Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 113
of alkyl halide. The stirring was continued overnight. The
reaction mixture was quenched by pouring it into 20 mL of
diluted hydrochloric acid and extracted with methylene chlo-
ride. The organic phase was dried over Na2SO4 and the solvent
removed in vacuo leaving behind a clear, yellowish oil which
solidified on standing. The crude products were purified by
either recrystallization or column chromatography. Synthesis
information and analytical data of a representative compound
(3a ) in the series are given below; see Supporting Information
for those of 3b-f.
3-Ben zyloxy-17â-m eth oxyestr a -1,3,5(10)-tr ien e (3a ): re-
crystallization from methanol, 63% yield, yellowish solid; mp
92-94 °C; TLC Rf 0.83; 1H NMR (CDCl3) δ 7.48-7.32 (m, 5H,
C6H5 of benzyl), 7.27 (d, J ) 8.7 Hz, 1H, 1-CH), 6.81 (dd, J )
8.7 and 2.1 Hz, 1H, 2-CH), 6.73 (d, J ) 2.4 Hz, 1H, 4-CH),
5.05 (s, 2H, OCH2 of benzyl)), 3.39 (s, 3H, 17â-OCH3), 3.33 (t,
1H, J ) 8.7 Hz, 17R-CH), 2.83 (m, 2H, 6-CH2), 1.22-2.34 (m,
13H), 0.80 (s, 3H, 13-CH3); MS m/z 377 [M + H]+.
Gen er a l P r oced u r e for P r ep a r a tion of 17â-Alk oxy-
estr a -1,3,5(10)-tr ien es 4a -f. To a solution of 2.0 mmol of
3a -f in 10 mL of methanol were added 0.2 g of Pd/C (10%)
and ammonium formate (1.00 g, 16 mmol). The reaction
mixture was stirred at room temperature for 1 h. Pd/C was
then removed by filtration and solvent was evaporated in
vacuo. To the oily residue water was added and the resulting
solid was collected by filtration. Either recrystallization or
column chromatography was used for purification. Synthesis
information and analytical data of a representative compound
(4a ) in the series are given below; see Supporting Information
for those of 4b-f.
17â-Meth oxyestr a -1,3,5(10)-tr ien -3-ol (4a ): recrystalli-
zation from methanol, 50% yield, white solid; mp 242-244 °C;
TLC Rf 0.48; 1H NMR (DMSO) δ 7.05 (d, J ) 8.40 Hz, 1H,
1-CH), 6.51 (dd, J ) 8.40 and 2.10 Hz, 1H, 2-CH), 6.45 (d, J )
2.40 Hz, 1H, 4-CH), 3.30 (s, 3H, 17â-OCH3), 3.28 (t, J ) 8.25
Hz, 1H, 17R-CH), 2.73-2.72 (m, 2H, 6-CH2), 2.56-1.22 (m,
13H), 0.74 (s, 3H, 13-CH3); 13C NMR (DMSO) δ 156.7(C-3)
139.3 (C-5 or C-10), 132.7 (C-10 or C-5), 128.0 (C-1), 116.8 (C-2
or C-4), 114.5 (C-4 or C-2), 92.2, 58.7 (OCH3), 51.7 (C-17), 45.6,
44.6, 40.2, 39.8, 31.1, 29.2, 28.8, 28.1, 24.4, 13.6 (13-CH3); MS
m/z 287 [M + H]+, 255 [M - OCH3]+. Anal. C, H.
were further incubated for 24 h before sodium glutamate in a
solution of phosphate buffer was added. Cell viability was
quantified 2 h later by the Calcein AM assay in a phosphate
buffer solution.
Sta tistica l An a lysis. ANOVA was used to determine the
significance of differences among groups. Comparison between
groups were done using the Tukey test. A p < 0.05 was
considered significant.
Ack n ow led gm en t. This project has been supported
by the National Institute on Aging (Grant No. PO1
10485) and Apollo BioPharmaceutics, Inc. Funds for the
mass spectrometer used in the study were provided by
the National Center for Research Resources (Grant No.
SS10 RR12023) and by the University of Florida. K.A.A.
wishes to acknowledge the National Science Foundation
and the University of Florida for funding the purchase
of X-ray equipment.
Su p p or tin g In for m a tion Ava ila ble: Detailed informa-
tion on the synthesis and characterization of 3b-f, 4b-f, and
5c; X-ray crystallographic data of 4d . This material is available
Refer en ces
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Gen er a l P r oced u r e for P r ep a r a tion of 3-Alk oxyestr a -
1,3,5(10)-tr ien es 5b,c. To a suspension of compound 1 (0.5
g, 1.8 mmol) and potassium carbonate (1.00 g, 7.2 mmol) in 5
mL of dry acetone was added 10 mmol of 1-bromoalkane. The
mixture was refluxed overnight then allowed to cool and it was
filtered. The acetone was removed in vacuo and the oily residue
was purified. Synthesis information and analytical data of a
representative compound (5b) in the series are given below;
see Supporting Information for those of 5c.
3-Bu toxyestr a -1,3,5(10)-tr ien -17â-ol (5b): recrystalliza-
tion from methanol:water (1:1, v/v), 68% yield, white solid; mp
1
86-88 °C; TLC Rf 0.62; H NMR (CDCl3) δ 7.17 (d, J ) 8.7
Hz, 1H, 1-CH), 6.70 (dd, J ) 8.4 and 2.40 Hz, 1H, 2-CH), 6.62
(d, J ) 2.4 Hz, 1H, 4-CH), 3.93 (t, J ) 6.30 Hz, 2H, OCH2 of
3-OC4H9), 3.71 (t, J ) 8.1 Hz, 1H, 17R-CH), 2.86-2.80 (m, 2H,
6-CH2,), 2.20-1.10 (m, 17H, including (CH2)2 of 3-OC4H9), 0.96
(t, J ) 7.4 Hz, 3H, CH3 of 3-OC4H9); 13C NMR (CHCl3) δ 156.9
(C-3), 137.7 (C-5 or C-10), 132.3 (C-10 or C-5), 126.1 (C-1),
114.4, 111.9, 81.7, 67.5 (OCH2 of 3-OC4H9), 49.9, 43.8, 43.1,
38.7, 36.6, 31.3, 30.4, 29.7, 27.2, 26.3, 23.0, 19.2, 13.7 (CH3 of
3-OC4H9), 10.9 (13-CH3); MS m/z 311 [M - OH]+.
Cytotoxicity Stu d ies. HT-22 cells (generously provided by
Dr. David Schulbert, Salk Institute, La J olla, CA) were
cultured in Dulbecco’s modified Eagle’s media (DMEM) supple-
mented with 10% fetal bovine serum under the usual condi-
tions. All wells in the 96-well culture plate contained approxi-
mately 5000 cells as determined by a Neubauer hemacytometer
and the cells were incubated for 24 h before the compounds
were added. The estradiol derivatives purified by recrystalli-
zation or column chromatography were free from 1 as deter-
mined by HPLC. All agents were dissolved in absolute ethanol
and diluted, with the culture media, to a final concentration
of 0.01, 0.1, 1.0, and 10 µM in their respective wells. The cells
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