Sinularia leptoclados has been identified as a source of bioactive 9,11-secosteroids. This study adopted a targeted isolation approach to the discovery and analysis of five 9,11-secosteroids, including two novel compounds named sinleptosterols A (1) and B (2) as well as five known analogues , 8βH-3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (4), leptosterol A (5), (24S)-3β,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9-one (6), and 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (7)) in terms of 1H-NMR patterns and potency against neutrophilic inflammation. The structure of secosteroids 1 and 2 was deduced from general spectroscopic analysis and an examination of NMR spectra. Among the above-mentioned isolates, compound 4 had the most pronounced effect in inhibiting elastase release and superoxide anion generation, with the IC50 values of 2.96 and 1.63 μM, respectively.Octocoral  Inflammation is one aspect of the regular host reaction to injury or infection caused by toxic chemicals, dead cells, pathogens, irritants, or allergens. The important role of neutrophils in a variety of infectious and inflammatory diseases makes them an attractive target for therapeutic interventions . NumerouSinularia leptoclados belongs to the order Alcyonacea, which has been shown to produce a remarkable diversity of steroids in large quantities [Soft corals are reported to produce a variety of secondary metabolites with diverse pharmacological activities. Most of the metabolites from soft corals are sesquiterpenes, diterpenes, and steroids . The octantities ,5,6. Theantities ,8. The pantities ,10,11 moS. leptoclados displayed notable anti-inflammatory activity (superoxide anion generation: IC50 3.97 μg/mL) following the isolated targeting of 9,11-secosteroids based on 1H-NMR and bioassay information. Column chromatography revealed two novel 9,11-secosteroids (sinleptosterols A (1) and B (2)) in addition to five known metabolites 3–7 -induced superoxide anion generation and elastase release.In this study, an ethyl acetate (EtOAc) extract of ites 3–7 . Most ofS. leptoclados. Comprehensive chemical and biological profiles of all fractions (fractions A–V) were then constructed through 1H-NMR analysis and an examination of anti-inflammatory activity  and potent anti-inflammatory activities . Consecutive column chromatographic processes (reverse phase) resulted in the isolation of two novel 9,11-secosteroids, (sinleptosterols A (1) and B (2)) as well as 8αH-3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (3) [βH-3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (4) [5) [S)-3β,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9-one (6) [β,11-dihydroxy-9,11- secogorgost-5-en-9-one (7) [Primary silica gel chromatographic fractionation was used to probe anti-inflammatory 9,11-secosteroids within the organic extract from activity . Fractio1 was obtained as a colorless oil. Positive mode high resolution electrospray ionization mass spectrum ((+)-HRESIMS)) of 1 revealed a sodiated adduct ion peak at m/z 451.31811, which established the molecular formula C28H44O3 , indicating seven degrees of unsaturation. IR absorption observed at 3369, 2958, and 1708 cm−1 suggested the presence of hydroxy, alkene, and ketonic groups (see 13C and distortionless enhancement by polarization transfer (DEPT) spectroscopic data revealed 28 carbon signals in this compound, (3 methylenes (including an oxymethylene), one sp2 methylene, six sp3 methines (including one oxymethine), two sp3 quaternary carbons, three sp2 methines, and three sp2 quaternary carbons (including two olefin carbons and one ketonic carbonyl) (see C 217.5 (C-9) and the proton shifts at δH 1.40 , 3.03 , 3.51 , and 5.47  were similar to those of 3-hydroxy-9,11-seco-9- oxosterols . A disubstituted alkene was recognized within the carbon signals at δC 134.6 (CH-22) and 130.2 (CH-23), and was further confirmed by two olefin proton signals at δH 5.65  and 5.93  , 1.06 , and 1.07  can respectively be attributed to the Me-21, Me-26, and Me-27 methyl groups. Two sharp methyl singlets for H3-18 and H3-19 respectively appeared at δH 0.69  and 1.40 . Taken together, these findings identify compound 1 as a tricyclic compound.Compound oups see . The 13Compound, , includinyl) see . It was z, H-23) . Three m1H NMR coupling information within the correlation spectroscopy (COSY) data of 1 enabled identification of separate spin systems for H2-1/H2-2/H-3/H2-4, H2-11/H2-12, H-6/H2-7/H-8/H-14/H2-15/H2-16/H-17/H-20/H-22/H-23, H-20/H3-21, and H-25/H3-26/H3-27  experiment, and through a comparison of NMR data between 1 and known secosterol 5. The results suggest that these two compounds possess the same 9,11-secosterol skeleton as well as the same core A-, B-, and D-rings [1 were found to be identical to those of 5. Key NOESY correlations for 1 displayed interactions between H-8/H3-18 and H-8/H3-19. Thus, H-8 should be located on the β-face (J = 16.0 Hz) indicated a trans relationship between H-22 and H-23. All these data allowed to identify compound (1) as depicted in The relative stereochemistry of e β-face . A large2) was the same as that of 1 (C28H44O3), as determined by (+)-HRESIMS at m/z 451.3239 , with seven degrees of unsaturation. Its IR bands revealed the presence of hydroxy (3406 cm−1) and ketone (1708 cm−1) groups  resembled those of 1; however, the signals corresponding to the disubstituted alkene between C-22/23 in 1 were replaced by aliphatic methylenes in 2, and one of the methyl groups at C-25 in 1 (Me-26) was replaced by an exocyclic carbon-carbon bond in 2.The molecular formula of compound  was designated as structure 2. The relative configurations at C-3, C-8, C-10, C-13, C-14, and C-17 of 2 were found to be the same as those of 1 in the core rings A–C. Note that the stereogenic carbons were identical to those of 1, which were in agreement with the observed 1H and 13C NMR chemical shifts and proton coupling constants. Therefore, compound (2) was unambiguously identified as presented in The observed HMBC correlations fully supported the locations of the functional groups. An olefinic bond was located at C-25/26 from HαH-3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (3), 8βH-3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9-one (4), leptosterol A (5), (24S)-3β,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9-one (6), and 3β,11-dihydroxy-9,11- secogorgost-5-en-9-one (7).Through a comparison of NMR spectroscopic data with those reported in the literature ,12, the 3 and 4 were elucidated by comparing their 1H NMR data [H 2.69/δC 48.6 (compound 3) to δH 3.02/δC 43.8 (compound 4). Moreover, the 1H- and 13C-NMR spectroscopic features for positions 7 and 11 were also found to be different between these two compounds  and δH 2.60 /δC 30.4 (C-7), 3.72 /δC 59.2 (C-11); 4:δH 1.98  and δH 2.40 /δC 33.0 (C-7), δH 3.83  and δH 3.68 /δC 59.3 (C-11)) (It is worth noting that the configurations of H-8 for compounds NMR data . A large (C-11)) .1–7 were characterized by assessing the inhibition of superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB  are a family of G-protein coupled receptors involved in the switching on of leucocyte responses during inflammation. Human FPR1 is expressed primarily in neutrophils, monocytes, and macrophages. It also initiates immune reactions in response to several formyl peptide ligands derived from bacteria or mitochondria [chondria . Researcchondria ,15,16.1–7 possess binding affinity to FPR1, the receptor-binding assay of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (fNLFNYK), an FPR1-specific fluorescent analog, on the surface of neutrophils. The results revealed that fMLF (10 μM) entirely inhibited the binding of fNLFNYK (2 nM), whereas only compound 2 presented a low affinity toward the FPR1 receptor at a concentration of 10 μM  using the residual CHCl3 signal (δH 7.26 ppm) as an international standard for 1H NMR and CDCl3 (δC 77.1 ppm) for 13C NMR, respectively. ESIMS and HRESIMS were recorded using a mass spectrometer . Silica gel  was used for open-column chromatography. Reverse-phase MPLC (RP-MPLC) was performed using a system comprising a gradient controller Model 601  and two Dual Pumps Model 204 . Reversed-phase HPLC (RP-HPLC) was performed using a system comprising of Nexera-i LC-2040C 3D  with photodiode-array (PDA) detector and a Rheodyne 7725i injection port . Post-run analysis was performed using Labsolutions 5.90. A Biotage® SNAP Ultra C18 (12 g) flash cartridge  was used for RP-MPLC and a reverse phase column  was used for RP-HPLC.The specific rotation of compounds was measured using a Jasco P-1010 digital polarimeter . IR spectra were acquired using a Jasco FT/IR-4100 spectrometer . NMR spectra were recorded on a Jeol Resonance ECZ400S NMR spectrometer (at 400 and 100 MHz for Sinularia leptoclados were obtained by hand using self-contained underwater breathing apparatus (SCUBA) off the coast of Pingtung, Taiwan. The samples were stored in a freezer at −20 °C until extraction. A specimen voucher was deposited with the Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Taiwan (specimen No.: CGUST-C004-2018-NOV).In November 2018, samples of the soft coral 50 3.97 μg/mL; elastase release: IC50 > 10 μg/mL) was separated on silica gel and eluted using n-hexane/EtOAc  to yield 22 fractions A−V. Fraction P  was further separated by RP-MPLC using a mixture of MeOH/ddH2O (v:v = 90:10 of volume ratio at a flow rate of 8.0 mL/min) to obtain compound 7 (82.5 mg) and nine subfractions, P1−P9. Subfraction P3  was repurified by RP-HPLC using a mixture of acetonitrile/ddH2O (volume ratio of v:v = 90:10 at a flow rate of 2.0 mL/min) to derive 1 (2.6 mg), 2 (2.0 mg), 3 (3.5 mg), 4 (100.4 mg) and 5 (13.3 mg). Subfraction P4 was purified by RP-HPLC using a mixture of MeOH/ddH2O (volume ratio of v:v = 90:10 at a flow rate of 2.0 mL/min) to yield compound 6 (6.8 mg).Soft coral material  was cut into small pieces prior to ethyl acetate (EtOAc) extraction at room temperature. The EtOAc layer : colorless oil; [αc 0.095, CHCl3); IR (neat) νmax 3369, 2958, 1708 cm−1; 1H  and 13C  NMR data .Sinleposterol A : colorless oil; [αc 0.1, CHCl3); IR (neat) νmax 3406, 2954, 1708 cm−1; 1H  and 13C  NMR data . Sinleposterol B  was used as the enzyme substrate for the detection of elastase release [Blood samples were collected from healthy adult donors (20–32 years) via venipuncture in accordance with the standard protocol approved by the local institutional review board. Human neutrophils were isolated from peripheral blood through dextran sedimentation, centrifugation in a Ficoll–Hypaque gradient, and hypotonic lysis of red blood cells. In accordance with the above mentioned protocol, assays were performed to measure superoxide anion generation based on the superoxide dismutase-inhibitable reduction of ferricytochrome C. Note that methoxy-succinyl-alanyl-alanyl-prolyl-valine- release ,18. Receptor binding assays were performed via FACScan analysis of fNLFNYK binding, a fluorescent analog of fMLF, as described in . Neutropt-test, and a p value < 0.05 was considered statistically significant. Sigma Plot software  was used for all statistical analysis [All experiments were conducted at least three times and the results are presented as the mean ± SEM. Statistical analysis was performed using the Student’s analysis . Pseudopterogorgia americana in 1972 [Sinularia leptoclados. The effectiveness of compounds 1–7 in inhibiting fMLP/CB-induced neutrophilic inflammation was also assessed. Compound 3 and 4 presented the most potent anti-inflammatory activities in terms of superoxide anion production and elastase emancipation. Cell free binding assays also demonstrated that the anti-inflammatory properties of these 9,11-secosteroids did not follow the pathway of conventional FPR1 antagonists.Marine 9,11-secosteroid was first isolated from the gorgonian  in 1972 ,21. In r in 1972 ,23,24,25


Lasianthus verticillatus (Lour.) Merr., belonging to family Rubiaceae. A new iridoid glycoside, lasianoside F (1), and three new bis-iridoid glycosides, lasianosides G–I (2–4), together with four known compounds (5–8) were isolated. The structures were established by spectroscopic methods, including 1D and 2D NMR experiments  in combination with HR-ESI-MS and CD spectra. A series of iridoid glycosides were isolated from the leaves of  Lasianthus species  22D − 65.5. The molecular formula was deduced to be C21H28O11 from HR-ESI-MS , which suggested eight degrees of unsaturation. The UV spectrum showed absorption maxima at 234 nm, indicating the presence of an enone system, and IR absorption bands at 3406, 1733, 1658, and 1634 cm−1 that corresponded to hydroxy, carbonyl, and olefinic groups. The 1H-NMR spectrum of 1 . The 13C-NMR spectrum of 1 showed 21 signals, of which six signals could be attributed to a glucopyranosyl unit , ten signals for an iridoid skeleton  which were similar to those reported for asperuloside (5)  22D − 55.0. Its molecular formula C36H44O22, from its HR-ESI-MS , indicating 15 degrees of unsaturation. The UV spectra of 2 exhibited absorption maxima at 236 nm, characteristic of an enol ether system. Similarly, IR spectra displayed absorption bands corresponding to hydroxy, carbonyl, and olefinic groups at 3309, 1736, 1541 cm−1, respectively. Duplication of the signals in both 1H and 13C-NMR spectra  and 4.92  supported the presence of two glucosyl units in 2  and 7.70 , which are characteristic for C-3 protons confirmed the presence of two iridoid moieties having an enol ether function. Consistent with these observations, the 13C-NMR spectrum showed 36 signals comprising four carbonyl carbons , eight olefinic carbons , six oxygenated carbons , four sp3 methine carbons  together with two anomeric carbons  and oxygenated carbons arising in the region of δc 62.7–78.6 belonging to two glucose moieties (2 were referred to as units “A” and “B” and determined to be asperuloside (5) and asperulosidic acid  22D − 59.9. The molecular formula was assigned as C36H44O22 by HR-ESI-MS at m/z 851.2212 [M + Na]+ , indicating that 3 was also another positional isomer of 8. Comparison of 1H and 13C-NMR data  of unit “A” and C-11(δc 168.6) of unit “B” in HMBC spectrum  was isolated as a colorless amorphous powder, with a specific optical rotation of [α]22D − 60.1. It has molecular formula of C36H44O22 established from its 13C-NMR data and positive mode HR-ESI-MS [m/z 851.2215 [M + Na]+ ]. The 13C-NMR data showed signals resembling those of 8, except the presence of two sp3 methines, C-4 at δc 44.4 and C-3 at 97.4 ppm in unit “B” of 4, instead of resonances of two olefinic carbons at the same position of 8, in addition to lower field shift of C-11 and C-6 to δc 176.9 and 87.9 ppm, respectively  to C-11 (δc 176.9)  -5-(β-D-glucopyranosyloxy)-2a,4a,5,7b-tetrahydro-1H-2,6-dioxacyclopent[cd]inden-1-one. Colorless amorphous powder [α]22D − 65.5 ; HR-ESI-MS (positive ion mode): m/z: 479.1521 [M + Na]+ ; CD λmax  nm (∆ε): 245 (−4.11); UV (MeOH) λmax nm (log ε) 234 (4.04); IR (film) νmax: 3406, 2960, 1733, 1658, 1634, 1292, 1183, 1164, 1077, 1017, 762 cm−1; 1H-NMR  and 13C : Lasianoside F (2): -4-[(acetyloxy)methyl]-5-[[2-O-[[-7-[(acetyloxy)methyl]-1-(β-D-glucopyranosyloxy)-1,4a,5,7a-tetrahydro-5-hydroxycyclopenta[c]pyran-4-yl]carbonyl]-β-D-glucopyranosyl]oxy]-2a,4a,5,7b-tetrahydro-1H-2,6-dioxacyclopent[cd]inden-1-one. Colorless amorphous powder [α]22D − 55.0 ; HR-ESI-MS (positive ion mode): m/z: 851.2214 [M + Na]+ ; CD λmax  nm (∆ε): 235 (−8.04); UV (MeOH) λmax nm (log ε) 236 (4.10); IR (film) νmax: 3309, 2925, 1736, 1716, 1541, 1260, 1162, 1057, 1033, 669 cm−1; 1H-NMR  and 13C : Lasianoside G (3): -4-[(acetyloxy)methyl]-5-[[3-O-[[-7-[(acetyloxy)methyl]-1-(β-D-glucopyranosyloxy)-1,4a,5,7a-tetrahydro-5-hydroxycyclopenta[c]pyran-4-yl]carbonyl]-β-D-glucopyranosyl]oxy]-2a,4a,5,7b-tetrahydro-1H-2,6-dioxacyclopent[cd]inden-1-one. Colorless amorphous powder [α]22D − 59.9 ; HR-ESI-MS (positive ion mode): m/z: 851.2212 [M + Na]+ ; CD λmax  nm (∆ε): 245 (−5.73); UV (MeOH) λmax nm (log ε) 235 (4.31); IR (film) νmax: 3388, 2932, 1730, 1658, 1632, 1261, 1158, 1075, 1044, 788 cm−1; 1H-NMR  and 13C : Lasianoside H (4): -4-[(acetyloxy)methyl]-5-[[6-O-[[-4-[(acetyloxy)methyl]-5-(β-D-glucopyranosyloxy)-2a,4a,5,7,7a,7b-hexahydro-1H-2,6-dioxacyclopenta[cd]inden-1-one-7-yl]]-β-D-glucopyranosyl]oxy]-2a,4a,5,7b-tetrahydro-1H-2,6-dioxacyclopent[cd]inden-1-one. Colorless amorphous powder [α]22D − 60.1 ; HR-ESI-MS (positive ion mode): m/z: 851.2215 [M + Na]+ ; CD λmax  nm (∆ε): 245 (−3.48); UV (MeOH) λmax nm (log ε) 234 (4.23); IR (film) νmax: 3407, 2927, 1739, 1658, 1254, 1175, 1070, 1052, 1017, 758 cm−1; 1H-NMR  and 13C : Lasianoside I (5): Colorless amorphous powder, [α]24D − 170.6 ; HR-ESI-MS (positive ion mode): m/z: 437.1053 [M + Na]+ ; 13C-NMR  δC: 20.6 (CH3-CO-), 37.4 (C-5), 45.3 (C-9), 61.9 (C-10), 62.8 (C-6ʹ), 71.6 (C-4ʹ), 74.6 (C-2ʹ), 77.9 (C-5ʹ), 78.4 (C-3ʹ), 86.3 (C-6), 93.3 (C-1), 100.0 (C-1ʹ), 106.2 (C-4), 128.9 (C-7), 144.3 (C-8), 150.3 (C-3), 172.3 (C-11), 172.6 (CH3-CO-).Asperuloside (6): Colorless amorphous powder, [α]24D − 125.4 ; HR-ESI-MS: m/z: 395.0946 [M + Na]+ ; 13C-NMR  δC: 37.5 (C-5), 45.0 (C-9), 60.1 (C-10), 62.8 (C2-6ʹ), 71.6 (C-4ʹ), 74.6 (C-2ʹ), 77.9 (C-5ʹ), 78.4 (C-3ʹ), 86.7 (C-6), 93.3 (C-1), 99.3 (C-1ʹ), 106.5 (C-4), 125.7 (C-7), 149.8 (C-8), 150.3 (C-3), 172.9 (C-11).Deacetyl asperuloside (7): Colorless amorphous powder, [α]25D − 109.8 ; HR-ESI-MS: m/z: 499.1210 [M + Na]+ ; 13C-NMR  δC: 37.4 (C-5), 45.0 (C-9), 62.6 (C-10), 62.7 (C-6ʹ), 71.5 (C-4ʹ), 74.6 (C-2ʹ), 77.9 (C-5ʹ), 78.4 (C-3ʹ), 86.3 (C-6), 93.4 (C-1), 100.0 (C-1ʹ), 106.2 (C-4), 129.4 (C-7), 129.7 , 130.7 , 130.9 (C-1ʹʹ), 134.6 (C-4ʹʹ), 144.3 (C-8), 150.3 (C-3), 165.9 (C-7ʹʹ), 172.9 (C-11).Besperuloside (8): Colorless amorphous powder, [α]24D − 52.5 ; HR-ESI-MS: m/z: 851.2216 [M + Na]+ ; 13C-NMR  δC: 20.8, 20.8 (each CH3-CO-), 37.4 (C-5A), 42.8 (C-5B), 45.2 (C-9A), 46.3 (C-9B), 61.9 (C-10A), 62.9 (C-6ʹB), 63.8 (C-10B), 64.4 (C-6ʹA), 71.5 (C-4ʹA), 71.7 (C-4ʹB), 74.6 (C-2ʹA), 74.9 (C-2ʹB), 75.5 (C-6B), 75.8 (C-5ʹA), 77.6 (C-5ʹB), 77.8 (C-3ʹA), 78.5 (C-3ʹB), 86.4 (C-6A), 93.2 (C-1A), 99.9 (C-1ʹA), 100.5 (C-1ʹB), 101.4 (C-1B), 106.3 (C-4A), 108.1 (C-4B), 129.2 (C-7A), 131.8 (C-7B), 144.1 (C-8A), 146.0 (C-8B), 150.2 (C-3A), 155.8 (C-3B), 168.6 (C-11B), 172.2 (C-11A), 172.6 (2 × CH3-CO-).Compound (− form), then it was filtered. The filtrate was evaporated and partitioned between EtOAc: H2O mixture (1:1). The aqueous layer was analyzed by HPLC with an amino column  and a chiral detector (JASCO OR-2090plus). The peak that appeared at tR 8.15 min  supported the presence of D-glucose in the structures of iridoid glucosides (1–4) [Each compound (2 mg) was refluxed individually in 1 M HCL (1.0 mL) at 80 °C for 3 h. The solution was neutralized with Amberlite IRA96SB (OHes (1–4) .L. verticillatus was further investigated to lead the isolation of a new iridoid glycoside, lasianoside F (1) and three new bis-iridoid glycosides, lasianosides G–I (2–4), together with four known compounds (5–8). The structures of isolated compounds (1–8) were characterized by physical and spectroscopic data analyses, including one-dimensional (1D) and two-dimensional (2D) NMR, IR, UV, and high-resolution electrospray ionization mass spectra (HR-ESI-MS). The absolute configuration of the new compounds was determined by acid hydrolysis and the analysis of the CD cotton effect. In summary, the chemical composition of the leaves of


Combretum laxum yielded a new dihydrostilbene derivative, 4′-hydroxy-3,3′,4-trimethoxy-5--bibenzyl (1), two phenanthrenes (2–3), and three dihydrophenanthrenes (4–6), along with one lignan, three triterpenoids, one aurone, one flavone, one naphthoquinone, and two benzoic acid derivatives. Their structures were determined by 1D and 2D nuclear magnetic resonance (NMR) spectroscopic techniques and/or mass spectrometry data. The occurrence of dihydrostilbenoid, phenanthrene and dihydrophenanthrene derivatives is unprecedented in a Combretum species native to the American continent. 2,7-Dihydroxy-4,6-dimethoxyphenanthrene, 2,6-dihydroxy-4,7-dimethoxy-9,10-dihydrophenanthrene and 5-O-methyl apigenin are novel findings in the Combretaceae, as is the isolation of compounds belonging to the chemical classes of aurones and naphthoquinones, while (+)-syringaresinol is reported for the first time in the genus Combretum. Compounds 1–6 were also evaluated for their in vitro cytotoxicity against five human cancer cell lines, and radical-scavenging ability against 1,1-diphenyl-2-picryl-hydrazyl (DPPH). 6-Methoxycoelonin (4) was the most cytotoxic against melanoma cells (IC50 2.59 ± 0.11 µM), with a high selectivity index compared with its toxicity against nontumor mammalian cells (SI 25.1). Callosin (6), despite exhibiting the strongest DPPH-scavenging activity (IC50 17.7 ± 0.3 µM), proved marginally inhibitory to the five cancer cell lines tested, indicating that, at least for these cells, antioxidant potential is unrelated to antiproliferative activity.The chemical investigation of the roots and stems of  Combretum, found in tropical and subtropical areas, is the largest within the Combretaceae and most of its species are extensively used in the folk medicine of African and Asian countries for the treatment of a wide variety of health disorders +) data  found between δC 132 to 135, as well as by proton resonances in the region of δH 6.08 to 6.68 (C 61.0 and 61.3 +) data . The aro to 6.68  9]. The. The1 waand 2.76 , which iure of 1 ,11. FurtHz each) , which, 2 was deduced as C16H14O4 based on the [M + H]+ ion at m/z 271.0963 in the HRESIMS , and six methine carbons, in addition to two aromatic methoxy groups, evidenced at δH 4.02/δC 56.2 and δH 4.12/δC 56.0 , two isolated protons at δH 7.24 (s) and 9.11 (s), and a pair of ortho-coupled protons at δH 7.44 and 7.56 . The chemical shifts and splitting patterns of these last two protons were shown to be characteristic of H-9 and H-10 of a phenanthrene derivative + ion at m/z 301.1076 in the HRESIMS , instead of four as in compound 2, located at carbons C-2, C-3, C-4, C-6, and C-7 in the phenanthrene skeleton. The foregoing information were supported by a singlet at δH 7.04 ascribable to H-1, which showed a connectivity with the carbon signal at δC 109.8 in the HSQC spectrum, which in turn displayed a three-bond correlation with H-10  in the HMBC spectrum. Likewise, long-range connectivities were observed between H-1 and C-2 , C-3 , C-10 , and C-4a . Since the chemical shifts of two of the methoxy carbons (δC 60.5 and 61.5) revealed their sterically hindered nature, they were placed at C-3 and C-4 positions. Three-bond correlations between the methoxyl protons at δH 4.00, 3.98, and 3.99 and C-3, C-4 (δC 152.8), and C-7 (δC 148.4), respectively, determined the attachment of the methoxy groups to these corresponding aromatic carbons, therefore establishing the location of the hydroxy functions at C-2 (δC 150.4) and C-6 (δC 147.4). Further correlations discernible in the HMBC spectrum between H-5 (δH 8.90) and both C-6  and C-7 , H-8 (δh 7.27) and both C-6 (3J) and C-7 (2J), and H-9 (δH 7.50) and C-8 , together with NOE-cross peaks observed between H-8 and OCH3 at δH 3.99, and between H-5 and OCH3 at δH 3.98, reinforced these assignments. The foregoing data could be satisfactorily assembled to establish the structure of 3 as 2,6-dihydroxy-3,4,7-trimethoxyphenanthrene, formerly obtained as a plant constituent only from Combretum apiculatum, but with no full description of its 1H and 13C NMR data +) . Its 1H ose of 2 , except coelonin , previouoifolium ,19,20, iriaceae) , but scareroense .5 was assigned the molecular formula C17H18O5 on the basis of its HRESIMS  + ion at m/z 273.1121 in the HRESIMS  and 6.39 . Correlations in the HSQC spectrum between H-1/C-1 (δC 108.5) and H-3/C-3 (δC 99.3), as well as further correlations observed in the HSQC, HMBC and nuclear Overhauser effect spectroscopy (NOESY) spectra, lent support to these assignments. Compound 6 was thus identified as 2,6-dihydroxy-4,7-dimethoxy-9,10-dihydrophenanthrene, whose NMR data agreed with those reported for callosin, previously isolated only from two species of the Orchidaceae  and 3.84 , together with those belonging to a spin system at δH 4.70 , 3.13 , 3.70–3.80 , and 4.18–4.24 , suggested that 7 was a bistetrahydrofuran lignan. This assumption was supported by the eight signals observed in the 13C NMR, in which four of them were attributed to the symmetrically substituted aromatic rings, namely four methines (δC 104.5), six oxygenated carbons (δC 136.2 and 149.3), and two carbons linked to the bistetrahydrofuran moiety (δC 133.1). The remaining four signals were ascribed to the four methoxy groups at δC 56.8, and to the methine (δC 55.5 and 72.5) and methylene (δC 87.6) carbons of the foregoing bistetrahydrofuran residue. These assignments were further corroborated by HSQC and HMBC correlations, while the carbon resonances of the tetrahydrofuran rings were indicative of the pseudoequatorial orientation of the aromatic rings in the structure of 7, as well as established their linkage to C-7/C-7′ + ; 1H and 13C NMR data -bibenzyl (NMR data .2): amorphous solid; HRESIMS m/z 271.0963 [M + H]+ ; 1H and 13C NMR data : amorphous solid; HRESIMS m/z 301.1076 [M + H]+ ; 1H and 13C NMR data : amorphous solid; HRESIMS m/z 273.1118 [M + H]+ ; 1H and 13C NMR data (6-Methoxycoelonin (NMR data .5): amorphous solid; HRESIMS m/z 303.1235 [M + H]+ ; 1H and 13C NMR data : amorphous solid; HRESIMS m/z 273.1121 [M + H]+ ; 1H and 13C NMR data : amorphous solid; [α]D20 + 11.1 ; 1H-NMR (CD3OD): δ 3.13 ; 3.70-3.80 ; 3.84 ; 4.18-4.24 ; 4.70 ; 6.65 . 13C-NMR (CD3OD): δ 133.1 ; 104.5 ; 149.3 ; 136.2 ; 87.6 ; 55.5 ; 72.5 ; 56.8 .(+)-Syringaresinol (8): amorphous solid; 1H-NMR (acetone-d6): δ 6.58 ; 6.72 ; 6.79 ; 6.89 ; 7.27 ; 7.56 . 13C-NMR (acetone-d6): δ 147.2 (C-2), 182.5 (C-3); 126.3 (C-4); 113.8 (C-5); 167.7 (C-6); 99.4 (C-7); 169.0 (C-8); 114.4 (C-9); 112.4 (C-10); 125.2 (C-1′); 118.7 (C-2′); 146.4 (C-3′); 148.6 (C-4′); 116.6 (C-5′); 125.4 (C-6′).Sulfuretin (O-Methyl Apigenin (9): amorphous solid; 1H-NMR (CD3OD): δ 3.85 ; 6.34 ; 6.46 ; 6.48 ; 6.89 ; 7.78 . 13C-NMR (CD3OD): δ 161.6 (C-2); 106.2 (C-3); 180.2 (C-4); 162.4 (C-5); 98.7 (C-6); 163.5 (C-7); 97.2 (C-8); 123.4 (C-1′); 129.0 ; 117.0 ; 162.3 (C-4′); 56.3 (OCH3-5).5-10): amorphous solid; 1H-NMR (CDCl3): δ 1.67 ; 1.77 ; 3.29 ; 5.19 ; 7.66 ; 7.73 ; 8.05 ; 8.10 . 13C-NMR (CD3OD): δ 181.7 (C-1); 152.7 (C-2); 123.5 (C-3); 184.5 (C-4); 126.8 (C-5); 134.8 (C-6); 132.8 (C-7); 126.0 (C-8); 129.4 (C-9); 132.9 (C-10); 22.6 (C-1′); 119.6 (C-2′); 133.8 (C-3′); 25.7 (C-4′); 17.9 (C-5′).Lapachol (11): amorphous solid; 1H-NMR (CD3OD): δ 3.85 * ; 3.87 * ; 6.98 ; 7.54 ; 7.64 . 13C-NMR (CD3OD): δ 124.3 (C-1); 113.6 (C-2); 150.0 (C-3); 154.6 (C-4); 111.8 (C-5); 125.0 (C-6); 169.9 (C-7); 56.4 * (OCH3-3 and OCH3-4) * interchangeable signals.3,4-Dimethoxybenzoic Acid (12): amorphous solid; 1H-NMR (CD3OD): δ 3.89 ; 6.83 ; 7.55 ; 7.56 . 13C-NMR (CD3OD): δ 123.6 (C-1); 113.8 (C-2); 152.4 (C-3); 148.6 (C-4); 115.8 (C-5); 125.2 (C-6); 169.8 (C-7); 56.4 (OCH3-4).3-Hydroxy-4-methoxybenzoic Acid (13): amorphous solid; 1H-NMR (CD3OD): δ 0.68 ; 0.80 ; 0.91 ; 0.93 ; 1.01 ; 1.15 ; 2.84 ; 3.30 ; 3.33 ; 3.49 ; 3.68 ; 5.24 ; 13C-NMR (CD3OD): δ 47.6 (C-1); 69.7 (C-2); 78.2 (C-3); 44.1 (C-4); 48.2 (C-5); 19.1 (C-6); 33.8 (C-7); 40.5 (C-8); 47.9 (C-9); 39.0 (C-10); 24.0 * (C-11); 123.4 (C-12); 145.4 (C-13); 43.0 (C-14); 28.8 (C-15); 24.6 * (C-16); 47.6 (C-17); 42.7 (C-18); 47.2 (C-19); 31.6 (C-20); 34.9 (C-21); 33.3 (C-22); 66.4 (C-23); 13.9 (C-24); 17.8 (C-25); 17.6 (C-26); 26.5 (C-27); 181.9 (C-28); 33.6 (C-29); 24.0 (C-30) * interchangeable signals.Arjunolic Acid (14): amorphous powder; 1H- and 13C-NMR data in accordance with those of an authentic sample and with literature [Betulinic Acid (terature .15): amorphous solid; 1H-NMR (pyridine-d5): δ 0.93 ; 0.97 ; 0.98 ; 1.00 ; 1.06 ; 1.25 ; 1.26 ; 3.30 ; 3.38 ; 4.11 ; 5.45 . 13C-NMR (pyridine-d5): δ 46.4 (C-1); 68.6 (C-2); 83.8 (C-3); 39.8 (C-4); 55.9 (C-5); 18.8 (C-6); 33.2 (C-7); 39.8 (C-8); 48.1 (C-9); 38.5 (C-10); 23.7 (C-11); 122.4 (C-12); 144.8 (C-13); 42.2 (C-14); 28.2 (C-15); 23.7 (C-16); 46.6 (C-17); 42.0 (C-18); 46.4 (C-19); 30.9 (C-20); 34.2 (C-21); 33.2 (C-22); 29.3 (C-23); 17.6 (C-24); 16.8 (C-25); 17.4 (C-26); 26.1 (C-27); 180.2 (C-28); 33.3 (C-29); 23.7 (C-30).Maslinic Acid , 786-0 (kidney), UACC-62 (melanoma), NCI/ADR-RES , and Hep2 (larynx), all of which were kindly provided by Prof. João Ernesto de Carvalho, of the School of Pharmaceutical Sciences, CPQBA, Universidade Estadual de Campinas, Campinas, Brazil. VERO (monkey kidney) nonneoplastic cells were obtained from the Rio de Janeiro cell bank. To this end, a sulforhodamine B  assay was performed, as described elsewhere [−1). IC50 values were calculated from the differences in absorbance readings at 540 nm in untreated (negative control) and treated cells on a SpectraMax 190 microplate reader  [p < 0.05).Cytotoxicity of compounds lsewhere ,54. CispCA, USA)  through CA, USA) . The dat1–6 were determined using DPPH , employing the method of microdilution in 96-well microplates described by Zhang et al. and Yamaguchi et al. [control − Asample)/Acontrol. Radical-scavenging activities were assessed on the basis of their IC50 values determined by linear regression.The radical-scavenging activities of compounds i et al. ,55, withC. laxum from the Brazilian Pantanal. Among the one new and 14 known compounds comprising nine different classes of secondary metabolites, the isolation of dihydrostilbenoid, phenanthrene and dihydrophenanthrene derivatives is unprecedented in a Combretum species native to the American continent. Also remarkable is the presence of aurone and naphthoquinone representatives, since these chemical classes are being reported for the first time in the Combretaceae, as are the isolation of phenanthrene 2, dihydrophenanthrene 6, and flavone 9, and the first reported occurrence of lignan 7 in the genus Combretum. The results of the present study also revealed that, at least with respect to the effects of compounds 2–6 against melanoma (UACC-62) cells, the presence of methoxy and hydroxy groups at C-6 and C-7, respectively, as well as the lack of aromaticity of ring B can be considered as important structural features for cytotoxicity. On the other hand, when compared with their radical-scavenging ability against DPPH, cytotoxicity of 1–6 is unrelated to their antioxidant potential, at least for the five cancer cell lines tested.The foregoing results constitute new information on the chemical composition of a specimen of 50 and selectivity index values presented by 4 (6-methoxycoelonin) against melanoma (UACC-62) cells thus indicate that this dihydrophenanthrene derivative can be considered as a promising candidate for further investigation of its mechanism of action. Future research on association of 6-methoxycoelonin with current anticancer drugs aiming at the development of potential new drug combination therapies within clinical oncology is also strongly encouraged.The anticarcinogenic, antimetastatic, and chemopreventive potentialities of plant-derived compounds either isolated or in combination with chemotherapy drugs have been the subject of an increasing number of recent preclinical and clinical studies aiming at the development of new antineoplastic agents. These studies reveal that combination of synthetic chemotherapy drugs with selected plant constituents not only may improve pharmacological activity and simultaneously minimize toxic side effects of synthetic chemical drugs, but also delay or even overcome the development of drug resistance ,60,61,62


BPNs) 1–5 as α-phenyl-N-tert-butylnitrone analogues prepared from commercially available -4-carbaldehyde and -4,4′-dicarbaldehyde. The neuroprotection of BPNs1-5 has been measured against oligomycin A/rotenone and in an oxygen–glucose deprivation in vitro ischemia model in human neuroblastoma SH-SY5Y cells. Our results indicate that BPNs 1–5 have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN), and they are quite similar to N-acetyl-L-cysteine (NAC), which is a well-known antioxidant agent. Among the nitrones studied, homo-bis-nitrone BPHBN5, bearing two N-tert-Bu radicals at the nitrone motif, has the best neuroprotective capacity  as well as anti-necrotic, anti-apoptotic, and antioxidant activities (EC50 = 11.2 ± 3.94 μM), which were measured by its capacity to reduce superoxide production in human neuroblastoma SH-SY5Y cell cultures, followed by mononitrone BPMN3, with one N-Bn radical, and BPMN2, with only one N-tert-Bu substituent. The antioxidant activity of BPNs1-5 has also been analyzed for their capacity to scavenge hydroxyl free radicals (82% at 100 μM), lipoxygenase inhibition, and the inhibition of lipid peroxidation (68% at 100 μM). Results showed that although the number of nitrone groups improves the neuroprotection profile of these BPNs, the final effect is also dependent on the substitutent that is being incorporated. Thus, BPNs bearing N-tert-Bu and N-Bn groups show better neuroprotective and antioxidant properties than those substituted with Me. All these results led us to propose homo-bis-nitrone BPHBN5 as the most balanced and interesting nitrone based on its neuroprotective capacity in different neuronal models of oxidative stress and in vitro ischemia as well as its antioxidant activity.Herein, we report the neuroprotective and antioxidant activity of 1,1′-biphenyl nitrones ( The Reactive Oxygen Species (ROS) formed in the metabolism in living organisms play key roles acting at low to moderate concentrations in many important cell processes; however, at high concentrations, they produce negative effects in key physiological biomolecules, such as DNA, proteins, and lipids . As a reN-tert-butylnitrone (PBN)  , which p placebo . In spit placebo  are stil placebo ,11, as w placebo ,13,14,15 placebo , and ind placebo  derived bis-nitrone W-AZN -1,1′-bis(N-benzylmethanimine oxide) (HBN6)  (HBN6)  as a potN6) (PBN .BPNs), as PBN analogues derived from -4-carbaldehyde, such as -4-carbaldehyde mononitrones (BPMNs) 1–3, and from -4,4′-dicarbaldehyde, such as -4,4′-dicarbaldehyde homo-bis-nitrones (BPHBNs) 4–6  4–6 . As a reBPMNs 1-3 has been carried out as shown in N-alkyl hydroxylamine hydrochloride, in THF, in the presence of sodium bicarbonate and sodium sulfate, by irradiation  as shown in BPHBNs 4–6 were obtained in good yields (The synthesis of d yields . Z isomers at the double PhC=N(O)R bond, as usual, and they gave analytical and spectroscopic data in good agreement with their structures as well as with the previously described data in the literature for known nitrones BPMN1 -4-carbaldehyde (1 equiv), in dry THF (0.05M), Na2SO4 (3 equiv), anhydrous NaHCO3 (3 equiv), and the appropriate hydroxylamine hydrochloride (1.2 equiv) were added, and the mixture was irradiated (90 ºC/ 15 bar) and stirred for the indicated time in each case. When the reaction was complete , the solvent was removed, and the crude was purified by flash-chromatography. (Z)-1--N-methylmethanimine oxide (BPMN1) -4-carbaldehyde  was treated with NaHCO3 , Na2SO4 , and MeNHOH.HCl , in THF (20 mL), and irradiated for 3 h, to give, after purification , nitrone BPMN1 , as a white solid: mp 134-6 ºC; IR (KBr) ν 3392, 1406, 1155 cm−1; 1H NMR  δ 8.30 , 7.65 , 7.64 , 7.46 , 7.41 , 7.40 , 3.91 ; 13C NMR  δ 143.0 , 140.2 , 134.9 , 129.5 , 129.0 , 127.9 , 127.1 , 54.5 (CH3); MS (ESI) m/z: 212 [M+1]+, 234 [M+Na]+, 445 [2M+Na]+. Anal. Calcd. for C14H13NO.2/7 H2O: C, 77.70; H, 6.32; N, 6.47. Found: C, 77.72; H, 6.14; N, 6.52. (BPMN1) . FollowiZ)-1--N-(tert-butyl)methanimine oxide (BPMN2) -4-carbaldehyde  was treated with NaHCO3 , Na2SO4  and tert-BuNHOH.HCl , in THF (20 mL), and irradiated for 21 h to give, after purification , nitrone BPMN2 , as a white solid: mp 109-111 ºC; IR (KBr) ν 3434, 1130, 1122 cm-1; 1H NMR  δ 8.38 , 7.65 , 7.64 , 7.60 , 7.46 , 7.38 , 1.67 ; 13C NMR  δ 142.6 , 140.4 , 130.1 , 129.6 , 129.3 , 128.9 , 127.8 , 127.2 *, 127. 1 *, 70.9 [N(O)C(CH3)3], 28.5 ; MS (ESI) m/z: 254 [M+1]+, 276 [M+Na]+, 529 [2M+Na]+}. Anal. Calcd. for C17H19NO: C, 80.60; H, 7.56; N, 5.53. Found: C, 80.72; H, 7.76; N, 5.43.Z)-1--N-benzylmethanimine oxide (BPMN3). was treated with NaHCO3 , Na2SO4 , and BnNHOH.HCl , in dry THF (20 mL), and irradiated for 4 h, to give, after purification , nitrone BPMN3 , as a white solid: mp 157-9 ºC; IR (KBr) ν 3435, 1148 cm-1; 1H NMR  δ 8.30 , 7.66 , 7.63 , 7.55-7.35 , 5.09 ; 13C NMR  δ 143.0 , 140.3 , 134.0 , 133.4 , 129.5 , 129.4 , 129.2, 129.1, 129.0 , 127.9 , 127.2 *, 127.1 *, 71.2 [C6H5CH2(N)O]; MS (ESI) m/z: 288 [M+1]+, 310 [M+Na]+, 597 [2M+Na]+. Anal. Calcd. for C20H17NO: C, 83.59; H, 5.96; N, 4.87. Found: C, 83.34; H, 5.99; N, 4.89.General method for the synthesis of bis-nitrones 4-6. To a solution of -4,4′-dicarbaldehyde (1 mmol) in dry THF (0.05M), Na2SO4 (4 mmol), anhydrous NaHCO3 (3 mmol), and the appropriate hydroxylamine hydrochloride (3 mmol), were added, and the mixture was irradiated (90 ºC/ 15 bar) and stirred for the indicated time in each case. When the reaction was complete , the solvent was removed, and the crude was purified by flash chromatography.Z,1′Z)-1,1′-bis(N-methylmethanimine oxide) (BPHBN4) -4,4′-dicarbaldehyde  was treated with NaHCO3 , Na2SO4 , and MeNHOH.HCl  in THF (20 mL), and irradiated for 1 h, to give, after purification , nitrone BPHBN4 , as a white solid: mp > 220 ºC; IR (KBr) ν 3422, 1164 cm−1; 1H NMR  δ 8.32 , 7.91 ), 7.83 , 3.81 ; 13C NMR  δ 140.4 , 133.9 , 131.0 , 128.8 , 126.9 , 54.60 ; MS (ESI) m/z: 269 [M+1]+, 291 [M+Na]+, 559 [2M+Na]+. Anal. Calcd. for C16H16N2O2: C, 71.62; H, 6.01; N, 10.44. Found: C, 71.69; H, 6.27; N, 10.33. Z,1′Z)-1,1′-bis(N-tert-butylmethanimine oxide) (BPHBN5) was treated with NaHCO3 , Na2SO4 , and tert-BuNHOH.HCl , in THF (20 mL), and irradiated for 8 h, to give, after purification , nitrone BPHBN5 , as a white solid: mp > 220 ºC; IR (KBr) ν 3435, 1361, 1125 cm−1; 1H NMR  δ 8.37 , 7.71 , 7.60 , 1.64 ; 13C NMR  δ 141.6 , 130.6 , 129.5 , 129.5 , 127.1 , 71.0 , 28.49 ; MS (ESI) m/z: 353 [M+1]+, 375 [M+Na]+, 727 [2M+Na]+}. Anal. Calcd. for C22H28N2O2: C, 74.97; H, 8.01; N, 7.95. Found: C, 74.60; H, 8.44; N, 7.57. Z,1′Z)-1,1′-bis(N-benzylmethanimine oxide) (BPHBN6). was treated with NaHCO3 , Na2SO4  and BnNHOH.HCl , in THF (20 mL), and irradiated for 3 h 30 min, to give, after purification , nitrone BPHBN6 , as a white solid: mp > 220 ºC; IR (KBr) ν 3444, 1151 cm−1. Anal. Calcd. for C28H24N2O2: C, 79.98; H, 5.75; N, 6.66. Found: C, 79.64; H, 6.09; N, 6.59.Neuroblastoma cell cultures. The human neuroblastomas cell line SH-SY5Y were cultured in Dulbecco’s: Ham’s F12, 1:1 [vol/vol] containing 3.15 mg/mL glucose, 2.5 mM Glutamax and 0.5mM sodium pyruvate DMEM/F-12, GlutaMAX™; GIBCO, Life Technologies, Madrid (Spain), 1% antibiotic–antimitotic  , 1% gentamicin 15 mg/mL  and 10% Fetal Calf Serum (FCS)  as described . Linear regression of fluorescence data (expressed as arbitrary fluorescence units (AFU)) was calculated for each condition, and the slopes (a) of the best fitting lines (y = ax) were considered as an index of O2•− production. SNP was used as a positive control of superoxide production [ribed 22.. Linear Statistical Analysis. Data were expressed as mean ± SEM of results obtained from at least three independent experiments from different cultures, each of which was performed in triplicate. Statistical comparisons between the different experimental conditions were performed using one-way analysis of variance (ANOVA), followed by Holm−Sidak’s post-test when the analysis of variance was significant. A P value <0.05 was considered statistically significant. Fit curves for EC50 determinations were performed according to the program of SigmaPlot v.11 .Materials and Methods. Nordihydroguaiaretic acid (NDGA), Trolox, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) Soybean LOX linoleic acid sodium salt were purchased from the Aldrich Chemical Co. Milwaukee, WI, (USA). Phosphate buffer (0.1 M and pH 7.4) was prepared mixing an aqueous KH2PO4 solution , and an aqueous of NaOH solution ; the pH (7.4) was adjusted by adding a solution of KH2PO4 or NaOH). For the in vitro tests, a Lambda 20 (Perkin–Elmer-PharmaSpec 1700) UV–Vis double beam spectrophotometer was used.Inhibition of linoleic acid peroxidation [xidation . For iniInhibition of soybean lipoxygenase [14 The tested compounds  dissolved in DMSO were incubated at room temperature with sodium linoleate (0.1 mM) and 0.2 mL of enzyme solution . The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded and compared with the appropriate standard inhibitor NDGA (IC50 0.45 μM and 93% at 100 μM).xygenase . The oxiHydroxyl radicals scavenging activity [3+/ascorbic acid system. EDTA (0.1 mM), Fe 3+ (167 μM), DMSO (33 mM) in phosphate buffer , the tested compounds (0.1 mM), and ascorbic acid (10 mM) were mixed in test tubes. The solutions were incubated at 37 °C for 30 min. The reaction was stopped by CCl3CO2H (17% w/v), and the percentage of scavenging activity of the tested compounds for hydroxyl radicals was given. Trolox was used as a reference compound.activity . The hyd+·-Decolorization assay for antioxidant activityABTS [+•), ABTS stock solution in water (7 mM) was mixed with potassium persulfate (2.45 mM) and left in the dark at room temperature for 12–16 h before use. The results are recorded after 1 min of the mixing solutions at 734 nm. The results were compared to the appropriate standard inhibitor Trolox.activity . In orde1–5  Among the nitrones studied, homo-bis-nitrone BPHBN5, bearing two N-tert-Bu radicals at the nitrone motif, has the best neuroprotective, anti-necrotic, anti-apoptotic, and antioxidant activities, followed by mononitrone BPMN3, with one N-Bn radical, and BPMN2, with only one N-tert-Bu substituent; and (3) BPNs bearing N-tert-Bu and N-Bn groups show better neuroprotective and antioxidant properties than those substituted with Me.To sum up, we conclude the following: (1) Taken together, our results indicate that


Penicillium sp. SY2107 was isolated from a sample of Mariana Trench sediment collected at a depth of 11000 m and an extract prepared from the culture of this fungus in rice medium showed antimicrobial activities. Chemical investigation on this active extract led to the isolation of 16 compounds, including one novel meroterpenoid, named andrastone C. Structure of the new compound was elucidated based on high-resolution electrospray ionization mass spectroscopy (HRESIMS) data, extensive nuclear magnetic resonance (NMR) spectroscopic analyses and a single crystal X-ray diffraction. The crystal structure of a known meroterpenoid andrastone B was also reported in this study. Both andrastones B and C exhibited antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans with minimum inhibitory concentration (MIC) values in a range from 6 to 13 μg/mL.Mariana Trench sediments are enriched in microorganisms, however, the structures and bioactivities of their secondary metabolites are not very known. In this study, a fungus  Marine natural products are important sources for the discovery of novel bioactive agents and drug leads ,2,3,4,5.Salinospora actinomycetes are found in tropical and subtropical marine sediments at the depth of up to 1100 m –  and 501.2482 [M + H]+ . The infrared radiation (IR) spectrum showed characteristic bands for hydroxy (νmax 3526 cm−1) and carbonyl  functional groups. The downfield 13C NMR spectrum showed eight signals for four carbonyls  and two pairs of double bonds  (1 had five rings. In the correlation spectroscopy (COSY) spectrum, three spin systems of H2-2  with H2-1  and H-3 , H-6  with H-5  and H-7 , and H-9  with H-11  were observed (δH 0.79) with C-2 (δC 22.4), C-10 (δC 51.5), and C-21 (δC 207.7); H-3 with C-1 (δC 28.5), C-5 (δC 45.8), and C-26 (δC 169.6); H-5 with C-4 (δC 36.8), C-6 (δC 66.0), C-10, C-19 (δC 23.0), and C-21; H-6 with C-7 (δC 91.9), C-8 (δC 45.6), and C-10; H-7 with C-5, C-6, C-8, and C-9 (δC 50.1); H-9 with C-11 (δC 120.5) and C-12 (δC 133.9); H-11 with C-10, C-13 (δC 52.9), and C-22 (δC 20.2); H3-18  with C-3 (δC 77.4), C-4, C-5, and C-19; H3-19  with C-3, C-4, C-5, and C-18 (δC 25.3); H3-20  with C-7, C-8, C-9, and C-17 (δC 68.5); H3-21  with C-1, C-10, and C-20 (δC 15.5); H3-22  with C-11, C-12, and C-13; H3-23  with C-12, C-13, and C-17; and H3-27  with C-26. These COSY and HMBC correlations established the partial structure of rings A–C (a 6/6/6 tricyclic fusion) with an acetyl group at C-3, two oxymethines at C-6 and C-7, an aldehyde group at C-21, and five methyls at C-18, C-19, C-20, C-22, and C-23, respectively. In addition, HMBC correlations of H3-23 with C-12, C-13, C-14 (δC 201.1), and C-17, H3-24  with C-14, C-15 (δC 111.6), C-16 (δC 185.3), and C-25 (δC 168.3), and H3-28  with C-25 were also observed, indicating the C/D/E ring juncture with a keto at C-14, a methyl at C-24, and a methoxy at C-28. Although no HMBC correlation of H-7 with C-16 was observed, the downfield chemical shifts at δC 91.9 for C-7 and δC 185.3 for C-16 suggested a five-membered ether ring for E, which was confirmed by the crystal structure  . These Nobserved . Heteronobserved : H-1 (δHtructure  of 1 obt1 was assigned by nuclear Overhauser effect spectroscopy (NOESY) experiment. NOE correlations (δH 0.79) with H-5 and H-9 and H-5 with H-6, H-9, and H3-18 indicated a β-orientation for these protons, while NOE correlations of H-7 with H3-19 and H3-20, H3-21 with H3-19 and H3-20, and H3-28 with H3-20 and H3-23 were suggestive of a α-orientation for these protons. The relative configuration was confirmed and the absolute configuration of 1 was determined as 3S,5R,6R,7R,8S,9R,10R,13R,17R by a single crystal X-ray diffraction analysis  , named andrastone C. Its 13C and 1H NMR data (The relative configuration of elations  of H-1β 0.00(6)] . Based oNMR data  were ass1–16 were tested for their antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans by the micro-broth dilution method D20 + 60° ; UV (MeOH) λmax (log ε) 256 (3.58) nm; IR (MeOH)νmax 3526, 1736, 1716, 1698, 1660, 1030 cm−1; 13C NMR data , 1H NMR data , m/z 499.2340 [M − H]− , 501.2482 [M + H]+ , and 523.2308 [M + Na]+ .Andrastone C (1): C28H36O8 (M = 500.57 g/mol): monoclinic, space group P21 (no. 4), a = 8.58899(11) Å, b = 8.86131(11) Å, c = 16.7937(2) Å, β = 96.8005(12)°, V = 1269.17(3) Å3, Z = 2, T = 293(2) K, μ(Cu Kα) = 0.784 mm−1, Dcalc = 1.310 g/cm3, 7640 reflections measured (5.3° ≤ 2Θ ≤ 147.088°), 4316 unique  which were used in all calculations. The final R1 was 0.0311 (I > 2σ(I)) and wR2 was 0.0866 . The crystal data and structure refinement parameters of 1 were also reported in 1) has been deposited at the Cambridge Crystallographic Data Centre (CCDC Number: 1979103). Copies of the data can be obtained free of charge from Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K. D20 −88° ; 13C NMR data , 1H NMR data , m/z 515.2286 [M − H]−  and 539.2253 [M + Na]+ .Andrastone B (2): C28H38O10 (M = 534.58 g/mol): orthorhombic, space group P212121 (no. 19), a = 9.1880(3) Å, b = 13.2582(5) Å, c = 22.8448(6) Å, V = 2782.87(16) Å3, Z = 4, T = 293(2) K, μ(Cu Kα) = 0.802 mm−1, Dcalc = 1.276 g/cm3, 13131 reflections measured (7.71° ≤ 2Θ ≤ 147.056°), 5414 unique  which were used in all calculations. The final R1 was 0.0438 (I > 2σ(I)) and wR2 was 0.1180 . The crystal data and structure refinement parameters of 3 were also reported in 2) has been deposited at the Cambridge Crystallographic Data Centre (CCDC Number: 1976943). Copies of the data can be obtained free of charge from Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K. [fax (+44)1223-336-033; or e-mail: data_request@ccdc.cam.ac.uk).Crystal data of andrastone B (Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans were tested by the micro-broth dilution method as describe in the previous study [8 cfu/mL of culture was added and the plates were incubated at 37 °C for 12 h overnight. Minimum inhibitory concentration (MIC) that inhibited the growth of microorganisms and minimum bactericidal concentration (MBC) that completely killed microorganisms were recorded. Finally, based on results obtained, specific concentrations of each compound were prepared to get more accurate values of MIC and MBC.The antimicrobial activities of all isolated compounds against methicillin-resistant us study  with lit2 humidified incubator at 37 °C and the cultured cells after the third generation were used for the experiments.The Sulforhodamine B (SRB) assay  was applPenicillium sp. SY2107 in rice medium. Andrastone C and most of the known compounds showed antimicrobial activities against MRSA, E. coli, and C. albicans. (Z)-N-(4-hydroxystyryl) formamide exhibited antiglioma activity. Data from this study enriched the chemical and bioactive diversities of the secondary metabolites from the Mariana Trench-sourced microorganisms.One novel meroterpenoid, named andrastone C, and fifteen known compounds with diverse structural classes, were discovered and characterized from the culture of a Mariana Trench sediment-associated fungus


H-chromen-4-yl)-N-substituted acetamides IIIa–h was planned as a step in the development of new modified nonsteroidal antiestrogens. The purity of target compounds IIIa–h was checked by thin-layer chromatography (TLC), and their structures were confirmed using various spectroscopic tools including IR, 1H-NMR, 13C-NMR, and MS spectroscopy. Viability tests were applied using 3--2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic effect of the synthesized compounds against two breast cancer cell lines, MCF-7 and MDA-MB-231. Compound IIIb proved the most active against MCF-7 cells, with an IC50 value of 0.32 μM. The results of an analysis of in vitro antiestrogenic activity indicated that only compound IIIb exhibited antiestrogenic activity; its IC50 value of 29.49 μM was about twice as potent as that of the reference compound, MIBP. The aromatase activity was evaluated for the synthesized target compounds IIIa–g and the intermediates Ib and IIa. A significant aromatase inhibition was observed for the intermediate Ib and compound IIIe, with IC50 values of 14.5 and 17.4 μM, respectively. Compound IIIb, namely 7-methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one, could be used as an antiestrogen and/or cytotoxic agent with selective activity against tumor cells.The preparation of certain 2-(2-oxo-2 Breast cancer is one of the most common and devastating cancers in women worldwide. According to researchers in the United States, the number of new cases of female breast cancer is 124.9 per 100,000 women per year ,4. The dCoumarouna odorata Aube, from which it was isolated for the first time in 1820  carbodiimide HCl (EDCI.HCl) reagent, which was added to a mixture of the appropriate aromatic amine and the acids IIa,b in dimethyformamide (DMF).The direct reaction of a carboxylic acid with an amine proceeds smoothly with aliphatic secondary amines like piperidine producing the target compounds IIIa–h were confirmed by their IR spectra, in which the appearance of C–N bands at 1026 cm−1 and 1024 cm−1 indicates compounds IIIa and IIIb, respectively. The disappearance of broad –OH bands suggested the formation of a new amide bond for compounds IIIc–h. The 1H-NMR spectra of compounds IIIa,b did not show –NH amide protons as they are tertiary amides. In addition, the 1H-NMR spectra of compounds IIIc–h were characterized by the appearance of –NH peaks that resonate at 10.08–10.53 ppm as singlets. Moreover, the 13C-NMR spectra of compounds IIIc–h were characterized by the presence of six new aromatic carbons in the range of 108.2–147.9 ppm. The 13C-NMR and DEPT spectra of the compound IIIa were well resolved and confirmed the presence of all carbon atoms in the molecule. On other hand, the HSQC spectrum of the compound IIIa specified its proton–carbon coupling.The structures of compounds Ia–b and IIa–b acetate (Ib) was highly cytotoxic towards both breast cancer cell lines.The biological investigation started with the evaluation of the cytotoxic activity of the intermediates nd IIa–b . CompounIIIa–h was evaluated in vitro against the human breast adenocarcinoma MCF7 (ER+ breast cancer cell line) and the MDA-MB-231 , using camptothecin as a pyranone-bearing reference standard. Treatment of the ER+ MCF-7 and MDA-MB-231 cell lines with 0.39, 1.56, 6.25, 25, and 100 μM concentrations of the synthesized target compounds IIIa–h resulted in a significant cytotoxic effect. The most active compounds against the MCF-7 breast cancer cell line were subjected to in vitro antiestrogenic activity and in vitro aromatase inhibition activity.The cytotoxic activity of the synthesized compounds N-substituted coumarin-4-acetamides IIIa–h . In addition, compound IIIe is the most active candidate against the MDA-MB-231 cell line with an IC50 value of 2.14 μM, while camptothecin displayed an IC50 value of 19.24 μM against the same cell line.Considering the cytotoxicity of s IIIa–h , compounIIIb and IIIf–h showed a greater in vitro cytotoxic effect against MCF-7 cells as compared with their positional isomers 6-methoxycoumarin derivatives IIIa and IIIc–e. The most active compounds are in the following order: IIIb > IIIg > IIIa > IIId, with IC50 values of 0.32, 0.72, 1.82, and 2.80 μM, respectively. On the other hand, the results of the cytotoxic evaluation of the acetamides IIIa–h against MDA-MB-231 human breast cancer cells indicated that compounds IIIc, e, and f had a highly cytotoxic effect, with IC50 values of 4.6, 2.1, and 6.9 μM, respectively  aromatase inhibitory activity with an IC50 value of 14.5 μM , letrozole, as a reference standard. Compound  14.5 μM .IIIa–g showed low or no aromatase inhibition activity , alongside its promising activity towards the MDA-MB-231 human breast cancer cell line .Moreover, acetamides activity , except −1) on an IR affinity-1s Fourier transform infrared spectrophotometer. The 1H-NMR and 13C-NMR spectra were determined on a Bruker (700 MHz)  and Agilent Technologies (600 MHz)  spectrometer. Correlation spectroscopy: 1H, 13C, HSQC, and DEPT spectra were recorded on a Bruker (700 MHz) spectrometer. Chemical shifts are expressed as δ values (ppm), using tetramethylsilane (TMS) as an internal reference. Signals are indicated by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Mass spectra (MS) were obtained on a GCMS QP 2010 Ultra/SE (Ver. 4.20), GCMS-TQ Series (Ver. 4.20) Shimadzu apparatus . Elemental analyses were carried out at Research Center, College of Pharmacy, King Saud University, Saudi Arabia, and the results agreed favorably with the proposed structures within ± 0.4% of the theoretical values. Follow-up of the reaction and checking on the homogeneity of the compounds were made by performing thin-layer chromatography (TLC) on pre-coated (0.25 mm) (GF 254) silica gel plates. Routinely used developing solvents were volume to volume: C6H14:EtOAc (6:4); C6H14:EtOAc (5:5); MeOH:CHCl3 (1:9). Visualization of the spots was performed by exposure to a UV lamp at 254 nm or to iodine vapors. All statistical analyses were carried out using GraphPad Prism  version 6.0 software. Statistical analysis was conducted using one-way ANOVA, followed by multiple Tukey–Kramer post hoc tests at p < 0.05, which was considered a marker of statistical significance.Melting points were determined in open glass capillaries on an electrothermal melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded for potassium bromide discs ν  were added, each in three equal portions, in a cold environment (temperature not exceeding 10 °C). The resulting reaction mixture was stored at 0 °C overnight, then poured into ice-cold water (40 mL). The white precipitate that formed was filtered off and re-crystallized from methanol to give the respective methyl 2-(2-oxo-2H-chromen-4-yl)acetate derivatives Ia,b + 248 (3.82).Yield (route A): 0.5 g (20%); White glittery crystals m.p.: 153–154 °C; IR (KBr): ν (cmH-chromen-4-yl)acetate (Ib):Methyl (7-methoxy-2-oxo-2−1):1724 , 1734 , 2966 , 3093 ; 1H-NMR (DMSO-6d): δ ppm (600 MHz) 3.62 , 3.84 , 3.97 , 6.30 , 6.94 , 7.00  and 7.87 ; 13C-NMR (DMSO-6d): δ ppm (150 MHz) 37.1 (CH2CO), 52.7 (ester CH3), 56.4 (OCH3), 101.4 (C-8), 112.8 (C-6), 113.5 (C-3), 114.8 (C-4a), 127.0 (C-5), 149.9 (C-8a), 155.4 (C-4), 160.5 (C-7), 162.9 (C-2), 170.1 (C=O); MS m/z (% relative abundance): [M]+ 248 (0.33).Yield by route A: 1.2 g (48%) and by route B: 0.7 g (70%); White crystals m.p.: 116–118 °C; IR (KBr): ν (cm2SO4 (5.6 mL) was stirred at room temperature for 60 min, then slowly heated (rate of heating governed by foaming) to 70 °C. After 35 min at this temperature, with stirring throughout, the evolution of carbon monoxide had slackened, and the clear solution was rapidly cooled to 0 °C. Then the appropriate methoxyphenol  and concentrated H2SO4 (2.24 mL) were added, each in three equal portions, to the stirred solution at such a rate that the internal temperature did not exceed 10 °C. The resulting reaction mixture was stored at 0 °C for 16 h, poured into ice cold water (40 mL), and the resulting crystalline precipitate filtered off and washed thoroughly with H2O. The collected solid was dissolved under stirring in 1N Na2CO3 solution (20 mL), heated for 15 min at 65 °C, and the insoluble matter was filtered off and washed with water (2 × 10 mL). The combined filtrate and washings were acidified with concentrated HCl to give the respective coumarin-4-acetic acid derivatives IIa,b + 234 (0.58), [M + 1] 235 (12.06).Yield by route A: 0.23 g (6.1%) and by route B: 0.84 g (89%); light brown fluffy powder m.p.: 178–179 °C; IR (KBr): ν (cmH-chromen-4-yl)acetic acid (IIb):7-Methoxy-2-oxo-2−1): 1664 , 1718 , 2926 , 3439 ; 1H-NMR (DMSO-6d): δ ppm (600 MHz) 3.82 , 6.27 , 6.93 , 6.97 , 7.58 , 12.81 ; 13C-NMR (DMSO-6d): δ ppm (150 MHz) 37.7 (CH2CO), 56.3 (OCH3), 101.3 (C-8), 112.7 (C-6), 112.9 (C-3), 113.3 (C-4a), 127.0 (C-5), 150.6 (C-8a), 155.4 (C-4), 160.5 (C-7), 162.8 (C-2), 171.1 (C=O); MS m/z (% relative abundance): [M]+ 234 (1.89), [M + 1] 235 (4.09).Yield (route A): 3.2 g (85%); White crystals m.p.: 186–187 °C acetate Ia,b  and piperidine  was heated to reflux in toluene (10 mL) in the presence of a catalytic amount of p-toluene sulfonic acid (p-TSOH) for 4 h. The solvent was concentrated under reduced pressure and the precipitated solid was filtered off, washed with toluene, and dried to yield the respective target compounds IIIa,b + 301 (0.48).Yield: 0.21 (70%); Fluffy pale yellow powder m.p.: 167–168 °C; IR (KBr): ν (cmH-chromen-2-one (IIIb):7-Methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2−1): 1024 (C–N), 1718 , 1734 , 2949 , 3095 ; 1H-NMR (DMSO-6d) δ ppm (700 MHz):1.44–1.47 , 1.56–1.57 ,1.61–1.63 , 3.45 , 3.51 , 3.87 , 3.98 , 6.19 , 6.96 , 7.01, 7.57 ; 13C-NMR (DMSO-6d): δ ppm (175 MHz) 24.4 (CH2-piperdine), 25.8 (CH2-piperdine), 26.5 (CH2-piperdine), 31.2 (CH2CO), 42.7 (CH2-piperdine), 46.8 (CH2-piperdine), 56.4 (OCH3), 101.1 (C-8), 112.6 (C-6), 125.9 (C-3), 127.3 (C-4a), 128.5 (C-5), 152.6 (C-8a), 155.3 (C-4), 160.6 (C-7), 162.7 (C-2), 166.7 (C=O); MS m/z (% relative abundance): [M]+ 301 (10.33).Yield: 0.21 g (70%); White crystals m.p.: 135–136 °C; IR (KBr): ν (cmH-chromen-4-yl)acetic acid derivative IIa,b  and the proper arylamine (1.2 mmol) in DMF (15 mL) at 0 °C. After 2 h, the reaction mixture was warmed to room temperature and stirring was continued for an additional 24 h. Thereafter, the reaction mixture was poured into H2O (22 mL). The separated solid was filtered off under suctioning, washed repeatedly with H2O, and dried to give compounds IIIc–h.1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide HCl  was slowly added to a stirred solution containing the appropriate 2-oxo-2H-chromen-4-yl)-N-phenylacetamide (IIIc):2-(6-Methoxy-2-oxo-2−1) 1043 (C–N), 1078–1213 (C–O), 1660 , 1734 , 2960 , 3059 , 3286 ; 1H-NMR (DMSO-6d): δ ppm (700 MHz) 3.80 , 3.99 , 6.54 , 7.08 , 7.26 , 7.32–7.34 , 7.40 , 7.71 , 10.42 ; 13C-NMR (DMSO-6d): δ ppm (175 MHz) 31.3 (CH2CO), 56.3 (OCH3), 108.8 (C-5), 117.2 (C-3), 118.2 (C-7), 119.4 (C-4a), 119.7 (C-2′ & C-6′), 120.1 (C-8), 124.1 (C-4′), 129.3 (C-3′ &C-5′), 139.2 (C-1′), 147.9 (C-8a), 150.8 (C-4), 156.0 (C-6), 160.3 (C-2), 167.0 (C=O); MS m/z (% relative abundance): [M]+ 309 (0.73).Yield: 0.27 g (87%); White powder m.p.: 219–220 °C; IR (KBr): ν (cmN-(4-Bromophenyl)-2-(6-methoxy-2-oxo-2H-chromen-4-yl)acetamide (IIId):−1) 600 (C-Br), 1031 (C–N), 1070–1246 (C–O), 1653 , 1730 , 2978 , 3088 , 3228 ; 1H-NMR (DMSO-6d): δ ppm (700 MHz) 3.82 , 4.05 , 6.53 , 7.16 , 7.26 , 7.39 , 7.51 , 7.56 , 10.53 ; 13C-NMR (DMSO-6d): δ ppm (175 MHz) 31.2 (CH2CO), 56.2 (OCH3), 108.8 (C-5), 115.7 (C-3), 117.3 (C-7), 118.2 (C-2′ &C-6′), 119.4 (C-4a), 119.7 (C-4′), 121.7 (C-8), 132.2 (C-3′ &C-5′), 138.6 (C-1′), 147.9 (C-8a), 149.7 (C-4), 156.0 (C-6), 160.2 (C-2), 169.6 (C=O); MS m/z (% relative abundance): [M]+ 388 (0.48).Yield: 0.19 g (48%); Silver powder m.p.: 202–203 °C; IR (KBr): ν (cmH-chromen-4-yl)acetamide (IIIe):N-(3-Hydroxy-4-methoxyphenyl)-2-(6-methoxy-2-oxo-2−1): 1028 (C–N), 1043–1257 (C–O), 1685 , 1734 , 2935 , 3083 , 3253 , 2446–3527 (OH); 1H-NMR (DMSO-6d): δ ppm (700 MHz) 3.72 , 3.80 , 3.92 , 6.51 , 6.84 , 6.93 , 7.13 , 7.25 , 7.32 , 7.40 , 9.10 , 10.15 ; 13C-NMR (DMSO-6d): δ ppm (175 MHz) 31.2 (CH2CO), 56.3 (OCH3), 108.3 (C-2′), 108.9 (C-5), 110.5 (C-3), 112.9 (C-5′), 117.1 (C-6′), 118.2 (C-7), 119.4 (C-4a), 120.1 (C-8), 132.7 (C-1′), 144.5 (C-3′), 146.9 (C-8a), 147.8 (C-4′), 151.0 (C-4), 156.0 (C-6), 160.3 (C-2), 166.4 (C=O); MS m/z (% relative abundance): [M]+ 355 (0.59), [M + 1] 356 (35.71).Yield: 0.21 g (59%); Brown powder m.p.: 173–174 °C; IR (KBr): ν (cmH-chromen-4-yl)-N-phenylacetamide (IIIf):2-(7-Methoxy-2-oxo-2−1): 1028 (C–N), 1647 , 1728 , 2978 , 3074 , 3265 ; 1H-NMR (DMSO-6d): δ ppm (700 MHz) 3.87 , 3.95 , 6.34 , 7.00 , 7.03 , 7.07 , 7.32 , 7.58 , 7.75 , 10.35 ; 13C-NMR (DMSO-6d): δ ppm (175 MHz) 39.6 (CH2CO), 56.4 (OCH3), 101.4 (C-8), 112.7 (C-6), 113.1 (C-3), 113.4 (C-4a), 119.7 (C-2′ & C-6′), 124.1 (C-5), 127.0 (C-4′), 129.3 (C-3′ & C-5′), 139.3 (C-1′), 151.3 (C-8a), 155.4 (C-4), 160.5 (C-7), 162.9 (C-2), 167.0 (C=O); MS m/z (% relative abundance): [M + 1] 310 (33).Yield: 0.2 g (65%); Yellow-brown powder m.p.: 217–218 °C; IR (KBr): ν (cmN-(4-Bromophenyl)-2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide (IIIg):−1): 500 (C-Br), 1066 (C–N), 1653 , 1728 , 2935 , 3111 , 3248 ; 1H-NMR (DMSO-6d): δ ppm (700 MHz) 3.87 , 3.95 , 6.34 , 7.00 , 7.04 , 7.51 , 7.55 , 7.73 , 10.48 ; 13C-NMR (DMSO-6d): δ ppm (175 MHz) 31.2 (CH2CO), 56.4 (OCH3), 101.4 (C-8), 112.7 (C-6), 113.1 (C-3), 113.5 (C-4a), 115.6 (C-2′ & C-6′), 121.6 (C-4′), 127.0 (C-5), 132.1 (C-3′ & C-5′), 139.6 (C-1′), 151.1 (C-8a), 155.4 (C-4), 160.5 (C-7), 162.9 (C-2), 167.3 (C=O); MS m/z (% relative abundance): [M]+ 388 (1.39), [M + 1] 389 (2.09), [M+2] 390 (1.29).Yield: 0.26 g (51%); Beige powder m.p.: 212–213 °C; IR (KBr): ν (cmN-(3-Hydroxy-4-methoxyphenyl)-2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide (IIIh):−1): 1028 (C–N), 1653 , 1707 , 2985 , 3100 , 3228 , 3327 (br OH); 1H-NMR (DMSO-6d): δ ppm (700 MHz) 3.72 , 3.88 , 6.31 , 6.84 , 6.94 , 7.00 , 7.02 , 7.12 , 7.74 , 9.09 , 10.08 ; 13C-NMR (DMSO-6d): δ ppm (175 MHz) 31.2 (CH2CO), 56.3 (2 × OCH3), 101.4 (C-8), 108.2 (C-2′), 110.4 (C-6), 112.7 (C-3), 112.9 (C-5′), 113.1 (C-4a), 113.3 (C-6′), 127.0 (C-5), 132.8 (C-1′), 144.5 (C-3′), 146.9 (C-4′), 151.5 (C-8a), 155.4 (C-4), 160.6 (C-7), 162.9 (C-2), 166.4 (C=O); MS m/z (% relative abundance): [M + 2] 357 (27.25).Yield: 0.26 g (73%); Brown powder m.p.: 188–189 °C; IR (KBr): ν  and MDA-MB-231  was determined using camptothecin as a pyranone-bearing reference standard [The cytotoxicity of compounds standard . The detThe antiestrogenic activity of the test compounds was examined by performing a 3--2,5-diphenyl tetrazolium bromide (MTT) assay of the MCF-7 cell line. In this experiment, MCF-7 cells were treated with 17β-estradiol (+ve cell proliferation compound). The effect of various concentrations of the tested compounds on cell proliferation in the presence of 17β-estradiol was measured . The detSandwich enzyme immunoassay was adopted for aromatase inhibition assessment . The detH-chromen-4-yl)-N-substituted acetamide derivatives IIIa–h have been prepared, characterized, and tested for their in vitro cytotoxic and antiestrogenic, as well as aromatase inhibition, activities. The target compounds IIIa–h showed variable cytotoxic activity against two breast cancer cell lines, MCF-7 and MDA-MB-231. 7-methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one (IIIb) was the most potent cytotoxic compound against MCF-7, being about 14-fold more potent than the reference standard, camptothecin. It also manifested high in vitro antiestrogenic activity (IC50 = 29.49 μM). These findings indicate that a cyclic aliphatic lipophilic substitution in compound IIIb produced obvious antiestrogenic and cytotoxic activities. Thus, it might have high affinity to ER. Unfortunately, the tested compounds show moderate to low aromatase inhibition activity, except for compound IIIe, which showed moderate inhibitory activity against aromatase with an IC50 value of 17.38 μM.In conclusion, 2-(2-oxo-2


Melicope latifolia have previously been reported to have anti-HCV activity and are a potential source of bioactive compounds for future novel drug development. This study aimed to evaluate the efficacy of the extract of M. latifolia fruit to treat HCV and to isolate its active compounds.New agents for developing alternative or complementary medicine to treat the hepatitis C virus (HCV) are still needed due to high rates of HCV infection globally and the current limitations of available treatments. Treatment of HCV with a combination of direct acting antivirals have been shown to be approximately 90% effective but will be limited in the future due to the emergence of drug resistance and high cost. The leaves of M. latifolia fruit was extracted using methanol and purified using vacuum liquid chromatography (VLC) and Radial Chromatography. The anti-HCV activity was analyzed using cell culture lines Huh7it-1 and JFH1 (genotype 2a). Time-of-addition and immunoblotting studies were performed to identify the mode of action of the isolated active compounds. The structures of the active compounds were determined using nuclear magnetic resonance (NMR) spectra, UV, IR, and Mass Spectra.M. latifolia fruit: O-methyloktadrenolon, alloevodionol, isopimpinellin, alloxanthoxyletin, methylevodionol, and N-methylflindersine. N-methylflidersine was the most active compound with IC50 value of 3.8 μg/ml while methylevodionol, isopimpinellin, and alloevodionol were less active. O-methyloktadrenolon and alloxanthoxyletin were moderately active with IC50 values of 10.9 and 21.72 μg/ml, respectively. N-methylflidersine decreased level of HCV NS3 protein expression in the cells.Six known compounds were isolated from M. latifolia possesses anti-HCV activity through post-entry inhibition and suppressed NS3 protein expression.The alkaloid compound, N-methylflindersine which was isolated from  The hepatitis C virus (HCV) is a pathogen that causes liver inflammation that can develop into cirrhosis and or hepatocellular carcinoma. No vaccine is presently available against HCV due to their extreme genetic variability and lack of animal models for vaccine testing , 2. SeveThe current treatment of HCV uses direct acting antivirals (DAAs) which increased sustained virologic response (SVR) > 90% of people in clinical trials. DAAs treatment is divided into four classes: protease inhibitors, non-nucleoside polymerase inhibitors, nucleoside/tide polymerase inhibitor, and NS5A inhibitors –5. HowevRuta angustifolia, exhibited anti-HCV activity by reducing the virus particle production and decreasing HCV NS3 protein level + positive ion mode. The molecular structures of isolated compounds are described in Fig. Six known compounds were isolated from the max nm (log ε): 217 (2.95); 223 (2.80); 278 (3.12); 289 (3.05) and 355 (2.14). IR spectrum showed a band at νmax (cm− 1): 3055; 2974; 2935; 1697; 1639–1579; and 1184. The mass spectrum of the compound showed a quasi-molecular ion [M + H]+ at m/z 315.1595 corresponding to the molecular formula C19H23O4.The compound showed a UV maximum absorption (MeOH) at λ1H-NMR (CDCl3) δH ppm: 5.51 , 6.48 , 5.53 , 6.59 , 2.50 , 1.42 , 1.41 , 3.75 . 13C-NMR (CDCl3) δC ppm: 201.1 (C-11), 153.7 (C-5), 151.5 (C-6a), 150.6 (C-10b), 127.9 (C-3), 127.8 (C-9), 116.8 (C-10), 116.2 (C-4), 117.7 (C-6), 108.2 (C-4a), 106.6 (C-10a), 77.2 (C-8), 76.9 (C-2), 32.7 (C-12), 28.0 (C-13/14), 27.9 (C-15/16), 63.7 (5-OCH3). The NMR spectra data are consistent with published data + at m/z 259.0979. The molecular formula was determined to be C15H15O4 .The compound showed a UV maximum absorption (MeOH) at λ1H-NMR (CDCl3) δH ppm: 6.16 , 6.61 , 7.96 , 5.5 , 6.35 , 3.87 . 13C-NMR (CDCl3) δC ppm: 161.6 (C-2), 158.3 (C-7), 155.9 (C-9), 150.3 (C-5), 138.7 (C-4), 111.2 (C-3), 106.6 (C-6), 127.6 (C-3′), 116,1 (C-4′), 103.8 (C-10), 91.6 (C-8), 77.8 (C-2′), 28.0 (C-5′/6′), 56.0 (7-OCH3). The NMR spectra data are consistent with published data + at m/z 263.1288. The molecular formula was determined to be C15H19O4 .The compound showed a UV maximum absorption (MeOH) at λ1H-NMR (CDCl3) δH ppm: 5.51 , 6.46 , 6.18 , 2.48 , 1.41 , 3.74 , 3.76 . 13C-NMR (CDCl3) δC ppm: 201.9 (C-11), 157.6 (C-8), 156.0 (C-10), 154.2 (C-6), 127.8 (C-3), 118.3 (C-7), 116.5 (C-4), 108.0 (C-5), 96.1 (C-9), 76.9 (C-2), 32.6 (C-12), 27.9 (C-13/14), 55.8 (6-OCH3), 55.8 (6-OCH3). The NMR spectra data are consistent with published data + at m/z 242.1180. The molecular formula was determined to be C15H16NO2 .The compound showed a UV maximum absorption (MeOH) at λskeleton . IR spec1H-NMR (CDCl3) δH ppm: 5.52 , 6.74 , 7.30 , 7.53 , 7.95 , 7.21 , 1.50 , 3.68 . 13C-NMR (CDCl3) δC ppm: 161.0 (C-5), 155.2 (C-10b), 130.9 (C-8), 139.3 (C-6a), 126.4 (C-3), 123.1 (C-10), 121.7 (C-9), 117.9 (C-4), 116.1 (C-10a), 114.1 (C-7), 105.8 (C-4a), 78.6 (C-2), 29.3 (N-CH3), 28.2 (C-11/12). The NMR spectra data are consistent with published data [50 value of 3.8 ± 2.7 μg/ml, followed by moderate HCV inhibition activity of O-methyloktadrenolon and alloxanthoxyletin with IC50 values of 10.9 ± 1.2 μg/ml and 21.72 μg/ml, respectively. While, alloevodionol possess a moderate inhibition and the two remaining compounds, isopimpinellin, and methylevodionol did not show any inhibitive effect on HCV infection. The cellular viability assay demonstrated that all compounds had a negligible cytotoxicity effect with a CC50 > 90 μg/ml, except for O-methyloktadrenolon that had a CC50 of 63 ± 2.3 μg/ml  and reported to possess an inhibitory activity against the enzyme adenine phosphoribosyltransferase (APRT) from Leishmania spp. [Pilocarpus goudotianus (Rutaceae) [Melicope lunu-akenda was found to contain ecodione (38.9%), (E)-β-ocimene (12.4%), isolycodolin (11.7%) and alloevadionol (10.6%) as major constituents. This oil exhibited antibacterial activity against Gram negative and Gram positive bacteria, in particular against Salmonella typhi and Klebsiella pneumonia, which are both human pathogens [Micromelum falcatum and showed strong toxicity towards brine shrimp larvae with a LD50 value of 1.39 μg/ml [Zanthoxylum integrifoliolum, another species belonging to the Rutaceae family. N-methylflindersine has been found to inhibit N-formylmethionylleucylphenylalanine-induced superoxide production with an IC50 < 12 μM [Melicope denhamii. In the same study, they showed moderate activity against murine leukemia P-388 cells with an IC50 value of 21.06 μg/ml and 11.98 μg/ml respectively [50 value of 3.8 ± 2.7 μg/ml, followed by O-methyloktadrenolon with an IC50 value of 10.9 ± 1.2 μg/ml. Alloevodionol and alloxanthoxyletin demonstrated a moderate activity with IC50 values of 41.1 ± 3.1 and 21.72 ± 2.2 μg/ml, respectively. On the other hand, methylevodionol and isopimpinellin, did not reveal anti-HCV activities at the highest concentration examined, 50 μg/ml  . An oil athogens . A quino39 μg/ml . It was  < 12 μM . N-methyM. latifolia fruit mediated a strong anti-HCV activity through post-entry inhibition and HCV NS3 protein reduction. This result suggests that M. latifolia is a potential candidate for developing an anti-HCV agent.The alkaloid compound, N-methylflindersine which was isolated from the


Their antitumoral potential was evaluated in two triple negative breast cancer (TNBC) cell lines—MDA-MB-231 and MDA-MB-468, by sulforhodamine B assay. Their effects on the non-tumorigenic MCF-12A cells were also evaluated. The results demonstrated that three compounds caused growth inhibition in both TNBC cell lines, with little or no effect against the non-tumorigenic cells. The most promising compound was further studied concerning possible effects on cell viability (by trypan blue exclusion assay), cell proliferation (by bromodeoxyuridine assay) and cell cycle profile (by flow cytometry). The results demonstrated that the GI50 concentration of compound 2e (13 μM) caused a decreased in MDA-MB-231 cell number, which was correlated with a decreased in the % of proliferating cells. Moreover, this compound increased G0/G1 phase and decreased S phases, when compared to control cells . Interestingly, compound 2e also reduced tumor size using an in ovo CAM  model. This work highlights the potential antitumor effect of a novel methyl 3-arylthienopyridine-2-carboxylate derivative.A series of novel functionalized methyl 3-(hetero)arylthieno[3,2- Regardless the great number of research studies worldwide to improve our understanding and effort to develop new therapies, breast cancer is still the most frequent diagnosed cancer and the main cause of cancer death in women . Nowadayb]pyridines and their potential as inhibitors of the vascular endothelial growth factor receptor 2 (VEGFR-2), which is a receptor of tyrosine kinase that triggers signalization pathways that cause endothelial cell proliferation and tumor vascularization (angiogenesis) [b]pyridine-6-carbonitriles, which act by inhibiting the non-receptor tyrosine kinase Src, a kinase implicated in cancer, osteoporosis and ischemic diseases when is overexpressed or overactivated [b]pyridine phenylacetylthioureas inhibit VEGFR-2 and c-Met (receptor for hepatocyte growth factor) both implicated in angiogenesis acting synergistically pyridines, either on the pyridine ring by C-C Suzuki-Miyaura pyridin-7-ylthio)phenyl]ureas with hydrophobic groups m- or p-F, m-CH3, m-CF3, and m-CF3/p-Cl (substitution pattern of Sorafenib) relative to the urea moiety in the terminal aryl ring, as antiangiogenic compounds using several assays in Human Umbilical Endothelial Cells (HUVEC) pyridine derivatives have already demonstrated their potential as anticancer and/or antiangiogenic agents. In summary, some thienopyridine-2-carboxylates by C-C Suzuki Miyaura cross-coupling is presented. Their antitumor potential was further evaluated in two different TNBC cell lines. Three of the thienopyridine derivatives presented antitumor activity against the TNBC MDA-MB-231 and MDA-MD-468 cells, without showing relevant toxicity against a non-tumorigenic mammary epithelial cell line (MCF-12A). Moreover, compound 2e significantly decreased the number of viable cells and cellular proliferation of MDA-MB-231 cells, as well as, increased G0/G1 phase and decreased S phase . Importantly, compound 2e also decreased the size of in ovo grafts of chick chorioallantoic membrane (CAM) with MDA-MD-231 cells. Thus, this work points to the antitumor potential of a series of novel thienopyridine derivatives, highlighting one compound as a promising molecule against TNBC.Herein the synthesis of novel methyl 3-(hetero)arylthienopyridine-2-carboxylate 1 was prepared according to a known procedure, from the corresponding 3-amino compound pyridine-2-carboxylates 2a–2h in moderate to high yields (35–84%) after column chromatography  and electron withdrawing groups—EWGs— in the para-position of the phenyl ring relative to the C-C bond formed or with an electron-deficient ring (pyridine) or an electron-rich one (furan).The precursor methyl 3-bromothieno+ (100%). Elemental Anal. calcd for C15H11NO2S: C 66.89%, H 4.12%, N 5.20%, S 11.91%. Found: C 67.08%, H 4.09%, N 5.46%, S 12.16%.From compound 1  and phenylboronic acid pinacol ester  following the general procedure for 3 h; purification by column chromatography using 50% ether/petroleum ether, gave compound 2a as a white solid , m.p. 152–154 °C. 1  and potassium p-tolyltrifluoroborate  and heating for 4.5 h, after purification by column chromatography using 40% ether/petroleum ether, compound 2b was obtained as a white solid , m.p. 153–154 °C. 1H-NMR  δ = 2.44 , 3.85 , 7.33 , 7.39 , 7.42 , 8.24 , 8.80  ppm. 13C-NMR  δ = 21.5 (Me), 52.4 (OMe), 121.1 (6-CH), 128.7 (3′ and 5′-CH), 130.1 (2′ and 6′-CH), 130.12 (C), 130.7 (7-CH), 131.2 (C), 134.9 (C), 138.2 (C), 144.0 (C), 148.5 (5-CH), 154.6 (C), 162.7 (C=O) ppm. MS (ESI) m/z (%): 284.07 [M + H]+ (83%). Elemental Anal. calcd for C16H13NO2S: C 67.82%, H 4.62%, N 4.94%, S 11.32%. Found: C 68.15, H 4.35, N 5.20, S 11.69%.From compound 1  and potassium 4-methoxyphenyltrifluoroborate , reacting for 4 h, after purification by column chromatography using 25% ether/petroleum ether, compound 2c was obtained as a white solid , m.p. 190–191 °C. 1H-NMR  δ = 3.85 , 3.89 , 7.05 , 7.39 , 7.50 , 8.23 , 8.80  ppm. 13C-NMR  δ = 52.5 (OMe), 55.2 (OMe), 113.4 (3′ and 5′-CH), 121.1 (6-CH), 125.2 (C), 130.7 (7-CH), 130.9 (C), 131.6 (2′ and 6′-CH), 134.9 (C), 143.6 (C), 148.5 (5-CH), 154.2 (C), 159.7 (4′-C), 162.9 (C=O) ppm. MS (ESI) m/z (%): 300.06 [M + H]+ (100%). Elemental Anal. calcd for C16H13NO3S: C 64.20%, H 4.38%, N 4.68%, S 10.71%. Found: C 64.20%, H 4.45%, N 4.84%, S 10.33%.From compound 1  and potassium 4-(trifluoromethyl)phenyltrifluoroborate , reacting for 4 h, after purification by column chromatography using 40% ether/petroleum ether, compound 2d was obtained as a white solid. This was recrystallized from ether to give a colorless crystals , m.p. 170–171 °C. 1H-NMR  δ = 3.85 , 7.43 , 7.66 , 7.78 , 8.28 , 8.80  ppm. 13C-NMR  δ = 52.7 (OMe), 120.6 (6-CH), 124.8 , 124.9 , 130.3 , 130.8 (2′ and 6′-CH), 131.0 (7-CH), 132.3 (C), 135.0 (C), 137.0 (C), 142.1 (C), 148.8 (5-CH), 153.3 (C), 162.4 (C=O) ppm. 19F-NMR : δ = −62.63  ppm. MS (ESI) m/z (%): 338.05 [M + H]+ (100%). Elemental Anal. calcd for C16H10F3NO2S: C 56.97%, H 2.99%, N 4.15%, S 9.51%. Found: C 56.62%, H 2.65%, N 4.35%, S 9.81 %.From compound 1  and potassium (4-chlorophenyl)trifluoroborate  reacting for 3 h, purification by column chromatography using 50% ether/petroleum ether, compound 2e was obtined as a white solid , m.p. 195–196 °C. 1H-NMR  δ = 3.85 , 7.43 , 7.49 , 8.27 , 8.81  ppm. 13C-NMR  δ = 52.6 (OMe), 121.3 (6-CH), 128.3 (2 × CH), 131.3 (7-CH), 131.7 (2 × CH), 132.2 (C), 134.7 (C), 135.1 (C), 142.1 (C), 148.4 (5-CH), 153.4 (C), 157.3 (C), 162.5 (C=O) ppm. HRMS (EI) Calcd. for C15H1035ClNO2S [M + 35Cl] 303.0121; found: 303.0104. Calcd. for C15H1037ClNO2S [M+37Cl] 305.0091; found: 305.0099.From compound 1  and potassium (4-cyanophenyl)trifluoroborate  reacting for 3 h, gave a solid which was submitted to dry flash till 90% ether/petroleum ether to give compound 2f as a white solid , m.p. 198-200 °C. 1H-NMR  δ = 3.86 , 7.44 , 7.65 , 7.80 , 8.27 , 8.78  ppm. 13C-NMR  δ = 52.7 (OMe), 112.2 (C), 118.9 (C), 121.5 (6-CH), 131.0 (7-CH), 131.3 (2 × CH), 131.6 (2 × CH), 132.6 (C), 135.0 (C), 138.1 (C), 141.4 (C), 148.8 (5-CH), 153.4 (C), 162.2 (C=O) ppm. HRMS (EI) M+ Calcd. for C16H10N2O2S: 294.0463; found: 294.0474.From compound 1  and 4-pyridine boronic acid , of PdCl2(dppf).CH2Cl2 4 mol% and heating for 4 h, a solid was obtained and submitted to dry flash chromatography till 80% ether/petroleum ether to give compound 2g as a white solid , m.p. 192-193 °C. 1H-NMR  δ = 3.90 , 7.50 , 7.90 , 8.32 , 8.79–8.82  ppm. 13C-NMR  δ = 53.1 (OMe), 122.0 (6-CH), 127.8 (3′ and 5′-CH), 131.1 (7-CH), 134.4 (C), 135.1 (C), 137.7 (C), 139.3 (C), 143.0 (2′ and 6′-CH), 149.3 (5-CH), 152.6 (C), 161.7 (C=O) ppm. MS (ESI) m/z (%): 271.04 [M + H]+ (100%). Elemental Anal. calcd for C14H10N2O2S: C 62.21%, H 3.73%, N 10.36%, S 11.86%. Found: C 61.90%, H 3.69%, N 10.06%, S 11.79%.From compound 1 , potassium 3-furanylboronic acid , PdCl2(dppf).CH2Cl2 4%mol and heating for 4.5 h, a solid was obtained and submitted to dry flash chromatography till 30 % ether/petroleum ether to give compound 2h as a white solid , m.p. 130–132 °C. 1H-NMR  δ = 3.94 , 7.02–7.03 , 7.40 , 7.56–7.57 , 8.21 , 8.25–8.26 , 8.81  ppm. 13C-NMR  δ = 52.6 (OMe), 112.3 (CH), 116.7 (C), 121.2 (6-CH), 130.0 (C), 130.6 (7-CH), 134.5 (C), 134.6 (C), 141.8 (CH), 144.3 (CH), 148.1 (5-CH), 153.7 (C), 162.8 (C=O) ppm. MS (ESI) m/z (%): 260.04 [M + H]+ (100%). Elemental Anal. calcd for C13H9NO3S: C 60.22%, H, 3.50%, N 5.40%, S 12.37%. Found: C 60.27%, H 3.33%, N 5.69%, S 12.48%.From compound Fetal bovine serum (FBS) and phosphate buffered saline (PBS) were purchased from Biowest and Gibco, respectively. Acetic acid, dimethyl sulfoxide (DMSO), ethylene diamine tetraacetic acid (EDTA), sulforhodamine B (SRB), trypan blue reagent, doxorubicin, trichloroacetic acid (TCA), Tris base and bromodeoxyuridine (BrdU) were purchased from Sigma-Aldrich. Stock solutions of the synthesized compounds were prepared with the solvent DMSO as vehicle, at stock concentration of 60 mM and kept at −20 °C. The other working stock solutions of 40 mM, 20 mM and 5 mM were also prepared in DMSO and kept at −20 °C.2a–2h were freshly prepared from the previous stock solutions in DMSO for the cell-based assays. The effect of the vehicle solvent (DMSO) on the growth of the cell lines was evaluated by exposing untreated control cells to the maximum concentration of DMSO used in each compound for each assay. Appropriate dilutions of compounds 2. Regularly, all cells were observed using an inverted light microscope (Leica DMi1). All the experiments were carried out with cells at the exponential phase of growth and with more than 90% viability. Two human triple negative breast cancer (TNBC) cell lines were used in this work: MDA-MD-231 and MDA-MB-468. Both TNBC cell lines were cultured in RPMI 1640 medium  with 25 mM of glutamine and 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (from Biowest). These media were supplemented with 5% FBS for the cell growth inhibition assay (SRB) or with 10% FBS for the remaining experiments. The non-tumorigenic cell line MCF-12A was cultured in RPMI 1640 medium supplemented with hEGF , hydrocortisone (500 ng/μL), 5% FBS and 1% Penicillin-Streptomycin (Gibco), as previously described . All theNeubauer Chamber) using the trypan blue reagent, which distinguishes alive (bright) from dead cells or non-viable cells (blue ones).The cell number and viability were assessed by counting the cells with a hemocytometer (4 cells per mL for MDA-MD-231 and MDA-MB468 cells) by adding 100 μL of cells per well and then incubated at 37 °C for 24 h. All experiments were performed in two 96 well-plates, a T0 plate to be analyzed at 0 h (time of treatment with the compounds) and a T48 plate to be analyzed 48 h later. Then, cells were treated with five serial dilutions of each compound (ranging from 150 µM to 9.4 µM) by adding 100 μL of compound per well, and incubated at 37 °C for further 48 h. The effect of the vehicle (DMSO) on cell growth was also evaluated (control) by exposing untreated cells to the maximum concentration of DMSO . The maximum concentration tested for each compound was 150 mM. However, for some compounds, precipitation was detected within the tested range of concentrations. In those cases, the GI50 concentration was not possible to determine and was indicated as “higher than” the highest concentration tested without causing precipitation. Doxorubicin (ranging from 100 nM to 6.25 nM) was used as positive control. Following 48 h incubation of cells with the compounds in the T48 plate (or 0 h in the T0 plate), cells were fixed by adding 10% (w/v) of cold TCA  during 1 h at 4 °C. Subsequently, after washing with water, air-dried cells were stained with 0.4% of SRB  for 30 min in the dark at room temperature (RT). At the end, cells were washed with 1% (v/v) of acetic acid and the bound dye was solubilized with 10 mM Tris base solution. The absorbance was measured at 510 nm in a microplate reader (BioTek’s SynergyTM Mx) using the Gen5 software (BioTek) pyridine-2-carboxylates 2a–2h were prepared in moderate to high yields by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothienopyridine-2-carboxylate with different boronated compounds. These were tested against TNBC cell lines and the potential of three novel thienopyridine derivatives 2e, 2f, 2h as anticancer agents in TNBC cell lines was demonstrated. Importantly, no toxicity was observed in non-tumorigenic breast cells. One of the most promising compounds, 2e, presented activity against TNBC both in vitro (by decreasing viable cell number and cell proliferation and interfering with the cell cycle profile) and in ovo (by reducing grafted tumor size). However, further studies must be carried out to unravel the underlying mechanisms of action of this compound. Furthermore, the anticancer effect of compound 2e should be also assessed in vivo, using xenografted mouse models of human TNBC cell lines. Although the present work is focused on compound 2e, the other promising compounds (2f and 2h) will be also explored in the future. Therefore, this work highlights the antitumor potential of a novel thienopyridine derivative against an aggressive type of cancer, TNBC. Eight novel methyl 3-(hetero)arylthieno[3,2-