Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1) and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen- 4-one (2), together with eleven known compounds: 4-hydroxy-2-[tridecanoyl] cyclohexane-1,3-dione (3), oleiferinone (4), 4-hydroxy-2-[undecanoyl]cyclohexane-1,3-dione (5), 4-hydroxy-2-[(11-phenyl- undecanoyl)cyclohexane-1,3-dione (6), proctorione C (7), surinone C (8), 5-hydroxy- 7,8,4'-trimethoxyflavone (9), 5-hydroxy-7,8,3',4'-tetramethoxyflavone (10), 5-hydroxy- 7,3',4'-trimethoxyflavone (11), 5,8-dihydroxy-7,3',4'-trimethoxyflavone (12) and cepharanone B (13) were isolated from the whole plant of Peperomia dindygulensis Miq. Their structures were elucidated by spectroscopic methods, including 2D-NMR techniques. Compounds 2, 3, 5 and 8 inhibited human umbilical vein endothelial cell (HUVEC) proliferation and compounds 5 and 8 sharply suppressed HUVEC tube formation.Two new polyketides: 2 Peperomia dindygulensis Miq. (Piperaceae), a widespread herb in the south of China, is used in the folk medicine to treat cough, asthma, phthisis, and stomach, lung, mammary and liver cancers  cyclohexane-1,3-dione (3)  and five methylenes  were found to be connected to two moieties, 6CH2–7CH2–8CH2 and O–4CH–3CH2–2CH(O)–1'CH2 using the 1H–1H COSY spectrum. The above two moieties were linked by the two quaternary olefinic carbons δC 173.4 (C-8a) and 114.7 (C-4a) which showed HMBC cross-peaks with H-4, H-7, H-8, and with H-3, H-8, respectively. The location of carbonyl carbon δC 200.6 (C-5) could be confirmed by the HMBC correlations with H-6 and H-7. Thus, the moiety were determined as O=5C–6CH2–7CH2–8CH2–8aC=4aC–4CH(O)–3CH2–2CH(O)–1'CH2 and further confirmed by the homoallylic coupling between H-4 and H-8 in the 1H-1H COSY spectrum. The downfield chemical shift of C-8a and the remaining two degrees of unsaturation suggested that the oxygen at C-2 connected to C-8a, and C-5 to C-4a. The 5-hydroxytetrahydrochromen moiety was also confirmed by the mass spectrum which showed a fragment at m/z 167 (1 was elucidated as 2Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one. The NOE correlation between H-2 and H-4 indicated their cis-configuration.Compound spectrum . Two olespectrum  suggestespectrum , HMBC an spectra . The cisδC 26.9) . Two oxy m/z 167 . Thus, t2 had the molecular formula C26H42O3 as deduced from the HRESI-MS peak at m/z 425.3029 [M+Na]+ . The UV maxima at 249 nm, and the IR absorptions at 1,661, 1,605 cm−1 suggested the presence of a γ-pyrone ring, which was 2,3,5-trisubstituted according to the olefinic signals at δC 112.7 (C-3) and δH 6.10  in the NMR spectra –6CH2–7CH2–8CH2, and a alkyl chain from the 1H-NMR, 13C-NMR, and 1H-1H COSY and HMBC spectra  showed correlation with the carbonyl carbon (δC 180.7), C-4a (δC 123.2), C-6 (δC 29.5), C-7 (δC 18.1) and C-8 (δC 27.6), indicating that C-5 is connected with C-4a. The protons of the methylene [δH 2.46  and 1.98 ] gave cross peaks with the carbons at C-5, C-6, C-7, C-4a and C-8a, but not with C-4, suggesting that C-8 is attached to C-8a. Surprisingly, the weak four bond reciprocal H-5/C-8 and H-8/C-5 HMBC correlations signals were observed. Although these may be considered unusual, such long-range correlations have been reported, especially in constrained ring systems . Si. Si2 had spectra . In the  systems ,22,23. T3~13 were confirmed by detailed NMR and MS data comparison with those in the literature  to give four fractions, and the second fraction (700 mg) was applied to preparative silica gel TLC using n-hexane/acetone/acetic acid (5:1:0.1) as eluent to afford 4 (40 mg), 5 (100 mg) and 6 (12 mg). Fraction 7C (7.6 g) was chromatographed over silica gel columns (3 × 50 cm) and eluted with n-hexane/EtOAc (3:1) to obtain five fractions, the second fraction (154 mg) was applied to preparative silica gel TLC using toluene/EtOAc (9:1) as eluent to give 11 (4 mg). Fraction 8 (4.5 g) was chromatographed over silica gel columns (3 × 50 cm) eluted with CHCl3/MeOH (50:1) to obtain four fractions (8A–D). Fraction 8B (205 mg) was recrystallized from CHCl3/MeOH (1:1) to afford 9 (51 mg). Fraction 8C (600 mg) was recrystallized from CHCl3/MeOH (1:1) to afford 10 (246 mg). Fraction 8D (1.4 g) was chromatographed over silica gel columns (3 × 50 cm) and eluted with n-hexane/acetone/acetic acid (100:10:1) to give 2 (277 mg) and 6 (36 mg). Fraction 9 (18.9 g) was separated repeatedly on silica gel columns (5 × 50 cm) using n-hexane/acetone (4:1) as eluent to obtain eight fractions (9A–H). Fraction 9B (1.5 g) was further separated repeatedly on silica columns (3 × 50 cm) and eluted with CHCl3/acetone (200:1) to give three fractions, and the second fraction was subjected to preparative silica gel TLC using n-hexane/acetone/acetic acid (100:10:1) as eluent to give 8 (20 mg). Fraction 9D (1.8 g) was chromatographed on silica gel columns (3 × 50 cm) using n-hexane/acetone (5:1) as eluent to obtain three fractions, and the last fraction (222 mg) was further purified by Sephadex LH-20 and eluted with CHCl3/MeOH (1:1) to give 13 (5 mg). Fraction 9E (12.2 g) was separated repeatedly on silica gel columns (5 × 50 cm) using CHCl3 as eluent to obtain six fractions, and the last fraction was separated repeatedly by Sephadex LH-20 and eluted with CHCl3/MeOH (1:1) to give 12 (12 mg).The air-dried whole plant (10 kg) of 2Z-(Heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1). Yellowish oil; [α]D13 126° ; UV λmax (MeOH) nm (log ε): 258 (4.44); IR (NaCl) νmax: 3466, 2925, 2854, 1613, 1427, 1370, 1249, 1188, 1083, 1054 cm−1; EI-MS m/z 404 [M]+ (9.1), 386 (100.0), 167 (24.1), 139 (50.7), 125 (43.1), 111(41.9), 97 (5.2), 43 (21.5); 1H- and 13C-NMR data: see m/z 427.3185.2-(Heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen-4-one (2). Yellowish oil; [α]D13 34° ; UV λmax (MeOH) nm (log ε): 216 (3.66), 249 (3.68); IR(NaCl) νmax: 3431, 3003, 2926, 2854, 1716, 1661, 1605, 1436, 1176, 1086, 950, 859, 722 cm−1; 1H- and 13C-NMR data: see m/z 425.3029 .in vitro angiogenesis using the Chemicon in vitro angiogenesis assay kit (ECM625). Details of the assays were provided in a previous report [The effect of isolated compounds on the proliferation of HUVEC was evaluated by CCK-8. HUVEC tube formation was conducted for the assay of s report .Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1), and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen-4-one (2), were isolated from Peperomia dindygulensis, together with eleven known compounds 3–13. Compounds 2, 3, 5 and 8 inhibited human umbilical vein endothelial cells (HUVEC) proliferation and compounds 5 and 8 sharply suppressed HUVEC tube formation.Two new polyketides: 2


A number of representative nucleophilic additions (RMgX and RLi) on 2 afforded α-ketoacetals 3a–j in 70–99% yield. These compounds represent a versatile arrangement of functional groups of significant synthetic value, as demonstrated in the synthesis of (±)-salbutamol.The Weinreb amides  They offer the possibility of performing the selective functionalization of a keto group over the more reactive aldehyde, as the latter is protected as an acetal. For instance, α-ketoacetals are key intermediates in the preparation of chiral cyanohydrins  in anh. THF at −78 °C gave WA 2a in 30% yield (R = Me), while 2b (R = Et) was only obtained in trace amounts  . Thus, tyl amine ,23,24 , a , a 28], 3c was reduced in quantitative yield to the corresponding secondary carbinol, which was hydrolyzed to the cyclic dimer of the corresponding α-hydroxyaldehyde 6. The latter was directly treated, without isolation, with primary amines followed by reduction of the imine intermediate 6 with NaBH4 to afford the corresponding 1,2-aminoalcohols 7a–d in 82–93% yield. This protocol was then successfully implemented for the total synthesis of (±)-salbutamol, a β2-adrenergic receptor agonist used for the treatment of chronic obstructive pulmonary disease. Thus, starting from 6-bromosalicylic acid (8), bromobenzodioxane 9 was obtained after reduction of the carboxyl group of 8 and after the successive formation of the dioxane functionality -salbutamol was obtained in 81% yield.For instance, α-ketoacetal ionality . Treatme1H and 13C-NMR spectra were recorded on Varian spectrometers at 500/125 and 300/75 MHz using CDCl3 as solvent and TMS as internal standard. Chemical shift values (δ) are reported in ppm . Proton spectra are reported as follows: δ . Multiplicities are indicated by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), st (sextet), m (multiplet), bs . Infrared spectra were recorded on a Perkin-Elmer Spectrum 2000 spectrophotometer. High Resolution Mass Spectra (HRMS) were determined with a JEOL GCmate spectrometer by Electronic Impact (EI) ionization. Thin-layer chromatograms (TLC) were done on precoated TLC sheets of silica gel Merck 60F-254. Spots on TLC were revealed by using UV lamp, ceric sulfate, iodine chamber or 2,4-dinitrophenylhydrazine. Purification of compounds was performed by column chromatography on silica gel (Merck 230–400 mesh). A Kugelrohr SEV 200 apparatus was also used for liquid samples. THF was freshly distilled from a purple solution of sodium and benzophenone under nitrogen atmosphere. Some reagents were purchased from Sigma-Aldrich Chemical Co. and used without further purification.N,O-Dimethylhydroxylamine hydrochloride  in THF (60 mL) was cooled for 10 min. at −78 °C in a bath of acetone-dry ice. A freshly prepared solution of isopropylmagnesium chloride  was slowly added to the above solution, maintaining a vigorous stirring for 30 min under nitrogen atmosphere. To the resulting reaction mixture, methyl dimethoxyacetate  was added dropwise and the reaction mixture was stirred for 1h at −78 °C, then quenched with 20 wt % NH4Cl. The reaction was extracted with dichloromethane (3 × 30 mL) and the organic layer was dried over anhydrous Na2SO4 and evaporated to dryness. The product was purified using a Kugelrohr apparatus at 40 °C (0.5 mmHg), obtaining amide 2a  as a pale yellow oil. The same procedure was followed to prepare amide 2b, starting from ethyl diethoxyacetate (1b).1c  in CH2Cl2 (10 mL) at 0 °C; triphosgene  and triethylamine  were added. Then N,O-dimethylhydroxylamine hydrochloride  was added to the solution and the ice bath removed. The reaction mixture was stirred at room temperature for 1h. The reaction was extracted with EtOAc (3 × 10 mL). Then; the organic phase was dried with anhydrous Na2SO4 and concentrated in vacuum. The product was purified by column chromatography on silica gel using EtOAc-n-hexane (1:1) as eluent to give 2a . Rf = 0.13 n-hexane:EtOAc (4:1). νmax (film): 2940; 1679; 1456; 1196; 1066; 977 cm−1. 1H-NMR : δ 5.22  C-1; 3.75  MeON; 3.45  (OCH3)2; 3.20  N-CH3. 13C-NMR : δ 167.5 (C-1); 96.3 (C-2); 61.5 (N-OCH3); 53.4 ((CH3O)2); 32.1 (N-CH3). EI-HRMS: calculated for C6H13NO4 163.0845; observed 163.0852.To a stirred solution of the carboxylic acid 2,2-Diethoxy-N-methoxy-N-methyl-acetamide (2b). To a stirred solution of carboxylic acid 1d  in CH2Cl2 (35 ml) at 0 °C, triphosgene  and triethylamine  were added. Then N,O-dimethylhydroxylamine hydrochloride  was added to the solution and the ice bath removed. The reaction mixture was stirred at room temperature for 2h, and then filtered, dried with anh. Na2SO4 and concentrated in vacuo. The product was purified by column chromatography on silica gel using EtOAc-n-hexane (1:1) to give 2b . Rf = 0.33 n-hexane-EtOAc (6:4). νmax (film): 2977, 2934, 1683, 1444, 1146, 1062, 987 cm−1. 1H-NMR : δ 5.21  H-1, 3.61  NOCH3, 3.59  2 OCH2, 3.18  NCH3, 1.22  (CH3)2, 13C-NMR : δ 168.0 (C-1), 94.6 (C-2), 63.1 (OCH2), 62.2 (NOCH3), 33.3 (NCH3), 14.8 (CH3). EI-HRMS: peak for molecular ion (C6H12NO4) not observed. Calculated for [M−OMe]+ 160.0974 (C7H14NO3); observed 160.0974.2a  in THF (6 mL), cooled at −78 °C, the organometallic reagents (1.5–2.0 eq.) were slowly added, maintaining vigorous stirring under nitrogen atmosphere for 1 h. Then, the reaction was quenched with a saturated solution of NH4Cl. The reaction was extracted with dichloromethane (3 × 4 mL). The organic layer was dried over anh. Na2SO4 and evaporated to dryness. The crude reaction was flash chromatographed (silica gel) using a mixture of n-hexane-EtOAc (8:2) as eluent.To a solution of amide 1,1-Dimethoxypropan-2-one (3a). (A) Following the General Procedure described above, N-methoxy-N-methylacetamide  was treated with MeMgBr (0.92 mmol), affording α-ketoacetal 3a  as a pale yellow liquid. (B) N-methoxy-N-methylacetamide  was treated with MeLi (0.92 mmol), affording α-ketoacetal 3a  as a pale yellow liquid. Rf = 0.66 n-hexane-EtOAc (8:2). 1H-NMR : δ 4.47  H-1, 3.42  (OCH3)2, 2.22  H-3 +· (C11H9O3) 173.0603; observed 173.0607.1,1-Dimethoxy-2-(4-methylphenyl)-ethan-2-one (3g). Following the General Procedure described above, N-methoxy-N-methylacetamide  was treated with 4-Me-C6H4MgBr (0.92 mmol), affording α-ketoacetal 3g  as a pale yellow liquid. Rf = 0.46 n-hexane-EtOAc (4:1). 1H-NMR : δ 8.12  H-o, 7.36  H-m, 5.23  H-1, 3.48  (OCH3)2, 2.43  CH3. 13C-NMR : δ 193.0 (C-2), 144.6 (C-p), 131.3 (C-i), 129.6 (C-o), 129.2 (C-m), 103.1 (C-1), 54.4 (OCH3)2, 21.7 (CH3) dioxane (9). To a stirred solution of 8a , p-TsOH  and sodium sulfate (9.6 g) in acetone (95 mL) 2,2-dimethoxypropane (121 mmol) were added. The reaction was maintained with a vigorous stirring for 72 h at 40 °C. After that, the reaction was extracted with CH2Cl2 (120 mL) and washed with sat. NaHCO3 (3 × 30 mL). The organic layer was dried over anh. Na2SO4 and evaporated to dryness giving 9  as an amber liquid. 1H-NMR : δ 7.21  Hb, 7.03  Hc, 6.70  Ha, 4.78  CH2, 1.51  2CH3 [6H) 2CH3 .1--2,2-methoxyethanone (10). To a solution of 9  in THF (7 mL) at −78 °C, 1.6 M nBuLi (0.92 mL 1.47 mmol) was slowly added maintaining a vigorous stirring under nitrogen atmosphere for 45 min. This solution was added dropwise to a solution of 2a  in 10 mL of THF cooled at −78 °C. The reaction was stirred under nitrogen atmosphere for 1 h. Then the reaction was quenched with a saturated solution of NH4Cl. The reaction was extracted with dichloromethane (3 × 5 mL), the organic layer was dried over anh. Na2SO4 and evaporated to dryness giving 10  as a yellow syrup. νmax (film): 1693, 1497, 1375, 1272, 1204, 1110, 1067, 955, 433 cm−1. 1H-NMR : δ 7.98  H-2', 7.83  H-6', 6.85  H-3', 5.15  H-1, 4.88  H-7, 3.46  (OCH3)2, 1.56  2CH3. 13C-NMR : δ 191.9 (C-2), 156 (C-1'), 130.2 (C-5'), 127.1 (C-6'), 126.1 (C-4'), 118.9 (C-2'), 117.0 (C-3'), 103.6 (C-1), 100.5 (C-8'), 60.6 (C-7'), 54.5 ((OCH3)2), 24.7 (2 CH3). EI-HRMS: calculated for C14H18O5 266.1154; observed 266.1154.1--2,2-dimethoxyethanol (11). To a cooled solution (0 °C) of 10  in ethanol (10 mL) NaBH4  was added. The reaction mixture was stirred for 30 min. Then it was quenched with acetone, the solvent was evaporated and 5 mL of hot water was added to the reaction crude. The reaction was extracted with dichloromethane (3 × 5 mL), the organic layer was dried over anh. Na2SO4 and evaporated to dryness giving 11  as a yellow and viscous liquid. 1H-NMR : δ 7.18  H-2', 7.02  H-6', 6.78  H-3', 4.83  H-7', 4.50  H-2, 4.24  H-1, 3.45  OCH3, 3.25  OCH3, 2.90  OH, 1.52  2CH3. 13C-NMR : δ 150.7 (C-4'), 131.2 (C-1'), 126.9 (C-2'), 123.2 (C-6'), 118.9 (C-5'), 116.6 (C-3'), 107.5 (C-1), 99.3 (C-8'), 73.3 (C-2), 60.7 (C-7'), 55.7 (OCH3), 54.7 ((OCH3)2), 24.7 (2CH3), 24.4 (CH3). EI-HRMS: calculated for C14H18O5 268.1311; observed 268.1311.Salbutamol. To a solution of hydroxyacetal 11  37% HCl (0.16 mL) diluted in THF (1 mL) was added. After 15 min the reaction finished and the reaction mixture was washed with a solution of NaHCO3 sat (3 × 2 mL). The reaction was extracted with dichloromethane and (3 × 3 mL) the organic layer was dried over anh. Na2SO4 and evaporated to dryness, giving the corresponding α-hydroxyaldehyde (80 mg as crude). To this crude t-BuNH2  was added and the reaction mixture was stirred for 30 min. The reaction crude was dissolved in ethanol (3 mL) and treated with NaBH4  under vigorous stirring for 30 min. The reaction was then quenched with acetone and the solvent was evaporated. After this, hot water (5 mL) was added to the crude. The reaction was extracted with dichloromethane and (3 × 5 mL). The organic layer was dried over anh. Na2SO4 and evaporated to dryness giving (rac)-salbutamol . 1H-NMR : δ 7.11  H-2', 7.03  H-6', 6.8  H-3', 4.53  CHOH, 3.90  C2HOH, 2.90  NHC2H, 2.60  NHC2H, 2.2  NH and 3(OH), 1.10  t-Bu [(±)-9H) t-Bu .2a,b represent an efficient and practical alternative for obtaining a wide variety of α-ketoacetals, which in turn represent an array of functional groups in high demand in synthetic organic chemistry. A practical synthetic application of α-ketoacetals was developed for the synthesis of some 1,2-aminoalcohols, including the total synthesis of (±)-salbutamol.In conclusion, it has been shown that WAs


Panax ginseng (Korean ginseng) are unique triterpenoidal saponins that are considered to be responsible for most of the pharmacological activities of P. ginseng. However, the various linkage positions cause different pharmacological activities. In this context, we aimed to synthesize new derivatives of ginsenosides with unusual linkages that show enhanced pharmacological activities. Novel α-glycosylated derivatives of ginsenoside F1 were synthesized from transglycosylation reactions of dextrin (sugar donor) and ginsenoside F1 (acceptor) by the successive actions of Toruzyme®3.0L, a cyclodextrin glucanotransferase. One of the resultant products was isolated and identified as (20S)-3β,6α,12β-trihydroxydammar-24ene-(20-O-β-D-glucopyranosyl-(1→2)-α-D-glucopyranoside) by various spectroscopic characterization techniques of fast atom bombardment-mass spectrometry (FAB-MS), infrared spectroscopy (IR), proton-nuclear magnetic resonance (1H-NMR), 13C-NMR, gradient heteronuclear single quantum coherence (gHSQC), and gradient heteronuclear multiple bond coherence (gHMBC). As expected, the novel α-glycosylated ginsenoside F1 (G1-F1) exhibited increased solubility, lower cytotoxicity toward human dermal fibroblast cells (HDF), and higher tyrosinase activity and ultraviolet A (UVA)-induced inhibitory activity against matrix metalloproteinase-1 (MMP-1) than ginsenoside F1. Since F1 has been reported as an antiaging and antioxidant agent, the enhanced efficacies of the novel α-glycosylated ginsenoside F1 suggest that it might be useful in cosmetic applications after screening.Ginsenosides from  Panax ginseng Meyer, a commonly used traditional herbal medicine in Korea, China, and Japan. Ginsenosides have been reported to have antifatigue and antioxidant activities, improve brain function, enhance stamina, and regulate blood circulation with approval from the Korea Food and Drug Administration (KFDA), in addition to various other pharmacological activities including anticancer - in negative high-resolution fast atom bombardment-mass spectrometry (FAB-MS). The infrared spectrum showed strong absorbance from hydroxyl groups (3366 cm–1) and a double bond (1650 cm–1) in G1–F1 , three oxygenated methines , and eight singlet methyls (δH 1.98 (H-28), 1.58 (H-26), 1.56 (H-27), 1.55 (H-21), 1.45 (H-29), 1.08 (H-18), 1.01 (H-19), 0.98 (H-30)) were observed, indicating that G1–F1 has a protopanaxatriol-type triterpene moiety. Proton signals due to the sugar moiety, two anomeric proton signals at δH 5.81  and 5.04 , and several oxygenated methines and methylene proton signals at δH 3.72~4.56 were observed (13C NMR spectrum of G1–F1 (C 131.0 (C-25), one olefin methine carbon signal at δC 125.9 (C-24), one oxygenated quaternary carbon signal at 83.5 (C-20), three oxygenated methine carbon signals (δC 78.6 (C-3), 67.8 (C-6), 70.2 (C-12)), and eight methyl carbon signals (δC 32.0 (C-28), 25.7 (C-26), 22.3 (C-21), 17.8 (C-18), 17.6 (C-19), 17.5 , 16.3 (C-29)) were observed for the protopanaxatriol-type aglycone moiety. The chemical shifts of the sugar moieties signal (δC 98.1 (C-1′), 81.2 (C-2′), 78.5 (C-3′), 76.6 (C-5′), 75.5 (C-4′), 62.1 (C-6′)) suggested the presence of a glucopyranoside. The coupling constant of the anomeric proton signal  was 8.0 Hz, confirming β-D-glucopyranoside. Another sugar moiety (δC 103.0 (C-1′′), 75.2 (C-3′′), 74.6 (C-2′′), 74.4 (C-5′′), 71.9 (C-4′′), 62.8 (C-6′′) suggested the presence of glucopyranoside; the coupling constant of the anomeric proton signal  was 3.6 Hz, confirming that the glucopyranose had a α-glucosidic linkage. The connection between the β-D-glucopyranosyl unit (C-1′) and the C-20 of the aglycone and that of another α-D-glucopyranosyl unit (C-1′′) with C-2′ of the inner glucose was verified by the cross-peaks observed between the anomer proton signal at δH 5.04 (H-1′) and the oxygenated quaternary carbon signal at δC 83.5 (C-20) and between the anomer proton signal at δH 5.81 (H-1′′) and the oxygenated methine carbon signal at δC 81.2 (C-2′) in the HMBC spectrum, respectively (C 78.5 (C-3′)) and proton signals (δH 4.53 (H-3′)) due to the glycosylation effect. Ultimately, the structure of G1-F1 was determined to be (20S)-3β,6α,12β -trihydroxydammar-24-ene-(20-O-β-D-glucopyranosyl-(1→2)-α-D-glucopyranoside), which has not been reported previously  in a dose-dependent manner . The α-gTo investigate the tyrosinase inhibitory activity of G1–F1, the half maximal inhibitory concentration (IC50) values against mushroom tyrosinase were measured. The tyrosinase inhibitory activity of α-glycosylated ginsenoside F1 was higher than that of ginsenoside F1 but weaker than that of arbutin .It was previously reported that F1 can function as an anti-aging and antioxidant agent  and as acis-3,3′,4′,5,5′,7-Hexahydroxy-flavane-3-gallate (EGCG) was used as a positive control.Skin aging occurs as a result of collagen degradation through induction of MMPs by UV irradiation . The α-gIn addition to the number of sugars, their linkage positions and alpha vs. beta linkages affect pharmacological activities. For example, ginsenoside F1 and Rh1 have the same number of sugar moieties and the same molecular weight but different glucose attachment positions at C-20 and C-6, respectively. F1 showed significantly greater inhibition of viability than Rh1 in prostate cancer cell lines . The glyThe alpha isomers of glucose also exhibited significant activity, especially stronger inhibitory activity of α-arbutin on tyrosinase compared with β-arbutin . SimilarD-glcp-(1→2)-β-D-glcp sugar chain (G1–F1). The novel compound G1–F1 showed lower cytotoxicity and stronger inhibitory activity against tyrosinase and collagenase (MMP-1) than ginsenoside F1. This novel G1–F1 may be a potential pharmacological active compound. A single α-glucosylated F1 was purified in this study, and other new glycosylated spots remain to be characterized.This study describes for the first time the glycosylation of ginsenoside F1 by CGTase and identification of a novel α-glucosylated F1 with an unusual α-


Their structures were characterized as 1β,2β,3β,4β,5β,26-hexahydroxyfurost-20(22),25(27)-dien-5,26-O-β-d-glucopyranoside (1), 1β,2β,3β,4β,5β,6β,7α,23ξ,26-nona-hydroxyfurost-20(22),25(27)-dien-26-O-β-d-glucopyranoside (2), -spirost-25 (27)-en-1β,3β,5β-trihydroxy-1-O-β-d-xyloside (3), tupisteroide B (4) and 5β-furost-Δ25(27)-en-1β,2β,3β,4β,5β,7α,22ξ,26-octahydroxy-6-one-26-O-β-d-glucopyranoside (5), respectively, by extensive use of spectroscopic techniques and chemical evidence. Additionally, the in vitro cytotoxic activity of 1–4 was evaluated on human A549 and H1299 tumor cell lines, and compound 3 exhibited cytotoxicity against A549 cells (IC50 86.63 ± 2.33 μmol·L−1) and H1299 cells (IC50 88.21 ± 1.34 μmol·L−1).Two new furostanol saponins  Tupistra chinensis Baker., a species in the Tupistra genus of the Liliaceae family, is used as an endemic herbal medicine, known as “Kai-Kou-Jian”, in the Qinba Mountains of Shaanxi Province in China −. The 1H-NMR spectrum showed three methyl protons at δH 0.67 , 1.70  and 1.58 , two exo-methylene protons  and 5.04 ), as well as signals for two anomeric protons at  and 4.89 ). The 13C-NMR spectrum displayed 39 carbon signals, 27 of which belonged to the aglycone carbons, while the remaining signals were assignable to two glucosyl moieties . Among carbon signals of the aglycone, δC 146.2 and 111.6 were due to an olefinic bond group, δC 14.3, 13.7 and 11.7 were due to three methyl groups, and δC 77.8, 68.1, 75.2, 67.6, 87.4, 84.4, 64.5 and 71.7 were due to eight oxygenated carbon groups, which indicated that 1 was a furostanol saponin with multiple hydroxyl groups. The structure of 1 was finally determined by analysis of its 2D NMR data (see 1. HMQC correlations of (δH 5.35 (H-27a) and 5.04 (H-27b)) to δC 111.6, showed the appearance of a terminal olefinic bond at C-27. Then, HMBC correlations of H-27/C-24, C-25 and C-26, H-24/C-22, C-23, C-25 and C-26, H-26/C-24, C-25 and C-27, indicated that the appearance of an isopentene group, linked at C-22 of the tetrahydrofuran ring of the furostanol saponin. Moreover, HMBC correlations of H-19/C-1, C-5, C-9 and C-10, H-3/C-1, C-2 and C-5, and H-6/C-4 and C-5, indicated that all hydroxyl groups were linked at C-1–C-5 of the A ring of the furostanol saponin ,25(27)-dien. In addition, the HMBC correlation signals of H-Glc-1′/C-5 and H-Glc-1′′/C-26, indicated that glucosyl groups were connected as (Glc-1′′-O-C-26) and (Glc-1′-O-C-5) ,25(27)-dien-5,26-O-β-d-glucopyranoside.Compound data see . The HMQonin see . Furtheronin see . TherefoC-5) see . The twomination , and judrved see , indicate-21 see . Therefo2 was obtained as a white amorphous powder, which showed positive reactions in the Liebermann-Burchard, Ehrlich, and Molisch tests, suggesting that 2 was a furostanol glycoside. The molecular formula C33H52O15 was deduced from the HR-ESI-MS peak at m/z 711.3198 [M + Na]+. Comparison of the HR-ESI-MS and NMR data of 2 and 1, indicated almost similar NMR spectroscopic features, except for the number of oxygenated methine groups. In the 13C-NMR spectrum of 2, only one glucosyl moiety  was recognized, however, nine oxygenated carbon groups of the aglycone at δC 79.1, 67.7, 76.1, 70.2, 78.6, 74.0, 72.5, 64.8 and 72.7 were identified. Meanwhile, the spectroscopic features of 2 were similar to those of tupisteroide B (4), indicating that seven hydroxyl groups were linked at C-1–C-7 of the furostanol saponin, which was confirmed by the 1H-1H COSY correlation of H-1/H-2/H-3/H-4 and H-6/H-7 and the HMBC correlation of H-19/C-1, C-5, C-9 and C-10, and H-6/C-4 and C-5  at δC 34.3 in 4, which was correlated with a proton signal at δH 5.13  in the HMQC spectrum, and the correlation signals of H-23/H-24 in the 1H-1H COSY spectrum, the correlation signals of H-23/C-20, C-22, C-24 and C-25, H-24/C-22, C-23, C-25, C-26 and C-27, and H-27/C-24, C-25 and C-26 in the HMBC spectrum , according to the same protocol as that described for 1. Thus, the planar structure of 2 was deduced as 1,2,3,4,5,6,7,23,26-nonanolfurost-20(22),25(27)-dien-26-O-β-d-glucose. In the NOESY spectrum of 2, the NOE correlations of Me-19/H-8, H-4/H-2, H-3 and H-9, and H-2/H-1 were observed, indicating α-axial configurations of H-1, H-2, H-3, and H-4, and β-orientation of Me-19, 1-OH, 2-OH, 3-OH, 4-OH and 5-OH, which supported the A/B cis ring junction pattern ,25(27)-dien-26-O-β-d-glucopyranoside.Compound  C-5 see . The 26- C-5 see . The remtrum see . In additern see . Besidestern see . Finallye-21 see . Therefo3 was obtained as a white amorphous powder, and the molecular formula of C32H50O9 was established by the HR-ESI-MS signal at m/z 579.3590 [M + H]+. The 13C-NMR spectrum exhibited 32 carbon signals, 27 of which were attributed to the aglycone carbons, while the remaining signals were assignable to a characteristic of a xylosyl moiety , which was identified as β-d-xylose by the coupling constant analysis of the anomeric proton (J = 7.2 Hz), the acid hydrolysis procedure, TLC comparison, and the optical rotation determination. Among the aglycone carbon signals, the quaternary carbon signal at δC 109.9 ] of the xylose was correlated with δC 82.5, which was confirmed as C-1 for the HMQC correlation of δH 4.26 (H-1)/δC 82.5 (C-1), 1H-1H COSY correlations of H-1/H-2/H-3/H-4, and HMBC correlations of H-19/C-1, C-5, C-9 and C-10 -en-1,3,5-trihydroxy-1-O-β-d-xyloside. In the NOESY spectrum of 3 -1β,3β,5β-trihydroxyspirost-25(27)-en-5-O-β-d-glucopyranoside − . A white amorphous powder, IR (KBr) νmax: 3475, 2980, 1742, 1062, 904, 804 cm−1. 1H-NMR  and 13C-NMR  spectral data, see m/z 711.3198 [M + Na]+ .A white amorphous powder, IR (KBr) νmax: 3306, 2922, 1650, 1042, 989, 917, 892, 876 cm−1; 1H-NMR  and 13C-NMR  spectral data, see m/z 579.3590 [M + H]+ .A white amorphous powder; IR (KBr) ν1 (3 mg), 2 (3 mg) and 3 (5 mg) were hydrolyzed with 2N HCl (5 mL) for 5 h at 80 °C, respectively. The reaction mixtures were concentrated and dried by N2, and then water (5 mL) was added and the mixtures were extracted with EtOAc (3 × 5 mL). The aqueous layers of 1 and 2 were subjected to CC over silica gel eluted with MeCN–H2O (8:1) to yield d-glucose, which was determined by TLC comparison  with the authentic sugar and the optical rotation determination c 0.16, H2O). The aqueous layer of 3 was subjected to CC over silica gel eluted with MeCN–H2O (8:1–15:1) to yield d-xylose, which was identified by TLC comparison with the authentic sugar and the optical rotation determination c 0.14, H2O).The solutions of compounds in vitro, using 5-fluorouracil as positive control. Briefly, 1 × 104 mL−1 cells were seeded into 96-well plates and allowed to adhere for 24 h. Compounds 1–4 were dissolved in DMSO and diluted with complete medium to five concentration levels (from 0.001 mmol·L−1 to 0.1 mmol·L−1) for inhibition rate determination. After incubation at 37 °C for 4 h, the supernatant was removed before adding DMSO (100 μL) to each well. 5-Fluorouracil (5-Fu) was used as positive control. The inhibition rate (IR) and IC50 were calculated. Values are mean ± SD, n = 3, ** p < 0.01 vs. DMEM control. Compound 3 exhibited cytotoxicity against A549 cells (IC50 86.63 ± 2.33 μmol·L−1) and H1299 cells (IC50 88.21 ± 1.34 μmol·L−1), while the positive control of 5-Fu exhibited cytotoxicity against A549 cells (IC50 42.78 ± 1.63 μmol·L−1) and H1299 cells (IC50 38.65 ± 1.59 μmol·L−1), respectively, (see The cytotoxic activity assays towards the A549 and H1299 tumor cell lines were measured by the MTT method ely, see .


S,6S,7S,9S,10S,11R,13S-abiet-8(14)-enepenta-6,7,9,11,13-ol, 1), was isolated from a rhizome extract of Kaempferia angustifolia Rosc. along with the known compounds crotepoxide, boesenboxide, zeylenol, 2′-hydroxy-4,4′,6′-trimethoxychalcone, (24S)-24-methyl-5α-lanosta-9(11),25-dien-3β-ol, β-sitosterol and β-sitosterol-3-O-β-D-glucopyranoside. The structures of all compounds were elucidated on the basis of mass spectroscopic and NMR data. Zeylenol (2), the major constituent of the plant, was derivatized into diacetate, triacetate and epoxide derivatives through standard organic reactions. The cytotoxic activity of compounds 1, 2 and the zeylenol derivatives was evaluated against the HL-60, MCF-7, HT-29 and HeLa cell lines.A new abietene diterpene, kaempfolienol (5 Kaempferia angustifolia, which is also known as Kunci pepet, Kunci menir or Kunci kunot can be found growing wild in forests of Western and Central Java of Indonesia and in parts of Thailand. The pleasant-smelling plant is usually used as a remedy for colds, stomach ache and dysentery, while its rhizomes are used for coughs and as a masticatory + corresponding to the molecular formula C20H34O5Na was observed in the HRESIMS spectrum, requiring four unsaturation equivalents. The absorptions at 3,438 cm-1 and 1,018 cm-1 in the IR spectrum were attributed to hydroxyl group and C-O, respectively, whereas a weak absorption band at 1,628 cm-1 was due to olefinic C=C stretching. These data indicated that compound 1 is an unsaturated alcohol derivative. The 1H-NMR spectrum was integrated for 29 protons. The presence of five methyl signals at δ 1.27, 1.21, 1.02, 0.96 and 0.93, of which the latter two, due to an isopropyl group, resonated as a doublet with J = 7.0 Hz, indicating that compound 1 is an abietane-type diterpene.Compound folienol , was isodd, J = 11.3, 4.9 Hz), 4.18  and δ 3.89  in the lower field region were also observed. The proton at δ 4.18 showed a cross peak with the signal at δ 3.89 in the COSY spectrum, revealing that they were linked to adjacent carbons. The occurrence of an isopropyl group was also supported by the COSY correlations between two methyls at δ 0.96 (H-16) and 0.93 (H-17) with the methine proton (H-15) at δ 1.71 (m)  and three methine protons at δ 4.35 (1.71 (m) . 13C-NMR spectrum, 19 carbon resonances which corresponded to six methine, four methylene, five methyl and four quartenary carbons from DEPT analysis were observed. One of the carbon signals at δ 45.0 was possibly due to overlapping of two carbon resonances since the intensity of the peak was about one-fold higher than the rest and the assignments remained to be clarified. A pair of olefinic carbons was observed at δ 139.9 (q) and 135.8 (methine), while crowded signals in the high-field region δ 15-50 were typical for a terpene skeletons.From the 3J correlation with the methine carbon at δ 44.3 (C-5).The HMQC spectrum showed carbons and protons connected by one bond whilst HMBC correlations determined two and three-bond carbon-proton couplings . Two quaFurthermore, H-5 (δ 1.96) displayed a correlation to another quartenary methyl (C-20: δ 21.2), and its protons (H-20: δ 1.21) were in turn correlated to the δ 37.1 methylene carbon (C-1), oxygenated quartenary carbon δ 78.0 (C-9) and the signal at δ 45.0 in the HMBC spectrum. Therefore, the carbon resonance at δ 45.0 was assigned to the quartenary carbon C-10 in addition to C-3. Correlations between H-5 and δ 4.18 (H-6) and between H-6 and δ 3.89 (H-7) were demonstrated in the COSY spectrum, thus revealing the connectivities of C5-C7. w-coupling) which also displayed a cross peak with the oxygenated methine proton H-11 (δ 4.35). The connectivities shown from the evidences mentioned above revealed the double-bond at C-8 (δ 139.9) and C-14 (δ 135.8) and the placement of isopropyl group at C-13. Methyl protons (H-16 and H-17) and the methine proton H-15 from the isopropyl fragment were correlated to the oxygenated quartenary carbon at δ 73.3 (C-13). H-15 also demonstrated long-range correlations to the methylene carbon δ 37.6 (C-12) and the olefinic carbon δ 135.8 (C-14). Again, the elucidation of structure was substantiated by COSY correlations between the olefinic proton H-14 at δ 5.78 and H-12 at δ 1.61 (via a 1 was identified as abiet-8(14)-enepenta-6,7,9,11,13-ol and named kaempfolienol. The NMR spectral data of this compound are summarized in Additional HMBC correlations of H-11/C-10, H-14/C-7 (δ 81.2) and H-1 (δ 1.54)/C-9 (78.0) established the linkage between three cyclic rings. Proton signals for hydroxyl groups were not observed, but the placement of hydroxyl groups at C-6, C-7, C-9, C-11 and C-13 was reasonable since the respective carbons resonated in a more deshielded region (δ 65 – 81) and conform to the type of carbons in DEPT spectrum. Consequently, compound 1, such as the ambiguity around the stereochemistry of chiral centres, led us to perform an X-ray diffraction analysis of the compound. The single crystal XRD data has been deposited with the Cambridge Crystallographic Data Centre (CCDC)  and the corresponding carbonyl carbons (δ 170.3 and 169.7) could also be observed in the 13C-NMR spectrum. The formation of diacetate derivative 3  could be1H-NMR spectrum of zeylenol triacetate , except for the presence of three extra singlet peaks at δ 1.96, 2.03 and 2.13 which can be assigned to the acetoxy methyls 2-OCOCH3, 6-OCOCH3 and 1-OCOCH3, respectively. The oxymethine protons H-2, H-3 and H-6 appeared more deshieded, whereas the inequivalent methylene protons of H-7 resonated at lower field upon acetylation compared to the starting material 2. Besides, the formation of triacetoxy derivative could be evidenced from 13C-NMR shifts by having three methyl signals at δ 20.7 (2-OCOCH3), 20.8 (6-OCOCH3) and 21.6 (1-OCOCH3) as well as three carbonyl groups at δ 169.2 (1-OCOCH3), 169.5 (6-OCOCH3) and 169.9 (2-OCOCH3). The etate 4,  is simil5, H 5.81 (δC 127.8) and δH 5.94 (δC 131.3) corresponding to the olefinic group of zeylenol (2), were no longer present. Instead, two doublet of doublet peaks were observed at the higher field region at δ 3.79 (H-4) and 3.67 (H-5) in the 1H-NMR spectrum. In the 13C-NMR spectrum, two additional methine carbon resonances at δ 57.0 (C-4) and 54.8 (C-5) were observed. Furthermore, 3J correlations between H-4 and C-2 as well as H-5 versus C-1 in HMBC spectrum proved the assignments of C-4 and C-5. The 13C-NMR spectral data of compounds 3 and 5 are reported for the first time in this paper.As for the elucidation of zeylenol epoxide 5, , the sig1-5 were evaluated against HL-60 (human promyelocytic leukemia), MCF-7 (human breast cancer), HT-29 (human colon cancer) and HeLa  cell lines and the results are summarized in 1) demonstrated moderate inhibition against HL-60 and MCF-7 cell lines with IC50 values 24.22 ± 0.30 µg/mL and 23.50 ± 1.70 µg/mL, respectively. Zeylenol (2) was selectively active towards inhibition of leukemic cancer cell (HL-60) with IC50 value of 11.65 ± 0.52 µg/mL. Acetylation of the hydroxyl groups of zeylenol (2) resulted in the elimination of its antileukemic activity for compounds 3 and 4 while weaker activities were also observed for the epoxide derivative (5) (IC50 20.91 ± 1.28 µg/mL). As for zeylenol diacetate (3), the anticancer property against HT-29 cell line (IC50 6.73 ± 0.68 µg/mL) was better than its starting material [zeylenol (2)]. To the best of our knowledge, this is the first report of the cytotoxic activities of (+)-zeylenol (2) and its derivatives (3-5). The cytotoxic activities of compounds 1H-NMR and 13C-NMR spectra were recorded with a JEOL FT NMR spectrometer (500 MHz and 125 MHz for compound 1 and 400 MHz/100 MHz for other compounds) using TMS as internal standard. Silica gel  was used for column chromatography. TLC was performed on silica gel plates (Merck DC-Alufolien 60 F254) and the spots were visualized under UV  followed by spraying with 10% aqueous H2SO4 and further heating on a hot plate. Melting points were determined using a Barnstead Electrothermal IA 9100 series melting point equipment and were uncorrected. Optical rotations were measured on a JASCO P-2000 series polarimeter. UV spectra were obtained by using Shimadzu 1650 PC spectrophotometer. IR spectra were recorded using a Perkin Elmer FTIR spectrophotometer model Spectrum BX. The HRESIMS mass spectra were obtained from a Bruker Daltonics micrOTOF-Q ESI-Qq-TOF mass spectrometer. Kaempferia angustifolia Rosc. were collected from Yogyakarta, Indonesia in 2001 and identified by Sugeng Riyanto from Gadjah Mada University, Indonesia. A voucher specimen was deposited in the herbarium of the institution.Rhizomes of Kaempferia angustifolia Rosc. were air-dried and ground into powder form. The plant sample (765 g) was extracted with methanol three times for three days each to give crude methanol extract which was then partitioned with hexane, water with methanol (10%) and chloroform (1 L each) to give hexane-soluble fraction (10.60 g) and chloroform-soluble fraction (42.50 g). The aqueous with methanol fraction was further discarded. The hexane extract (8.00 g) was subjected to vacuum column chromatography and eluted with mixtures of hexane, ethyl acetate and methanol to give 85 fractions (200 mL each) which were then combined according to their similar TLC profiles. Upon purification, (24S)-24-methyl-5α-lanosta-9(11),25-dien-3β-ol (40 mg), β-sitosterol (35 mg), 2′-hydroxy-4,4′,6′-trimethoxychalcone (7 mg), crotepoxide (10 mg) and β-sitosterol-3-O-β-D-glucopyranoside (8 mg) were obtained. Part of the chloroform extract (40.00 g) was fractionated in the similar manner to obtain 100 fractions (200 mL each) which were combined into major fractions based on their TLC profiles. 2′-Hydroxy-4,4′,6′-trimethoxychalcone (10 mg), boesenboxide (16 mg), crotepoxide (0.79 g), zeylenol (0.97 g) and kaempfolienol (abiet-8(14)-enepenta-6,7,9,11,13-ol, 1, 20 mg) were obtained. Rhizomes of 2, 0.10 g; 2.604 × 10-4 mole) was subjected to acetylation using acetic anhydride  and pyridine (2 mL) with stirring for 2 hours at room temperature. The reaction was monitored by using thin-layer chromatography. Upon completion, the reaction crude product was poured into iced-water (10 mL) and extracted with dichloromethane (2 × 10 mL). The organic layer was then washed with 20% hydrochloric acid and further dried by adding anhydrous sodium sulphate. The crude product (0.17 g) was obtained after filtering off the drying agent and evaporating the solvent under reduced-pressure. Purification of the product using silica gel 9385 column chromatography was carried out. Eluent with a solvent system of hexane-ethyl acetate (70:30 → 60:40) gave zeylenol diacetate .Zeylenol (-2 mole) was added to zeylenol  in pyridine (4.0 mL) and the mixture refluxed for six hours. The reaction product was worked-up similarly to 3 to give crude product (0.20 g). The yellowish solid was then chromatographed on a silica gel column to yield a pale yellow oil which solidified at room temperature and was recrystallized with methanol to afford colourless needle-shaped crystals of zeylenol triacetate .Acetic anhydride  was reacted with meta-chloroperbenzoic acid (MCPBA)  in dichloromethane (10.0 mL) by reluxing for 24 hours. The solvent was evaporated from the reaction product under reduced-pressure and the crude mixture (94 mg) was purified by silica-gel column chromatography. Elution with hexane-ethyl acetate (70:30) afforded zeylenol epoxide .Zeylenol (Kaempfolienol (1). Colourless needle-shaped crystals. m.p. 278-280 °C. [α]D = + 1.8 ° . UV (EtOH) λmax nm (log ε): 273 (2.70), 202 (4.06). IR  νmax : 3,438, 2,934, 2,884, 1,628, 1,436, 1,384, 1,342, 1,018. For 1H- and 13C-NMR spectra, see m/z : 377.2746 , 355.3210 .Zeylenol (2). White amorphous solid. m.p. 108-110 °C  [Lit.: [α]D = + 121.6 ° ] D = + 24.8 °  [Lit. [D = + 17 ° ]. UV (CHCl3) λmax nm (log ε): 282 (3.60), 275 (3.67), 240 (4.52). IR  νmax : 3,464, 2,962, 1,728, 1,602, 1,448, 1,372, 1,264, 1,110, 712. 1H-NMR : δ 7.99 , 7.56 , 7.42 , 6.01 , 5.95 , 5.88 , 5.71 , 5.51 , 4.65 , 4.46 , 3.16 , 2.12 , 2.09 . 13C-NMR : δ 170.3 (2-OCO), 169.7 (6-OCO), 166.7 (7-OCO), 165.9 (3-OCO), 133.5 (C-4″), 133.3 (C-4′), 129.2 (C-5), 125.8 (C-4), 129.7 , 129.3 (C-1″), 129.0 (C-1′), 128.5 (C-3″/5″), 128.4 (C-3′/5′), 74.1 (C-1), 71.7 (C-2), 70.9 (C-3), 70.3 (C-6), 65.9 (C-7), 20.9 (2-OCOCH3), 20.8 (6-OCOCH3). HR-CIMS m/z : 469.1497 .3) [Lit. : [α]D = Zeylenol triacetate (4). Colourless needle-shaped crystals. m.p. 150-151 °C  [Lit. [D = + 24 ° ]. UV (CHCl3) λmax nm (log ε): 283 (3.24), 275 (3.35), 242 (4.14). IR  νmax: 3,062, 2,986, 2,954, 1,752, 1,726, 1,604, 1,454, 1,374, 1,104, 712. 1H-NMR : δ 8.03 , 7.57 , 7.47 , 7.43 , 6.19 , 6.06 , 5.99 , 5.96 , 5.89 , 5.09 , 4.82 , 2.13 , 2.03 , 1.96 . 13C- NMR : δ 169.9 (2-OCOCH3), 169.5 (6-OCOCH3), 169.2 (1-OCOCH3), 166.0 (7-OCO), 165.5 (3-OCO), 133.4 (C-4″), 133.3 (C-4′), 130.0 (C-5), 129.8 (C-2″/6″), 129.6 (C-2′/6′), 129.4 (C-1″), 129.2 (C-1′), 128.5 , 125.3 (C-4), 81.4 (C-1), 70.9 (C-3), 70.7 (C-2), 67.9 (C-6), 63.1 (C-7), 21.6 (1-OCOCH3), 20.8 (6-OCOCH3), 20.7 (2-OCOCH3). HR-CIMS m/z (% intensity): 511.1607 .°C (Lit. : 158-1593) [Lit. : [α]D = Zeylenol epoxide (5). Colourless needle-shaped crystals. m.p. 164-166 °C  [Lit. [D = + 68 ° ]. UV (CHCl3) λmax nm (log ε): 283 (3.14), 276 (3.23), 242 (4.05). IR  νmax : 3,476, 2,924, 1,696, 1,600, 1,450, 1,279, 1,069, 709. 1H-NMR : δ 8.10 , 8.04 , 7.59 , 7.44 , 5.71 , 4.84 , 4.58 , 4.16 , 4.02 , 3.79 , 3.67 , 3.39 , 2.96 , 2.61 . 13C-NMR : δ 167.6 (7-OCO), 167.1 (3-OCO), 133.5 (C-4′/4″), 130.0 (C-2′/6′), 129.8 (C-2″/6″), 129.4 (C-1″), 129.3 (C-1′), 128.5 , 77.1 (C-1), 74.1 (C-3), 68.9 (C-2), 68.1 (C-6), 66.6 (C-7), 57.0 (C-4), 54.8 (C-5). HRFAB-MS m/z : 401.1242 . °C (Lit. : 173-1753) [Lit. : [α]D = 50 (μg/mL) ± standard deviation from three separate experiments, each performed in triplicate. Compounds were assayed for cytotoxic test against HL-60, MCF-7, HT-29 and HeLa cell lines using the MTT method [Kaempferia angustifolia Rosc., a new abietene diterpene identified as 5S,6S,7S,9S,10S,11R,13S-abiet-8(14)-enepenta-6,7,9,11,13-ol to which the trivial name kaempfolienol has been assigned was isolated. Cyclohexane derivatives, chalcone, triterpenes and glycoside were also obtained from the plant. Several derivatives of zeylenol were synthesized and the cytotoxic properties against some selected cancer cell lines of these compounds were studied. From the rhizome extract of


Aquilaria sinensis, known as agarwood (Chen Xiang in Chinese), is traditionally used for the treatment of abdominal pain, vomiting, circulatory disorders, and dyspnea. Four new 2-(2-phenylethyl)-4H-chromen-4-one derivatives, namely 7-methoxy-2-[2-(4′-hydroxy-phenyl)ethyl]chromone (1), 7-hydroxy-2-[2-(4′-methoxyphenyl)ethyl]chromone (2), 5,6-dihydroxy- 2-[2-(3′-hydroxy-4′-methoxyphenyl)ethyl]chromone (3), and 6-hydroxy-5-methoxy-2-(2-phenyl-ethyl)chromone (4), have been isolated from the resinous wood of A. sinensis, together with nine known compounds. The structures of these compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, neopetasan, 7-methoxy-2-(2-phenylethyl)-chromone, 6,7-dimethoxy-2-(2-phenylethyl)chromone, and 6,7-dimethoxy-2-[2-(4′-methoxy-phenyl)ethyl]chromone inhibited NF-κB activation in LPS-stimulated RAW 264.7 macrophages with relative luciferase activity values of 0.55 ± 0.09, 0.54 ± 0.03, 0.31 ± 0.05, and 0.38 ± 0.14, respectively, versus that of vehicle control (1.03 ± 0.02). In addition, 5,6-dihydroxy-2-[2-(3′-hydroxy-4′-methoxyphenyl)ethyl]chromone, 7-methoxy-2-(2-phenylethyl)chromone, 7-dimethoxy-2-(2-phenylethyl)chromone, and 6,7-dimethoxy-2-[2-(4′-methoxyphenyl)ethyl]chromone could suppress LPS-induced NO production in RAW 264.7 cells and did not induce cytotoxicity against RAW 264.7 cells after 24-h treatment.The resinous wood of  Aquilaria sinensis (Lour.) Gilg. (Thymelaeaceae) is an evergreen tree endemic to China. The fragrant resin-infused wood derived from the wounded trees of Aquilaria species is called ‘agarwood’ or ‘eaglewood’. Agarwood has been widely used as a traditional sedative, analgesic, and digestive medicine in China chromone (1), 7-hydroxy-2-[2-(4′-methoxyphenyl)ethyl]chromone (2), 5,6-dihydroxy-2-[2-(3′-hydroxy-4′-methoxyphenyl)ethyl]chromone (3), and 6-hydroxy-5-methoxy-2-(2-phenylethyl)- chromone (4), and nine known compounds 5–13 have been isolated from the resinous wood of A. sinensis and identified. Their structures are depicted in 1 through 4 and the inhibitory activities of all isolates on LPS-induced NF-κB activation of macrophages.Aberrant activation of macrophages is related to many inflammatory disorders such as sepsis, neurodegenerative disorders, osteoporosis, cardiovascular and metabolic diseases . NuclearA. sinensis on a silica gel column and preparative thin-layer chromatography (TLC) afforded four new (1–4) and nine known compounds (5–13) (Chromatographic purification of the EtOAc-soluble fraction of a MeOH extract of resinous wood of s (5–13) .1 was obtained as brown plates. The molecular formula, C18H16O4, was deduced from a proton adduct ion at m/z 297.11224 [M + H]+  in the HR-ESI-MS spectrum (positive-ion mode) and was supported by the 1H-, 13C-, and DEPT NMR data. IR absorptions for OH (3418 cm−1) and γ-pyrone (1634 and 1590 cm−1) functions were observed. The 1H-NMR spectrum of 1 showed the presence of a methoxy group at δH 3.91 , four methylene protons at δH 2.86  and 2.98  and a olefinic proton at δH 6.07 . Additionally a set of typical ABX coupling systems at δH 6.83 , 6.95  and 8.08  and para-disubstituted benzene protons at δH 6.76  and δH 7.06  were observed. The 13C-NMR spectrum of 1 displayed the presence of two methylene groups at δC 32.0 and 36.3, a trisubstituted double bond at δC 109.8 and 168.5, a methoxyl group at δC 56.1, and a carbonyl group at δC 177.9. Based on the above, 1 was deduced to be a 2-(2-phenylethyl)chromone derivative with a methoxyl and a hydroxyl groups. Two signals at δC 164.1 and 154.5 arising from O-bearing aromatic C-atoms were attributable to C-7 and C-4′ respectively, by related HMBC and comparison with a structurally similar compound 10. The HMBC correlations (H 3.91) and C-7 (δC 164.1), as well as NOESY correlations (H 3.91) and H-8 (δH 6.83) and H-6 (δH 6.95), revealed that the methoxyl group was supposed to be positioned at C-7. Thus, the hydroxyl group should be linked to the last open position at C-4′. Consequently, the structure of 1 was determined to be 7-methoxy-2-[2-(4′-hydroxyphenyl)ethyl]chromone.Compound elations  observedelations  observed2 was isolated as a yellowish amorphous powder. The ESI-MS afford the quasi-molecular ion [M + H]+ at m/z 297, implying a molecular formula of C18H17O4, which was confirmed by the HR-ESI-MS . The IR spectrum showed the presence of OH (3210 cm−1) and γ-pyrone (1635 and 1600 cm−1) groups. The 1H- and 13C-NMR data of 2 were similar to those of 7-methoxy-2-[2-(4′-hydroxyphenyl)ethyl]chromone (1), except that the 7-OH and 4′-OMe [δH 3.79 ] groups of 2 replaced the 7-OMe and 4′-OH groups of 1. This was supported by NOESY correlations between OMe-4′ (δH 3.79)/H-3′ (δH 6.83) and OMe-4′ (δH 3.79)/H-5′ (δH 6.83) and by HMBC correlations between OMe-4′ (δH 3.79)/C-4′ (δC 158.3), H-5 (δH 8.02)/C-7 (δC 156.1), and H-6 (δH 7.09)/C-7 (δC 156.1). According to the above data, the structure of 2 was elucidated as 7-hydroxy-2-[2-(4′-methoxyphenyl)ethyl]chromone. This was further confirmed by the 1H–1H-COSY, NOESY  and this was supported by the 1H, 13C, and DEPT NMR data. The IR spectrum showed the presence of OH (3418 cm−1) and γ-pyrone (1634 and 1590 cm−1) groups. The 1H- and 13C-NMR data of 3 were similar to those of 3′,6-dihydroxy-4′-methoxy-2-(2-phenylethyl)chromone [H 12.50 ] of 3 replaced H-5 of 3′,6-dihydroxy-4′-methoxy-2-(2-phenylethyl)chromone chromone.Compound chromone , except chromone . This waC, NOESY , and HMBC, NOESY  spectral4 was obtained as a yellowish amorphous powder. The molecular formula C18H16O4 was deduced from a sodium adduct ion at m/z 319.09395 [M + Na]+  in the HRESI mass spectrum. The presence of a conjugated carbonyl group was revealed by the band at 1633 cm−1 in the IR spectrum, which was confirmed by the resonances at δC 177.5 in the 13C-NMR spectrum. The IR spectrum also revealed a hydroxy absorption at 3340 cm−1. Comparison of the 1H- and 13C-NMR data of 4 with those of 6-hydroxy-2-(2-phenylethyl)chromone (corylifol A)  of 4 replaced H-5 of 6-hydroxy-2-(2-phenylethyl)chromone D, and MS) with corresponding authentic samples or literature values, and this included four sesquiterpenes, neopetasane (5) chro-mone (12) can significantly inhibit LPS-induced NF-κB activation and did not show cytotoxicity against RAW 264.7/Luc-P1 cells after 24 h treatment (except compound 6 with dose-dependent cytotoxicity in RAW 264.7/Luc-P1 cells) (3), 7-methoxy-2-(2-phenylethyl)chromone (10), 7-dimethoxy-2-(2-phenylethyl)chromone (11), and 6,7-dimethoxy-2-[2-(4′-methoxyphenyl)ethyl]chromone (12) could suppress LPS-induced NO production in RAW264.7 macrophages (5), 7α-H-9(10)-ene-11,12-epoxy-8-oxoeremphilane (6), dehydrokaranone (7) displayed no inhibitory activity on NO production in RAW264.7 macrophages (11) (with a 6-methoxy moiety) exhibited stronger inhibition than its analogue, 6-hydroxy-7-methoxy-2-(2-phenylethyl)chromone (13) (with a 6-hydroxy group); (e) 7-methoxy-2-(2-phenylethyl)chromone (10) (without the 4′-hydroxy moiety) exhibited more effective inhibition than its analogue, 7-methoxy-2-[2-(4′-hydroxyphenyl)ethyl]chromone (1) (with a 4′-hydroxy group); (f) 2-(2-phenylethyl)chromone (9) (without any substituents) exhibited no inhibitory activity; (g) among the sesquiterpene analogues 5, 6, and 8, neopetasane (5) (with a prop-1-en-2-yl moiety at C-7) exhibited stronger inhibition than the analogues 6 and 8; (h) 6,7-dimethoxy-2-(2-phenylethyl)chromone (11) was the most effective among the isolated compounds, with a relative luciferase activity value of 0.31 ± 0.05, against LPS-induced NF-κB activation in RAW 264.7/Luc-P1 cells.LPS-activated macrophage serves as an in vitro model system to study inflammation . A LPS-riferase) . This RAiferase) ,19. Furtiferase) . Thus, N1 cells) A,C; (b) rophages B and didrophages D; (c) amrophages B and didrophages D; (d) 6,5, 6, and 7 in It is noted that the inhibition on NO production is not always in a tight correlation with NF-κB activation  spectra were obtained on a Jasco UV-240 spectrophotometer. Infrared (IR) spectra (KBr or neat) were recorded on a Perkin Elmer 2000 FT-IR spectrometer . Nuclear magnetic resonance (NMR) spectra, including nuclear Overhauser effect spectrometry (NOESY), correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) experiments, were acquired using a Varian VNMRS-600 or a Varian Inova 500 spectrometer  operating at 600 or 500 MHz (1H) and 150 or 125 MHz (13C), respectively, with chemical shifts given in ppm (δ) using tetramethylsilane (TMS) as an internal standard. Electrospray ionisation (ESI) and high-resolution electrospray ionization (HRESI)-mass spectra were recorded on a VG Platform Electrospray ESI/MS mass spectrometer  or a Bruker APEX II . Silica gel  was used for column chromatography (CC). Silica gel 60 F-254  was used for preparative thin-layer chromatography (PTLC) and thin-layer chromatography (TLC).Melting points were determined on a Yanaco micro-melting point apparatus  and were uncorrected. Optical rotations were measured using a Jasco DIP-370 polarimeter  in CHClAquilaria sinensis (Lour.) Gilg. (Thymelaeaceae) were collected from Chiayi County, Taiwan, in August 2015 and identified by J.-J. Chen. A voucher specimen (AS-201308) was deposited in the Faculty of Pharmacy, National Yang-Ming University, Taipei, Taiwan.The resinous wood of A. sinensis was pulverized and extracted three times with MeOH (15 L each) for 3 days. The extract was concentrated under reduced pressure at 35 °C, and the residue (110 g) was partitioned between EtOAc and H2O (1:1) to provide the EtOAc-soluble fraction . Fraction A (65 g) was purified by CC  to afford 9 fractions: A1–A9. Fraction A1 (3.8 g) was subjected to CC  to give 10 subfractions: A1-1–A1-10. Part (70 mg) of fraction A1-6 was further purified by preparative TLC  to obtain neopetasane  (Rf = 0.69). Part (85 mg) of fraction A1-10 was further purified by preparative TLC  to afford 7α-H-9(10)-ene-1,12-epoxy-8-oxoeremphilane  (Rf = 0.67). Fraction A2 (4.8 g) was subjected to CC  to give 10 subfractions: A2-1–A2-10. Fraction A2-2 (327 mg) was purified by CC , 250 mL-fractions) to give eight sub-fractions: A2-2-1–A2-2-8. Fraction A2-2-2 (35 mg) was further purified by preparative TLC  to yield dehydrokaranone  (Rf = 0.60) and 2-(2-phenylethyl)chromone  (Rf = 0.36). Fraction A2-8 (235 mg) was purified by CC , 200 mL-fractions) to give nine subfractions: A2-8-1–A2-8-9. Fraction A2-8-4 (30 mg) was further purified by preparative TLC  to afford 7-methoxy-2-(2-phenylethyl)chromone  (Rf = 0.43). Fraction A4 (7.3 g) was subjected to CC  to afford 12 subfractions: A4-1–A4-12. Part (125 mg) of fraction A4-7 was further purified by preparative TLC  to yield 6,7-dimethoxy-2-(2-phenylethyl)chromone  (Rf = 0.48) and ligudicin C  (Rf = 0.35). Fraction A5 (7.7 g) was subjected to CC  to give 11 subfractions: A5-1–A5-11. Part (110 mg) of fraction A5-6 was further purified by preparative TLC  to obtain 6,7-dimethoxy-2-[2-(4′-methoxyphenyl)ethyl]chromone  (Rf = 0.62). Fraction A7 (7.2 g) was subjected to CC  to give 10 subfractions: A7-1–A7-10. Fraction A7-7 (515 mg) was purified by CC , 500 mL-fractions) to give eight subfractions: A7-7-1–A7-7-8. Fraction A7-7-2 (33 mg) was further purified by preparative TLC  to obtain 7-methoxy-2-[2-(4′-hydroxy-phenyl)ethyl]chromone  (Rf = 0.53), 7-hydroxy-2-[2-(4′-methoxyphenyl)ethyl]chromone  (Rf = 0.47), and 6-hydroxy-7-methoxy-2-(2-phenylethyl)chromone  (Rf = 0.43). Part (95 mg) of fraction A7-9 was further purified by preparative TLC  to yield 5,6-dihydroxy-2-[2-(3′-hydroxy-4′-methoxyphenyl)ethyl]chromone  (Rf = 0.55). Fraction A8 (8.6 g) was subjected to CC  to afford 13 subfractions: A8-1–A8-13. Fraction A8-2 (620 mg) was purified by CC , 400 mL-fractions) to give seven subfractions: A8-2-1–A8-2-7. Part (82 mg) of fraction A8-2-6 was further purified by preparative TLC  to obtain 6-hydroxy-5-methoxy-2-(2-phenylethyl)chromone  (Rf = 0.48).The dried resinous wood (600 g) of 7-Methoxy-2-[2-(4′-hydroxyphenyl)ethyl]chromone (1): brown plates; m.p. 155–157 °C (CH2Cl2-MeOH); UV (MeOH): λmax (log ε) = 219 (4.64), 247 (4.41), 282 (4.31), 302 (4.21) nm; IR (KBr): υmax = 3418 (OH), 1634, 1590 (γ-pyrone) cm−1; 1H-NMR : see 13C-NMR : see m/z = 297 [M + H]+; HR-ESI-MS: m/z = 297.11224 [M + H]+ .7-Hydroxy-2-[2-(4′-methoxyphenyl)ethyl]chromone (2): yellowish amorphous powder; UV (MeOH): λmax (log ε) = 222 (4.69), 251 (4.42), 303 (4.16) nm; IR (KBr): υmax = 3210 (OH), 1635, 1600 (γ-pyrone) cm−1; 1H-NMR : see 13C-NMR : see m/z = 297 [M + Na]+; HR-ESI-MS: m/z = 297.11227 [M + H]+ .5,6-Dihydroxy-2-[2-(3′-hydroxy-4′-methoxyphenyl)ethyl]chromone (3): yellowish amorphous powder; UV (MeOH): λmax (log ε) = 204 (4.83), 235 (4.52), 358 (3.68) nm; IR (KBr): υmax = 3418 (OH), 1634, 1590 (γ-pyrone) cm−1; 1H-NMR : see 13C-NMR : see m/z = 329 [M + Na]+; HR-ESI-MS: m/z = 329.10186 [M + H]+ .6-Hydroxy-5-methoxy-2-(2-phenylethyl)chromone (4): yellowish amorphous powder; UV (MeOH): λmax (log ε) = 203 (4.40), 238 (4.37), 334 (3.71) nm; IR (KBr): υmax = 3340 (OH), 1633, 1609 (γ-pyrone) cm−1; 1H-NMR : see 13C-NMR : see m/z = 319 [M + Na]+; HR-ESI-MS: m/z = 319.09395 [M + Na]+ .The inhibitory effects of the isolated compounds on LPS-induced NF-κB activation in RAW 264.7/Luc-P1 macrophage were evaluated by measuring the luminescence resulted from luciferase activity in a concentration-dependent manner. The purity of the tested compounds was >98% as identified by NMR and MS.2 incubator in DMEM supplemented with 10% heat-inactivated BCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 μM L-glutamine, and 1 mM sodium pyruvate.RAW 264.7/Luc-P1 cell was an LPS-responsive cell line with an integrated reporter gene (pELAM1-Luc). The cell line was cultured under conditions as described previously . The RAW5 cells in 24-well plates) were treated with isolated compounds, vehicle (0.1% DMSO) or the positive control (30 μM andrographolide) for 1 h and then LPS (10 ng/mL) for 23 h, collected, and analyzed using luciferase assays . Cell lysates (20 μL) were then mixed with 100 μL luciferin right before luminescence detection [® 200 PRO .The RAW 264.7/Luc-P1 cells (1.5 × 10etection . The lum4 cells) were seeded into 96-well plates and treated with compounds  and 0.1% DMSO for 24 h.The MTT assay was used according to a previously described method . RAW 2644 cells) were seeded into 96-well plates and treated with compounds  and 0.1% DMSO for 1 h, then incubated with LPS (1 μg/mL) for 23 h. The 100 μL of cell culture medium was incubated with 100 μL of Griess reagent at room temperature for 10 min. The absorbance was measured at 550 nm against a calibration curve with sodium nitrite (NaNO2) standards.RAW 264.7 cells  are worthy of further biomedical investigation and could be developed as potential candidates for the treatment or prevention of various inflammatory diseases. The structure-and-activity relationship (SAR) of these isolated compounds in term of anti-inflammatory activity certainly merits further investigation.Thirteen compounds, including four new 2-(2-phenylethyl)-4


Orthosiphon stamineus leaf extracts and to identify the active compound(s) contributing to its anti-inflammatory activity using a developed HPLC method. Active chloroform extract of O. stamineus was fractionated into three fractions using a dry flash column chromatography method. These three fractions were investigated for anti-peritoneal capillary permeability, in vitro nitric oxide scavenging activity, anti-inflammatory and nitric oxide (NO) inhibition using carrageenan-induced hind paw edema method. The flavonoid rich chloroform extract fraction (CF2) , significantly reduced rat hind paw edema, NO and decreased dye leakage to peritoneal cavity at p < 0.05. IC50 of in vitro NO scavenging of CF2 was 0.3 mg/mL. These results suggest that the anti-inflammatory properties of these CF2 may possibly be due to the presence of flavonoid compounds capable of affecting the NO pathway.The aim of the present study was to verify the anti-inflammatory activity of  Orthosiphon stamineus (OS), Benth. (Lamiaceae) has been used as a medicinal herb for many centuries in Southeast Asian countries including Indonesia and Malaysia. It is used as a remedy for catarrh of the bladder and as medicine for various disorders such as nephritis, nephrolithiasis, hydronephrosis, vesical calculi, arteriosclerosis, gout and rheumatism , 60m × 0.25 mm, i.d., with a HP 5 methysilicone film (film thickness 0.25 µm). The conditions were as follows; injector temperature, 250 ºC; carrier gas, Helium; column flow rate, 0.9 mL/min; splitting ratio, 10:1; injection volume, 2 µL in all experiment; oven temperature was programmed from 70 ºC to 320 ºC at 22.5 ºC/min. The total ion chromatograms (TICs) and mass spectra were recorded using an Agilent Network 5973N mass engine with enhanced chemstation in the electron impact ionization mode at 2200 eV. The transfer line is maintained at 280 ºC, the source temperature at 230 ºC (maximum 250 ºC) and quadrupole temperature at 150 ºC (maximum 200 ºC). All melting points (uncorrected) were measured with a Gallenkamp (England) melting point apparatus.The CF2 fraction (0.2 g) was subjected to silica gel chromatography and eluted with chloroform/ethanol (85:15). The smaller fractions were combined and further purified by preparative TLC on silica gel 60 FEupatorin: Yellow powder. Mp: 197-199°C. GC-MS : 344 [M+]. FTIR (KBr): 3430 cm-1 (OH), 2945 cm-1 (CH), 1653 cm-1 (C=O), 1497-1566 cm-1 (C=C aromatic) 1605 (C=C stretch). 1H-NMR (CDCl3) δ: 6.58 , 12.78 , 6.61 , 7.45 , 5.83 , 6.98 , 7.48 , 3.96 , 4.00 , 4.02 . 13C-NMR (CDCl3) δ: 164.2 (C2), 104.9 (C3), 182.6 (C4), 153.4 (C5), 133.8 (C6), 158.5 (C7), 90.0 (C8), 153.1 (C9), 104.7 (C10), 124.9 (C1’), 111.1 (C2’), 146.4 (C3’), 149.9 (C4’), 112.7 (C5’), 119.4 (C6’), 61.2 (OMe)*, 56.6 (OMe)*, 56.6 (OMe)*. Sinensetin: Colorless solid. Mp: 174-176°C. GC-MS: 372 [M+]. FTIR (KBr): 2996 cm-1 (CH), 1633 cm-1 (C=O), 1450=1546 cm-1 (C=C aromatic). 1H-NMR (CDCl3) δ: 6.61 , 6.81 , 7.35 , 6.99 , 7.52 , 4.00 , 3.98 , 3.89 , 3.94 , 4.01 . 13C-NMR (CDCl3) δ: 162.1 (C2), 108.4 (C3), 178.1 (C4), 152.9 (C5), 141.4 (C6), 157.8 (C7), 97.3 (C8), 154.8 (C9), 108.1 (C10), 121.2 (C1’), 109.8 (C2’), 149.9 (C3’), 150.3 (C4’), 112.2 (C5’), 120.6 (C6’), 63.2 (OMe)*, 62.5 (OMe)*, 57.3 (OMe)*, 57.2 (OMe)*, 57.1 (OMe)* (* these assignments may be interchanged). Eupatorin and sinensetin gave a positive TLC flavone test which showed a dark blue colouration after being sprayed with NP/PEG and observed under UV 365 nm (data not shown). The UV spectrum showed λmax (log ε) at 243 nm (1.06) and 343 nm (1.36) for eupatorin . Such anti-inflammatory properties may be ascribed to inhibition of prostaglandin synthesis and reduced NO production . It is i


BisP1: 1,5-diphenyl-3,7-bis(2-hydroxyethyl)-3,7-diazabicyclo[3.3.1]nonan-9-one; BisP2: 3,7-bis-(2-(S)-amino-4-methylsulfanylbutyryl)-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride; BisP3: [2-{7-[2-(S)-tert-butoxycarbonylamino-3-(1H-indol-3-yl)-propionyl]-9-oxo-1,5-diphenyl-3,7-diazabicyclo[3.3.1]non-3-yl}-1-(S)-(1H-indol-3-ylmethyl)-2-oxoethyl]-carbamic acid tertbutyl ester; BisP4: 3,7-bis-[2-(S)-amino-3-(1H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride) was assessed against PC cell lines . Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) colorimetric assay, while apoptotic cell death was confirmed using fluorescence microscopy and flow cytometry. Initial viability screening revealed significant cytotoxic activity from BisP4 treatment (1 µM–100 µM) on all three cell lines, with IC50 values for MiaPaca-2, BxPC-3, and CFPAC-1 16.9 µM, 23.7 µM, and 36.3 µM, respectively. Cytotoxic treatment time-response  revealed a 24 h treatment time was sufficient to produce a cytotoxic effect on all cell lines. Light microscopy evaluation (DAPI staining) of BisP4 treated MiaPaca-2 PC cells revealed dose-dependent characteristic apoptotic morphological changes. In addition, flow cytometry confirmed BisP4 induced apoptotic cell death induction of activated caspase-3/-7. The bispidinone derivative BisP4 induced an apoptosis-mediated cytotoxic effect on MiaPaca-2 cell lines and significant cytotoxicity on CFPAC-1 and BxPC-3 cell lines. Further investigations into the precise cellular mechanisms of action of this class of compounds are necessary for potential development into pre-clinical trials.Pancreatic cancer (PC) is a complex, heterogeneous disease with a dismal prognosis. Current therapies have failed to improve survival outcomes, urging the need for discovery of novel targeted treatments. Bispidinone derivatives have yet to be investigated as cytotoxic agents against PC cells. The cytotoxic effect of four bispidinone derivatives ( Pancreatic cancer (PC) remains a devastating disease; it is the fourth most common cause of cancer death in the Western world  and is pDrug development from synthetic compounds and synthetic analogues of pharmacophores in natural products provides the benefit of structural modification of drug candidates to improve potency, selectivity, and bioavailability ,6. PlantThe ability of the bispidine ring to adopt different conformational forms in solution  has been a point of significant research interest, prompting a number of spectroscopic studies ,13. ThisBispidine moieties have been investigated as potential scaffolds for inducing secondary structures in peptides, selected because of their rigidity and lipophilic surface area, potentially enabling interaction with cellular/viral proteins and cell membranes . BispidiN,N′-substituted bispidinones can be prepared using a simple one-pot Mannich reaction nonan-9-ones —are a synthetic bispidine derivatives, distinguished by incorporation of a carbonyl unit at C9. Symmetric reaction ,18, whilapproach ,20. The approach . 3)  .BisP1 was accessed via a “twin double Mannich reaction” from 1,3-diphenylacetone, ethanolamine, and aqueous formaldehyde  on three pancreatic cancer cell lines  revealed that derivative BisP4 was the most effective in reducing cell viability. The parent 1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one was not examined in our assays as it failed our preliminary in silico analysis displaying predicted BBB penetration. BisP4 showed significant cytotoxic activity (<50 µM) when compared to the control , followed by BxPC-3 (23.7 µM) and CFPAC-1 (36.3 µM) (BisP4.The cytotoxicity of the bispidinone derivatives was determined using Cell Counting Kit-8 (CCK-8) colorimetric assays after 48 h of treatment. Results from the initial screening of the four bispidinone derivatives  . ConsequBisP4 treated MiaPaca-2 cells. MiaPaca-2 cells treated with vehicle  induced a dose-dependent increase in the proportion of apoptotic MiaPaca-2 cells . The bispidinone 3,7-bis-[2-(S)-amino-3-(1H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride (BisP4), containing tryptophan pendant arm substituents, was identified to be the most effective of the derivatives evaluated in reducing viability of PC cells. BisP4 showed statistically significant cytotoxic activity, with MiaPaca-2 cells being the most sensitive, showing significant activity at concentrations well below 20 µM.This study investigated the anti-pancreatic cancer activity of four rationally synthesized bispidinone derivatives  group present on BisP3 serves as a protection group during synthesis, preventing chemical transformation of the α-amino group of tryptophan nonan-9-oneBisP2: 3,7-Bis-(2-(S)-amino-4-methylsulfanylbutyryl)-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochlorideBisP3: [2-{7-[2-(S)-tert-Butoxycarbonylamino-3-(1H-indol-3-yl)-propionyl]-9-oxo-1,5-diphenyl-3,7-diazabicyclo[3.3.1]non-3-yl}-1-(S)-(1H-indol-3-ylmethyl)-2-oxoethyl]-carbamic acid tert-butyl ester BisP4: 3,7-Bis-[2-(S)-amino-3-(1H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride1,5-Diphenyl-3,7-bis(2-hydroxyethyl)-3,7-diazabicyclo[3.3.1]nonan-9-one (BisP1)BisP1. Yield: 3.54 g, 93%. m.p. 182–184 °C. IR (cm−1): 3465, 3056, 2945, 2825, 1721, 1600, 1494, 1445, 1344, 1296, 1272, 1208, 1183, 1117, 1080, 1039, 933, 836. To a mixture of 1,3-diphenylacetone  and ethanol (20 mL) was added ethanolamine  and aqueous formaldehyde . The resultant mixture was heated under reflux overnight. After this time, the reaction mixture was chilled and the resulting white precipitate collected and washed with diethylether to afford 1H NMR (CDCl3) (300 MHz): δ 2.73–2.77 , 3.19–3.27 , 3.63–3.71 , 3.74–3.78 , 4.94 , 7.30–7.41 .13C NMR (CDCl3) (75 MHz): δ 54.7 (CPh), 58.3 (CH2OH), 58.6 (exocyclic NCH2), 65.3 (ring CH2), 127.2 (aryl), 127.3 (aryl), 128.0 (aryl), 138.1 (aryl), 209.8 (CO).m/z 380 (11%) M+, 362 (17), 307 (45), 292 (86), 276 (19), 248 (13), 234 (42), 204 (8), 190 (27), 176 (24), 144 (14), 115 (57), 103 (76), 91 (68), 88 (100), 77 (33), 72 (36), 58 (70), 44 (66), 42 (58). LRMS (ESI M + 1): 381Mass Spectrum (EI): 23H29N2O3: calculated 381.2173, found 381.2171HRMS (ESI M + H) for CS)-amino-4-methylsulfanylbutyryl)-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride (BisP2)3,7-Bis-(2-(N-(tert-butoxycarbonyl)-l-methionine  and 1-hydroxy benzotriazole (HOBt) hydrate  in CH2Cl2 (30 mL), was added a solution of 1,3-dicyclohexylcarbodiimide (DCC)  in CH2Cl2 (20 mL). The mixture was stirred overnight and cooled to 4 °C. The precipitate was collected and washed with citric acid , saturated sodium bicarbonate (2 × 100 mL), and brine (100 mL). The organic layer was dried (MgSO4) and evaporated in vacuo. The residue was purified by flash chromatography (EtOAc:CH2Cl2 1:20) to afford {1-(S)-nonane-3-carbonyl]-3-methylsulfanylpropyl}-carbamic acid tert-butyl ester .To a rapidly stirred mixture of 1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one , S)-nonane-3-carbonyl]-3-methylsulfanylpropyl}-carbamic acid tert-butyl ester  from above and stirred at room temperature overnight. The mixture was evaporated in vacuo to dryness and cold ether (5 mL) was added. The resulting precipitate was collected and dried under high vacuum (0.01 mm Hg) for 48 h to afford BisP2 as an off-white solid . m.p. 199 °C decomp., IR (cm−1): 3334, 2927, 2852, 1731, 1634, 1653, 1627, 1635, 1559, 1490, 1379, 1296, 1257, 1054, 760, 698, 617, 510. A 4M HCl solution in dioxane (5 mL) was added to the freshly prepared {1-(1H NMR (CDCl3) (300 MHz): δ 1.89–1.96 , 2.11 , 2.41–2.51 , 3.42–3.52 , 3.87–3.96 , 4.02–4.06 CH), 4.62–4.71 , 5.47–5.57 , 7.19 –7.44 , 8.55 .13C NMR (CDCl3) (75 MHz): δ 18.3 (SCH3), 31.0 (CH2S), 35.7 (CHCH2CH2S), 50.6 (C(O)CH), 52.6 (ring CH2), 53.5 (CPh), 56.4 (ring CH2), 127.7 (aryl), 128.5 (aryl), 128.7 (aryl), 135.9 (aryl), 173.3 (NC(O)CH), 207.2 (CO).−): m/z 555 (M−H)− (100%), 157 (35).Mass Spectrum (ESLRMS (ESI M + 1): 55529H39N4O3S2: calculated 555.2458, found 555.2468HRMS (ESI M + H) for CS)-tert-Butoxycarbonylamino-3-(1H-indol-3-yl)-propionyl]-9-oxo-1,5-diphenyl-3,7-diazabicyclo[3.3.1]non-3-yl}-1-(S)-(1H-indol-3-ylmethyl)-2-oxoethyl]-carbamic acid tert-butyl ester (BisP3)nonan-9-one , 1H NMR (CDCl3) (300 MHz): δ 1.60 , 1.95–2.03 , 2.64–2.74 , 2.87–2.95 , 3.25–3.35 , 3.85–3.93 , 4.89–4.99 , 5.10–5.14 , 5.95 , 6.22–6.26 , 7.13–7.30 , 7.68–7.74 , 8.07 .13C NMR (CDCl3) (75 MHz): δ 29.5 (bocCH3), 32.7 (CH2-indole), 51.5 CONHCH), 53.1 (ring CH2), 53.7 (CPh), 55.7 (ring CH2), 80.5 (C(CH3)), 112.0 (indole CH), 119.9 (indole CH), 120.8 (indole CH), 123.1 (indole CH), 127.5 (aryl), 128.3 (indole CH), 128.8 (aryl), 129.0 (aryl), 135.1 (indole-C), 136.7 (aryl), 156.2 (NC(O)O), 172.0 (NC(O)CH), 207.6 (CO).m/z 864 (1%) M+, 764 (3), 734 (1), 677 (1), 633 (1), 604 (2), 577 (6), 563 (1), 535 (3), 478 (9), 447 (1), 291 (2), 261 (2), 248 (18), 234 (7), 205 (1), 185 (6),144 (5), 132 (6), 130 (3), 116 (24), 103 (13), 101 (100), 91 (38), 77 (8), 57 (63), 44 (51).Mass Spectrum (EI): LRMS (ESI M + 1): 86551H57N6O7: calculated 865.4283, found 865.4289HRMS (ESI M + H) for CS)-amino-3-(1H-indol-3-yl)-propionyl]-1,5-diphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one dihydrochloride (BisP4)3,7-Bis-, using GraphPad Prism software Version 6.0b .The 50% inhibitory concentration  and imaged every 30 min for a total of 20 h at 20× magnification using an Axiovert 200M inverted microscope  equipped with an incubation chamber set at 37 °C/5% CO2. Images were viewed and analysed using AxioVision LE, Version 4.9.1.0 software .MiaPaca-2 cells were seeded in 6-well plates at 1 × 104 cells per well and allowed to adhere for 24 h at 5% CO2/37 °C. Cells were then treated with either 0.1% DMSO (control) or BisP4 at concentrations of 17.5 µM, 26 µM, or 35 µM, and further incubated for 24 h before staining.To visually observe the effect of bispidinone treatment on the viability of pancreatic cancer cells, two different fluorescent cellular staining techniques were used. In preparation for both cell staining techniques, MiaPaca-2 cells were seeded into 12-well plates containing round coverslips, at a density of 5 × 10After treatment, DAPI staining was prepared by gently washing the cells with 3.7% formaldehyde in PBS solution for 5 min. Cells were then permeabilized in 1mL of permeabilization buffer , left for 5 min before aspiration, followed by another 5 min wash in 3.7% formaldehyde/PBS solution. The DAPI 4’,6-diamino-2-phenylindole stain was prepared immediately prior to use  and protected from light. DAPI stain (1000 µL) was added to each well and left for 5 min at room temperature, protected from light. The stain was then removed, followed by a final wash with the 3.7% formaldehyde/PBS solution. The coverslips were then mounted and viewed (20x objective) with an Axiovert 200M inverted microscope . Images were viewed and analyzed using AxioVision LE, Version 4.9.1.0 software.® staining was prepared by gently washing the cells in 1000 µL of cold PBS. The coverslips were removed and placed face up on parafilm, ready for staining. The two dye reagents were combined and diluted in PBS  immediately before use and protected from light. The coverslips were covered with 100µL of the prepared reagent, covered, and incubated for 30 min at room temperature. Following incubation, 4 µL of reagent was placed on a clean microscope slide, before carefully inverting and mounting the wet coverslip, then viewed (20× objective) with an Axiovert 200M inverted microscope . Images were viewed and analyzed using AxioVision LE, Version 4.9.1.0 software .LIVE/DEAD5 cells per well and incubated for 24 h at 37 °C with 5% CO2 for cells to adhere. Cells were treated for 24 h at 37 °C with 0.1% DMSO as the control and a range of BisP4 concentrations . Following treatment, cells were assessed for apoptosis using the MuseTMCaspase-3/7 Kit  as per manufacturer’s instructions. Briefly, cells were dissociated with trypsin, resuspended in Assay Buffer and incubated for 30 min at 37 °C, protected from light in Muse™ Caspase 3/7 working reagent. After incubation, 150 µL of Muse™ Caspase 7-AAD working reagent was added, mixed thoroughly, and incubated for a further 5min at room temperature, protected from light, and analyzed with the Muse™ Cell Analyser (Merck Millipore Corp.) using Muse™ software Version 1.4.MiaPaca-2 cells were seeded into 6-well plates at a concentration of 1 × 10p < 0.05.GraphPad Prism Version 6.0b software  was used for statistical and graphical evaluations. Results are expressed as the mean ±SD of at least six replicates, unless otherwise stated. Statistical comparisons between the differences in cell viability of treatment groups compared to controls were performed using a one-way ANOVA and a post-hoc Dunnett’s Multiple Comparisons test. Statistical significance was set at BisP4 to improve potency and targetability. The versatility and relatively straightforward synthesis of bispidinone derivatives provides the potential for further discovery and development of bispidinone derivatives. The necessary demand for novel pancreatic cancer drug treatments and the results of this study of apoptosis-mediated cytotoxicity from in vitro treatment with a bispidinone derivative, makes this an important effort towards the development of potential novel lead compounds to target pancreatic cancer.Further work is needed to elucidate the detailed molecular mechanisms of the cytotoxic effect. This will lead to potential opportunities of structural optimization of bispidinone derivative


Muntingia calabura (Tiliaceae) is commercially used in healthcare for the improvement of hypertension, myocardial infarction, spasm, and inflammatory conditions. Its fruits can be processed into jam and the leaves can be used for making tea. In the work reported herein a new biflavan, (M),(2S),(2''S)-,(P),(2S),(2''S)-7,8,3',4',5',7'',8'',3''',4''',5'''-decamethoxy-5,5'' biflavan (1), a new flavone, 4'-hydroxy-7,8,3',5'-tetramethoxyflavone (2), and a new dihydrochalcone, (R)-2',β-dihydroxy-3',4'-dimethoxydihydrochalcone (3), have been isolated from the stem wood of M. calabura, together with 12 known compounds (4–15). The structures of these new compounds were elucidated by the interpretations of extensive spectroscopic data. Among the isolated compounds, 5-hydroxy-7-methoxyflavone (5), quercetin (6), and (2S)-7-hydroxyflavanone (10) exhibited potent inhibition of fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 1.77 ± 0.70, 3.82 ± 0.46, and 4.92 ± 1.71 μM, respectively. Muntingia calabura L. (Tiliaceae) is an evergreen tree originally distributed in tropical America , three aromatic protons , an oxymethine proton [δ 5.02 ], and four methylene protons . Comparison of the 1H-, 13C-NMR, and MS data of 1 with those of (M),(2S),(2''S)-, (P),(2S),(2''S)-8,5',8''-trihydroxy-7,3',4',7'',3''',4''',5'''-heptamethoxy-5,5''-biflavan (4) , 5'-methoxy , and 8′′-methoxy  groups of 1 replaced the 8,5',8''-trihydroxy groups of 4 + ion at m/z 381.0952 . The presence of a carbonyl group was revealed by a band at 1630 cm−1 in the IR spectrum, and was confirmed by the resonance at δ 178.0 in the 13C-NMR spectrum. The 1H-NMR spectrum of 2 showed the presence of four methoxy groups , five aromatic protons , and a hydroxy group [δ 5.88 ]. The 1H- and 13C-NMR data of 2 were similar to those of 7,8,3',4',5'-pentamethoxyflavone (7)  of 2 replaced the 4'-methoxy group  of 7. This was supported by HMBC correlation observed between H-2'/H-6' (δ 7.22) and C-4' (δ 136.5). On the basis of the evidence above, the structure of 2 was elucidated as 4'-hydroxy-7,8,3',5'-tetramethoxyflavone, which was further substantiated through 2D-experiments, including HSQC, 1H-1H COSY, HMBC -2',β-Dihydroxy-3',4'-dimethoxydihydrochalcone (3) was isolated as amorphous powder. The ESI-MS of 3 afforded an [M + Na]+ ion at m/z 325, implying a molecular formula of C17H18O5, which was confirmed by HR-ESI-MS . The IR spectrum showed the presence of OH (3412 cm−1) and carbonyl (1625 cm−1) groups. Comparison of the 1H-NMR data of 3 with those of 3'-methoxy-2',4',β-trihydroxydihydrochalcone (3a) [3 replaced the 5'-hydroxy group [δ 6.41 ] of 3'-methoxy-2',4',β-trihydroxydihydrochalcone. This was supported by the NOESY correlations between OMe-4' (δ 3.91) and H-5' (δ 6.46) and by the HMBC correlations between OMe-4' (δ 3.91) and C-4' (δ 158.5). Furthermore, the absolute configuration of 3 was proposed to be R by comparing specific rotation data (3 with that reported for (R)-β-hydroxydihydrochalcone -2',β-dihydroxy-3',4'-dimethoxydihydrochalcone.(7 +60.8) . By the nd NOESY  techniqu1H-NMR, [α]D, and MS) with corresponding authentic samples or literature values, and this included a biflavan, (M),(2S),(2''S)-,(P),(2S),(2''S)-8,5',8''-trihydroxy-7,3',4',7'',3''',4''',5'''-heptame-thoxy-5,5''-biflavan (4) +. HR-ESI-MS: m/z = 741.2889 [M + Na]+ .4'-Hydroxy-7,8,3',5'-tetramethoxyflavone (2). Yellowish needles (MeOH); m.p. 221–223 °C. UV (MeOH): λmax (log ε) = 242 (4.23), 269 (4.29), 312 (4.28) nm. IR (KBr): υmax 3290 (OH), 1630 (C=O) cm−1. 1H-NMR : δ = 3.99 , 4.02 (OMe-8), 4.05 (OMe-7), 6.72 (H-3), 7.07 , 7.22 , 7.96 . 13C-NMR : δ = 56.3 (OMe-3'), 56.3 (OMe-5'), 56.4 (OMe-7), 61.4 (OMe-8), 104.1 (C-2'), 104.1 (C-6'), 106.4 (C-3), 110.0 (C-6), 118.5 (C-10), 121.0 (C-5), 126.9 (C-1'), 136.5 (C-4'), 136.8 (C-8), 147.8 (C-3'), 147.8 (C-5'), 150.4 (C-9), 156.6 (C-7), 162.7 (C-2), 178.0 (C-4). ESI-MS: m/z = 381 [M + Na]+. HR-ESI-MS: m/z = 381.0952 [M + Na]+ .(R)-2',β-Dihydroxy-3',4'-dimethoxydihydrochalcone (3). Amorphous powder. c 0.16, CHCl3). UV (MeOH): λmax (log ε) = 211 (4.05), 230 , 287 (3.85) nm. IR (KBr): υmax = 3412 (OH), 1625 (C=O) cm−1. 1H-NMR spectroscopic data, see 13C-NMR : δ = 52.6 (C-α), 70.2 (C-β), 56.1 (OMe-4'), 60.6 (OMe-3'), 102.9 (C-5'), 114.1 (C-1'), 125.9 (C-4), 126.0 (C-6'), 127.8 (C-2 and C-6), 128.7 (C-3 and C-5), 136.3 (C-3'), 143.2 (C-1), 156.9 (C-2'), 158.5 (C-4'), 200.6 (C=O). ESI-MS: m/z = 325 [M + Na]+. HR-ESI-MS: m/z = 325.1053 [M + Na]+ .The effect of the isolated compounds on neutrophil pro-inflammatory response was evaluated by monitoring the inhibition of superoxide anion generation in fMLP-activated human neutrophils in a concentration-dependent manner. The purity of the tested compounds was >98% as identified by NMR and MS.2+)-free HBSS buffer at pH 7.4 and were maintained at 4 °C prior to use. The protocol was approved by the Institutional Review Board at Chang Gung Memorial Hospital. All donors gave written consent. The Medical Ethics Committee of Chang Gung Memorial Hospital approved this consent procedure.Human neutrophils from venous blood of healthy, adult volunteers (20–30 years old) obtained by venipuncture were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes . Purifie2•− generation was based on the SOD-inhibitable reduction of ferricytochrome c [6 cells/mL) pretreated with the various test agents at 37 °C for 5 min were stimulated with fMLP (1 μmol/L) in the presence of ferricytochrome c (0.5 mg/mL). Extracellular O2•− production was assessed with a UV spectrophotometer at 550 nm . The percentage of superoxide inhibition of the test compound was calculated as the percentage of inhibition = {(control − resting) − (compound − resting)}/(control − resting) × 100. The software, SigmaPlot was used for determining the IC50 values.The assay for measurement of Ochrome c . In briet-test. A probability of 0.05 or less was considered significant. The software SigmaPlot was used for the statistical analysis.Results were expressed as the mean ± SEM, and comparisons were made using Student’s 1–3, were isolated from the stem wood of M. calabura. The structures of these compounds were established on the basis of spectroscopic data. Reactive oxygen species (ROS) , hydrogen peroxide) produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. The effects on neutrophil pro-inflammatory responses of isolates were evaluated by suppressing fMLP/CB-induced O2•− generation by human neutrophils. The results of anti-inflammatory experiments indicate that compounds 2, 4–6, 8, 10, and 12 can significantly inhibit fMLP-induced O2•− generation. 5-Hydroxy-7-methoxyflavone (5), quercetin (6), and (2S)-7-hydroxyflavanone (10) are the most effective among the isolated compounds, with IC50values of 1.77 ± 0.70, 3.82 ± 0.46, and 4.92 ± 1.71 μM, respectively, against fMLP-induced superoxide anion generation. Our study suggests M. calabura and its isolates  could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.Fifteen compounds, including three new compounds


A metal-free, mild and efficient method for the synthesis of 2-methylquinolines was successfully developed by condensation of anilines with vinyl ethers in the presence of catalytic amount of iodine. Modification of both pyridine and benzene moieties was easily achieved by changing only the vinyl ether and aniline. In this reaction, the iodine species was revealed to show dual behavior; molecular iodine serves as an oxidant, while its reduced form, hydrogen iodide, activates the vinyl ether. The redox reaction between these iodine species enables the use of a catalytic amount of iodine in this synthetic method. Iodine-catalyzed reactions have attracted much attention as environmentally sustainable alternatives to transition metal catalysis in industrial chemistry for producing commodity and specialty chemicals, foods, medicines, and pharmaceuticals . Iodine 2 receptor antagonist [Staphylococcus aureus [Meanwhile, the importance and usefulness of quinoline derivatives has considerably increased in the pharmaceutical industry. Among them, 2-methylquinoline derivatives have versatile pharmacological properties such as antibacterial , antimaltagonist , and thetagonist  and an as aureus . In addis aureus ,22.Despite their great importance, 2-methylquinolines are commonly prepared using traditional methods developed by Doebner-von Miller , Skraup H-pyran [Recently, an iodine-mediated reaction was applied to the synthesis of quinoline derivatives. Wang and co-workers reported a molecular iodine-catalyzed reaction for the synthesis of substituted quinolines from imines and aldehydes . FurtherH-pyran . In thisp-methoxyaniline 1a with ethyl vinyl ether 2a in dichloromethane was used as model reaction to optimize conditions (1a recovered (entry 1). On the other hand, quinoline 3a [3a (entry 2). Among the three solvents tested, benzene was found to be the most suitable for this reaction (entries 2–4). While 5 mol % iodine was enough for the reaction to operate efficiently, the yield of quinoline 3a decreased along with a 71% recovery of 1a, when the catalyst loading was reduced to 1 mol % (entries 4−6). Consequently, the reaction conditions used in entry 4 were determined to be the optimal conditions.Initially, the reaction of nditions . The reaoline 3a  was succ3b–h [1b–h . In contrast, aniline possessing both o- and m-methoxy groups (1d) had a higher product yield, indicating that high electron density on the benzene ring overcomes the aforementioned disadvantages (entry 4). In the case of aminophenol 1e, no detectable 3e was produced due to side reactions, such as the oxidation of 1e (entry 5). Anilines connected to another electron-donating group, 1f and 1g, reacted efficiently to afford aminoquinoline 3f and methylquinoline 3g, respectively (entries 6 and 7). Unsubstituted aniline 1h was also a suitable substrate in this reaction, but with somewhat lower efficiency.With the optimized conditions in hand, we applied the reaction to the syntheses of 2-methylquinolines 3b–h ,48,49,50–h [1b–h . The rea2a–f, were subjected to the reaction with p-methoxyaniline 1a , indicating that the pyridine moiety in quinolines 3 was derived from the vinyl group. Indeed, propenyl ether 2c afforded 2-ethyl-3-methylquinoline 4 [2d was used, a quinoline possessing acetal functions, 5, was synthesized (See the 6 and 7 were not detected when electron-poor vinyl ethers 2e and 2f were employed (entries 5 and 6).To further demonstrate the scope of this reaction, various vinyl ethers, iline 1a . Interesnoline 4  in good noline 4 . When 2, See the . Desiredo-methoxyaniline 1d with vinyl ether 2a, several products were formed. In order to gain insight into the mechanism, the reaction mixture was subjected to column chromatography and the products were identified as 4-ethoxy-1,2,3,4-tetrahydro-8-methoxy- 2-methylquinoline (8) as single diastereomer [N-ethyl-2-methoxyaniline (9) [8 was attempted by 1H-NMR, it was not achieved due to two protons at the 3-position having the same coupling constant (12.0 Hz) with a proton at the 2-position. Considering steric repulsion, the stereochemistry was predicted to be the cis-isomer because the ethoxy and methyl groups are located at the equatorial position. Indeed, DFT calculation using B3LYP 6-31G revealed that the cis-isomer was more stable than the trans-isomer, with 1.887 kcal/mol. Interestingly, tetrahydroquinoline 8 was converted to 3d in 77% yield by heating in the presence of iodine. In contrast, no reaction was observed for 9 under the same conditions. These results suggest that 8 is an intermediate in the formation of 3d, and that N-alkylaniline 9 is a by-product (In the reaction of tereomer  and N-etline (9)   at 400 MHz with TMS as an internal standard. The 13C-NMR spectra were measured on a Bruker Ascend-400 at 100 MHz, and assignments of 13C-NMR spectra were performed by DEPT experiments. The high resolution mass spectra were measured on a AB SCIEX Triple TOF 4600 . All the reagents and solvents were commercially available and used as received. The 2a  in benzene (10 mL), were added p-methoxyaniline  and iodine , and the resultant mixture was heated at 80 °C for 2 h. The reaction mixture was washed with saturated sodium thiosulfate solution (1 × 10 mL) to remove unreacted iodine, and dried over magnesium sulfate. After removal of solvent, the residue was subjected to silica gel column chromatography (eluent: hexane/ethyl acetate = 95/5) to afford 3a . To a solution of vinyl ether 1 with other vinyl ether 2 were performed in a similar manner.The reactions of the aniline 6-Methoxy-2-methylquinoline (3a) [1H-NMR : δ 7.94 , 7.91, 7.33 , 7.23 , 7.04 , 3.91 , 2.70 .ine (3a) : 1H-NMR 7-Methoxy-2-methylquinoline (3b) [1H-NMR : δ 7.95 , 7.63 , 7.37 , 7.14 , 7.13 , 3.93 , 2.71 .ine (3b) : 1H-NMR 8-Methoxy-2-methylquinoline (3c) [1H-NMR : δ 8.00 , 7.38 , 7.34 , 7.31 , 7.03 , 4.08 , 2.80 .ine (3c) : 1H-NMR 5,8-Dimethoxy-2-methylquinoline (3d) [1H-NMR : δ 7.45 , 7.09 , 6.87 , 6.66 , 3.90 , 3.75 , 2.68 .ine (3d) : 1H-NMR 6--2-methylquinoline (3f) [1H-NMR : δ 7.98 , 7.76 , 7.37 , 7.22 , 6.88 , 2.97 , 2.56 .ine (3f) : 1H-NMR 2,6-Dimethylquinoline (3g) [1H-NMR : δ 7.96 , 7.95 , 7.52 , 7.51 , 7.24 , 2.74 , 2.51 .ine (3g) : 1H-NMR 2-Methylquinoline (3h) [1H-NMR : δ 8.04 , 8.02 , 7.77 , 7.68 , 7.48 , 7.28 , 2.75 .ine (3h) : 1H-NMR 2-Ethyl-6-methoxy-3-methylquinoline (4) [1H-NMR : δ 7.91 , 7.69 , 7.25 , 6.94 , 3.87 , 2.94 , 2.43 , 1.35 .line (4) : 1H-NMR 5:Compound 1H-NMR : δ 7.89 , 7.81 , 7.27 , 6.99 , 5.14 , 4.01−3.93 , 3.91 , 3.88−3.67 , 3.54−3.48 , 3.07−3.00 , 2.13−2.06 , 2.03−1.78 ; 13C-NMR  23.4 (CH2), 23.5 (CH2), 29.3 (CH2), 32.1 (CH2), 32.3 , 32.4 (CH2), 55.4 (CH3), 66.8 , 103.8 (CH), 103.9(CH), 104.7 (CH), 121.0 (CH), 127.9 (C), 130.0 (CH), 130.9 (C), 134.6 (CH), 142.8 (C), 157.2 (C), 158.9 (C); HRMS Calcd for C23H31NO5: 402.2275. Found: 402.2272. Cis-4-Ethoxy-1,2,3,4-tetrahydro-8-methoxy-2-methylquinoline (8) [1H-NMR : δ 7.00 , 6.64 , 4.70 , 4.1 , 3.82 , 3.69–3.55 , 3.51 , 2.21 , 1.70 , 1.28 , 1.27 .line (8) : 1H-NMR N-Ethyl-2-methoxyaniline (9) [1H-NMR : δ 6.87 , 6.76 , 6.65 , 6.60 , 4.08 , 3.84 , 3.16 , 1.28 .line (9) : 1H-NMR 3 from substituted anilines 1 and vinyl ethers 2 in the presence of an iodine catalyst. This protocol can be performed with simple manipulations in one step under mild conditions for both the reaction and work-up. Furthermore, no transition metals are used, which eliminates the need for a product decontamination step, thus considerably reducing the cost. Hence, this is a new synthetic method for quinoline derivatives that can be applied in various fields.In conclusion, we have successfully developed an environmentally benign and efficient method for the construction of quinolines Furthermore, the roles of iodine species were also studied, finding that the iodine species had dual behavior, with molecular iodine serving as an oxidant, and its reduced form, hydrogen iodide, activating the vinyl ether. The redox reaction between these iodine species enables the use of a catalytic amount of iodine in this reaction.