Journal of Natural Products
Article
mmol) in MeOH (3 mL), and the solution was stirred at 55 °C for 10
min. Acetic acid (0.3 mL) was added, and the reaction mixture was
diluted with brine (100 mL) and extracted with CH Cl (2 × 100
mL). The combined organic phases were washed with brine (100 mL),
dried, and concentrated to yield a dark brown residue. The residue
Prof. J. Pezzuto and T. Kondratyuk (University of Hawaii at
Hilo) for conducting cytotoxic and aromastase inhibition
assays. We are grateful to S. Shaw for technical assistance. Dr.
E. Ballard is thanked for helpful discussions.
2
2
(
1.25 g) was purified on silica gel with elution in 20% ethyl acetate in
REFERENCES
hexane to afford formestane (1) (0.452 g, 31%) [mp 195−200 °C
(
spectra, respectively, of semisynthetic formestane (1).
■
15
1
13
(
(
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lit. 199−202 °C)]. See Figures S1 and S2 for H and C NMR
Baptista, T.; Alcaro, S.; Costa, G.; Carvalho, R. A.; Teixeira, N. A. A.;
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Bioconversion with R. oryzae. Formestane (1) (1 g) was pulse
fed to R. oryzae. Workup yielded broth and mycelial extracts, which
were pooled and purified on silica gel. Elution in 25% ethyl acetate in
hexane afforded 4β,5α-dihydroxyandrostane-3,17-dione (5) (86 mg)
17
1
[
mp 165−166 °C (lit. 168−170 °C)]. See Figures S3 and S4 for H
13
and C NMR spectra, respectively, of metabolite 5.
Biotransformation with B. bassiana. The bioconversion of
formestane (1) (1 g) with B. bassiana provided mycelial and broth
extracts that were combined and chromatographed on silica gel.
Elution in 20% ethyl acetate in hexane yielded untransformed
formestane (1) (95 mg), while elution in 25% ethyl acetate in hexane
(6) Ghosh, D.; Lo, J.; Morton, D.; Valette, D.; Xi, J.; Griswold, J.;
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afforded 4,17β-dihydroxyandrost-4-en-3-one (6) (53 mg) [mp 199−
8
2
04 °C (lit. 205−210 °C)]. The metabolite 3α,17β-dihydroxy-5β-
8
androstan-4-one (7) (9 mg) [mp 180−183 °C (lit. 168−170 °C)]
was obtained in 30% ethyl acetate in hexane, and elution in 50% ethyl
acetate in hexane provided 4,11α,17β-trihydroxyandrost-4-en-3-one
(
7
8) (0.24 g). See Figures S5−S15 for NMR spectra of compounds 6,
, and 8.
,11α,17β-Trihydroxyandrost-4-en-3-one (8): amorphous solid;
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2
0
[
α] +57.5 (c 0.73, CHCl ); UV (EtOH) λ (log ε) 280 (1.20)
D 3 max
−1 1
nm; IR νmax 3350, 1648, 1614, 1386, 1159 cm ; H NMR (CDCl ,
3
1
3
3
3
3
00 MHz) and C NMR (CDCl , 75 MHz), see Table 1; ESIMS m/z
(12) Martin, G. D. A. Curr. Org. Chem. 2010, 14, 1−14.
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3
+
21 [M + H] (100), 239 (28), 228 (67), 220 (24); HRMS(ESI) m/z
19.1912 [M − H] (calcd for C H O , 319.1915).
SRB Assay. The cytotoxic potential of formestane (1) and its
−
19
27
4
(14) Huang, C.-L.; Chen, Y.-R.; Liu, W.-H. Enzyme Microb. Tech.
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metabolites (5−8) toward MCF-7 cancer cells was determined using
1
8
the previously described SRB assay. These experiments were
performed in triplicate. Test compounds (dissolved in DMSO) were
(15) Ciattini, P. G.; Morera, E.; Ortar, G. Synth. Commun. 1992, 22,
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transferred to 96-well plates and incubated in a CO incubator for 72 h
́
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2
at 37 °C. The incubation was terminated with trichloroacetic acid. The
cells were washed, air-dried, and stained with SRB solution, and optical
densities were determined at 515 nm using a microplate reader. In
each case, a zero-day control was performed by adding an equivalent
number of cells to several wells, incubating at 37 °C for 30 min, and
processing as described above. Percent of cell survival was calculated
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(
986, 24, 607−617.
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J.; Marler, L.; Rostama, B.; Wright, A. D. Nat. Prod. Commun. 2009, 4,
1717−1728.
using the formula (ODcells+tested compound − ODday0)/(OD
−
cells+10%DMSO
1
8
ODday0) × 100. Vinblastin was used as the positive control.
Aromatase Assay. Compounds 1 and 5−8 were evaluated for
aromatase inhibition according to a previously established protocol.
(19) Azerad, R. Adv. Biochem. Eng./Biotechnol. 1999, 63, 169−218
1
8
and references therein.
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(
ASSOCIATED CONTENT
Supporting Information
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(
*
S
Heeremans, C. E. M.; Van der Hoeven, R. A. M.; Niessen, W. M.
A.; van der Greef, J. J. Chromatogr. B 1992, 576, 235−244.
AUTHOR INFORMATION
Corresponding Author
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partly funded by a grant provided by the
Biological Chemistry Summer Research Program (Merck/
AAAS), the American Chemical Society (ACS) Project SEED
endowment, the Hillsborough County Public Schools Academ-
ic Programs, and the ACS Tampa Bay local section. We thank
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dx.doi.org/10.1021/np400585t | J. Nat. Prod. 2013, 76, 1966−1969