Design, synthesis, and biological evaluation of steroidal analogs as estrogenic/anti-estrogenic agents

Article abstract of DOI:10.1016/j.steroids.2016.11.005

Series of estrone based analogs were synthetically investigated at positions C-9, C-11, C-16, and C-17 positions, to be biologically evaluated via assessment of cell proliferation, cytotoxicity, and estrogenic/anti-estrogenic activity. LA-7 and LA-10 revealed their potential to exhibit inhibitory estrogenic profile. This was further validated by Estrogen Receptor-α (ER-α) and Estrogen Receptor-β (ER-β) competitive binding assays to reveal the high selective affinity of LA-7 towards ER-α at 5.49 μM, while LA-10 did not show any binding affinity towards neither ER-α nor ER-β; suggesting another mechanism for inhibition. This was validated by in silico molecular docking simulations of LA-7 to reveal the optimum binding affinity of LA-7 towards ER-α.

Full text of DOI:10.1016/j.steroids.2016.11.005

Steroids 118 (2017) 32–40
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Design, synthesis, and biological evaluation of steroidal analogs as
estrogenic/anti-estrogenic agents
b
a
c
a
a
Abdulrhman Alsayari , Lucas Kopel , Mahmoud Salama Ahmed , Adam Pay , Taylor Carlson ,
Fathi T. Halaweish
a
a,
⇑
Department of Chemistry and Biochemistry, South Dakota State University, Box 2202, Brookings, SD 57007, USA
Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Saudi Arabia
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The British University in Egypt, Al-Sherouk City, Cairo, Egypt
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of estrone based analogs were synthetically investigated at positions C-9, C-11, C-16, and C-17
positions, to be biologically evaluated via assessment of cell proliferation, cytotoxicity, and estrogenic/
anti-estrogenic activity. LA-7 and LA-10 revealed their potential to exhibit inhibitory estrogenic profile.
Received 19 September 2016
Received in revised form 13 November 2016
Accepted 17 November 2016
Available online 20 November 2016
This was further validated by Estrogen Receptor-
binding assays to reveal the high selective affinity of LA-7 towards ER-
not show any binding affinity towards neither ER- nor ER-b; suggesting another mechanism for inhibi-
tion. This was validated by in silico molecular docking simulations of LA-7 to reveal the optimum binding
affinity of LA-7 towards ER-
a
(ER-a
) and Estrogen Receptor-b (ER-b) competitive
a
at 5.49 M, while LA-10 did
l
a
Keywords:
Breast cancer
Estrone
Estrogenic/anti-estrogenic activity
Breast cancer (MCF-7) cell lines
a
.
Ó 2016 Published by Elsevier Inc.
1
. Introduction
Breast cancer is one of the most frequently diagnosed cancers
ERb, have been identified and they are encoded by two unique
genes [8]. ER b shares a high homology to ER
the DNA Binding Domain (DBD) (95%) and the Ligand Binding
Domain (LBD) (55%) [9]. ER alpha-positive (ER +) breast cancer is
a, in particular in
among women. It is the second leading cause of death, after lung
cancer [1]. Several studies have revealed that estrogen receptor
a
the most common type of breast cancer and consequently depends
on estrogen for growth and development. Therefore, targeting
estrogen and its receptor is one of the major strategies in the treat-
ment of breast cancer [8]. Tamoxifen (Nolvadex), a non-steroidal
estrogen antagonist, is the drug of choice for the treatment of
(
ER), progesterone receptor (PR), and human epidermal growth
factor receptor two (HER2) are among the most frequently found
biomarkers in breast cancer tissue, playing an important role in
the growth of human breast cancer [2,3]. Estrogens are known
group of steroid hormones that are synthesized from cholesterol
precursors [4]. In premenopausal women, estrogens are mainly
produced by ovaries, while in postmenopausal women, estrogens
are synthesized in peripheral tissues, such as the adrenal gland,
adipose tissue and others tissues such as the breast itself [5]. The
most common naturally occurring estrogen forms are Estrone
ERa+ breast cancer with both premenopausal and postmenopausal
women [10]. Tamoxifen is a selective estrogen receptor modulator
(SERM) that competes with estrogens in binding to ERs; thereby
blocks the transactivation AF-2 site of ER and inhibits estrogen
action [11]. Despite the beneficial effects of tamoxifen in the treat-
ment of breast cancer, its promoter effect on the gynaecological
tract may lead to the development of potential side effects such
as hot flashes, vaginal discharge and bleeding, suppression of men-
struation, and gastrointestinal disturbances [11]. The second line
(
E1), Estradiol (E2) and Estriol (E3), where Estradiol (E2, 17b-estra-
diol) is the most abundant estrogen form in the tissues [6]. Estro-
gens have been recognized as promoters of cancer cell proliferation
and increased tumour invasiveness through stimulating the secre-
tion of protease [6]. Estrogen action is regulated via its nature as
endogenous substrate for estrogen receptors (ER), a member of
the nuclear hormone receptor family of hormone activated tran-
treatment option for ER
a+ breast cancer is fulvestrant (Faslodex,
ICI 182, 780), a steroidal 7
a-alkyl sulfinyl analogue of estradiol
[12]. Fulvestrant is a selective estrogen receptor down regulator
(SERD), that prevents ER dimerization and down-regulates the
ER. Unlike tamoxifen, fulvestrant is a pure anti-estrogen in all tis-
sues without known estrogenic activities; however its bioavailabil-
ity is poor [12]. The major problems associated with hormonal
treatment of breast cancer are drug resistance and side effects
scription factors [7]. Two types of estrogen receptor, ER
a and
⇑
Corresponding author.
E-mail address: fathi.halaweish@sdstate.edu (F.T. Halaweish).
http://dx.doi.org/10.1016/j.steroids.2016.11.005
039-128X/Ó 2016 Published by Elsevier Inc.
0

A. Alsayari et al. / Steroids 118 (2017) 32–40
33
[
13]. Therefore, novel potential drug candidates are always
addition of a solution of alkene LA-5 (1.20 g, 4.06 mmol) in DCM
(13.1 mL) to the reaction mixture at 0 °C. The reaction was allowed
to warm to room temperature and stirred for 18 h. The reaction
required to overcome the resistance arising from chemotherapeu-
tic agents. Minor modifications on the estrone/estradiol scaffold
result in significant changes in the biological activity profile, such
as 2-methoxyestradiol that showed anticancer activities and
induction of apoptosis via microtubule disruption and angiogene-
sis inhibitory effect; without targeting LBD of ER [14]. Recently,
2 3
was cooled to 0 °C and 10% Na SO (40 mL) was added to the mix-
ture. The aqueous layer was extracted with DCM (3ꢀ 75 mL). The
combined organic layers were washed with saturated NaCl
2 4
(30 mL), dried with Na SO , filtered and concentrated in vacuo.
installation of 23, 24
a
, b unsaturated side chain to steroidal scaf-
The crude material was purified by silica gel column chromatogra-
fold exhibited significant inhibition for mitogen activated protein
kinase (MAPK) pathway [15]. Herein, starting with estrone scaf-
fold, several analogs were synthetically investigated installing
functional group moieties at C-9, C-11, C-16 and C-17, to be fol-
lowed by biological screening.
phy using a gradient of hexanes/EtOAc (9:1–4:1) to yield alcohol
LA-7 (0.94 g, 74%) as a white solid, with ethyl acetate as solvent
1
residual. H NMR (400 MHz, CDCl
3
) d 7.20 (d, J = 8.6 Hz, 1H), 6.70
(dd, J = 8.7, 2.8 Hz, 1H), 6.62(d, J = 2.7 Hz, 1H), 5.51 (qd, J = 7.2,
2.0 Hz, 1H), 4.54 (d, J = 6.04 Hz, 1H), 3.77 (s, 3H), 2.94–2.85 (m,
2
1
H), 2.40–2.24 (m, 3H), 1.94–1.86 (m, 1H), 1.79–1.67 (m, 2H),
.69 (dt, J = 7.2, 2.0 Hz, 3H), 1.61–1.23 (m, 5H), 0.9 (s, 3H); 13
) d 157.5, 155.1, 137.9, 132.7, 126.2, 119.4,
13.8, 111.5, 74.2, 55.1, 51.5, 44.7, 43.8, 37.8, 37.4, 34.9, 29.6,
C
2
. Experimental section
NMR (100 MHz, CDCl
3
1
2
3
2
2
4
.1. Chemistry
7.6, 26.8, 17.6, 13.3; HRMS: Calcd for C21
12.20892.
28 2
H O : 312.2084, found
.1.1. Material and methods
LA-8 Alcohol: Alcohol LA-7 (100 mg, 0.32 mmol) was added to
a suspension of 5% Pd/C (140 mg) in EtOH (1.30 mL) and DCM
0.60 mL). The reaction was then stirred for 18 h under an atmo-
sphere of H gas using a balloon. The reaction was diluted with
1H and 13C NMR spectra were acquired on a Bruker AVANCE-
3
00 MHz NMR spectrometer, in CDCl using TMS (d = 0 ppm) as
(
1
the internal standard for H NMR and CDCl3 (d = 77.16 ppm) for
1
3
2
C NMR, with the reporting of coupling constants in Hz and the
signal multiplicities are reported as singlet (s), doublet (d), triplet
t), quartet (q), doublet of doublets (dd), doublet of triplets (dt),
EtOAc (10 mL), filtered through a pad of silica gel and concentrated
in vacuo. The crude material was purified by silica gel column
chromatography using a gradient of hexanes/EtOAc (9:1–4:1) to
(
multiplet (m), or broad (br). HRMS data was obtained using EI ion-
ization on a ThermoFinnigan MAT 95 XL mass spectrometer. TLC
analysis was performed using pre-coated silica gel PE sheets. Prod-
ucts were purified via column chromatography using silica gel 40–
1
yield alcohol LA-8 (88 mg, 87%) as a white solid. H NMR
(
400 MHz, CDCl
3
) d 7.20 (d, J = 8.6 Hz, 1H), 6.70 (dd, J = 8.7,
.8 Hz, 1H), 6.62(d, J = 2.7 Hz, 1H), 4.08–3.98 (m, 1H), 3.77 (s,
H), 2.96–2.78 (m, 2H), 2.47–2.17 (m, 2H), 1.97–1.87 (m, 2H),
2
3
1
6
3
l
m (230–400 mesh), normal phase preparative TLC plates. TLC
13
.79–1.32 (m, 11H), 1.24(m, 1H), 1.06 (t, 3H), 0.62 (s, 3H);
) d 157.5, 138.0, 132.7, 126.2, 113.8, 111.5,
6.0, 72.4, 55.4, 50.2, 45.9, 43.4, 38.9, 37.7, 31.4, 29.8, 27.7, 26.2,
2.9, 18.6, 14.0; HRMS: Calcd for C21
14.22491.
C
plates were visualized by ultraviolet at 254 nm. TLC plates were
stained by Iodine, Vanillin, and Ceric Ammonium Molybdate
NMR (100 MHz, CDCl
3
8
2
3
(
CAM) stain. All reagents and solvents were obtained from com-
30 2
H O : 314.2240, found
mercial suppliers and used as received. All chemical reactions
requiring anhydrous conditions were performed with oven-dried
glassware under an atmosphere of nitrogen.
LA-9 Acetate: To a stirred solution of alcohol LA-7 (0.10 g,
.32 mmol) in DCM (1.1 mL) was added pyridine (0.155 mL,
.92 mmol), DMAP (8 mg, 0.64 mmol) and acetic anhydride
0
1
2.1.2. Synthetic procedures and structural elucidation
(0.121 mL, 1.28 mmol). The reaction mixture was stirred for 18 h.
LA-2: Procedures were executed as reported as Ref. [16].
LA-3: Procedures were executed as reported as Ref. [17].
LA-4: Procedures were executed as reported as Ref. [18].
LA-5 Alkene: Procedures were executed as reported as Ref. [16].
LA-6 Diol: To a stirred solution of alkene LA-5 (50 mg,
The reaction was cooled to 0 °C and quenched by the addition of
saturated NaHCO
O (5 mL) and EtOAc (10 mL). The aqueous layer was extracted
SO , filtered and concen-
3
(5 mL). The reaction mixture was diluted with
H
2
with EtOAc (3ꢀ 25 mL), dried with Na
2
4
trated in vacuo. The crude material was purified by silica gel col-
umn chromatography using a gradient of hexanes/EtOAc (95:5–
0
.17 mmol) in t-BuOH (0.85 mL) and H
2
O (0.85 mL) was added
1
AD-mix-
a
(0.24 g) and methane sulfonamide (16 mg, 0.17 mmol).
9:1) to yield LA-9 acetate (95 mg, 84%) as a white solid. H NMR
The reaction was stirred for 15 h at room temperature. To the reac-
tion mixture was added THF (0.40 mL) and the solution was stirred
for 21 h at room temperature. Solid sodium sulfite (100 mg) was
added to the reaction and the mixture was stirred for 30 min.
(400 MHz, CDCl3) d 7.20 (d, J = 8.6 Hz, 1H), 6.70 (dd, J = 8.7,
2.8 Hz, 1H), 6.62(d, J = 2.7 Hz, 1H), 5.58–5.52 (m, 2H), 3.77 (s,
3H), 2.93–2.80 (m, 2H), 2.46–2.27 (m, 3H), 2.07 (s, 3H), 1.92–1.81
(m, 2H), 1.79–1.65 (m, 7H), 1.64–1.34 (m, 4H), 0.9 (s, 3H); 13
C
The reaction was diluted with H
The resulting aqueous layer was extracted with EtOAc (3ꢀ
5 mL). The combined organic layers were dried with MgSO and
2
O (10 mL) and EtOAc (10 mL).
NMR (100 MHz, CDCl3) d 171.2, 157.5, 149.8, 138.0, 132.5, 126.1,
121.3, 113.8, 111.6, 76.7, 55.3, 52.0, 44.7, 43.7, 37.8, 37.2, 32.9,
1
4
29.8, 27.6, 26.8, 21.7, 17.7, 13.4; HRMS: Calcd for C23
354.2189, found 354.21976.
30 3
H O :
concentrated in vacuo. The crude material was purified by silica
gel column chromatography using a gradient of hexanes/EtOAc
LA-10 Ethyl Ether: To a stirred solution of alcohol LA-7 (0.10 g,
0.32 mmol) in THF (2.15 mL) at 0 °C was added 60% sodium
hydride (63 mg, 0.96 mmol). The reaction was stirred for 10 min
and then ethyl iodide (0.13 mL, 1.60 mmol) was added. The reac-
tion was warmed to room temperature and stirred for 15 h. The
reaction was cooled to 0 °C and quenched by the addition of satu-
1
(
2:1–1:1) to yield diol LA-6 (45 mg, 80%) as a white solid.
H
3
NMR (400 MHz, CDCl ) d 7.18 (d, J = 8.7 Hz, 1H), 6.70 (dd, J = 8.5,
2
3
1
.7 Hz, 1H), 6.62 (d, J = 2.6 Hz, 1H), 3.77–3.68 (m, 1H), 3.76 (s,
H), 2.94–2.77 (m, 2H), 2.32–2.12 (m, 2H), 1.83–1.68 (m, 1H),
.68–1.18 (m, 8H), 1.25 (d, 3H), 0.69 (s, 3H); 13C NMR (100 MHz,
CDCl
5
1
3
) d 157.5, 138.0, 132.7, 126.2, 113.8, 111.5, 86.0, 72.4, 55.4,
0.2, 45.9, 43.4, 38.9, 37.7, 31.4, 29.75, 27.73, 26.2, 22.9, 18.6,
4.0; HRMS: Calcd for C21 : 330.2195, found 330.2193.
LA-7 Alcohol: solution of selenium dioxide (0.225 g,
.03 mmol) in DCM (26.0 mL) was cooled to 0 °C and 70% t-BuOOH
rated NH
(5 mL) and EtOAc (10 mL). The aqueous layer was extracted with
SO , filtered and concentrated
in vacuo. The crude material was purified by silica gel column
chromatography using a gradient of hexanes/EtOAc (98:2 to 95:5
to 9:1) to yield ethyl ether LA-10 (89 mg, 93%) as a white solid.
4 2
Cl (5 mL). The reaction mixture was diluted with H O
H
30
O
3
EtOAc (3ꢀ 25 mL), dried with Na
2
4
A
2
(
0.780 mL). The solution was stirred for 1 h at 0 °C, followed by the

3
4
A. Alsayari et al. / Steroids 118 (2017) 32–40
1H NMR (400 MHz, CDCl
.8 Hz, 1H), 6.62(d, J = 2.7 Hz, 1H), 5.59 (qd, J = 7.2, 1.5 Hz, 1H), 4.15
d, J = 6.29 Hz, 1H), 3.77 (s, 3H), 3.61–3.54 (m, 1H), 3.48–3.40 (m,
) d 7.20 (d, J = 8.6 Hz, 1H), 6.70 (dd, J = 8.7,
give the crude material that was purified by silica gel column chro-
3
2
(
matography using a gradient of hexanes/EtOAc (9:1–4:1–7:3) to
1
yield ketone LA-13 (45 mg, 90%) as a white solid. H NMR
1
1
0
1
3
C
H), 2.85–2.83 (m, 2H), 2.38–2.23 (m, 3H), 1.93–1.81 (m, 3H),
3
(400 MHz, CDCl ) d 7.20 (d, J = 8.6 Hz, 1H), 6.70 (dd, J = 8.7,
.78 (dd, J = 7.30, 1.26 Hz, 3H), 1.59–1.39 (m, 5H), 1.23 (t, 3H),
2.8 Hz, 1H), 6.62(d, J = 2.7 Hz, 1H), 6.54 (q, 1H,), 3.77 (s, 3H),
2.96–2.78 (m, 2H), 2.47–2.17 (m, 5H), 1.97–1.88 (m, 1H), 1.69
(dt, J = 7.2, 2.0 Hz, 3H), 1.61–1.23 (m, 5H), 0.98 (s, 3H); 13C NMR
1
3
3
.91 (s, 3H); C NMR (100 MHz, CDCl ) d 151.6, 149.8, 138.0,
32.8, 126.3, 119.7, 113.6, 111.5, 81.3, 64.6, 55.3, 52.0, 44.7, 43.7,
8.0, 37.1, 31.1, 29.9, 27.6, 26.8, 17.7, 15.7, 13.4; HRMS: Calcd for
3
(100 MHz, CDCl ) d 206.2, 157.6, 148.0, 137.7, 132.1, 129.1,
23
H
32
O
2
: 320.2402, found 320.2405.
LA-11 Methyl Ether: To a stirred solution of alcohol LA-7
0.20 g, 0.64 mmol) in THF (4.2 mL) at 0 °C was added 60% sodium
126.0, 113.8, 111.7, 55.2, 49.20, 43.59, 43.54, 37.7, 37.5, 36.3,
29.7, 27.8, 26.5, 17.8, 13.3; HRMS: Calcd for C21
found 310.19321.
26 2
H O : 310.1927,
(
hydride (77 mg, 1.92 mmol). The reaction was stirred for 10 min
and then methyl iodide (0.20 mL, 3.20 mmol) was added. The reac-
tion was warmed to room temperature and stirred for 15 h. The
reaction was cooled to 0 °C and quenched by the addition of satu-
LA-14 Alcohol: To a stirred solution of ketone LA-13 (69 mg,
0.22 mmol) in MeOH (2.7 mL), iPrOH (0.9 mL) and THF (0.9 mL)
was added NaBH
for 15 h at room temperature. To the reaction was added 1 M HCl
(0.5 mL), followed by saturated NaHCO (10 mL). The resulting
solution was stirred for 15 min and then diluted with H O (5 mL)
and EtOAc (20 mL). The aqueous layer was extracted with EtOAc
(3ꢀ 30 mL). The combined organic layers were washed with H
(15 mL), saturated NaCl (15 mL), dried with Na SO , filtered and
4
(60 mg, 1.59 mmol). The reaction was stirred
rated NH
5 mL) and EtOAc (10 mL). The aqueous layer was extracted with
SO , filtered and concentrated
4
Cl (5 mL). The reaction mixture was diluted with H
2
O
3
(
2
EtOAc (3ꢀ 25 mL), dried with Na
2
4
in vacuo. The crude material was purified by silica gel column
chromatography using a gradient of hexanes/EtOAc (98:2–95:5–
2
O
2
4
9
:1) to yield Methyl ether LA-11 (190 mg, 91%) as a white solid.
concentrated in vacuo. The crude material was purified by silica
gel prep-plate chromatography, developing 3x with hexanes/EtOAc
(4:1) to yield alcohol LA-14 (46 mg, 67%) as a white solid. H NMR
1
H NMR (400 MHz, CDCl
J = 8.7, 2.8 Hz, 1H), 6.62(d, J = 2.7 Hz, 1H), 5.61 (qd, J = 7.2, 2.0 Hz,
H), 4.00 (d, J = 6.04 Hz, 1H), 3.77 (s, 3H), 3.34 (s, 3H), 2.94–2.85
m, 2H), 2.40–2.24 (m, 3H), 1.94–1.86 (m, 1H), 1.79–1.67 (m,
3
) d 7.20 (d, J = 8.6 Hz, 1H), 6.70 (dd,
1
1
(
3
(400 MHz, CDCl ) d 7.20 (d, J = 8.6 Hz, 1H), 6.70 (dd, J = 8.7, 2.8 Hz,
1H), 6.62(d, J = 2.7 Hz, 1H), 5.54 (qd, J = 7.2, 2.0 Hz, 1H), 4.40 (t, 1H),
3.77 (s, 3H), 2.96–2.78 (m, 2H), 2.47–2.17 (m, 5H), 1.97–1.88 (m,
1H), 1.79–1.67 (m, 2H), 1.69 (dt, J = 7.2, 2.0 Hz, 3H), 1.61–1.23
2
H), 1.69 (dt, J = 7.2, 2.0 Hz, 3H), 1.61–1.23 (m, 5H), 0.9 (s, 3H);
1
3
3
C NMR (100 MHz, CDCl ) d 157.5, 155.2, 137.9, 132.7, 126.2,
1
3
1
3
20.5, 113.8, 111.5, 83.5, 56.7, 55.1, 51.9, 44.2, 43.8, 37.8, 37.2,
0.5, 29.6, 27.6, 26.8, 17.6, 13.3.
3
(m, 4H), 1.07 (s, 3H); C NMR (100 MHz, CDCl ) d 157.5, 154.8,
137.9, 132.6, 126.2, 118.8, 113.8, 111.6, 75.4, 55.2, 50.7, 44.0,
43.8, 37.6, 37.0, 34.3, 29.8, 27.6, 26.7, 18.2, 13.3; HRMS: Calcd for
LA-12a/12b Alcohol: A borane tetrahydrofuran complex solu-
tion (1.0 M in THF, 7.4 mL, 7.4 mmol) was added to a solution of
alkene LA-11 (600 mg, 1.84 mmol) in THF (7.4 mL). The reaction
was stirred for 15 h at 35 °C. After cooling to 0 °C, 15 mL of 10%
21 28 2
C H O : 312.2089, found 312.2094.
LA-15: Procedures were executed following Ref. [19].
LA-16: Procedures were executed following Ref. [20].
LA-17: Procedures were executed following Ref. [21].
LA-18 Ketone: To a stirred solution of ketal LA-17 (32 mg,
2 2
NaOH and 20 mL 30% H O were slowly added and stirred for 1 h
at 0 °C, followed by 1 h at room temperature. The aqueous layer
was extracted with EtOAc (3ꢀ 60 mL) and the combined organic
layers were washed with saturated sodium thiosulfate (25 mL),
0.086 mmol) in acetone (1.5 mL) was added 10% HCl (0.50 mL).
The reaction was stirred until TLC showed all of the starting mate-
2
H O (25 mL), dried with Na
2
SO
4
and concentrated in vacuo. The
rial was consumed. To the reaction was added saturated NaHCO
3
crude material was purified by silica gel column chromatography
using a gradient of hexanes/EtOAc (9:1–4:1), followed by silica
gel prep-plate chromatography, developing 2ꢀ with hexanes/
EtOAc (9:1) to yield alcohol LA-12A (82 mg, 13%) and LA-12B
until no further gas evolution was observed, followed by dilution
with EtOAc (30 mL). The aqueous layer was extracted with EtOAc
(3ꢀ 30 mL). The combined organic layers were dried with Na
2 4
SO ,
filtered and concentrated in vacuo. The crude material was purified
by silica gel column chromatography using a gradient of hexanes/
EtOAc (60:40 to 1:1–1:2) to yield ketone LA-18 (24 mg, 83%) as a
white solid.
(
68 mg, 11%) as white solids.
1
3
H NMR LA-12A (400 MHz, CDCl ) d 7.20 (d, J = 8.6 Hz, 1H), 6.70
(
dd, J = 8.7, 2.8 Hz, 1H), 6.62(d, J = 2.7 Hz, 1H), 4.22 (t, 1H), 4.04 (m,
1H), 3.76 (s, 3H), 3.72 (m, 1H), 3.32 (s, 3H), 2.85 (m, 2H), 2.31 (m,
2H), 2.11 (m, 1H), 1.90–1.80 (m, 3H), 1.70 (dd, J = 12.59, 2.77 Hz,
1
H), 1.48 (m, 5H), 1.34 (m, 3H), 0.77 (s, 3H); 13C NMR (100 MHz,
1H NMR (400 MHz, CDCl
3
): d 6.89 (d, J = 8.7 Hz, 1H), 6.67 (dd,
J = 8.7 Hz, 2.8 Hz, 1H), 6.62 (d, J = 2.74 Hz, 1H), 4.19–4.01 (m, 1H),
3.77 (s, 3H), 3.58–3.44 (m, 1H), 3.36–3.28 (m, 1H), 2.90 (m, 1H),
2.70 (m, 1H), 2.50–2.45 (m, 3H), 2.30–2.20 (m, 3H), 2.20–2.02
CDCl
3
) d 157.5, 138.0, 133.1, 126.2, 113.8, 111.4, 85.2, 67.9, 59.1,
7.2, 50.2, 45.9, 43.4, 38.9, 37.7, 31.40, 29.8, 27.7, 26.2, 22.9,
8.6, 14.0; HRMS: Calcd for C22 : 344.2351, found 344.2347.
) d 7.20 (d, J = 8.6 Hz, 1H), 6.70 (dd,
J = 8.7, 2.8 Hz, 1H), 6.62(d, J = 2.7 Hz, 1H), 4.04 (m, 1H), 3.94 (t, 1H),
5
1
(m, 3H), 1.98–1.90 (m, 1H), 1.80–1.70 (m, 1H), 0.920(s, 1H); 13
C
H
32
O
3
3
NMR (100 MHz, CDCl ) d 217.4, 213.2, 158.7, 137.0, 129.0, 123.8,
1
H NMR 12B(400 MHz, CDCl
3
114.9, 112.8, 66.3, 58.8, 55.2, 50.1, 47.2, 41.9, 35.8, 32.5, 24.6,
22.1, 19.4, 14.4; HRMS: Calcd for C20H24O: 328.1675, found
3
2
2
.67 (s, 3H), 3.72 (m, 1H), 3.32 (s, 3H), 2.95 (br, 1H), 2.77 (m, 1H),
.31 (m, 2H), 2.11 (m, 1H), 1.90–1.80 (m, 3H), 1.70 (dd, J = 12.59,
.77 Hz, 1H), 1.48 (m, 5H), 1.34 (m, 3H), 0.77 (s, 3H); 13C NMR
328.1670.
2.2. Biological screening
2.2.1. Reagents
(
100 MHz, CDCl
3
) d 157.5, 138.0, 133.1, 126.2, 113.8, 111.4, 85.2,
6
2
3
7.9, 59.1, 57.2, 50.2, 45.9, 43.4, 38.9, 37.7, 31.40, 29.8, 27.7,
6.2, 22.9, 18.6, 14.0; HRMS: Calcd for C22
44.2347.
H
32
O
3
: 344.2351, found
Dimethyl sulfoxide (DMSO), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium-bromide (MTT), sodium dodecyl sul-
phate (SDS), 4-hydroxytamoxifen and estradiol (E2) were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine
serum (FBS) was purchased from HyClone (Logan, UT, USA). Dul-
becco’s modified Eagle medium with phenol red (DMEM), Dul-
becco’s modified Eagle medium with no phenol red (DMEM),
charcoal stripped FBS, antibiotics, phosphate buffered saline
LA-13 Ketone: To a stirred solution of alcohol LA-7 (50 mg,
0
.16 mmol) in DSM (1.6 mL) was added 4 Å powdered molecular
sieves (140 mg), NaOAc (63 mg, 0.77 mmol), and PCC (69 mg,
.32 mmol). The reaction mixture was stirred for 18 h and then
Et O was added and the mixture was filtered through a pad of silica
gel, washing with Et O. The filtrate was concentrated in vacuo to
0
2
2

A. Alsayari et al. / Steroids 118 (2017) 32–40
35
(
PBS) 1X solution and trypsin were purchased from Gibco (Grand
of each well was measured using the Synergy 2 multi-mode micro-
plate reader (BioTek) with 535 nm excitation filters and 590 nm
emission filters. Three controls were included in the assay: (1)
Island, NY, USA). PolarScreen ER alpha and beta competitor assay
kit, red were purchased from Life Technologies (Grand Island, NY,
USA).
assay maximum polarization control (10
complex solution and 10 l of ER red assay buffer with 4% DMSO,
provides maximum polarization value for assay); (2) assay mini-
mum polarization control (10 l of ER/Fluromone TM complex
solution and 10 l of E2 (20 M), provides bottom baseline); (3)
free Fluoromone TM tracer control (10 l Fluoromone TM tracer
and 10 l of ER Red assay buffer with 4% DMSO, provides absolute
ll of ER/Fluoromone TM
l
2
.2.2. Cell cultures
Human cancer breast cell line (MCF-7) was obtained from
l
American Type Cell Culture (ATCC, Rockville, MD, USA). MCF-7
was maintained at 37 °C in a humidified atmosphere of 5% CO in
a DMEM medium containing 10% fetal bovine serum and antibi-
otics (100 IU/mL penicillin and 100 g/ml).
l
l
2
l
l
l
minimum polarization value). The polarization values were nor-
malized to percent inhibition using the following equation:
2
.2.3. Cell proliferation assay
I% ¼ ðA
0
ꢁ AÞ=ðA
0
ꢁ A100Þ ꢀ 100
= A at 0% inhibition, A100 = A at 100%
A
cell proliferation assay was performed, as previously
described, with a modification [22,23]. The MCF-7 cells were
grown and maintained in DMEM supplemented with 10% FBS
and transferred to phenol-red free DMEM supplemented with
I% = the percent inhibition, A
inhibition, and A = observed A value.
The percent inhibition values are plotted against the concentra-
tion of test compounds and analyzed by a non-linear regression
curve fitting. The concentration of the test compounds needed to
displace half of the bond ligand equals IC50 of the test compound.
0
3
1
0% charcoal stripped FBS. Cells (5 ꢀ 10 cells/well) were seeded
into a 96-well plate and allowed to attach to the well overnight.
Compounds were dissolved in acetone diluted with phenol-red
free DMEM and added at different concentrations (0, 0.16, 0.8, 4,
20, lM). The culture was continued for 8 days, and the cell growth
2
.3. Molecular modeling
was measured using MTT. Cell proliferation was expressed as a
percentage, where the control with 1% acetone alone was taken
as 100%.
This was done using OpenEyeÓ molecular Modeling software.
LA-7 was energy minimized using MMFF94 force field, followed
by generation of multi-conformers using OMEGA application. The
whole energy minimized library will be docked along with the pre-
pared ER
physical property (
ligand-receptor complex [27,28]. Vida application can be employed
as a visualization tool to show the potential binding interactions of
the ligands to the receptor of interest.
2.2.4. Cytotoxicity assay
Cytotoxicity was determined by the MTT method, as previously
4
a
(PDB ID: 1GWR) using FRED application to generate a
G) reflecting the predicted energy profile of
described, with a modification [24–26]. Briefly, cells (1 ꢀ 10 cells/
well) were seeded into a 96-well plate and allowed to attach to the
well overnight. Compounds were then added at different concen-
D
trations (0, 0.16, 0.8, 4, 20,
ther 24 h. After incubation, 10
added to the cells for 4 h at 37 °C, followed by the addition of
00 L of 10% SDS in 0.01 N HCl as a solubilizing agent. The absor-
lM) and cells were incubated for a fur-
lL of 5 mg/mL of MTT dye was
1
l
3. Results and discussion
bance at 570 nm was recorded using an ELISA microplate reader.
The results of viability were expressed as a percentage of the
control.
3.1. Synthetic Route strategy
Starting from Estrone, eighteen analogs were synthesized; 3-
methoxy Estrone (LA-2) was synthesized via methylation in alka-
line media using methyl iodide (MeI/KOH). Alternatively, a three
step procedure involving the protection of the C-17 ketone as the
ketal, methylation of the C-3 hydroxyl and removal of the ketal
provided the same product (LA-2), but allowing for easier purifica-
tion [11]. Methylation at C-3 is a key structural modification pur-
sued in all of the shown analogs. Additionally, structural changes
to the estrone scaffold at C-16 and C-17 produced LA-3 via reduc-
2
.2.5. Anti-estrogenic assay
An anti-estrogenic assay was performed, as previously
described, with a modification [22,23]. Briefly, MCF-7 cells were
grown and maintained in DMEM supplemented with 10% FBS
and transferred to phenol-red free DMEM supplemented with
3
1
0% charcoal stripped FBS. Cells (5 ꢀ 10 cells/well) were seeded
into a 96-well plate and allowed to attach to the well over night.
Compounds were dissolved in acetone, diluted with phenol-red
free DMEM and added at different concentrations (0, 0.16, 0.8, 4,
4 2
tion using NaBH [12], LA-4 via a-bromination using CuBr [13],
ꢁ9
2
0,
lM) in the presence of 10 M of 17 b-estradiol. The culture
and LA-5 via a Wittig reaction using EtPPh3Br [11]. LA-5 offered
further potential synthetic modifications for analogs investigation
by addition of a diol across the double bond, giving LA-6. Also, a
hydroxyl group at C-16 was installed using LA-5 to assemble LA-
7 via a selective allylic oxidation using selenium dioxide and
hydrogen peroxide. Consequently, LA-7 offered the ability for fur-
ther structural modifications by allowing for assembly of LA-8 via
the reduction of the double bond and oxidation of hydroxyl group
at C-16 to a ketone, giving LA-13. The resulting ketone of LA-13 can
further be reduced to the alcohol providing LA-14, the C-16 epimer
of LA-7. The hydroxyl group at C-16 of LA-7 was an attractive site
for acetylation (LA-9), ethylation (LA-10), and methylation (LA-
11); followed by non-selective addition of hydroxyl group across
the double bond, generating a diastereomeric mixture of com-
pounds LA-12a/b, separable by chromatography. Further synthetic
modifications were carried out at positions C-9 and C-11 of 3-
methoxy Estrone (LA-2), following literature precedence to assem-
ble LA-17 [19]. Insertion of the double bond was performed using
was continued for 8 days, and the cell proliferation was measured
using the MTT method as described in the cytotoxicity assay. The
stimulatory effect of 10 M of 17 b-estradiol was taken as 100%
and the effects of different concentrations of compounds were
expressed as a percentage of stimulation as related to 17 b-
estradiol.
ꢁ9
2.2.6. Estrogen receptor competitive binding assay
ERa
and ERb competitive binding assays were performed using
TM
Polar Screen ER
a and ERb competitor assays according to the
manufacturer‘s instruction. A stock solution of each compound
was serially diluted using DMSO, and the10 l of each concentra-
tion was transferred to the assay plate (Corning, 384-well low vol-
ume black round bottom) followed by adding 10 l human
recombinant ER and ERb/Fluoromone TM complex solution. After
l
l
a
mixing and allowing at least 2-h incubation at room temperature
with light protection of the reagents, the fluorescence polarization

3
6
A. Alsayari et al. / Steroids 118 (2017) 32–40
either DDQ or a two-step process involving benzylic oxidation with
DMDO [20], followed by elimination [21] to give LA-16 [20]. Pro-
tection of the C-17 ketone as a ketal was followed by the epoxida-
tion of the olefin with DMDO. The resulting epoxide was subjected
ER. On the other hand, LA-6, LA-7, LA-12A, LA-12B, and LA-19 dis-
played moderate to weak estrogenic activity, indicating these com-
pounds may act as a partial agonist for the ER. Interestingly, LA-10
did not stimulate MCF-7 cell proliferation at any tested concentra-
tions, indicating that it may act as a pure antagonist for the ER.
4
to LiClO in refluxing benzene to provide rearranged product LA-
1
7. Alkylation of the ketone in LA-17 with formaldehyde supplied
LA-18. Removal of the ketal at C17 provided LA-19, as shown in
3.2.2. Cytotoxicity and anti-estrogenic assays
Scheme 1.
The cytotoxicity of these compounds must be determined in
order to interpret their anti-estrogenic activities since anti-estro-
genic activities was determined as the inhibition of the prolifera-
tive effects of estradiol on MCF-7 cells. Cytotoxicity was
determined using an MTT assay for 24 h, as shown in Fig. 2.
3.2. Biological evaluation
MCF-7 cell lines are characterized by ER positive and HER2 neg-
ative, and exhibit estrogen responsive cell growth. The synthesized
Estrone analogs, including 4-hydoxytamoxifen, the active metabo-
lite of tamoxifen, were tested for their estrogenic, cytotoxicity, and
anti-estrogenic activities at various concentrations using MCF-7
cells.
LA-4 showed marked cytotoxicity at a concentration of 20 lM
among all Estrone analogs while LA-8, LA-14, and LA-19 showed
a small cytotoxic effect (cell viability% ranged from 87 ± 6 to
90 ± 5%). However, the rest of the compounds were not cytotoxic
within the range of concentrations tested. As mentioned earlier,
anti-estrogenic activities can be determined as the inhibition of
the proliferative effects of estradiol on MCF-7 cells at various con-
centrations where a compound shows no cytotoxicity. Cell prolifer-
ation in the presence of estradiol alone was taken as 100% and the
effect of test compounds at various concentrations were expressed
as percentages of stimulation by estradiol. For comparison pur-
poses and to validate that our assay was working, 4-hydroxyta-
moxifen, a partial agonist-antagonist for an ER and the drug of
choice for treatment of ER positive breast cancer, was tested for
its estrogenic, cytotoxicity, and anti-estrogenic activities at various
concentrations using MCF-7 cells. The 4-hydroxytamoxifen
showed estrogenic activities (partial agonist) at concentrations
3.2.1. Cell proliferation assay
In order to estimate the estrogenic activities, cell proliferation
of estrogen dependent MCF-7 cells was determined after 8 days
of treatment with various test compounds. The cell proliferation
without estradiol and test compounds was taken as 100%, while
the cell proliferations of estradiol and test compounds were
expressed as a percentage of the control, as shown in Fig. 1. The
estradiol showed the maximum proliferation of cells at a concen-
ꢁ9
tration of 10 M, with growth more than 300% of the control (3-
fold stimulation). Among all examined compounds, LA-2, LA-3,
LA-13, LA-14, LA-16, LA-17, and LA-18 showed potent stimulation
of cell proliferation in a dose-dependent manner in all tested con-
centrations, while LA-4, LA-8, and LA-9 were found to have the
ranging from 0.0064
ities (partial antagonist) were observed in a concentration range
from 4 M to 100 M, with 26–40% inhibition under concentra-
tions where no cytotoxicity was observed. Our results for 4-
lM to 0.8 lM, while its anti-estrogenic activ-
same stimulatory effect, but in concentrations ranging from 4
lM
l
l
to 0.8 M, suggesting that these compounds are agonist for the
l
Scheme 1. Synthetic Route for the synthesized analogs.

A. Alsayari et al. / Steroids 118 (2017) 32–40
37
Fig. 1. Cell Proliferation Assay for the synthesized analogs and 4-hydroxytamoxifen.
hydroxytamoxifen were in agreement with the data published by
Okubo et al. [22,23]. The anti-estrogenic activities of estrone ana-
logs were tested in the presence of 10 M of estradiol for 8 days.
Our results revealed that LA-10 and LA-7, among all estrone ana-
logs examined, indicated greater inhibition of cell growth stimu-
lated by estradiol in a dose-dependent manner at concentration
tions. LA-4, LA-6, LA-8, LA-9, and LA-12B were less potent than
4-hydroxytamoxifen, since they exhibited their anti-estrogenic
ꢁ
9
activities at a concentration of 20
59% to 74%). LA-4 showed inhibition of cell growth stimulated by
estradiol at 20 M (85% inhibition of the control), however its
anti-estrogenic activities cannot be interpreted since it showed sig-
nificant cytotoxicity at 20 M.
lM (the inhibition ranged from
l
ranges from 0.8 to 20
and 4–20 M for LA-7 (73–81% inhibition of the control), when
compared with tamoxifen (60–74% inhibition at a concentration
of 4–100 M). The above data suggest that LA-10 and LA-7 could
be potential ER antagonists. However, LA-7 showed estrogenic
activities at a low concentration (0.16–0.8 M), whereas LA-10
did not stimulate MCF-7 cell proliferation at all tested concentra-
l
M for LA-10 (62–79% inhibition of control)
l
l
l
3.2.3. Estrogen receptor competitive binding assay
In order to evaluate the binding affinity Estrone analogs to ERs,
TM
l
competitive binding assays were performed using a Polar Screen
ERa and ERb competitor assay kit. The assay used fluorescence
polarization to monitor displacement of a Fluromone TM Tracer

3
8
A. Alsayari et al. / Steroids 118 (2017) 32–40
Fig. 2. Cytotoxicity and anti-estrogenic activity of synthesized analogs and 4-hydroxytamoxifen.
from the ER/Fluromone TM Tracer complex. If the compounds do
not displace the Fluromone TM Tracer from the complex, the polar-
ization value remains high, while the compounds displaced the
Fluromone TM Tracer cause a low polarization value. The shift in
the polarization value is used to estimate the binding affinity of
the compounds to ER. Estradiol and 4-hydroxytamoxifen were
included in the binding affinity study for comparison purposes,
and they showed binding affinities to ER
Table 1.
The competitive ligand binding assay indicated that LA-7 and
LA-8, of the compounds showing anti-estrogenic activities, exhib-
a and ERb, as shown in
ited selective binding affinities to ER
a with IC50 5.49 lM and
48.46 M, respectively. However, their binding affinities were
l
low when compared with 4-hydroxytamoxifen. On the other hand,
LA-3 and LA-16, among the compounds showing estrogenic activ-

A. Alsayari et al. / Steroids 118 (2017) 32–40
39
Table 1
group at C-3 and the amino acid residue ARG: 394, filling the entire
hydrophobic pocket of LBD of ERa; as shown in Fig. 3.
Binding affinity (IC50) of estrone analogs and 4-hydroxytamoxifen.
Compounds
IC50
ER
(lM)
a
ERb
4. Conclusions
LA-2
LA-3
LA-4
LA-6
LA-7
LA-8
LA-9
LA-10
LA-12A
LA-12B
LA-13
LA-14
LA-16
LA-17
LA-18
LA-19
>100
0.42
>100
48.46
5.49
>100
0.5
In this study, novel Estrone analogs were synthesized to be
evaluated for their binding affinity potential to ER- using molec-
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
19.19
>100
>100
>100
0.004
0.003
a
ular modeling and ER competitive-binding assay. The biological
evaluation of these Estrone analogs demonstrated that LA-10 and
LA-7, among all Estrone analogs examined, showed potent inhibi-
tion of cell growth stimulated by estradiol, suggesting that they
may have potential for treatment of breast cancer. Further studies
are needed to investigate the mechanism of action of LA-10 since it
did not stimulate the MCF-7 cell proliferation and has a weak bind-
ing affinity to ligand binding domain of ERs.
>100
>100
>100
>100
>100
>100
>100
4.09
>100
>100
>100
0.0075
0.0069
Acknowledgements
4
-Hydroxytamoxifen
The authors would like to acknowledge King Khalid University,
Abha, Saudi Arabia for financial support, OpeneyeÓ molecular
modeling software for supporting an academic license, and South
Dakota Board of Regents-2010 BCAAP (Biological Control and Anal-
ysis by Applied Photonics) Center for financial support.
Estradiol
ities, displayed binding affinities to ER
.09 lM, respectively for ERa and 0.5, 19.19 lM, respectively for
a
and ERb with IC50 0.42 and
4
ERb. However, LA-10 was the most active anti-estrogenic com-
pound in this study, without any estrogenic activities, and demon-
Appendix A. Supplementary material
strated very weak binding affinities to LBD of ER
a and ERb,
indicating that the compound inhibits cell proliferation through a
different mechanism. This finding is supported by the mechanism
of action of 2-methoxyestradiol, estradiol metabolites and anti-
cancer drug in clinical trials, which have been reported to possess
anticancer and anti-angiogenesis activities through microtubule
disruption and up regulation of apoptotic pathways without tar-
geting LBD of ER (500 and 3200 fold lower than estradiol binding
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2016.11.
005.
References
[
1] W. Shao, M. Brown, Advances in estrogen receptor biology: prospects for
improvements in targeted breast cancer therapy, Breast Cancer Res. 6 (2004)
for ER
low binding affinities at the concentrations examined and their
IC50 values were >100 M.
a and ERb, respectively) [19]. Other compounds showed very
39–52.
[2] M. Cristofanilli, G. Hortobagyi, Molecular targets in breast cancer: current
status and future directions, Endocr. Relat. Cancer 9 (2002) 249–266.
l
[
[
[
3] J. Bange, E. Zwick, A. Ullrich, Molecular targets for breast cancer therapy and
prevention, Nat. Med. 7 (2001) 548–552.
4] S.L. Larsen, Identification of Kinases Driving Growth of Anti-Estrogen Resistant
T47D Breast Cancer Cell Lines, 2013.
5] K. Rau, H. Kang, T. Cha, S. Miller, M. Hung, The mechanisms and managements
of hormone-therapy resistance in breast and prostate cancers, in: Endocrine-
related Cancer 12 (3) (2005) 511–532.
3.3. In silico molecular modeling validation
Molecular modeling study was conducted for validation for the
binding affinity of the synthesized analogs towards ER co-crys-
a
[6] S. Sasson, C. Notides, Estriol and estrone interaction with the estrogen
receptor. II. Estriol and estrone-induced inhibition of the cooperative binding
of [3H] estradiol to the estrogen receptor, J. Biol. Chem. 258 (1983) 8118–8122.
tallized with estradiol [20]. The molecular docking results showed
that the LA-7 possess a very similar binding mode to the estradiol
forming a hydrogen bonding interaction between the methoxy
[
7] M. Marino, P. Galluzzo, P. Ascenzi, Estrogen signaling multiple pathways to
impact gene transcription, Curr. Genom. 7 (2006) 497–508.
(A)
(B)
Fig. 3. Visual representation of (A) Estradiol on LBD of human ERa, where the red arrows show the formation of two hydrogen bonds between the hydroxy group at C-3 and
the hydroxy group at C-17 Arg 394 and His529, respectively. (B) LA-7 on LBD of human ER
a, where the red arrow shows the formation of hydrogen bonds between the
methoxy group at C-3 and Arg 394.

4
0
A. Alsayari et al. / Steroids 118 (2017) 32–40
[
8] J. Macgregor, V.C. Jordan, Basic guide to the mechanisms of anti-estrogen
action, Pharmacol. Rev. 50 (1998) 151–196.
[18] B. Schönecker, C. Lange, M. Kötteritzsch, W. Günther, J. Weston, E. Anders, H.
Görls, Conformational design for 13 -steroids, JOC 65 (2000) 5487.
a
[
9] J. Hoffmann, A. Sommer, Anti-Hormone Therapy: Principles of Endocrine
Therapy of Cancer, Springer, 2007. pp. 19–82.
[19] L. Kürti, B. Czakó, E.J. Corey, A short, scalable synthesis of the carbocyclic core
of the anti-angiogenic cortistatins from (+)-estrone by B-ring expansion, Org.
Lett. 10 (2008) 5247.
[20] P. Bovicelli, P. Lupattelli, E. Mincione, T. Prencipe, R. Curcii, Oxidation of
natural targets by dioxiranes. Oxyfunctionalization of steroids, JOC 57 (1992)
2182–2184.
[
[
[
10] J. Peng, S. Sengupta, V.C. Jordan, Potential of selective estrogen receptor
modulators as treatments and preventives of breast cancer, Anti-Cancer
Agents Med. Chem. 9 (2009) 481–499.
11] S. Martinkovich, D. Shah, S.L. Planey, J.A. Arnott, Selective estrogen receptor
modulators: tissue specificity and clinical utility, Clin. Interv. Aging 9 (2014)
[21] J.J. Harburn, G.C. Loftus, B.A. Marples, Synthesis of novel steroidal inhibitors of
HIV-1 protease, Tetrahedron 54 (1998) 11907–11924.
1
437–1452.
12] (A) E. Wardell, J.R. Marks, D.P. McDonnell, The turnover of estrogen receptor
a
[22] T. Okubo, T. Suzuki, Y. Yokoyama, K. Kano, I. Kano, Estimation of estrogenic and
anti-estrogenic activities of some phthalate diesters and monoesters by MCF-7
cell proliferation assay in vitro, Biol. Pharm. Bull. 26 (2003) 1219–1224.
[23] T. Okubo, Y. Yokoyama, K. Kano, Y. Soya, I. Kano, Estimation of estrogenic and
antiestrogenic activities of selected pesticides by MCF-7 cell proliferation
assay, Arch. Environ. Contam. Toxicol. 46 (2004) 445.
by the selective estrogen receptor degrader (SERD) fulvestrant is a saturable
process that is not required for antagonist efficacy, in: Biochem. Pharmacol. 82
(
(
2011) 122–130;
B) S.E. Jones, A new estrogen receptor antagonist – an overview of available
data, in: Breast Cancer Res. Treat. 75 (2002) 19–21.
[
13] (A) X. Long, M. Fan, M. Bigsby, P. Nephew, Apigenin inhibits antiestrogen-
[24] H.O. Saxena, Synthesis of chalcone derivatives on steroidal framework and
their anticancer activities, Steroids 72 (2007) 892.
resistant breast cancer cell growth through estrogen receptor-
a-dependent
and estrogen receptor- -independent mechanisms, in: Mol. Cancer Therap. 7
a
[25] R. Tedesco, J.A. Thomas, B.S. Katzenellenbogen, J.A. Katzenellenbogen, The
estrogen receptor: a structure-based approach to the design of new specific
hormone–receptor combinations, Chem. Biol. 8 (2001) 277–287.
[26] M.S. Ahmed, F.T. Halaweish, Cucurbitacins: potential candidates targeting
mitogen-activated protein kinase for treatment of melanoma, J. Enzyme Inhib.
Med. Chem. 29 (2014) 162–167.
[27] (A) OMEGA 2.5.1.4: OpenEye Scientific Software, Santa Fe, NM. http://www.
eyesopen.com. (B) P.C.D. Hawkins, A.G. Skillman, G.L. Warren, B.A. Ellingson,
M.T.Stahl, Conformer generation with OMEGA: algorithm and validation using
high quality structures from the protein databank and Cambridge Structural
Database, Chem. Inf. Model. 50 (2010) 572–584.
(
(
2008) 2096–2108;
B) C.K. Osborne, A. Wakeling, R.I. Nicholson, Fulvestrant: an oestrogen
receptor antagonist with a novel mechanism of action, in: Br. J. Cancer 90
2004) S2–S6.
(
[
14] A.O. Mueck, H. Seeger, 2-Methoxyestradiol–biology and mechanism of action,
Steroids 75 (2010) 625–631.
15] M.S. Ahmed, L. Kopel, F. Halaweish, Structural optimization and biological
screening of a steroidal Scaffold possessing cucurbitacin-like functionalities as
B-Raf inhibitors, ChemMedChem 9 (2014) 1361–1367.
16] L. Kopel, M.S. Ahmed, F.T. Halaweish, Synthesis of novel estrone analogs by
incorporation of thiophenols via conjugate addition to an enone side chain,
Steroids 78 (2013) 1119.
[
[
[28] M. McGann, FRED Pose Prediction and Virtual Screening Accuracy, J. Chem. Inf.
Model. 5 (2011) 578–596.
[
17] F. Balssa, M. Fischer, Y. Bonnaire, An easy stereoselective synthesis of 5(10)-
estrene-3b, 17
2014) 1–4.
a-diol, a biological marker of pregnancy in the mare, Steroids 86
(