Among the tested compounds, AC-10 showed maximum cytotoxic effect on MCF-7 cell line with IC50 value 74.7 ± 3.5 µM. On HCT-116 cells, AC-13 exhibited maximum cytotoxicity with IC50 value 42.1 ± 4.0 µM followed by AC-14 and AC-10 with IC50 values 62 ± 2.3 µM and 95.4 ± 1.7 µM respectively. All tested compounds were found to be safe on Vero cell line with IC50 value more than 200 µM. Based on their highest efficacy on HCT-116, AC-10, AC-13 and AC-14 were selected for mechanistic study on this cell line by evaluating changes nucleomorphological characteristics using acridine orange-ethidium bromide (AOEB) dual stain and by analyzing cell cycle with flow cytometry using propidium iodide stain. In AOEB staining, all three tested compounds showed significant (p < 0.05) increase in percentage apoptotic nuclei compared to control cells, with highest increase in apoptotic nuclei by AC-13 treatment (31 %). Flow cytometric studies showed cell cycle arrest by AC-10 and AC-14 treatment in G0/G1 phase and by AC-13 in G0/G1 and G2/M phase. The study reflected the potential of AC-10, AC-13 and AC-14 to be the lead molecules for further optimization.A series of 5'-amino-2'-hydroxy-1,3-diaryl-2-propen-1-ones (AC1-AC15) were synthesized by Claisen-Schmidt condensation of 5'-acetamido-2'-hydroxy acetophenone with various substituted aromatic aldehydes. The synthesized compounds were characterized by FTIR,  Cancer is rapid and uncontrolled growth of abnormal cells. It is the major cause of death, after cardiovascular diseases all over the world , has been demonstrated to possess many bioactive properties including anti-parasitic, estrogenic, antimalarial and antitumor activities  is suggested to exhibit broad spectrum anticancer properties against different types of human cancer cells primarily through inhibition of proliferation and induction of human cancer cell apoptosis . Xanthoann, 1983). Flavokann, 1983; Gold anann, 1983; Kawabatann, 1983) in kavaann, 1983).th position, and hydroxyl group at the 2nd positions of ring A of chalcones have not been reported as of our knowledge. In view of the wide spectrum of medicinal applications of chalcone derivatives, our research continues to explore the anticancer potential of flavonoids having free amino group/s. The present study includes the synthesis, characterization and mechanistic insight into anticancer activities of some new 5'-amino-2'-hydroxy-1,3-diaryl-2-propen-1-ones. Introduction of amino groups into various heterocyclic systems had led to very effective therapeutic agents. 5-Amino flavones showed antitumor activity highly selective to the ER-positive breast cancer cell line . 6-Amin1H NMR spectra were recorded by Bruker AV400 (400MHz) spectrometer in deuterated dimethyl sulphoxide using tetramethyl silane as internal standard. Mass spectra were scanned on a Shimadzu LCMS (ESI) 2010A spectrometer. 5'-Acetamido-2'-hydroxy acetophenone (2)   on pre-coated silica gel aluminium backed plates (Kieselgel 60 F254 Merck (Germany)). The IR spectra in KBr pellets were recorded using Shimadzu FTIR 8400S spectrophotometer.  Figure 1 was prep Figure 1 were preA mixture of concentrated hydrochloric acid and water (1:1) was added to 5'-acetamido-2'-hydroxy chalcone, and boiled for one hour. The reaction progress was monitored with TLC using petroleum ether-ethyl acetate solvent system in the ratio 2:3. When the reaction was complete, the reaction mixture was cooled to room temperature and ice cold water was added. The solution was basified by adding 10 % sodium bicarbonate solution. The product obtained was filtered and purified by recrystallization from ethanol to get amino chalcones Figure 1.(E)-1-(5-Amino-2-Hydroxyphenyl)-3-phenylprop-2-en-1-one (AC-1): Light Brown solid, yield: 72 %; m.p. 126 °C; IR (KBr) νmax/cm−1: 3425, 3340 (NH2), 3565 (OH), 1690 , 3026 (Ar-CH), 1136 (C-N ), 1510 (C=C); 1H NMR : δ ppm 5.43 , 6.60 , 6.71 , 6.90 , 7.47 , 7.51 , 7.71 , 7.79 , 8.17 , 11.70 ; LCMS (m/z): 239 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-one (AC-2): Dark Brown solid, yield: 70 %; m.p. 138 °C; IR(KBr) νmax/cm−1 : 3450, 3341 (NH2), 3588 (OH), 1683 , 3070 (Ar-CH), 1130 (C-N), 1510 (C=C); 1H NMR : δppm 5.34 , 6.97 , 7.24 , 7.63 , 7.70 , 7.78 , 7.84 , 8.18 , 11.81 ; LCMS (m/z): 273 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-fluorophenyl)prop-2-en-1-one (AC-3): Pale brown solid, yield: 68 %; m.p. 154 °C; IR(KBr) νmax/cm−1: 3430, 3329 (NH2), 3572 (OH), 1690 , 3062 (Ar -CH), 1130 (C-N), 1510 (C=C); 1H NMR : δppm; 5.48 , 6.97 , 7.32 , 7.71 , 7.73 , 7.78 , 7.90 , 8.18 , 11.73 ; LCMS (m/z): 257 (M+).E)-1-(5-Amino-2-Hydroxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one (AC-4): Brown solid, yield: 50 %; m.p. 112 °C; IR (KBr) νmax/cm−1: 3420, 3319 (NH2), 3570 (OH), 1676  3060 (Ar-CH), 1130 (C-N), 1510 (C=C); 1H NMR : δppm: 5.40 , 6.97 , 7.46 , 7.88 , 7.93 , 8.01 , 8.26 , 8.30 , 11.86 ; LCMS (m/z): 284 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-ethoxyphenyl)prop-2-en-1-one (AC-5): Brown solid, yield: 68 %; m.p. 135 °C; IR(KBr) νmax/cm−1: 3420, 3329 (NH2), 3568 (OH), 1692 , 3060 (Ar-CH), 1130 (C-N), 1510 (C=C); 1HNMR : δppm: 1.40 , 4.23 , 5.43 , 6.75 , 6.86 , 6.98 , 7.06 , 7.46 , 7.771 , 7.84 , 11.8 ; LCMS (m/z): 283 (M+). (E)-1-(5-Amino-2-Hydroxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one (AC-6): Brown solid, yield: 79 %; m.p. 110 °C; IR (KBr) νmax/cm−1: 3415, 3329 (NH2), 3588 (OH), 1672 , 3060 (Ar -CH), 1130(C-), 1510(C=C); 1H NMR : δppm: 3.81 , 5.43 , 6.96 , 7.04 , 7.37 , 7.71 , 7.72 , 7.79 , 8.14; LCMS (m/z): 269 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (AC-7): Brown solid, yield: 61 %; m.p. 136 °C; IR (KBr) νmax/cm−1: 3446, 3301 (NH2), 3591 (OH), 1680 , 3078 (Ar-CH), 1138 (C-N), 1520 (C=C); 1H NMR : δppm: 3.89 , 5.37 , 7.84 , 7.77 , 7.46 , 7.06 , 6.98 , 6.86 , 6.75 , 11.8 ; LCMS (m/z): 285 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one (AC-8): Pale brown solid, yield: 61 %; m.p. 136 °C; IR (KBr) νmax/cm−1: 3446, 3301 (NH2), 3589 (OH), 1680 , 3078 (Ar-CH), 1138 (C-N), 1520 (C=C); 1H NMR : δppm: 3.81 , 5.40 , 7.84 , 7.77 , 7.46 , 7.06 , 6.98 , 6.86 , 6.75 , 11.5 ; LCMS (m/z): 285 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-prop-2-en-1-one (AC-9): Brown solid, yield: 73 %; m.p. 90 °C; IR(KBr) νmax/cm−1: 3430, 3329 (NH2), 3588 (OH), 1690 , 3062 (Ar-CH), 1130 (C-N), 1510 (C=C); 1H NMR : δppm: 3.78 , 5.48 , 6.97 , 7.32 , 7.71 , 7.73 , 7.78 , 7.90 , 8.18 , 11.73 ; LCMS (m/z): 299 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(2-hydroxyphenyl)prop-2-en-1-one (AC-10): Dark brown solid, yield: 68 %; m.p. 130-132 °C; IR(KBr) νmax/cm−1: 3448, 3320 (NH2), 3576 (OH), 1692 , 3062 (Ar -CH), 1138 (C-N), 1510 (C=C); 1H NMR : δppm.; 5.39 , 6.97 , 7.32 d, 2H,J = 8.9 Hz), 7.64 , 7.75 , 7.78 , 7.90 , 8.18 , 11.80 ; LCMS (m/z): 255 (M+).((E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (AC-11): Brown solid, yield: 72 %; m.p. 102-104 °C; IR (KBr) νmax/cm−1: 3450, 3344 (NH2), 3591(OH), 1678 , 3070 (Ar-CH), 1138 (C-N), 1515 (C=C); 1H NMR : δppm: 3.84 , 5.43 , 7.84 , 7.771 , 7.46 , 7.06 , 6.98 , 6.86 , 6.75 , 11.8 ; LCMS (m/z): 269(M+).E)-1-(5-Amino-2-Hydroxyphenyl)-3-prop-2-en-1-one (AC-12): Brown solid, yield: 70 %; m.p. 138 °C; IR(KBr) νmax/cm−1: 3450, 3341 (NH2), 3580 (OH), 1690 , 3070 (Ar-CH), 1130 (C-N), 1510 (C=C); 1H NMR : δppm: 5.34 , 7.01 , 7.24 , 7.63 , 7.70 , 7.78 , 7.86 , 8.18 , 11.75 ; LCMS (m/z): 308 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-prop-2-en-1-one (AC-13): Pale brown solid, yield: 81 %; m.p. 128 °C; IR (KBr) νmax/cm−1: 3446, 3301 (NH2), 3591 (OH), 1680 , 3078 (Ar-CH), 1138 (C-N), 1520 (C=C); 1H NMR : δppm: 5.40 , 6.034 , 6.90 , 6.96 , 6.99 , 7.06 , 7.46 , 7.70 , 7.84 , 11.5 ; LCMS (m/z): 283 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-prop-2-en-1-one(AC-14): Light Brown solid, yield: 81 %; m.p. 136-138 °C; IR (KBr) νmax/cm−1: 3430, 3329 (NH2), 3588 (OH), 1690 , 3062 (Ar-CH), 1130 (C-N), 1510 (C=C); 1H NMR : δppm: 3.85 , 5.48 , 7.50 , 7.32 , 7.71 , 7.73 , 7.78 , 7.93 , 11.73 ; LCMS (m/z): 329 (M+).(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-benzyloxyphenyl)prop-2-en-1-one (AC-15): Dark Brown solid, yield: 73 %; m.p. 136-138 °C; IR (KBr) νmax/cm−1: 3425, 3340 (NH2), 3565 (OH), 1690 , 3026 (Ar-CH), 1136 (C-N), 1510 (C=C); 1H NMR : δppm: 5.25 , 5.50 , 7.11 , 7.29 , 7.47 , 7.71 , 7.77 , 7.79 , 7.82 , 7.87 , 8.01 , 8.17 , 11.70 ; LCMS (m/z): 345 (M+). cells and human breast cancer (MCF-7) cells and normal kidney epithelial cells (Vero). These cells were originally procured from National Cancer Center for Cell Science, Pune, India and cultured in our lab using Dulbecco's modified Eagles medium (DMEM) containing 10 % fetal bovine serum (FBS) at 37 °C in an atmosphere containing 5 % CO4 cells in 0.1 mL was seeded in a 96 well plate for 24 h. Test compounds were serially diluted with the medium to get a stock solution of 400 µM, 200 µM, 100 µM, 50 µM. After 24 h of incubation, 100 µL of test solution from respective stocks was added in triplicate and incubated for 48 h. After the treatment, drug-containing media was removed and 20 µL of MTT reagent (5 mg/mL in PBS) was added to each well. After 4 h of incubation at 37 °C, the MTT reagent was removed and 100 µL of 100 % DMSO was added to each well to solubilize formazan crystals. The optical density was measured using an ELISA plate reader at 540 nm and percentage cytotoxicity was calculated. .3 cells were seeded per well in 24-well plates with DMEM, containing 10 % FBS. After 24 h, cells were treated with selected concentrations of test compounds and incubated for 24 h. The media was removed and plate was washed with phosphate buffer saline . Cells were fixed in ice-cold methanol for 20 min, washed with PBS again and stained with acridine orange and ethidium bromide stain (20/30 µg/ml). After incubation for 20 min at 37 °C, and washing with PBS thrice, the plate was observed under a fluorescent microscope for morphological changes in nucleus such as condensed chromatin and fragmented nuclei. The apoptotic index (AI) was calculated as % of apoptotic cells from randomly counted 100 cells in each treatment group  and Ethidium bromide (EB)  were used for the morphological detection of apoptotic cells. 5 x 10al., 2015).6) were seeded in 25 cm2 flasks and, after overnight adherence, incubated with test compounds. Then cells were detached by trypsinization and mixed with floating cells, centrifuged and washed with PBS. The cell pellets were fixed in 70 % ice-cold methanol and stored at -20 °C for 24 h. After that cell pellets were washed with PBS and isotonic PI solution  was added. The stained cells were analyzed using Accuri C6 flow cytometer  with excitation at 488 nm and emission at 575/40 nm , CA, USA, where p<0.05 was considered to be significant.th position and hydroxyl substituent at the 2nd position of ring A and different substituents in the 2nd, 3rd, 4th, 5th positions of ring B were synthesized. The acetamido chalcones were synthesized by Claisen-Schmidt condensation of substituted benzaldehydes and 5'-acetamido-2'-hydroxy acetophenone. All the compounds were obtained in excellent yields (70-90 %). The acetamido chalcones were hydrolysed in acidic medium to obtain amino chalcones in good yields (50-81 %)  displayed high cytotoxicity (close to 200 µM including SEM) against HCT-116 and five compounds  against MCF-7 cells. Most of the compounds were free from cytotoxicity to normal kidney epithelial cells (Vero) at < 200 µM concentration, which indicated the selectivity of the compounds towards tested tumor cells. The results are presented in Table 150 value i.e, 100, 50 and 50 µM, respectively. All tested compounds produced morphological changes in the nuclei and showed significant (p < 0.05) increase in apoptotic nuclei. AC-13 showed maximum increase in percentage apoptotic nuclei (31 ± 4.16 %) followed by AC-10 (27 ± 3.21 %) and AC-14 (24.3 ± 3.53 %)  but not necrosis (orange colour) , accumulated 7.4 % cells in G2/M phase as compared with that of normal control suggesting cell cycle arrest at G2/M phase. The test compounds AC-10 (100 µM), AC-13 (50 µM), and AC-14 (50 µM), caused accumulation of cells , in G0/G1, phase which indicated the arrest of cell cycle in this phase. In addition AC-13 also showed accumulation of cells in G2/M phase (25.1 %) .1H NMR and mass spectroscopy. The FT-IR spectra of the newly synthesized chalcones displayed a strong absorption band due to α,β-unsaturated C=O stretching at 1692-1672 cm-1, 3591-3565 cm-1 (Ar-OH), 3078-3026 cm-1 (Ar-H), 3344-3319 cm-1 (NH2). Inspection of the 1H-NMR spectra suggested that the chalcones presented trans configurations (J = 15-17 Hz). The other expected peaks were in the region of 11.50-11.81 ppm , 5.34-5.50 ppm , 6.90-8.18 ppm (Ar-H). The OCH3 protons resonated at 3.85 ppm and were observed as a singlet. The mass spectra of all newly synthesized amino chalcones showed molecular ion peaks, which were in accordance with their respective molecular masses.The structures of synthesized compounds were confirmed with FT-IR and 50 62 ± 2.3 µM and 196.6 ± 7.9 µM against HCT-116 and MCF-7 respectively whereas compounds having one methoxy group at 3 or 4 positions of ring B  and two methoxy substituents at 3 and 4 positions of ring B (AC-1) were inactive. It is observed that AC-9 with no substituent on ring B was more active than compounds having electron withdrawing groups like NO2, F or Cl at the 4 position of ring B. The compound AC-8 with OH substitution at 3 and OMe substitution at 4 positions of ring B showed significant cytotoxicity. However, when the positions of OH and OMe were interchanged, as in AC-7, the cytotoxicity was reduced.The results revealed that the most potent compounds were AC-13, AC-10 and AC-14. The 3,4-methylene substitution (AC-13), 2-hydroxy substitution (AC-10), and 3,4,5-trimethoxy substitution (AC-14) on ring B of the chalcone increase the cytotoxic activity. Regarding the structure activity relationship, it appeared that the number of methoxy groups in ring B is important for cytotoxicity. AC-14 with three methoxy groups at 3,4 and 5 positions of ring B showed good activity with IC50 values namely, AC-10, AC-13 and AC-14, were selected for evaluation of apoptotic changes on the colon cancer cells (HCT-116) using acridine orange/ethidium bromide double staining technique. Treatments showed increased apoptosis compared to untreated control cells. The nucleomorphological changes such as apoptotic nuclei in the form of nuclear condensation and fragmentation were significantly (p<0.05) high in the treated cells compared to control cells. AC-13 treatment was found to be more effective with about five times increase in apoptotic nuclei compared to medium control . AO/EB Figures 3.0/G1, S and G2/M to ensure proper division of the cells. Any abnormality detected in the cell cycle leads to arrest further growth with the help of check-points. In the present study, the regulation of cell cycle check-points in HCT cancer cells was studied by cell cycle analysis using propidium iodide staining. The effect of the compounds on cell cycle phase was assessed and the results of the same were shown as the percentage of cells in G0/G1, S, and G2/M phase. The standard, doxorubicin (1 µM), showed 7.4 % more accumulation of cells in G2/M phase as compared to normal control. This suggests that cell cycle arrest is at G2/M phase. The test compounds, AC-10 and AC-14, caused accumulation of cells in G0/G1 phase while AC-13 produced accumulation of cells in G0/G1 and G2/M phase. These accumulations of cells indicated the arrest of cell cycle by AC-10, and AC-14 in G0/G1 phase and by AC-13 in G0/G1 and G2/M phase. The results obtained were in accordance with the earlier reports on chalcones, which state that cell cycle arrest by chalcones happens in either G0/G1 phase or G2/M phase . The heal., 2007).0/G1 and G2/M phase and AC-10 and AC-14 arrested G0/G1 phase of the cell cycle.In this study, a series of fifteen 5'-amino-2'-hydroxy-1,3-diaryl-2-propen-1-ones were synthesized and characterized. The compound AC-13 exhibited maximum cytotoxicity in HCT-116 and moderate cytotoxicity in MCF-7. AC-10 exhibited best cytotoxicity in MCF-7. The AO/EB staining showed the extent of apoptotic damage. AC-13 displayed highest % of apoptotic nuclei and arrested cell cycle in GThe authors acknowledge Manipal Institute of Technology and Manipal College of Pharmaceutical Sciences for providing research facilities. We thank Dr. N. Gopalan Kutty, Professor, Department of Pharmacology, MCOPS, Manipal University Manipal, for his support in drafting manuscript.


The strain was derived from a crown-of-thorns starfish, Acanthaster planci, collected from Chuuk, Federated States of Micronesia. The structures of violapyrones were elucidated by the analysis of 1D and 2D NMR and HR-ESIMS data. Violapyrones (1–4) exhibited cytotoxicity against 10 human cancer cell lines with GI50 values of 1.10–26.12 μg/mL when tested using sulforhodamine B (SRB) assay. This is the first report on the cytotoxicity of violapyrones against cancer cell lines and the absolute configuration of violapyrone C.Two new α-pyrone derivatives, violapyrones H ( Streptomyces sp. 112CH148 from a crown-of-thorns starfish, Acanthaster planci. A. planci has a long history in the scientific literature but only few studies have been done on its microbial symbionts + peak at m/z 261.1466  in the HR-ESIMS, which required four degrees of unsaturation. The IR absorptions at 3341 and 1674 cm−1 indicated the presence of hydroxyl (OH) and carbonyl (CO) groups, respectively. The UV maximum at 290 nm and 13C NMR data indicated typical α-pyrone moiety + peak at m/z 247.1313  in the HR-ESIMS. Preliminary, the NMR analysis showed a close similarity between the spectra of 1 and 2 (2 less than 1) and the observation of different splitting pattern of the methyl signal H-13 at δH 0.90 . In addition, a lack of one methyl carbon was observed in the 13C NMR data of 2 compare to 1. A detailed analysis of 1D and 2D spectra of 2 revealed the existence of a rigid α-pyrone ring same to 1. Furthermore, an aliphatic chain was assigned by COSY and HMBC correlations, consisting of 6 methylenes with a terminal methyl proton H-13 resonated at δH 0.90 . Finally, the aliphatic chain was connected to the α-pyrone ring and complete assignments of the atoms in the structure of violapyrone I (2) were achieved by 1H–1H COSY and HMBC experiments. Thus, the structure of 2 was determined as a new 3,4,6-trisubstituted α-pyrone, and 2 was named violapyrone I.Violapyrone I  was not determined in the previous report D27 +49°  and [α]D27 −53° , respectively. The absolute stereochemistry of C-11 in violapyrone C (4) was determined to be S, because the optical rotation value [α]D27 +50°  and 1H and 13C NMR data were consistent with those of synthetic (S)-violapyrone C , I (2), B (3) and C (4) was assessed by sulforhodamine B (SRB) assay +.Violapyrone H (2): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 289 (0.70) nm; IR (MeOH) νmax 3345 (br), 2926, 1670 cm−1; 1H and 13C NMR data (CD3OD), m/z 247.1313 [M + Na]+.Violapyrone I (3): Yellowish amorphous solid; UV (MeOH) λmax (log ε) 286.5 (1.34) nm; IR (MeOH) νmax 3347 (br), 2943, 1674 cm−1; 1H NMR (CD3OD) δH 5.97 , 2.45 , 1.85 , 1.62 , 1.54 , 1.35 , 1.23 , 0.88 ; 13C NMR (CD3OD) δC 169.4 (C-2), 168.8 (C-4), 164.9 (C-6), 101.5 (C-5), 98.9 (C-3), 39.9 (C-10), 34.4 (C-7), 29.2 (C-11), 28.3 (C-8), 27.9 (C-9), 23.1 (C-12), 8.4 (Me-3); HR-ESIMS m/z 225.1485 [M + H]+.Violapyrone B (4): Yellowish amorphous solid; [α]D27 +50 ; UV (MeOH) λmax (log ε) 288.0 (0.91) nm; IR (MeOH) νmax 3343 (br), 2925, 1674 cm−1; 1H NMR (CD3OD) δH; 5.98 , 2.47 , 1.85 , 1.62 , 1.36 , 1.34 , 1.13 , 1.33 , 1.17 , 1.32 , 0.88 , 0.87 ; 13C NMR (CD3OD) δC; 169.5 (C-4), 169.2 (C-2), 164.9 (C-6), 101.8 (C-5), 98.8 (C-3), 37.5 (C-12), 35.7 (C-11), 34.4 (C-7), 30.7 (C-10), 28.4 (C-8), 27.6 (C-9), 19.7 (Me-11), 11.9 (C-13), 8.4 (Me-3); HR-ESIMS m/z 261.1461 [M + Na]+.Violapyrone C (2. The growth inhibition assay against human cancer cell lines was carried out according to a sulforhodamine B (SRB) assay [1), I (2), B (3) and C (4) including vehicle control (0.1% DMSO) and positive control (adriamycin). After being incubated for 48 hours, cultures were fixed with 50% trichloroactetic acid (50 μg/mL) and stained with 0.4% sulforhodamine B in 1% acetic acid. Unbound dye was removed by washing with 1% acetic acid, and protein-bound dye was extracted with 10 mM Tris base (pH 10.5) for determination of optical density. The absorbance at 540 nm was determined using a VersaMax microplate reader . GI50 values were calculated using GraphPad Prism 4.0 software .Human cancer cell lines, HeLa (cervix), ACHN , HCT-15 (colon), HCT-116 (colon), MDA-MB-231 (breast), NCI-H23 (lung), NCI-H460 (lung), NUGC-3 (stomach), Hep-G2 (liver) and PC-3 (prostate), were purchased from American Type Culture Collection . The cell lines were cultured RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Cell cultures were maintained at 37 °C under a humidified atmosphere of 5% COB) assay . In brie1) and I (2) and two known B (3) and C (4), from the culture broth of Streptomyces sp. 112CH148. These violapyrones exhibited the cytotoxicity against 10 human cancer cell lines . Consequentially, these compounds could be new frontiers for the development of anticancer agents. Further studies are needed to clearly elucidate the mechanism of structure-activity relationship.As a result, we isolated two new 3,4,6-trisubstituted α-pyrone derivatives, violapyrones H (


The structures of the isolated compounds were identified on the basis of MS and NMR data analysis. The 13C NMR assignment of spirocyclohexadienylisoxazoline moieties of 1 and 2 were confirmed by an 1,1-ADEQUATE experiment. Compounds 1 and 2 showed a mild to moderate cytotoxic activities against KB-31 and FS4-LTM cell lines. Only aplysinin A (2) exhibited cytotoxicity against MCF-7 cells.Three new bromotyrosine-derived alkaloids 14-debromo-11-deoxyfistularin-3 ( The molecular formula of C31H31Br5N4O10 was deduced from HRMS–ESI(+) at m/z 1036.7844 [M + Na]+  which required 16 double bond equivalents (DBEs). The 13C NMR spectrum of 1 indicated two amide groups at δ 158.9 (C-9´) and 159.5 (C-9), 14 olefinic carbons, two hetero-olefinic carbons at δ 154.5 (C-8´) and 155.0 (C-8), and 5 ring systems to fulfill the DBEs. The comparison of the 1H and 13C NMR data from positions C-1, C-1´ to C-9, C-9´(1 with those of aerothionin (11) + (1:4:6:4:1) indicating a tetrabrominated compound. HRMS–ESI(+) of 2 at m/z 793.8320 [M + Na]+ suggested a molecular formula of C23H25Br4N3O7 . The 1H and 13C NMR spectra of 2 resemble to 1 except that the spectrum of 2 showed one extra methyl group  and one aromatic proton less . According to one set of δH 3.17 (H-7), 3.61 (H-7), 3.93 (H-1), 6.56 (H-5), δC 39.7 (C-7), 73.5 (C-1), 90.3 (C-6), 113.1 (C-2), 120.9 (C-4), 131.1 (C-5), 147.1 (C-3), 154.3 (C-8), and 159.0 (C-9) together with 1H,13C-HMBC correlations revealed that 2 consist of only one spirocyclohexadienylisoxazoline moiety in comparison with 1. The structure determination of 2 was accomplished based on 1H,1H-COSY and 1H,13C-HMBC correlations in the same manner as for 1. Once again, 1H,1H-COSY revealed propanamine [H-18 /H-19 /H-20 (δ 3.25)/20-NH (δ 7.86)] and hydroxyethylamine [9-NH (δ 8.39)/H-10 /H-11 (δ 4.67)/11-OH (δ 5.72)] substructures. The signals at δH 7.57 , δC 117.3 , 130.4 , 142.5 (C-12), and 151.4 (C-15) and 1H,13C-HMBC correlations among those signals showed a 1,2,4,6-dibromophenyl moiety. The substructures were assembled by 1H,13C-HMBC correlations from H-10 to C-9 and C-12, from H-11 to C-12 and C-13 as well as from δH H-18 to C-15. The terminal of side chain was connected to an acetamide moiety according to 1H,13C-HMBC correlations from H-20, 20-NH, and δH 1.80  to δC 169.1 (C-21). Compound 2 was named aplysinin A. The structure of 2 is similar to right-side portions of 1 and 8 (start at C-10). However, compound 2 contained an acetamide in the left-side portion instead of a ring system in comparison with 8 and showed great similarity with hexadellin B (10) isolating from the same organism. Hexadellin B (10) was originally isolated from the sponge Hexadella sp. The spectroscopic data of 10 was coincidently obtained from diacetylhexadellin B  and 3.61 (C-7) as well as between 6.56 (H-6) and 3.17 (H-7) suggesting a trans-hydroxyspiroisoxazoline ring similar to compound 1. An NOE was also observed between δH 5.72 (11-OH) and 1-OH indicating the same planar alignment of both hydroxy groups. The absolute configuration of spiroisoxazoline moiety was confirmed as 1-(R),6-(S) by positive Cotton effects  comparing to (+)-aerothionin (11) in the same manner of 1  whi whi2 wasner of 1 . The arr3 was isolated together with N-methylaerophobin-2 (15) as a mixture (approximate ratio 1:5). The ESIMS spectrum exhibited a 1:2:1 ion cluster at m/z 457/459/461, indicating the presence of two bromine atoms. The HRMS–ESI(+) spectrum revealed a pseudomolecular ion [M + H]+ at m/z 456.9892, which indicated a molecular formula of C16H18Br2N4O2 , containing nine DBEs. Two singlet aromatic protons δH 7.88 (δC 131.7) suggested a tetrasubstituted benzene pattern which was confirmed by 1H,13C-HMBC correlations; from δH 7.88  to δC 118.5 , δC 154.4 (C-4), δC 131.7 , and δC 135.7 (C-7), from δH 3.82 (4-OMe) to C-4. The connection of the benzene fragment to the E-vinyl moiety was confirmed by HMBC correlations from two olefinic protons δH 7.33  and δH 6.66  to δC 134.9 (C-1). 1H,1H-COSY correlations in between 9-NH (δ 8.17)/H-10 /H-11 /H-12  indicated a propanamine fragment which was connected to a 2-aminoimidazole moiety according to 1H,13C-HMBC correlations from H-12 to δC 126.8 (C-13) and 109.2 (C-14) . The structure of 3 is very similar to compound 21  . The twopound 21  which wagula sp. . In comp1) and aplysinin A (2) were tested for their antimicrobial activity against different Gram-positive and Gram-negative bacteria, fungi, and for their antiproliferative activity. Aplysinin B (3) was not subjected to any biological activity test due to the minute amount and its existence as the minor compound of a mixture. The results showed that 1 and 2 exhibited mild cytotoxic activity against KB-31 epidermoid carcinoma cells . Only 2 showed mild toxicity against the breast cancer cell line MCF-7 and to FS4-LTM conditional immortalization human fibroblasts . The cytotoxicities of the known compounds (4–17) are also listed in The new compounds 14-debromo-11-deoxyfistularin-3  and a Bruker Avance NMR spectrometer . All NMR experiments were measured at a temperature of 303 K using DMSO-d6  as internal standard. HPLC separation was achieved by Jasco PU-1580 using a Kromasil RP18 column  and a Kromasil RP18 column  and was eluted with gradient H2O (0.1% TFA) and MeCN (0.1% TFA).UV spectra were recorded during HPLC separation with a DAD detector (JASCO MD-2010 Plus). CD spectra were recorded on a JASCO J-810 spectropolarimeter. Low and high resolution ESIMS was performed with a Bruker micrOTOFAplysina lacunosa was collected by SCUBA diving at a depth of 8 m from Stirrup Cay in the Bahamas in June 2008. The sample was immediately frozen and kept at −20 °C until extraction. A voucher specimen of this species is deposited in AG Köck, Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung (voucher number: Aplysina lacunosa 08/21). The freeze-dried sponge (200 g) was extracted three times with CH2Cl2/MeOH  at room temperature. The filtrates were pooled and evaporated to yield 24.3 g of crude extract which was further partitioned between n-hexane and MeOH. The MeOH extract was then partitioned between EtOAc and H2O. The EtOAc fraction was further purified by vacuum liquid chromatography using silica gel eluting with stepwise gradient from 100:0 to 80:20 . The fraction eluted with 97:3  was concentrated and further purified by HPLC using an RP C18 column  to yield 5 (198.0 mg), 11 (263.0 mg), 12 (445.0 mg), 13 (55.0 mg), 14 (7.9 mg), 16 (15.0 mg), 17 (9.1 mg), and two other fractions. The first fraction was purified using reversed-phase HPLC  to obtain 7 (3.2 mg), 8 (4.5 mg), 9 (9.6 mg) and following with analytical RP18 column  yielding 1 (0.8 mg). The other fraction was purified using RP18 HPLC  and follow with an analytical RP18 column  yielding 2 (0.8 mg) and 18 (5.3 mg). The 94:6  fraction was purified by HPLC using a RP C18 column  yielding 6 (10.7 mg), 10 (25.5 mg), 15 (5.0 mg), and one fraction which was purified using reversed-phase HPLC  to get a mixture of 3 and 15 (3.5 mg) as well as 4 (3.0 mg).The sponge Straptococcus aureus (MRSA and MSSA) and Micrococcus luteus; Gram-negative: Peumonia aruginosa and Klebsiella pneumonia] and antifungal Candida albicans using microdilution technique. The MIC was defined as lowest concentration that shows 50% growth inhibition after 24 hour incubation.The antimicrobial activities of isolated compounds were evaluated against five microorganisms + .14-Debromo-11-deoxyfistularin-3 (2): white solid; UV (DAD) λmax 225 nm; CD (MeOH) λmax 252 nm (∆ε +4.77), 283 nm (∆ε +3.34); 1H NMR and 13C NMR see m/z = 793.8320 [M + Na]+ .Aplysinin A (3): white solid; UV (DAD) λmax 227 nm; 1H NMR and 13C NMR see m/z = 456.9892 [M + H]+ .Aplysinin B (File 11D, 2D NMR, and CD spectra of three new compounds. 1D NMR, mass and CD spetra of all known isolated compounds.


Their structures were elucidated by extensive physicochemical and spectroscopic properties, besides comparison with literature data. The absolute configurations of compounds 1–3 were corroborated by chemical transformation, GC analysis and X-ray crystallographic analysis. Meanwhile, the absolute configuration of compound 4 and the planar structure of compound 6 were also determined based on the X-ray diffraction analysis. The cytotoxicity of compounds 1–10, antifungal and anti-HIV activities of compounds 1–5 and the in vitro assay for glucose consumption of compounds 1–3 were done in the anti-diabetic model, whereas none showed obvious activity.Five new compounds, including a benzopyran ribonic glycoside, daldiniside A ( Marine fungi are known as a rich source of structurally diverse and biologically active secondary metabolites, including polyketides, steroids, terpenes and alkaloids. Nevertheless, the potential chemical investigations on marine fungi are limited. In recent decades, bioactive natural products obtained from the marine-derived fungi have attracted the rising attention of organic chemists for discovering new drugs .Daldinia eschscholzii was well-known to produce abundant polyketides as a mantis-associated fungus , [H)-one  [10) [9]1 was obtained as colorless crystal. Its molecular formula was determined as C15H20O7 by HRESIMS at m/z 335.1097 [M + Na]+ , indicating the presence of six degrees of unsaturation. The IR spectrum of 1 showed absorptions of hydroxyl (3423 cm−1) and aromatic  functionalities. The 1H-NMR spectrum of 1 , 7.12  and 6.49 , ascribed to one set of the typical 1,2,3-trisubstituted aromatic ring. Additionally, the 1H NMR spectrum of 1 also revealed the signals of one methyl group  and two oxygen-bearing methines at 5.70  and 5.04 . The 13C-NMR spectrum showed one methyl, two methylenes (one oxygenated), nine methines (three aromatic and six oxygenated) and three aromatic quaternary carbons. Furthermore, a series of proton signals at δH 3.64–5.70 and their corresponding carbons at δC 63.5, 71.4, 73.9, 88.5 and 103.4 might suggest a pentose moiety.Compound rum of 1  showed s1H-1H COSY and HMBC correlations  suggested that the sugar moiety was linked to the C-5 of aglycone. The remaining one degree of unsaturation, together with the 1H-1H COSY correlations of H-9/H-2, H-2/H-3 and H-3/H-4 and the HMBC correlations from H-9 to C-2, C-3 and from H-4 to C-2, C-4a, C-5 and C-8a, indicating that a pyranoid ring was linked to C-4a and C-8a, and the methyl and hydroxyl groups were located at C-2 and C-4, respectively. Thus, the planar structure of 1 was established.Analysis of the key elations  was used1 gave the sugar motif, and then, it was unambiguously established as d-ribose by chemical transformation and GC analysis. The coupling constant of the anomeric proton at δH 5.70  in the 1H NMR spectrum of 1 indicated the d-ribose unit to be in the α-configuration + . UV absorption bands at 210, 243, 257 and 300 nm and IR absorption bands at 3394, 3325 and 1607 cm−1 implied the presence of amine, hydroxy and conjugated carbonyl functionalities. In the 1H NMR spectrum , 8.21 , 7.19 , 7.23  and 7.49  indicated the presence of an unsubstituted indole aromatic ring, which was inferred by the 1H-1H COSY correlations of H-4/H-5, H-5/H-6 and H-6/H-7 (2(10)OH- subunit was established by analysis of the 1H-1H COSY correlation of H-9/H-10 and HMBC correlation from H-10 to C-8 and C-9 and linked to the indole moiety by C-3, determined by the HMBC correlations from H-2 to C-3 and C-8. The configuration at C-9 was unequivocally established to be R by the single-crystal X-ray diffraction using Cu Kα radiation (4 was established and named 1-(3-indolyl)-2R,3-dihydroxypropan-1-one.Compound spectrum , the sig H-6/H-7 . The -COadiation . Consequ5 was isolated as a yellow oil with the molecular formula C10H12N2O4 as determined by the HRESIMS peak at m/z 275.0998 [M + Na]+ . In the 2D NMR spectra of 5 , RP-C18 silica gel  and Sephadex LH-20 . Semi-preparative HPLC was conducted on an Agilent 1100 liquid chromatography with a YMC-Pack ODS-A  column. GC analysis was performed with an Agilent Technologies 6890N gas chromatography system. Solvents were distilled prior to use, and spectroscopic grade solvents were used. TLC was performed with silica gel 60 F254  and RP-C18 F254 plates . Peptone  was purchased from Beijing Shuangxuan Microbial Medium Plant .UV spectra were measured on a Varian Cary 50 spectrophotometer or a Shimadzu UV-2401A spectrophotometer. Optical rotations were recorded on a Perkin-Elmer PE-341LC polarimeter. IR spectra were determined on a Bruker Vertex 70 FT-IR spectrophotometer. The D. eschscholzii was isolated from the branches of Scaevola sericea Vahl, collected from the mangrove forest nature reserve in Haikou, Hainan province, China. The fungus was identified by sequence analysis of the ITS region of its rDNA, as described previously + .Daldiniside A (2): Yellowish powder; c = 0.67 mg/mL, MeOH); UV (MeOH) λmax (log ε): 236 (4.56), 243 (4.57) and 329 (3.69) nm; IR (KBr) νmax: 3429, 2926, 1689, 1644, 1624, 1573, 1506, 1385, 1354, 1235, 1172, 1074, 1043, 694 cm−1; 1H and 13C NMR data, see m/z 347.0731 [M + Na]+ .Daldiniside B (3): Yellowish powder; c = 0.13 mg/mL, MeOH); UV (MeOH) λmax (log ε): 203 (3.62), 244 (3.92) and 334 (3.06) nm; IR (KBr) νmax: 3427, 2924, 1686, 1626, 1505, 1384, 1240, 1171, 1127, 1083, 1045, 695 cm−1; 1H and 13C NMR data, see m/z 391.0994 [M + Na]+ .Daldiniside C : Colorless crystal; c = 0.70 mg/mL, MeOH); UV (MeOH) λmax (log ε): 210 (4.19), 243 (3.82), 257 (3.70) and 300 (3.82) nm; IR (KBr) νmax: 3394, 3325, 1607, 1520, 1442, 1156, 1090, 986, 742, 705 cm−1; 1H and 13C NMR data, see m/z 228.0628 [M + Na]+ .1-(3-indolyl)-25): Yellow oil; UV (MeOH) λmax (log ε): 210 (3.94), 243 (3.18) and 279 (3.86) nm; IR (KBr) νmax: 2975, 2935, 1714, 1452, 1392, 1251, 1177, 1122 cm−1; 1H and 13C NMR data, see m/z 275.0998 [M + Na]+  and m/z 253.1178 [M + H]+ .3-ethyl-2,5-pyrazinedipropanoic acid  Å, b = 10.3774(4) Å, c = 26.8183(9) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1480.91(9) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.943 mm−1, 10,224 reflections measured, 2578 independent reflections (intR = 0.1191). The final 1R values were 0.1218 (I > 2σ(I)). The final wR(F2) values were 0.3053 (I > 2σ(I)). The final 1R values were 0.1580 . The final wR(F2) values were 0.3760 . The goodness of fit on F2 was 1.445. Flack parameter = 0.0 (7).Crystal data for 4: C11H11NO3, M = 205.21, monoclinic, a = 4.7449(5) Å, b = 5.4635(5) Å, c = 17.8653(16) Å, α = 90.00°, β = 95.820(5)°, γ = 90.00°, V = 460.75(8) Å3, T = 100(2) K, space group P21, Z = 2, μ(Cu Kα) = 0.903 mm−1, 3,878 reflections measured, 1,498 independent reflections (Rint = 0.0490). The final 1R values were 0.0523 (I > 2σ(I)). The final wR(2F) values were 0.1467 (I > 2σ(I)). The final 1R values were 0.0527 . The final wR(2F) values were 0.1470 . The goodness of fit on 2F was 1.109. Flack parameter = −0.4(4). The Hooft parameter is 0.02(13) for 567 Bijvoet pairs.Crystal data for 6: C10H12N2O4, M = 224.22, monoclinic, a = 5.6215(9) Å, b = 13.275(2) Å, c = 7.0331(11) Å, α = 90.00°, β = 105.791(2)°, γ = 90.00°, V = 505.04(14) Å3, T = 100(2) K, space group P21/n, Z = 2, μ(Mo Kα) = 0.116 mm−1, 5,086 reflections measured, 1415 independent reflections (intR = 0.0238). The final 1R values were 0.0368 (I > 2σ(I)). The final wR(F2) values were 0.0918 (I > 2σ(I)). The final 1R values were 0.0380 . The final wR(F2) values were 0.0926 . The goodness of fit on F2 was 1.088.Crystal data for 1 (deposition No. CCDC 989294), 4 (deposition No. CCDC 981181), and 6 (deposition No. CCDC 981180) have been deposited in the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK .The crystallographic data for 1 (1.2 mg) was hydrolyzed with 2 M aqueous CF3COOH (2.0 mL) at 90 °C for 6 h. The reaction mixture was evaporated to dryness; Then, the residue and l-cysteine methyl ester hydrochloride (2.5 mg) were dissolved in dry pyridine (1.0 mL) and kept at 65 °C for 2 h. The reaction mixture was dried, and then, trimethylsilylimidazole (0.2 mL) was added to the residue, followed by stirring at 65 °C for 1 h [n-hexane, and then, the organic phase was submitted to GC analysis by using an HP-5MS capillary column ; column temperature, 230 °C; injection temperature, 250 °C; detector FID, detector temperature, 250 °C. A peak at the retention time of 12.61 min for compound 1 was observed. When the corresponding ribose was prepared by the same reaction, the retention times of presilylated d-ribose and l-ribose were 12.66 and 14.09 min, respectively. Hence, the sugar in compound 1 was determined to be d-ribose.Compound  for 1 h . In the 2 (1.5 mg) and 3 (9.8 mg) were subjected to a similar treatment as compound 1, and the retention times of ribose were 12.63 and 12.66 min, respectively. Therefore, the sugar in compounds 2 and 3 were determined to be d-ribose. In addition, the reaction mixture of compound 3 was diluted with H2O (1.5 mL) and extracted with CHCl3. The CHCl3 layer was dried to yield the aglycone, whose NMR and optical rotation data were identical to de-O-methyldiaporthin  [Compounds 9, MeOH) .1–10 against HL-60, SMMC-7721, A-549, MCF-7 and SW-480 was studied using the MTT method [50 > 40 μg/mL. In addition, compounds 1–5 were tested for antifungal activities against Candida albicans (ATCC32354 and ATCC10231) at a concentration of 128 μg/mL and anti-HIV activity according to the described method [in vitro assay for glucose consumption of compounds 1–3 was done in the anti-diabetic model with DMEM-induced 3T3 fibroblasts [The cytotoxicity of T method , and thed method ,15; Unforoblasts , whereas1), two new isocoumarin glycosides, daldinisides B (2) and C (3), and two new alkaloids, 1-(3-indolyl)-2R,3-dihydroxypropan-1-one (4) and 3-ethyl-2,5-pyrazinedipropanoic acid (5), together with five known compounds, 2,5-pyrazinedipropanoic acid (6), cyclo-(Phe-Tyr) (7), de-O-methyldiaporthin (8), 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (9) and orcinotriol (10), were discovered from the marine-associated fungus, D. eschscholzii. Natural products embodying the α-d-ribose moiety were quite scarce to be reported. To the best of our knowledge, compounds 2 and 3 were hitherto the first example of isocoumarins containing the α-d-ribose moiety in natural products. Interestingly, the nonenzymatic cyclic dimerization of 5-aminolevulinic acid (5-ALA) might be the key reaction to form a pyrazine nucleus, and a series of derivatives might lead to the formation of 5 and 6 [A new benzopyran glycoside, daldiniside A ( 5 and 6 .


Dendrobium brymerianum led to the isolation of eight phenolic compounds. Among the isolated compounds (1–8), moscatilin (1), gigantol (3), lusianthridin (4), and dendroflorin (6) showed appreciable cytotoxicity against human lung cancer cell lines with IC50 values of 196.7, 23.4, 65.0, and 125.8 μg/mL, respectively, and exhibited antimigratory property at nontoxic concentrations. This study is the first report on the biological activities of this plant.Chromatographic separation of a methanol extract prepared from the whole plant of  High incidence of cancer metastasis in lung cancer makes this type of cancer among the most severe in the world . Althoug Dendrobium (Orchidaceae) have long been used in traditional Chinese medicine as tonics +; 1H NMR  δ: 2.78 , 3.75 , 3.76 , 6.48 , 6.64 , 6.75 , 6.78 ; 13C NMR  δ: 38.3 (C-α′), 38.8 (C-α), 56.1 (MeO-3′), 56.5 , 106.7 , 112.9 (C-2′), 115.4 (C-5′), 121.6 (C-6′), 133.1 (C-1), 134.1 (C-1′), 134.8 (C-4), 145.3 (C-4′), 147.9 (C-3′), 148.3 . Flavanthrinin ( 2). Brown amorphous solid; C15H12O3; ESI-MS m/z 241 [M+H]+; 1H NMR  δ: 4.15 , 6.98 , 7.06 , 7.08 , 7.40 , 7.42 , 7.49 , 7.62 ; 13C NMR  δ: 58.5 (MeO-4), 102.5 (C-3), 107.7 (C-1), 114.0 (C-4a), 116.9 (C-6), 119.9 (C-4b), 121.0 (C-8), 126.9 (C-10), 127.4 (C-5), 129.7 (C-9), 134.9 (C-8a), 137.0 (C-10a), 155.2 (C-7), 156.3 (C-2), 157.3 (C-4). Gigantol ( 3). Brown amorphous solid; C16H18O4; ESI-MS m/z 275 [M+H]+; 1H NMR  δ: 2.78 , 3.69 , 3.78 , 6.22 , 6.28 , 6.30 , 6.64 , 6.69 , 6.79 ; 13C NMR  δ: 37.9 (C-α′), 39.0 (C-α), 55.2 (MeO-3), 56.0 (MeO-3′), 99.6 (C-4), 106.2 (C-6), 108.8 (C-2), 112.8 (C-5′), 115.4 (C-2′), 121.5 (C-6′), 134.0 (C-1′), 145.1 (C-4′), 145.4 (C-1), 147.9 (C-3′), 159.1 (C-3), 161.7 (C-5). Lusianthridin ( 4). Brown amorphous solid; C15H14O3; ESI-MS m/z 243 [M+H]+; 1H NMR  δ: 3.72 , 6.36 , 6.42 , 6.67 , 6.70 , 8.22 ; 13C NMR  δ: 30.6 (C-9), 31.4 (C-10), 55.2 (MeO-2), 101.5 (C-3), 105.8 (C-1), 113.4 (C-6), 114.9 (C-8), 115.7 (C-4a), 125.8 (C-4b), 129.8 (C-5), 139.7 (C-8a), 141.3 (C-10a), 155.8 (C-4), 155.9 (C-7), 159.2 (C-2).Nobilone ( 5). Red amorphous solid; C14H10O4; ESI-MS m/z 243 [M+H]+; 1H NMR  δ: 4.13 , 6.78 , 6.80 , 6.93 , 7.10 , 7.13 ; 13C NMR  δ: 57.5 (MeO-4), 105.9 (C-1), 106.2 (C-3), 116.7 (C-8), 122.6 (C-4a), 125.0 (C-6), 128.0 (C-4b), 130.2 (C-5), 135.8 (C-8a), 137.2 (C-9a), 151.6 (C-7), 153.5 (C-4), 160.9 (C-2), 193.2 (C-9).Dendroflorin ( 6). Red amorphous solid; C14H10O5; ESI-MS m/z 259 [M+H]+; 1H NMR  δ: 4.10 , 6.58 , 6.76 , 6.79 , 6.87 ; 13C NMR  δ: 57.4 (MeO-4), 105.6 (C-1), 106.1 (C-3), 117.4 (C-8a), 119.7 (C-7), 122.4 (C-4a), 124.3 (C-4b), 128.9 (C-6), 137.4 (C-9a), 145.1 (C-5), 152.8 (C-8), 154.1 (C-4), 160.9 (C-2), 195.3 (C-9).Denchrysan B ( 7). Red amorphous solid; C14H12O4; ESI-MS m/z 245 [M+H]+; [α]D20  −7.9 ; 1H NMR  δ: 4.06 , 5.37 , 6.59 , 6.73 , 6.83 , 7.03 , 7.07 ; 13C NMR  δ: 57.0 (MeO-4), 75.4 (C-9), 100.3 (C-3), 107.2 (C-1), 116.9 (C-6), 117.0 (C-8), 118.9 (C-4a), 124.7 (C-4b), 128.4 (C-7), 148.5 (C-8a), 150.8 (C-9a), 151.4 (C-5), 153.0 (C-4), 159.6 (C-2).Tristin ( 8). Brown amorphous solid; C15H16O4; ESI-MS m/z 261 [M+H]+; 1H NMR  δ: 2.74 , 3.78 , 6.17 , 6.20 , 6.63 , 6.69 , 6.79 ; 13C NMR  δ: 37.9 (C-α′), 38.9 (C-α), 56.1 (MeO-3′), 101.0 (C-4), 107.8 , 112.8 (C-2′), 115.5 (C-5′), 121.5 (C-6′), 134.1 (C-1′), 145.1 (C-4′), 145.5 (C-1), 148.0 (C-3′), 159.2 . l-glutamine, and 100 units/mL penicillin/streptomycin in a 5% CO2 environment at 37°C. 3--2,5-diphenyltetrazolium bromide (MTT) and other chemicals were obtained from Sigma Chemical, Inc. .Lung cancer H460 cells were obtained from the American Type Culture Collection . Cells were cultured in RPMI 1640 containing 5% fetal bovine serum, 2 mM4 cells/well in a 96-well plate and allowed to attach for 12 h, treated with different concentrations of compounds in DMSO  for 24 h, and then incubated with 100 μL of 500 μg/mL MTT solution for 4 h at 37°C. Then, MTT solution was removed, and 100 μL of 99.9% DMSO was added to dissolve the formazan crystal. The intensity of formazan product was measured at 570 nm using a microplate reader. All analyses were performed in at least three independent replicate cultures. The cell viability was calculated as follows:To determine cytotoxicity, cell viability was determined by modified MTT assay  which me4 cells/well was cultured in 96-well plates, and a wound space was created by a 1 mm width tip. After rinsing with PBS, the cell monolayers were treated with nontoxic concentration of compounds (0.1 μg/mL) and allowed to migrate for 0–48 h. Micrographs were taken under a phase-contrast microscope , and wound spaces were measured from 10 random fields of view using Olympus DP Controller software. Quantitative analysis of cell migration was performed by using an average wound space from random fields of view, and the relative migration was calculated.Migratory activity of the cancer cells was evaluated by wound-healing assay . BrieflyP value of less than 0.05 (P < 0.05) was considered statistically significant. The IC50 value was obtained using the GraphPad Prism software.All results from three or more independent experiments were expressed as means ± SD. Statistical differences between means were determined using the analysis of variance (ANOVA) and the post hoc test. A μL of the plant extract solution (0.5 mg/mL in ethanol) was added to 1 : 10 diluted Folin-Ciocalteu reagent (100 μL). After 5 minutes, 100 μL of Na2CO3 (75 g/100 mL in deionized water) was added. After incubation at room temperature for 1 hr, the absorbance was measured at 765 nm using a Perkin-Elmer Victor3 1420 multilabel counter. Gallic acid (Sigma) was used as a reference standard, and the total phenolic content was expressed as mg of gallic acid equivalent (GAE)/g dried plant material.The total phenolic content (TPC) was determined by Folin-Ciocalteu method . Assays  Dendrobium nobile [1) [2) [3) [4) [5) [6) [7) [8) [1H NMR, 13C NMR, and MS data in comparison with previously reported values. Compounds 1, 2, 4, 5, 7, and 8 were not found in the previous study [The total phenolic content of the dried plant material was found to be 1.13 mg GAE/g, which was in the same level as that ofm nobile . Chromatbile [1) , flavant [1) [2) , giganto [2) [3) , lusiant [3) [4) , nobilon [4) [5) , dendrof [5) [6) , denchry [6) [7) , and tri [7) [8) . The strus study . 1–8 were evaluated for cytotoxicity against human lung cancer H460 cells using established protocols [1) which has been earlier reported for cytotoxic and antimigratory effects [3), lusianthridin (4), and dendroflorin (6) exhibited appreciable cytotoxic properties, whereas the others were inactive (IC50 > 200 μg/mL). From these preliminary limited data it could be speculated that the stronger cytotoxicity of the extract, as compared with the individual components, was probably due to the additive or synergistic effects from the eight phenolic (1–8) and/or other unisolated compounds. Compounds 3, 4, and 6 were further investigated for antimigratory potential in comparison with 1, using a modified method of the wound-healing assay [1) showed inhibition against the migration of breast MDA-MB-231 cells [μg/mL) to avoid the possible interfering cytotoxic effect. The human lung cancer H460 cells were then allowed to migrate in the presence or absence of the test compound for 0, 6, 12, 24, and 48 h, after which the migratory activity was measured. The results, as shown in Figures 1, 3, 4, and 6) inhibited the migration of the cells across the wound space in a time-dependent manner. During the first 24 h, dendroflorin (6) appeared to be the strongest antimigratory compound; however, after 48 h, moscatilin (1) became the most potent compound, as a result of the substantial increase of its activity during the period of 24–48 h. It should be mentioned that the antimigratory effects of moscatilin (1) and gigantol (3) have been earlier demonstrated in other cancer cells, for example, human lung H23 and breast MDA-MB-231 cells [3) has been shown to suppress lung cancer cell migration through a caveolin-1-dependent pathway [3) was the weakest antimigratory agent despite its strong cytotoxicity, suggesting that no linear correlation exists between cytotoxicity and antimigratory activity.Compoundsrotocols , and the effects , only ging assay . In a pr31 cells  and thuse IC50 > 00 μg/mL.31 cells , 10, 26.It is known that cell migration may be regulated by the caveolin-1 and/or the protein kinase B (Akt) signaling pathway . In cert Dendrobium brymerianum. To the best of our knowledge, this is the first report on the biological activities of this plant. Our findings on the antimigratory activities of moscatilin (1) and gigantol (3) are in agreement with earlier reports. In addition, our preliminary data revealed for the first time the antimigratory potential of lusianthridin (4) and dendroflorin (6). The biological data obtained in the present study should be useful for the future development of new antimetastatic drugs.In summary, in this communication we provide new information on the cytotoxic and antimigratory effects of the phenolic compounds isolated fromLyon/Geneva, 12 December 2013 – The International Agency for Research on Cancer (IARC), the specialized cancer agency of the World Health Organization, today released the latest data on cancer incidence, mortality, and prevalence worldwide.1 The new version of IARC's online database, GLOBOCAN 2012, provides the most recent estimates for 28 types of cancer in 184 countries worldwide and offers a comprehensive overview of the global cancer burden.GLOBOCAN 2012 reveals striking patterns of cancer in women and highlights that priority should be given to cancer prevention and control measures for breast and cervical cancers globally. Global burden rises to 14.1 million new cases and 8.2 million cancer deaths in 2012 According to GLOBOCAN 2012, an estimated 14.1 million new cancer cases and 8.2 million cancer-related deaths occurred in 2012, compared with 12.7 million and 7.6 million, respectively, in 2008. Prevalence estimates for 2012 show that there were 32.6 million people (over the age of 15 years) alive who had had a cancer diagnosed in the previous five years. The most commonly diagnosed cancers worldwide were those of the lung , breast , and colorectum . The most common causes of cancer death were cancers of the lung , liver , and stomach . Projections based on the GLOBOCAN 2012 estimates predict a substantive increase to 19.3 million new cancer cases per year by 2025, due to growth and ageing of the global population. More than half of all cancers (56.8%) and cancer deaths (64.9%) in 2012 occurred in less developed regions of the world, and these proportions will increase further by 2025.


Natural products present in low quantity in herb medicines constitute an important source of chemical diversity. However, the isolation of sufficient amounts of these low abundant constituents for structural modification has been a challenge for several decades and subsequently halts research on the utilization of this important source of chemical entities for drug discovery and development. And, pro-angiogenic therapies are being explored as options to treat cardio-cerebral vascular diseases and wound healing recently. The present study investigates the pro-angiogenic potential of tanshinone derivatives produced by one-pot synthesis using zebrafish model.Salvia miltiorrhiza. This led to the isolation of ten new imidazole-tanshinones (Compounds 1–10) and one oxazole-tanshinone (Compound 11), the structures of which were characterized by spectroscopic methods in combination with single-crystal X-ray crystallographic analysis. The angiogenesis activities of the new tanshinone derivatives were determined in an experimental model of chemical-induced blood vessels damage in zebrafish. Of all the tested new derivatives, compound 10 exhibited the most potent vascular protective and restorative activity with an EC50 value of 0.026 µM. Moreover, the mechanism underlying the pro-angiogenesis effect of 10 probably involved the VEGF/FGF-Src-MAPK and PI3K-P38 signalling pathways by gene expression analysis and a blocking assay with pathways-specific kinase inhibitors.In order to address the difficulty of chemical modification of low abundant constituents in herb medicines, a novel one-pot combinatorial modification was used to diversify a partially purified tanshinone mixture from 10 than other tanshinones and revealed 10 has potential for development as a pro-angiogenic agent for diseases associated with insufficient angiogenesis. Our results highlighted the great potential of adopting a newly modified one-pot approach to enhance the chemical diversity and biological activities of constituents from natural products regardless of their abundances.Taken together, our study demonstrated the more distinctive pro-angiogenic properties of  Over 50% of commercial available drugs originated from the optimization of lead compounds isolated from natural products Recently, there has been increasing interest in the application of the one-pot synthesis approach to create chemical structural diversity, in which chemical reactions were carried out by mixing starting materials including parental compounds and reactants into a single container to undergo multiple chains of modifying reactions without isolating intermediates. One-pot synthesis is also regarded as “green” mild approach as it improves the efficiency of chemical reactions and minimizes chemical waste generation by eliminating complicated separation and purification processes of the intermediate chemical compounds. For instance, even compounds with complex structure and multiple stereocentres, such as Oseltamivir, an anti-viral drug which is regarded as the last resort for controlling H5N1 infuenza pandemic, is successfully produced by sequential one-pot syntheses Salvia miltiorrhiza (Danshen), is widely used botanical material in traditional Chinese medicine for treating cerebral and cardiovascular diseases such as stroke Salvia species and could be considered as a natural combinatorial library Salvia miltiorrhiza remain an unexplored natural source of starting materials for the discovery and development of new therapeutics.de novo blood vessels formation are important steps in the recovery process following severe ischemic and traumatic situations Angiogenesis is a complex and critical step of various physiological and pathological processes. Many diseases such as cancers, psoriasis associated with excessive angiogenesis in vitro assays which can precisely reflect the pharmacodynamic change of a specific mode of action to a specific cell type and/or molecular target, is that these assays do not facilitate the assessment of absorption, distribution, metabolism, and excretion (ADME) Danio rerio) has become an ideal environmentally friendly and economical in vivo drug screening model for improving various drug-like properties such as pharmacodynamic, absorption and metabolism simultaneously Tg(fli-1a:EGFP)y1 zebrafish embryo provides a powerful tool for the discovery of novel angiogenesis modulating agents allowing observation of the responses of live embryo to drugs in real time. Moreover, our previous study established a novel chemical-induced blood vessels loss model which can mimic certain pathological conditions relating to angiogenesis deficiencies. Thus, a more physiologically relevant assay is required for identifying pro-angiogenesis agents against diseases asscoiated with angiogenesis deficiencies Salvia miltiorrhiza using the zebrafish angiogenesis assays.One of major limitations of commonly used In present study, new tanshinone derivatives were synthesized by mixing total natural tanshinones with reactants using a one-pot synthesis approach. A series of new tanshinone derivatives were isolated and subjected to both structural elucidation and zebrafish angiogenesis assays. Furthermore, the involvement mechanism underlying the angiogenesis effect of the new tanshinone derivatives was investigated using real-time qPCR analysis and testing for blocking effects with pathway-specific inhibitors.3Cl3 on a Jasco V-550 UV/vis spectrophotometer. ESI-MS spectra were carried out on a Finnigan LCQ Advantage Max ion trap mass spectrometer. HR-ESI-MS data were obtained on an Agilent 6210 ESI/TOF mass spectrometer. Optical rotation was recorded in CH3Cl3 on Jasco P-1020 polarimeter at room temperature. Infrared (IR) spectra were measured on a Jasco FT/IR-480 plus Fourier Transform infrared spectrometer using KBr pellet. Nuclear magnetic resonance (NMR) spectra were measured on Bruker AV-300 or AV-400 spectrometers. Thin-layer chromatography (TLC) analyses were carried out using pre-coated silica gel GF254 plates . Column chromatographies were performed on silica gel , reverse-phase C18 silica gel  and Sephadex LH-20 . The solvents used in column chromatography and high-performance liquid chromatography (HPLC) were of analytical  grade and chromatographic grade , respectively.Ultraviolet (UV) spectra were determined in CHSalvia miltiorrhiza were purchased from Qingping Herbal Medicine Market  and identified by Prof. Guang-xiong Zhou in Jinan University. A voucher specimen (No. 20090901) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, China.The dried roots of Salvia miltiorrhiza (10 kg) was crushed and extracted using 95% ethanol. The crude extract was suspended in distilled water and partitioned with dichloromethane to afford the dichloromethane fraction 178.5 g, which was revealed to be the total tanshinones  by HPLC analysis.The dried roots of 1 (10.4 mg) and 2 (5.1 mg) were directly crystallized from Fr. 4 and Fr. 1, respectively. Fr. 2 was separated by Sephadex LH-20 eluted with chloroform-methanol (1∶1) and preparative HPLC eluted with acetonitrile-water (70∶30) to afford 6 (25.5 mg) and 4 (4.2 mg). 3 (7.0 mg) was obtained from Fr. 7 by preparative HPLC eluted with acetonitrile-water (75∶25). Similarly, compounds 5 (2.8 mg) and 7 (6.5 mg) were obtained from Fr. 8 by preparative HPLC with the same gradient system as 3. Fr. 5 was chromatographed on silica gel eluted with gradient cyclohexane-acetic ether  to give subfractions Fr. 5a- 5f. Fr. 5c was further purified by preparative HPLC eluted with acetonitrile-water (85∶15) to afford 9 (22.9 mg) and 10 (2.2 mg). Similarly, Fr. 5d was separated by preparative HPLC with the same mobile phase as Fr. 5c to afford 8 (5.5 mg) and 11 (4.2 mg). The purity of compounds was determined to be ≥97% by HPLC analysis. A stack plot showing the HPLC chromatograms of individual compounds were shown in The extract of dichloromethane fraction (10.0 g) was dissolved in ethanol (300 ml), benzaldehyde  and ammonium acetate  were added to the solution. The solution was stirred for 24 hours under 35°C. Then the solvent was removed in vacuum and the residue was washed 2 times by waters to remove the excess ammonium acetate and afford the semi-synthetic mixture (17.1 g). The semi-synthetic mixture was subjected to silica gel (200–300 mesh), eluted with cyclohexane-acetic ether  to give 8 fractions (Fr. 1 to Fr. 8). Compounds 1: m.p. 288–290°C; ESI-MS m/z: 363.4 [M+H+], 361.4 [M-H−]; HR-ESIMS m/z [M+H]+ 363.1510, calculated for C25H18N2O requires 363.1492. UV-Vis (MeOH): 374.0, 355.8, 307.8, 265.8, 203.6 nm; IR (KBr): 3435, 2962, 2930, 1628, 1455, 1367, 1289, 775, 694 cm−1. 1H-NMR δH10.87 , 7.72 , 7.54 , 8.13 , 8.37 , 8.02 , 2.66 , 2.78 , 8.45 , 7.65 , 7.54 , 13.04 ; 13C-NMR δC 126.7 (C-1), 126.2 (C-2), 127.0 (C-3), 133.9 (C-4), 130.4 (C-5), 121.8 (C-6), 118.9 (C-7), 115.6 (C-8), 119.2 (C-9), 130.5 (C-10), 130.3 (C-11), 126.2 (C-12), 112.8 (C-13), 148.8 (C-14), 115.4 (C-15), 142.0 (C-16), 9.7 (C-17), 19.9 (C-18), 136.5 (C-1′), 127.0 , 128.8 , 129.4 (C-4′), 149.0 (C-7′).Compound 2: m.p. 210–212°C; ESI-MS m/z: 365.5 [M+H+], 363.8 [M-H−]; HR-ESIMS m/z [M+H]+ 365.1685, calculated for C25H20N2O requires 365.1648; UV-Vis(MeOH): 370.2, 352.4, 336.8, 275.8, 202.8 nm; IR(KBr): 3215, 2878, 2828, 1605, 1482, 1452, 1348, 1024, 696 cm−1. δH 4.25 , 2.36 , 7.55 , 7.50 , 8.14 , 7.90 , 2.60 , 2.15 , 8.31 , 7.58 , 7.47 , 12.85 . 13C-NMR δC 22.8 (C-1), 24.8 (C-2), 120.4 (C-3), 133.1 (C-4), 132.6 (C-5), 125.5 (C-6), 118.4 (C-7), 118.1 (C-8), 121.7 (C-9), 132.1 (C-10), 130.6 (C-11), 125.5 (C-12), 112.3 (C-13), 147.1 (C-14), 115.5 (C-15), 141.4 (C-16), 9.7 (C-17), 20.0 (C-18), 136.1 (C-1′), 126.6 , 128.7, 129.0 (C-4′), 148.8 (C-7′).Compound 3: m.p. 250–251°C; ESI-MS m/z: 365.5 [M+H+], 363.4 [M-H−]; HR-ESIMS m/z [M+H]+ 365.1663, calculated for C25H20N2O requires 365.1648; UV-Vis(MeOH): 390.6, 371.2, 306.2, 244.6, 204.2 nm; IR(KBr): 3438, 3053, 2961, 2930, 1632, 1454, 1386, 773, 689 cm−1. 1H-NMR δH10.74 , 7.68 , 7.51 , 7.96 , 8.20 , 4.07 , 4.96 , 4.50 , 1.53 , 2.75 , 8.37 , 7.62 , 7.51 , 13.07 ; 13C-NMR δC 126.7 (C-1), 126.0 (C-2), 127.3 (C-3), 133.7 (C-4), 130.4 (C-5), 120.5 (C-6), 120.5 (C-7), 114.7 (C-8), 121.6 (C-9), 130.8 (C-10), 130.1 (C-11), 126.0 (C-12), 111.7 (C-13), 148.4 (C-14), 36.3 (C-15), 78.8 (C-16), 19.8 (C-17), 19.9 (C-18), 135.6 (C-1′), 126.3 , 129.0 , 129.3 (C-4′), 152.4 (C-7′).Compound 4: ESI-MS m/z: 365.6 [M+H+], 363.6 [M-H-]; HR-ESIMS m/z [M+H]+ 365.1655, calculated for C25H20N2O requires 365.1648; UV-Vis(MeOH): 368.2, 353.0, 272.2, 224.4, 203.8 nm; IR(KBr): 3200, 2942, 2851, 1618, 1450, 1386, 693 cm−1. δH 4.09 , 2.04 , 2.61 , 7.90 , 8.10 , 7.94 , 2.61 , 5.07 , 5.65 , 8.32 , 7.59 , 7.48 , 12.90 ; 13C-NMR δC 30.3 (C-1), 23.6 (C-2), 32.6 (C-3), 150.5 (C-4), 144.3 (C-5), 121.5 (C-6), 117.9 (C-7), 118.3 (C-8), 122.1 (C-9), 134.4 (C-10), 131.6 (C-11), 125.4 (C-12), 112.3 (C-13), 147.6 (C-14), 115.4 (C-15), 141.7 (C-16), 9.71 (C-17), 109.5 (C-18), 136.5 (C-1′), 126.7 , 128.7 , 129.0 (C-4′), 148.6 (C-7′).Compound 5: ESI-MS m/z: 399.5 [M+H+], 397.5 [M-H-]; UV-Vis(MeOH): 368.2, 353.0, 272.2, 224.4, 203.8 nm; IR(KBr): 3395, 2925, 1624, 1385, 1026, 694 cm−1. δH 2.12 , 1.95 , 3.57 , 4.14 , 7.83 , 8.12 , 7.91 , 2.61 , 4.01 , 3.77 , 8.32 , 7.58 , 7.47 , 12.83 ; 13C-NMR δC 29.9 (C-1), 20.4 (C-2), 38.5 (C-3), 69.2 (C-4), 141.8 (C-5), 123.9 (C-6), 117.6 (C-7), 117.6 (C-8), 122.0 (C-9), 132.5 (C-10), 130.7 (C-11), 125.2 (C-12), 112.0 (C-13), 147.2 (C-14), 115.3 (C-15), 141.3 (C-16), 9.5 (C-17), 71.3 (C-18), 136.4 (C-1′), 126.6 , 128.6 , 128.8 (C-4′), 148.7 (C-7′).Compound 6: m.p.128–130°C; ESI-MS m/z: 381.5 [M+H+], 379.5 [M-H−]; HR-ESIMS m/z [M+H]+ 381.1981, calculated for C26H24N2O requires 381.1961; UV-Vis(MeOH): 352.4, 383.2, 260.6, 204.2 nm; IR(KBr): 3500, 2924, 2854, 1608, 1455, 1386, 1260, 691 cm−1. δH 3.96 , 1.95 , 1.75 , 7.61 , 8.08 , 7.88 , 2.66 , 1.37 , 1.37 , 8.31 , 7.57 , 7.47 , 12.80 ; 13C-NMR δC 30.8 (C-1), 19.6 (C-2), 38.5 (C-3), 34.3 (C-4), 142.7 (C-5), 123.8 (C-6), 117.8 (C-7), 117.1 (C-8), 122.6 (C-9), 132.8 (C-10), 130.8 (C-11), 125.4 (C-12), 111.8 (C-13), 147.1 (C-14), 115.3 (C-15), 141.2 (C-16), 9.7 (C-17), 32.0 (C-18), 32.0 (C-19), 136.4 (C-1′), 126.6 , 128.7 , 128.9 (C-4′), 148.8 (C-7′).Compound 7: ESI-MS m/z: 397.5 [M+H+], 395.6 [M-H−]; HR-ESIMS m/z [M+H]+ 397.1952, calculated for C26H24N2O2 requires 397.1911; UV-Vis(MeOH): 353.0, 337.4, 261.0, 203.8 nm; IR(KBr): 3361, 2929, 2866, 1608, 1452, 1397, 1028, 694 cm−1. δH 3.97 , 2.00 , 1.55 , 7.60 , 8.07 , 7.89 , 2.61 , 1.31 , 3.54 , 3.62 , 8.32 , 7.58 , 7.47 , 12.80 ; 13C-NMR δC 30.7 (C-1), 19.2 (C-2), 32.3 (C-3), 34.5 (C-4), 139.9 (C-5), 124.1 (C-6), 117.4 (C-7), 117.1 (C-8), 122.6 (C-9), 134.2 (C-10), 130.8 (C-11), 125.4 (C-12), 111.8 (C-13), 147.1 (C-14), 115.2 (C-15), 141.1 (C-16), 9.5 (C-17), 26.8 (C-18), 69.7 (C-19), 136.3 (C-1′), 126.6 , 128.6 , 128.8 (C-4′), 148.8 (C-7′).Compound 8: ESI-MS m/z: 425.4 [M+H+], 423.4 [M-H−]; HR-ESIMS m/z [M+H]+ 425.1875, calculated for C27H24N2O3 requires 425.1860; UV-Vis(MeOH): 353.2, 337.4, 262.4, 203.8 nm; IR(KBr): 3392, 2946, 2860, 1726, 1610, 1451, 1384, 1256, 694 cm−1. δH 4.02 , 2.25 , 1.88 , 7.34 , 8.09 , 7.93 , 2.61 , 1.61 , 3.60 , 8.33 , 7.59 , 7.48 , 12.91 ; 13C-NMR δC 30.1 (C-1), 19.6 (C-2), 34.2 (C-3), 46.9 (C-4), 148.6 (C-5), 124.7 (C-6), 118.0 (C-7), 117.6 (C-8), 122.5 (C-9), 133.5 (C-10), 130.7 (C-11), 125.6 (C-12), 112.3 (C-13), 145.8 (C-14), 115.3 (C-15), 141.7 (C-16), 9.9 (C-17), 27.6 (C-18), 177.5 (C-19), 52.5 (COCH3), 136.3 (C-1′), 126.7 , 128.7 , 129.0 (C-4′), 148.6 (C-7′).Compound 9: ESI-MS m/z: 383.4 [M+H+], 381.5 [M-H−]; HR-ESIMS m/z [M+H]+ 383.2111, calculated for C26H26N2O requires 383.2118; UV-Vis(MeOH): 367.4, 354.4, 259.0, 236.6, 205.2 nm; IR(KBr): 3408, 3061, 2956, 2924, 1604, 1454, 1406, 1382, 696 cm−1. δH 3.91 , 1.93 , 1.73 , 7.46 , 7.76 , 3.97 , 4.87 , 4.42 , 1.46 , 1.33 , 1.33 , 8.25 , 7.55 , 7.42 , 12.83 ; 13C-NMR δC 30.6 (C-1), 19.6 (C-2), 38.5 (C-3), 34.2 (C-4), 142.8 (C-5), 122.6 (C-6), 119.4 (C-7), 116.3 (C-8), 129.1 (C-9), 132.0 (C-10), 130.9 (C-11), 124.8 (C-12), 110.0 (C-13), 146.4 (C-14), 36.2 (C-15), 78.7 (C-16), 19.8 (C-17), 31.8 (C-18), 31.8 (C-19), 135.3 (C-1′), 125.8 , 128.7 , 128.4 (C-4′), 152.0 (C-7′).Compound 10: ESI-MS m/z: 357.4 [M+H+], 355.4 [M-H−], 379.4 [M+Na+], 735.1 [2M+Na+]; UV-Vis (MeOH): 368.2, 353.0, 272.2, 224.4, 203.8 nm; IR(KBr): 3213, 2930, 2865, 1644, 1563, 1472, 1450, 1384, 1278, 702 cm−1. HR-ESIMS m/z [M+H]+ 357.1597, calculated for C23H20N2O2 requires 357.1598. δH 3.54 , 1.84 , 1.67 , 7.52 , 7.76 , 1.31 , 1.31 , 8.24 , 7.56 , 7.54 , 14.01 ; 13C-NMR δC 29.8 (C-1), 19.1 (C-2), 37.6 (C-3), 34.7 (C-4), 154.4 (C-5), 127.1 (C-6), 127.4 (C-7), 131.6 (C-8), 137.1 (C-9), 135.1 (C-10), 128.8 (C-11), 168.1 (C-12), 182.0 (C-13), 180.4 (C-14), 31.5 (C-18), 31.5 (C-19), 136.5 (C-1′), 126.8 , 129.0 , 129.0(C-4′), 145.3 (C-7′).Compound 11: ESI-MS m/z: 384.3 [M+H+], 382.4 [M-H−], 789.2 [2M+Na+]; UV-Vis (MeOH): 368.2, 353.0, 272.2, 224.4, 203.8 nm; IR(KBr): 3358, 2922, 2851, 1466, 1384, 1289, 775, 693 cm−1. HR-ESIMS m/z [M+H]+ 384.1588, calculated for C25H21NO3 requires 384.1594. 1H-NMR δH 10.25 , 7.72 , 7.56 , 8.04 , 8.38 , 3.89 , 4.01 , 4.04 , 1.51 , 2.77 , 8.33 , 7.68 , 7.66 ; 13C-NMR δC 126.3 (C-1), 126.0 (C-2), 127.9 (C-3), 133.9 (C-4), 130.8 (C-5), 121.6 (C-6), 121.3 (C-7), 113.4 (C-8), 121.8 (C-9), 129.4 (C-10), 129.2 (C-11), 149.2 (C-12), 113.4 (C-13), 159.5 (C-14), 33.8 (C-15), 64.9 (C-16), 16.1 (C-17), 19.7 (C-18), 126.9 (C-1′), 126.8 , 129.4 , 131.2 (C-4′), 149.4 (C-7′).Compound All animal experiments were conducted in accord with the ethical guidelines of the Animal Ethics Committee, Institute of Chinese Medical Sciences, University of Macau and the protocol was approved by Animal Ethics Committee, Institute of Chinese Medical Sciences, University of Macau prior to the initiation of the experiments.3--2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were acquired from Sigma (USA). VEGFR tyrosine kinase inhibitor II (VRI), FGFR inhibitor (SU5402), EGFR inhibitor (PD 153035), non-selective eNOS inhibitor (L-NAME), Src inhibitor (PP2), P38 MAP kinase Inhibitor III (P38i), Raf kinase inhibitor IV (Rafi), MEK1/2 inhibitor (MEK1/2i), ERK1/2 inhibitor (ERK1/2i), PI3K inhibitor (LY 294002), HIF-1 inhibitor (HIFi) and three subclasses of Akt inhibitor (Akti) were purchased from Calbiochem , and each compound was dissolved in their recommended solvent, as per manufacturers. Tan IIA, CPT and Tan I were purchased from Jiangsu Yongjian Co., Ltd . Tanshinone derivatives and three tanshiones were dissolved in DMSO at a concentration of 50 mM. Purified water was prepared using a Milli-Q purification system from Millipore .Tg(fli-1a:EGFP)y1 and Wide-type, were maintained as previously described in our previous paper The zebrafish strains, including Zebrafish embryos were generated by natural pair-wise mating (3–12 months old) and were raised at 28°C in embryo medium. Healthy, hatched zebrafish were selected at 21 hour post-fertilization (hpf) and distributed into a 12-well microplate (15 embryos per well for fluorescence observation) or 6-well microplate (30 embryos per well for RNA extract). The embryos which received embryo medium with 0.1% DMSO (v/v) only served as vehicle controls. The medium in all treatment groups was refreshed every 24 h and all groups were incubated at 28 °C.Restorative treatment plan in VRI-treated zebrafishEmbryos at 21 hpf were pre-treated with 500 ng/ml VRI for 3 h. The VRI was then washed out and replaced with either 0.1% DMSO (v/v) embryo medium or three tanshinones and eleven derivatives for another 24 h and 48 h.b. Specific inhibitors assay in VRI-treated zebrafish10 (0.3 µM) and 10 (0.3 µM) plus co-treatment with inhibitors for 24 h.Embryos at 21 hpf were pretreated with 500 ng/ml VRI for 3 h. The VRI was then washed out and replaced with either 0.1% DMSO (v/v) embryo medium or only compound At 48 hpf or 72 hpf, zebrafish embryos were removed from microplates and observed for viability and gross morphological changes under a fluorescence microscope  equipped with a digital camera . Images were analyzed with Axiovision 4.2 and Adobe Photoshop 7.0. In the control group, intersegmental vessels (ISVs) showing sprouting and elongating from dorsal aorta (DA) or posterior cardinal vein (PCV) to dorsal longitudinal anastomotic vessels (DLAVs) were defined as intact. And in the VRI-only treatment group or some drug treatment groups, some of the ISVs observed sprouting from DA or PCV, but did not form complete ISVs, were defined as defective. The number of intact and defective ISVs in each zebrafish embryo was counted.kdrl (flk-1A), kdr (flk-1B) and flt1  in the ABI 7500 Real-Time PCR System . The expression of kdrl, kdr and flt-1 was normalized to the amount of bactin1, using the relative quantification method described by the manufacturer.The wild-type zebrafish embryos were treated with the protocols described above. At 48 hpf, total RNA was extracted from 30 embryos in each treatment group, using the RNeasy Mini Kit  in accordance with the manufacturer's instructions. RNA was reverse transcribed into single-strand cDNA using III First-Strand Synthesis System for RT-PCR , followed by real-time PCR using the Universal PCR Master Mix, and 250 nM custom primers for zebrafish bactin1 primers were 5′-CAAGATTCCATACCCAGGAAGGA -3′ (F) and 5′-CAAGATTCCATACCCAGGAAGGA-3′ (R). The zebrafish kdrl primers were 5′-GACCATAAAACAAGTGAGGCAGAAG-3′ (F) and 5′- CTCCTThe zebrafish kdr primers were 5′-CAAGTAGGTTTGACAGAGCGATA-3′ (R). The zebrafish 5′-GGTCTGCTACACAACGCATTACTCGTTTTCTCAACCTAAGC-3′ (F) and flt-1 primers were 5′-AACTCACAGACCAGTGAATAAC- 3′ (R). The zebrafish 5′-GCCCTGTAACGTGTGCACTAAA-3′ (R).ACAAGATC- 3′ (F) and 2 tissue culture flasks in a humidified atmosphere containing 5% CO2, at 37°C.Cancer cells were maintained in suitable medium  , supplemented with 10% heat-inactivated fetal bovine serum (FBS) , and 1% antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin). Cells were grown in 75 cm5 cells/well overnight to allow for cell attachment. The working solutions of compound 10 and tanshinones were dispensed appropriately into the partitioned 96-well plates then incubated for another 48 h. The cells were also incubated with DMSO (0.1%) which served as the vehicle control. Then the medium was discarded and cells were incubated for 4 h at 37°C in MTT solution . The solution was then replaced by 100 µl DMSO to dissolve the violet formazan crystals in intact cells. The absorbance was measured at a wavelength of 570 nm by a SpectraMaxR M5 Multi-Mode Microplate Readers . Cell viability was expressed as a percentage of the vehicle control.The cancer cells were plated into a 96-well plate at a concentration of 0.8–1×10Each experiment was done in triplicate at least. The mean and standard deviations were compared using Students' t-test with the following statistical criteria: p<0.05, significant; p<0.01, highly significant; p<0.001 extremely significant. All relevant data are presented as mean±SEM.Salvia miltiorrhiza and identified by HPLC analysis. One-pot synthesis was then carried out by reacting the total tanshinones fraction with benzaldehyde and ammonium acetate under 35°C to obtain a semi-synthetic mixture  fraction was isolated and enriched from an ethanol extract of  mixture . A compa mixture . The seme , suggesting that the phenylimidazole might be replaced by a phenyloxazole ring, which was further confirmed by the absence of a proton signal for the NH group.The last miscellaneous group included compounds 1H, 13C and DEPT-NMR spectra, see Section 3 of 1, 2, 3, 6 and 10 were further confirmed by single crystal X-ray analysis  and one oxazole derivative (11), through two different pathways. Based on this hypothetical reaction mechanism, we deduced that 1–11 are derived from different natural parental tanshinones including Tan I (1a) 2a) 3a) 4a) 5a) 6a) 7a) 8a) 9a) 10a) 11a) 6a), CPT (9a) and Tan I (1a) which are generally regarded as major representative components, most of the remaining tanshinones are present in low abundance in Salvia miltiorrhiza. Based on the natural parental tanshinones, these new derivatives could be accorded the trivial names phenylimidazoletanshinone I (1), 1,2-dihydrophenylimidazoletanshinone I (2) 3), methylenephenylimidazoletanshinquinone (4) 5), phenylimidazoletanshinone IIA (6) 7), methyl phenylimidazoletanshinonate (8) 9), phenylimidazoleneocryptotanshinone II (10), and phenyloxazoleneocryptotanshinone (11).Compounds 3, 9 and 11, and at C-4 for 5, 7 and 8. The absolute configurations of these natural tanshinones were well established R for 3a, 9a and 11a; 4S for 7a and 8a; 4R for 5a). Furthermore, final refinement of the CuKα X-ray diffraction data of 3 resulted in a small Flack parameter 0.1, confirming the absolute configuration of this compound.It should be noted that there is a chiral atom at C-16 for 1–11 were determined in a chemical-induced blood vessels loss zebrafish model 1–11 and three major tanshinone controls for another 24 h and 48 h, the VRI-induced blood vessels loss in ISVs, DLAVs and SIVs regions were restored to different degrees. Of 1–11, compound 10 (EC50 =  0.026 µM) and 3 (EC50 =  4.03 µM) exhibited significantly potency and efficacy in the recovery of blood vessels loss in VRI-treated zebrafish  for 3 h, and then treated with 10  for 24 h. Total RNA from different treatment groups of embryos was isolated and reverse transcribed to cDNA. The relative mRNA expressions of flt-1 encoding the VEGFR1 as well as kdrl and kdr encoding VEGFR2 were determined using real-time qPCR. The gene expression results clearly showed that increasing dosages of 10 exhibited a significant concentration-dependent recovery of down-regulation of kdrl, kdr and flt-1 mRNA expression in VRI-treated zebrfish. Compared to the VRI-only treatment group, the relative change in gene expression level in 1 µM 10 treatment group was 2.01-fold for kdrl (p<0.05), 1.80-fold for kdr (p<0.01) and 2.98-fold for flt1 (p<0.01) (10 reversed the VRI-induced down-regulation of the expression of three key VEGF receptors in zebrafish.In order to confirm the vascular phenotypic effects observed in (p<0.01) . Thus, t10 also involved epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling pathways, the effects of specific small molecule inhibitors on 10-induced angiogenesis were studied. SU5402, a cell-permeable small molecule compound, is an ATP-competitive inhibitor of the FGFR1 tyrosine kinase 10 for 24 h, almost entirely inhibited the restorative effect of 10 on blood vessels loss in VRI-treated zebrafish . The results revealed that 10 exhibited cytotoxic activities against MCF-7 (IC50 =  3.3 µM), MDA-231 (IC50 =  6.5 µM) and A549 (IC50 =  17.9 µM)  . It is nectively .In our study, a series of new tanshinone derivatives were successfully synthesized from a mixture of natural tanshinones through one-pot synthesis approach. The present pilot study, for the first time, proved the concept of feasibility using a modified one-pot synthesis to modify minor tanshinones, which are hardly to be isolated with sufficient quantity for chemical modification by conventional organic synthesis approach. We found that some of the new tanshinone derivatives exhibited strong protective and restorative effects against blood vessels loss in zebraifsh. And the newly identified derivatives could serve as new lead candidates for further development as angiogenesis therapeutic agents.Salvia miltiorrhizaTanshinones are a group of diterpenoid quinones with unique structural scaffolds and promising biological activities. However, a substantial number of tanshinones which are present in very low quantities in 10 as the most potent and effective angiogenic compound for restoring blood vessels damage which may have been the result of improvement in either one or a combination of various drug-like properties. Further studies to investigate the change of pharmacodynamic and pharmacokinetic properties of 10 were warranted. In addition, the new tanshinone derivatives exhibited limited close relatedness in their structures as they evolved from the random modification of different natural tanshinones. Therefore, no conclusive relationship between their structures and angiogenesis activity in VRI-treated zebrafish could be drawn.Angiogenesis deficiencies are associated with various human diseases (e.g. cardio-cerebrovascular problems and ulcers). Our zebrafish angiogenesis assay which was produced by inactivation of the VEGF signaling pathway was more physiological relevant assay for identifying pro-angiogenesis agents against diseases associated with angiogenesis deficiencies 10-treatment, these results alone did not entirely reflect the activation of the downstream signaling targets  involved in VEGF/FGF-induced angiogenesis because their activation usually occurs via phosphorylation in post-translation regulation 10. Our results demonstrated that 10 probably promoted angiogenesis through VEGF and/or FGF by activating Src, PI3K, P38, Raf, MEK1/2 and ERK1/2 kinases but not HIF-1, eNOS and Akt kinases. Taken together, all these evidences showed that the angiogenesis activity of 10 probably involved the interaction of VEGF/FGF-Src-MAPK and PI3K-P38 signaling pathways in VRI-treated zebrafish  ebrafish .10, the most potent new angiogenic tanshinone derivative identified without any promoting proliferation effects in three cancer cells, had potential for further development as a therapeutic agent for diseases associated with imbalanced angiogenesis such as ischemic heart disease and traumatic injuries. In addition, both the mRNA expression assay and blocking assay of specific kinases inhibitors provided insights into the mechanism underlying the angiogenesis effect of 10. It is worthy of note that the zebrafish screening model used in this study is based on embryonic development, but not a real pathology model. Further investigation of the pharmacodynamic data of 10 based on the vascular injury pathological model was warranted. In addition, physiological angiogenesis is a vital process in growth and development of organism. Since the process is complicated, and typically consists of cell proliferation, migration, invasion and tube formation 10 could directly act on either one of the biological steps in angiogenesis on human umbilical vein endothelial cells (HUVECs). In our results, 10 could significantly promote the migration and invasion of HUVECs in a concentration-dependent manner, as effective as the effects of VEGF. However, the HUVECs proliferation stimulating effect of 10 was mild when compared with that of VEGF Click here for additional data file.


Organoselenium compounds find applications due to their biological activity and useful synthetic properties  are usually prepared from alkyl halides and Seous NaOH , liquid ous NaOH , sodium ous NaOH , and sodous NaOH  were repous NaOH  or selenous NaOH , are les2) and 1,3-bis-2-selena propane (4).Herein we report improved methods for the preparation of pyrazole- and benzotriazole-containing selenoethers 1,5-bis-3-selena pentane  in aqueous NaOH  (5) was obtained in high yield (84%); therefore selenoether 2 and not some other impurity acted as a ligand and the structure of the complex can be used for the elucidation of compound 2 structure.In order to establish the structures of compounds 5 crystallizes in a monoclinic crystal system; crystallographic parameters and details of the diffraction experiment are given in  5 it is evident that compound 2 is indeed a selenoether and not a diselenide. The lengths of C–C and C–N bonds in pyrazole rings are within the usual range +), 203 ([M-PzCH2CH2]+), 109 ([PzCH2]+). Anal. Calc'd for C14H22N4Se (325.31): C, 51.59; H, 6.82; N, 17.22. Found: C, 51.97; H, 7.01; N, 17.70.A suspension of selenium , sodium formaldehydesulfoxylate dihydrate , and NaOH  in water (5 mL) was stirred at room temperature, until the initially formed red solution turned colorless and white precipitate of Na 4 was prepared similarly to compound 2 from 2.10 g (12.54 mmol) chloro-derivative 1, 0.50 g (6.27 mmol) of selenium, 2.31 g (15.0 mmol) of sodium formaldehydesulfoxylate dihydrate, and 1.38 g (34.5 mmol) of NaOH in 6 mL of water and 15 mL of acetonitrile. Yield 1.94 g (90%), colorless crystals, mp 182-183°C (DMF). IR  1612, 1496, 1453 (νBta), 754 (νC–Se). 1H NMR (DMSO-d6): 6.19 , 7.44 , 7.58 , 7.97 , 8.08 . 13C NMR (DMSO-d6): 42.5 (CH2), 111.1 (7-C-Bta), 119.3 (4-C-Bta), 124.4 (5-C-Bta), 127.5 (6-C-Bta), 131.9 (8-C-Bta), 145.4 (9-C-Bta). Anal. Calc'd for C14H12N6Se (343.25): C, 48.99; H, 3.52. Found: C, 49.30; H, 3.83.Selenoether 2  in acetone (0.2 mL), solution of Cu (NO3)2·3H2O  in acetone (0.2 mL) was added. After standing for 2 hours, deep-green crystals of the complex were formed, which were filtered, washed with acetone, and dried. The crystals were suitable for X-ray crystal structure determination. Yield 0.086 g (84%). IR  1556 (νPz), 1026 (Pz breating), 811 (νC–Se). Anal. Calc'd for C14H22CuN6O6Se (512.87): C, 32.79; H, 4.32; N, 16.39. Found: C, 33.04; H, 4.50; N, 15.96.To a solution of selenoether4 and 5.Supplementary Material contains crystallographic information files (CIF) for compounds


Sanrafaelia ruffonammari Verd and Ophrypetalum odoratum Diels that belongs to the rare genera confined to East African coastal forests led to the isolation of enantiomeric styrylpyrone dimer, (±)-5-methoxy-7-phenyl-[4-methoxy-2-pyronyl]-1-(E)-styryl-2-oxabicyclo-[4.2.0]-octa-4-en-3-one (1) alongside (+)-6-styryl-7,8-epoxy-4-methoxypyran-2-one (2) and the enantiomeric (+)- (3) and (−)-6-styryl-7,8-dihydroxy-4-methoxypyran-2-ones (4). Their structures were established by means of spectroscopic methods. In this paper we reveal for the first time the occurrence of styrylpyrones in East African biodiversity. (+)-6-Styryl-7,8-epoxy-4-methoxypyran-2-one (2) and the dihydroxystyrylpyrone enantiomer (3) showed in vitro antifungal activity against Candida albicans at a concentration of 24.4 and 26.2 µM with zones of inhibition of 17 and 9 mm, respectively. Compound 2 exhibited strong activity in the brine shrimp test with LC50 = 1.7 µg/mL. Their high cytotoxic and antifungal activities render them candidates for further scientific attention for drug development programs against cancer and microbial infections.Phytochemical investigation of  Sanrafaelia ruffonammari Verd and Ophrypetalum odoratum Diels belong to the rare genera that are confined to East African coastal forests, of which S. ruffonammari was only recently described by Verdcourt, 1999 -1-(E)-styryl-2-oxabicyclo-[4.2.0]-octa-4-en-3-one (1), (+)-6-styryl-7,8-epoxy-4-methoxypyran-2-one (2) and the enantiomeric (+)- (3) and (−)-6-styryl-7,8-dihydroxy-4-methoxypyran-2-ones (4), while the root bark of S. ruffonammari yielded (+)-6-styryl-7,8-epoxy-4-methoxypyran-2-one (2) and (−)-6-styryl-7,8-dihydroxy-4-methoxypyran-2-one (3). rt, 1999 . The plaS. ruffonammari and stem and root barks, and leaves of O. odoratum were separately fractionated by vacuum liquid chromatography (VLC) over silica gel and repeated column chromatography on silica gel and Sephadex LH-20 column to afford the enantiomeric styrylpyrone dimer, (±)-5-methoxy-7-phenyl-[4-methoxy-2-pyronyl]-1-(E)-styryl-2-oxabicyclo-[4.2.0]-octa-4-en-3-one (1), (+)-6-styryl-7,8-epoxy-4-methoxypyran-2-one (2) and the enantiomeric (+)- (3) and (−)-6-styryl-7,8-dihydroxy-4-methoxypyran-2-ones (4) from the stem and root barks, and leaves of O. odoratum while the root bark of S. ruffonammari yielded (+)-6-styryl-7,8-epoxy-4-methoxypyran-2-one (2) and (−)-6-styryl-7,8-dihydroxy-4-methoxypyran-2-one (3). The structures of styrylpyrones were elucidated by extensive spectroscopic methods and comparison with literature data  and gradient elution was applied. The vacuum was generated from a membrane pump.Optical rotations were measured on Jasco-P-1020 polarimeter. Infrared (IR) spectra, taken in chloroform solutions were recorded on a Shimadzu Model IR-435 spectrophotometer with absorptions given in wave numbers  over silica gel and repeated column chromatography on silica gel and Sephadex LH-20 yielded the enantiomeric styrylpyrone dimer, (±)-5-methoxy-7-phenyl-[4-methoxy-2-pyronyl]-1-(E)-styryl-2-oxabicyclo-[4.2.0]-octa-4-en-3-one (1), (+)-6-styryl-7,8-epoxy-4-methoxypyran-2-one (2) and the enantiomeric (+)- (3) and (−)-6-styryl-7,8-dihydroxy-4-methoxypyran-2-ones (4) from the stem and root barks, and leaves of O. odoratum while the root bark of S. ruffonammari yielded (+)-6-styryl-7,8-epoxy-4-methoxypyran-2-one (2) and (−)-6-styryl-7,8-dihydroxy-4-methoxypyran-2-one (3).The air-dried powdered plant materials were soaked consecutively in petrol, chloroform and ethanol for 2 × 48 h. The extract from the root bark of Artemia salina) larvae as test organisms was carried out using the method on Meyer et al. +), 228 , 200 , 185 (25), 157 , 129 (25), 103 , 77 .Yield: 39 mg; m.p. 191 °C. Anisaldehyde: Pink. [α]D = + 136.2° .Anisaldehyde: Pink. IR νmax 3400, 3083, 2939, 1717, 1643 and 1567 cm−1. MS, m/z (% rel. int.) 245 , 244 (30), 228 (17), 215 (17), 188 (18), 187 (60), 157 (12), 155 (20), 138 (100), 125 (40), 110 (60), 112 (22), 95 (65), 80 (40), 77 (47), 69 (81), 59 (20) and 52 (30). UV, νmax 211, 224, 241 and 287 nm. 1H NMR and 13C NMR see Tables Yield: 88 mg from root bark, 60 mg from stem bark; m.p. 110 °C. [α]D = + 126.9° . UV, νmax 210 and 290 nm, IR, νmax 3573, 3353, 1708, 1664, 1567, 1452, 1409, 1245, 1031, 811 and 699 cm−1. 1H MS, m/z (% re int) 262 (M+), 156 , 127 (30), 107 , 79 (25), 69 (18), 59 (10) and 39 (10). 1H NMR and 13C NMR data see Tables Overall yield 317 mg (0.02 % based on dry leaves) m.p. 137 °C. [α]D = + 126.9° . UV, λmax 210, 226 and 290 nm, IR, υmax 3573, 3353, 1708, 1664, 1567, 1452, 1409, 1245, 1031, 811 and 699 cm−1. MS, m/z (% rel. int.) 262 , 156 , 127 (30), 107 , 79 (25), and 69 (18). 1H NMR and 13C NMR see Tables Yield: 317 mg from leaves, m.p. 137 °C. Anisaldehyde: Pink. [α]


The structures of these metabolites were determined by HR-MS and 1D and 2D NMR. The absolute configurations of compounds 1, 5 and 6 were assigned by the modified Mosher’s method and by CD data. Sargassopenilline C (3) inhibited the transcriptional activity of the oncogenic nuclear factor AP-1 with an IC50 value of 15 µM.Seven new 6,6-spiroketals, sargassopenillines A–G ( Penicillium thomii KMM 4645 and Penicillium lividum KMM 4663 associated with the marine brown alga Sargassum miyabei + and was in accordance with 13C NMR data  as for sargassopenilline A (1). The HMBC correlations from H-4 (δH 5.75) to 4-Ac (δC 171.1), from 4-Ac  to C-4 (δC 66.1), 4-Ac (δC 171.1) and downfield chemical shift of H-4 indicated replacement the alcohol group at C-4 by the acetoxy group. The COSY-45 data and HMBC correlations H-9 (δH 5.07)/C-3 (δC 96.5), 9-Ac (δC 170.3), C-10 (δC 24.2), C-11 (δC 26.8); H3-14 (δH 1.13)/C-11, C-12 (δC 68.4) and H-12 (δH 3.98)/C-3 established the structure of the 9-acetoxy-12-methylpyran ring and indicated the presence of spirocyclic system in 2. The NOE correlation H-12/ H-1b (δH 4.57) indicated a β-orientation for the 14-methyl group and suggested the configuration of C-3 to be S. The small coupling constants of the H-9 signal at δ 5.07  and biogenetic relationship between sargassopenilline A and 2 suggested an α-orientation for the 9-acetoxy group. Unfortunately, the correlations observed in the NOESY spectrum did not allow us to unequivocally establish the relative configuration at C-4. Compound 2 was named sargassopenilline B.The molecular formula of NMR data . The str3 was determined to be C21H26O11 by a HRESIMS peak at m/z 477.1365 [M + Na]+ and was in accordance with 13C NMR data. The 1H and 13C NMR spectra of 3  and three acetoxy  groups, three methylenes  including one oxygen-bearing, four oxygenated methines , two carbonyl functions (δC 192.2 and 198.0), one tetrasubstituted double bond (δC 130.2 and 139.8) and one double oxygenated quaternary carbon (δC 98.4). The HMBC correlations H3-15 (δH 1.57)/C-6 (δC 198.0), C-7 (δC 84.7), C-8 (δC 192.2); H-5a (δH 3.40)/C-4 (δC 64.6), C-4a (δC 130.2), C-6, C-7 and H-1a (δH 4.70)/C-3 (δC 98.4), C-4, C-4a, C-8 and C-8a (δC 139.8) indicated the presence of the bicyclic isochromene core in 3 with carbonyl functions at C-6 and C-8 and a methyl group at C-7. The locations of the acetoxy groups at C-4 and C-7 were evident from the HMBC correlations from 4-Ac (δH 2.08) to C-4 (δC 64.6), 4-Ac (δC 170.5) and from 7-Ac (δH 2.16) to C-7 (δC 84.7), 7-Ac (δC 169.5). The interpretation of the COSY and HSQC data revealed one isolated spin system: (-CH(O)(9)-CH(O)(10)-CH2(11)-CH(CH3)(12)-). These data and the HMBC correlations H-9 (δH 4.99)/C-3 (δC 98.4), 9-Ac (δC 169.3), C-10 (δC 65.8), C-11 (δC 34.7); H-11a (δH 1.74)/C-10; H-11b (δH 1.71)/C-12 (δC 63.5); H3-14 (δH 1.23)/C-3, C-11 and C-12 established the presence of the 9-acetoxy-10-hydroxy-12-methylpyran ring in 3.The molecular formula of tra of 3  by DEPT C 63.5) in comparison with the spectra of 2 (δC 68.4) (γ-effect) indicated that 3 contained a secondary alcohol function with an α-orientation. Furthermore, NOE correlations H-1b (δH 4.25)/H-12 indicated a β-orientation for the 14-methyl group and suggested the configuration of C-3 to be S. We observed a strong NOE correlation between H-9 and H-10 and small coupling of the H-9 signal at δ 4.99  proposed to be an α-orientation for the 9-acetoxy group. The configurations at C-4 and C-7 have not been determined. Compound 3 was named sargassopenilline C.The small coupling of the H-10 signal at δ 3.90  and downfield chemical shift of C-12 (δ4 was determined to be C19H22O9 by a HRESIMS peak at m/z 417.1157 [M + Na]+ and was in accordance with 13C NMR data. The 1H and 13C NMR (3-15 (δH 1.95)/C-6 (δC 151.2), C-7 (δC 117.4), C-8 (δC 186.0); 6-OH (δH 6.95)/C-5 (δC 180.6), C-6, C-7, H-1a (δH 4.73)/C-3 (δC 97.1), C-4a (δC 144.2), C-8, C-8a (δC 130.7) and H-4 (δH 5.92)/C-3, C-4a, C-5 and C-8a indicated the presence of the bicyclic isochromene core in 4 with carbonyl functions at C-5 and C-8 and hydroxyl and methyl groups at C-6 and C-7, respectively. The location of the acetoxy group at C-4 was evident from the HMBC correlations from 4-Ac (δH 1.97) to C-4 (δC 59.4) and 4-Ac (δC 168.3). The structure of the 9-acetoxy-12-methyl pyran ring and the presence 6,6-spiroring system in 4 was found by extensive NMR spectroscopy  as for sargassopenilline B (2). The NOE correlation H-12/ H-1b (δH 4.35) indicated a β-orientation for the 14-methyl group and suggested the configuration of C-3 to be S. The small coupling constants of the H-9 signal at δ 4.95  and biogenetic relationship between sargassopenilline A and 4 suggested an α-orientation for the 9-acetoxy group. The configuration at C-4 has not been determined. Compound 4 was named sargassopenilline D.The molecular formula of  13C NMR , DEPT an5 was determined to be C15H22O6 by a HRESIMS peak at m/z 299.1483 [M + H]+ and was in accordance with 13C NMR data. The 1H and 13C NMR spectra , 149.5 (C) and 126.6 (C), suggested the presence of a carbonyl carbon and one tetrasubstituted double bond. The 1H and 13C data observed for the 3,4,5,6,7,8-hexahydroisochromene core resemble those reported for pestafolide A + and by 13C NMR analyses. The 1H and 13C NMR spectra of 7  and two acetoxy  groups, four methylenes  including one oxygen-bearing, three oxygenated methines  and one double oxygenated quaternary carbon (δC 97.0). The remaining functionalities, corresponding to the carbon signals at δC 214.4 (C), 141.8 (C) and 127.3 (C), suggested the presence of a ketone function and one tetrasubstituted double bond.The molecular formula of compound tra of 7  by DEPT H 2.92)/C-4a (δC 127.3), C-6 (δC 214.4), C-7 (δC 77.9) and C-7a (δC 141.8); H3-14 (δH 1.36)/C-6, C-7 and C-7a revealed the presence of 2-hydroxy-2-methylcyclopent-3-enone moiety in 7. Furthermore, HMBC correlations H-1a (δH 4.43)/C-3 (δC 97.0), C-4 (δC 64.3), C-4a, C-7a and H-4 (δH 5.25)/C-3, C-4a, C-5 (δC 40.1), C-7a and 4-Ac (δC 170.4) show that 7 contains an unusual tetrahydrocyclopentapyranone ring system in the molecular structure. The structure of the 9-acetoxy-12-methylpyran ring and the presence 6,6-spiroring system in 7 was found by extensive NMR spectroscopy  as for sargassopenilline B (2). Thus compound 7 is a new spiroketal-containing natural compound and it was named sargassopenilline G.The HMBC correlations H-5b (δH 4.23) (2.854 Å) indicated a β-orientation for the 13-methyl group and suggested the configuration of C-3 to be S. The small coupling constants of the H-8 signal at δ 5.02  and biogenetic relationship between sargassopenilline A and 7 suggested an α-orientation for the 8-acetoxy group. These data and the observed NOE correlations H3-14/H-1b (δH 4.23), H-5a (δH 3.02) and H-5a/H-4 indicated the relative configuration of 7 (The NOE correlation H-11/H-1b (δion of 7 .1–3 and 7 were assayed for their cytotoxic activity against MDA-MB-231 and JB6 Cl41 cell lines. None of the compounds exhibited cytotoxicity (IC50 < 100 μM).Sargassopenillines 1–3 and 7 on the basal AP-1-dependent transcriptional activity was also studied using JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by an AP-1-DNA binding sequence 20D −45 ; UV (MeOH) λmax (log ε) 235 (3.22), 282 (3.07) nm; CD  λmax (Δε) 233(+0.06), 247 (−0.04), 282 (+0.04), 350 (+0.01) nm; 1H and 13C NMR data, see m/z 319.1157 [M + Na]+ .Sargassopenilline A (2): Amorphous solid; [α]20D −137 ; UV (MeOH) λmax (log ε) 219 (3.77), 284 (3.25) nm; CD  λmax (Δε) 244 (−0.86), 274 (−0.35), 308 (+0.20), nm; 1H and 13C NMR data, see m/z 403.1366 [M+Na]+ .Sargassopenilline B (3): Amorphous solid; [α]20D −84 ; UV (MeOH) λmax (log ε) 208 (4.25), 250 (3.76), 283 (3.82) nm; CD  λmax (Δε) 240 (−2.46), 318 (+0.38), 380 (−0.27), nm; IR (CHCl3) νmax 3610, 2928, 2855, 1742, 1690, 1648, 1603, 1456, 1373, 1253, 1164, 1092, 1067 cm−1; 1H and 13C NMR data, see m/z 477.1365 [M + Na]+ .Sargassopenilline C (4): Amorphous solid; [α]20D −37 ; UV (MeOH) λmax (log ε) 196 (3.29), 268 (2.91) nm; CD  λmax (Δε) 198 (−2.51), 210 (−2.15), 254 (+1.51), 282 (−8.43), 340 (−0.35) nm; 1H and 13C NMR data, see m/z 417.1157 [M + Na]+ , 393.1207 [M − H]+ .Sargassopenilline D (5): Amorphous solid; [α]20D −107 ; UV (MeOH) λmax (log ε) 242 (3.01) nm; CD  λmax (Δε) 216 (+7.57), 248 (−5.07), 310 (+0.29), 350 (−0.05) nm; 1H and 13C NMR data, see m/z 321.1296 [M + Na]+ , 299.1483 [M + H]+ .Sargassopenilline E (6): Amorphous solid; [α]20D −45 ; UV (MeOH) λmax (log ε) 206 (3.05), 221 (3.02), 247 (2.70), 268 (2.63) nm; CD  λmax (Δε) 220 (+4.60), 248 (−6.50), 328 (+0.39) nm; 1H and 13C NMR data, see m/z 379.1379 [M + Na]+ , 357.1548 [M + H]+ .Sargassopenilline F (7): Amorphous solid; [α]20D −158 ; UV (MeOH) λmax (log ε) 215 (3.69) nm; CD  λmax (Δε) 242 (−0.64), 306 (−0.39), 347 (+0.14), nm; 1H and 13C NMR data, see m/z 391.1367 [M + Na]+ .Sargassopenilline G (R)-MTPACl (20 μL) were added to a solution of the 1 (4.0 mg) in pyridine and stirred at room temperature (25 °C) for 24 h. After evaporation of the solvent, the residue was passed through a silica gel column (7% EtOAc–hexane) to generate the (S)-MTPA ester (1a). The (R)-MTPA ester (1b) was prepared in a similar manner using (S)-MTPACl. 1H and COSY data, 1am/z 729.39 [M + H]+ and of 1bm/z 729.35 [M + H]+.4-Dimethylaminopyridine  and (R)-MTPACl (20 μL) were added to a solution of the 5 (4.0 mg) in pyridine and stirred at room temperature (25 °C) for 24 h. After evaporation of the solvent, the residue was passed through a silica gel column (7% EtOAc–hexane) to generate the (S)-MTPA ester (5a). The (R)-MTPA ester (5b) was prepared in a similar manner using (S)-MTPACl. 1H and COSY data, 5am/z 731.42 [M + H]+ and of 5bm/z 7315.44 [M + H]+.4-Dimethylaminopyridine  and (R)-MTPACl (20 μL) were added to a solution of the 6 (3.0 mg) in pyridine and stirred at room temperature (25 °C) for 24 h. After evaporation of the solvent, the residue was passed through a silica gel column (15% EtOAc–hexane) to generate the (S)-MTPA ester (6a). The (R)-MTPA ester (6b) was prepared in a similar manner using (S)-MTPACl. 1H and COSY data, 6am/z 573.21 [M + H]+ and of 6bm/z 573.30 [M + H]+.4-Dimethylaminopyridine  and  was immediately injected into the peritoneal cavity and the body intensively palpated for 1–2 min. Then, the peritoneal fluid was aspirated with a syringe and transferred to Petri dishes. Petri dishes with the fluid were incubated at 37 °C for 1–2 h to facilitate attachment of peritoneal macrophages to the dish. Then, a cell monolayer was triply flushed with PBS (pH 7.4) to delete attendant lymphocytes, fibroblasts and erythrocytes. Subsequently, macrophages were removed from the surface of the dishes with a scraper and flow of a saline solution, and then placed on an ice bath until use. The working concentration of cells was usually 1–2 × 10d method  with somTwenty milliliters of test compound solutions (at non-toxic concentration 10 μM) and 200 mL of macrophage suspension were added to 96-well flat-bottom plates. After incubation for 2 h at 37 °C, cells were fixed with 70% ethanol solution (100 mL per well) for 15 min at RT. Then, cells were washed with 200 mL of PBS, and a 0.05% solution of Trypan blue (100 mL per well) was added to each well and further incubated at 37 °C for 15 min. Next, media was removed and the cells were gently washed with cold PBS (3 × 200 mL). The cells were then lysed with 50 mL of 1% SDS for at least 4 h and gently triturated. Finally, the SDS/trypan blue solution absorbance was detected spectrophotometrically at 590 nm using a plate reader. The percentage of adhered cells compared to the control level of Trypan blue absorbance was calculated. All samples were assayed in triplicates.50 value 21.3 μM.The experiments were performed as previously reported . Ascorbi1–7) have been isolated from the lipophilic extracts of the marine-derived fungi Penicillium thomii and Penicillium lividum. Sargassopenillines A (1) and B (2) are new members of the rare class of natural products that contain an aryl ring fused to the 6,6-spiroketal part [4) and G (7) are the presence of p-benzoquinone and cyclopentenone moieties as their core skeletons. Sargassopenilline C (3) inhibited the transcriptional activity of the oncogenic nuclear factor AP-1 with an IC50 value of 15 µM.Seven new polyketides, named sargassopenillines A–G 1– have beetal part ,22. Nota