steroids 7 3 ( 2 0 0 8 ) 1252–1261
1255
methylene chloride (50 mL). The organic phase was combined,
washed with a saturated sodium chloride solution (100 mL),
and dried over anhydrous sodium sulfate. Flash chromatogra-
phy afforded compound 2 (14.6 g, 94% yield).
(1H, brS), 5.29 (1H, m), 4.59 (1H, m), 3.85 (2H, m), 3.67 (1H, t,
J = 8.4 Hz), 3.55 (1H, m), 3.43 (1H, m), 2.81 (2H, m), 2.21 (2H, m),
1.92–1.12 (38H, m), and 0.73 (3H, s). MS (TOF MS ES+, C35H55O4N,
M+ 553): 555 (M+1) (base peak). HRMS (M+1): 554.4209 (calcu-
lated) and 554.4208 (observed). Compound 5a. 1H NMR (300 Hz,
DMSO-d6): 9.15 (1H, brs), 8.20 (3H, brs), 7.07 (1H, d, J = 8.4 Hz),
6.57 (1H, d, J = 8.4 Hz), 6.51 (1H, s), 4.94 (1H, brs), 3.59 (1H, t,
J = 7.8 Hz), 2.77 (3H, m), 2.65 (1H, d, J = 16.8Hz), 2.27 (2H, m),
1.97–1.11 (21H, m), and 0.72 (3H, s). MS (C25H39NO2, M 385):
385 (base peak). HRMS (M): 385.2981 (calculated) and 385.2984
(observed).
To synthesize compound 3, a 1.0 M solution of potassium
t-butoxide (t-BuOK) in THF (20 mL, 20 mmol) was added to a
cooled solution (at 0 ◦C) of compound 2 (1.2 g, 2.67 mmol) in
THF (20 mL). The reaction mixture was stirred at 0 ◦C for 40 min
and then cooled to −78 ◦C. 7-Iodoheptanenitrile (compound
9, 2.0 g, 7.1 mmol) was added dropwise to the solution. The
reaction mixture was allowed to warm to 0 ◦C and stirred for
2 h, and then to room temperature and stirred overnight. The
reaction was quenched with water and extracted with ethyl
acetate. The organic phase was dried over sodium sulfate and
the solvent was evaporated in vacuo. Flash chromatography
(hexane:ethyl acetate = 4:1, v:v) afforded compound 3 in 62%
yield (0.93 g). 1H NMR (300 Hz, CDCl3): 7.61 (1H, brS), 7.15–7.25
(2H, m), 5.39 (1H, m), 4.62 (1H, m), 3.80 (2H, m), 3.68 (1H, t,
J = 8.4 Hz), 3.56 (1H, m), 3.42 (1H, m), 2.59 (1H, m), 2.39 (2H, m),
2.24 (2H, t, J = 7.2Hz), 1.16–2.02 (31H, m), and 0.73 (3H, s). MS
(Scan ES+, C35H49O5N, M+1 564): 564 (base peak), 586 (M+Na).
HRMS (M+1): 564.3689 (calculated) and 564.3682 (observed).
2.1.9. Synthesis of 7˛-{7-[8-(biotinamido)-octanamido]-
heptyl}-estradiol (compound 7)
The crude preparation containing mostly compound
5
(42 mg, 0.11 mmol) was dissolved in 10 mL DMF. Then
0.053 g (0.11 mmol) of N-hydroxysuccinimide ester of 8-N-
biotinylaminooctanoic acid (compound 13) and 47 L of TEA
were added. The mixture was stirred at room temperature
for 24 h. The solvents were removed under high vacuum, and
the raw product was dissolved in a mixture of acetic acid,
methanol and water (4:2:1, v:v:v) and stirred for 24 h at room
temperature. The solvents were removed in vacuo, and the raw
product was purified by HPLC to afford compound 7.
2.1.7. Synthesis of 6-hydroxy-7˛-(6-cyanohexyl)-
estradiol-3,17ˇ-di-tetrahydropyranyl ether (compound 4)
1H NMR (300 Hz, DMSO-d6): 8.98 (1H, s), 7.72 (2H, m), 7.04
(1H, d, J = 8.7 Hz), 6.49 (1H, dd, J = 8.7, 2.7 Hz), 6.43 (1H, s), 6.41
(1H, d, J = 2.7 Hz), 6.36 (1H, s), 4.48 (1H, d, J = 4.8 Hz), 4.29 (1H,
m), 4.13 (1H, m), 3.53 (1H, m), 3.09 (1H, m), 3.0 (4H, m), 2.80
(2H, m), 2.62 (2H, m), 2.26 (2H, m), 2.0 (4H, m), 190–1.05 (38H,
m), and 0.66 (3H, s). MS (TOF MS ES+, C43H68N4O5S, M+ 752.5):
753.5 (M+1) (base peak). HRMS (M+1): 753.4988 (calculated) and
753.4982 (observed).
Compound
3 (0.51 g, 0.91 mmol) was dissolved in 20 mL
methanol. To this solution was added sodium borohydride
(0.69 g, 18.1 mmol). The reaction mixture was stirred at room
temperature for 4 h. The methanol was removed in vacuo.
The residue was then dissolved in water (20 mL). The aque-
ous solution was extracted with methylene chloride three
times (3 × 20 mL). Organic fractions were combined, dried over
sodium sulfate, and concentrated. The crude product was
purified by flash chromatography (ethyl acetate:hexane = 1:3,
v:v) to afford compound 4 (0.5 g, 98%). 1H NMR (300 Hz, CDCl3):
7.4 (1H, d, J = 2.4 Hz), 7.09 (1H, d, J = 8.8 Hz), 6.84 (1H, m), 5.35 (1H,
m), 4.81 (1H, m), 4.57 (1H, m), 3.84 (2H, m), 3.68 (1H, t, J = 7.6 Hz),
3.52 (1H, m), 3.42 (1H, m), 2.18–2.31 (4H, m), 1.87–2.07 (4H, m),
1.76 (4H, m), 1.20–1.65 (25H, m), and 0.73 (3H, s). MS (TOF MS
ES+, C35H51O5N, M+ 565): 540, 548, 566, 583 (M+NH4+) (base
peak), 588 (M+Na+). HRMS [588 (M+Na+)]: 588.3665 (calculated)
and 588.3660 (observed).
2.2.
ER˛- and ERˇ-binding assays
The ER␣ and ER competitive binding assays were performed
according to the method described in our recent study [15].
The ER-binding buffer used for dilution of the receptor prepa-
rations consisted of 10% glycerol, 2 mM dithiothreitol, 1 mg/mL
BSA, and 10 mM Tris–HCl (pH 7.5). The ER␣ washing buffer con-
tained 40 mM Tris–HCl and 100 mM KCl (pH 7.4), but the ER
washing buffer contained only 40 mM Tris–HCl (pH 7.5). The
50% (v/v) hydroxylapatite (HAP) slurry was prepared by using
the 50 mM Tris–HCl solution (pH 7.4). The reaction mixture
contained 50 L of varying concentrations of the test com-
pound in the ER-binding buffer, and 45 L of [3H]E2 solution
(at 22.22 nM). Then 5 L of ER␣ or ER protein was added and
mixed gently. Nonspecific binding by [3H]E2 was determined
by addition of a 400-fold concentration of the nonradioactive
E2 (8 M). The binding mixture was incubated at room tem-
perature for 2 h. At the end of the incubation, 100 L of the
HAP slurry was added and the tubes were incubated on ice for
15 min with three times of brief vortexing. An aliquot (1 mL)
of the washing buffer was added, mixed, and centrifuged
at 10,000 g for 1 min, and the supernatants were discarded.
This wash step was repeated twice. The HAP pellets were
then resuspended in 200 L ethanol, and the content was
transferred to scintillation vials for measurement of the 3H-
radioactivity in a liquid scintillation counter (Packard Tri-CARB
2900 TR; Downers Grove, IL). The data obtained from dupli-
2.1.8. Synthesis of
7˛-(7-aminoheptyl)-estradiol-3,17ˇ-di-tetrahydropyranyl
ether (compound 5)
Compound 4 (0.4 g, 0.71 mmol) and 10% palladium hydrox-
ide on carbon (400 mg) were added to 50 mL of ethanol. The
reaction vessel was evacuated of air and filled to a pres-
sure of 45 psi with hydrogen on a Parr apparatus, and then
shaken for 8 h at 60 ◦C. The mixture was then filtered through
celite, and the filtrate was evaporated under reduced pressure
to give a mixture of compound 5 and compound 5a [7␣-(7-
aminoheptyl)-estradiol] by a ratio of 5:1 based on 1H NMR (total
0.39 g, ∼100%). This mixture was not further purified for the
next synthetic step.
For spectrometric analysis, a fraction of the samples was
used for separation of compounds 5 and 5a by flash chro-
matography (hexane:ethyl acetate = 4:1, v:v). Compound 5. 1
NMR (300 Hz, CDCl3): 7.11 (1H, m), 6.77 (1H, d, J = 8.4 Hz), 6.68
H