Synthesis and biological evaluation of 17β-alkoxyestra-1,3,5(10)-trienes as potential neuroprotectants against oxidative stress

Article abstract of DOI:10.1021/jm000280t

17β-O-Alkyl ethers (methyl, ethyl, propyl, butyl, hexyl, and octyl) of estradiol were obtained from 3-O-benzyl-17β-estradiol with sodium hydride/alkyl halide, followed by the removal of the O-benzyl protecting group via catalytic transfer hydrogenation. A

Full text of DOI:10.1021/jm000280t

110
J . Med. Chem. 2001, 44, 110-114
Syn th esis a n d Biologica l Eva lu a tion of 17â-Alk oxyestr a -1,3,5(10)-tr ien es a s
P oten tia l Neu r op r otecta n ts Aga in st Oxid a tive Str ess
Laszlo Prokai,*,† Su-Min Oon,§ Katalin Prokai-Tatrai,‡ Khalil A. Abboud, and J ames W. Simpkins‡,#
Center for Drug Discovery, College of Pharmacy, Department of Anesthesiology, College of Medicine, and
Center for Neurobiology of Aging, College of Pharmacy, University of Florida, Gainesville, Florida 32610-0497, and
Department of Chemistry, University of Florida, Gainesville, Florida 32611
Received October 12, 2000
17â-O-Alkyl ethers (methyl, ethyl, propyl, butyl, hexyl, and octyl) of estradiol were obtained
from 3-O-benzyl-17â-estradiol with sodium hydride/alkyl halide, followed by the removal of
the O-benzyl protecting group via catalytic transfer hydrogenation. An increase compared to
estradiol in the protection of neural (HT-22) cells against oxidative stress due to exposure of
glutamate was furnished by higher (C-3 to C-8) alkyl ethers, while methyl and ethyl ethers
decreased the neuroprotective effect significantly. Lipophilic (butyl and octyl) ethers blocking
the phenolic hydroxyl (3-OH) of A-ring were inactive.
In tr od u ction
activities.6,8 All neuroprotective derivatives or analogues
of 1 possess, however, a common structural element
which is an intact, unsubstituted phenolic A-ring with
its free 3-hydroxyl group.9 Since lipophilic phenols also
protect neural cells against glutamate and peroxide-
mediated oxidative damage and cell death,10 we decided
to functionalize the 17-hydroxyl function of 1 by prepar-
ing alkyl ethers having a wide range of lipophilicity. We
hypothesized that these ethers not only retain but also
eventually increase neuroprotection compared to 1. (In
the meantime, these 17â-O-ethers also are expected to
have a significantly less “feminizing” effect than 1.) As
controls, lipophilic 3-O-alkyl ethers have also been
prepared and evaluated to further confirm the struc-
tural requirements of a phenolic A-ring for neuropro-
tection.
Estrogens have long been recognized as antioxidants
in a variety of in vitro and in vivo models. The antioxi-
dant action is believed to be due to their ability to
scavenge free radicals that cause neuronal cell death.
Oxidative stress has been linked to neuronal cell death
resulting from either acute insults due to ischemia,
trauma, or chronic neurodegenerative diseases such as
Alzheimer’s disease (AD)2 characterized by a progressive
loss of memory and cognitive function. Although the
histopathogenesis of AD is yet to be fully understood,
one theory hypothesizes that the oxidative microenvi-
ronment surrounding the accumulated amyloid-â-pep-
tide (Aâ) plaques is responsible for peroxidation of cell
membrane lipids leading to cell lysis and death.3
Another oxidative stressor suggested in AD’s pathogen-
esis is the amino acid glutamate, the major excitatory
neurotransmitter in the central nervous system.4
Population studies have shown that estrogen replace-
ment therapy in postmenopausal women can decrease
the incidence of AD or delay its onset.4 It has been
demonstrated5 that the most biologically active estro-
gen, 17â-estradiol (1), is a potent antioxidant and has
neuroprotective activity; however, the mechanism of
action is still unclear. The direct neuroprotective effects
of 1 on SK-N-SH human neuroblastoma cells under
serum deprivation were first reported in 1994.6 Numer-
ous recent studies have demonstrated similar effects of
1 against a variety of toxicities, including oxidative
stress,7 in different types of neuronal cells. Interestingly,
an enantiomer of estradiol (“ent-estradiol”), its epimer
(17R-estradiol), and estratrien-3-ol are equipotent to 1
in protection of neural cells against oxidative damage,
although they have significantly reduced estrogenic
Ch em istr y
The previously reported preparation of 17â-methyl-
estradiol11 could have offered an attractive and conve-
nient entry to a series of 17â-alkylestradiols. Unfortu-
nately, we were unable to reproduce the procedure.
Since alkylation on the phenolic 3-hydroxyl group
proceeds under much milder conditions than that at the
17-position, we decided to protect the 3-OH selectively
(and reversibly) before alkylating on the 17-position
under strong basic conditions with the relevant alkyl
halide. However, the initially prepared 3-tert-butyldi-
methylsilylated estradiol proved to be too unstable in
both very mild acidic or basic conditions against alkyl
halides, which resulted in a rapid desilylation followed
by alkylation of the 3-OH. Similarly, the commercially
available 3-benzoylestradiol was also unsuitable for the
preparation of 17-alkylated ethers, since the phenolic
ester group also hydrolyzed rapidly under the condition
of our attempted alkylations. Therefore, the protection
of the 3-OH of 1 as benzyl (Bz) ether12 (2) was employed,
followed by elaboration of the 17â-OH to the corre-
sponding 17â-alkoxyl congeners 3a -f. The 17-OH group
was successfully alkylated with the corresponding alkyl
halide in the presence of sodium hydride in DMF.
However, the subsequent removal of the 3-benzyl pro-
* To whom correspondence should be addressed. Tel: +1 352 392
3421. Fax: +1 352 392 3421. E-mail: lprokai@grove.ufl.edu.
† Center for Drug Discovery.
§ Department of Anesthesiology.
‡ Center for Neurobiology of Aging.
Department of Chemistry.
Present address: Department of Pharmacology and Neuroscience,
#
University of North Texas Health Science Center, Fort Worth, TX
76107-2699.
10.1021/jm000280t CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/23/2000

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 111
Sch em e 1. Synthesis of 17- and 3-Alkyl Ethers of
Estradiol
F igu r e 1. ORTEP plot of the X-ray crystal structure of 17-
O-butylated 17â-estradiol 4d . Thermal ellipsoids are shown
at the 30% probability level.
When testing the neuroprotective potency of estro-
gens, the ED50 for estrogen neuroprotection is in the
nanomolar range by dye exclusion techniques,17 mor-
phological criteria/cell counting,18 and vital dyes (Cal-
cein AM),19 whereas ED50s in the micromolar range
were observed when MTT reduction is used as an
endpoint. We have abandoned the frequently used
trypan blue exclusion assay, as it requires the applica-
tion of an additional stress - the harvesting of cells to
conduct counts. This additional stress confounds the
data. On the other hands, lactic acid dehydrogenase
(LDH) release from dead or dying cells was highly
variable in our hands and was too cumbersome to be
used for screening in this study.
Compared to 1, only 4c-f of the six 17â-O-alkylestra-
diols tested improved neuroprotection in a dose-depend-
ent manner against the glutamate-induced oxidative
damage in murine HT-22 cells at concentrations of 0.1
µM and higher (Figure 2). These compounds were
essentially equipotent at 1 µM (approximately twice as
many cells were viable compared to the control) and
showed no apparent relationship with a single molecular
property such as lipophilicity (based on the calculated
log P20) or water solubility (according to our solubility
studies all of the tested compounds are essentially
water-insoluble). The butyl (4d ) and octyl (4f) ethers
were, for example, no more neuroprotective at a con-
centration of 10 µM than at 1 µM. The parent compound
(1) was effective only at 10 µM but was clearly super-
seded by 4c,e at this concentration. 17â-Methylestradiol
(4a ) was also ineffective below 10 µM and decreased cell
viability compared to 1 at 10 µM, while 17â-ethylestra-
diol (4b) were ineffective even at 10 µM. Similarly to
earlier observations regarding the inability of 3-O-
methylestradiol to exert neuroprotection,11 5b,c ethers
blocking the phenolic hydroxyl in the A-ring also were
inactive.
tecting group was extremely slow in the usual fashion
by using a Parr hydrogenator with Pd/C as the catalyst
in glacial acetic acid. On the other hand, the 3-Bz
protecting group was removed rapidly under ambient
conditions by catalytic transfer hydrogenation using
ammonium formate resulting in the desired products
4a -f.13,14 3-O-Butyl and -octyl ethers of 1 (5b,c; Scheme
1) as controls were prepared directly from 1 by using
alkyl halide in the presence of potassium carbonate.
In addition to NMR, mass spectrometry, chromato-
graphic, and combustion analyses to characterize the
compounds prepared, crystallography data were ob-
tained for two representative 17â-ethers (4a ,d ). The
solid-state conformation (ORTEP-type plot) of 4d is
shown in Figure 1. The crystals were monoclinic and
belong to the P2(1) space group, and this confirmed that
the 17-methoxy and -butoxy groups assumed a â-orien-
tation in the D-ring.
Resu lts a n d Discu ssion
Cytotoxicity studies on the compounds involved were
done on mouse clonal hippocampal HT-22 cells, and the
Calcein AM assay was used to quantify cell viability
because it is robust, reproducible, has high capacity, and
reliably determines cell viability against a variety of
insults.15 We have previously found this assay to be the
most reliable and reproducible of all of the viability
assays used. [For example, in an exhaustive assessment
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) versus Calcein AM in measuring toxicity
induced by Aâ, we observed that Calcien AM, but not
MTT, accurately measured viability - an observation
similar to that reported by others;16 both assays were
equally effective in detecting H2O2-induced toxicity on
the other hand.17]
The complex relationship of neuroprotection and 17-
alkoxy chain length was surprising. A comparison of the
solid-state conformation of 4a ,d revealed no apparent
differences in the preferred geometry of the steroid
backbone between a representative “active” (4d ) and an

112 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
Brief Articles
(Milwaukee, WI). Estradiol (1) and 3-O-methyl-17â-estradiol
(5a ) were purchased from Sigma (St. Louis, MO). Sodium
hydride was used as a 60% dispersion in mineral oil. Melting
points were determined on a Fisher-J ohns melting point
apparatus and uncorrected. Thin-layer chromatography (TLC)
was done on Whatman silica gel plates (on aluminum backing)
containing UV fluorescence indicator by using ethyl acetate:
hexane (1:4, v/v) eluent. All chromatographic purifications
were done on gravity columns with 230-435 mesh neutral
silica gel with ethyl acetate:hexane (1:4, v/v) as an eluent.
Elemental analyses were performed by the Atlantic Microlab,
Inc. (Norcross, GA). NMR spectral data were recorded for all
compounds using a Varian XL-300 spectrometer and TMS as
an internal standard. Mass spectral data were obtained by
using atmospheric-pressure chemical ionization (APCI) on a
quadrupole ion trap instrument (LCQ, Finnigan MAT, San
J ose, CA). Analytical reversed-phase high-performance liquid
chromatography was performed on
a ThermoSeparation/
SpectraPhysics (Fremont, CA) system consisting of an SP8810
isocratic pump, a Rheodyne (Cotati, CA) model 7125 injector
valve equipped with a 20-µL sample loop, an SP8450 variable
wavelength UV/VIS detector operated at 280 nm, and an
SP4290 computing integrator. A 15-cm × 4.6-mm i.d. octade-
cylsilica column (Phase Sep S5 ODS2, Queensferry, Clwyd,
U.K.) and a mobile phase of acetonitrile containing 1% acetic
acid at a flow rate of 1.0 mL/min were used for the analyses.
X-ray crystallography data were collected at 173 K on a
Siemens SMART PLATFORM equipped with A CCD area
detector and a graphite monochromator utilizing Mo KR
radiation (λ ) 0.71073 Å). Cell parameters for each structure
were refined using up to 8192 reflections and a hemisphere of
data (1381 frames) was collected using the ω-scan method (0.3°
frame width). The first 50 frames were remeasured at the end
of data collection to monitor instrument and crystal stability
(maximum correction on I was <1%). Absorption corrections
by integration were applied based on measured indexed crystal
faces. Both structures were solved by the Direct Methods in
SHELXTL521 and refined using full-matrix least-squares. The
non-H atoms were treated anisotropically, whereas the hy-
drogen atoms were calculated in ideal positions and were
riding on their respective carbon atoms, except the hydroxyl
protons H18 in 4a and H18 and H26 in 4d . These protons were
obtained from a difference Fourier map and refined without
any constraints. While no solvent crystallized with 4a , a
methanol molecule was found in the general position in the
lattice of 4d . A total of 196 parameters of 4a were refined in
the final cycle of refinement using 2961 reflections with I >
2σ(I) to yield R1 and wR2 of 5.03% and 12.66%, respectively.
For 4d , a total of 247 parameters were refined in the final
cycle of refinement using 3294 reflections with I > 2σ(I) to
yield R1 and wR2 of 3.71% and 8.90%, respectively. Refinement
was done using F2. Tables of geometric data, indicating
H-bonding interactions, are available as Supporting Informa-
tion.
F igu r e 2. HT-22 cell viability in vitro after glutamate
exposure (20 mM) following treatment with estradiol (1), its
17â-alkyl ethers (4a -f), and 3-butylestradiol (5b, as a typical
representative of the 3-alkyl ethers). Statistically significant
differences between groups were tested by analysis of variance
(ANOVA) followed by post hoc Tukey test: *significant in-
crease (p < 0.05) vs vehicle control, **significant increase (p
< 0.05) vs vehicle control but decrease compared to 10 µM
estradiol (1), ***significant increase (p < 0.05) vs vehicle
control and statistically significant increase compared to 10
µM estradiol (1).
“inactive” (4a ) ether derivative of 1. One possible
explanation is that the interaction of the alkyl chain of
the 17â-substituent with the target site or the lipoidal
cell membrane plays an important role in the efficacy
of the derivative as a neuroprotectant. Thus, 4a ,b
having a compact alkyl group may not have the flex-
ibility (i.e., sufficient degrees of freedom for bond
rotation) to embed into a cell membrane effectively;
however, a longer alkyl chain (C g 3) may provide this
property. We also noted that the two “inactive” 17-ethers
(4a ,b) were compounds with high melting points, while
the active ones have significantly lower melting points.
Further studies correlating neuroprotection by phenolic
A-ring steroids (including 4a -f) with their effect on
membrane fluidity are underway.
3-Ben zyloxyestr a -1,3,5(10)-tr ien -17â-ol (2).12 To 5 g (18
mmol) of 1 and 10 g (72 mmol) of potassium carbonate in 100
mL of dry acetone was added 5.7 g (4.0 mL, 34 mmol) of benzyl
bromide. The mixture was refluxed overnight under nitrogen
atmosphere. Upon cooling the solid was removed by filtration.
The filtrate was collected and acetone was removed in vacuo
leaving behind clear yellowish oil, which solidified on standing.
Recrystallization from ethyl acetate/hexane gave 6.1 g (93%
1
yield) of a white fluffy solid: mp 61-63 °C; TLC Rf 0.23; H
NMR (CDCl3) δ 7.44-7.19 (m, 5H, C6H5 of benzyl), 7.12 (d, J
) 8.6 Hz, 1H, 1-CH), 6.78 (dd, J ) 8.7 and 2.7 Hz, 1H, 2-CH),
6.72 (d, J ) 2.4 Hz, 1H, 4-CH), 5.05 (s, 3H, OCH2 of benzyl),
3.37 (tr, J ) 8.4 Hz, 1H, 17R-CH), 2.87-2.82 (m, 2H, 6-CH2),
2.34-1.18 (m, 13H), 0.78 (s, 3H, 13-CH3); MS m/z 363 [M +
H]+.
In summary, our results indicate that higher 17â-
alkyl ethers of estradiol (4c-f) show a dose-dependent
neuroprotection in vitro against oxidative stress in HT-
22 cells. Moreover, this effect is manifested at lower
concentration (<1 µM) than that of the parent com-
pound (1).
Gen er a l P r oced u r e for P r ep a r a tion of 3-Ben zyloxy-
17â-a lk oxyestr a -1,3,5(10)-tr ien es 3a -f. Compound 2 (0.8 g,
2.2 mmol) was dissolved in 5 mL of anhydrous DMF and, then,
sodium hydride (0.3 g) was added. The mixture was stirred at
room temperature for 30 min before the addition of 20 mmol
Exp er im en ta l Section
In str u m en ts a n d Ma ter ia ls. All solvents and materials
were obtained from Fisher Scientific (Atlanta, GA) or Aldrich

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
free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Glenner, G. G. Alzheimer’s disease: its proteins and genes. Cell
1988, 52, 307-308.
(2) Yankner, B. A. Mechanism of neuronal degeneration in Alzhe-
imer’s disease. Neuron 1996, 16, 921-936.
(3) Maher, P.; Davis, J . The role of monoamine metabolism in
oxidative glutamate toxicity. J . Neurosci. 1996, 15, 6394-6401.
(4) (a) Paganni-Hill, A.; Henderson, V. Estrogen deficiency and risk
of Alzheimer’s disease in women. Am. J . Epidemiol. 1994, 140,
256-261. (b) Kawas, C.; Resnick, S.; Morrison, A.; Brookmeyer,
R.; Corrasa, M.; Zonderman, A.; Basal, C.; Lingle, D. D.; Metter,
E. A prospective study of estrogen replacement therapy and the
risk of developing Alzheimer’s disease: the Baltimore longitu-
dinal study of aging. Neurology 1997, 48, 1517-1521.
(5) Gridley, K. E.; Green, P. S.; Simpkins, J . W. A novel, synergistic
interaction between 17â-estradiol and glutathione in the protec-
tion of neurons against â-amyloid 25-35-induced toxicity in
vitro. Mol. Pharmacol. 1998, 54, 874-880.
(6) Bishop, J .; Simpkins, J . W. Estradiol treatment increases
viability of glioma and neuroblastoma cells in vitro. Mol. Cell
Neurosci. 1994, 5, 303-308.
(7) (a) Behl, C.; Widmann, M.; Trapp, T.; Holspoer, F. 17â-estradiol
protects neurons from oxidative stress-induced cell death in
vitro. Biochem. Biophys. Res. Commun. 1995, 216, 473-482. (b)
Sawada, H.; Ibi, M.; Kihara, T.; Urushitani, M.; Akaike, A.;
Shimohama, S. Estradiol protects mesencephalic dopaminergic
neurons from oxidative stress-induced neuronal death. J . Neu-
rosci. Res. 1998, 54, 707-719.
(8) (a) Green, P. S.; Bishop, J .; Simpkins, J . W. 17R-estradiol exerts
neuroprotective effects in SK-N-SH cells. J . Neurosci. 1997, 17,
511-515. (b) Behl, C.; Skutella, T.; Lezoualch, F.; Post, A.;
Widmann, M.; Newton, C. J .; Holsber, F. Neuroprotection
against oxidative stress by estrogens. Structure-activity rela-
tionship. Mol. Pharmacol. 1997, 51, 535-541.
(9) Green, P. S.; Gordon, K.; Simpkins, J . W. Phenolic A ring
requirements for the neuroprotective effects of steroids. J .
Steroid Biochem. Mol. Biol. 1997, 63, 229-235.
(10) Moorsmann, B.; Behl, C. The antioxidant neuroprotective effects
of estrogens and phenolic compounds are independent from their
estrogenic properties. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
8867-8872.
(11) Coombs, M.; Roderick, H. 17â-O estradiol 17-methyl ether.
Steroids 1965, 6, 841-4.
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
(12) Qian, X.; Abul-Hajj, Y. J . Synthesis and biological activity of 17â-
substituted estradiol. J . Steroid Biochem. 1988, 29, 657-664.
(13) Anwer, M.; Spatola, A. An advantageous method for the rapid
removal of hydrogenolysable protecting groups under ambient
conditions; synthesis of leucine-enkephalin. Synthesis 1980,
929-932.
(14) Elamin, B.; Anantharamaiah, G.; Royer, G.; Means, G. Removal
of benzyl-type protecting groups from peptides by catalytic
transfer hydrogenation with formic acid. J . Org. Chem. 1979,
44, 3442-3444.

114 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1
Brief Articles
(15) Green, P. S.; Perez, E. T.; Calloway, T.; Simpkins, J . W. Estradiol
attenuation of â-amyloid-induced toxicity: A comparison of MTT
and Calcein assays. J . Neurocytol., in press.
(16) (a) Behl, C.; Davis, J . B.; Lesley, R.; Schubert, D. Hydrogen-
peroxide mediates amyloid-beta protein toxicity. Cell 1994, 77,
817-827. (b) Shearman, M. S.; Ragan, C. I.; Iversen, L. L.
Inhibition of PC12 cell redox activity is a specific, early indicator
of the mechanism of beta-amyloid-mediated cell-death. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 470-1474.
(17) (a) Green P. S.; Gridley K. E.; Simpkins, J . W. Estradiol protects
against â-amyloid (25-35)-induced toxicity in SK-N-SH human
neuroblastoma cells. Neurosci. Lett. 1996, 218, 165-168. (b)
Green P. S.; Gridley K. E.; Simpkins, J . W. Nuclear estrogen
receptor-independent neuroprotection by estratrienes: A novel
interaction with glutathione. Neuroscience 1998, 84, 7-10.
(18) Mook-J ung, I.; J oo, I.; Sohn, S.; Kwon, H. J .; Huh, K.; J ung, M.
W. Estrogen blocks neourotoxic effect of â-amyloid (1-42) and
induces neurite extension on B103 cells. Neurosci. Lett. 1997,
235, 101-104.
(19) Pike, J . Estrogen modulates neuronal Bcl-xl expression and
â-amyloid-induced apoptosis. Relevance to Alzheimer’s disease.
J . Neurochem. 1999, 72, 1552-1563.
(20) The logarithm of the 1-octanol/water partition coefficient (log
P) was calculated by an atom fragment method implemented in
the molecular modeling package HyperChem version 6.0 (Hy-
percube, Gainesville, FL): Ghose, A. K.; Pritchett, A.; Crippen,
G. M. Atomic physicochemical parameters for 3-dimensional
structure directed quantitative structure-activity-relationships.
3. Modeling hydrophobic interactions. J . Comput. Chem. 1988,
9, 80-90. The obtained log P values were as follows: 4.01 (1),
4.29 (4a ), 4.63 (4b), 5.10 (4c), 5.49 (4d ), 6.29 (4e), and 7.08 (4f).
The calculated log P values for the 3-alkylestradiols were 4.09
(5a ), 5.25 (5b), and 6.83 (5c).
(21) Sheldrick, G. M. SHELXTL5; Bruker-AXS: Madison, WI, 1998.
J M000280T