Journal of Medicinal Chemistry
Article
standard (CDCl3, δ = 77.16, CD3CN, δ = 1.30, center line, acetone-d6,
δ = 29.80, center line). High resolution mass spectra were obtained at
the University of Illinois Mass Spectrometry Laboratory. The purities
of target compounds were ≥95%, measured by HPLC using a Waters
1525 binary HPLC pump equipped with a Waters in-line degasser AF,
Waters 2487 Dual λ absorbance detector and a Symmetry C18 5 μm
4.6 mm × 150 mm column (part no. WAT045905). Chiral high
pressure liquid chromatographic (HPLC) analysis was performed
using a Waters 1525 binary HPLC pump equipped with a Waters in-
line degasser AF, Waters 2487 Dual λ absorbance detector, and a Regis
Technologies (R,R)-Whelk-O 2 column (particle size, 10 μm, 100 Å;
column dimensions, 25 cm × 4.6 mm, cat. no. 786315). Optical
rotations were obtained using a JASCO DIP-370 digital polarimeter
and a 3.5 mm × 50 mm cell and are reported as follows: concentration
(c = g/100 mL), solvent. Melting points were recorded on a Thomas-
Hoover Uni-Melt 6427-F10 capillary melting-point apparatus. [3H]-
17β-Estradiol, specific activity 89 Ci/mmol (3293 GBq/mmol), was
purchased from Perkin-Elmer Life Science (Boston, MA). 17β-
Estradiol (17β-E2) was obtained from Sigma (St. Louis, MO). Purified
full-length human ERα and ERβ were purchased from Pan Vera
(Madison, WI). The thiol reactive fluorophore, 5-iodoacetamido
fluorescein, and terbium-labeled streptavidin were obtained from
Molecular Probes/Invitrogen (Eugene, CA). Thiol reactive biotin
derivative (MAL-dPEG4-biotin) was from Quanta BioDesign (Powell,
OH).
The resulting white suspension was stirred at 0 °C for 3 h before being
quenched with cold 0.1 M HCl (20 mL).20 The residue was extracted
with EtOAc (2 × 100 mL), and the combined organic extracts were
dried over MgSO4 and concentrated in vacuo. Purification by column
chromatography (EtOAc:Hex, 2:1) afforded 9 (0.63 g, 96.6%) as an
off-white solid; mp 117−119 °C. Rf = 0.21 (Hex:EtOAc, 2:1). [α]D23
138.61 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ 7.21 (d, J = 8.8
Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 6.77 (d, J
= 8.6 Hz, 2H), 3.82−3.71 (m, 7H), 3.31 (dd, J = 13.9, 8.4 Hz, 1H),
2.95 (dd, J = 13.8, 7.2 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 179.4,
159.0, 158.1, 130.8, 130.0, 129.9, 129.1, 114.0, 113.7, 55.2, 55.2, 52.8,
38.4. HRMS (ESI) calcd for C17H18O4Na [M + 1] 309.1103; found
309.1103.
(S)-2,3-Bis(4-hydroxyphenyl)propanenitrile (2). To a solution of 9
(50.1 mg, 0.18 mmol) in triethylamine (36.6 μL, 0.26 mmol) and THF
(4 mL) at −20 °C was added isobutyl chloroformate (45.5 μL, 0.35
mmol). The resulting solution was stirred for 20 min at −20 °C,
followed by the addition of ammonia (2.0 M in IPA, 0.88 mL, 1.75
mmol), and left to stir for an additional 20 min at −20 °C before being
quenched by passing through a Celite plug. The crude solution was
concentrated in vacuo, redissolved in THF (1.5 mL), and cooled to 0
°C, followed by addition of pyridine (60.9 μL, 0.75 mmol) and
trifluoroacetic anhydride (51.1 μL, 0.37 mmol). The mixture was left
to stir at 0 °C for 5 min before being quenched passing through a
Celite plug and evaporation of solvent. The crude was redissolved in
DCM (1.5 mL) and cooled to −78 °C, and BBr3 (1.0 M in DCM, 1.5
mL, 1.50 mmol) was added dropwise over 5 min. The resulting
mixture was left to warm to room temperature over 3 h before being
quenched upon slow addition of MeOH at 0 °C. The crude solution
was passed through a Celite plug, concentrated in vacuo, and
recrystallized (Hex:EtOAc, 1:1) to afford 2 (26.5 mg, 63.2% over 3
steps) as an off-white solid; mp 190−192 °C. [α]D23 1.386 (c 1.1,
(S)-4-Benzyl-3-(2-(4-methoxyphenyl)acetyl)oxazolidin-2-one
(6). To a mixture of 4-methoxyphenylacetic acid (4, 2.80 g, 16.9
mmol) and (S)-4-benzyl-2-oxazolidinone (5, 1.50 g, 8.46 mmol) in
PhCH3 (15 mL) at room temperature was added triethylamine (4.72
mL, 33.9 mmol).18 The clear solution was heated to 80 °C for 10 min,
and then a solution of pivaloyl chloride (2.08 mL, 16.9 mmol) in
PhCH3 (3.5 mL) was added dropwise. After full addition, the reaction
mixture was refluxed for 14 h before being cooled to room
temperature and quenched with 1 M HCl (20 mL) and extracted
with EtOAc (2 × 50 mL), and the combined organic extracts were
washed with 5% NaHCO3 solution (15 mL), dried over MgSO4, and
concentrated in vacuo. Purification by column chromatography
(Hex:EtOAc, 2:1, to Hex:EtOAc:MeOH, 1:1:0.1) and recrystallization
(PhCH3:Hex, 1:1) afforded 6 (2.07 g, 75.2%) as a white solid; mp 80−
1
MeOH). Rf = 0.73 (Hex:EtOAc, 1:2). H NMR (500 MHz, acetone-
d6) δ 7.19 (d, J = 8.6 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.6
Hz, 2H), 6.75 (d, J = 8.1 Hz, 2H), 4.16 (t, J = 7.6 Hz, 1H), 3.11−3.00
(m, 2H). 13C NMR (126 MHz, CD3CN) δ 157.7, 157.0, 131.4, 129.9,
129.4, 128.2, 122.2, 116.6, 116.1, 41.4, 39.4. HRMS (ESI) calcd for
C15H13NO2Na 262.0844; found 262.0846.
Protein Expression, Purification, and Labeling of ERα-417,
ERβ-369, and SRC3. The pET15b bacterial expression plasmids
encoding six-His fusion proteins of human ER LBDs, ERα-417 (amino
acids 304−554), and ERβ-369 (amino acids 256−505), each with a
single reactive cysteine at C417 or C369, respectively, and the nuclear
receptor interaction domain (NRID) of human steroid receptor
coactivator 3 (SRC3) encompassing 3 NR boxes (amino acids 627−
829) have been described previously, as have the methods for protein
expression and purification.32,40 ER LBDs and the SRC3 fragment
were respectively labeled with MAL-dPEG4-biotin and 5-iodoaceta-
mido fluorescein, according to the previously published procedure.40
Radiometric Competitive Binding Assay to Determine
Relative Ligand-Binding Affinity (RLA). RLAs (previously re-
ferred to as relative binding affinities, RBAs) were determined by
competitive radiometric binding assays using 0.5 nM full length human
ERα or ERβ in the presence of 2 nM [3H]-17β-E2 and various
concentrations of unlabeled 17β-E2, rac-DPN, R-DPN, and S-DPN as
previously described.26,27 The concentrations of unlabeled 17β-E2 and
different DPNs required to reduce the binding of [3H]-17β-E2 by 50%
(IC50) were obtained from the displacement curves. The RLA values
of rac-DPN, R-DPN, and S-DPN were determined using the following
equation:
1
82 °C. Rf = 0.31 (Hex:EtOAc, 2:1). H NMR (500 MHz, CDCl3) δ
7.31−7.22 (m, 5H), 7.13 (d, J = 6.6 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H),
4.69−4.61 (m, 1H), 4.30−4.13 (m, 4H), 3.79 (s, 3H), 3.25 (dd, J =
13.3, 3.2 Hz, 1H), 2.74 (dd, J = 13.4, 9.5 Hz, 1H). 13C NMR (126
MHz, CDCl3) δ 171.6, 158.8, 153.4, 130.8, 129.4, 128.9, 127.3, 125.4,
114.0, 66.1, 55.3, 55.2, 40.7, 37.7. HRMS (ESI) calcd for C19H20NO4
[M + 1] 326.1392; found 326.1392.
(S)-4-Benzyl-3-((S)-2,3-bis(4-methoxyphenyl)propanoyl)-
oxazolidin-2-one (8). To a solution of 6 (0.10 g, 0.31 mmol) in THF
(1 mL) at −78 °C was added NaHMDS (1.0 M in THF, 0.33 mL, 0.33
mmol) dropwise and left to stir at this temperature for 1 h.19 4-
Methoxybenzyl bromide (7, 90 μL, 0.61 mmol) was then added at
−78 °C dropwise and left to stir to room temperature over 5 h before
being quenched with H2O (10 mL). The crude reaction was extracted
with EtOAc (2 × 20 mL), and the combined organic extracts were
dried over MgSO4 and concentrated in vacuo. Purification by column
chromatography (Hex:EtOAc, 3:1) and recrystallization (Hex:EtOAc,
1:1) afforded 8 (0.11 g, 79.4%, dr > 99:1) as a white solid; mp 169−
1
171 °C. Rf = 0.54 (Hex:EtOAc, 2:1). [α]D23 145.9 (c 1.2, CHCl3). H
NMR (500 MHz, CDCl3) δ 7.36 (d, J = 8.8 Hz, 2H), 7.29−7.22 (m,
5H), 7.17 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 8.6
Hz, 2H), 5.38 (dd, J = 9.5, 5.7 Hz, 1H), 4.60−4.53 (m, 1H), 4.04−
3.99 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.44 (dd, J = 13.1, 9.4 Hz,
1H), 3.04−2.92 (m, 2H), 2.59 (dd, J = 13.5, 8.8 Hz, 1H). 13C NMR
(126 MHz, CDCl3) δ 173.7, 158.9, 158.1, 152.8, 135.0, 131.1, 130.3,
130.3, 129.7, 129.4, 128.8, 127.2, 114.0, 113.7, 65.5, 55.3, 55.2, 55.2,
49.6, 39.7, 37.5. HRMS (ESI) calcd for C27H28NO5 [M + 1] 446.1967;
found 446.1970.
RLA(DPN) = {IC (17β − E )/IC (DPN)} × 100
50
2
50
SRC3 Titration Assay: Determination of Relative Coactiva-
tor-Binding Affinity (RCA). These assays were performed as
recently described.31 Different concentrations of fluorescein-labeled
SRC3 fragment (Fl-SRC3) were prepared in buffer A (50 mM Tris pH
7.9, containing 10% glycerol, 0.01% Nonidet P-40, 50 mM KCl, 2 mM
β-mercaptoethanol, 2% dimethylformamide, and 0.3 mg/mL ovalbu-
min). Streptavidin−terbium (SA-Tb) and biotinylated-ERα or ERβ
LBD were premixed in buffer A. Ligand dilutions were made in buffer
(S)-2,3-Bis(4-methoxyphenyl)propanoic Acid (9). To a solution of
8 (1.01 g, 2.27 mmol) in THF:H2O (120 mL, 5:1) at 0 °C was added
H2O2 (30 wt % in H2O, 14.3 mL) and LiOH (54.3 mg, 2.27 mmol).
535
dx.doi.org/10.1021/jm201436k | J. Med. Chem. 2012, 55, 528−537