714 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Rescifina et al.
[(3RS,5RS)-2-Methyl-3-pyren-1-ylisoxazolidin-5-yl]metha-
nol (8c): reaction time 20 min; yield 30%; brown solid, mp 58-
61 °C. Anal. (C21H19NO2) C, H, N.
[(3RS,5SR)-2-Benzyl-3-pyren-1-ylisoxazolidin-5-yl]metha-
nol (7d): reaction time 20 min; yield 52.3%; yellow solid, mp 45-
47 °C. Anal. (C27H23NO2) C, H, N.
[(3RS,5RS)-2-Benzyl-3-pyren-1-ylisoxazolidin-5-yl]metha-
nol (8d): reaction time 20 min; yield 32.7%; yellow solid, mp 63-
65 °C. Anal. (C27H23NO2) C, H, N.
The capability of some compounds to induce cell death was also
evaluated using the trypan blue exclusion test and by detection of
apoptosis using morphological analysis following staining with
acridine orange, as previously described.20 Evaluation of fas-induced
apoptosis was performed by adding anti-fas antibody (50 ng/mL;
CH11, Upstate Biotechnology Inc., Lake Placid, NY) to the
untreated or treated cells in 96-well plates.
Evaluation of Antiviral Activity. Antiviral activity was deter-
mined by previously established procedures.21
[(3RS,5SR)-2-Benzyl-3-(9-phenanthryl)isoxazolidin-5-yl]meth-
anol (7e): reaction time 20 min; yield 23.3%; yellow sticky foam.
Anal. (C25H23NO2) C, H, N.
[(3RS,5RS)-2-Benzyl-3-(9-phenanthryl)isoxazolidin-5-yl]meth-
anol (8e): reaction time 20 min; yield 15.7%; yellow solid, mp
41-43 °C. Anal. (C25H23NO2) C, H, N.
This work was partially supported by M.I.U.R. (progetto P.R.I.N.
2002 and F.I.R.B.). The authors are grateful to Dr. Maria Rosaria
Brigida Pino for her technical assistance.
Supporting Information Available: Elemental analyses data
1
and H and 13C NMR spectroscopic data of all compounds. This
Synthesis of Isoxazolidines 9-11f. General Procedure. A
solution of nitrone 6 (0.28 mmol) in methyl acrilate (5 mL, 55.5
mmol) in a pressure tube equipped with a stir bar was inserted into
the cavity of a Discover Microwave System apparatus and heated
at 90 W for 60 min (internal temperature 150-160 °C). The mixture
was evaporated and the resulting solid was purified by flash
chromatography on a silica gel with cyclohexane/ethyl acetate (95:
5) as eluent.
Methyl (3RS,5SR)-3-(9-anthryl)-2-benzylisoxazolidine-5-car-
boxylate (9f): yield 24.2%; dark yellow solid, mp 90-95 °C. Anal.
(C26H23NO3) C, H, N.
Methyl (3RS,5RS)-3-(9-anthryl)-2-benzylisoxazolidine-5-car-
boxylate (10f): yield 10.0%; dark yellow solid, mp 105-108 °C.
Anal. (C26H23NO3) C, H, N.
material is available free of charge via the Internet at http://
pubs.acs.org.
References
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Synthesis of Isoxazolidines 7f, 8f, and 12f. General Procedure.
To a solution of isoxazolidine 9-11f (20 mmol) in a 1:1 dioxane/
water mixture (30 mL) was added sodium borohydride (200 mmol),
at room temperature, and then the reaction was magnetically stirred
for 20 h. Successively, the reaction mixture was extracted with
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[(3RS,5SR)-3-(9-Anthryl)-2-benzylisoxazolidin-5-yl]metha-
nol (7f): yield 91.48%; brown solid, mp 91-95 °C. Anal. (C25H23-
NO2) C, H, N.
[(3RS,5RS)-3-(9-Anthryl)-2-benzylisoxazolidin-5-yl]metha-
nol (8f): yield 93.5%; brown solid, mp 85-88 °C. Anal. (C25H23-
NO2) C, H, N.
[(3RS,4RS)-3-(9-Anthryl)-2-benzylisoxazolidin-4-yl]metha-
nol (12f): yield 68.5%; dark yellow solid, mp 129-134. Anal.
(C25H23NO2) C, H, N.
Evaluation of Cytotoxicity. The cytotoxicity of all compounds
was evaluated by a commercial viability assay (CellTiter 96
Aqueous One Solution Assay, Promega Co., Madison WI), accord-
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principle that cells, at death, rapidly lose the ability to reduce MTS
tetrazolium. Briefly, MOLT-3, U-937, THP-1, and Vero cells were
cultured under optimal culture conditions for 20 h in 96-well plates,
in the absence of the compounds or in their presence, at concentra-
tions ranging from 10 to 500 µM. At the end of the incubation
time, the MTS-tetrazolium-based reagent was added to each well.
After a further incubation of 1 h at 37 °C in a humidified, 5% CO2
atmosphere, the absorbance of the samples was recorded at 490
nm using a 96-well spectrophotometer. The assays were performed
in triplicate. The IC50 were calculated as the concentrations of the
compounds required to cause 50% reduction of absorbance values.
For compounds 7c and 7e, which showed low IC50 values with
reference to the range of concentrations utilized, a nonlinear
regression was the most adequate for their calculation, while for
the other compounds a linear regression was used. The 95%
confidence interval for IC50 was also calculated.
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