 , 1.39 , 1.45 (H-8), 1.50 (H-9), 1.50 (H-21), 1.54 (H-5), 1.56 (H- 12), 1.57 (H-20), 1.72 (H’-8), 1.76 (H’-12), 1.77 (H’-9), 1.80 (H’-21), 1.81 (H’-5), 1.82 (H-13), 1.99 (H’-13), 2.10 (H-4), 2.23 (H’-4), 2.23 (H- 16), 2.27 (H’-20), 2.40 (H’-16), 3.07 , 3.41 , 3.89 , 4.06 (H- 22), 4.23 , 4.54 (H-18), 4.83 , 5.01 . 13C NMR : δ 19.6 (C-27), 20.3 (C-26), 21.3 (C-24), 21.3 (C-30), 21.8 (C-12), 23.1 (C-8), 23.7 (C-25), 25.3 (C- 29), 26.6 (C-13), 28.1 (C-21), 28.4 (C-4), 28.4 (C-16), 30.9 (C-1), 30.9 (C-17), 31.5 (C-20), 37.4 (C-5), 38.7 (C-9), 59.0 (C-3), 72.3 (C-23), 72.5 (C-14), 72.8 (C-10), 74.2 (C-6), 74.8 (C-2), 78.7 (C-11), 83.6 (C- 18), 85.4 (C-19), 86.5 (C-7), 87.1 (C-22), 109.2 (C-28), 151.4 (C-15).An SO2Cl2 (0.7 mL). The resulting mixture was stirred for 3 h at room temperature (27 °C). Afterwards, the solution was filtered and concentrated to give a solid residue that was chromatographed using HPLC , affording 18-keto-dehydrothyrsiferol  (yield 28%). 18-Keto-dehydrothyrsiferol: 3); 1H NMR:  δ 1.15 , 1.20 , 1.22 , 1.25 , 1.27 , 1.34 , 1.40 , 1.44 (Hα-9), 1.49 (Hβ-8), 1.52 (Hα-5), 1.60 (Hβ-12), 1.73 (H-20), 1.76 (Hα-8), 1.77 (Hβ-9), 1.80 (Hβ-5), 1.81 (Hα-12), 1.84 (H2-21), 1.86 (Hβ-13), 2.11 (Hα-13 and H’-20), 2.12 (H-16), 2.13 (Hα-4), 2.26 (Hβ-4), 2.40 (H’-16), 2.70 , 2.93 , 3.08 , 3.42 , 3.77 , 3.89 , 4.29 , 4.86 , 5.03 . 13C NMR:  δ 19.3 (C-27), 20.2 (C-26), 21.9 (C-12), 23.0 (C-8), 23.5 (C-25), 24.3 (C-24), 24.5 (C-29), 26.3 (C-21), 26.6 (C-13), 27.7 (C-30), 28.0 (C-4), 29.8 (C-16), 31.1 (C-1), 35.0 (C-20), 35.5 (C-17), 37.1 (C-5), 38.9 (C-9), 59.1 (C-3), 70.5 (C-23), 72.5 (C-14), 72.7 (C-10), 74.3 (C-6), 75.0 (C-2), 78.8 (C-11), 86.7 (C-7), 87.6 (C-22), 89.0 (C-19), 109.9 (C-28), 151.4 (C-15), 215.3 (C-18).Pyridinium chlorochromate  was added to a solution of DT (1)  in CH1)  in dry CH2Cl2 (1 mL), and the resulting mixture was stirred for 5 h at room temperature. The solution was filtered and concentrated under reduced pressure to give a mixture of isomeric iododerivatives of saiyacenol A and B. Afterwards, the reaction mixture was then concentrated in vacuo and purified by HPLC , affording 28-iodosaiyacenol A  (yield 22.6%) and B  (yield 43.0%).Iodine  and sodium bicarbonate  were added sequentially to a stirred solution of DT (4 and dehydrothyrsiferol (1)  in 2 mL acetone were added to N-methylmorpholine-N-oxide  in acetone:H2O mixture . The reaction was stirred for 24 h at room temperature. The excess OsO4 was quenched by addition of NaHCO3 , and the solvent mixture was removed in vacuo. The residue was purified by Sephadex LH-20 column (CHCl3:MeOH 1:1) followed by an HPLC  to give the mixture of 28-hydroxytyrsiferol  (yield 70%). 28-hydroxytyrsiferol: 1H NMR : δ 1.14 , 1.16 , 1.20 , 1.21 , 1.23 , 1.28 , 1.35 (H-17), 1.40 , 1.43 (H-8), 1.52 (H-16), 1.53 (H-9), 1.53 (H-12), 1.54 (H-5), 1.60 (H-20), 1.65 (H0 -17), 1.75 (H’-16), 1.76 (H’-8), 1.78 (H’-9), 1.79 (H-13), 1.81 (H’-5), 1.84 (H2-21), 1.92 (H’-12), 1.96 (H’-13), 2.11 (H-4), 2.11 (H’-20), 2.25 (H’-4), 3.07 , 3.45 , 3.48 , 3.61 , 3.73 , 3.77 , 3.90 , 3.96 . 13C NMR  δ 20.1 (C-26), 21.0 (C-12), 21.1 (C-13), 21.4 (C-27), 23.0 (C-8), 23.6 (C-25), 23.9 (C-24), 23.9 (C-29), 24.8 (C- 17), 26.3 (C-21), 27.7 (C-30), 28.3 (C-4), 30.6 (C-16), 31.0 (C-1), 31.7 (C-20), 37.0 (C-5), 38.5 (C-9), 59.0 (C-3), 65.3 (C-28), 70.7 (C-23), 72.7 (C-10), 74.4 (C-6), 75.0 (C-2), 75.3 (C-15), 75.9 (C-11), 76.1 (C- 14), 77.5 (C-18), 86.1 (C-19), 86.7 (C-7), 87.6 (C-22).A catalytic OsO8 and 9, these were also prepared from DT (1). m-Chloroperbenzoic acid (1.5 equiv) was added into a CH2Cl2 (0.5 mL) solution containing 1.5 mg of DT (1) (0.0025 mmol). The resulting mixture was stirred for 3 h at rt. Afterward, the solution was filtered and concentrated to give a solid residue, which was chromatographed using HPLC , affording 0.6 mg of 28-hydroxysaiyacenol A (8)  and 0.8 mg of 28-hydroxysaiyacenol B (9) . All spectroscopic data matched with those of natural compounds.Due to the small amount of sample available from natural source of compounds Acanthamoeba castellanii Neff (ATCC 30010) type strain from the American Type Culture Collection. This strain was axenically incubated in PYG medium (0.75% (w/v) proteose Peptone, 0.75% (w/v) Yeast extract, and 1.5% (w/v) Glucose) supplemented with 10 µg/mL gentamicin . As for the cytotoxicity assays, murine macrophages J774A.1 (ATCC TIB-67) were used, cultivated in DMEM medium  with 10% supplement of fetal bovine serum (FBS) and 10 µg/mL gentamicin at 37 °C in a 5% CO2 incubator.The antiamoeboid activity of the compounds was evaluated against A.griffini, genotype T3 obtained in a previous study [A. polyphaga genotype T4 ATCC 30461. Those strains were grown axenically in the same medium as A. castellanii Neff.Subsequently, DT was tested against two clinical isolates, us study  and A. pAs positive controls, chlorhexidine , voriconazole , and amphotericin B  were used.Laurencia were evaluated using a previously [50) was calculated through nonlinear regression analysis with 95% confidence limits using the software Sigma Plot 12.0 . The experiments were performed three times each, allowing for standard deviation.The in vitro activities of the different compounds of eviously  developeA. castellanii Neff were prepared as previously described [4·7H2O, 0.4 mM CaCl2·2H2O, 1 mM NaHCO3, 20 mM ammediol , pH 8.8 at 25 °C) and were cultured in this medium with gentle shaking for a week in order to obtain mature cysts. After that, mature cysts were harvested and washed twice using PYG medium.The cysticidal activity of DT was determined by the alamarBlue™ assay and confirmed visually by inverted microscopy. Cysts of escribed . Briefly4 mature cysts of Acanthamoeba/mL were added. After 7 d of incubation with the drugs, the plate was centrifuged at 3000 rpm for 10 min. The supernatant was removed and replaced with 100 µL of fresh PYG medium in each well. Finally, 10 μL of the Alamar Blue Assay Reagent  was placed into each well, and the plates were then incubated for 144 h at 28 °C. The emitted fluorescence was periodically examined with an Enspire microplate reader  at 570/585 nm.A serial dilution of the tested molecules was made in PYG. The in vitro susceptibility assay was performed in sterile 96-well microtiter plates (Corning™). To these wells, containing 50 µL of drug dilutions, 5 × 10v/v) inactivated FBS and with 10 µg/mL gentamicin. In this assay, the macrophages were counted and seeded into a 96-well microtiter plate, adding 50 µL to each well from a stock solution of 2 × 105 cells/mL. Cells were then left to adhere for 15 min to the plate bottom and were checked with the inverted microscope Leica DMIL. After that, 50 µL of a serial dilution of the compounds was added to the 96-well microtiter plate. Finally, alamarBlue™ was added into each well at an amount equal to 10% of the final volume and incubated at 37 °C in the presence of CO2 at 5% for 24 h. After this period of time, as in the in vitro anti-Acanthamoeba assay, the fluorescence was determined with an EnSpire Multimode Plate Reader  at a wavelength of 570/585 nm. The cytotoxicity concentration to inhibit the growth of 50% of murine macrophages (CC50) was calculated through nonlinear regression analysis with 95% confidence limits using the software Sigma Plot 12.0 . Experiments were performed three times each, allowing for standard deviation.The cytotoxicity of the tested compounds was determined in vitro with murine macrophages (J774A.1). For this assay, the DMEM culture medium was changed from RPMI without phenol red  to L-Glutamine supplemented with 10%  will show a higher level of blue fluorescence (as chromatin condenses), and dead cells will show low-blue and high-red fluorescence (as the propidium iodide stain enters the nucleus).A double-stain apoptosis detection kit (Hoechst 33342/PI)  and an EVOS FL Cell Imaging System AMF4300  were used. The experiment was carried out by following the manufacturer’s recommendations, and 10® Deep Red fluorescent probe . The cells were treated with IC90 of DT for 24 h and exposed to CellROX® Deep Red  at 26 °C in the dark. Cells were observed in a Leica TSC SPE confocal microscope equipped with inverted optics at λexc 633 and λem 519 nm.Generation of intracellular ROS was detected using the CellROX90 of the test compound for 24 h, the cells were centrifuged (1000 rpm × 10 min) and resuspended in JC-1 buffer. Images were taken on an EVOS FL Cell Imaging System AMF4300  from Life Technologies . The staining pattern allows the identification of two groups in a cellular population: live cells will show only red fluorescence; cells with low mitochondrial potential, (undergoing PCD) will show a higher level of green and red fluorescence.Collapse of the electrochemical gradient across the mitochondrial membrane during apoptosis was detected with the JC-1 mitochondrial membrane potential detection kit . After being treated with IC® Luminescent Cell Viability Assay . This required adding a single reagent , which relies on the properties of a proprietary thermostable luciferase, generating a stable proportional luminescent signal to the amount of ATP present, which is directly proportional to the number of cells present in culture. After incubation, treated amoebas were mixed with the kit reagent into a white-wall 96-well microtiter plate  following the manufacturer’s instructions for measurement of the luminescence on a PerkinElmer spectrophotometer. The effect of the drug on the ATP production was evaluated by incubating 105 cells/mL with the previously calculated IC90 of DT following the manufacturer´s recommendations. As a negative control, an A. castellanii culture incubated with PYG medium was used.The ATP level was measured using a Cell Titer-Glo5 trophozoites were washed and incubated in saline solution with the SYTOX Green at a final concentration of 1 μM (Molecular Probes) for 15 min in the dark. Subsequently, DT solution was added (IC90). After 24 h of treatment, cells were observed in a Leica TSC SPE confocal microscope equipped with inverted optics at λexc 482 nm and λem 519 nm.The SYTOX Green assay was performed to detect alterations of the membrane permeability in treated cells. Briefly, 10p values of <0.05 were considered significant. Statistical analyses were carried out using the GraphPad Prism8.0.2 software program .All experiments were performed three times each in duplicate. The results obtained were analyzed using one-way ANOVA, and Acanthamoeba castellanii Neff trophozoites. Based on the in vitro amoebicidal activity and cytotoxicity assays, iubol (3) was the most active substance; it also turned out to be the most toxic against murine macrophages. The other active substance, the main metabolite dehydrothyrsiferol (1), was chosen as a scaffold for structure–activity relationship studies. The SAR analysis showed that the introduction of a hydroxyl group at C-15 ) or at C-22 ) maintained the activity range but eliminated the toxic effects against macrophages, making them candidates for further activity and mechanisms of action studies. In addition, our studies using dehydrothyrsiferol (1) suggest that these substances cause inhibition of trophozoites by damages at the mitochondrial level. In this paper, we describe the activities against amoebae of a group of triterpenes produced by red algae of the genus Laurencia, establishing a new leader structure on which it is possible to design amoebicidal substances.All tested oxasqualenoids molecules, with natural or semisynthetic origin, demonstrated activity against


S-sanchirhinosides A1 (1), A2 (2), A3 (3), A4 (4), A5 (5), and A6 (6), and sanchirhinoside B (7) were obtained as minor constituents from the root extract of Panax notoginseng , which showed protection effects against antimycin A induced mitochondrial oxidative stress. Their structures were elucidated by chemical and spectroscopic methods . Among them, compounds 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury.Seven new protopanaxatriol type saponins, 20 Reactive oxygen species (ROS) cause protein and DNA injuries and further induce pathological changes, such as heart failure , neuronaPanax notoginseng , have been cultivated in China for more than 400 years. As a traditional Chinese medicine, whose root components have several medicinal properties and are used for stenching the blood, dispersion of gore and reduction of the pain caused by blood diseases, etc. The main components in this plant were identified to be saponins, flavonoids, dencichine and polysaccharides 25D + 12.6° (MeOH). The IR spectrum showed absorption bands at 3,365, 1,717, and 1,654 cm−1 ascribable to hydroxyl, α,β-unsaturated ester, and olefin functions, respectively. The molecular formula, C40H66O10 of 1 was determined by positive-ion HRESI-TOF-MS . The 1H-NMR spectrum of 1 (5D5N) showed signals assignable to nine methyls , three methines bearing oxygen functions , one trisubstituted olefin [δ 5.33 ], one α,β-unsaturated ester moiety , together with an anomeric proton signal at δ 5.06 . The 13C-NMR spectrum displayed 40 carbons, including 30 carbons for the aglycon, six carbons for the sugar unit and four for a butenoyl group. Taken together the 1H- and 13C-NMR spectra suggested that 1 was a dammarane-type triterpene saponin derivative. The chemical shift of δC 61.5 (C-5) indicated that 1 was a protopanaxatriol type saponin . In conjunction with analysis of the HSQC spectrum, the 1H- and 13C-NMR data for 1 were assigned as shown in 5D5N) and 3OD). The 1H 1H COSY experiment on 1 indicated the presence of the partial structure written in bold lines. In HMBC experiment, long-range correlations were observed between the following protons and carbons: H3-18 and C-7−9, 14; H3-19 and C-1, 5, 9, 10; H3-21 and C-17, 20, 22; H3-26 and C-24, 25, 27; H3-27 and C-24−26; H3-28 and C-3−5, 29; H3-29 and C-3−5, 28; H3-30 and C-8, 13−15; H-1' and C-6; H-6' and C-1''; H-2'', 3'' and C-1''; H3-4'' and C-2'', 3'' , 27.0 (C-21), 27.1 (C-16), 35.9 (C-22), 48.3 (C-13), 54.8 (C-17), 126.3 (C-24)] with those of similar 20-epimers of the dammarane type compounds, 20R-gensenoside Rh1   D25 + 7.4°). The molecular formula, C43H72O14, of 2 was determined by positive-ion HRESI-TOF-MS . Acid hydrolysis of 2 yielded D-glucose and D-xylose, which was identified by the same method as 1 ; a β-D-xylopyranosyl [δ 5.76 ]; together with an acetyl group . Furthermore, in the HMBC experiments, long-range correlations between the following protons and carbons were observed: H-1' and C-6; H-1'' and C-2'; H-6' and C-1'' (2 was determined and named as 20(S)-sanchirhinoside A2.C C5D5N,  and vari aglycon  . On the other hand, the 13C-NMR chemical shift of the carbon in the 20-position was shifted from 73.0 , a β-D-glucopyranoside [δ 5.20 ], and an α-L-arabipyranoside [δ 4.98 ]. In the HMBC experiments, long-range correlations between H-1' and C-6, H-1'' and C-20 were observed (3 and 4 were elucidated as 20(S)-sanchirhinosides A3 and A4, respectively, as shown in rabinose . Compareside Rh1  showed iC C5D5N,  data of R C5D5N,  and variobserved . On the 520(S)-Sanchirhinosides A (5) and 6A (6) were both isolated as white powders with positive optical rotations . The molecular formula, C47H80O18, of 5 was determined from positive-ion HRESI-TOF-MS . On the other hand, the molecular formula, C53H90O23, of 6 , was determined from HRESI-TOF-MS, too. Acid hydrolysis of 5 and 6 with 1 M HCl liberated D-glucose (from 5 and 6), D-xylose (from 6), and L-arabinose (from 5) 25D + 14.7° (MeOH), was isolated as a white powder. The molecular formula, C42H70O13, of 7 was determined by positive-ion HRESI-TOF-MS . The 1H-, 13C-NMR  and δH 1.74, 2.81  was observed  and δC 80.0 (C-6); δH 4.98 (H-1'') and δC 77.1 (C-12). Finally, acid hydrolysis of 7 only liberated D-glucose 25D P. notoginseng 70% EtOH extract and new compounds 1–7 against antimycin A-induced mitochondrial oxidative stress were determined. The 70% ethanolic extract and compounds 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury , silica gel , Sephadex LH-20 , and ODS . A Cosmosil 5C18-MS-II  preparative HPLC (PHPLC) column was used to purify the constituents. TLC plates pre-coated with silica gel GF254  were used to detect the purity of isolates by spraying with 10% aqueous H2SO4-EtOH, followed by heating.Optical rotations were measured on a Rudolph AutopolP.notoginseng  were collected from Wenshan, Guangxi province, China and identified by Dr. Li Tianxiang. The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 20120505).The dried roots of P. notoginseng (5.0 kg) were refluxed twice with 70% ethanol-water (volume) for 2 times. Evaporation of the solvent under reduced pressure provided a 70% ethanol-water extract (480.2 g). The residue was dissolved in H2O, then subjected to D101 CC [EtOH-H2O  to afford three fractions (Fr. 1−3). Fraction 3 (120.0 g) was subjected to silica gel CC [CHCl3 → CHCl3-MeOH  → CHCl3-MeOH-H2O ] to give 12 fractions (Fr. 1–12). Fraction 7 (8.0 g) was subjected to normal phase silica gel CC [CHCl3 → CHCl3-MeOH-H2O  → MeOH] to yield fourteen fractions (Fr. 7-1-1–7-1-14). Fraction 7-6 (97.9 mg) was purified by prepared HPLC (PHPLC) [MeOH-H2O ], and sanchirhinoside A1  was obtained. Fraction 8 (4.0 g) was isolated by ODS CC  to yield sanchirhinosides A4 , B , and A2 , respectively. Fraction 9 (16.0 g) was subjected to ODS CC [MeOH-H2O ] to afford nine fractions (Fr. 9-1–9-9). Fraction 9-7 (113.8 mg) was purified by PHPLC [MeOH-H2O ], and sanchirhinoside A3  was obtained. Fraction 10 (3.6 g) was separated by ODS CC [MeOH-H2O ] to afford 15 fractions (Fr. 10-1–10-15). Fraction 10-7 (393.8 mg) was purified by PHPLC [MeOH-H2O ] to give sanchirhinoside A5 . Fraction 12 (10.0 g) was subjected to ODS CC [MeOH-H2O ] to give 13 fractions (Fr.12-1–12-13). Fraction 12-9 (107.8 mg) was further purified by silica gel CC [CHCl3-MeOH-H2O  to yield sanchirhinoside A6 .The dried roots of 120S-Sanchirhinoside A (1): White powder. [α]D25 + 12.6° ; IR νmax (KBr) cm−1: 3,365, 2,928, 2,872, 1,717, 1,654, 1,457, 1,375, 1,316, 1,188, 1,085, 1,045. 1H-NMR  and 13C-NMR  spectroscopic data, see 1H-NMR  and 13C- NMR  spectroscopic data, see m/z 729.4543 [M + Na]+ ; Negative-ion mode m/z 741.4364 [M + Cl]− .220S-Sanchirhinoside A (2): White powder. [α]D25 +7.4° ; IR νmax (KBr) cm−1: 3,367, 2,931, 2,876, 1,733, 1,642, 1,456, 1,373, 1,242, 1,160, 1,075, 1,043. 1H NMR  and 13C-NMR  spectroscopic data, see m/z 835.4832 [M + Na]+ ; Negative-ion mode m/z 847.4568 [M + Cl]− .320S-Sanchirhinoside A (3): White powder. [α]D25 + 19.7° ; IR νmax (KBr) cm−1: 3,367, 2,927, 2,875, 1,647, 1,457, 1,386, 1,253, 1,074, 1,027. 1H-NMR  and 13C-NMR  spectroscopic data, see m/z 793.4720 [M + Na]+ ; Negative-ion mode m/z 815.4779 [M + COOH]− .420S-Sanchirhinoside A (4): White powder. [α]D25 + 23.2° ; IR νmax (KBr) cm−1: 3,366, 2,929, 2,872, 1,643, 1,457, 1,386, 1,255, 1,127, 1,073, 1,043. 1H-NMR  and 13C- NMR  spectroscopic data, see m/z 793.4716 [M + Na]+ ; Negative-ion mode m/z 815.4468 [M + COOH]− .520S-Sanchirhinoside A (5): White powder. [α]D25 + 105.3° ; IR νmax (KBr) cm−1: 3,367, 2,930, 2,875, 1,647, 1,457, 1,386, 1,310, 1,073, 1,042. 1H-NMR  and 13C- NMR  spectroscopic data, see m/z 955.5248 [M + Na]+, calcd. for C47H80O18Na 955.5237); Negative-ion mode m/z 967.4950 [M + Cl]− .620S-Sanchirhinoside A (6): White powder. [α]D25 + 3.1° ; IR νmax (KBr) cm−1: 3,367, 2,929, 2,878, 1,645, 1,456, 1,386, 1,307, 1,074, 1,043. 1H-NMR  and 13C-NMR  spectroscopic data, see m/z 1117.5725 [M + Na]+, calcd. for C53H90O23Na 1117.5765); Negative-ion mode m/z 1093.5731 [M - H]− .Sanchirhinoside B (7): White powder. [α]D25 + 14.7° ; IR νmax (KBr) cm−1: 3,367, 2,927, 2,874, 1,653, 1,457, 1,395, 1,151, 1,072, 1,024. 1H-NMR  and 13C-NMR  spectroscopic data, see m/z 805.4700 [M + Na]+, calcd. for C42H70O13Na 805.4709); Negative-ion mode m/z 817.4518 [M + Cl]− .1–7 (each 1.5 mg) in 1 M HCl (1 mL) was heated under reflux for 3 h, respectively. The reaction mixture was neutralized with Amberlite IRA-400 (OH− form) and removed by filtration. The aqueous layer was subjected to the HPLC analysis under the following condition, respectively: HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. × 250 mm ; detection, optical rotation [Chiralyser ]; mobile phase, CH3CN-H2O ; flow rate 1.0 mL/min. As results, D-xylose , L-arabinose (from 3–5), D-glucose (from 1–7) and were confirmed by comparison of the retention times with the authentic samples .A solution of new compounds 4 cells/well in Dulbecco’s modified Eagle’s medium  supplemented with 10% calf serum (Thermo Scientific) in a 96-well plate and were incubated at 37 °C for 24 h. Cells were treated with or without 10 μmol/L sample DMSO solution . One hour later, medium was removed and 100 μg/mL antimycin A  in 200 μL DMEM was added to each well, The MTT assay was performed 24 h later to detect the cell survival rate. Probucol was used as positive control. Antimycin A was used to induce mitochondrial oxidative stress . Brieflyp < 0.05 was considered to indicate statistical significance.Values are expressed as mean ± S.D. All the grouped data were statistically performed with SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA) and Tukey’s Studentized range test was used for post hoc evaluations. P. notoginseng roots, seven new protopanaxatriol type saponins, 20S-sanchirhinosides A1–A6 (1–6), and sanchirhinoside B (7) were obtained. Among the new compounds, 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury. This research will benefit investigation of trace bioactive chemical constituents of P. notoginseng root. On the basis of the activity screening results, further studies of the antioxidant mechanisms of compounds 1–7 are necessary. Antimycin A is known to cause the leakage of superoxide radicals from cell mitochondria by inhibiting mitochondrial electron transport . Compare


The optimization of the reaction was performed varying parameters, such as type of base/number of reagent equivalents, solvent, temperature and reaction time. The yield of the reaction was improved to 87%. The new synthetic route is versatile as several chromone-2-carboxylic acids (compounds 4B–10B) were obtained with good yields (54–93%). Only in the case of the nitro substituent (compound 11B), an ester was obtained instead of the desired carboxylic acid. Following this synthetic route chromone carboxylic acids can be attained with a high degree of purity, without the need of the tedious and expensive purification processes through column chromatography. The reaction is safe, cost-effective, fast and robust, and can be used in the development of concise and diversity-oriented libraries based on chromone scaffold. The overall study can be looked as a step forward to speed-up the discovery of chromone-based multitarget-directed ligands.6-Bromochromone-2-carboxylic acid ( Chromones are oxygen-containing heterocyclic compounds with a benzoannelated γ-pyrone ring widely distributed in nature and present in notable amounts in several species ,2. They H-chromen-4-one; 4H-1-benzopyran-4-one) is a privileged structure used in drug discovery programs, being the scaffold recognized as a pharmacophore of a great number of bioactive molecules [Chromone , which are versatile synthons owing to its ability to participate in an extensive spectrum of chemical reactions ,3,10.H-chromene-2-carboxylic acid and its derivatives, which are often used in medicinal chemistry to develop small libraries for structure-activity studies  was initially chosen for the optimization study. As the chromone is commercially available it was used as standard allowing a rapid confirmation of its formation along the reaction by thin-layer chromatography (TLC).The synthesis of 6-bromochromone-2-carboxylic acid (3) was carried out using 5′-bromo-2′-hydroxyacetophenone (1) as starting material and reaction conditions similar to the classic reaction described in the literature [The synthesis of 6-bromochromone-2-carboxylic acid (terature .3) by a MW-assisted synthesis (2) and a base; (2) addition of chloridric acid (HCl) and the studies performed for its optimization, which include diverse parameters-type of base/number of reagent equivalents, solvent, temperature and reaction time.Herein we describe a method to obtain 6-bromochromone-2-carboxylic acid  and of the ethyl oxalate lead to an improvement of the yield of compound 3 , Entry 63 , Entry 8Additional modifications in the temperature and time of the reaction were performed. The increase of the temperature of the first reaction step to 140 °C resulted in a decrease of the yield to 19% , Entry 9To improve the acetophenone solubilization the solvent of the reaction was also changed to a less polar  to obtain chromone-2-carboxylic acid derivatives (B) having different type of substituents (After the reaction optimization its versatility was verified using dissimilar 2′-hydroxyacetophenone derivatives (tituents . The sel4A) was used, a decrease in yield  allowed to obtain the desired chromone with a slight lower yield  a similar reaction performance to compound 3, with yields between 81–93%  a reduction of the reaction yield to 62% was observed , instead of the desired chromone carboxylic acid, the corresponding methyl ester was obtained. This shortcoming can be related to the presence of a highly deactivating substituent that can preclude ester hydrolysis by stabilizing the reaction intermediate or by bring about a compound with low solubility in the reaction media.Finally, when the aromatic ring possesses a nitro group in position 6 . 1H- and 13C-NMR spectra were recorded on an AMX 400 NMR spectrometer . Spectra were recorded at room temperature in 5 mm outside diameter (o.d.) tubes. Tetramethylsilane (TMS) was used as internal standard, chemical shifts are expressed in ppm (δ) and J in Hz. For the DEPT sequence, the width of the 90° pulse for 13C was 4 μs, and that of the 90° pulse for 1H was 9.5 μs; the delay 2JC,H −1 was set to 3.5 ms . The melting point were carried out on a Stuart Scientific Melting Point SMP1 apparatus .Microwave-assisted synthesis was performed in a BiotageAll reagents were purchased from Sigma-Aldrich Química S.A.  and TCI Chemicals Europe . All solvents were pro analysis grade from Merck , Carlo Erba Reagents  and Scharlab .Thin layer chromatography (TLC) was performed on precoated silica gel 60 F254 acquired from Merck with layer thickness of 0.2 mm. Reaction control was monitored using ethyl acetate and/or ethyl acetate:methanol (9:1) and spots were visualized under UV detection at 254 and 366 nm.1 or A, 1.16 mmol) was dissolved in dioxane (2 mL) in a MW vial. Then diethyl oxalate  and a solution of MeONa in MeOH  were added. The resulting solution was heated to 120 °C for 20 min. Then, a solution of HCl  was added and the reaction was heated to 120 °C for 40 min. The reaction was decanted over water (50 mL) and the solid formed was filtered and washed with water. The solid was then dried, washed with dichloromethane and dried again.The appropriate hydroxyacetophenone (6-Bromo-4-oxo-4H-chromene-2-carboxylic acid (3): η = 87%, 1H-NMR (DMSO-d6) δ: 8.12 , 8.03 , 7.74 , 6.95 . 13C-NMR (DMSO-d6) δ 176.4 (C4), 160.9 (COOH), 154.4 (C2), 153.6 (C8a), 137.7 (C6), 126.9 (C5), 125.2 (C4a), 121.5 (C3), 118.4 (C6), 113.4 (C8). M.p.: 263–267 °C.4-Oxo-4H-chromene-2-carboxylic acid (4B): η = 54%, 1H-NMR (MeOD) δ: 8.17 , 7.88 , 7.72 , 7.55 , 7.08 . 13C-NMR (MeOD-d4) δ:179.4 (C4), 161.5 (COOH), 156.2 (C2), 153.9 (C8a), 135.1 (C7), 125.9 (C5), 124.9 (C6), 123.8 (C4a), 118.6 (C3), 113.3 (C8). M.p.: >285 °C dec.6-Chloro-4-oxo-4H-chromene-2-carboxylic acid (5B): η = 71%, 1H-NMR (DMSO-d6) δ: 8.08 , 7.83 , 7.71 , 7.06 . 13C-NMR (DMSO-d6) δ: 176.6 (C4), 161.2 (COOH), 154.1 (C2), 153.6 (C8a), 135.0 (C7), 130.5 (C6), 124.8 (C5), 123.9 (C2a), 121.4 (C3), 113.3 (C8). M.p.: 280–284 °C.6-Methyl-4-oxo-4H-chromene-2-carboxylic acid (6B): η = 64%, 1H-NMR  δ: 7.92 , 7.68 , 7.58 , 7.02 , 2.48 . 13C-NMR (MeOD-d4) δ: 180.8 (C4), 163.0 (COOH), 156.0 (C2), 155.1 (C8a), 137.8 (C7), 137.7 (C6), 125.5 (C5), 125.0 (C4a), 119.8 (C3), 114.6 (C8), 21.0 (CH3). M.p.: >279 °C dec.7-Methoxy-4-oxo-4H-chromene-2-carboxylic acid (7B): η = 83%, 1H-NMR (DMSO-d6) δ: 7.95 , 7.22 , 7.12 , 6.86 , 3.94 . 13C-NMR (DMSO-d6) δ: 177.2 (C4), 165.1 (C7), 161.9 (COOH), 158.0 (C2), 153.6 (C8a), 126.9 (C5), 118.2 (C4a), 116.1 (C3), 114.2 (C6), 101.5 (C8), 56.8 (OCH3). M.p.: 283–287 °C.6-Methoxy-4-oxo-4H-chromene-2-carboxylic acid (8B): η = 81%, 1H-NMR (DMSO-d6) δ: 7.71 , 7.47 , 7.42 , 6.89 , 3.88 . 13C-NMR (DMSO-d6) δ: 177.3 (C4), 161.5 (COOH), 157.0 (C2), 153.0 (C6), 150.2 (C8a), 124.5 (C4a), 124.4 (C7), 120.5 (C3), 112.5 (C8), 104.5 (C5), 55.8 (OCH3). M.p.: 293–294 °C.5-Methoxy-4-oxo-4H-chromene-2-carboxylic acid (9B): η = 93%, 1H-NMR (MeOD) δ: 7.73 , 7.20 , 7.03 , 6.91 , 3.95 . 13C-NMR (MeOD-d4) δ: 179.1 (C4), 161.4 (COOH), 159.6 (C2), 158.1 (C5), 151.8 (C8a), 135.4 (C7) 115.0 (C3), 114.2 (C4a), 110.1 (C6), 107.0 (C8), 55.3 (OCH3). M.p.:> 256°C dec.7,8-Dimethoxy-4-oxo-4H-chromene-2-carboxylic acid (10B): η = 62%, 1H-NMR (DMSO-d6) δ: 7.77 , 7.31 , 6.82 , 3.95 , 3.91 . 13C-NMR (DMSO-d6) δ: 176.9 (C4), 161.4 (COOH), 156.8 (C2), 152.9 (C7), 149.7 (C8a), 136.5 (C8), 120.1 (C3), 118.3 (C4a), 112.9 (C5), 111.4 (C7), 60.9 (OCH3), 56.5 (OCH3). M.p.: >287 °C dec.Methyl 6-nitro-4-oxo-4H-chromene-2-carboxylate (11B): η = 83%, 1H-NMR (DMSO-d6) δ: 8.72 , 8.61 , 8.00 , 7.03 , 3.57 . 13C-NMR (DMSO-d6) δ: 176.9 (C4), 160.9 (COOCH3), 158.5 (C2), 154.0 (C8a), 144.7 (C6), 129.2 (C7), 123.7 (C4a), 121.2 (C5), 120.9 (C3), 113.7 (C8), 66.3 (OCH3). M.p.: 274–279 °C.Details of apparatus and NMR spectra of the synthesized compounds are available in 3) using 5′-bromo-2′-hydroxy-acetophenone (1) as starting material and ethyl oxalate (2) was successfully accomplished using a MW-assisted process. For the optimization process a number of parameters were adjusted, such as type of base/number of reagent equivalents, solvent, temperature and reaction time. The yield of the reaction was improved to 87%.The synthesis of 6-bromochromone-2-carboxylic acid (4B–10B) were obtained with good yields (54–93%). Only in the case of the nitro substituent (compound 11B), an ester was obtained instead of the desired carboxylic acid.The new synthetic route is versatile as several chromone-2-carboxylic acids (compounds Following this synthetic route, a variety of chromone carboxylic acids can be attained with a high degree of purity, without the need of the tedious and expensive purification processes through column chromatography. This is a great advantage for the scale-up of the process.The reaction is safe, cost-effective, fast, robust, and can be used in the development of concise and diversity-oriented libraries based on chromone scaffold. The overall study can be looked as a step forward to speed-up the progress of chromone-based multitarget-directed ligands.


S,6R,7S,8R)-5,8-dichloro-6,7-dihydroxy-2-phenylethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (1), and four known ones (2–5), were isolated from 150 mM NaCl-elicited Aquilaria sinensis cell suspension cultures. In addition, three feruloyl amides (6–8), six nucleosides (9–14), (+)-syringaresinol (15), indole-3-carboxaldehyde (16), and two glycosides (17–18) were also obtained. The structures were unambiguously identified by analysis of their UV, IR, NMR, and HRESIMS data. The absolute configuration of the new 2-(2-phenylethyl)chromone (1) was established by a dimolybdenum tetraacetate-induced circular dichroism experiment. Compared to un-elicited cell lines, the appearance of 2-(2-phenylethyl)chromones in NaCl-treated cells occurred on the 3rd and 5th days of their treatment. 2-(2-Phenylethyl)chromones, feruloyl amides, nucleosides, and lignins have been reported to be closely related to plant defense; therefore, the identification of these compounds from NaCl-elicited A. sinensis cell suspension cultures would be useful for further exploring the mechanism of agarwood formation.Five 2-(2-phenylethyl)chromones including a new one, (5 Aquilaria, Gonystulus, and Gyrinops species (Thymelaeaceae) in response to biotic or abiotic stress. Hitherto, more than 100 congeners of 2-(2-phenylethyl)chromones have been reported, and many of them have potentially anti-inflammatory + at m/z 355.0484, in accordance with a empirical molecular formula C17H16O4Cl2 . The ratio of [M + H]+ isotope peaks, 3:2, at m/z 355/357 clearly indicated the presence of two chlorine atoms in 1. In the IR spectrum of 1, absorptions at 3417 cm−1 and 1659 cm−1 suggested the presence of hydroxyl groups and carbonyl group in 1. The 1H-NMR spectrum of 1 showed the presence of a phenylethyl moiety at δH 7.19–7.29 , 2.94–3.04 , a singlet at δH 6.17, and four consecutive methine protons at δH 4.94 , 4.19 , 4.36 , and 4.95 . The 13C-NMR exhibited the presence of 17 carbons including two methylenes at δC 33.8 and 36.2, and four methines at δC 52.0, 58.3, 72.8, and 74.3. Comparison of the NMR data with those of the known Compound 2  were homogeneously mixed, and the induced circular dichroism (ICD) spectrum was recorded. In the ICD spectrum of 1 -5,8-dichloro-6,7-dihydroxy-2-phenylethyl-5,6,7,8-tetrahydro-4H-chromen-4-one  and other 13 known compounds (6–18) were identified as: -8-chloro-5,6,7-trihydroxy-2-phenylethyl-5,6,7,8-tetrahydro-4H-chromen-4-one (2) + ; 1H-NMR  and 13C-NMR  data, see 1 (0.27 mg) was added to a stock solution of 0.6–0.7 mg/mL Mo2(AcO)4  in absolute anhydrous dimethylsulfoxide (DMSO) to achieve the ligand-to-metal ratio is approximately 0.6/0.8 up to 1.0/1.2. CD spectra of Compound 1 were recorded using a Jasco J810 spectropolarimeter, with a 0.1-cm cell in DMSO  and at room temperature, see According to the published procedure , CompounA. sinensis. Most of these compounds have been previously demonstrated to be closely related to plant defense. Thus, it would be meaningful to further explore and understand the mechanism of pharmaceutically important and commercially valued agarwood formation.We have firstly reported five 2-(2-phenylethyl)chromones including a new one, together with 13 other known compounds from NaCl-elicited cell suspension cultures of


All derivatives were tested for antimicrobial activity against a selection of Gram-positive cocci, Gram-negative rods and yeasts. Three compounds, III, IV, and VI, showed antimicrobial activity against Gram-positive bacteria (MIC 50 to 200 μg/mL). Compounds VI and III exhibited antifungal activity against the Candida strains C. albicans and C. parapsilosis (MIC – 100 μg/mL).Halogen derivatives of selected 3-benzofurancarboxylic acids were prepared using 6-acetyl-5-hydroxy-2-methyl-3-benzofuranocarboxylic acid as starting material.  It is well known that many of heterocyclic compounds containing oxygen display important biological properties such as antiarrhytmic, spasmolitic, antiviral, anticancer, antifungal and anti-inflammatory activity ,4,5,6,7.1, for example, shows high affinity for adrenergic receptors and possesses antidepressive activity  stabilizes the conformation of the molecule.The geometry of the benzofuran ring is comparable to that found in other benzofuran derivatives ,24,25,26VI, the packing of the molecules is determined by intermolecular C-H···O hydrogen bonds, Cl···O, C···O interactions and stacking forces. The molecules are linked by C14-H14A···O17 hydrogen bonds forming chains along the a axis, then adjacent chains are connect via C10-H10A···O15 and O1···Cl1 interactions to form layers parallel to the (001) plane  plane .Cohesion between sheets results in Cl···O, C···O interactions and stacking forces. The geometric parameters of all intra- and intermolecular interactions are given in III, IV, VI were active against Gram-positive cocci: Staphylococcus aureus NCTC 4163, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, Micrococcus luteus ATCC 9341, Micrococcus luteus ATCC 10240. Compounds III, VI showed antifungal activity against Candida albicans ATCC 10231, Candida albicans ATCC 90028 and Candida parapsilosis ATCC 22019. The results are summarized in All the synthesized 6-acetyl-5-hydroxy-2-methyl-3-benzofuranocarboxylic acid derivatives were tested for antimicrobial activity. The microorganisms used in this study included Gram-positive cocci, Gram-negative rods and yeasts. Compounds I, II showed no microbiological activity. The change of microbiological activity appears when a halogen is introduced into a benzofuran structure. Derivatives in which an acetyl group hydrogen was substituted by one halogen  were inactive, in contrast to derivatives in which two acetyl group hydrogens were substituted by halogens . These compounds were active against Gram-positive cocci. Additionally, derivatives containing a halogen in the aromatic ring  showed antifungal activity (Unsubstituted esters activity .1H-NMR) were recorded in CDCl3 on a Varian UNITY-plus 300 spectrometer operating at 300 MHz. Chemical shift values are expressed in ppm (parts per million) in relation to tetramethylsilane as an internal standard. Mass spectral ESI (Electrospray Ionization) measurements were carried out on a Mariner PE Biosystems instrument with TOF detector. The spectra were obtained in the positive ion mode with a declustering potential 140–300 V. Elemental analyses were recorded with CHN model 2400 Perkin-Elmer. Chromatographic columns were filled with Merck Kieselgel 0.05–0.2 mm reinst (70–325 mesh ASTM) silica gel. Reactions were monitored by TLC on silica gel , using chloroform-methanol 98:2 and 95:5 as eluents.Melting points were determined in a capillary in Electrothermal 9100 apparatus and are uncorrected. The proton nuclear magnetic resonance spectra (Methyl 6-acetyl-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (I). A mixture of 6-acetyl-5-hydroxy-2-methyl-1-benzofuran-3-carboxylic acid (0.02 mole), K2CO3 (0.02 mole) and (CH3O)2SO2 (0.02 mole) in acetone (30 mL) was refluxed for 5 h. When the reaction was complete, the mixture was filtered and the solvent was evaporated. The residue was purified by column chromatography on silica gel, eluent: chloroform. Yield: 87%; m.p. 170–171 °C; 1H-NMR δ (ppm): 12.17 , 7.76 , 7.46 , 3.93 , 2.78 , 2.67 ; Anal. Calc. for C13H12O5: 62.90% C; 4.87% H; found: 62.87% C, 4.87% H; ESI MS m/z: 247.1, 248.8 (100%).Methyl 6-acetyl-5-methoxy-2-methyl-1-benzofuran-3-carboxylate (II). A mixture of 6-acetyl-5-hydroxy-2-methyl-1-benzofuran-3-carboxylic acid (0.02 mole), K2CO3 (0.02 mole) and excess (CH3O)2SO2 (0.06 mole) in acetone (30 mL) was refluxed for 10 h. When the reaction was complete, the boiling mixture was filtered and the solvent was evaporated. The residue was purified by column chromatography on silica gel, eluent: chloroform. Yield: 78%; m.p. 118–119 °C; 1H-NMR δ (ppm): 7.83 , 7.48 , 3.87 , 3.95 , 2.76 , 2.65 ; Anal. Calc. for C14H14O5: 64.12% C; 5.38% H; found 64.19% C; 5.39% H.I or II (0.02 mole) was dissolved in CHCl3 (20 mL) then a solution of bromine (0.04 mole) in CHCl3 (10 mL) was added dropwise with stirring for 0.5 h. Stirring was continued for 8 h at room temperature. When the reaction was complete the solvent was evaporated. The residue was purified by column chromatography on silica gel, eluent: chloroform.The appropriate ester Methyl 4-bromo-6-(dibromoacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (III). Yield: 77%; m.p. 149–150 °C; 1H-NMR δ (ppm): 11.98, 8.00 , 6.72 , 3.95 , 2.63 ; Anal. Calc. for C13H9O5Br3: 32.20% C; 1.87% H; found 32.38% C; 2.09% H; ESI MS m/z: 506.8:508.7 [M+Na]+ (100%).Methyl 6-(dibromoacetyl)-5-methoxy-2-methyl-1-benzofuran-3-carboxylate (IV). Yield: 78%; m.p. 135–138 °C; 1H-NMR δ (ppm): 7.95 , 7.54 , 7.22 , 4.05 , 3.98 , 2.80 ; Anal. Calc. for C14H12O5Br2: 40.03% C; 2.88% H; found 39.94% C; 2.77% H.I or II (0.02 mole) was dissolved in CHCl3 (20 mL). Next chlorine, obtained in the reaction of KMnO4 with concentrated HCl, was passed through the solution. When the reaction was complete the solvent was evaporated. The residue was purified by column chromatography on silica gel, eluents: chloroform and chloroform/methanol 100:0.5.The appropriate ester Methyl 4-chloro-6-(chloroacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (V). Yield: 23%; m.p. 187–188 °C; 1H-NMR δ (ppm): 12.11 , 7.74 , 4.74 , 3.96 , 2.68 ;; Anal. Calc. for C13H10O5Cl2: 49.24% C; 3.18% H; found 49.14% C; 3.24% H; ESI MS m/z: 339.6:341[M+Na]+ (100%).Methyl 4-chloro-6-(dichloroacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (VI). Yield: 36%; m.p. 171–172 °C; 1H-NMR δ (ppm): 11.70 , 7.98 , 6.73 , 3.96 , 2.68 ; Anal. Calc. for C13H9O5Cl3: 44.41% C; 2.58% H; found 44.60% C; 2.71% H; ESI MS m/z: 350.8:352.6 [M+H]+ (100%).Methyl 6-(chloroacetyl)-5-methoxy-2-methyl-1-benzofuran-3-carboxylate (VII). Yield: 54%; m.p. 130–131 °C; 1H-NMR δ (ppm): 7.69 , 7.19 , 4.75 , 3.88 , 2.60 ; Anal. Calc. for C14H13O5Cl*2H2O: 50.53% C; 3.90% H; found 50.60% C; 3.90% H.VI suitable for X-ray analysis were grown from acetic acid solution by slow evaporation. All details of the measurements, crystal data and structure refinement are given in MoKα radiation. The unit cell parameters were determined by least-squares treatment of setting angles of highest-intensity reflections chosen from the whole experiment. Intensity data were corrected for the Lorentz and polarization effects [o|2-|Fc|2) differences. The function Σw(|Fo|2-|Fc|2)2 was minimized with w‑1 = [σ2(Fo)2+(0.0474P)2+0.6944P], where P = (Fo2+2Fc2)/3. All non-hydrogen atoms were refined with anisotropic thermal parameters. The coordinates of the hydrogen atoms were calculated in idealized positions and refined as a riding model with their thermal parameters calculated as 1.2 (1.5 for methyl group) times Ueq of the respective carrier carbon atom. An empirical extinction correction was also applied according to the formula Fc’ = kFc[1+(0.001χFc2λ3/sin2θ)]-1/4 -1/4 , and theStaphylococcus aureus NCTC 4163, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, Enterococcus hirae ATCC 10541, Micrococcus luteus ATCC 9341, Micrococcus luteus ATCC 10240; Gram-negative rods: Escherichia coli ATCC 10538, Escherichia coli ATCC 25922,Escherichia coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseudomonas aeruginosa ATCC 15442, Pseudomonas aeruginosa NCTC 6749, Pseudomonas aeruginosa ATCC 27863, Bordetella bronchiseptica ATCC 4617 and yeasts: Candida albicans ATCC 10231, Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019. The antimicrobial activity of compounds was tested against a series of microorganisms obtained from the collection of the Department of Pharmaceutical Microbiology, Medical University of Warsaw (Poland), including: Gram-positive cocci: 4 CFU mL-1 (colony forming units per mL), except the final inoculum for E. hirae ATCC 10541, which was 105 CFU mL-1. The results were read after 18 h (for bacteria) or 24 h (for fungi) of incubation at 35 °C. The results are summarized in Antimicrobial activity was examined by the disc-diffusion method and the MIC method under standard conditions using Mueller-Hinton II agar medium (Becton Dickinson) for bacteria or RPMI agar medium (Sigma) with 2% glucose for fungi, according to CLSI guidelines ,32. For This study describes the synthesis and preliminary microbiological investigations of some 3-benzofurancarboxylic acid derivatives. Seven derivatives were examined. It was noticed that compounds not possessing halogen in their structure did not show any activity during the experiments, whereas compounds possessing halogens in their structure showed such activity. Halogens were introduced to alkyl substituent as well as to the aromatic ring. Derivatives in which the halogen, without the regard for the kind, was introduced in the aromatic ring, showed better activity, as these compounds displayed both antimicrobial and antifungal activity. Basing on these findings, as well as on results from our earlier investigations, we can confirm that the search for biologically active compounds in the area of halogen derivatives of benzofurans remains an interesting field from the biological point of view.