Prangos ferulacea (L.) Lindl. (Apiaceae) is a medicinal plant distributed in Mediterranean regions, Caucasia and southwest of Asia. In the present study phytochemical constituents of the extract obtained from the aerial parts of P. ferulacea were investigated using various chromatographic and spectroscopic methods. Essential oil of the plant aerial parts was also analyzed using GC-MS. Five phenolic derivatives, isoimperatorin (1), ferudenol (2), caffeic acid glucosyl ester (3), isorhamnetin-3-O-β-D-glucopyranoside (4) and quercetin-3-O-β-D-glucopyranoside (5) were isolated from the aerial parts of P. ferulacea and their structures were elucidated using 1H-NMR, 13C-NMR, EI-MS and UV spectral analyses. Twenty-seven compounds were also identified in the essential oil of plant aerial parts, of which β-pinene (43.1%), α-pinene (22.1%) and -δ3-carene (16.9%) were characterized as main compounds. The results of this study introduce P. ferulacea as a source of potentially bioactive phenolic compounds and suggest it as an appropriate candidate for further studies.  Prangos Lindl. from Apiaceae family, consists of about 30 species, mainly distributed in Irano-Turanian phytogeographic region (Prangos ferulacea (L.) Lindl. (The genus c region . In flor) Lindl. .P. ferulacea is a perennial plant with up to 1.5 m height which grows in Eastern Europe, Turkey, Caucasia and southwestern Asia  reported the isolation of four coumarins, herniarin, umbelliferone, scopoletin, and osthenol, along with two furanocoumarins, xanthotoxin and imperatorin from the aerial parts of P. ferulacea , ferudenol (2), caffeic acid glucosyl ester (3), isorhamnetin-3-O-β-D-glucopyranoside (4) and quercetin-3-O-β-D-glucopyranoside (5) from the aerial parts of General procedures1H-NMR and 13C-NMR spectra were acquired in CDCl3 and DMSO-d6 on a Bruker Avance DRX 400 spectrometer. EI-MS spectra were obtained on a Hewlett-Packard model 5973 HP system. UV spectra were recorded in methanol (and after the addition of shift reagents) on a CECIL 7250 spectrophotometer. 254 (Merck) plates. Pre-coated silica gel GF254 aluminum sheets (Merck) were used for TLC and monitoring of the spots were carried out under UV (254 and 366 nm) and by spraying with anisaldehyde-H2SO4 reagent followed by heating at 120°C for 5 min. All of the solvents were also obtained from Merck chemical company.Silica gel , fully endcappedRP-18  and Sephadex LH-20 (Fluka) were applied for column chromatographies. Preparative thin layer chromatography (PTLC) was also performed on handmade silica gel 60 GFPlant materialP. ferulacea were collected from the Razi border, located in Khoy County  in June 2014. The plant was identified by botanist Dr. Yousef Ajani, Institute of Medicinal Plants, Academic Center for Education, Culture and Research (ACECR), Karaj, Iran.The flowering aerial parts of Extraction and fractionationP. ferulacea (1kg) were grinded and extracted exhaustively with MeOH by maceration method at the room temperature (10×12 L and 48 h each time). The combined extracts were concentrated by a rotary evaporator at 45°C. The obtained total extract (274.58 g) was fractionated successively with petroleum ether and chloroform using liquid-liquid extraction, to get three main fractions, petroleum ether, chloroform and methanol (residue) fractions.The air-dried aerial parts of Isolation and purification of compounds The petroleum ether fraction (12 g) was dissolved in petroleum ether (200 ml) and transferred into a flask. Following the addition of ethanol (30 ml) to above solution, a white precipitate was appeared which was then separated by filtration and named fraction E2.n-hexane-EtOAc (9:1 to 5:5) to get fourteen fractions (E2A-E2N). Fraction E2B (52 mg) was chromatographed on silica gel PTLC with n-hexane-EtOAc (8:2), to give compound 1 (9 mg). Compound 2 (5 mg) was also obtained from the fraction E2I (18 mg) by chromatography on silica gel PTLC . A part of methanol fraction (6 g) was moved in two divided portions over a sephadex LH-20 column (4×25 cm) and eluted with MeOH-H2O (8:2), to get three fractions (M1-M3). Reversed phase C18 column chromatography (3×15 cm) of the fraction M2 (2 g) with ACN (acetonitrile)-H2O (1:9-3:7) yielded eleven fractions (M2A-M2K). Chromatography of the fraction M2A (250 mg) on a RP-18 column (1.5×20 cm) using ACN-H2O (0.5:9.5) resulted in isolation of compound 3 (18 mg). Fraction M2H (38 mg) was rechromatographed on a RP-18 column (1×20 cm) with ACN-H2O (1:9-2:8) to obtain compound 4 (21 mg). Compound 5 (7 mg) was also achieved from the fraction M2I (34 mg) through RP-18 column chromatography (1×20 cm) using ACN-H2O (1.5:8.5) as eluent and its impurities were removed over a sephadex LH-20 column .The fraction E2 (7 g) was moved over a silica gel column (4.5×30 cm) and eluted with Isolation of essential oil Essential oil of the plant aerial parts was extracted using hydro-distillation for 4 h by a Clevenger-type apparatus. The prepared oil was subsequently dried over anhydrous sodium sulfate and stored at 4°C in the dark until analysis.GC-MS analysis-1. The oven temperature was raised from 50 °C to 240 °C at a rate of 3 °C per minute and then raised to 300 °C at a rate of 15 °C and finally maintained at 300 °C for 3 min. The injection temperature was 290 °C, and the oil sample (1.0 µL) was injected with a split ratio of 1:30. The Kovats retention indices (KI) of the compounds were calculated using a homologous series of n-alkanes (C8-C30) injected in conditions equal to the samples. Identification of the chemical constituents was performed using Wiley7n.l online library, as well as by direct comparison of their MS spectra and KIs with data published in the literature for standard compounds , 0.25 µm film thickness) equipped with a MS detector  was applied for the essential oil analysis. The flow rate of carrier gas (Helium) was 0.5 ml minResultsP. ferulacea yielded the isolation of five compounds. The structures of isolated compounds were established as isoimperatorin (1), ferudenol (2), caffeic acid glucosyl ester (3), isorhamnetin-3-O-β-D-glucopyranoside (4) and quercetin-3-O-β-D-glucopyranoside (isoquercetin) (5)  (5)  using thterature , 30-33. Spectral data of the isolated compounds Compound 1; Isoimperatorin: White crystals, 1H-NMR : δ 8.17 , 7.60 , 7.15 , 6.97 , 6.26 , 5.55 , 4.93 , 1.81 , 1.71 ; 13C-NMR ; 161.1 (C2), 158.2 (C7), 152.6 (C9), 148.8 (C5), 144.8 (C2'), 139.7 (C3''), 139.4 (C4), 119.2 (C2''), 114.9 (C6), 112.3 (C3), 107.3 (C10), 105.1 (C3'), 95.1 (C8), 69.8 (C1''), 25.8 (C5''), 18.3 (C4''); UV (MeOH) λ max: 249, 260, 309 (30).Compound 2; Ferudenol: White crystals, 1H-NMR : δ 8.52 , 7.65 , 7.37 , 6.88 , 6.27 , 4.89 , 4.62 , 3.96 , 3.28 , 3.17 ,1.92 ; UV (MeOH) λ max: 210, 253 (sh), 322.; EI-MS (m/z): 276 [M+], 258, 219, 203, 189, 175, 131 (27).Compound 3; Caffeic acid glucosyl ester: Light brown solid, 1H-NMR : δ 7.46 , 7.04 , 6.95 , 6.75 , 6.23 , 4.10 , 2.8-3.8 ; 13C-NMR : δ 170.1 (C9), 150.2 (C4), 149.6 (C3), 146.3 (C7), 128.3 (C1), 125.1 (C6), 118.4 (C8), 117.5 (C5), 115.1 (C2), 102.4 (C1'), 79.1 (C3'), 74.6 , 69.1 (C4'), 60.7 (C6'); UV (MeOH) λ max: 206, 245 (sh), 300 (sh), 328., +AlCl3: 206, 259, 304 (sh), 366., +AlCl3/HCl: 206, 272 (sh), 302 (sh), 342 (31).Compound 4; Isorhamnetin-3-O-β-D-glucopyranoside: Yellow powder, 1H-NMR : δ 8.06 , 7.40 , 6.91 , 6.29 , 6.09 , 5.50 , 3.82 , 3.0-4.0 ; 13C-NMR : δ 177.0 (C4), 167.3 (C7), 161.1 (C5), 156.6 (C2), 155.2 (C9), 149.8 (C3'), 145.9 (C4'), 133.4 (C3), 122.8 (C6'), 121.2 (C1'), 115.6 (C5'), 111.3 (C2'), 103.6 (C10), 101.0 (C1''), 99.5 (C6), 93.9 (C8), 77.5 (C3''), 76.5 (C5''), 74.1 (C2''), 69.9 (C4''), 60.9 (C6''), 55.6 (OCH3); UV (MeOH) λ max: 253, 335, 360 (sh)., +AlCl3: 365, 266., +AlCl3/HCl: 266, 305 (sh), 355, 404 (sh)., +NaOMe: 272, 329 (sh), 395., +NaOAc: 332, 269; EI-MS (m/z): 299 [Aglycon+], 149, 137 (32).Compound 5; Quercetin-3-O-β-D-glucopyranoside (Isoquercetin): Yellow powder, 1H-NMR : δ 8.28 ; 7.35 ; 6.82 ; 6.40 ; 6.20 ; 5.23  3.0-4.0 ; 13C-NMR : δ 177.3 (C4), 164.5 (C7), 161.2 (C5), 156.3 (C2), 156.1 (C9), 148.5 (C4'), 144.8 (C3'), 133.2 (C3), 121.5 (C6'), 121.1 (C1'), 116.1 (C5'), 115.2 (C2'), 103.8 (C10), 100.8 (C1''), 98.7 (C6), 93.5 (C8), 77.5 (C5''), 76.4 (C3''), 74.0 (C2''), 69.9 (C4''), 60.9 (C6''); UV (MeOH) λmax: 259, 368., +AlCl3: 269, 304 (sh), 421., + NaOMe: 272, 411., +NaOAc: 262, 368 (33).P. ferulacea essential oil, among them β-pinene (43.1%), α-pinene (22.1%) and δ-3-carene (16.9%) were characterized as main compounds  pale yellowish oils. Twenty seven compounds  were identified as a result of GC-MS analysis of ompounds . The resP. ferulacea aerial parts. These compounds (1 and 2) have been previously reported from the roots of P. ferulacea  and direct inhibition of CYP1A activity  and ferudenol (2) were isolated from the petroleum ether fraction of erulacea , 27, 28. extract . In 2006activity . It has  and feruactivity , 37. Furrculosis  and its rculosis  have beerculosis  and querrculosis . Caffeicrculosis . A revierculosis , 41, 42.mpound 3  43). Ca. CaP. feimganica .  Two isranoside , have beranoside , 46, α-granoside , 46, antranoside , 48, antranoside  and antiranoside  activitiranoside .P. ferulacea aerial parts resulted to the identification of twenty seven compounds, mainly β-pinene (43.1%), α-pinene (22.1%), δ-3-carene (16.9%)  . Ostholetraction . Previouet al. (1998) reported β-pinene (22.9%), δ-3-carene (16.0%) and α-pinene (12.6%) as the main compounds of the essential oil of P. ferulacea aerial parts from Isfahan (center of Iran)  revealed that α-thujene, α-pinene, camphene, β-pinene, β-myrcene, δ-3-carene, α-terpinene, p-cymene, (Z)-β-ocimene, (E)-β-ocimene, γ-terpinene, β-caryophyllene and α-bisabolol are the compounds present in both essential oil samples  reported the level of monoterpene hydrocarbons at 64.0 and 2.7%, respectively -ane samples . The pre3-carene .0% and αectively . Genetical parts .1-5) from the aerial parts of P. ferulacea emphasize the therapeutic potentials of this medicinal species and suggest it as an appropriate candidate for further biological and pharmacological research. This study also introduces P. ferulacea as a plant with monoterpene rich oil and report β-pinene (43.1%) as the main compound of its aerial parts oil. The results of present study on isolation of bioactive phenolic derivatives (


The molecule comprises a tetrasubstitued phenyl ring linked to a toluenesulfonate through one of its O atoms. In the crystal, C—H⋯O and C—H⋯π interactions link the molecules, forming a three-dimensional network.The title compound, C The reaction mixture was heated to reflux for two h. The end of the reaction was controlled by TLC. The reaction mixture was washed with a hydrochloric acid solution (0.1 M) to neutral pH, extracted three times with ethyl ether (3 × 20 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using hexane/ethyl acetate (94:6) as eluent to give 360 mg  of 2-isopropyl-4-methoxy-5-methylphenyl 4-methylbenzenesulfonate 2. X-ray quality colourless crystals were obtained by slow evaporation of a petroleum ether solution of the title compound.In a 100mL flask, 430 mg (2.33mmol) of NMR data for compound 2:1H NMR : 6.66 , 6.84 , 3.45 , 1.09 , 2.13 , 3.72 , 7.33 , 7.75 , 2.43  ppm. 13C NMR : 156.4 (C-1), 124.0 (C-2), 107.5 (C-3), 145.2 (C-4), 139.8 (C-5), 124.1 (C-6),15.7 (C-7), 26.8 (C-8), 23.1 , 55.4 (OCH3), 145.2 (C-1′), 129.7 (C-2′), 128.4 (C-3′), 139.7 (C-4′), 21.5 (C-5′) ppm.Uiso(H) = 1.2Ueq(CH and CH2) or Uiso(H) = 1.5Ueq(CH3).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018003110/xu5919sup1.cifCrystal structure: contains datablock(s) I, global. DOI: Click here for additional data file.10.1107/S2056989018003110/xu5919Isup3.cmlSupporting information file. DOI: 10.1107/S2056989018003110/xu5919Isup3.hklStructure factors: contains datablock(s) I. DOI: 1825267CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the battle against cancer discovery of new and novel chemotherapeutic agent demands extreme obligation. Development of anticancer compounds with higher potency and reduced side-effects is timely and challenging.in vitro evaluation. β-tubulin was considered as possible molecular target and in vitro anticancer evaluation was conducted against SiHa, B16F10, K562 and Chang cell lines. Caspase-3 activation assay and hematoxylin/eosin staining of the cells were also accomplished.A small series of fourteen diastereomeric β-lactams (seven pairs) were synthesized through multi-step process exploring [2+2] ketene-imine cycloaddition as the key step. Comparative stereochemical preferences were studied through computational docking and validated by cis- over the trans-β-lactams indicated favorable β-lactam—β-tubulin interactions in cis-geometry. In vitro (IC50) evaluation confirmed better anticancer activity of the cis-diastereoisomers. Apoptosis-induced cell death was supported by caspase-3 activation study. A cis-β-lactam  was found to be more active (in vitro) than the marketed natural drug colchicine against SiHa and B16F10 (six times higher potency) cell lines. Reduced toxicity (compared to colchicine) in Chang cells confirmed better site-selectivity (accordingly less side-effects) of 6C than colchicine. Aside from 6C, most of the reported molecules demonstrated good to strong in vitro anticancer activity against SiHa and B16F10 cancer cell lines.Better docking scores of the cis-β-lactams over their trans-counterparts, toward the molecular target β-tubulin, was confirmed by docking studies and in vitro anticancer evaluation. Apoptosis was identified as the cause of cell death. The lead 6C exhibited higher potency and selectivity than the marketed drug colchicine both in silico as well as in vitro.Stereochemical preferences of the  Cancer is one of the leading causes of death globally and is expected to increase approximately 70% in the next two decades . Althougtrans-β-lactams have been reported  ketene-imine cycloaddition reaction  was perfA total of 14 β-lactam derivatives (seven pairs of diastereomers) have been synthesized  through The structure of the 2-azetidinone derivatives were elucidated by extensive spectroscopic analyses as follows:Cis-3-amino-1,4-bis(4-methoxyphenyl) azetidin-2-one (1C). Brown solid (78%); Formula Weight: 298.34; MP 102 ºC; IR (KBr) 3401, 3012, 1715,1507, 1246, 826 cm-1; 1H NMR  δ 7.32 , 7.20 , 6.95 , 6.81 , 5.21 , 4.57 , 3.82 , 3.77 . Formula: C16H16N2O2; Anal. calcd for: C, 71.6%; H, 6.0%; N, 10.4%.(±)-Cis-3-amino-4-(4-methoxyphenyl)-1-phenyl-azetidin-2-one (2C). Brown solid (88%); Formula Weight: 268.32; MP 106 ºC; IR (KBr) 3341, 2928,1728, 1499, 1248, 746 cm-1; 1H NMR  δ 7.29 – 7.08 , 7.03 – 6.93 , 6.86 – 6.77 , 4.56 , 3.96 , 3.71  1.93 . Anal. calcd for: C17H18N2O3 Composition: C, 68.4%; H, 6.1%; N, 9.4%.(±)-Cis-3-amino-4--1-(4-methoxyphenyl) azetidin-2-one (3C). Brown solid (68%); Formula Weight: 328.36; MP 92 ºC; IR (KBr) 3379, 2956, 1738, 1509,1237, 828 cm-1; 1H NMR  δ 6.83 – 6.80 , 6.79 – 6.76 , 6.75 – 6.74 ,, 5.10 , 4.48 , 3.80 - 3.68 . Anal. calcd for: C18H20N2O4; Composition: C, 65.8%; H, 6.1%; N, 8.5%.(±)-Cis-3-amino-1-(4-methoxyphenyl)-4-(2-nitrophenyl) azetidin-2-one (4C). Brown solid (52%); Formula Weight: 313.31; MP 185 ºC; IR (KBr) 3325, 2952, 1747, 1509, 1248, 822 cm-1; 1H NMR  δ 8.08 – 8.03 , 7.79 – 7.76 , 7.75 – 7.70 , 7.70 – 7.66 , 7.62 – 7.56 , 6.08 , 6.05 , 5.35 – 5.27 , 3.83 . Anal. calcd for: C16H15N3O4; Composition: C 61.3%, H 4.8%, N 13.4%.(±)-Cis-3-amino-1-(4-methoxyphenyl)-4-(p-tolyl) azetidin-2-one (5C). Brown solid (80%); Formula Weight: 282.34; MP 103 ºC; IR (KBr)3359, 2934, 1730, 1510, 1245, 823 cm-1; 1H NMR  δ 7.24 – 7.20 , 7.14 , 7.07 , 6.74 – 6.70 , 5.13 , 4.50 , 3.68  2.36 – 2.32 . Anal. calcd for: C17H18N2O2; Composition: C, 72.3%; H, 6.4%; N, 9.9%.(±)-Cis-3-amino-1-phenyl-4-(p-tolyl) azetidin-2-one (6C). Brown solid (84%); Formula Weight: 252.31; MP 130 ºC; IR (KBr) 3356, 2925, 1731, 1502, 748 cm-1; 1H NMR  δ 7.73 , 7.71 – 7.65 , 7.43 – 7.35 , 7.26 , 6.94 – 6.87 , 5.68 , 5.46 , 3.82 . Anal. calcd for: C16H16N2O; Composition: C, 76.2%; H, 6.4%; N, 11.1%.(±)-Cis-3-amino-1-(4-methoxyphenyl)-4-(2-pyridyl)azetidin-2-one (7C). Brown solid (51%); Formula Weight: 269.30; MP 120 ºC; IR (KBr) 3373, 2952, 1721, 1509, 1239, 828 cm-1; 1H NMR  δ 8.71 , 7.74 , 7.33 , 7.31 – 7.27 , 6.82 , 5.32 , 4.76 , 3.77 . Anal. calcd for: C15H15N3O2; Composition: C, 66.9%; H, 5.6%; N, 15.6%.(±)-Trans-3-amino-1,4-bis(4-methoxyphenyl) azetidin-2-one (1T). Brown solid (71%); Formula Weight: 268.32; MP 96 ºC; IR (KBr) 3341, 2953, 1724,1510, 1245, 823 cm-1; 1H NMR  δ 7.19 , 7.14 , 6.81 , 6.69 , 4.57 , 3.96 , 3.72 , 3.66 . Anal. calcd for: C16H16N2O2; Composition: C, 71.6%; H, 6.0%; N, 10.4%.(±)-Trans-3-amino-4-(4-methoxyphenyl)-1-phenyl-azetidin-2-one (2T). Brown solid (72%); Formula Weight: 298.34; MP 102 ºC; IR (KBr) 3342, 3065,1730, 1513, 1249, 747 cm-1; 1H NMR  δ 7.29 – 7.08 , 7.03 , 6.86 , 4.56 , 3.96 , 3.71  1.84 . Anal. calcd for: C17H18N2O3Composition: C, 68.4%; H, 6.1%; N, 9.4%.(±)-Trans-3-amino-4--1-(4-methoxyphenyl) azetidin-2-one (3T). Brown solid (93%); Formula Weight: 328.36; MP 104 ºC; IR (KBr) 3301, 3001, 1733, 1508,1237, 877 cm-1; 1H NMR  δ 7.32 , 7.25 , 6.91 , 6.87 , 6.85 – 6.83 , 6.81 , 6.76 , 4.61 , 4.07 , 3.89 – 3.77 . Anal. calcd for: C18H20N2O4; Composition: C, 65.8%; H, 6.1%; N, 8.5%.(±)-Trans-3-amino-1-(4-methoxyphenyl)-4-(2-nitrophenyl) azetidin-2-one (4T). Brown solid (28%); Formula Weight: 313.31; MP 195 ºC;IR (KBr) 3282, 3064, 1745, 1509, 1243, 860 cm-1; 1H NMR  δ 7.17 – 7.13 , 7.09 , 6.72 – 6.67 , 4.89 , 4.84 , 4.55 , 3.66 . Anal. calcd for: C16H15N3O4; Composition: C 61.3%, H 4.8%, N 13.4%.(±)-Trans-3-amino-1-(4-methoxyphenyl)-4-(p-tolyl)azetidin-2-one (5T). Brown solid (95%); Formula Weight: 282.34; MP 110 ºC; IR (KBr) 3294, 2952, 1730, 1509, 1242, 824 cm-1; 1H NMR  δ 7.23 – 7.18 , 7.15 , 7.10 , 7.06 , 6.74 – 6.68 , 4.52 , 3.93 , 3.66  2.36 – 2.32 . Anal. calcd for: C17H18N2O2; Composition: C, 72.3%; H 6.4%; N, 9.9%.(±)-Trans-3-amino-1-phenyl-4-(p-tolyl)azetidin-2-one (6T). Brown solid (62%); Formula Weight: 252.31; MP 135 ºC; IR (KBr) 3354, 3028, 1731, 1501, 792 cm-1; 1H NMR  δ 7.21 , 7.18 – 7.14 , 7.11 , 7.00 – 6.94 , 4.57 , 3.96 , 2.32 – 2.23 , 1.78 . Anal. calcd for: C16H16N2O; Composition: C, 76.2%; H, 6.4%; N, 11.1%.(±)-Trans-3-amino-1-(4-methoxyphenyl)-4-(2-pyridyl)azetidin-2-one (7T). Brown solid (60%);Formula Weight: 269.30; MP 114 ºC;IR (KBr) 3371, 2953, 1729, 1510, 1294, 820 cm-1; 1H NMR  δ 8.71 , 7.74 , 7.33 , 7.31 – 7.27 , 6.82 , 5.32 , 4.76 , 3.77 . Anal. calcd for: C15H15N3O2; Composition: C, 66.9%; H, 5.6%; N, 15.6%.(±)-By means of computational chemistry, a molecular docking assay was performed targeting the protein β-tubulin. The drug-protein interaction of all the fourteen 3-amino-azetidin-2-one derivatives (1C–7C and 1T–7T) was studied on the three available and known binding sites  of β-tubulin . VinblasCis-3-amino-1-phenyl-4-(p-tolyl)azetidin-one (6C) is shown in Figure The docking interactions of (±)-50 values of the fourteen 2-azetidinone derivatives (seven pair of diastereomers) were calculated from the concentrations of 10 to 0.312 μM for each compound (triplicate) against SiHa , B16F10 (murine melanoma), K562  and Chang cell lines. IC50 values of the azetidin-2-ones are shown in Table The ICThe cytotoxic effect of the compound 6C in SiHa and Chang cells has been studied Figure . In a Hetrans diastereomers exhibited moderate to poor (IC50: 4.79 to >10.00 in SiHa and 5.16 to > 10.00 in B16F10 cell lines) anticancer activity against the tested cancer cell lines as well as in Chang  cells  as positive control  [1H NMR spectra of the compounds (±)-trans-3-amino-1,4-bis(4-methoxyphenyl)azetidin-2-one (1T) and (±)-cis-3-amino-1,4-bis(4-methoxyphenyl)azetidin-2-one (1C) could be considered  and 4.57 ppm (J = 1.6 Hz) respectively whereas in the compound (1C), the coupling constant values for both the protons H-3 and H-4 are the same (J = 5.0 Hz). In fact, the diastereoselectivity in β-lactam formation depends on the generation of zwitterionic intermediate and the extent of conrotatory cyclization (ring-closure) of the intermediate to produce cis or trans β lactams [It is always challenging to control the diastereoselectivity in β-lactam synthesis. The oisomer) . As a re lactams , 20.Rule of 5’ (RO5) in hits’. The in vitro anticancer evaluation has been carried out against SiHa , B16F10 (murine melanoma), K562  and Chang cell lines. It has also been confirmed that the mechanism of anticancer activity was apoptosis-induced cell death. Finally, through a molecular docking analysis, a better and clearer understanding of the plausible β-lactam–β-tubulin interaction was explained, considering the protein β-tubulin as the therapeutic target.It is well-known that stereochemistry of a drug molecule is greatly responsible for its pharmacological activity. Accordingly, the stereoisomer that produces better therapeutic activity is known as eutomer and the less potent (sometimes inactive or even toxic) stereoisomer is known as distomer. The drugability of the newly synthesized terminal β-lactams (1C–1T and 1T–7T) has been validated by modified ‘cis and trans β-lactams on the proposed target β-tubulin do not match with that of two widely used natural anticancer drugs taxol and vincristine rather the values match with that of another anticancer alkaloid colchicine. This indicates that the binding sites of the β-lactams and colchicine are similar in nature. Accordingly, the detail mechanism of action of the β-lactams might be similar to that of colchicine. The docking score of the compound 6C is better than the natural drug colchicine  in murine melanoma (B16F10) cell lines and IC50 = 2.34 μM in human cervical carcinoma SiHa cell lines and qualified as the lead with considerable anticancer activity in comparison to colchicine. The trans-β-lactams 1T, 2T, 4T, 5T, and the cis-β-lactams 2C, 3C, 4C and 7C showed good in vitro anticancer activity (IC50 = 5 – 10 μM) against SiHa cancer cell lines whereas the β-lactams 1T, 2T, 5T and 4C demonstrated good in vitro anticancer activity (IC50 = 5 –10 μM) against B16F10 cancer cell lines. On the other hand, two compounds 2C and 3C showed strong in vitro anticancer activity (IC50 < 5 μM) against B16F10 cancer cell lines. Besides the lead compound 6C, two other compounds 1C and 5C demonstrated strong in vitro anticancer activity (IC50 = 5–10 μM) against SiHa and B16F10 cancer cell lines. Interestingly, two cis-compounds 3C and 7C showed good in vitro anticancer activity (IC50 = 5–10 μM) against K562 cancer cell lines. All the fourteen compounds are less toxic (more selective) than the positive control colchicine, a well-known cancer chemotherapeutic agent. In Chang cells 6C showed 10 times lesser toxicity in comparison to the drug colchicine  and 13C NMR (150 MHz) spectra were obtained at room temperature with Bruker superconducting Ultrashield Plus 600 MHz NMR spectrometer with central field 14.09 T, coil inductance 89.1 H, and magnetic energy 1127.2 kJ using CDCl3 as solvent. All the solvents were purchased from Fisher-Scientific throughout the investigation. Dichloromethane and triethylamine were dried following the standard procedures. Deionized water was used for the preparation of all aqueous solutions.Melting points were determined in a Fisher Scientific electrochemical Mel-Temp* manual melting point apparatus (Model 1001) equipped with a 300ºC thermometer. FT-IR spectra were recorded on a Bruker Alpha modular Platinum-ATR FT-IR spectrometer with OPUS software, using the samples directly (neat) without making pallets. Amine and aldehyde were mixed in equimolar (1:1) ratio and refluxed in dry toluene using a Dean-Stark water separator. After completion of the reaction , the solvent was removed under reduced pressure distillation (by rotavapor) and the pure imine was isolated by crystallization from dichloromethane/hexanes.N-phthaloylglycine) (1.5 mmol) in anhydrous dichloromethane (10 mL) was added (dropwise during 30 minutes) to another solution consisted of 2-chloro-1-methylpyridinium iodide  and triethylamine (6 mmol) in 10 mL dry dichloromethane at 0 to (–5)°C temperature under inert (argon) atmosphere. This mixture was stirred for 2 hours maintaining the same temperature. Imine (1 mmol) in dry dichloromethane (10 mL) was then added to this mixture at the same temperature. The reaction mixture was then stirred overnight at room temperature and monitored by thin layer chromatography (TLC). After completion of the reaction, the reaction mixture was washed with saturated sodium bicarbonate solution (10 mL), brine (10 mL) and deionized water successively. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to obtain the crude product. Column chromatography over silica gel was performed using ethyl acetate/hexanes mixtures to isolate the cis- and trans-3-phthalimido β-lactams in pure form. The cis:trans ratio was approximately 2:1 in all the cases.A representative experimental procedure is described as follows: A solution of phthalimidoacetic acid (via column chromatography over silica gel using ethyl acetate/hexanes as the eluent.Ethylenediamine (2 mmol) was added to a solution of diastereomerically pure 3-phthalimido β-lactam (1 mmol) in anhydrous ethyl alcohol (5 mL) under argon atmosphere. This mixture was stirred at room temperature and the progress of the reaction was monitored by TLC. After completion of the reaction (15 min–1 h) the ethanol was removed by reduced pressure distillation and the crude mass was extracted with ethyl acetate (25 mL). The ethyl acetate layer was washed with brine and water successively (10 mL), and dried over anhydrous sodium sulfate. The pure products were obtained One of the major cancer related proteins, β-tubulin, was considered as the theoretical target to perform docking studies of the azetidin-2-ones (β-lactams) as possible antiproliferative compounds. The hydrogens and charges on receptor and ligand were assigned with the Chimera program . The pdbIn vitro anticancer assays were performed in triplicate for the determination of mean values. The human cervical carcinoma SiHa, murine melanoma B16F10, chronic myelogenous leukemia (CML) K562, and Chang cell lines were cultured in minimum essential medium of Eagle at 37 °C in a 95% O2/5% CO2 atmosphere with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin. Cells were trypsinized and seeded at a density of 5 × 104 cells/mL in a 96-well plate and incubated at 37 °C, 95% O2/5% CO2 atmosphere for 24 h. Thereafter the cells were treated with azetidin-2-one derivatives (dissolved in DMSO) at various concentrations between 312 nM–10 μM, and re-incubated for further 72 h. Wells containing the equal volume of the vehicle DMSO (1% v/v) were used as background (negative control). The culture medium was then removed and the cells washed with 100 μL phosphate buffered saline (PBS); then 10 μL WST-1  was added. Cells were incubated for 2 h in absence of light at 37°C, and absorbance was read subsequently [50) using Graph-Pad Prism software.equently . The abs4) were grown on coated poly-lysine coverslips which were placed in 6-well microplates overnight and then incubated for 24 h in the presence of compound 6C or taxol, subsequently stained with hematoxylin and eosin. Coverslips were then observed by Light microscopy of bright field (40X).Cells (10 × 106 cells/mL (B16F10) was taken and incubated for 24 h at 37 °C in 5% CO2 environment. Podophyllotoxin (apoptosis inducing agent) was used as positive control. After incubation for 24 h the cells were re-suspended and 50 μL of cell lysis reagent was added, then cells were incubated for 10 min in ice bath and centrifuged. The supernatant (cytosol extract) was transferred into a new effendrof. Cytosol extract was diluted with cell lysis reagent to a concentration of 2 μg/μL, in a total volume of 50 μL. Then 50 μL of 2X reaction buffer and 5 μL of tetrapeptide-p-nitroaniline (DEVD-pNA) were added. It was incubated at 37 °C for 2 h (in dark), and finally the optical density at 405 nm was measured with an ELISA Reader [The mechanism of cell death was identified as apoptosis. It was validated with the compound 6C. Enzyme activity associated with the induction of apoptosis was determined by caspase-3 assay  as described in the protocol . A concecis- and trans-diastereomers) diversely substituted diastereomeric β-lactam derivatives have been successfully synthesized. In vitro anticancer evaluation showed that one of these newly synthesized cis-β-lactam derivatives (6C) possesses higher potency in human cervical carcinoma SiHa cell lines, six times higher potency in murine melanoma B16F10 cell lines and 10 times less toxicity in normal liver Chang cell lines compared to the positive control colchicine, a well-known anticancer alkaloid. Besides the cis-β-lactam 6C, most of the newly synthesized β-lactams (both diastereomers) exhibited moderate to strong anticancer activity against SiHa and B16F10 cancer cell lines. The binding sites of the compound on β-tubulin was found to be similar with that of colchicine and apoptotic cell death through caspase activation was found as the mechanism of action. It was supported by the microscopic observation. Molecular coupling of the compound 6C on β-tubulin colchicine site, exhibited binding interactions with the Cys 239, Lys 252, Val 323, Met 257, Lys 350, Leu 246 y Ala 248 amionacid residues by molecular docking assay. The molecule 6C may find its application in anticancer drug discovery process.In conclusion, a series of fourteen .


The synthesized compounds were screened for cytotoxicity against breast cancer cells and showed IC50 values of 7.4 μM and higher. Compounds were tested against prostate cancer cells and exhibited antiproliferative activity with IC50 values of 9.4 μM and higher comparable to that of cisplatin. Lead compound 4a displayed selectivity in ERα-positive breast cancer cells. At 10 μM concentration, this heterosteroid inhibited 50% of the E2-mediated ERα activity and led to partial ERα down-regulation. The ERα reporter assay and immunoblotting were supported by the docking study, which showed the probable binding mode of compound 4a to the estrogen receptor pocket. Thus, heterosteroid 4a proved to be a selective ERα modulator with the highest antiproliferative activity against hormone-dependent breast cancer and can be considered as a candidate for further anticancer drug development. In total, the synthesized heterosteroids may be considered as new promising classes of active anticancer agents.Most breast and prostate tumors are hormone-dependent, making it possible to use hormone therapy in patients with these tumors. The design of effective endocrine drugs that block the growth of tumors and have no severe side effects is a challenge. Thereupon, synthetic steroids are promising therapeutic drugs for the treatment of diseases such as hormone-dependent breast and prostate cancers. Here, we describe novel series of steroidal pyrimidines and dihydrotriazines with anticancer activities. A flexible approach to unknown pyrimidine and dihydrotriazine derivatives of steroids with selective control of the heterocyclization pattern is disclosed. A number of 18-nor-5α-androsta-2,13-dienepyrimidine, androsta-2-enepyrimidine, Δ Breast cancer is the most common cancer in women worldwide, with more than 1.5 million new cases recorded every year; it is also the fifth highest cause of cancer death . Splitting patterns are designated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, double doublet; ddd, double double doublet; dt, doublet triplet. The coupling constants (J) are in Hertz. The structures of compounds were established using 1D NMR  and 2D NMR  spectroscopy. Infrared spectra were measured on a FT-IR spectrometer in KBr pellets. High-resolution mass spectra (HRMS) were measured using electrospray ionization (ESI) in positive ion mode ; the mass range was from m/z 50 to 3,000 Da; external/internal calibration was performed using an electrospray calibrant solution. A syringe injection was used for solutions in CH3CN (flow rate 3 ml/min). Nitrogen was applied as a dry gas and the interface temperature was set at 180°C. Melting points were measured on a Boetius capillary melting point apparatus and are uncorrected. Analytical thin-layer chromatography (TLC) was carried out on silica gel plates ; the visualization was accomplished with an UV lamp (254/365 nm) and using chemical staining with [Ce(SO4)2/H2SO4]. Column chromatography was performed on silica gel 60 . Androst-5-en-3β-ol-17-one-3β-acetate, 17β-hydroxy-5α-androstan-3-one, estrone, phosphorus oxychloride, guanidine salts, and acetimidamide hydrochloride were commercially available and were used as purchased. The spectroscopic data for steroidal chlorovinyl aldehydes 2a–c are consistent with those reported previously -estratetraen-3-ol 2a  was added to a suspension of acetamidine hydrochloride  and potassium carbonate  in DMF (4 mL). The mixture was stirred at 80°C for 9 h until the complete conversion of the intermediates (TLC monitoring). The resulting mixture was cooled to room temperature and diluted with water (30 mL). The precipitate that formed was filtered, dried, and washed with water (3 × 5 mL). The product was isolated by column chromatography using petroleum ether/ethyl acetate, 1:1, to obtain colorless solid . Rf 0.27 ; m.p. 264–266°C. 1H NMR , δ: 0.92 , 1.35–1.41 , 1.50–1.56 , 1.61–1.69 , 1.70–1.76 , 1.90–1.95 , 2.14–2.18 , 2.21–2.27 , 2.37–2.42 , 2.48–2.54 , 2.56 , 2.73–2.85 , 6.47 , 6.53 , 7.07 , 8.46 , 9.00 . 13C NMR , δ: 17.1 (18-CH3), 25.5 (2′-CH3), 25.8 (11-CH2), 26.9 (7-CH2), 27.0 (15-CH2), 28.9 (6-CH2), 32.7 (12-CH2), 37.2 (8-CH), 43.7 (9-CH), 45.8 (13-C), 54.3 (14-CH), 112.8 (2-CH), 115.0 (4-CH), 125.8 (1-CH), 130.1 (10-C), 130.5 (16-C), 137.0 (5-C), 151.8 (4′-CH), 155.0 (3-C), 165.1 (2′-C), 181.0 (17-C). IR (KBr), cm−1: 3179 (OH), 2986, 2929, 2859 (CH), 1607, 1585, 1555, 1501 . HRMS (ESI) for C21H25N2O ([M+H]+): calcd 321.1961, found 321.1951.Chloro-16-formyl-Δ1,3,5(10)-estratetraen-3-ol 2a  was added to a suspension of guanidine acetate  and potassium carbonate  in methanol (10 mL). The mixture was refluxed for 6 h until the complete conversion of the intermediates (TLC monitoring). The resulting mixture was cooled to room temperature and the solvent was removed under reduced pressure. The solid reside was washed with water (10 mL) and dried. The workup afforded the analytically pure product as colorless solid . Rf 0.31 . The spectral data are consistent with those reported by Forgo and Vincze , δ: 0.89 , 1.32–1.36 , 1.45–1.50 , 1.54–1.64 , 1.65–1.68 , 1.86–1.90 , 2.03–2.08 , 2.20–2.25 , 2.31–2.38 , 2.62 , 2.69 , 2.77 , 6.36 , 6.40 , 6.45 , 6.98 , 8.00  . 13C NMR , δ: 17.1 (CH3), 26.0 (11-CH2), 26.6 (15-CH2), 27.1 (7-CH2), 29.1 (6-CH2), 32.9 (12-CH2), 37.4 (8-CH), 43.9 (9-CH), 45.8 (13-C), 54.4 (14-CH), 113.5 (2-CH), 115.6 (4-CH), 121.0 (16-C), 125.6 (1-CH), 128.2 (10-C), 136.7 (5-C), 152.6 (4′-CH), 157.5 (3-C), 162.9 (2′-C), 182.0 (17-C). IR (KBr), cm−1: 3361, 3181 (OH), 2930, 2858 (CH), 1637 (NH2), 1608, 1560 . HRMS (ESI) for C20H24N3O ([M+H]+): calcd 322.1914, found 322.1903.17-Chloro-16-formyl-Δd Vincze ; m.p. 282b  was added to a suspension of acetamidine hydrochloride  and potassium carbonate  in DMF (5 mL). The mixture was stirred at 80°C for 6.5 h until the complete conversion of the intermediates (TLC monitoring). The resulting mixture was cooled to room temperature and the solvent was removed under reduced pressure. The crude product was isolated by column chromatography using petroleum ether/ethyl acetate, 1:2, to obtain colorless solid . Rf 0.47 . The spectral data are consistent with those reported by Gogoi et al. , δ: 0.91 , 1.05 , 1.07–1.13 , 1.49 , 1.53–1.65 , 1.67–1.73 , 1.74–1.82 , 1.86 , 1.99 , 2.05–2.12 , 2.28–2.32 , 2.47 , 2.54 , 2.72 , 4.43–4.48 , 5.39–5.40 , 8.43 . 13C NMR , δ: 16.7 (18-CH3), 18.9 (19-CH3), 20.0 (11-CH2), 21.0 (CH3), 25.4 (2′-CH3), 27.3 (2-CH2), 27.3 (15-CH2), 30.1 (8-CH), 30.6 (7-CH2), 32.4 (12-CH2), 36.3 (10-C), 36.3 (1-CH2), 37.7 (4-CH2), 45.4 (13-C), 49.8 (9-CH), 55.0 (14-CH), 73.1 (3-CH), 121.6 (6-CH), 130.5 (16-C), 139.8 (5-C), 151.8 (4′-CH), 165.1 (2′-C), 169.7 (CO), 180.8 (17-C). IR (KBr), cm−1: 2941, 2903, 2858 (CH), 1732 (CO), 1589, 1556 . HRMS (ESI) for C24H33N2O2 ([M+H]+): calcd 381.2537, found 381.2535.3β-Acetoxy-17-chloro-16-formylandrosta-5,16-diene i et al. . m.p. 163c  and potassium carbonate  in MeOH (8 mL) was refluxed for 3 h until the complete conversion of the starting material (TLC monitoring). The resulting mixture was cooled to room temperature and the solvent was removed under reduced pressure. The crude product was washed with water (6 mL) and dried to get colorless solid . Rf 0.28 ; m.p. 182–183°C. 1H NMR , δ: 0.92 , 1.03 , 0.99–1.09 , 1.32–1.38 , 1.48 , 1.50–1.55 , 1.58–1.73 , 1.74–1.82 , 2.04–2.19 , 2.46 , 2.55 , 2.72 , 3.22–3.32 , 5.28–5.30 , 8.39  . 13C NMR , δ: 16.7 (18-CH3), 19.1 (19-CH3), 20.0 (11-CH2), 25.3 (2′-CH3), 27.4 (15-CH2), 30.2 (8-CH), 30.7 (7-CH2), 31.3 (2-CH2), 32.5 (12-CH2), 36.3 (10-C), 36.8 (1-CH2), 42.2 (4-CH2), 45.4 (13-C), 50.1 (9-CH), 55.2 (14-CH), 69.9 (3-CH), 119.8 (6-CH), 130.4 (16-C), 141.6 (5-C), 151.5 (4′-CH), 164.9 (2′-C), 180.7 (17-C). IR (KBr), cm−1: 3378 (OH), 2965, 2938, 2858, 2818 (CH), 1599, 1554 . HRMS (ESI) for C22H31N2O ([M+H]+): calcd 339.2431, found 339.2431.A mixture of steroidal pyrimidine 2b  was added to a suspension of guanidine acetate  and potassium carbonate  in methanol (10 mL). The resulting mixture was refluxed for 6 h until the complete conversion of the intermediates (TLC monitoring). The resulting mixture was cooled to room temperature. The precipitate that formed was filtered, washed with H2O (5 mL), and dried. The workup afforded the analytically pure product as colorless solid . Rf 0.56 . The spectral data are consistent with those reported by Matsumoto et al. , δ: 0.90 , 1.03 , 0.98–1.10 , 1.34–1.42 , 1.42–1.50 , 1.44–1.52 , 1.55–1.83 , 1.99–2.09 , 2.09–2.22 , 2.29 , 2.55 , 3.24–3.32 , 4.41 , 5.30–5.32 , 6.11 , 7.98 . 13C NMR , δ: 16.4 (18-CH3), 18.8 (19-CH3), 19.9 (11-CH2), 26.6 (15-CH2), 30.1 (7-CH2), 30.5 (8-CH), 31.2 (2-CH2), 32.5 (12-CH2), 36.1 (10-C), 36.6 (1-CH2), 42.0 (4-CH2), 45.0 (13-C), 50.0 (9-CH), 55.1 (14-CH), 69.7 (3-CH), 119.6 (6-CH), 121.0 (16-C), 141.5 (5-C), 152.2 (4′-CH), 162.6 (2′-C), 181.5 (17-C). IR (KBr), cm−1: 3535 (OH), 3368, 3314, 3159 (NH2), 2935, 2893, 2844 (CH), 1647 (NH2), 1608, 1559 . HRMS (ESI) for C21H30N3O ([M+H]+): calcd 340.2383, found 340.2382. Anal. calcd for C21H29N3O:C, 74.30; H, 8.6; N, 12.38. Found: C, 73.83; H, 8.65; N, 12.15.3β-Acetoxy-17-chloro-16-formylandrosta-5,16-diene o et al. . m.p. 342c  was added to a suspension of guanidine acetate  and potassium carbonate  in methanol (10 mL). The mixture was refluxed for 4 h until the complete conversion of the intermediates (TLC monitoring). The resulting mixture was cooled to room temperature. The precipitate that formed was filtered, washed with water (5 mL), and dried. The workup afforded analytically pure product as colorless solid . Rf 0.67 . The spectral data are consistent with those reported by De Ruggieri et al. (lit > 300°C). 1H NMR , δ: 0.68 , 0.69 , 0.78–0.84 , 0.86–0.95 , 0.98–1.06 , 1.14–1.29 , 1.32–1.42 , 1.49–1.56 , 1.58–1.64 , 1.64–1.70 , 1.77–1.82 , 1.83–1.89 , 2.16 , 2.24 , 2.43–2.56 , 3.47 , 4.18 , 5.90 , 7.91 . 13C NMR , δ: 10.8 (18-CH3), 10.9 (19-CH3), 20.2 (11-CH2), 22.7 (15-CH2), 27.8 (6-CH2), 29.6 (16-CH2), 30.5 (7-CH2), 34.5 (10-C), 35.0 (8-CH), 35.2 (4-CH2), 36.3 (12-CH2), 38.4 (1-CH2), 40.8 (5-CH), 42.1 (13-C), 50.4 (14-CH), 53.0 (9-CH), 79.8 (17-CH), 116.7 (2-C), 157.9 (4′-CH), 161.6 (2′-C), 164.2 (3-C). IR (KBr), cm−1: 3322, 3169 (NH2), 2969, 2923, 2905, 2849 (CH), 1659 (NH2), 1596, 1561 . HRMS (ESI) for C21H32N3O ([M+H]+): calcd 342.2540, found 342.2538.3-Chloro-2-formyl-17β-formyloxy-5α-androstane i et al. . m.p. > 1,3,5(10)-estratetraen-3-ol 2a  was added to a suspension of acetamidine hydrochloride  and potassium carbonate  in DMF (4 mL). The mixture was stirred at 80°C for 8 h until the complete conversion of the intermediates (TLC monitoring). The resulting mixture was cooled to room temperature and diluted with water (30 mL). The precipitate that formed was filtered and washed with water (5 mL) and hot benzene (5 mL). The workup afforded the analytically pure product as colorless solid . Rf 0.37 ; m.p. 209–210°C. 1H NMR , δ: 0.84 , 1.30–1.36 , 1.37–1.50 , 1.58–1.62 , 1.75–1.83 , 1.80 , 1.82 , 1.95 , 2.14 , 2.18–2.22 , 2.31–2.36 , 2.68–2.78 , 5.35 , 6.44 , 6.51 , 7.02 , 9.03 , 9.37 . 13C NMR , δ: 15.0 (18-CH3), 20.2 , 25.8 (11-CH2), 26.7 (7-CH2), 28.9 , 33.6 (12-CH2), 36.9 (8-CH), 43.7 (9-CH), 47.9 (13-C), 53.1 (14-CH), 67.8 (2′-CH), 112.7 (2-CH), 115.0 (4-CH), 125.6 (1-CH), 130.2 (10-C), 135.4 (17-C), 137.0 (5-C), 138.6 (16-C), 151.6 , 155.0 (3-C). IR (KBr), cm−1: 3198 (NH), 2929, 2857 (CH), 1703, 1611 , 1499, 1456, 1435, 1378, 1287, 1248. HRMS (ESI) for C23H29ClN3O ([M+H]+): calcd 398.1994, found 398.1995.17-Chloro-16-formyl-Δ2b  was added to a suspension of acetamidine hydrochloride  and potassium carbonate  in DMF (4 mL). The mixture was stirred at 80°C for 8 h until the complete conversion of the intermediates (TLC monitoring). The resulting mixture was cooled to room temperature and diluted with water (30 mL). The precipitate that formed was filtered, washed with water (5 mL), and dried. The crude product was purified by column chromatography using chloroform/MeOH, 10:1, to obtain colorless solid . Rf 0.45 ; m.p. 158–160°C. 1H NMR , δ: 0.84 , 1.01 , 1.02–1.11 , 1.32 , 1.36–1.41 , 1.43–1.49 , 1.51–1.65 , 1.69–1.73 , 1.76–1.84 , 1.79 , 1.81 , 1.88 , 1.92–1.97 , 1.98 , 2.07 , 2.26–2.31 , 4.42–4.48 , 5.32 , 5.34–5.36 , 9.40 . 13C NMR , δ: 14.8 (18-CH3), 18.8 (19-CH3), 19.9 (11-CH2), 20.3 , 20.9 (CH3COO), 27.3 (2-CH2), 29.2 (15-CH2), 29.9 (8-CH), 30.3 (7-CH2), 33.4 (12-CH2), 36.3 (1-CH2), 36.3 (10-C), 37.6 (4-CH2), 47.5 (13-C), 49.8 (9-CH), 53.6 (14-CH), 67.4 (2′-CH), 73.1 (3-CH), 121.8 (6-CH), 135.4 (17-C), 138.5 (16-C), 139.8 (5-C), 152.0 , 169.7 (CO). IR (KBr), cm−1: 3183 (NH), 2945, 2857 (CH), 1735 (COO),1704, 1629 . HRMS (ESI) for C26H37ClN3O3 ([M+H]+): calcd 458.2569, found 458.2558.3β-Acetoxy-17-chloro-16-formylandrosta-5,16-diene 2c  was added to a suspension of acetamidine hydrochloride  and potassium carbonate  in DMF (5 mL). The mixture was stirred at 60–65°C for 6 h until the complete conversion of the intermediates (TLC monitoring). The resulting mixture was cooled to room temperature and the solvent was removed under reduced pressure. The product was purified by column chromatography using chloroform/MeOH, 6:1, to obtain colorless solid . Rf 0.30 ; m.p. 210–212°C. 1H NMR , δ: 0.62 , 0.68 , 0.65–0.71 , 0.78–0.88 , 0.90–0.96 , 1.11–1.18 , 1.22–1.39 , 1.40–1.50 , 1.60 , 1.64 , 1.71 , 1.80 , 1.82 , 2.00–2.05 , 2.06 , 2.14–2.19 , 3.39–3.43 , 4.40 , 5.48 , 9.50 . 13C NMR , δ: 11.1 (18-CH3), 11.6 (19-CH3), 20.3 , 20.4 (11-CH2), 23.0 (15-CH2), 27.2 (6-CH2), 29.8 (16-CH2), 30.6 (7-CH2), 34.1 (10-C), 35.0 (8-CH), 36.4 (12-CH2), 38.1 (4-CH2), 39.1 (1-CH2), 42.4 (13-C), 42.5 (5-CH), 50.4 (14-CH), 53.1 (9-CH), 70.7 (2′-CH), 80.0 (17-CH), 123.0 (3-C), 134.0 (2-C), 152.2 . IR (KBr), cm−1: 3205 (NH), 2926, 2872 (CH), 1708, 1665 , 1498, 1469, 1444, 1380, 1380, 1338, 1250. HRMS (ESI) for C24H37ClN3O ([M+H]+): calcd 418.2620, found 418.2610.3-Chloro-2-formyl-17β-formyloxy-5α-androstane 2 and 80–85% humidity (NuAir CO2 incubator). The cell growth was evaluated by the modified MTT  (Applichem) test  in 900 μL of the medium. The tested compounds were dissolved in DMSO (Applichem) to 10 mM before experiments and then were diluted in the medium to the required concentrations. Compounds with different concentrations in 100 μL of the medium were added 24 h after the seeding, and the cells were grown for 72 h. After incubation with the compounds, the medium was removed, and the MTT reagent dissolved in the medium was added to the final concentration of 0.2 mg/mL to each well and incubated for 3 h. Then the cell supernatants were removed and the MTT formazan purple crystals were dissolved in 100% DMSO (350 μL per well). Plates were gently shaken and the absorbance was measured at 571 nm with a MultiScan reader (ThermoFisher). The viability of the cells was assessed after subtraction of the blank value (the absorbance in the well w/o cells) from all wells. Dose-response curves were analyzed by regression analysis using sigmoidal curves (Log(concentration) vs. normalized absorbance). The half maximal inhibitory concentrations (IC50) were determined with GraphPad Prism.The MCF-7 and MDA-MB231 human breast cancer cell lines and the PC3 and 22Rv1 prostate cancer cell lines were purchased from the ATCC collection. Cells were cultured in standard high glucose DMEM medium (Hyclone) supplemented with 10% fetal calf serum (FCS) (HyClone) and 0.1 mg/ml sodium pyruvate (Santa Cruz) at 37°C, 5% COp-nitrophenyl β-D-galactopyranoside (ONPG). Briefly, cell lysates were mixed with 0.1 M phosphate buffer (pH 7.5) containing 1.0 mM MgCl2, 3.3 mM ONPG and 53 mM β-mercaptoethanol. After incubation for 1 h at 37°C, the absorbances at 405 nm were measured on the MultiScan FC reader (ThermoFisher). The luciferase activity was calculated in arbitrary units evaluated as the ratio of the luciferase activity to the galactosidase activity.To determine the transcriptional activity of the estrogen receptor α (ERα), MCF-7 cells were transfected with the plasmids containing luciferase reporter gene under the control of the promoter containing estrogen responsive elements. Assay was performed in steroid-free conditions (phenol red-free DMEM medium supplemented with 10% DCC serum). The reporter plasmid ERE-TK-LUC used in this work was kindly provided by Reid et al. . The traThe cells were removed from the dishes with 1.2 ml of phosphate buffer, twice washed, and incubated for 10 min on ice in the modified lysis buffer containing 50 mM Tris-HCl, pH 7.5, 0.5% Igepal CA-630, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mM sodium orthovanadate and aprotinin, leupeptin, pepstatin (1 μg/mL each) as described earlier  were separated in 10% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane (SantaCruz), and processed according to the standard protocol. To prevent nonspecific absorption, the membranes were treated with 5% nonfat milk solution in TBS buffer  with 0.1% Tween-20 and then incubated with primary antibodies overnight at 4°C.Primary antibodies to ERα were purchased from Sigma-Aldrich (Merck); the antibodies against α-tubulin  were added to standardize loading. Goat anti-rabbit IgGs (Jackson ImmunoResearch) conjugated to horseradish peroxidase were used as secondary antibodies. Signals were detected using the ECL reagent as described in Mruk and Cheng  and an It-test was used to evaluate the significance of differences in comparisons. P-value of < 0.05 was considered statistically significant.Each biology experiment was repeated three times. Statistical analysis was performed using Microsoft Excel and GraphPad Prism. Results were expressed as mean ± S.D. . Student's In silico docking was performed using Autodock Vina  by the general two-step sequence involving: (1) the Vilsmeier–Haack reaction giving steroidal β-chlorovinyl aldehydes 2a–c, (2) the condensation of the former with amidines  techniques and HRMS.The heterocyclization pattern was directed to dihydrotriazines by increasing amidine excess up to 4–5 equivs. The dihydrotriazine ring was constructed from two acetimidamide molecules and one steroid molecule 50  are given in Table The antiproliferative activity of all the synthesized compounds was evaluated against the human estrogen-responsive MCF-7 breast cancer cell line and ERα-negative MDA-MB231 cells using the MTT assay Figures , S2. Cis3a,b containing the D-ring-fused pyrimidine moiety proved to be inactive, while their androstene analogs 3c–e were active with the IC50 values in the range of 12.0–21.6 μM. It is remarkable that the IC50 value for compound 3d containing the 3-OH group is higher than that for compound 3c possessing the 3-OAc protected group. The solubility of steroidal A-ring annulated pyrimidine 3f in DMSO is too low to perform the MTT assay.Most of the tested heterosteroids showed remarkable anticancer activity against ERα-positive MCF-7 cancer cells. Estranes 4a–c proved to be more active against MCF-7 cancer cells compared to steroidal fused pyrimidines. Androstene derivative 4c bearing the dihydrotriazine moiety at C-2 had the IC50 value of 14.8 μM, while the IC50 value for compound 4b modified at 16-C reached 11.2 μM. The 16-C dihydrotriazine-modified estrane 4a was shown to be the most active derivative. Moreover, only steroidal dihydrotriazines 4a, 4b, and 4c were active against ERα-negative MDA-MB231 cells; their IC50 values vary in the range of 12.2–19.1 μM.Steroidal dihydrotriazines 4a, 4b, and 4c displayed antiproliferative activity. Estrane derivative 4a inhibited the growth of PC3 and 22Rv1 prostate cancer cells with IC50 of 13.5 and 11.7 μM, respectively, while androstene derivative 4b displayed cytotoxicity comparable to that of cisplatin in hormone-dependent 22Rv1 prostate cancer cells. Androstene derivative 4c was less active against prostate cancer cells than compounds 4a and 4b, and revealed the IC50 value about 20 μM . Thus, ERα activity was correlated to luciferase activity measured in treated or control cells.3a,b containing the D-ring-fused pyrimidine moiety did not inhibit ERα activity at 10 μM concentration. Moreover, these 3a and 3b stimulated ERα activity at low (10 nM) concentration acting as partial receptor agonists  and EEu (PDB: 2YAT) are known to modulate the dynamics of the ERα helix 12 . However, the major binding modes of compound 4a for 2P15 and 2YAT are as follows: the A-ring phenolic hydroxyl group points away the binding pocket of ER and the dihydrotriazine moiety points inward the binding pocket. The critical hydrogen-bonding interactions between the steroid estradiol, TFMPV-E2 and the A-ring phenolic hydroxyls of EEu with Arg-394/Glu-353 were not found for compound 4a  by the standard MTT assay. The compounds showed moderate to good activity and proved to be nontoxic to normal PBM cells. One of the synthesized compounds was found to be active against all three cancer cell lines but more specific against the MDA-MB231 cells with IC50 of about 9 μM, which is similar to our data on the activity of the compounds against breast cancer cells. Finally, the authors discussed the ability of steroidal compounds to interact with the protein HSA involved in drug delivery.Other promising steroidal pyrimidines were discussed by Ali et al. . The antMetastatic bone tumors occur at particularly high rates in cancers of the breast, prostate, and lung, accounting more than 70% of all patients. Treatment of skeletal metastasis and development of new specific “blockers” of bone resorption are relevant. Pyrimidine-fused betulinic acid may be considered as promising compounds for the design of novel inhibitors of osteoclast differentiation and bone resorption. Jun Xu et coworkers synthesized over 20 heterocyclic ring-fused betulinic acid derivatives and evaluated their inhibition on RANKL-induced osteoclast formation in preosteoclast RAW264.7 cells  were transformed into the corresponding A- and D-modified steroidal pyrimidines and dihydrotriazines in moderate to high yields (29–89%) using a two-step sequence involving the Vilsmeier–Haack reaction and condensation with amidines, such as guanidine and acetimidamide. The new compounds showed remarkable cytotoxic activity against breast and prostate cancer cells. Furthermore, lead compounds demonstrated selectivity toward ERα in MCF-7 breast cancer cells. Compound 4a inhibits 50% of ERα activity at its cytotoxic concentration. Using immunoblotting, partial ERα downregulation was observed in compound 4a-treated MCF-7 cells. Docking approaches confirmed the ability of compound 4a to bind to ERα. Thus, compound 4a may be considered as a candidate for future anticancer drug design, in particular, for ERα-positive breast cancers.Here, we describe novel series of steroidal anticancer agents. In summary, this study demonstrates that the cyclization of steroidal β-chlorovinyl aldehydes with bis-nucleophilic amidines provides an easy approach to various novel heterosteroids. Natural hormones Despite a limited number of compounds in series, it provides significant novel insight into the structure–activity relationship of heterosteroids as anticancer agents. Biological studies show that annulation of androst-5-ene core with pyrimidine is efficient for development of novel selective compounds for treatment of hormone-dependent breast cancer. Moreover, installation of dihydrotriazine pendant at A- and D-rings of estrane and androst-5-ene cores results in strong antiproliferative activities against breast and prostate cancer cells comparable with cisplatine. The two-fold gain in cytotoxicity of 16-C dihydrotriazine-modified estrane against ERα-positive breast cancer cells vs. ERα-negative cells and its high activity as ERα inhibitor were shown while similar androstene derivative was less selective.These results offer new knowledge about the binding site and receptor flexibility of ERα. The described heterosteroids will be useful lead agents for the development of more potent and selective SERMs.in silico analysis, and managed the project; All authors read and approved the final manuscript.AS carried out the immunoblotting, performed the statistical analysis and the transient transfection, drafted and prepared the manuscript for submission, worked with cell cultures; AVK synthesized steroidal compounds; ASK synthesized steroidal compounds, prepared the supporting information; MY worked with cell cultures; OA performed the reporter analysis, worked with plasmids; VS managed the project; IZ managed the project; AH managed the project; YV wrote the manuscript, conceived of the study, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-acetylneuraminic acid-derived compounds bearing anomeric sulfo functional groups are described. These novel compounds, which we refer to as sulfo-sialic acid analogues, include 2-decarboxy-2-deoxy-2-sulfo-N-acetylneuraminic acid and its 4-deoxy-3,4-dehydrogenated pseudoglycal. While 2-decarboxy-2-deoxy-2-sulfo-N-acetylneuraminic acid contains no further modifications of the 2-deoxy-pyranose ring, it is still a more potent inhibitor of avian-origin H5N1 neuraminidase (NA) and drug-resistant His275Tyr NA as compared to the oxocarbenium ion transition state analogue 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The sulfo-sialic acid analogues described in this report are also more potent inhibitors of influenza NA (up to 40-fold) and bacterial NA (up to 8.5-fold) relative to the corresponding anomeric phosphonic acids. These results confirm that this novel anomeric sulfo modification offers great potential to improve the potency of next-generation NA inhibitors including covalent inhibitors.The design, synthesis and application of  N-acetylneuraminic acid  or N-glycolylneuraminic acid  with an α2-3 or α2-6 ketosidic bond to galactose. NA is found in many types of pathogens including Trypanosoma cruzi, Clostridium perfringens, Streptococcus pneumoniae, Vibrio cholerae, mumps virus, parainfluenza virus and influenza virus. By destroying sialic acid-containing glycosides, NA facilitates the movement of pathogens through biological environments rich in sialic acid receptors, including mammalian tissues and mucus. Therefore inhibition of NA can hinder the spread of sialic acid binding pathogens, and NA inhibitors offer great potential as anti-microbial agents. In the case of sialic acid binding viruses, NA also facilitates the release of progeny virions from infected cells. Accordingly, NA inhibitors have been especially successful as anti-influenza pharmaceuticals2.Neuraminidase , also referred to as sialidase, is a retaining glycosidase that hydrolyzes α-ketosidic linkages to sialic acids. Common substrates recognized by NA contain terminal N-acetylneuraminic acid (Neu5Ac2en or DANA). Currently, all clinically used NA inhibitors contain an anomeric carboxy group, which forms strong electrostatic interactions with three structurally conserved arginine residues in the NA active site -derived peracetylated octoses 2a and 2b, first reported by Potter and von Itzstein, were selected as the substrate for sulfur addition14. The decarboxylated Neu5Ac derivatives 2a and 2b were synthesized based on the method of Shie et al. with modifications5.Sulfo-sialic acid analogues were synthesized via oxidation of a mixture of acetylthio intermediates according to Fig. 2 anomeric position with HSAc was mediated by either BF3·Et2O or TMSOTf, using CH3CN, CHCl3, or 1,2-dichloroethane  as solvent , and characterization of the oxidation products 6b and 8b. The dominance of the axial acetylthio orientation of this pseudoglycal could be explained by the anomeric effect. Less polar reaction conditions were used to produce a higher ratio of 4b to 3  in AcOH buffered with KOAc, based upon the method of Reddie19. Workup was somewhat difficult due to the presence of excess salt, so the procedure was modified with NH4OAc in place of KOAc for the oxidation of 3 to 5, which appeared to be quantitative. Yields for the oxidation of 4 to 6 were low due to concurrent oxidation of the pseudoglycal ring double bond by the excess Oxone. During HRMS analysis of polar Oxone oxidation products, negative ions m/z 452.0826 (6 + O), m/z 454.0985 (6 + O + 2H) and m/z 470.0949 (6 + 2O + 2H) were detected, indicating that 6 was further oxidized. Sulfonates 5a and 5b could also be converted to the corresponding methyl esters by reaction with trimethylsilyldiazomethane, a toxic reagent that should be handled with caution. Active sulfo-sialic acid analogues, 7a, 7b and 8b were obtained after deprotection of the precursors in the presence of NaOH.Oxidation of the acetylthio functionality of 5-8 were determined using HRMS,1H, GCOSY, NOESY and13C data. H-1, H-2 and H-5 are all shifted downfield in the axial anomeric sulfonates 5b and 7b relative to the equatorial isomers 5a and 7a, which is also observed in 2, 3, and the phosphonic acid analogues of 5 and 75. The H-1 to H-2 coupling constants of 5a, 5b, 7a and 7b match those of the corresponding phosphonic acids and carboxylic acids as well20. A clear H-1 to H-5 NOE correlation is observed in the equatorial sulfo compound 7a. In contrast, the lack of any NOE correlation between H-1 and H-5 in 8b is consistent with the axial anomeric functional group orientations of 4b, 6b and 8b. A clear H-1 to H-2 NOE correlation in 8b further supports the equatorial orientation of H-1. Synthesis of psuedoglycals with anomeric sulfones has also been reported with axial anomeric stereoselectivity21. Isomerization of the anomeric position was never observed for any of the sulfo-sialic acid analogues.The structures of sulfo-sialic acid analogues 5 and 7 are shifted downfield relative to the corresponding carboxylic acids and phosphonic acids  and 7b  were synthesized based upon the methods of Shie et al. with modifications5. Inhibition of influenza A/Anhui/1/2005 (H5N1) NA (N1), A/RI/5+/1957(H2N2) NA (N2), Clostridium perfringens NanJ NA (CpNA) and Streptococcus 6646K NA (StrepNA), was quantified using a fluorogenic assay based upon the method by Potier et al. with modifications22. Inhibition of influenza and bacterial NA by sulfo-sialic acid analogues confirms that the sulfo group enables more potent NA inhibition relative to the analogous phosphonic acids  NA, 7a inhibits H5N1 N1 and its drug-resistant His275Tyr form at the same level 27. Therefore, our sulfo modification in combination with other modifications may offer potential to produce sub-nanomolar level NA inhibitors. In conclusion, this report proves the concept that sulfo-sialic acid analogues are the most potent NA inhibitors with regards to anomeric functionality. Further development of novel sulfo-sialic acid based inhibitors with increased potency and efficacy is currently underway.The clinically used NA inhibitors zanamivir, oseltamivir, peramivir and laninamivir inhibit influenza NA at a low nanomolar level2-4 dissolved in CDCl3, and 5-8 dissolved in CD3OD. NMR chemical shifts (δ) are provided in parts per million (ppm).1H-NMR coupling constants (J) are listed in Hz. Residual peaks of chloroform (7.26 ppm) and methanol (3.31 ppm) were used as1H-NMR references. The residual peaks of chloroform (77.0 ppm) and methanol (49.0 ppm) were also used as13C-NMR references. For 2D experiments, the standard pulse sequences programmed into the Varian NMR systems were used. 2D-NOESY experiments were performed at room temperature with a spectral width of 9615.4 Hz (sw), an acquisition time of 0.15 seconds (at), 2844 data points (np), a filter bandwidth of 4000 Hz (fb), a relaxation delay of 2.000 seconds (d1), 4 transients (nt), 128 or 200 experiments (ni), a pulse width of 9.700 or 10.900 microseconds (pw) and a mixing time 0.500 seconds (mixN).Compounds were analyzed using Varian 400 MHz and 600 MHz systems (now Agilent) at the Okayama University Collaboration Center, and also using a JEOL ECZ400 spectrometer at the Kobe University Department of Chemical Science and Engineering. Measurements were taken with Compound identities were confirmed with HRMS, which was performed using an Agilent 6520 Accurate-Mass Q-TOF at the Okayama University Collaboration Center.et al.22 with modifications. 4-methylumbelliferyl-α-d-Neu5Ac (4-MU-Neu5Ac) substrate was utilized at a final concentration of 120 μm for inhibition of N2 and Streptococcus 6646K NA (Amsbio). For inhibition of Clostridium perfringens NanJ NA (Sigma) the 4-MU-Neu5Ac concentration was 60 μm when using 7a and 120 μm when using eqPO3H. For IC50 determination, 5–8 inhibitor concentrations were used with 1–4 replicates for each condition. For N1 Ki determination, 5 inhibitor concentrations and 3 substrate concentrations were used. NA and inhibitor were pre-incubated for 20 minutes at room temperature before starting each assay with addition of 4-MU-Neu5Ac. Fluorescence of liberated 4-methylumbelliferyl  was detected at room temperature using a Thermo Scientific Varioskan™ Flash or a Molecular Devices SpectraMax Paradigm microplate reader. IC50 values and confidence intervals were calculated by analyzing inhibition data using the ‘log(inhibitor) vs. response (three parameters)’ function of Prism 7 software. N1 Ki values were calculated using the Michaelis-Menten function of Prism 7, and N2 Ki values were calculated using the Cheng-Prusoff equation23. A/Anhui/1/2005 (H5N1) NAs were purchased from Sino Biological. Influenza A/RI/5+/1957(H2N2) NA was produced according to our previously reported methods3.Inhibition of neuraminidase (NA) activity was quantified using a fluorogenic assay based upon the method by Potier 7a was built in the A/RI/5+/1957(H2N2) N2 (PDB ID: 45H2) and A/Vietnam/1203/04 (H5N1) His275Tyr N1 (PDB ID: 3CKZ) active sites using the MOE Builder function. This was followed by running Structure Preparation, Protonate 3D, and Energy Minimization with the Amber10:EHT forcefield. Binding of 7a to wildtype N1 was performed in the same manner as that of the N1-His275Tyr structure (PDB ID: 3CKZ) after substitution of Try275 with histidine using the MOE Protein Builder function.Sulfo-sialic acid analogue binding to NA active sites was modeled with Molecular Operating Environment (MOE) version 2016.0802 from Chemical Computing Group. The structure of O-acetyl-2,4-dideoxy-d-glycero-d-galacto-octopyranose (2) was synthesized based upon the methods of Shie et al.5 with a 49% yield. 2  was dissolved in distilled 1,2-dichloroethane  or CH3CN dried with excess molecular sieves. After cooling, HSAc and Lewis acid (BF3·Et2O or TMSOTf) were carefully added to the solution according to Table 2 and the temperature was slowly raised to room temperature. After completion, the organic layer was combined with CHCl3 and washed with aqueous solutions of NaHCO3 and NaCl. The washed organic layer was dried over MgSO4, filtered through diatomaceous earth and evaporated under reduced pressure. Acetylthio reaction products 3 and 4 were isolated using normal phase column chromatography with EtOAc as the mobile phase. When using the polar conditions of Table 3a was obtained from 426 μmol 2 (14% yield); when using the non-polar conditions of Table 3b was obtained from 1.97 mmol 2 (39% yield); and when using the more non-polar conditions of Table 4b was obtained from 673 μmol 2 (26% yield).The known compound 4-acetamido-1,3,6,7,8-penta-3a (α-Anomer): Rf = 0.4 (EtOAc); 1H-NMR : 1.89 , 2.0-2.13 , 2.34 , 3.77 , 4.0 , 4.36 , 5.36 ; + ESI HRMS calcd for C20H29NNaO11S: 514.1359, found: m/z 514.1393 [M + Na]+.3b (β-Anomer): Rf = 0.4 (EtOAc); 1H-NMR : 1.89 , 1.98–2.12 , 2.16–2.19 , 2.30–2.36 , 2.37 , 3.91 , 4.00–4.05  4.27 , 5.00 , 5.12 , 5.31 , 5.41 , 6.14 ; 13C-NMR : 20.67, 20.69, 20.8, 20.9, 21.05, 23.15 (C=OCH3), 36.1 (C-2), 50.1, 62.1, 67.3, 69.6, 69.9, 72.5 (C-3–C-8), 79.0 (C-1), 169.4, 170.0, 170.13, 170.16, 170.5, 171.0 (C=OCH3); + ESI HRMS calcd for C20H29NNaO11S: 514.1359, found: m/z 514.1393 [M + Na]+.4b (β-Anomer): Rf = 0.4 (EtOAc):1H-NMR : 1.98–2.12 , 2.35  3.83 , 4.11 , 4.30 , 4.50 , 5.21 , 5.31 , 5.53  5.80  6.27 ; 13C-NMR : 20.70, 20.73, 20.95, 21.1, 23.4 (5 C=OCH3), 43.6, 62.1, 67.4, 69.3, 70.1 (C-4–C-8), 78.7, (C-1), 125.7, 131.1 , 169.6, 169.8, 170.2, 170.53, 170.58 (5 C=OCH3); + ESI HRMS calcd for C18H25NNaO9S: 454.1148, found: m/z 454.1177 [M + Na]+.3  and 4b  were dissolved in AcOH. NH4OAc  and Oxone  were mixed in to form a slurry. After 21 hours, MeOH was added to the reaction mixture, which was then filtered through a fritted funnel. The crude mixture was evaporated under reduced pressure, followed by purification of 5a and 5b  with normal phase chromatography using a gradient of EtOAc and EtOH.5a (α-Anomer): Rf = 0.39 (EtOAc:EtOH = 2:1); 1H-NMR : 1.8–2.0  1.85 , 1.97–2.08 , 2.49 , 3.81 , 3.97 , 4.21 , 4.26 , 4.53 , 5.00 , 5.30 , 5.39 ; 13C-NMR : 20.69, 20.79, 20.82, 20.82, 22.7 (5 C=OCH3), 33.6 (C-2), 50.5, 63.2, 69.4, 72.6, 73.2, 78.6 (C-3–C-8), 87.3 (C-1), 171.7, 171.8, 172.0, 172.5, 173.4 (5 C=OCH3); -ESI HRMS calcd for C18H26NO13S−: 496.1130, found: m/z 496.1142 [M − H]−.5b (β-Anomer): Rf = 0.4 (EtOAc:EtOH = 2:1);1H-NMR : 1.84  1.9–2.1 , 1.97, 2.00, 2.07, 2.08 , 2.68 , 3.96 , 4.12 , 4.42 , 4.66 , 4.77 , 5.21 , 5.39 , 5.58 ; 13C-NMR : 20.7, 20.8, 20.9, 21.3, 22.6 (5 C=OCH3), 31.5 (C-2), 50.7, 63.1, 69.1, 71.0, 71.0, 73.0 (C-3–C-8), 85.8 (C-1), 171.7, 172.0, 172.2, 172.6, 173.5 (5 C=OCH3); -ESI HRMS calcd. for C18H26NO13S−: 496.1130, found: m/z 496.1142 [M − H]−.4b  was dissolved in AcOH (3 mL). KOAc  and Oxone  were mixed in to form a slurry. After 4.66 hours MeOH was added to the reaction mixture, which was then filtered through a fritted funnel. The crude mixture was evaporated under reduced pressure, followed by purification of 6b  using normal phase column chromatography.6b (β-Anomer): Rf = 0.39 (EtOAc:EtOH = 2:1);1H-NMR : 1.92, 2.01, 2.08 , 4.21 , 4.52 , 4.60 , 4.91 , 5.28 , 5.40 , 5.83 , 6.11 ;13C-NMR : 20.7, 21.3, 22.6, 23.7 (4 C=OCH3), 43.9, 63.1, 69.2, 71.5, 71.7 (C-4–C-8), 86.1 (C-1), 124.8, 131.9 , 171.6, 172.2, 172.6, 173.2 (4 C=OCH3); -ESI HRMS calcd for C16H22NO11S−: 436.0919, found: m/z 436.0929 [M − H]−.5a (10 mg), 5b (10 mg), and 6b (4 mg) were treated with a solution of 0.1 m NaOH in MeOH. After deprotection reached completion, the pH was neutralized with AcOH and solvent was evaporated under reduced pressure resulting in quantitative final product yields. 7a, 7b and 8b were purified to homogeneity using an Inertsil® ODS-3 semi-preparative HPLC column (GL Sciences) with a mobile phase of 10–15% MeOH supplemented with NH4OAc.To prepare active sulfo-sialic acid analogues, 7a (α-Anomer): Rf = 0.6 (acetone:butan-1-ol:H2O = 70:27:3); 1H-NMR : 1.79 , 2.00 , 2.45 , 3.47 , 3.62–3.65 , 3.74–3.89 , 4.28 ; 13C-NMR : 22.8 (C=OCH3), 36.4 (C-2), 53.7, 64.9, 70.2, 70.8, 71.4, 78.6 (C-3–C-8), 87.0 (C-1), 174.8 (C=OCH3); -ESI HRMS calcd for C10H18NO9S−: 328.0708, found: m/z 328.0727 [M − H]−.7b (β-Anomer): Rf = 0.65 (acetone:butan-1-ol:H2O = 70:27:3); 1H-NMR : 1.92 , 2.01 , 2.62 , 3.44 , 3.57–3.60 , 3.75 , 3.79–3.84 , 4.25 , 4.40 , 4.70 ; 13C-NMR : 22.6 (C=OCH3), 34.3 (C-2), 54.3, 64.6, 67.2, 70.2, 72.4, 74.3 (C-3–C-8) 86.7 (C-1), 175.3 (C=OCH3); -ESI HRMS calcd for C10H18NO9S−: 328.0708, found: m/z 328.0727 [M − H]−.8b (β-Anomer): Rf = 0.6 (acetone:butan-1-ol:H2O = 70:27:3); 1H-NMR : 1.98 , 3.50 , 3.62 , 3.83 , 3.92 , 4.35 , 4.68 , 4.96 , 5.93 , 6.13 ; 13C-NMR : 22.6 (C=OCH3), 44.6, 64.9, 70.1, 72.4, 73.0 (C-4–C-8), 86.3 (C-1), 124.9, 132.0 , 173.9 (C=OCH3); -ESI HRMS calcd for C10H16NO8S−: 310.0602, found: m/z 310.0578 [M − H]−.The datasets generated and analyzed during the current study are available from the corresponding authors upon reasonable request.


Their structures were elucidated by extensive spectroscopic analysis. Compound 1 possesses an unprecedented isopropyl-branched bicyclo [6.3.0] undecane carbon skeleton with unique endoperoxide moiety, and a plausible biosynthetic pathway of it was postulated. According to the reported biological properties of endoperoxide, the antimalarial, cytotoxic, antiviral, and target inhibitory activities of 1 were tested. Compound 1 showed mild in vitro antimalarial activity against Plasmodium falciparum 3D7, weak cytotoxic activities toward Jurkat, MDA-MB-231, and U2OS cell lines, inhibitory effects against influenza A viruses H1N1 and PR8, as well as mild target inhibitory activity against acetylcholinesterase. The other compounds were evaluated for cytotoxicities against HeLa, HCT-116, and A549 tumor cell lines and target inhibitory activities against protein tyrosine phosphatase 1B (PTP1B). Compound 20 exhibited cytotoxicities against HeLa and HCT-116, and compounds 5, 11, and 15 showed mild target inhibitory activities against PTP1B.Three new sesquiterpenoids (sinuketal ( Sinularia consists of almost 170 species  undecane sesquiterpenoid, which is different from tehranolide, the only similar analogue, because of the position of the methyl and isopropyl groups + , indicating four degrees of unsaturation. The infrared radiation (IR) spectrum showed the presence of a hydroxyl group (3321 cm−1), which was supported by an electrospray ionization mass spectrometry (ESIMS) fragment at m/z 235 [MH − H2O]+. The 1H NMR spectrum of 1  and 0.95 ), an AX coupling system  and 3.14 ), and two terminal olefinic protons  and 4.93 ). Its 13C NMR and DEPT spectroscopic data ), and 1 terminal double bond (δC 113.0 (CH2) and 149.4 (C)), accounting for one degree of unsaturation. The above NMR signals suggested the structure of a tricyclic sesquiterpenoid. All the connections of H and protonated C were assigned by detailed analysis of heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectra. The 1H-1H correlation spectroscopy (1H-1H COSY) experiment displayed two spin systems, H2-6/H2-7 and H2-2/H2-1/H-11(H-12/H3-13(H3-14))/H-10/H2-9  and anomeric C-5 (δ 107.4), a (C-3)–O–O–(C-5)–OH moiety should be present. Thus, the planar structure of 1 was established as shown in Sinuketal (rum of 1  showed tpic data  exhibite-10/H2-9 . This wa1 to C-2 . HMBC co1 was established by nuclear Overhauser effect spectroscopy (NOESY) experiment . In order to gain further support for determination of the relative configuration of 1, 13C NMR chemical shifts were calculated by the Gaussian 09 program package. The stable conformations of all the eight isomers of 1 were calculated by DFT gauge-including atomic orbitals (GIAO) model at RB3LYP/6-31+G level + . The 13C NMR spectra  and 9.49 ; δC 157.4 (CH), 143.7 (C) and 192.7 (CH)) and a ketone (δC 213.2 (C)). Thus, a bicyclic sesquiterpenoid structure of 2 was revealed. From the 1H-1H COSY spectrum (2-5/H2-6 and H-8/H-9/H-10/(H-11/H3-12(H3-13))/H2-1/H2-2, were established. Key HMBC correlations from H3-14 to C-2, C-3, C-4 and C-9, from H2-5 to C-4, from H-15 to C-6 and C-7, and from H-8 to C-15 assembled the planar structure of 2 -2 matched well with the calculated ECD curve of 3R,9R,10R  , was obt spectra  of 2 ind spectra  implied spectrum , two parure of 2 , which hperiment . IrradiaR,9R,10R a.3) was isolated as a colorless, amorphous solid with molecular formula of C15H24O3 on the basis of its HRESIMS at m/z 253.1798 [M + H]+ , requiring four degrees of unsaturation. 1H and 13C NMR spectral data (δC 213.0 (C)) and a terminal double bond  and 5.05 ; δC 142.9 (C) and 110.6 (CH2)). According to the molecular formula, 13C NMR and DEPT spectroscopic data -3 was assigned as 5S,6R,7R,10R on the basis of the experimental ECD curve of 3, and gave good overall mirror-imagine agreement with the calculated ECD curve of 5S,6R,7R,10R , a colorless oil, was established as C23H30O8 from the HRESIMS data [M + Na]+ at m/z 457.1836 . Analysis of the 1H and 13C NMR , 4.79 , and 1.81 ; δC 145.9 (C), 111.4 (CH2), and 20.8 (CH3)), a trisubstituted furane ring ; δC 161.0 (C), 149.0 (C), 114.6 (C), and 112.2 (CH)), two methyl esters  and 3.72 ; δC 164.0 (C), 167.3 (C), 51.4 (–OCH3), and 52.1 (–OCH3)), a methoxy group ; δC 57.8 (–OCH3)), a ketone (δC 208.6 (C)), a quaternary methyl ; δC 27.9 (CH3)) and a trisubstituted double bond ; δC 144.1 (CH) and 125.1 (C)). Their positions were confirmed by HMBC correlations from H2-16 and H3-17 to C-1, from H2-2 to C-3 and C-4 and from H-7 to C-6, from H3-21 to C-18, from H3-23 to C-20, from H3-22 to C-7, from H2-9 and H2-11 to C-10, from H3-19 to C-8, and from H2-11 to C-12 and C-13 , a colorless oil, showed a pseudomolecular ion peak [M + H]+ at m/z 363.1801 , consistent with the molecular formula C20H26O6, and eight degrees of unsaturation. 13C NMR and DEPT spectrum (δC 207.1 (C), 212.1(C), and 205.7 (C)), an isopropenyl group , 4.75 , and 1.75 ; δC 144.9 (C), 112.2 (CH2), and 21.2 (CH3)), and one α,β-unsaturated methyl ester , and 3.71 ; δC 140.9 (CH), 128.6 (C), 167.3 (C), and 52.0 (CH3)), implying bicyclic structure of 5. The 1H-1H COSY correlations H-13/H2-14/H-1/H2-2 and H2-4/H-5 with the assistance of key HMBC correlations from H3-17 to C-1, C-15, and C-16, from H2-2 and H-5 to C-3, from H-5 and H2-7 to C-6, from H3-18 to C-7, C-8, and C-9, from H2-9 and H2-11 to C-10, and from H-13 to C-11, C-12, and C-19 (16) + .Sinuketal (2): colorless oil; c 0.10, MeOH); IR (KBr) νmax 2956, 2926, 2971, 1698, 1458, 1385, 1204 cm−1; 1H and 13C NMR data, see m/z 257.1514 [M + Na]+ .Sinulin A (3): colorless amorphous solid; c 0.10, MeOH); IR (KBr) νmax 3297, 2969, 2941, 1709, 1653, 1452, 1381 cm−1; 1H and 13C NMR data, see m/z 253.1798 [M + H]+ .Sinulin B (4): colorless oil; c 0.10, MeOH); IR (KBr) νmax 3447, 2932,1439, 1384, 1211, 1081 cm−1; 1H and 13C NMR data, see m/z 457.1836 [M + Na]+ .Sinulin C (5): colorless oil; c 0.10, MeOH); IR (KBr) νmax 3396, 2927, 2854, 1759, 1710, 1644, 1436, 1381, 1331, 1281, 1260, 1199, 1167, 1180 cm−1; 1H and 13C NMR data, see m/z 363.1801 [M + H]+ .Sinulin D  and oscillator strengths (fn) of the lowest excited states of stable conformers were calculated using TD-DFT method with the basis set RB3LYP/DGDZVP. Solvent effects of methanol solution were evaluated at the same DFT level by using the SCRF/PCM method in agreement with the experiment condition. Electronic transitions were expanded as Gaussian curves with a full width at half maximum (FWHM) for each peak of 0.32 eV. The ECD spectra were combined after Boltzmann weighting according to their population contribution.The quantum chemical calculations were carried out by Gaussian 09  softwareP. falciparum (3D7) using an SYBR-Green assay  undecane carbon skeleton with unique endoperoxide moiety. In bioactivity assays, compound 1 showed mild in vitro antimalarial activity against P. falciparum 3D7, weak cytotoxic activities toward Jurkat, MDA-MB-231, and U2OS cell lines, inhibitory effects against influenza A viruses H1N1 and PR8, as well as mild target inhibitory activity against acetylcholinesterase. Compound 20 exhibited cytotoxicities against HeLa and HCT-116, and compounds 5, 11, and 15 showed mild target inhibitory activities against PTP1B.The chemical study of soft coral


Fragaria vesca var. nubicola Lindl. ex Hook.f. The structures of these compounds were elucidated to be 5-(4-hydroxy-3-methoxyphenethyl)-7-methoxy-2H-chromen-3-ol (1) and 5-(4-hydroxy-3-methoxyphenethyl)-4,7-dimethoxy-2H-chromen-3-ol (2) based on spectroscopic data through IR, UV, 1H-NMR, 13C-NMR along with two dimensional (2D) techniques HMBC, HMQC, and COSY. Both compounds 1 and 2 were studied in tail suspension and forced swim tests for antidepressant like effects. A significant dose dependent antidepressant like effect was observed by causing spontaneous anti-immobility at various test doses upon intraperitoneal administration.Two new benzyl derivatives were isolated from ethyl acetate fraction of wild strawberry,  Fragaria vesca var. nubicola Lindl. ex Hook.f.; commonly known as wild strawberry is synonym of Fragaria nubicola (Lindl. ex Hook.f.) Laciata. It is a perennial herb belonging to family Rosaceae that mostly grows along roadsides and in forests. The family Rosaceae comprises of 85 genera and nearly about 3000 species widely distributed in Europe and North America while represented by 27 genera and about 160 species in Pakistan  using tetramethyl silane (TMS) as internal standard. The TLC analysis was done by using pre coated silica gel plates while columns were packed using E. Merck 230–400 mesh and 70–230 mesh and UV active compounds were detected by using ceric sulfate in 10% H2SO4 solution.The EI-MS and HR-EI-MS analysis were done using double focusing Varian MAT-312 spectrometer and Bruker AMX-500 MHz spectrometer was used for The IR and UV spectra were recorded through Hitachi JASCO-320-A and Hitachi UV-3200 spectrophotometer respectively.F. vesca var. nubicola (6 kg) was collected from Hazara division of Khyber Pakhtunkhwa, Pakistan in May 2015 and a voucher specimen (no 8473) has been deposited in herbarium Department of Botany Postgraduate College, Abbottabad Pakistan.The whole plant of n-hexane (120 g), chloroform (75 g), ethyl acetate (150 g), and n-butanol (65 g). The ethyl acetate fraction was selected on the basis of TLC analysis and subjected to column chromatography by using n-hexane as gradient of ethyl acetate to 100% followed by methanol. Total 12 sub-fractions (A–L) were obtained and depending on TLC analysis four sub-fractions (G–J) were re-subjected to column chromatography to obtain compound 1 (12 mg) at n-hexane: ethyl acetate (40:60). Similarly compound 2 (7.8 mg) was obtained from re-column chromatography of sub-fractions (D–F) at polarity of n-hexane: ethyl acetate (45:55) .The whole shade dried plant was ground into fine powder and extracted with methanol at room temperature and filtered thrice. The vacuum rotary evaporator was used to get crude extract from filtrate. The crude extract (450 g) was partitioned into four fractions as max 222 (2.9), 252 (3.6), 298 (3.9), 324 (4.6) nm; IR (KBr) υmax 3372, 2920, 1640, 1610, 1230, 1019 cm-1; EI-MS m/z: 328 [M]+ (100), 310 (80), 296 (60), 292 (62), 264 (50), 260 (39), 228 (71), 150 (30) and 120 (45); HR-EI-MS: m/z [M+] Calcd. 328.1311 for Mol. formula C19H20O5; Observed 328.1305; 1H NMR  δ (ppm): 6.68 , 6.89 , 6.72 , 2.76 , 2.94 , 6.54 , 6.40 , 6.30 , 4.28 , 3.80 , 3.74 , 3.65 ; 13C NMR  δ (ppm): 137.2 (C-1), 119.2 (C-2), 149.9 (C-3), 148.3 (C-4), 115.7 (C-5), 124.8 (C-6), 40.2 (C-7), 38.9 (C-8), 145.8 (C-9), 107.9 (C-10), 162.1 (C-11), 102.3 (C-12), 160.6 (C-13), 109.7 (C-14), 104.4 (C-15), 150.6 (C-16), 64.3 (C-17), 58.4 (3-OCH3), 56.4 (11-OCH3).A colorless oil; UV (MeOH) λmax 212 (3.6), 240 (2.8), 296 (4.1), 310 (3.9) nm; IR (KBr) υmax 3380, 2960, 1650, 1230, 1102 cm-1; EI-MS m/z: 358 [M]+ (100), 340 (90), 326 (75), 322 (62), 294 (50), 290 (60), 264 (49), 258 (70), 226 (39), 148 (35), and 118 (43); HR-EI-MS: m/z [M+] Calcd. 358.1416 for Mol. formula C20H22O6; Observed 358.1409; 1H NMR  δ (ppm): 6.60 , 6.90 , 6.76 , 2.62 , 2.82 , 6.50 , 6.45 , 4.20 , 3.81 , 3.74 , 3.59 , 3.57 ; 13C NMR  δ (ppm): 138.4 (C-1), 115.4 (C-2), 150.1 (C-3), 144.6 (C-4), 117.7 (C-5), 123.8 (C-6), 37.4 (C-7), 36.8 (C-8), 146.3 (C-9), 111.6 (C-10), 162.4 (C-11), 99.6 (C-12), 159.3 (C-13), 107.8 (C-14), 116.4 (C-15), 136.6 (C-16), 60.9 (C-17), 57.1 (3-OCH3), 55.1 (11-OCH3), 56.9 (15-OCH3).A colorless oil; UV (MeOH) λ1 and 2 was determined by using swiss albino mice, according to the method described by Irwin (n = 9). One group served as a control and received vehicle orally. Briefly, compounds 1 and 2 each were administered at the dose level of 50,100, 200, and 300 mg/kg to each mouse orally. Each animal was subjected to various parameters including writhing, convulsions, aggressiveness, hypersensitivity, salivation, lacrimation, spontaneous activity, ataxia, and catalepsy 30 min prior to injection (baseline) and then at 0 (straight after injection), 30 and 60 min, 24, 48, and 72 h and 1 week after administration for any kind of behavioral, physical, and pharmacological toxic effects.The acute toxicity of compounds by Irwin . The aniMice were hung by their tail on the tail hanger using sticky tape for tail fixation, at approximately 1 cm from the end of the tail. The hanger was fixed in the black plastic box (20 cm × 20 cm × 45 cm) with the opening at the top front. The distance between the hanger and floor was approximately 40 cm. The mouse was suspended in the air by its tail and the immobility time was recorded over a period of 5 min. The duration of immobility was defined as the absence of all movements except for those required for respiration .1 and 2 like 10, 30, and 100 mg/kg i.p. and standard Imipramine (60 mg/kg) were administered during two swim sessions: an initial 15-min pretest followed 14 days later by a 5-min test.For forced swim test in mice, the method reported by It is certified that the Departmental Research Ethics Committee (DREC) reviewed the National Research Program for Universities (NRPU) research grants application of the project entitled “Anxiolytic and Antidepressant Activities of Selected Natural product (Glycosides and Flavonoids).” The principal investigator of the project is Dr. Nasiara Karim, Assistant Professor, Department of Pharmacy, University of Malakand.The Committee approves (DAEC/Pharm/2017/01) the study to be conducted in the present form, and expects to be inform about any revision in the protocol and subject/patient information/informed consent (where applicable).post hoc Dunnett’s test for multiple comparisons was used to assess behavior in tests. The P-value < 0.05 was considered as significant.Data are presented as mean ± SEM of six mice. A one-way factorial analysis of variance (ANOVA) followed by F. vesca var. nubicola through successive column chromatography, while characterization was made through various spectroscopic techniques and comparison with literature. The isolated compounds 1 and 2 exhibited significant antidepressant like effects in tail suspension and forced swim tests.Two new benzyl derivatives have been isolated from ethyl acetate fraction of 1 was isolated as colorless oil with molecular formula C19H20O5 as suggested by HR-EI-MS with highest molecular ion peak at m/z 328.1305 [M+] and m/z values obtained were 310, 296, 292, 264, 260, 228, 150, and 120. The UV spectrum showed absorption band at λmax 222 (2.9), 252 (3.6), 298 (3.9), 324 (4.6) and presence of various groups like hydroxyl group, alkane, alkene, aromatic moiety, and C-O were suggested by absorption bands in IR spectrum at υmax 3372, 2920, 1640, 1610, and 1230, respectively.Compound 1H-NMR spectrum revealed the presence of five aromatic protons at chemical shift values of δH 6.54 , 6.40 , 6.68 , 6.89 , and 6.72 . Similarly two methylene proton signals appeared at δH 2.76  and 2.94 , while a methine proton resonated at δH 6.30  along with two oxidized protons at δH 4.28 and 3.80 showing germinal coupling with each other  as given in Table 1. The 1H-NMR spectrum also suggested the presence of two methoxy groups at δH 3.74  and δH 3.65 .The 13C-NMR and DEPT spectrum revealed the presence of nineteen 19 carbon atoms including two methyl, three methylene, six methine, and eight quaternary carbon atoms. Two methoxy carbons resonated at δC 56.4 (C-11) and 58.4 (C-3) while three methylene carbons were observed at δC 40.2 (C-7), 38.9 (C-8), and 64.3 (C-17). 13C-NMR showed five aromatic methine carbon atoms at δC 102.3 (C-12), 107.9 (C-10), 119.2 (C-2), 115.7 (C-5), and 124.8 (C-6) with another methine carbon at δC 104.4 (C-15) as given in Table 1. The quaternary carbons resonated at δC 149.9 (C-3), 148.3 (C-4), 137.2 (C-1), 145.8 (C-9), 162.1 (C-11), 160.6 (C-13), 109.7 (C-14), and 150.6 (C-16).The 1. The HMBC correlation of methoxy protons at δH 3.74 showed strong correlation with C-3, C-4, and C-2 confirming its position at C-3 similarly second methoxy group (δH 3.65) was placed at position C-11 showing HMBC correlation with C-10, C-11, and C-12. The placement of two hydroxyl groups at C-4 and C-16 were established from HMBC correlations, like H-2 (δH 6.68) and H-6 (δH 6.72) showed connectivity with C-4 and correlation of H-17 and H-15 with C-16 respectively. The methylene protons at position C-7 showed strong HMBC correlations with C-1, C-2 and C-6, while protons at position C-8 showed HMBC correlations with C-9 and C-10 suggesting presence of bibenzyl group in compound 1 as depicted in Figure 2 -7-methoxy-2H-chromen-3-ol on the basis of all spectral data and literature comparison , 240 (2.8), 296 (4.1), 310 (3.9) and IR spectrum showed absorption bands at υmax 3380, 2960, 1650, 1610, and 1230 suggesting presence of hydroxyl group, alkane, alkene, aromatic moiety, and C-O respectively.Compound 1H-NMR and 13C-NMR data of compound 2 is quite identical to compound 1. Compound 2 had one additional methoxy group which was evident from all spectral data. The 1H-NMR showed chemical shift value for three methoxy groups at δH 3.74  and 3.59  at position C-3 and C-11 similar to compound 1 with additional methoxy group at position C-15 having chemical shift value of δH 3.57 . The 1H-NMR spectrum showed presence of five aromatic protons at δH 6.60 , 6.90 , 6.76  for ring A along with δH 6.50  having meta coupling with proton at δH 6.45  at ring B.The 13C-NMR spectrum and DEPT showed presence of twenty carbon atoms including three methoxyl group, three methylene, five methine, and nine quaternary carbons given in Table 1. The HMBC correlations of substituents were quite identical to those for compound 1, the only difference observed was a methoxy group at position C-15 showing HMBC connectivity of protons with C-15, C-16 and C-14. On the basis of all spectral data and literature comparison the structure established for compound 2 was 5-(4-hydroxy-3-methoxyphenethyl)-4,7-dimethoxy-2H-chromen-3-ol.1 and 2 at the dose level of 50–300 mg/kg did not produce any significant change in the behavior of the animals as observed by lack of convulsions, respiratory distress, writhing, changes to reflex activity, or mortality. A slight sedation was observed at 300 mg/kg, however, the animals remained alert. At 24 h to 1 week, all animals seemed well with no observable changes in behavior or appearance. No deaths were observed up to 1 week of study.Acute toxicity studies showed that the administration of compounds 1 and 2 in tail suspension tested are shown in Table 2. Both compounds 1 and 2 exhibited marked dose dependent antidepressant effects by reducing immobility time.The effects of both compounds 1 and 2 showed dose dependent antidepressant effect as given in Table 3. Both isolated compounds caused significant immobility time and thus showed their potential as antidepressant.Forced swim test is another useful and commonly used assay for the determination of antidepressant like effects of compounds . Both coThe acute toxicity and behavioral test is mandatory for further detail studies. In our study both compounds were found safe with no behavior changes in the specified time, nor any mobility or mortality.The TST and FST are reported to be the most commonly used predictive tests of antidepressant effects sensitive to the acute administration of antidepressant substances . These b1 and 2 may serve as leads for the development of newer antidepressant agents from natural sources. However, further detail studies are needed to ascertain their clinical status.The antidepressant like potential of natural isolated compounds is well-documented . Herbal in vivo studies. JH analyzed the data. UF and AK wrote the manuscript with inputs and comments from all co-authors. All authors have read and approved the final version of the manuscript.AK and UF conceived and designed the study. SN performed isolation and NK performed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.