110
A.F. Barrero et al. / Phytochemistry 66 (2005) 105–111
4
.5. 7a,18-Diacetoxy,9b,13b-epi-dioxiabiet-8(14)-ene (2)
4.10. Cell proliferation assay
Colorless oil, [a]D +6.2ꢁ (c 0.13, CHCl ); IR (film)
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium (MTT; Sigma Chemical Co., St. Louis, MO) dye
reduction assay in 96-well microplates was used, essen-
tially as described (Mosmann, 1983). The assay is depen-
dent on the reduction of MTT by mitochondrial
dehydrogenases of viable cell to a blue formazan prod-
uct which can be measured spectrophotometrically.
3
À1
mmax 2932, 2872, 1736, 1465, 1377, 1240, 1037 cm
;
HRFABMS m/z 443.2410 (calcd for C H O Na,
2
4
36
6
4
43.2409).
4
.6. 7a,18-Diacetoxyabiet-8(14)-en-13b-ol (3)
3
Colorless oil, [a]D +14.7ꢁ (c 1.0, CHCl ); IR (film)
BAE cells (8 · 10 cells in a total volume of 200 ll of
3
3
mmax 3452, 2929, 2854, 1731, 1654, 1466, 1370, 1242,
DMEM/20% FBS) and tumor cells (4 · 10 A-549 cells
À1
3
3
3
1038 cm ; HRFABMS m/z 429.2631 (calcd for
C H O Na 429.2617).
or 6 · 10 H-116, 6 · 10 PSN1, 6 · 10 SKBR3 and
3
6 · 10 T98G cells in a total volume of 200 ll of com-
2
4
38
5
plete medium) were incubated in each well with serial
dilutions (5, 2.5, 1, 0.5, 0.1, 0.05, 0.01 lg/ml) of the
tested compounds. After 2 days of incubation (37 ꢁC,
4
.7. 7a,18-Diacetoxy-13b-methoxyabiet-8(14)-ene (4)
Colorless oil, [a]D +43.7ꢁ (c 0.1, CHCl ); IR (film)
5% CO in a humid atmosphere) 50 ll of MTT (5 mg/
3
2
mmax 2955, 2927, 2854, 1736, 1456, 1370, 1240, 1038
cm ; HRFABMS m/z 443.2789 (calcd for C H O Na
4
ml in PBS) were added to each well and the plate was
incubated for a further 2 h (37 ꢁC). The resulting forma-
zan was dissolved in 100 ll DMSO and read at 490 nm.
All determinations were carried out in triplicate. IC50
value was calculated as the concentration of drug yield-
ing a 50% of cell survival.
À1
2
5
40
5
43.2773).
4
.8. 13b-Hydroxyabiet-8(14)-en-7-one (5)
Colorless oil, [a]D +45.0ꢁ (c 0.34, CHCl ); IR (film)
3
mmax 3464, 2952, 2927, 2853, 1682, 1613, 1462, 1386,
À1
Acknowledgements
1251, 1175 cm ; HRFABMS m/z 327.2294 (calcd for
C H O Na 343.2300).
2
0
32
2
This work was supported by a grant from CICYT
SAF2001-2639), the Spanish Ministerio de Ciencia y
Tecnolog ´ı a (Project BQU 2002-03211), from the
ESRSFC Ministry of the Moroccan Government (PRO-
TARS P2T2/07), and from the Junta de Andaluc ´ı a
Consejer ´ı a de Presidencia, Programa de Cooperaci o´ n
Andaluc ´ı a-Marruecos). We also thank Instituto Biomar,
S.A. for the antitumor tests.
(
4
en-18-oate
.9. Oxidation of 1 to methyl 9a,13a-epidioxiabiet-8(14)-
To a solution of 1 (20 mg, 0.06 mmol) in 1 ml of dry
(
DCM was added 76 mg (0.20 mmol) of PDC were
added. The mixture was stirred at room temp. for 5 h.
Then, it was filtered trough a pad of 3 cm of SiO using
2
t-BuOMe, washed with brine, dried over Na SO and
2
4
concentrated to give 19 mg of aldehyde. A solution of
2 mg of NaClO (0.50 mmol), 52 mg of NaH PO
4
References
5
2
2
(
0.37 mmol) and 1 ml of H O was added over a solution
2
Barrero, A.F., Sanchez, J.F., Alvarez-Manzaneda, E.J., Mu n˜ oz
Dorado, M., Haidour, A., 1993. Terpenoids and sterols from the
wood of Abies pinsapo. Phytochemistry 32, 1261–1265.
of the aldehyde (19 mg, 0.06 mmol) in 1 ml of t-BuOH
and 0.5 ml of 2-methyl-2-butene. The mixture was stir-
red at room temp. for 4 h. Then, after removing
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Dorado, M., Haidour, A., 1991. Endoperoxide diterpenoids and
other terpenoids of the wood of Abies marocana. Phytochemistry
t-BuOH, 1 ml of H O was added, and the resulting mix-
2
ture extracted with t-BuOMe. The extracts were washed
with brine, dried over Na SO and concentrated to give
3
5, 1271–1274.
2
4
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Antifungal activity of essential oils from several medicinal plants
against four postharvest citrus pathogens. Phytopathol. Mediterr.
1
5 mg of the corresponding acid. To a solution of 15 mg
of this acid (0.05 mmol) in 0.1 ml of dried MeOH and
.2 ml of dried benzene was added under inert atmo-
4
2, 251–256.
0
Cousins, D.J., 1994. Medicinal, Essential Oil, Culinary Herb and
Pesticidal Plant of the Labiatae, Parts I and II. CAB International,
Wallingford.
sphere a solution of 0.11 ml of TMSCHN (0.08 mmol)
2
and 0.2 ml of dried benzene. After stirring for 12 h, the
volatile components were evaporated. The crude was
cromatographed (n-hexane/t-BuOMe, 4:1) to give 11
mg (0.03 mmol) of methyl 9a,13a-epidioxiabiet-8(14)-
en-18-oate.
Erdtman, H., Westfelt, L., 1963. Neutral diterpenes from pinewood
resins. Acta Chem. Scand. 17, 1826–1827.
Fetizon, M., Moreau, G., Moreau, N., 1968. Determination of the
configuration of aldehydes in the 4b-position of the di- or
triterpenic series. Bull. de la Soc. Chim. de France 8, 3295–3301.