42 Journal of Natural Products, 2007, Vol. 70, No. 1
Saladino et al.
1
Compound 4: colorless oil; [R]25 +94 (c 0.5, CHCl3); H NMR
(2) Teles, H. L.; Hemerly, J. P.; Pauletti, P. M.; Pandolfi, J. R. C.; Araujo,
A. R.; Valentini, S. R.; Young, H. C. M.; Bolzani, V. Da S.; Dulce,
H. S. Nat. Prod. Res. 2005, 19, 319-323.
D
(CDCl3, 200 MHz) δ (ppm) 6.88-6.70 (3H, m, Ar-H), 5.97 (2H, s,
OCH2O), 4.88 (1H, m, H-7′), 4.05 (2H, m, CH2, H-9′), 3.72 (2H, m,
CH2, H-9), 2.6 (2H, m, H-8+H-8′); 13C NMR (CDCl3, 200 MHz) δ
(ppm) 178.20 (C-7′), 147.75 (C-3), 147.60 (C-4), 133.45 (C-1), 120.81
(C-6), 108.19 (C-5), 101.53 (OCH2O), 84.56 (C-7), 69.90 (C-9′), 61.09
(C-9), 47.79 (C-8′), 46.78 (C-8); EIMS m/z (relative %) 266 [M+] (100);
HREIMS m/z 266.0790 (calcd for C13H14O6, 266.0790).
Compound 5: colorless oil (characterized as acetate derivative);
1H NMR (CDCl3, 200 MHz) δ (ppm) 7.59 (dd, J ) 8.1, 1.7 Hz, 1H),
7.20 (d, J ) 1.7 Hz, 1H), 7.04 (d, J ) 1.6 Hz, 1H), 6.94 (d, J ) 8.1
Hz, 1H), 6.80 (dd, J ) 8.0, 1.6 Hz, 1H), 6.65 (d, J ) 8.0 Hz, 1H),
6.06 (s, 2H), 5.95 (s, 2H), 5.05 (m, 1H), 4.44 (m, 1H), 4.03 (m, 1H),
4.0 (m, 1H), 3.71 (m, 2H), 3.66 (m, 1H), 3.0 (m, 1H), 1.75 (s, 3H);
13C NMR (CDCl3, 200 MHz) δ (ppm) 198.4 (C-7′), 170.7 (CO), 152.2
(C), 148.6 (C), 148.4 (C), 147.7 (C), 133.4 (C), 130.0 (C), 124.9 (CH),
119.9 (CH), 108.8 (CH), 108.0 (CH), 108.0 (CH), 107.0 (CH), 102.0
(OCH2), 101.0 (OCH2), 84.9 (C-7), 71.0 (C-9′), 62.0 (C-9), 49.0 (C-
8), 48.7 (C-8), 20.8 (CH3CO); EIMS m/z (relative %) 412 [M+] (100);
HREIMS m/z 412.3892 (calcd for C22H20O8, 412.3894).
(3) Jeng, K. C. G.; Hou, R. C. W. Curr. Enzyme Inhib. 2005, 1, 11-20.
(4) (a) Min, B.-S.; Na, M.-K.; Oh, S.-R.; Ahn, K.-S.; Jeong, G.-S.; Li,
G.; Lee, S.-K.-; Joung, H.; Lee, H.-K. J. Nat. Prod. 2004, 67, 1980-
1984. (b) Yamauchi, S.; Ina, T.; Kirikihira, T.; Masuda, T. Biosci.
Biotech. Biochem. 2004, 68, 183-192. (c) Moritani, T. In NoVel
Compounds from Natural Products in the New Millenium; Tan, B.
K.-H., Bay, B.-H., Zhu, Y.-Z., Eds.; World Scientific Publishing:
Singapore, 2004; pp 196-204.
(5) Marchand, P. A.; Zajicek, J.; Lewis, N. G. Can. J. Chem. 1997, 75,
840-849.
(6) Nikaido, T.; Ohmoto, T.; Kinoshita, T.; Sankawa, U.; Nishibe, S.;
Hisada, S. Chem. Pharm. Bull. 1981, 29, 3586-3592.
(7) Hirata, F.; Fujita, K.; Ishikura, Y.; Hosoda, K.; Ishikawa, T.;
Nakamura, H. Atherosclerosis 1996, 22, 135-147.
(8) Brown, R. C. D.; Swain, N. A. Synthesis 2004, 6, 811-827.
(9) (a) Yamauchi, S.; Hayashi, Y.; Nakashima, Y.; Kirikihira, T.;
Yamada, K.; Masuda, T. J. Nat. Prod. 2005, 68, 1459-1470. (b)
Yamauchi, S.; Ina, T.; Kirikihira, T.; Masuda, T. Biosci. Biotech.
Biochem. 2004, 68, 183-192.
Cytotoxicity Assay. Cytotoxicity of compounds 3A and 4 was
evaluated using the murine fibroblast cell line (3T3 cells), the
plasmocytoma murine cell line (NSO cells), normal human lymphocytes
PHA-stimulated, and the human lymphoblastoid cell line (Daudi cells)
and compared with that of the parent terahydrofurofuran lignan, asarinin
1. Data were obtained by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) test aimed to analyze cell proliferation
in cells cultured in the presence of different compounds.25,26 No
difference was observed for cytotoxicity between data obtained by using
the MTT test and DNA synthesis. The data processing included the
Student’s t-test with p < 0.05 taken as significance level.
Cell Lines. All cell lines were obtained from ATCC. The cells were
cultured in RPMI 1640 supplemented with 5% FCS, 0.1 mM glutamine,
1% penicillin, and streptomycin. Cells were grown in Nunc clone plastic
bottles (TedNunc, Roskilde, Denmark) and split twice weekly at
different cell densities according to the standard procedure. 3T3 cells
were grown as a monolayer and were split by using trypsin. Peripheral
blood mononuclear cells (MNC) were separated from heparinized whole
blood cells obtained from a healthy donor on a Fycoll-Hypaque gradient
as previously described.41 MNC thus obtained were washed twice with
RPMI 1640 supplemented with 10% FCS, glutamine, and antibiotics,
suspended at 200.000 viable cells/mL in medium containing, as mitogen,
5 µg/mL PHA (Sigma) and used in toxicity tests.
Chemicals. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide) was purchased from Aldrich. It was dissolved at a
concentration of 5 mg/mL in sterile PBS at room temperature, and the
solution was further sterilized by filtration and stored at 4 °C in a dark
bottle. SDS was obtained from Sigma. Lysis buffer was prepared as
follows: 20% w/v of SDS was dissolved at 37 °C in a solution of
50% each of DMF and demineralized H2O; the pH was adjusted to 4.7
by adding 2.5% of an 80% acetic acid and 2.5% 1 N HCl solution.
General Procedure. Cells were plated at different concentrations
on flat bottom 96-well microplates (0.1 mL/well). Lymphocytes were
plated out at 20 000 cells/well. 3T3 cells (murine fibroblast line) were
plated at 10 000 cells/well. NSO cells (plasmocytoma murine cell line)
were plated out at 3000 cells/well, and Daudi cells (human lympho-
blastoid cell line) were plated at 300 cells/well. Twelve hours after
plating, different concentrations of each compound were added to each
well. After 48 h, the MTT assay was performed to analyze the
cytotoxicity of the different compounds. Some experiments were
performed by using confluent cells: compounds were added to the 3T3
monolayer 3 days after plating. Tests were then run as described above.
(10) For some examples of the effect of cytochrome P 450-dependent
metabolic process on lignans’ biological activity see: (a) Sinha, B.
K.; Eliot, H. M.; Kalayanaraman, B. FEBS Lett. 1988, 227, 240. (b)
Niemeyer, H. B.; Metzler, M. Anal. Technol. Biomed. Life Sci. 2002,
777, 321-327.
(11) Mincione, E.; Sanetti, A.; Bernini, R.; Felici, M.; Bovicelli, P.
Tetrahedron Lett. 1998, 39, 8699-8702.
(12) (a) Owens, C. S.; Arias, J.; Abu-Omar, M. M. Catal. Today 2000,
55, 317-363. (b) Roma˜o, C. C.; Ku¨hn, F. E.; Herrmann, W. A. Chem.
ReV. 1997, 97, 3197-3246. (c) Beattie, I. R.; Jones, P. J. Inorg.
Chem. 1979, 18, 2318.
(13) Bianchini, G.; Crucianelli, M.; De Angelis, F.; Neri, V.; Saladino,
R. Tetrahedron Lett. 2004, 45, 2351-2353.
(14) (a) Crestini, C.; Pro, P.; Neri, V.; Saladino, R. Bioorg. Med. Chem.
2005, 13 (7), 2569-2578. (b) Bernini, R.; Mincione, E.; Cortese,
M.; Aliotta, G.; Saladino, R. Tetrahedron Lett. 2001, 42/32, 5401-
5404. (c) Saladino, R.; Neri, V.; Mincione, E.; Marini, S.; Coletta,
M.; Fiorucci, C.; Filippone, P. J. Chem. Soc., Perkin Trans. 1 2000,
581-586. (d) Lin, J.; Saha-Mo¨ller, L. R.; Shimizu, M. J. Mol. Catal.
1995, 97, 15. (e) Adam, W.; Herrmann, W. A.; Lin, J.; Saha-Mo¨ller,
L. R. J. Org. Chem. 1994, 59, 8281.
(15) For the preparation of heterogeneous poly(4-vinylpyridine) and
polystyrene/MTO catalysts see: (a) Saladino, R.; Pelliccia, A. R.;
Neri, V.; Caminiti, R.; Sadun, C. J. Org. Chem. 2002, 67, 1323-
1332. For some recent examples of their application in oxidative
chemistry see: (b) Saladino, R.; Mincione, E.; Attanasi, O. A.;
Filippone, P. Pure Appl. Chem. 2003, 75, 261-268. (c) Saladino,
R.; Neri, V.; Cardona, F.; Goti, A. AdV. Synth. Catal. 2004, 346,
639-647. (d) Saladino, R.; Andreoni, A.; Neri, V.; Crestini, C.
Tetrahedron 2005, 61, 1069-1075. (e) Bianchini, G.; Crucianelli,
M.; De Angelis, F.; Veronica, N.; Saladino, R. Tetrahedron Lett.
2005, 46 (14), 2427-2432.
(16) Herrmann, W. A.; Fischer, R. W.; Scherer, W.; Rouch, M. U. Angew.
Chem., Int. Ed. Engl. 1993, 32, 1157-1160.
(17) Gisdakis, P.; Antonczak, S.; Ko¨stlmeier, S.; Herrmann, W. A.; Ro¨sch,
N. Angew. Chem., Int. Ed. 1998, 37, 2211.
(18) Saladino, R.; Fiani, C.; Belfiore, M. C.; Gualandi, G.; Penna, S.;
Mosesso, P. Bioorg. Med. Chem. 2005, 13, 5949-5960.
(19) Gonzale`z, M. J. T. G.; Pinto, M. M. M.; Kijjoa, A.; Anantachoke,
C.; Herz, W. Phytochemistry 1993, 32, 433-438.
(20) Spatafora, C.; Tringali, C. Phytochem. Anal. 1997, 8, 139-142.
(21) (a) Takano, S.; Ohkawa, T.; Tamori, S.; Satoh, S.; Ogasara, K. J.
Chem. Soc., Chem. Commun. 1988, 189-191. (b) Marchand, P. A.;
Zajicek, J.; Lewis, N. G. Can. J. Chem. 1997, 75, 840-849.
(22) The structure of keto-lignan 5 was unambiguously assigned as an
acetate derivative by comparison with an authentic sample prepared
as reported in ref 11.
MTT/Formazan Extraction Procedure. A total of 20 µL of the 5
mg/mL stock solution of MTT was added to each well; after 2 h of
incubation at 37 °C, 100 µL of the extraction buffer was added. After
an overnight incubation at 37 °C, the optical densities at 570 nm were
measured using a Titer-Tech 96-well multiscanner, employing the
extraction buffer as the blank.
(23) For an example of spontaneous ring-opening of hydroxylated
furofurans to corresponding keto-lignans see: Rao, K. V.; Wu, W.
N. Lloydia 1978, 41, 56-62.
(24) Ju, Y.; Still, C. C.; Sacalis, J. N.; Li, J.; Ho, C. T. Phytother. Res.
2001, 15, 441-443.
(25) D’Atri, S.; Tentori, L.; Fuggetta, M.P.; Marini, S.; Bonmassar, E.
Int. J. Tissue React. 1986, 8, 383-389.
References and Notes
(1) For some exhaustive reviews on lignans and related compounds
see: (a) Ward, R. S. Nat. Prod. Rep. 1999, 16, 75-96. (b) Ayres,
D. C.; Loike, J. D. In Lignans. Chemical, Biological and Clinical
Properties; Cambridge University Press: Cambridge, 1990.
(26) Scatena, R.; Marini, S.; Tavazzi, B.; Lazzarino, G.; Giardina, B. Clin.
Chem. Enzymol. Commun. 1990, 3, 73-79.
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