Synthesis and biological evaluations of new analogs of 2-methoxyestradiol: Inhibitors of tubulin and angiogenesis

Article abstract of DOI:10.1016/j.ejmech.2014.08.002

The synthesis, cytotoxicity, inhibition of tubulin polymerization and anti-angiogenic effects of 15 analogs of 2-methoxyestradiol (1) are reported. The biological studies revealed that the position of nitrogen atom in the heterocyclic ring is important for inhibition of both tubulin polymerization and angiogenesis. The most potent inhibitors were compounds 11f and 13e, with a 6-substituted isoquinoline ring in the 17-position of the steroid skeleton. Moreover, low estrogen activity was observed for the analogs tested at 10 μM concentrations.

Full text of DOI:10.1016/j.ejmech.2014.08.002

European Journal of Medicinal Chemistry 85 (2014) 391e398
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluations of new analogs of 2-
methoxyestradiol: Inhibitors of tubulin and angiogenesis
,
Eirik Johansson Solum a, Jing-Jy Cheng b c, Irene B. Sørvik d, Ragnhild E. Paulsen d,
,
*
Anders Vik a, Trond Vidar Hansen a
a Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, PO Box 1068, Blindern, N-0316 Oslo, Norway
b National Research Institute of Chinese Medicine, Taipei, Taiwan
c Institute of Biophotonics, National Yang-Ming University, Taipei 112, Taiwan
d Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, cytotoxicity, inhibition of tubulin polymerization and anti-angiogenic effects of 15 analogs
of 2-methoxyestradiol (1) are reported. The biological studies revealed that the position of nitrogen atom
in the heterocyclic ring is important for inhibition of both tubulin polymerization and angiogenesis. The
most potent inhibitors were compounds 11f and 13e, with a 6-substituted isoquinoline ring in the 17-
position of the steroid skeleton. Moreover, low estrogen activity was observed for the analogs tested
Received 10 June 2014
Received in revised form
28 July 2014
Accepted 1 August 2014
Available online 2 August 2014
at 10 mM concentrations.
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
2-Methoxyestradiol
Anti-cancer
Tubulin inhibition
Anti-angiogenesis
Steroids
1. Introduction
posses a plethora of interesting anti-cancer effects without any
undesirable estrogen activity [5e9]. 2-Methoxyestradiol (1) has
entered several clinical trials [10]. In addition, the steroid 1 has
been employed as a lead compound in many structuraleactivity
relationship (SAR) studies [11]. One example that has emerged from
these efforts is ENMD-1198 (2) that has been the subject of clinical
trials and extensive biological evaluations [12].
The cortistatins, exemplified by cortistatins A (3), J (4) and G (5),
see Fig. 1, are also examples of natural occurring steroids that have
attracted great interest within anti-cancer research [13]. These
steroidal alkaloids were first isolated in 2006 from the marine
sponge Corticium simplex and showed potent anti-angiogenetic
effects [14]. It was also reported that cortistatin A (3) inhibits the
proliferation of human umbilical vein endothelial cells (HUVECs) in
the low nano-molar range [14]. The cortistatins have recently
attracted a great interest in the synthetic community as targets for
total synthesis [15]. In addition, several analogs of 3 with inter-
esting anti-cancer effects have been prepared [16]. These SAR-
studies have revealed that the presence of an isoquinoline group
is crucial for activity and that the two hydroxyl-groups on the A-
ring are removable. Along these lines, one of the most interesting
analogs reported so far is the amino steroid 6 reported by Corey and
co-workers [16].
Several natural products have been reported to be microtubule-
binding agents [1]. Such natural products and their derivatives have
been widely used in cancer chemotherapy [2]. These anti-cancer
agents can either stabilize microtubules and promote polymeriza-
tion, or destabilize the microtubules and promote depolymeriza-
tion by inhibition of tubulin [3]. Both types interfere with the
mitotic spindle polymerization during cell division that eventually
leads to cell death. Studies have shown that most microtubule-
binding agents have anti-vascular effects induced by vascular dis-
rupting or from anti-angiogenetic activities, or both [4].
The endogenous steroid 2-methoxyestradiol (2-ME, 1), see Fig.1,
is a natural product that exhibits anti-vascular effects [5] and anti-
angiogenetic activities [6]. 2-Methoxyestradiol (1) was for long a
period of time believed to be an inactive metabolite, but D'Amoto
and co-workers reported in 1994 that 1 was a tubulin polymeri-
zation inhibitor [7]. Several studies revealed that the steroid 1
* Corresponding author.
E-mail addresses: e.j.solum@farmasi.uio.no (E.J. Solum), verona@nricm.edu.tw
(J.-J. Cheng), i.b.sorvik@farmasi.uio.no (I.B. Sørvik), r.e.paulsen@farmasi.uio.no
(R.E. Paulsen), anders.vik@farmasi.uio.no (A. Vik), t.v.hansen@farmasi.uio.no
(T.V. Hansen).
http://dx.doi.org/10.1016/j.ejmech.2014.08.002
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

392
E.J. Solum et al. / European Journal of Medicinal Chemistry 85 (2014) 391e398
Fig. 1. Examples of steroids exhibiting anti-cancer effects.
Based on the previous SAR-studies, and our interest in analogs of
TBS-group was achieved with TBAF in THF yielding the analogs
13ae13g. In all cases, only one diastereomer was obtained after
chromatography.
natural products with anti-cancer activities [17], we became
interested in preparing analogs with either a pyridine, a quinoline
or an isoquinoline ring in the C-17 position of 2-ME (1). The syn-
thesis, cytotoxicity, inhibition of tubulin polymerization data and
anti-angiogenetic effects of such analogs are presented herein.
Some of the new analogs were also tested as agonists for the
3. Biological evaluation
To investigate the anti-cancer effects of the prepared com-
pounds, cell growth inhibition assay against the three different
human cancer cell lines K562, OVCAR-3 and WM35 were per-
formed [21]. The most cytotoxic compounds were also subjected to
a tubulin polymerization inhibition assay [22]. All new analogs
were also tested as inhibitors of angiogenesis [23]. Finally, some of
the compounds were also evaluated as agonists against the
estrogen receptor a.
2. Chemistry
The new analogs of 2-methoxyestradiol (1) were prepared as
depicted in Scheme 1. First, 1 was oxidized in an Oppenauer
oxidation to ketone 8a that was converted to the TBS-protected
ketone 8b Then ketone 8b was converted into the triflate 9 using
N-phenyl-bis(trifluoromethanesulfonimide). This afforded 9 in 69%
estrogen receptor
ERE-luc and ER. Cells were treated with 10
11d, 13a, 13c, 13g and 10 of 17
methoxyestradiol for comparison. Luciferase activity was
measured 24 after initiation of the treatments. 2-
b
[24,25]. PC-12 cells were co-transfected with
M of the analogs 11b,
-estradiol and 2-
m
mM
b
yield from 2-methoxyestradiol (1). Then compound
9 was
h
employed in SuzukieMiyaura reaction [18] with different
a
Methoxyestradiol (1) was included for reference in all assays.
commercially available boronic acids. In the presence of Pd(PPh3)4
and Cs2CO3 in THF/H2O (1:1), the TBS protected compounds
10ae10h were obtained (Scheme 1). The Stille reaction was also
attempted for the synthesis of 10c and 10d. However, this proved to
be a less efficient method, as demonstrated when the triflate 9 was
coupled with either 3- or 4-(tributylstannyl)pyridine in the pres-
ence of lithium chloride, copper chloride and Pd(PPh3)4 in DMSO.
The desired analogs 11ae11h were obtained after removal of the
TBS-group in 10ae10h under standard conditions [19].
4. Results and discussion
All compounds with a 16,17-double bond tested in the K562 cell
line, except 11a, 11b and 11g, exhibited cell growth inhibition in the
micromolar range, see Table 1.
The most potent analogs of the 16,17-unsaturated compounds in
the leukemia cell line K562 were 11c, 11d, and 11f with IC50-values
Reduction of the 16,17-double bond in analogs 11be11h was
performed in the presence of one atmosphere of hydrogen and Pd/C
with EtOAc as the solvent. The reduction of 11d was sluggish.
Reduction of the benzene ring in 11d was observed. After five days
the 4-substituted 5,6,7,8-tetrahydro-isoquinoline product 12c was
isolated in 36% yield after chromatography. The structure of 13c
was confirmed by 2D-NMR and MS-analyses, see Supporting
information. Examples of similar reductions of aromatic com-
pounds have been reported [20]. Reduction of the heterocyclic ring
in compound 11h was also observed; the 5-substituted 1,2,3,4-
tetrahydroquinoline compound 13g was obtained in 33% yield af-
ter chromatographic purification. The reduction of the 16,17-double
bond in the other analogs occurred smoothly; the TBS-protected
compounds 12aeb and 12de12f were obtained in 53e77% yields
after chromatographic purification. Again, deprotection [19] of the
of 0.9, 0.5 and 0.4
or slightly more active than the lead compound 2-
methoxyestradiol (1), IC50 0.8 M, see Table 1. The 5-
substituted isoquinoline analog 11e and the 5-substituted quino-
line analog 11h showed less potent inhibitory effects than 1. The
same trends were more or less observed both in the OVCAR-3 and
the WM35 cell assays. Noteworthy, in the ovarian cancer cell line
OVCAR-3, the 6-substituted isoquinoline analog 11f was six-fold
mM, respectively. All three analogs were as active
¼
m
more potent than 2-methoxyestradiol (1) with IC50 ¼ 0.2
m
M.
This analog was also the most potent compound (IC50 ¼ 1.0
mM)
subjected to the WM-35 melanoma cancer cell line assay. In both
the OVCAR-3 and WM35 cell assays, analog 11d also displayed
cytotoxic effects in the low mM range.
The analogs 13be13h were then tested for their cytotoxic ef-
fects. All analogs, except 13f, showed activity with IC50-values in

E.J. Solum et al. / European Journal of Medicinal Chemistry 85 (2014) 391e398
393
Scheme 1. Synthesis of analogs 11ae11h and analogs 13ae13g. Reagents and conditions: (a) cyclohexanone, Al(O-iPr)3, toluene,
D, 88%; (b) TBSCl, imidazole, DMF, rt., 92%; (c)
KHMDS, PhN(SO2CF3)2, THF, ꢀ78 ꢁC, 85%; (d) ArB(OH)2, Cs2CO3, Pd(Ph3P)4, THF:H2O (1:1), 60 ꢁC; (e) Pd/C, EtOAc, rt.; (f) TBAF, THF, rt.
the low micromolar range against the three human cancer lines.
The tetrahydroisoquinoline analog 13c showed cytotoxic activities
in the three cell lines similar to the 4-substituted isoquinoline
analog 11d. The 6-substituted isoquinoline compound 13e also
displayed potent cytotoxic effects in all cell lines, comparably to its
congener 11d. The analog 13c displayed potent activity
receptor using a 10 mM concentration of the compounds 11b, 11d,
13a, 13c, and 13g, see Fig. 2. Therefore, the compounds reported in
this study induce cell death independently of the estrogen
receptor b.
5. Conclusions
(IC50 ¼ 0.5
mM) against the ovarian cancer cell line. The absence of a
double bond in the 16,17-position of the 3-substituted pyridine
analog 13a proved beneficial, as the cytotoxicity enhanced in all cell
lines compared to compound 11b.
Corey and co-workers reported that the position of the nitrogen
atom in the isoquinoline group is essential for the potent anti-
angiogenesis activity of analog
6 [16]. Both the 3-pyridine
All analogs except 11a, 11b and 11g were tested in a tubulin
inhibition assay. In this assay, the analog 13f was inactive
substituted compound 13a and the 6-isoquinoline substituted an-
alogs 11f and 13e reported herein, displayed higher anti-
angiogenetic effects compared to 2-methoxyestradiol (1). The po-
sition of the nitrogen atom seems to be important for potent in-
hibition of angiogenesis for the compounds reported herein. The
three analogs 11f, 13a and 13e were all also inhibitors of tubulin
polymerization. Tubulin polymerization inhibitors leading to
decreased angiogenesis in cancer cells are of interest for the
development of new remedies against cancer [27]. The most potent
compounds presented herein provide new information for further
SAR-studies towards the development of new anti-cancer agents.
(IC50 > 10
were 11f (IC50
(IC50 ¼ 2.1 M). All three compounds were as potent as 2-
M). The other compounds sub-
mM). The most potent tubulin polymerization inhibitors
¼
2.2 M), 13c (IC50 2.5 M) and 13e
m
¼
m
m
methoxyestradiol (1) (IC50 ¼ 2.2
m
mitted to the tubulin polymerization inhibition assay were all less
active than the lead compound 1.
Analogs 11ae11h and 13be13h were also tested as inhibitors in
an angiogenesis growth assay. For the lead compound 1, an IC50
value of 3.2 M was obtained. Interestingly, most compounds
prepared, except 11a, 11b and 11g, were active as inhibitors in this
M), 11f
M), and 13e
M) were all more potent than 2-methoxyestradiol (1).
-
m
assay. Compounds 11c (IC50 ¼ 0.6
m
M), 11d (IC50 ¼ 0.8
m
6. Experimental
(IC50 ¼ 0.7
(IC50 ¼ 0.6
m
M), 13c (IC50 ¼ 1.1
m
M), 13d (IC50 ¼ 1.3
m
m
6.1. General methods
The other compounds tested showed IC50-values in the 2.4e5.2 mM
range. Apparently, the presence of the 6-isoquinoline heterocyclic
ring is important for both inhibition of tubulin and angiogenesis,
since the 6-isoquinoline substituted compounds 11f and 13e dis-
played strong inhibition in both assays. Moreover, the reduction of
the 16,17-double bond proved beneficial for the 3-pyridine
substituted analog 13a since its congener 11b was inactive in the
angiogenesis growth assay.
All reagents and solvents were used as purchased without
further purification unless stated otherwise. Melting points are
uncorrected. Analytical TLC was performed using silica gel 60 F254
aluminum plates (Merck). Flash column chromatography was per-
formed on silica gel 60 (40e63
mm) produced by Merck. NMR
spectra were recorded on a Bruker Avance DPX-300 MHz, DPX-
400 MHz or DPX-600 MHz spectrometer for 1H NMR, and 75 MHz,
101 MHz or 151 MHz for 13C NMR. Coupling constants (J) are re-
ported in hertz, and chemical shifts are reported in parts per million
relative to CDCl3 (7.26 ppm for 1H and 77.0 ppm for 13C). Mass
spectra were recorded at 70 eV with Fison's VG Pro spectrometer.
High-resolution mass spectra were performed with a VG Prospec
mass spectrometer and with a Micromass Q-TOF-2™. The HPLC
analyses were performed on an Agilent Technologies 1200 Series
2-Methoxyestradiol (1) has been reported to exhibit very weak
estrogenic activity with a 500-fold and 3200-fold lower affinity for
the estrogen receptor
a and b, respectively, compared to b-estradiol
[26]. Hence, the analogs 11b, 11d, 13a, 13c, and 13g were tested as
potential agonists towards the estrogen receptor. Estradiol was
included as positive control and 2-methoxyestradiol (1) for com-
parison. Low agonist activity was observed towards the b-estrogen

394
E.J. Solum et al. / European Journal of Medicinal Chemistry 85 (2014) 391e398
Table 1
Biological evaluation of compounds 11ae11h and 13ae13g.
Compound
K562 cell assay
OVCAR-3 cell assay
WM35 cell assay
Tubulin inhibition
Anti-angiogenesis
IC50
(m
M)a
IC50
(m
M)a
IC50
(m
M)a
IC50
(m
M)b
IC50 (m
M)a
11a
11b
11c
11d
11e
11f
11g
11h
13a
13b
13c
13d
13e
13f
>10
>10
0.9
0.5
3.1
0.4
>10
>10
1.2
0.4
3.9
0.2
>10
>10
1.0
0.7
5.1
1.0
n.d.c
n.d.
4.1
4.8
7.2
2.2
n.d.
3.1
5.1
5.8
2.5
8.1
2.1
>10
3.7
2.2
>10
>10
0.6
0.8
2.4
0.7
>10
>10
>10
>10
1.1
1.1
1.4
0.5
2.1
0.7
>10
1.3
0.8
1.2
0.8
1.0
0.4
3.0
0.5
>10
1.5
1.2
2.9
1.9
1.6
0.9
4.1
0.9
>10
1.9
2.1
2.0
5.2
4.2
1.1
1.3
0.6
3.3
3.3
3.2
13g
2-ME
a
Results of three experiments performed as triplicates.
Results of three experiments performed as duplicates.
n.d. ¼ not determined.
b
c
instrument with an Eclipse XDB-C18 (5 mm 4.6 ꢂ 150 mm) column.
2-Methoxyestrone (7) and 3-tert-butyldimethylsiloxy-2-
6.3. (8S,9S,13S,14S)-2-methoxy-13-methyl-17-phenyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol
methoxyestrone (8) were prepared as previously reported [25].
Protocols for the preparation, physical and spectral data of the in-
termediates 9 and 10ae10h and 13ae13g are presented in the
Supporting information.
(11a)
1H NMR (400 MHz, CDCl3):
d 7.49e7.41 (m, 2H), 7.35 (t,
J ¼ 7.5 Hz, 2H), 7.29 (d, J ¼ 7.6 Hz, 1H), 6.83 (s, 1H), 6.71 (s, 1H), 5.98
(m, 1H), 5.48 (s, 1H), 3.90 (s, 3H), 2.97e2.75 (m, 2H), 2.35 (m, 3H),
2.24 (m,1H), 2.16 (m,1H), 2.03e1.92 (m,1H),1.83 (m,1H),1.78e1.65
(m, 3H), 1.50 (m, 1H), 1.11 (s, 3H). 13C NMR (101 MHz, CDCl3):
6.2. General procedure for the preparation of analogs 11ae11h and
13ae13g
d
155.0, 144.7, 143.6, 137.5, 132.2, 129.7, 128.3, 127.3, 126.9, 126.8,
114.8, 108.0, 57.0, 56.2, 47.7, 44.5, 37.5, 35.7, 31.5, 29.1, 28.0, 27.0,
16.9. Eluent 20% EtOAc in hexane, Rf ¼ 0.57, yield 91 mg, 84%,
product: colorless solid, mp 135e137 ꢁC. HRMS calcd. for C25H28O2
[M]þ: 360.2089. Found 360.2078.
The TBS protected steroids 10aeh, 12aeg (0.3e0.6 mmol,
1 equiv.) were placed in a dry round-bottomed flask under argon
atmosphere, and dissolved in dry THF. Tetra-n-butylammonium-
fluoride (1 M in THF, 1.1 equiv.) was added dropwise. The reaction
mixture was stirred at room temperature (16e18 h). Upon
completion the reaction the mixture was poured into saturated
aqueous NaHCO3 (10 ml), and extracted with ethyl acetate
(4 ꢂ 5 ml). The combined organic extracts were dried (MgSO4) and
the solvent evaporated in vacuo. The residues were purified by
chromatography (silica gel, 5e70% ethyl acetate in hexane) to give
the pure products.
6.4. (8S,9S,13S,14S)-2-methoxy-13-methyl-17-(pyridin-3-yl)-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol
(11b)
1H NMR (400 MHz, CDCl3):
d
8.69 (d, J ¼ 2.4 Hz, 1H), 8.51 (dd,
J ¼ 4.6, 1.7 Hz, 1H), 7.72 (dt, J ¼ 7.9, 1.9 Hz, 1H), 7.30e7.27 (m, 1H),
6.82 (s, 1H), 6.70 (s, 1H), 6.06 (s, 1H), 5.98 (s, 1H), 3.89 (s, 3H),
2.97e2.66 (m, 2H), 2.44e2.28 (m, 2H), 2.25e2.11 (m, 2H), 2.02e1.92
(m, 1H), 1.90e1.78 (m, 1H), 1.78e1.62 (m, 3H), 1.55e1.37 (m, 1H),
1.08 (s, 3H). 13C NMR (101 MHz, CDCl3):
d 151.9, 147.8, 144.9, 143.8,
143.8, 134.0, 133.2, 131.8, 129.6, 129.4, 123.3, 115.1, 115.0, 108.1, 108.1,
56.9, 56.2, 47.8, 44.5, 37.4, 35.5, 31.6, 29.0, 27.9, 26.9, 16.9. Eluent
70% EtOAc in hexane, Rf ¼ 0.50, yield 90 mg, 83%, product colorless
solid, mp 203e208 ꢁC decomp. HRMS calcd. for C24H27NO2 [M]þ:
361.2033. Found 361.2035.
6.5. (8S,9S,13S,14S)-2-methoxy-13-methyl-17-(pyridin-4-yl)-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol
(11c)
1H NMR (400 MHz, CDCl3):
d
8.53 (s, 2H), 7.31 (d, J ¼ 4.7 Hz, 2H),
6.79 (s, 1H), 6.67 (s, 1H), 6.22 (s, 1H), 3.87 (s, 3H), 2.97e2.67 (m, 2H),
2.42e2.26 (m, 3H), 2.23 (d, J ¼ 8.4 Hz, 1H), 2.21e2.10 (m, 1H),
1.98e1.89 (m, 1H), 1.86e1.75 (m, 1H), 1.73e1.60 (m, 3H), 1.53e1.39
(m, 1H), 1.26 (s, 1H), 1.09 (s, 3H) 13C NMR (101 MHz, CDCl3):
d 152.7,
Fig. 2. Analogs show no agonist activity towards the estrogen receptor. Results are
corrected for internal standard (Renilla luciferase activity) and presented as fraction of
2-methoxyestradiol. Bars show mean SE from 2 independent experiments based on
n ¼ 3e5 replicates. *p < 0.05 compared to DMSO control (ANOVA on ranks, Dunn's post
hoc test).
149.5, 145.0, 144.9, 143.8, 131.9, 131.8, 129.6, 121.4, 114.9, 108.0, 56.9,
56.3, 47.7, 44.5, 37.4, 35.4, 31.7, 29.0, 27.9, 26.9, 16.9. Eluent 70%
EtOAc in hexane, Rf ¼ 0.47, yield 89 mg, 81% product light yellow

E.J. Solum et al. / European Journal of Medicinal Chemistry 85 (2014) 391e398
395
solid, mp 212e217 ꢁC decomp. HRMS calcd. for C24H27NO2 [M]þ:
361.2042. Found 361.2038.
6.10. (8S,9S,13S,14S)-2-methoxy-13-methyl-17-(quinolin-5-yl)-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol
(11h)
6.6. (8S,9S,13S,14S)-17-(isoquinolin-4-yl)-2-methoxy-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol
(11d)
1H NMR (400 MHz, CDCl3):
d
8.83 (d, J ¼ 3.9 Hz, 1H), 8.48 (d,
J ¼ 8.5 Hz, 1H), 8.13 (d, J ¼ 8.5 Hz, 1H), 7.67 (t, J ¼ 7.8 Hz, 1H),
7.46e7.29 (m, 2H), 6.65 (s, 1H), 6.58 (s, 1H), 5.73 (s, 1H), 5.53 (s, 1H),
3.74 (s, 3H), 2.88e2.65 (m, 2H), 2.49e2.35 (m, 1H), 2.30e2.12 (m,
3H), 1.96e1.79 (m, 2H), 1.70e1.30 (m, 5H), 0.92 (s, 3H). 13C NMR
1H NMR (400 MHz, CDCl3):
d 9.19 (s, 1H), 8.39 (s, 1H), 8.07 (d,
J ¼ 8.4 Hz, 1H), 8.01e7.97 (m, 1H), 7.77e7.65 (m, 1H), 7.67e7.57 (m,
1H), 6.76 (s, 1H), 6.69 (s, 1H), 5.91 (dd, J ¼ 3.0, 1.6 Hz, 1H), 5.81 (s,
1H), 3.84 (s, 3H), 2.96e2.75 (m, 2H), 2.55e2.44 (m, 1H), 2.43e2.21
(m, 3H), 2.09e1.93 (m, 2H), 1.79e1.67 (m, 2H), 1.64e1.47 (m, 3H),
(101 MHz, CDCl3):
d 151.3, 148.4, 144.8, 143.8, 137.3, 136.7, 131.9,
131.5, 129.9, 129.6, 128.3, 126.8, 120.7, 114.8, 108.0, 56.9, 56.2, 50.1,
44.7, 37.8, 35.3, 32.2, 29.1, 28.1, 26.9, 16.7. Eluent 70% EtOAc in
hexane, Rf ¼ 0.54, yield 56 mg, 91%, product light yellow solid, mp
207e213 ꢁC decomp. HRMS calcd. for C28H29NO2 [M]þ: 411.2198.
Found 411.2201.
1.03 (s, 3H). 13C NMR (101 MHz, CDCl3):
d 151.2, 149.7, 144.8, 143.8,
141.5, 135.8,132.0,132.0,130.3,129.6,127.9, 127.2, 125.7,114.9, 108.1,
77.5, 77.4, 77.2, 76.8, 56.8, 56.2, 50.1, 44.7, 37.9, 35.3, 32.3, 29.1, 28.2,
26.9, 16.6. Eluent 70% EtOAc in hexane, Rf ¼ 0.58, yield 74 mg, 90%,
product light yellow solid, mp 123e126 ꢁC. HRMS calcd. for
6.11. (8S,9S,13S,14S,17S)-2-methoxy-13-methyl-17-(pyridin-3-yl)-
7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-
3-ol (13a)
C
28H29NO2 [M]þ: 411.2203. Found 411.2205.
6.7. (8S,9S,13S,14S)-17-(isoquinolin-5-yl)-2-methoxy-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol
(11e)
1H NMR (300 MHz, CDCl3)
d
8.49 (d, J ¼ 5.9 Hz, 2H), 7.61 (d,
J ¼ 7.6 Hz,1H), 7.29 (s, 1H), 6.77 (s, 1H), 6.65 (s, 1H), 5.68 (s, 1H), 3.85
(s, 3H), 2.79 (s, 3H), 2.46e1.82 (m, 6H), 1.68 (d, J ¼ 8.4 Hz, 1H), 1.47
1H NMR (400 MHz, CDCl3):
d
9.26 (s, 1H), 8.51 (d, J ¼ 5.9 Hz, 1H),
(d, J ¼ 10.9 Hz, 6H), 0.53 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 150.1,
7.94e7.85 (m, 2H), 7.64e7.47 (m, 2H), 6.76 (s, 1H), 6.69 (s, 1H), 5.85
(dd, J ¼ 3.1, 1.6 Hz, 1H), 3.84 (s, 3H), 2.99e2.76 (m, 2H), 2.61e2.46
(m, 1H), 2.42e2.22 (m, 3H), 2.11e1.90 (m, 2H), 1.80e1.66 (m, 2H),
147.2, 145.1, 144.0, 137.2, 136.7, 132.0, 129.9, 123.4, 115.1, 108.5, 56.5,
55.7, 54.9, 45.2, 44.6, 39.6, 37.9, 29.5, 28.3, 27.0, 26.4, 24.6, 13.2.
Eluent 70% EtOAc in hexane Rf ¼ 0.43, yield 55 mg, 74%, product
colorless solid, mp 197e201 ꢁC decomp. HRMS calcd. for C24H29NO2
[M]þ: 363.2198. Found 363.2192.
1.66e1.46 (m, 3H), 1.02 (s, 3H). 13C NMR (101 MHz, CDCl3):
d 152.6,
151.3, 144.9, 143.8, 142.8, 135.6, 135.2, 131.9, 131.0, 129.8, 129.6,
126.7, 126.5, 119.5, 115.0, 108.1, 56.8, 56.2, 50.0, 44.7, 37.8, 35.3, 32.2,
29.1, 28.1, 26.9, 16.7. Eluent 70% EtOAc in hexane, Rf ¼ 0.55, yield
105 mg, 85%, product light yellow solid, mp 212e217 ꢁC decomp.
HRMS calcd. for C28H29NO2 [M]þ: 411.2203. Found 411.2203.
6.12. (8S,9S,13S,14S,17S)-2-methoxy-13-methyl-17-(pyridin-4-yl)-
7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-
3-ol (13b)
6.8. (8S,9S,13S,14S)-17-(isoquinolin-6-yl)-2-methoxy-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol
(11f)
1H NMR (300 MHz, CDCl3)
d
8.53 (d, J ¼ 4.2 Hz, 2H), 7.21 (d,
J ¼ 5.2 Hz, 2H), 6.79 (s, 1H), 6.67 (s, 1H), 3.87 (s, 4H), 2.79 (t,
J ¼ 8.1 Hz, 3H), 2.36e1.83 (m, 6H), 1.73 (d, J ¼ 9.4 Hz, 1H), 1.60e1.35
(m, 6H), 0.53 (s, 3H). 13C NMR (75 MHz, CDCl3)
d 151.6, 148.5, 144.8,
1H NMR (400 MHz, CDCl3):
d
9.25 (s, 1H), 8.50 (d, J ¼ 5.8 Hz, 1H),
143.7, 131.7, 129.6, 124.4, 114.8, 108.2, 56.6, 56.2, 55.6, 45.2, 44.3,
39.3, 37.8, 29.2, 28.0, 26.7, 25.7, 24.3, 13.0. Eluent 70% EtOAc in
hexane Rf ¼ 0.39, yield 65 mg, 78%, product colorless solid,
mp 203e206 ꢁC decomp. HRMS calcd. for C24H29NO2 [M]þ:
363.2198. Found 363.2207.
7.97 (d, J ¼ 8.6 Hz, 1H), 7.84 (s, 1H), 7.81e7.64 (m, 2H), 6.80 (s, 1H),
6.68 (s, 1H), 6.25 (s, 1H), 5.72 (s, 1H), 3.87 (s, 3H), 2.96e2.74 (m, 2H),
2.48e2.27 (m, 4H), 2.29e2.14 (m, 1H), 2.01e1.93 (m, 1H), 1.91e1.80
(m, 1H), 1.77e1.63 (m, 3H), 1.55e1.38 (m, 1H), 1.17 (s, 3H). 13C NMR
(101 MHz, CDCl3):
d 154.1, 150.5, 144.8, 143.8, 140.8, 140.7, 136.8,
131.8, 131.6, 129.6, 128.0, 128.0, 127.4, 123.0, 121.6, 114.9, 108.0, 57.0,
56.3, 48.0, 44.5, 37.4, 35.7, 31.8, 29.0, 27.9, 27.0, 17.0. Eluent 70%
EtOAc in hexane, Rf ¼ 0.56, yield 56 mg, 91%, product light yellow
solid, mp 248e255 ꢁC decomp. HRMS calcd. for C28H29NO2 [M]þ:
411.2190. Found 411.2190.
6.13. (8S,9S,13S,14S,17S)-2-methoxy-13-methyl-17-(5,6,7,8-
tetrahydroisoquinolin-4-yl)-7,8,9,11,12,13,14,15,16,17-decahydro-
6H-cyclopenta[a]phenanthren-3-ol (13c)
1H NMR (400 MHz, CDCl3)
d 8.29 (s, 1H), 8.07 (s, 1H), 6.69 (s, 1H),
6.58 (s, 1H), 3.76 (s, 3H), 3.03 (t, J ¼ 9.8 Hz, 1H), 2.85e2.47 (m, 6H),
6.9. (8S,9S,13S,14S)-17-(isoquinolin-7-yl)-2-methoxy-13-methyl-
7,8,9,11,12,13,14,15-octahydro-6H-cyclopenta[a]phenanthren-3-ol
(11g)
2.30e2.09 (m, 2H), 2.06e1.96 (m, 2H), 1.95e1.59 (m, 6H), 1.59e1.22
(m, 7H), 0.64 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 147.1, 146.4, 146.3,
144.9, 143.8, 134.9, 132.7, 131.7, 129.6, 114.9, 108.3, 56.2, 55.8, 48.9,
46.1, 44.4, 39.3, 38.6, 29.2, 28.8, 28.0, 27.2, 26.8, 26.7, 24.6, 23.0, 22.1,
13.5. Eluent 70% EtOAc in hexane Rf ¼ 0.42, yield 639 mg, 63%,
product colorless solid, mp 127e130 ꢁC. HRMS calcd. for C28H35NO2
[M]þ: 417.2668. Found 417.2662.
1H NMR (400 MHz, CDCl3):
d
9.27 (s, 1H), 8.48 (d, J ¼ 5.6 Hz, 1H),
7.99 (s, 1H), 7.82 (q, J ¼ 8.6 Hz, 2H), 7.69 (d, J ¼ 5.7 Hz, 1H), 6.81 (s,
1H), 6.68 (s, 1H), 6.18 (s, 1H), 5.85 (s, 1H), 3.87 (s, 3H), 2.96e2.71 (m,
2H), 2.49e2.27 (m, 4H), 2.27e2.12 (m, 1H), 2.02e1.92 (m, 1H),
1.92e1.80 (m, 1H), 1.79e1.65 (m, 3H), 1.55e1.37 (m, 1H), 1.17 (s, 3H).
6.14. (8S,9S,13S,14S,17S)-17-(isoquinolin-5-yl)-2-methoxy-13-
methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]
phenanthren-3-ol (13d)
13C NMR (101 MHz, CDCl3):
d 154.0, 151.7, 144.9, 143.8, 141.1, 136.9,
135.3, 131.8, 131.2, 129.9, 129.6, 128.8, 126.5, 124.3, 120.9, 114.9,
108.1, 57.0, 56.3, 47.9, 44.5, 37.4, 35.8, 31.7, 29.0, 27.9, 27.0, 17.0.
Eluent 70% EtOAc in hexane, Rf ¼ 0.55, yield 54 mg, 88%, product
light yellow solid, mp 253e261 ꢁC decomp. HRMS calcd. for
1H NMR (400 MHz, CDCl3)
d
9.07 (s, 1H), 8.34 (d, J ¼ 6.2 Hz, 1H),
7.89 (d, J ¼ 6.3 Hz,1H), 7.68 (d, J ¼ 8.1 Hz,1H), 7.59 (d, J ¼ 7.0 Hz,1H),
C
28H29NO2 [M]þ: 411.2203. Found 411.2203.
7.43 (t, J ¼ 7.7 Hz, 1H), 6.57 (s, 1H), 6.49 (s, 1H), 3.63 (s, 3H), 3.56 (t,

396
E.J. Solum et al. / European Journal of Medicinal Chemistry 85 (2014) 391e398
J ¼ 9.6 Hz, 1H), 2.77e2.51 (m, 2H), 2.20e1.90 (m, 4H), 1.90e1.68 (m,
2H), 1.60e1.10 (m, 8H), 0.44 (s, 3H). 13C NMR (101 MHz, CDCl3)
RPMI medium supplemented with FCS (10% v/v). The test com-
pound was dissolved in DMSO. A solution of 100 l in medium was
added to 100 l of cell solution (40,000 cells/ml) in a 96-well
microtitre testplate (4 l of the compound solution diluted in me-
dium in order to reach decreasing concentrations). This series of
dilutions was continued to afford samples at different concentra-
tions leaving one cell solution free of drug acting as a control. The
plates were incubated at 37 ꢁC (5% CO2 in air) for 5 days. The plate
m
d
153.0, 144.9, 143.8, 141.9, 137.0, 136.3, 131.7, 129.9, 129.5, 129.2,
m
126.6,126.4,117.6,115.0,108.3, 56.2, 56.0, 49.9, 45.9, 44.4, 39.5, 38.7,
29.2, 28.4, 28.0, 26.9, 24.6, 13.4. Eluent 70% EtOAc in hexane
Rf ¼ 0.47, yield 49 mg 70%, product colorless solid. mp 207e210 ꢁC
m
decomp. HRMS calcd. for
413.2361.
C
28H31NO2 [M]þ: 413.2355. Found
was then removed from the incubator and 50 ml of a solution of MTT
6.15. (8S,9S,13S,14S,17S)-17-(isoquinolin-6-yl)-2-methoxy-13-
methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]
phenanthren-3-ol (13e)
(3 mg/ml in PBS) was added to each well. After incubation (37 ꢁC,
5% CO2 in air, 3 h) the medium was carefully removed from each
well by suction and the resulting formazan precipitate re-dissolved
in 200
wavelengths (l
m
l DMSO. The optical density of each well was read at two
540 and 690 nm) using a Titretek Multiscan MCC/
1H NMR (400 MHz, CDCl3)
d
9.24 (s, 1H), 8.49 (d, J ¼ 5.7 Hz, 1H),
7.93 (d, J ¼ 8.3 Hz, 1H), 7.78e7.63 (m, 2H), 7.56 (d, J ¼ 8.4 Hz, 1H),
6.78 (s, 1H), 6.66 (s, 1H), 3.84 (s, 3H), 3.05e2.96 (m, 1H), 2.90e2.73
(m, 2H), 2.37e2.19 (m, 3H), 2.17e2.05 (m, 1H), 2.05e1.92 (m, 2H),
1.81e1.71 (m, 1H), 1.66e1.20 (m, 7H), 0.56 (s, 3H). 13C NMR
340 plate reader. After processing and analysis through the appli-
cation of an ‘in-house’ software package, the results enabled the
calculation of the drug dose required to inhibit cell growth by 50%
(IC50 value), determined by graphical means as percentage of the
control growth.
(101 MHz, CDCl3)
d 151.3, 145.3, 144.8, 143.7, 141.6, 136.2, 131.8,
129.9, 129.7, 127.6, 127.1, 125.3, 121.0, 114.8, 108.3, 57.7, 56.2, 55.6,
45.5, 44.4, 39.4, 38.0, 29.2, 28.0, 26.8, 26.4, 24.4, 13.2. Eluent 70%
EtOAc in hexane Rf ¼ 0.49, yield 50 mg, 67%, product colorless solid,
mp 235e237 ꢁC. HRMS calcd. for C28H31NO2 [M]þ: 413.2355. Found
413.2347.
6.19. Inhibition of tubulin polymerization
The method applied was that described by Lawrence and co-
workers [22]. Tubulin was isolated from porcine brain and stored
at ꢀ78 ꢁC. Samples were prepared directly in a 96-well microtitre
plate that was pre-incubated at 4 ꢁC in the fridge for 30 min and
6.16. (8S,9S,13S,14S,17S)-17-(isoquinolin-7-yl)-2-methoxy-13-
methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]
phenanthren-3-ol (13f)
contained Mes buffer [128
MgCl2, distilled water, pH 6.6)], GTP (20
tubulin (50 l, 11 mg/ml in Mes buffer) and the candidate drug
(20 l, with appropriate concentration of sample in DMSO). The
tubulin/drug samples were immediately placed in a 96-well plate
reader, alongside blank samples containing Mes buffer (198 l) and
350 nm)
ml (0.1 M Mes, 1 mM EGTA, 0.5 mM
m
l, 5 mM in Mes buffer),
m
1H NMR (400 MHz, CDCl3)
d
9.12 (s, 1H), 8.31 (d, J ¼ 5.7 Hz, 1H),
m
7.71 (s, 1H), 7.66 (d, J ¼ 8.5 Hz, 1H), 7.61e7.47 (m, 2H), 6.61 (s, 1H),
6.49 (s, 1H), 3.68 (s, 3H), 2.84 (t, J ¼ 9.7 Hz, 1H), 2.72e2.55 (m, 2H),
2.25e2.02 (m, 3H), 2.02e1.90 (m, 1H), 1.89e1.70 (m, 2H), 1.65e1.50
(m, 1H), 1.47e1.08 (m, 7H), 0.39 (s, 3H). 13C NMR (101 MHz, CDCl3)
m
the analogs (10 ml, same concentration). The absorbance (l
was recorded at 25 ꢁC temperature for a period of 60 min, and the
results were compared to untreated controls to evaluate the rela-
tive degree of change in optical density.
d
150.8, 144.8, 143.7, 142.0, 139.7, 135.4, 133.9, 131.8, 129.7, 128.5,
126.8,125.9,121.3,114.8,108.3, 57.4, 56.3, 55.5, 45.3, 44.4, 39.4, 38.0,
29.2, 28.0, 26.8, 26.5, 24.4, 13.1. Eluent 70% EtOAc in hexane
Rf ¼ 0.50, yield 35 mg 76%, product colorless solid, mp 241e248 ꢁC
6.20. Inhibition of angiogenesis
decomp. HRMS calcd. for
413.2362.
C
28H31NO2 [M]þ: 413.2355. Found
Endothelial cell tube formation assay was modified from a
method previously described [23]. Matrigel (12.5 mg/ml) was
6.17. (8S,9S,13S,14S,17S)-2-methoxy-13-methyl-17-(1,2,3,4-
tetrahydroquinolin-5-yl)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-
cyclopenta[a]phenanthren-3-ol (13g)
thawed at 4 ꢁC, and 50
ml were quickly added to each well of a 96-
well plate and allowed to solidify for 10 min at 37 ꢁC. Once solid, the
wells were incubated for 30 min with BAECs (30,000 cells/well).
After adhesion of the cells, the medium was removed and replaced
by fresh medium supplemented with compounds with five
1H NMR (400 MHz, CDCl3)
d
6.95 (t, J ¼ 7.8 Hz, 1H), 6.78 (s, 1H),
6.74 (d, J ¼ 7.5 Hz, 1H), 6.65 (s, 1H), 6.42 (d, J ¼ 7.5 Hz, 1H), 3.85 (s,
3H), 3.34e3.21 (m, 2H), 3.13 (t, J ¼ 9.7 Hz, 1H), 2.95e2.71 (m, 4H),
2.31e2.15 (m, 2H), 2.05e1.81 (m, 6H), 1.73e1.62 (m, 1H), 1.58e1.20
different concentrations ranging from 10 mM to 0.001 mM and
incubated at 37 ꢁC for 18 h. The tubes of growth were visualized
with an inverted ZEISS microscope at a magnification of 10. The
length of the capillary network was quantified with a map scale
calculator (KURABO Angiogenesis Image Analysis Software).
(m, 7H), 0.69 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 144.6,144.3,143.5,
140.4, 132.1, 129.7, 125.4, 121.7, 118.7, 114.7, 113.0, 108.2, 56.2, 55.8,
50.5, 45.9, 44.5, 41.7, 39.3, 38.5, 29.2, 29.0, 28.1, 26.9, 24.8, 24.6, 22.8,
13.6. Eluent 70% EtOAc in hexane Rf ¼ 0.46, yield 18 mg 66%,
product colorless solid, mp.131e133 ꢁC. HRMS calcd. for C28H31NO2
[M]þ: 417.2668. Found 417.2685.
6.21. Estrogen agonist assay
Rat pheochromocytoma PC-12 cells were grown in DMEM
supplemented with 10% fetal calf serum, 5% horse serum, sodium
6.18. Cancer cell growth inhibition (cytotoxicity)
pyruvate (1 mM), penicillin (100 IE/ml), and streptomycin (100 mg/
ml). Cells were kept at 37 ꢁC in a humidified atmosphere with 5%
CO2 and plated with a density of 7 ꢂ 104 cells/ml in plastic dishes.
Two days after plating cells were exposed to medium containing
The method applied was that described by Edmondson and
coworkers [21] and is exemplified for the K562 cell line. K562 hu-
man chronic myelogeneous leukemia cells were cultivated in RMPI
medium, free of antibiotics and containing 2-mercaptoethanol
1 pM, 10 pM, 50 pM, 0.1 nM, 1 nM and 10
2-methoxyestradiol and the other analogs concentrations of 1 nM,
100 nM, 500 nM, 1 M, 2.5 M 5 M and 10 M were used. 0.1%
mM of 17-b-estradiol. For
(2
mM) and
L-glutamine (2 mM), supplemented with fetal calf
m
m
m
m
serum (FCS) (10% v/v). The cells were adjusted to a concentration
depending on their observed doubling time (ca. 40,000 cells/ml), in
DMSO served as solvent control. Cells were harvested for mea-
surement of luciferase after 24 h.

E.J. Solum et al. / European Journal of Medicinal Chemistry 85 (2014) 391e398
397
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J. Milbrandt, Washington University, ST-Louis, MO) were transfected
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Acknowledgment
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The School of Pharmacy and the Faculty of Mathematics and
Natural Sciences, University of Oslo, is gratefully acknowledged for
Ph.D.-scholarships to E. J. S. and I. B. S, respectively.
(c) A. Aoki, Y. Watanabe, D. Tanabe, A. Setiawan, M. Arai, M. Kobyashi, Cor-
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Appendix A. Supplementary data
Supplementary data related to this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.ejmech.2014.08.002.
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