Synthesis, molecular modeling and biological evaluation of potent analogs of 2-methoxyestradiol

Article abstract of DOI:10.1016/j.steroids.2018.05.002

The endogenous steroid 2-methoxyestradiol (1) has attracted a great interest as a lead compound towards the development of new anti-cancer drugs. Herein, the synthesis, molecular modeling, anti-proliferative and anti-angiogenic effects of ten 2-ethyl and four 2-methoxy analogs of estradiol are reported. The ethyl group was introduced to the steroid A-ring using a novel Friedel-Crafts alkylation protocol. Several analogs displayed potent anti-proliferative activity with IC₅₀-values in the submicromolar range towards the CEM human leukemia cancer cell line. As such, all of these compounds proved to be more active than the lead compound 2-methoxyestradiol (1) in these cells. The six most cytostatic analogs were also tested as anti-angiogenic agents using an in vitro tube formation assay. The IC₅₀-values were determined to be in the range of 0.1 μM ± 0.03 and 1.1 μM ± 0.2. These six compounds were also modest inhibitors against tubulin polymerization with the most potent inhibitor was 14b (IC₅₀ = 2.1 ± 0.1 μM). Binding studies using N,N′-ethylene-bis(iodoacetamide) revealed that neither14a or 14b binds to the colchicine binding site in the tubulin protein, in contrast to 2-methoxyestradiol (1). These observations were supported by molecular modeling studies. Results from a MDA-MB-231 cell cycle assay showed that both 10e and 14b gave accumulation in the G2/M phase resulting in induction of apoptosis. The results presented herein shows that the novel analogs reported exhibit their anticancer effects via several modes of action.

Full text of DOI:10.1016/j.steroids.2018.05.002

Steroidsxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis, molecular modeling and biological evaluation of potent analogs
of 2-methoxyestradiol
Nora al-Kazaalea, Phuong T. Trana, Farhad Haidaria, Eirik Johansson Soluma,b, Sandra Liekensc,
Peter Vervaekec, Ingebrigt Sylted, Jing-Jy Chenge,f, Anders Vika, Trond Vidar Hansena,⁎
a
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068 Blindern, N-0316 Oslo, Norway
Faculty of Health Sciences, Nord University, 7801 Namsos, Norway
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Herestraat 49, Postbus 1043,
b
c
B-3000 Leuven, Belgium
d
Department of Medical Biology, Faculty of Health Sciences, UiT – The Arctic University of Norway, 9037 Tromsø, Norway
National Research Institute of Chinese Medicine, 155-1 Li-Nung Street, Section 2, Shih-Pai, Taipei, Taiwan
e
f
Institute of Biophotonics, National Yang-Ming University, Taipei 112, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords:
The endogenous steroid 2-methoxyestradiol (1) has attracted a great interest as a lead compound towards the
development of new anti-cancer drugs. Herein, the synthesis, molecular modeling, anti-proliferative and anti-
angiogenic effects of ten 2-ethyl and four 2-methoxy analogs of estradiol are reported. The ethyl group was
introduced to the steroid A-ring using a novel Friedel-Crafts alkylation protocol. Several analogs displayed
potent anti-proliferative activity with IC50-values in the submicromolar range towards the CEM human leukemia
cancer cell line. As such, all of these compounds proved to be more active than the lead compound 2-methox-
yestradiol (1) in these cells. The six most cytostatic analogs were also tested as anti-angiogenic agents using an in
2-Methoxyestradiol
Cytostatic activity
Anti-angiogenic effects
Tubulin inhibition
Anti-cancer agents
Friedel-Crafts alkylation
vitro tube formation assay. The IC50-values were determined to be in the range of 0.1 μM
0.03 and
1.1 μM
0.2. These six compounds were also modest inhibitors against tubulin polymerization with the most
potent inhibitor was 14b (IC50 = 2.1
0.1 μM). Binding studies using N,N′-ethylene-bis(iodoacetamide) re-
vealed that neither14a or 14b binds to the colchicine binding site in the tubulin protein, in contrast to 2-
methoxyestradiol (1). These observations were supported by molecular modeling studies. Results from a MDA-
MB-231 cell cycle assay showed that both 10e and 14b gave accumulation in the G2/M phase resulting in
induction of apoptosis. The results presented herein shows that the novel analogs reported exhibit their antic-
ancer effects via several modes of action.
1. Introduction
effects without any undesirable estrogen activity [9–12]. These anti-
cancer mechanisms have inspired several efforts of structure-activity
Steroids have extensively been used as drugs for treating several
human diseases and occupy a central part of the pharmacopeia today
[1]. For a long time the endogenous steroid 2-methoxyestradiol (2-ME,
1) was considered an inactive endogenous metabolite of estradiol (2)
[2–4]. In 1989 Seegers et al. reported that 2-ME (1) exhibits anti-pro-
liferative activity in MCF-7 cells, a human breast cancer cell line with
estrogen, androgen, progesterone and glucocorticoid receptors [2,3].
The anti-proliferative activity of 1 is the result of binding to tubulin,
leading to apoptosis of fast-growing tumor cells [5–8]. Later it was
reported that 2-ME (1) also exhibits effects towards angiogenesis and
causes vascular disruption [9]. Several biological and pharmacological
studies have revealed that 2-ME (1) displays its interesting anti-cancer
relationship studies aiming at the development of new anti-cancer
drugs [9–12]. Some examples are depicted in Fig. 1. Of note, the anti-
cancer drug abiraterone (3) contains a 3-pyridinyl substituent at C-17.
Some clinical trials have been conducted to evaluate the pharma-
cokinetics, systemic toxicity and anti-cancer effects of 2-ME (1) or
Panzem™ [13,14]. These trials revealed that 2-ME (1) is well tolerated
with some minor side effects [15–19], and the compound reduces tu-
mour growth. In a combination therapy study, 2-ME (1) resulted in
significant reduction of tumor growth [20]. Moreover, at therapeutic
doses, 2-ME (1) does not induce the many side effects usually observed
with chemotherapy [4,21]. One synthetic analog of 1 that has entered
clinical trials is ENMD-1198 (4) [13,14].
⁎
Corresponding author.
E-mail address: t.v.hansen@farmasi.uio.no (T.V. Hansen).
https://doi.org/10.1016/j.steroids.2018.05.002
Received 7 March 2018; Received in revised form 27 April 2018; Accepted 3 May 2018
0039-128X/©2018ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:Al-Kazaale,N.,Steroids(2018),https://doi.org/10.1016/j.steroids.2018.05.002

N. al-Kazaale et al.
Steroidsxxx(xxxx)xxx–xxx
(PPh3)4 (5 mol %) was added and the reaction mixture was stirred at
60 °C under argon atmosphere overnight. The reaction mixture was
then brought to room temperature, added brine (10 mL) and extracted
with ethyl acetate (4 × 5.00 mL). The combined organic phases were
dried over MgSO4 and evaporated in vacuo. The residue was purified by
flash column chromatography (silica gel, 10–20% EtOAc in heptane) to
obtain the pure TBS-ethers of 9a-9e or 13a-13b as light yellow oils.
N
OH
OH
H
H
H
MeO
H
H
H
H
H
H
HO
HO
HO
3
abiraterone (
N
)
1
2-methoxyestradiol (
)
2
estradiol (
)
2.3. General procedure for TBS-deprotection for the synthesis of 9a-9e and
13a-13b
H
MeO
H
In brief, the individual TBS-ether (1.0 equiv.) was placed in flame
dried 10 mL round bottomed flask and dissolved in dry THF (2.0 mL).
Tert-butylamoniumfluoride (1.0 M in THF, 1.1 equiv.) was added
dropwise. The reaction mixture was stirred at room temperature under
argon atmosphere overnight. The reaction mixture was poured into
saturated aqueous NaHCO3 (5.0 mL) and extracted with ethyl acetate
(4 × 3.0 mL). The combined organic extracts were dried over MgSO4
and evaporated in vacuo. The residues were purified by column chro-
matography (silica gel, 20–30% EtOAc in heptane) to give the pure
products 9a-9e, 13a and 13b as colorless solids.
H
H
H2N
H
H
H2NO2SO
O
4
ENMD-1198 (
)
5
pyridine analog (
)
Fig. 1. Structure of 2-ME (1), estradiol (2), the anti-cancer drug abiraterone (3)
and two synthetic analogs 4 and 5.
Previously we have reported the synthesis, molecular modeling and
biological evaluations of new tubulin inhibitors as putative anticancer
agents as exemplified by 5 (Fig. 1) [22–24]. This compound displayed
potent cytotoxic activity and modest inhibitory effects on the poly-
merization of tubulin. The latter effects were supported by molecular
modeling studies, which also revealed the 4-pyridinyl ring in 5 to be
amendable for substitutions. Moreover, potent anti-vascular effects
were also observed [22]. The aforementioned biological activities and
molecular modeling studies spurred our interest in the synthesis of
additional analogs of 1. The synthesis, biological evaluation and mo-
lecular modeling studies of 14 novel analogs are reported herein.
2.4. (8S,9S,13S,14S)-2-ethyl-13-methyl-17-(2-methylpyridin-4-yl)-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (9a)
1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 5.2 Hz, 1H), 7.17 (s, 1H),
7.12 (dd, J = 5.2, 1.7 Hz, 1H), 7.06 (s, 1H), 6.55 (s, 1H), 6.30 (s, 1H),
6.18 – 6.16 (m, 1H), 2.84 (m, 2H), 2.64 (q, J = 7.6 Hz, 2H), 2.57 (s,
3H), 2.47 – 2.25 (m, 3H), 2.25 – 2.09 (m, 2H), 1.97 – 1.89 (m, 1H), 1.84
– 1.61 (m, 5H), 1.52 – 1.36 (m, 1H), 1.24 (t, J = 7.6 Hz, 3H), 1.08 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 158.5, 153.3, 152.3, 149.1, 145.6,
135.6, 132.5, 131.5, 128.0, 126.4, 121.3, 118.9, 115.7, 57.2, 48.0,
44.6, 37.8, 35.8, 32.0, 29.6, 28.2, 27.0, 24.7, 23.6, 17.2, 14.9. Eluent
30% EtOAc in heptane, Rf = 0.18, [α]20 = +6.7 (c = 0.9, CHCl3), yield
43 mg (88%) as white solid, m.p.D229–231 °C. HRMS calcd. for
2. Experimental
2.1. Synthesis of (8R,9S,13S,14S,17S)-2-Ethyl-13-methyl-
7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-
3,17-diol (6)
C
26H31NO [M]·+ 373.2406. Found 373.2403.
Under an inert atmosphere, 17β-estradiol (2) (5.0 mmol, 1.4 g,
1.0 equiv.), acetaldehyde (15.0 mmol, 0.84 mL, 3.0 equiv.), copper(II)
trifluoromethanesulfonate (45 mg, 2.5 mol%) and 2,2,2-trifluroethanol
2.5. 8S,9S,13S,14S)-2-ethyl-17-(2-fluoropyridin-4-yl)-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (9b)
1H NMR (400 MHz, CDCl3) δ 8.13 (d, J = 5.3 Hz, 1H), 7.18 (m, 1H),
7.05 (s, 1H), 6.92 (s, 1H), 6.54 (s, 1H), 6.28 – 6.24 (m, 1H), 4.79 (s,
1H), 2.94 – 2.78 (m, 2H), 2.62 (q, J = 7.5 Hz, 2H), 2.48 – 2.25 (m, 3H),
2.24 – 2.12 (m, 2H), 1.98 – 1.90 (m, 1H), 1.85 – 1.76 (m, 1H), 1.72 –
1.61 (m, 3H), 1.52 – 1.40 (m, 2H), 1.24 (t, J = 7.5 Hz, 3H), 1.08 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 165.8, 163.5, 152.4, 152.3, 151.8, 150.7,
150.6, 147.7, 147.5, 135.7, 133.3, 132.8, 127.7, 126.4, 119.5, 119.4,
115.7, 106.9, 106.6, 57.1, 48.0, 44.5, 37.7, 35.7, 32.0, 29.5, 28.1, 27.0,
23.5, 17.2, 14.8. Eluent 30% EtOAc in heptane, Rf = 0.25,
[α]2D0 = +19.2 (c = 1.0, CHCl3), yield 48 mg (64%) as white solid, m.p.
217–219 °C. HRMS calcd. for C25H28FNO [M]·+ 377.2155. Found
377.2143.
(15 mL) were combined in
a round-bottomed flask. Ethanethiol
(30 mmol, 2.2 mL, 6.0 equiv.) was added and the reaction mixture was
stirred over night at 50 °C. Triethylsilane (15 mmol, 2.5 mL, 3.0 equiv.)
was added and the reaction mixture was allowed to stir for an addi-
tional 4 h at 50 °C. Removal of volatiles was performed in vacuo where
the exhaust was passed through a solution over basic potassium per-
manganate solution to quench excess ethanethiol. The residue was
purified by flash column chromatography (silica gel, 27% EtOAc in
heptane) to give 1.1 g of the desired product as a white solid in 68%
yield. 1H NMR (400 MHz, CDCl3) δ 7.06 (s, 1H), 6.50 (s, 1H), 4.74 (s,
1H), 3.74 (t, J = 9.0, 7.9 Hz, 1H), 2.87 – 2.72 (m, 2H), 2.60 (q,
J = 7.5 Hz, 2H), 2.39 – 2.28 (m, 1H), 2.23 – 2.06 (m, 2H), 2.00 – 1.92
(m, 1H), 1.91 – 1.81 (m, 1H), 1.76 – 1.65 (m, 1H), 1.57 – 1.42 (m, 3H),
1.41 – 1.28 (m, 3H), 1.27 – 1.08 (m, 5H), 0.79 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 151.1, 135.2, 132.3, 127.1, 126.1, 115.0, 81.8,
49.8, 43.8, 43.1, 38.7, 36.5, 30.4, 29.0, 27.1, 26.2, 22.9, 22.8, 14.2,
10.9. Rf = 0.23 (heptane/EtOAc 8:2, CAM-stain). HRMS calcd. for
2.6. (8S,9S,13S,14S)-2-ethyl-17-(2-methoxypyridin-4-yl)-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (9c)
1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 5.5, 1H), 7.05 (s, 1H),
6.91 (dd, J = 5.5, 1.5 Hz, 1H), 6.76 (s, 1H), 6.52 (s, 1H), 6.20 – 6.14
(m, 1H), 4.85 (s, 1H), 3.96 (s, 3H), 2.93 – 2.77 (m, 2H), 2.62 (q,
J = 7.5 Hz, 2H), 2.42 – 2.09 (m, 5H), 1.97 – 1.89 (m, 1H), 182 – 1.74
(m, 1H), 1.70 – 1.60 (m, 3H), 1.51 – 1.38 (m, 1H), 1.24 (t, J = 7.5 Hz,
3H), 1.06 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 164.9, 153.1, 151.7,
147.9, 146.6, 135.8, 133.0, 131.6, 127.7, 126.5, 115.7, 115.6, 108.2,
57.2, 54.0, 48.0, 44.5, 37.7, 35.7, 32.3, 31.9, 29.5, 28.2, 27.0, 23.5,
17.2, 14.9. Eluent 20% EtOAc in heptane, Rf = 0.29, yield 37 mg (60%)
as white solid, m.p. 142–144 °C. HRMS calcd. for C26H32NO2 [M]·+
C
20H28O2 [M]+ 300.2089, found 300.2083. The spectroscopic data for
6 was in agreement with that previously reported [22,25].
2.2. General procedure for the Suzuki-Miyaura cross-coupling reaction.
The TBS protected steroid triflate 722 or 1224 (1.00 equiv.), cesium
carbonate (2.00 equiv.) and the boronic acid (1.05 equiv.) was in-
dividually placed in a flame dried 10 mL round bottomed flask and
dissolved in a 1:1 mixture of water and THF (6.00–8.00 mL). Then Pd
2

N. al-Kazaale et al.
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389.2355. Found 389.2344.
dried 10 mL round bottomed flask and dissolved in dry di-
chloromethane (3–5 mL) under argon atmosphere. The reaction mixture
was cooled to 0 °C before sulfamoyl chloride (2.95 equiv.) was added
and stirred for additional 30 min. Then the reaction mixture was stirred
at room temperature overnight. The reaction mixture was added satu-
rated aqueous NaHCO3 (5 mL) and extracted with ethyl acetate
(3 × 10 mL). The combined organic extracts were dried over MgSO4
and evaporated in vacuo. The residues were purified by flash column
chromatography (silica gel, 30–50% EtOAc in heptane) to yield the
pure products 10a-10e and 14a-14b as colorless solids.
2.7. (8S,9S,13S,14S)-17-(2-chloropyridin-4-yl)-2-ethyl-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (9d)
1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 5.2, 1H), 7.35 – 7.30 (m,
1H), 7.22 (dd, J = 5.2, 1.5 Hz, 1H), 7.04 (s, 1H), 6.54 (s, 1H), 6.29 –
6.22 (m, 1H), 4.82 (s, 1H), 2.90 – 2.78 (m, 2H), 2.62 (q, J = 7.5 Hz,
2H), 2.45 – 2.25 (m, 3H), 2.23 – 2.12 (m, 2H), 2.01 – 1.88 (m, 1H), 1.84
– 1.75 (m, 1H), 1.71 – 1.61 (m, 3H), 1.51 – 1.41 (m, 1H), 1.24 (t,
J = 7.5 Hz, 3H), 1.07 (s, 3H). Eluent 20% EtOAc in heptane, Rf = 0.18,
[α]2D0 = +6.0 (c = 0.5, CH2Cl2), yield 9 mg (45%) as white solid, m.p.
238–240 °C. HRMS calcd. for C25H28ClNO [M]·+ 393.1859. Found
393.1855.
2.12. (8S,9S,13S,14S)-2-Ethyl-13-methyl-17-(2-methylpyridin-4-yl)-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-yl
sulfamate (10a)
2.8. (8S,9S,13S,14S)-17-(5-chloropyridin-3-yl)-2-ethyl-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (9e)
1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 5.3 Hz, 1H), 7.18 (m, 2H),
7.14 (m, 1H), 7.12 (s, 1H), 6.22 (t, J = 2.5 Hz, 1H), 5.16 (s, 2H), 2.93 –
2.87 (m, 2H), 2.71 (q, J = 7.5 Hz, 2H), 2.59 (s, 3H), 2.45 – 2.28 (m,
3H), 2.25 – 2.12 (m, 2H), 2.01 – 1.93 (m, 1H), 1.83 – 1.60 (m, 5H), 1.52
– 1.40 (m, 1H), 1.24 (t, J = 7.6 Hz, 3H), 1.08 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 157.5, 152.5, 146.2, 139.2, 135.8, 133.8, 133.6,
126.6, 121.5, 120.9, 118.5, 56.7, 47.4, 44.2, 36.8, 35.2, 31.5, 29.0,
27.4, 26.3, 23.9, 23.0, 16.7, 14.6. Eluent 50% EtOAc in heptane,
Rf = 0.16, [α]20 = +15.5 (c = 0.6, CH2Cl2), yield 15 mg (42%) as
white solid, mD.p. 180–182 °C. HRMS calcd. for C26H32N2O3S [M]·+
452.2134. Found 452.2131.
1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 1.3 Hz, 1H), 8.45 (d,
J = 2.3 Hz, 1H), 7.71 (s, 1H), 7.05 (s, 1H), 6.54 (s, 1H), 6.10 (dd,
J = 3.3, 1.7 Hz, 1H), 5.12 (s, 1H), 2.97 – 2.74 (m, 2H), 2.62 (q,
J = 7.5 Hz, 2H), 2.47 – 2.35 (m, 2H), 2.35 – 2.26 (m, 2H), 2.22 – 2.10
(m, 2H), 2.00 – 1.89 (m, 1H), 1.89 – 1.76 (m, 1H), 1.73 – 1.61 (m, 3H),
1.53 – 1.39 (m, 1H), 1.24 (t, J = 7.5 Hz, 3H), 1.05 (s, 3H).13C NMR
(101 MHz, CDCl3) δ 151.4, 150.4, 146.1, 145.1, 135.3, 134.4, 133.8,
132.3, 131.8, 131.1, 127.3, 126.0, 115.3, 56.7, 47.8, 44.1, 37.3, 35.3,
31.6, 29.1, 27.7, 26.5, 23.1, 16.7, 14.4. Eluent 30% EtOAc in heptane,
Rf = 0.27, [α]20 = +52.0 (c = 0.85, MeOH), yield 0.160 g (90%) as
white solid, m.Dp. 176–179 °C. HRMS calcd. for C25H28ClNO [Μ+Na]+
394.1930. Found 394.1932.
2.13. (8S,9S,13S,14S)-2-Ethyl-17-(2-fluoropyridin-4-yl)-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-yl
sulfamate (10b)
2.9. (8S,9S,13S,14S)-17-(2-chloropyridin-4-yl)-2-methoxy-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (13a)
1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 5.5 Hz, 1H), 7.17 (m, 2H),
7.11 (s, 1H), 6.91 (s, 1H), 6.31 – 6.23 (m, 1H), 5.05 (s, 2H), 2.97 – 2.86
(m, 2H), 2.71 (q, J = 7.5 Hz, 2H), 2.48 – 2.11 (m, 5H), 2.08 – 1.92 (m,
1H), 1.87 – 1.60 (m, 5H), 1.55 – 1.39 (m, 1H), 1.23 (t, J = 7.5, 3H),
1.08 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 165.4, 163.4, 152.2, 152.2,
150.6, 150.5, 147.7, 147.6, 146.7, 139.7, 136.2, 134.2, 133.3, 127.1,
121.9, 119.5, 119.4, 106.9, 106.5, 57.1, 47.9, 44.7, 37.2, 35.6, 32.0,
29.4, 27.9, 26.7, 23.5, 17.1, 15.0. Eluent 50% EtOAc in heptane,
Rf = 0.57, [α]20 = +24.4 (c = 0.84, CH2Cl2), yield 39 mg (81%) as
white solid, mD.p. 187–189 °C. HRMS calcd. for C25H29FN2O3S [M]·+
456.1883. Found 456.1865.
1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 5.3 Hz, 1H), 7.32 (s, 1H),
7.21 (dd, J = 5.1, 1.6 Hz, 1H), 6.78 (s, 1H), 6.67 (s, 1H), 6.25 (q,
J = 3.4, 1.8 Hz, 1H), 5.46 (s, 1H), 3.87 (s, 3H), 2.94 – 2.75 (m, 2H),
2.44 – 2.26 (m, 3H), 2.25 – 2.11 (m, 2H), 1.97 – 1.91 (m, 1H), 1.85 –
1.74 (m, 1H), 1.72 – 1.61 (m, 3H), 1.53 – 1.40 (m, 1H), 1.08 (s, 3H). 13
C
NMR (101 MHz, CDCl3) δ 151.7, 151.6, 149.3, 147.8, 144.6, 143.6,
133.1, 131.5, 129.4, 121.3, 119.9, 114.7, 107.8, 56.7, 56.1, 47.6, 44.3,
37.2, 35.2, 31.6, 28.8, 27.7, 26.7, 16.8. Eluent 30% EtOAc in heptane,
Rf = 0.27, [α]20 = +29.0 (c = 0.75, MeOH), yield 0.160 g (56% over
two steps) asDwhite solid, m.p. 176–179 °C. HRMS calculated for
2.14. (8S,9S,13S,14S)-2-Ethyl-17-(2-methoxypyridin-4-yl)-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-yl
sulfamate (10c)
C
24H26ClNO2 [M]+: 395.1652. Found 395.1658.
2.10. (8S,9S,13S,14S)-17-(3-chloropyridin-4-yl)-2-methoxy-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (13b)
1H NMR (400 MHz, DMSO‑d6) δ 8.09 (d, J = 5.4 Hz, 1H), 7.93 (s,
2H), 7.19 (s, 1H), 7.03 (s, 1H), 7.02 (dd, J = 5.4, 1.5 Hz, 1H), 6.75 (s,
1H), 6.34 – 6.29 (m, 1H), 3.85 (s, 3H), 2.88 – 2.78 (m, 2H), 2.69 – 2.57
(m, 2H), 2.46 – 2.37 (m, 1H), 2.36 – 2.06 (m, 4H), 1.94 – 1.86 (m, 1H),
1.77 – 1.51 (m, 4H), 1.48 – 1.33 (m, 1H), 1.13 (t, J = 7.5 Hz, 3H), 1.02
(s, 3H). 13C NMR (101 MHz, DMSO‑d6) δ 163.9, 151.7, 146.7, 146.5,
146.1, 138.0, 134.9, 133.4, 131.5, 126.1, 121.6, 114.8, 106.7, 56.2,
53.0, 46.8, 43.6, 36.4, 34.6, 30.9, 28.4, 26.9, 25.9, 22.4, 16.3, 14.7.
Eluent 30% EtOAc in heptane, Rf = 0.22, [α]20 = +9.0 (c = 1.0,
DMSO), yield 27 mg (90%) as white solid, m.pD.197–200 °C. HRMS
calcd. for C26H32N2O4S [M]·+ 468.2083. Found 468.2075.
1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 1.9 Hz, 1H), 8.46 (d,
J = 2.3 Hz, 1H), 7.74 (m, 1H), 6.78 (s, 1H), 6.67 (s, 1H), 6.16 – 6.11
(m, 1H), 5.46 (s, 1H), 3.87 (s, 3H), 3.01 – 2.70 (m, 2H), 2.45 – 2.29 (m,
3H), 2.23 – 2.10 (m, 2H), 2.00 – 1.90 (m, 1H), 1.81 (td, J = 11.5,
6.5 Hz, 1H), 1.73 – 1.62 (m, 3H), 1.52 – 1.39 (m, 1H), 1.06 (s, 3H). 13
C
NMR (101 MHz, CDCl3) δ 150.3, 146.0, 145.0, 144.6, 143.6, 134.4,
133.8, 131.9, 131.6, 131.3, 129.4, 114.7, 107.8, 56.7, 56.1, 47.8, 44.3,
37.2, 35.3, 31.6, 28.8, 27.7, 26.7, 16.8. Rf = 0.30. [α]25 = +45.9
(c = 0.45, MeOH), yield 135 mg (65% over two steps) as white solid,
m.p. 191–192 °C. HRMS calcd. for C24H26ClNO2 [Μ+Na]+ 418.1550.
Found 418.1544.
2.15. (8S,9S,13S,14S)-17-(2-chloropyridin-4-yl)-2-Ethyl-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-yl
sulfamate (10d)
2.11. General procedure for the synthesis of sulfamates 10a-10e and 14a-
14b
1H NMR (400 MHz, d4-MeOH) δ 8.31 (d, J = 5.3 Hz, 1H), 7.51 (s,
1H), 7.46 (dd, J = 5.2, 1.6 Hz, 1H), 7.25 (s, 1H), 7.14 (s, 1H),
6.47–6.44 (m, 1H), 2.99 – 2.91 (m, 2H), 2.76 (q, J = 7.5 Hz, 2H), 2.56 –
The individual estrogen of 9a-9e or 13a-13b (1.00 equiv.) and 2,6-
di-tert-butyl-4-methylpyridine (DBMP, 3.00 equiv.) was placed in flame
3

N. al-Kazaale et al.
Steroidsxxx(xxxx)xxx–xxx
2.20 (m, 6H), 2.08 – 2.00 (m, 1H), 1.92 – 1.82 (m, 1H), 1.78 – 1.67 (m,
3H), 1.60 – 1.44 (m, 1H), 1.25 (t, J = 7.5 Hz, 3H), 1.16 (s, 3H). 13C
NMR (101 MHz, d4-MeOH) δ 152.8, 152.4, 150.3, 149.8, 148.0, 139.7,
136.6, 135.2, 135.0, 127.4, 122.8, 122.5, 121.5, 58.0, 45.5, 38.4, 36.3,
32.5, 29.9, 28.6, 27.5, 24.0, 17.0, 15.1. Eluent 50% EtOAc in heptane,
Rf = 0.54, [α]20 = +20.5 (c = 0.8, CH2Cl2), yield 75 mg (81%) as
white solid, m.Dp. 190–192 °C. HRMS calcd. for C25H29ClN2O3S [M]·+
472.1587. Found 472.1579.
later, the cells were trypsinized and counted. Human T-cell leukemia
(CEM) cells were seeded in 96-well plates at 60,000 cells/well in the
presence of the compounds, allowed to proliferate for 4 days and then
counted. The 50% inhibitory concentration (IC50) was defined as the
compound concentration required to reduce cell proliferation by 50%
[26].
2.20. Tubulin binding
2.16. (8S,9S,13S,14S)-17-(5-chloropyridin-3-yl)-2-Ethyl-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-yl
sulfamate (10e)
Human breast carcinoma MDA-MB-231 cells were seeded in 6-well
plates at 500,000 cells/well. After 48 h, compounds were added to the
cells for 16 h before adding EBI (N,N′-ethylene-bis(iodoacetamide) at
100 µM. After 1.5 h, the cells were harvested and cell extracts were
prepared for western blot analysis. Twenty µg of proteins were sub-
jected to gel electrophoresis using 0.1% SDS (85% purity) and 10%
polyacrylamide gels. After electrophoresis, proteins were transferred to
pretreated Hybond-P polyvinylidene difluoride (PVDF) membranes
(Amersham Biosciences). The membranes were incubated for 1 h at
room temperature in blocking buffer (2.5% non-fat dry milk in PBS
containing 0.1% Tween) and subsequently for 16 h at 4 °C in blocking
buffer with primary antibodies raised against β-tubulin. After washing,
the membranes were incubated with the corresponding horseradish
peroxidase-conjugated secondary antibody in blocking buffer for 25′ at
room temperature. Next, the membranes were washed extensively.
Immunoreactive proteins were detected by chemiluminescence
(ECLplus, Bio-Rad). In living cells, EBI cross-links the cysteine residues
at positions 239 and 354 of β-tubulin. This β-tubulin adduct formed by
EBI is easily detectable by western blot as a second immunoreactive
band that migrates faster than β-tubulin. Colchicine was added as a
reference compound.
1H NMR (400 MHz, DMSO‑d6) δ 8.59 (d, J = 1.8 Hz, 1H), 8.52 (d,
J = 2.3 Hz, 1H), 7.94 (s, 2H), 7.88 (t, J = 2.1 Hz, 1H), 7.20 (s, 1H),
7.03 (s, 1H), 6.30 (dd, J = 3.4, 1.7 Hz, 1H), 2.91 – 2.77 (m, 2H), 2.63
(q, 2H), 2.48 – 2.39 (m, 1H), 2.37 – 2.26 (m, 2H), 2.20 – 2.03 (m, 2H),
1.97 – 1.86 (m, 1H), 1.80 – 1.71 (m, 1H), 1.69 – 1.38 (m, 3H), 1.49 –
1.36 (m, 1H), 1.13 (t, J = 7.5 Hz, 3H), 1.03 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 150.2, 146.7, 146.6, 145.8, 138.5, 135.5, 134.1, 134.0, 133.3,
131.7, 131.4, 126.7, 122.1, 56.7, 47.5, 44.1, 36.9, 35.0, 31.6, 29.0,
27.5, 26.4, 22.9, 16.7, 15.2. TLC, eluent 30% EtOAc in heptane,
Rf = 0.42, [α]20 = +43 (c = 0.66, MeOH), yield 73 mg (50%) as white
D
solid, m.p. 189–194 °C. HRMS calcd for C25H29ClN2O3S [Μ]+
472.1587. Found 472.1579.
2.17. (8S,9S,13S,14S)-17-(2-chloropyridin-4-yl)-2-methoxy-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (14a)
1H NMR (300 MHz, CDCl3): δ 8.29 (d, J = 5.3 Hz, 1H), 7.32 (s, 1H),
7.21 (dd, J = 5.3, 1.5 Hz, 1H), 6.78 (s, 1H), 6.67 (s, 1H), 6.26 – 6.24
(m, 1H), 5.54 (bs, 1H), 3.87 (s, 3H), 2.88 – 2.77 (m, 2H), 2.42 – 2.28
(m, 3H), 2.23 – 2.14 (m, 2H), 1.98 – 1.90 (m, 1H), 1.84 – 1.63 (m, 4H),
1.52 – 1.41 (m, 1H), 1.08 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 151.82,
151.69, 149.49, 147.87, 144.77, 143.72, 133.26, 131.64, 129.49,
121.43, 120.02, 114.82, 107.92, 56.79, 56.21, 47.68, 44.38, 37.26,
35.29, 31.72, 28.95, 27.84, 26.85, 16.89. TLC, eluent 30% ethyl acetate
2.21. Cell cycle analysis
MDA-MB-231 cells were seeded in 6-well plates at 125,000 cells/
well in DMEM with 10% FCS. After 24 h, the cells were exposed to
different concentrations of the compounds. After 16 h, the DNA of the
cells was stained with propidium iodide using the CycleTEST PLUS DNA
Reagent Kit (BD Biosciences, San Jose, CA). The DNA content of the
stained cells was assessed by flow cytometry on a FACSCalibur flow
cytometer and analyzed with CellQuest software (BD Biosciences)
within 3 h after staining. Cell debris and clumps were excluded from the
analysis by appropriate dot plot gating. Percentages of sub-G1, G1, S,
and G2/M cells were estimated using appropriate region markers.
Colchicine was added as reference compound.
in heptane, Rf = 0.27, [α]20 = +46.0 (c = 0.91, MeOH), yield 82 mg
D
(82%) as white solid, m.p. 192–195 °C. HRMS calculated for
C
24H26ClNO2 [M]+: 395.1652. Found 395.1658.
2.18. (8S,9S,13S,14S)-17-(2-chloropyridin-4-yl)-2-methoxy-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol (14b)
1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 1.9 Hz, 1H), 8.42 (d,
J = 2.2 Hz, 1H), 7.68 (t, 1H), 7.07 (s, 1H), 6.91 (s, 1H), 6.10 (m, 1H),
5.17 (s, 2H), 3.88 (s, 3H), 2.93 – 2.78 (m, 2H), 2.52 – 2.28 (m, 3H), 2.27
– 2.09 (m, 2H), 2.06 – 1.92 (m, 1H), 1.80 (td, J = 11.4, 6.4 Hz, 1H),
1.74 – 1.60 (m, 3H), 1.56 – 1.38 (m, 1H), 1.05 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 150.0, 149.0, 145.5, 144.4, 140.2, 136.9, 134.5,
134.4, 132.1, 131.7, 130.2, 124.3, 110.2, 56.7, 56.5, 47.7, 44.5, 36.7,
35.2, 31.6, 28.5, 27.4, 26.5, 16.7. TLC, eluent 30% EtOAc in heptane,
Rf = 0.42, [α]25 = +46.0 (c = 045, MeOH), yield 12 mg (52%) as a
white solid, m.p. 191–192 °C. HRMS calcd for C25H29ClN2O3S [Μ+H]+
473.1666. Found: 473.1659.
2.22. Inhibition of tubulin polymerization
The method applied was that described by Lawrence and coworkers
[27]. The assay was performed using a commercial kit (Cytoskeleton
Inc., Denver, USA). Briefly, samples were prepared directly in a 96-well
microtitre plate that was pre-incubated at 4 °C in the fridge for 30 min
and contained Mes buffer [128 μl (0.1 M Mes, 1 mM EGTA, 0.5 mM
MgCl2, distilled water, pH 6.6)], GTP (20 μl, 5 mM in Mes buffer), tu-
bulin (50 μl, 11 mg/ml in Mes buffer) and the test compound (20 μl in
DMSO). The tubulin and samples of test compounds were immediately
placed in a 96-well plate reader, alongside the blank samples containing
Mes buffer (198 μl) and the analogs (10 μl, same concentration). The
absorbance (λ 340 nm) was recorded at 25 °C temperature for a period
of 60 min.
2.19. Cell growth inhibition
Human cervical carcinoma (HeLa), Human colon carcinoma (HT29)
and Monkey kidney epithelial (Vero) cells were seeded in 48-well plates
at 10,000 cells/well. After 24 h, 5-fold dilutions of the compounds were
added. After 3 days of incubation, the cells were trypsinized and
counted by means of a Coulter counter (Analis, Belgium). Human
dermal microvascular endothelial (HMEC-1) cells were seeded on ge-
latin-coated 48-well plates at 20,000 cells/well. After overnight in-
cubation, 5-fold dilutions of the compounds were added. Three days
2.23. Inhibition of angiogenesis
The method applied was essential that described previously [28].
Endothelial cell tube formation assay was modified from a method
previously described [24]. Matrigel (12.5 mg/ml) was thawed at 4 °C,
and 50 μl was quickly added to each well of a 96-well plate and allowed
4

N. al-Kazaale et al.
Steroidsxxx(xxxx)xxx–xxx
to solidify for 10 min at 37 °C. Once solid, the wells were incubated for
30 min with endothelial cells (30,000 cells/well). After adhesion of the
cells, the medium was removed and replaced by fresh medium sup-
plemented with compounds with five different concentrations ranging
from 10 μM to 0.001 μM and incubated at 37 °C for 18 h. The tubes were
visualized with an inverted ZEISS microscope at a magnification of 10.
The tube formation areas were obtained using KURABO Angiogenesis
Image Analysis Software. The length of the capillary network was
quantified with a map scale calculator (KURABO Angiogenesis Image
Analysis Software). Inhibition curve was obtained between areas and
concentrations to get the IC50-value.
to literature [22], see Scheme 1. The synthetic route presented herein
reduces the number of steps for the preparation of 2-ethylestradiol (6)
compared to those previously reported. Moreover, this approach is also
amendable for large-scale preparations of 6. The 2-ethyl group should
be introduced prior to the modification at C-17 since making sub-
stituted pyridine analogs of 2 did not work in our hands. All compounds
were obtained as one isomer and displayed spectroscopic data in ac-
cordance with their structure (Supporting Information).
Based on the initial screening of cytostatic effects of 9a-9e and 10a-
10e four 2-methoxy analogs were also prepared (Scheme 2).
3.2. Biological evaluations
2.24. Docking studies
Compounds 9a-9e, 10a-10e, 13a, 13b, 14a and 14b were evaluated
for their effect on the proliferation of three human tumor cell lines T-
cell leukemia (CEM), cervical carcinoma (HeLa) and colon adeno-
carcinoma (HT29), African green monkey kidney epithelial cells
(VERO) and human dermal microvascular endothelial cells (HMEC-1),
see Table 1. The compounds that exhibited the most potent anti-pro-
liferative activity in all five cell lines were the compounds 10d, 10e,
14a and 14b, which all contain a sulfamate group in the 3-position of
the A-ring of the steroid skeleton and a 2- or a 3-chloro-substituted
pyridine ring. Interestingly, all compounds exhibited submicromolar
The Internal Coordinate Mechanics (ICM) program[29] was used for
docking of compounds 10d, 10e, 14a, 14b and 2-ME (1) into the β-
subunit of tubulin. The docking studies were performed with the tu-
bulin structure from two X-ray complexes of tubulin with colchicine
(PDB ids: 1SA0, 4O2B) [30,31]. Crystallographic water molecules, ions
and the co-crystallized inhibitor were removed from the X-ray com-
plexes and hydrogen atoms were added and optimized using the
ECEPP/3 force field of ICM. The compounds were built using ICM and
optimized before docking. A grid map that included the amino acids
within 5 Å of the co-crystallized inhibitors was calculated, and semi-
flexible docking with flexible ligands was performed. Each docking was
run in three parallels. The docking poses were scored using the Virtual
Ligand Scoring (VLS) module of the ICM program.
activities against CEM cells. The IC50-values were 0.40
0.07 µM,
0.05 µM for 10d, 10e and 14a, respec-
was even more potent with
0.001 µM. Potent cytostatic effects were also observed
0.26
tively.
IC50 = 0.039
0.05 µM and 0.11
Compound 14b
in the other cell lines for all four compounds (Table 1). In particular,
HT29 and VERO cells proved very sensitive to all compounds (Table 1).
In these two cell lines analog 14b displayed superior potent cytostatic
3. Results and discussion
3.1. Chemistry
effects with IC50-values of 0.11
0.05 µM and 0.03
0.02 µM, re-
spectively (Table 1).
Previously, we have based our synthesis of 2-ethyl-substituted
steroid analogs [22] on a highly selective ortho-formylation reaction of
phenols [32–34]. This approach also afforded also the undesired 4-ethyl
substituted isomer of 6 rendering careful and tedious chromatographic
purification necessary. Thanks to the recent work of Parnes and Pappo,
a simple and highly chemo- and regioselective method for introducing
primary alkyl substituents into phenols is now available [35]. We
therefore decided to use this new method for the preparation of 2-
ethylestradiol (6) (Scheme 1). In all attempts, we did not detect by GLC
and 1H NMR-analyses any of the 4-substituted isomer of 6. We en-
visaged that the protocol developed by Parnes and Pappo will be useful
for the synthesis of medicinal agents and phenolic natural products. The
syntheses of compounds 7 and 8 and the remaining steps towards the
preparation of the analogs 9a-9e and 10a-10e were achieved according
Among the phenolic steroid analogs 9a-9e, the most active com-
pound was 9e. This analog, containing a chlorine atom in the 5-position
of the pyridine ring, was particularly active towards the CEM cell line
(IC50 = 5.2
1.4 µM). The other analogs displayed modest anti-pro-
liferative effects. The cytostatic effects were less prominent when a
fluorine atom was present, as for 9b. Electron releasing substituents, as
in compounds 9a and 9c, provided only modest inhibitory effects.
These two compounds were also less cytostatic compared to compound
5 [22] (Table 1).
The microtubule system is a well-studied biological target for anti-
cancer drugs [36]. The steroid 2-ME (1) has been reported to inhibit
tumor growth by binding to the colchicine binding site of β-tubulin,
inhibiting tubulin-polymeriziation in vitro, resulting in cell cycle arrest
at the G2/M phase and eventually in cell death [37–40]. The analogs
Scheme 1. Synthesis of analogs 9a-9e and 10a-
10e. (i) (a) CH3CHO, Cu(OTf)2, EtSH,
CF3CH2OH, 50 °C, 16 h; (b) Et3SiH 50 °C, 4 h,
68%; (ii) cyclohexanone, Al(O-iPr)3, toluene, Δ,
77%; (iii) TBSCl, imidazole, DMF, 69%; (iv)
KHMDS, PhN(SO2CF3)2, THF, −78 °C, 64%; (v)
ArB(OH)2, Cs2CO3, Pd(Ph3P)4, THF:H2O (1:1),
60 °C, 53–90%; (vi) TBAF, THF, rt, 45–90%; (vii)
H2NSO2Cl, DBMP, CH2Cl2, 0 °C – rt., 42–90%.
O
OH
OH
(i)
(ii), (iii)
H
H
H
H
H
H
H
H
H
TBSO
HO
HO
2
6
7
(iv)
OTf
Ar
Ar
H
H
H
(v), (vi)
(vii)
H
H
H
H
H
H
TBSO
H2NO2SO
HO
10a-10e
9a-9e
8
N
N
F
O
N
Cl
N
Cl
b
: Ar =
d
: Ar =
a
c
: Ar =
: Ar=
e
: Ar =
N
5

N. al-Kazaale et al.
Steroidsxxx(xxxx)xxx–xxx
Scheme 2. Synthesis of analogs 13a-13b and 14a-
14b. (i) cyclohexanone, Al(O-iPr)3, toluene, Δ; (ii)
TBSCl, imidazole, DMF, 71% over two steps; (iii)
KHMDS, PhN(SO2CF3)2, THF, −78 °C, 86%; (iv)
ArB(OH)2, Cs2CO3, Pd(Ph3P)4, THF:H2O (1:1),
60 °C; (v) TBAF, THF, rt, (13a) 56% over two steps,
65% over two steps (13b); (v) H2NSO2Cl, DBMP,
CH2Cl2, 0 °C – rt., 82% (14a); 52% (14b).
O
OH
OTf
(iii)
(i), (ii)
H
H
H
O
O
O
H
H
H
H
H
H
TBSO
HO
TBSO
1
11
12
(iv), (v)
Ar
Ar
(vi)
H
H
O
O
H
H
H
H
HO
H2NO2SO
14a 14b
-
13a 13b
-
Cl
N
Cl
: Ar =
a
: Ar =
b
N
Table 1
Biological evaluations of analogs 9a-9e, 10a-10e, 13a, 13b, 14a and 14b.
Comp.
CEM
HeLa
HMEC-1
VERO
HT29
MDA-MB-231
IC50 (µM)a
(proliferation)
Tubulin polymerization inhibition
IC50 (µM)b
Anti-angiogenesis
IC50 (µM)b
IC50 (µM)a
(prolifer-ation)
IC50 (µM)a
(prolifer-ation)
IC50 (µM)a
(prolifer-ation)
IC50 (µM)a
(prolifer-ation)
IC50 (µM)a
(prolifer-ation)
9a
9b
9c
9d
56
78
35
90
5.2
1.7
1.3
2.0
0.40
0.26
0.89
2.0
0.11
0.039
8.0
6
17
4
32
≥100
39
≥100
34
4.1
1.9
6.7
1.6
1.6
9.3
72
0.33
0.37
≥100
0.4
4
5
1
> 100
> 100
38
28
≥100
≥100
38
1.7
2.0
1.6
0.39
1.3
9.1
30
0.28
0.03
≥100
2.1
6
7
55
36
≥100
≥100
11
2.9
8.4
1.5
1.6
0.43
9.7
3.1
0.43
0.11
≥100
6.7
13
11
30
0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3.6
2.3
2.6
> 5c
2.9
2.1
4.3
2.9
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.4
0.1
1.1
0.7
0.5
0.2
3.2
3.2
n.d.
n.d.
n.d.
n.d.
0.55
n.d.
n.d.
n.d.
n.d.
9.1
62
1
7
> 100
9e
1.4
0.1
0.4
0.4
61
1
2
0.2
0.3
0.1
2
10a
10b
10c
10d
10e
13a
13b
14a
14b
5d
2.6
9.0
4.5
10
0.3
2.7
0.4
0.6
0.2
0.1
0
2.2
0.4
0.1
0.01
0.14
0.1
0.2
0.4
0.1
0.2
16
0.01
0.3
1
0.07
0.05
0.12
2.3
2.3
14
0.5
0.6
0.1
0.3c
0.1c
0.1c
0.1
0.05
0.1
0.2
0.1
0.05
0.1c,d
0.4
0.2
0.3
12
1
0.3
0.7
0.04
0.03
0.1
0.05
0.001
1.4
0.9
64
41
0.07
0.02
15
0.48
0.40
n.d.
1.9
0.1
0.02
0.32
0.11
n.d.
0.8
0.1c
0.1c
0.1c,d
0.1c
2-ME
1.69
0.1
0.1
0.3
0.1
0.5
n.d., not determined.
a
Results of three experiments as duplicates.
Results of two experiments as duplicates.
Results of two experiments as triplicates.
Results are from Ref. [22].
b
c
d
10d, 10e, 14a and 14b showed inhibition of tubulin polymerization
binding site.
with IC50-values of 3.6
and 2.1 μM 0.1, respectively. Similar data (IC50 = 2.9
0.3 μM, 2.3
0.1 μM, 2.9 μM
0.1 μM
Next we tested the anti-angiogenic activity of 2-ME (1), 10d, 10e,
13a, 13b, 14a and 14b (Table 1), since the anti-angiogenic activity of 1
has been reported [5,9,41]. All six analogs exhibited anti-angiogenic
activity in the low micromolar range with IC50-values between
0.1 μM)
was observed for 2-ME (1) which is in accord with literature [7]. Cell
cycle analyses revealed that 2-ME (1), 14a and 14b induced apoptosis
and caused G2/M phase arrest in MDA-MB-231 breast cancer cells.
Compound 14b proved as potent as 2-ME, displaying activity at 0.3 µM.
Compound 14a induced G2/M arrest only at 10 µM and induction of
apoptosis at 2 µM.
0.1
these
(IC50 = 3.29 μM
0.05 μM and up to 1.1
analogs were more
0.04). In this assay, 10e was the most potent
0.1 μM. Interestingly, in this assay,
potent than 2-ME (1)
analog.
We also subjected 14a and 14b to a competition binding assay with
N,N′-ethylene-bis(iodoacetamide) (EBI) using the human breast cancer
MDA-MB-231. This assay is based upon adduct formation between EBI
and tubulin, which occurs at the colchicine-binding site. Thus, a com-
pound that interacts with the colchicine-binding site of tubulin will
prevent adduct formation, resulting in a single tubulin band, whereas a
compound that does not bind at this site will show two bands (higher
tubulin and lower tubulin-adduct band). Colchicine was used as a po-
sitive control and completely inhibited the EBI-tubulin band at 2 µM. In
contrast, 14a and 14b did not inhibit adduct formation even at 10 µM,
indicating that these compounds do not bind tubulin at the colchicine-
The steroid 2-ME (1) inhibits angiogenesis directly through anti-
proliferative and apoptotic effects, and indirectly through inhibition of
HIF-1α (hypoxia-inducible factor-1α) expression in endothelial and
tumor cells [42]. However, studies have reported that the inhibition of
HIF-1α required higher concentrations compared to the concentrations
needed to inhibit cell proliferation and induce apoptosis [43]. Hence,
we did not evaluate our novel compounds as inhibitors against HIF-1α.
Studies have shown that 2-ME (1) displays a low binding affinity to
the α-estrogen receptor compared to both 17α-estradiol and 17β-es-
tradiol (2). The affinity is even lower for the β-estrogen receptor
[44,45]. Previously we have shown that analogs where the alcohol
6

N. al-Kazaale et al.
Steroidsxxx(xxxx)xxx–xxx
Fig. 2. Above: Compound 10e (yellow) docked at the
putative binding site between α-tubulin (red) and β-
tubulin (blue) superimposed with the 4O2B X-ray
structure with colchicine (green) at the colchicine
binding site. α- and β-tubulin are shown as ribbon
representations. Below: A close up of the best docking
poses of compound 10d and 10e. Amino acids of α-
tubulin are marked in red and β-tubulin are marked
in blue. Color coding of atoms in 10d and 10e: blue;
nitrogen, red; oxygen, dark yellow; sulphur, yellow;
carbon, green; chloride.
functionality at C-17 in 2-ME (1) has been replaced with an aryl-group,
resulted in no α-estrogen receptor activation [24]. Similar observations
are expected for all novel analogs 9a-9e and 10a-10e reported herein.
To our delight none of the analogs 9e, 10e, 13b, 14a and 14b showed
any agonist effects towards the estrogen receptor GPR30 (Supporting
Information).
with the backbone carbonyl group of V351 (β-tubulin). The nitrogen
atom of chloro-substituted pyridine ring of 10e formed a hydrogen
bond with the backbone amino group of Y224 (α-subunit), while the
methoxy groups of 14a and 14b interacted with T353 and D329.
4. Conclusions
Herein 14 new analogs of 2-ME (1) have been reported. The 2-ethyl
substituted analogs were prepared using an improved and new syn-
thetic route. The introduction of a chloro-atom on the pyridine ring in
the 17-position of 2-ethyl estradiol enhanced the anti-proliferative ac-
tivity. The two most potent analogs 10e and 14b contain a sulfamate
group in the 3-position of the A-ring of the steroid skeleton. Analogs
10d and 14a have their chloro-substituent in the 2-position of the
pyridine ring. Different substitution patterns affect the physical-prop-
erties of pyridines [46]. All attempts to prepare other chloro-substituted
analogs failed using the Suzuki-Miyaura reaction. Electron releasing
substituents gave less cytostatic compounds, while the fluoro-analog
10b was less potent than both 10d and 10e. Modest effects were ob-
served towards inhibition of tubulin polymerization. Competitive
binding studies revealed that these analogs do not bind at the colchicine
binding-site, and these observations were supported by molecular
modeling studies. Docking studies indicate that the most interesting
analogs occupy a binding site close to the binding-site of colchicine and
2-ME, and that the α-subunit is more important for binding the analogs
than for binding colchicine and 2-ME (1).
Based on the biological results reported herein and in the literature
[2,3,13,47], it is likely that the novel analogs exhibit their anticancer
effects via several modes of action. Steroids and their synthetic analogs
are of interest for the development of new remedies against cancers
[48]. Recently inhibitors against stereoid sulfatase have attracted in-
terest from the biomedical research community [49,50]. The biological
results presented herein provide new information for further studies
towards the development of such anti-cancer agents.
3.3. Docking studies
The compounds were initially docked using grid maps that included
all amino acids within 5 Å of the co-crystallized compound colchicine in
the X-ray crystal structure complexes. The docking indicated that 10d,
10e, 14a and 14b bind to a site close to the colchicine site (Figs. 2 and
3), with Virtual Ligand Screening (VLS) scoring values of −15.3,
−19,5, −16.6 and −19.8 for 10d, 10e, 14a and 14b, respectively. 2-
ME (1) occupied the colchicine site with a VLS scoring value of −14.9.
Both the docking pose and scoring values of 2-ME (1) are in agreement
with our previous results [22]. In the next docking experiment, we
decreased the grid maps to include amino acids within 3 Å of co-crys-
tallized colchicine and in that way forcing the compounds into the
colchicine site. This docking gave VLS scoring values of approximately
−10 for 10d and 10e, and approximately −8 for 14a and 14b. Similar
results were obtained with both X-ray structures of tubulin with col-
chicine (PDBids: 1SA0, 4O2B). Altogether, this indicates that 10d, 10e,
14a and 14b bind to another site than colchicine and 2-ME (1), which is
in agreement with the completion binding assay with EBI for 14a and
14b. These sites are slightly overlapping (Fig. 2) and it seems that the
α-subunit is more important for binding of our compounds than for
binding of colchicine and 2-ME (1).
The binding poses of the four compounds were very similar. In their
best binding poses (Figs. 2 and 3), the sulfamate and the ethyl groups of
10d and 10e and the sulfamate and methoxy groups of 14a and 14b
interacted with V351, D329 and T353 in the β-subunit and P175, Y210
and V177 in the α-subunit. The pyridine ring interacted with T223 and
Y224 in the α-subunit and Q247 in the β-subunit. For 10d, favorable
hydrogen bonds are seen between the sulfamate NH2 group and the side
chain of D329 (β-tubulin) and backbone carbonyl group of P175 (α-
tubulin). For 10e, the sulfamate NH2 group has hydrogen bonds both
Acknowledgements
We thank Lizette van Berckelaer and Eef Meyen for excellent
7

N. al-Kazaale et al.
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Fig. 3. Above: Compound 14b (yellow) docked at the
putative binding site between α-tubulin (red) and β-
tubulin (blue) superimposed with the 4O2B X-ray
structure with colchicine (green) at the colchicine
binding site. α- and β-tubulin are shown as ribbon
representations. Below: A close up of the best docking
poses of compound 14a and 14b. Amino acids of α-
tubulin are marked in red and β-tubulin are marked
in blue. Color coding of atoms in 14a and 14b: blue;
nitrogen, red; oxygen, dark yellow; sulphur, yellow;
carbon, green; chloride.
technical assistance. The authors express their sincere gratitude to-
wards the European network “Challenging organic syntheses inspired
by nature – from natural products chemistry to drug discovery” (COST
Action CM 1407) for collaborations and fruitful scientific interactions.
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Supplementary data associated with this article can be found, in the
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