Synthesis, cytotoxic effects and tubulin polymerization inhibition of 1,4-disubstituted 1,2,3-triazole analogs of 2-methoxyestradiol

Article abstract of DOI:10.1016/j.steroids.2014.05.020

Thirteen 1,4-disubstituted 1,2,3-triazole analogs of 2-methoxyestradiol were prepared and tested for their cytotoxic and tubulin polymerization inhibition effects. Two compounds, 11j and 11k, exhibited anti-proliferative effects at low micromolar concentr

Full text of DOI:10.1016/j.steroids.2014.05.020

Accepted Manuscript
Synthesis, cytotoxic effects and tubulin polymerization inhibition of 1,4-disub-
stituted 1,2,3-triazole analogs of 2-methoxyestradiol
Eirik Johansson Solum, Anders Vik, Trond Vidar Hansen
PII:
S0039-128X(14)00133-0
http://dx.doi.org/10.1016/j.steroids.2014.05.020
STE 7582
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 March 2014
16 May 2014
25 May 2014
Please cite this article as: Solum, E.J., Vik, A., Hansen, T.V., Synthesis, cytotoxic effects and tubulin polymerization
inhibition of 1,4-disubstituted 1,2,3-triazole analogs of 2-methoxyestradiol, Steroids (2014), doi: http://dx.doi.org/
10.1016/j.steroids.2014.05.020
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Graphical Abstract
Synthesis, cytotoxic effects and tubulin polymerization Leave this area blank for abstract info.
inhibition of 1,4-disubstituted 1,2,3-triazole analogs of
2-methoxyestradiol
Eirik Johansson Solum, Anders Vik, Trond Vidar Hansen
OCF3
N
N
N
OH
H
IC50 = 0.7 µM (cytotoxicity)
O
IC50 = 5.9 µM (tubulin inhibition)
H
H
HO

Synthesis, cytotoxic effects and tubulin polymerization inhibition of 1,4-
disubstituted 1,2,3-triazole analogs of 2-methoxyestradiol
Eirik Johansson Solum, Anders Vik, Trond Vidar Hansen*
School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo,
PO Box 1068 Blindern, N-0316 Oslo, Norway.
E-mail: t.v.hansen@farmasi.uio.no
Phone: +47 22857450
Fax: + 47 22855947
Abstract:
Thirteen 1,4-disubstituted 1,2,3-triazole analogs of 2-methoxyestradiol were prepared
and tested for their cytotoxic and tubulin polymerization inhibition effects. Two
compounds, 11j and 11k, exhibited anti-proliferative effects at low micromolar
concentrations. The two analogs 11j and 11k also inhibited tubulin assembly with
IC50 values of 8.1 and 5.9 µM, respectively.
Keywords: estrogens; 2-methoxyestradiol, 1,2,3-triazoles; cytotoxicity; tubulin
inhibition; anti-cancer.
1. Introduction
Several steroids have been reported to exhibit anti-cancer effects [1] and some
examples are depicted in Figure 1. One such example is 2-methoxyestradiol (2-ME,
1), the endogen metabolite of 17β-estradiol. The estrogen 1 is oxidized by
cytochrome P450 (CYP1A2 and CYP3A) to 2-hydroxyestradiol, followed by 2-O-
methylation by catechol O-methyltransferase (COMT) [2]. It was previously believed
that 1 was inactive. However, in 1994 D´Amato, Folkman and co-workers
demonstrated that 2-ME (1) inhibits tubulin polymerization by interfering with the
colcichine binding-site [3]. It has been reported that these effects lead to inhibition of
angiogenesis in several cancer cell lines [4]. 2-ME (1) also showed potent in vivo anti-
angiogenetic effects leading to inhibition of proliferation, migration and invasion of
several cancer cell lines [5], [6]. In addition, it was also reported that 2-ME (1) down
regulates hypoxia-inducible factor-1 α (HIF-1 α) resulting in potent cytotoxic effects
in prostate and breast cancer cells [7]. These observations were later related to
intrinsic and extrinsic apoptotic pathways [8] that were confirmed in vivo [9]. In
addition, no toxic effects of 2-methoxyestradiol (1) have been observed in phase I/II
clinical trials [10].
The aforementioned activities have spurred a great interest in using 2-ME (1)
as a lead compound for the development of new anti-cancer agents [11]. The
compounds 2-4 depicted in Figure 1 are examples of such analogs.
[Insert Figure 1 Here]
Fig. 1. Some examples of steroids exhibiting anti-cancer effects.
Most analogs of 1 have been prepared with the aim of reducing the metabolic
oxidation of the hydroxyl group in the C-17 position, but also avoiding secondary

conjugation reactions at this and at the phenolic position [11], [12]. In connection with
our interest in the synthesis of potential new anti-cancer agents, several 1,2,3-triazole
analogs displayed tubulin polymerization inhibition, cytotoxic and anti-vascular
effects in the high nanomolar range [13a], [13b], [13c], [13d]. We wanted to
investigate if the introduction of a 1,4-disubstituted 1,2,3-triazole in the C-17 position
of 2-methoxyestradiol (1) would induce cell death by inhibiting tubulin assembly.
Such analogs should also reduce metabolic transformations. Modifications in this
position have been reported to afford compounds, such as 3 and 4, with anti-cancer
activities [1a]. The regioselective copper(I) catalyzed cycloaddition reaction between
terminal alkynes and azides affords 1,4-disubstituted-1,2,3-triazoles in high yields
[14]. This reaction has found many applications in medicinal chemistry [15],
including modifications of natural products [16]. Herein, the synthesis and the
biological evaluations of 13 1,4-disubstituted-1,2,3-triazoles analogs of the steroid 1
are reported.
2. Experimental
2.1. General
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
performed on silica gel 60 (40-63 µm) produced by Merck. NMR spectra were
recorded on a Bruker Avance DPX-300 MHz, DPX-400 MHz or DPX-600 MHz
1
spectrometer for H NMR, and 75 MHz, 101 MHz or 151 MHz for 13C NMR.
Coupling constants (J) are reported in hertz, and chemical shifts are reported in parts
1
per million relative to CDCl3 (7.26 ppm for H 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-2TM. The HPLC analyses were performed on an Agilent Technologies 1200
Series instrument with an Eclipse XDB-C18 (5 mm 4.6 x 150 mm) column. The
chemical purity of the products 11a-11k and 12a-12b was determined by HPLC, see
Supporting information for details.
2.2. 2-Methoxyestrone
2-Methoxyestradiol (1) (2.42 g, 8.0 mmol) was placed in a flame dried 250 mL two-
necked round-bottomed flask equipped with a 5 mL flame dried Dean-Stark trap and a
reflux condenser. Toluene (80 mL) was added to dissolve the starting material.
Aluminum iso-propoxide (8.17 g, 40.0 mmol) and cyclohexanone (33.1 mL, 320.0
mmol) were added, and the reaction mixture was heated at reflux for 20 h. The
reaction mixture was allowed to cool to room temperature. Then water (100 mL) and
1 M HCl (45 mL) were added. The organic material was extracted with ethyl acetate
(3 x 150 mL). The aqueous emulsion was acidified with 1 M HCl (50 mL) until the
emulsion separated. The aqueous layer was extracted once more with ethyl acetate
(100 mL). The combined organic extracts were dried (MgSO4) and evaporated in
vacuo. The residue was triturated with hexane to give the desired product in 88%
yield (2.11 g). Mp 188-190 °C; 1H NMR (400 MHz, CDCl3): δ 6.79 (s, 1 H), 6.66 (s,
1H), 5.48 (s, 1H), 3.86 (s, 3H), 2.88 – 2.72 (m, 2H), 2.51 (dd, J = 18.9, 8.6 Hz, 1H),
2.42 – 2.32 (m, 1H), 2.30 – 2.21 (m, 1H), 2.20 – 1.84 (m, 4H), 1.72 – 1.28 (m, 6H),
0.92 (s, 3H); 13C NMR (101MHz, CDCl3) δ 220.9, 144.7, 143.7, 131.1, 129.3, 114.7,
108.1, 56.1, 50.4, 48.0, 44.3, 38.4, 35.9, 31.6, 28.9, 26.6, 26.2, 21.6, 13.9.

2.3. 3-Tert-butyldimethylsiloxy-2-methoxyestrone (5)
2-Methoxyestrone (1.51 g, 5.0 mmol) was placed in a 100 mL round bottomed flask
and dissolved in dry DMF (25 mL). Imidazole (0.85 g, 12.5 mmol) and TBSCl (1.13
g, 7.5 mmol) were added. The reaction mixture was stirred at room temperature under
argon atmosphere for 5 h. Saturated aqueous NaCl (20 mL) was added, and the
mixture was extracted with ethyl acetate (3 x 20 mL). The combined organic extracts
were washed with brine (10 mL), aqueous AcOH (10%, 10 mL) and aqueous
NaHCO3 (10 mL) before it was dried (MgSO4) and evaporated in vacuo. The product
was purified by flash chromatography (silica gel, 20% ethyl acetate in hexane, Rf =
1
0.43) to give the pure product in 92% yield (1.91 g). Mp 163-166 °C; H NMR (300
MHz, CDCl3): δ 6.77 (s, 1H), 6.56 (s, 1H), 3.77 (s, 3H), 2.83 – 2.74 (m, 2H), 2.50 (dd,
J = 18.3, 8.4 Hz, 1H), 2.40 – 1.86 (m, 5H), 1.70 – 1.32 (m, 6H), 0.99 (s, 9H), 0.92 (s,
3H), 0.15 (s, 6H) ; 13C NMR (101 MHz, CDCl3) δ 221.1, 149.0, 143.3, 132.7, 128.9,
121.2, 110.0, 56.0, 50.7, 48.2, 44.5, 38.5, 36.1, 31.8, 28.9, 26.9, 26.3, 25.9, 21.8, 18.6,
14.1, -4.4, -4.3.
2.4. 3-Tert-butyldimethylsiloxy-2-methoxyethynylestradiol (6)
The TBS-protected estrone 5 (829 mg, 2.0 mmol) was dissolved in THF (20 mL) and
cooled to 0 °C. A 0.5 M solution of ethynylmagnesiumbromide in THF (20.0 mL, 6.7
mmol) was added dropwise to the mixture over 15 min. The reaction mixture was
stirred at 0 °C for additional 30 min., then brought to room temperature, and stirred
until no sign of starting material (TLC) (approx. 4 h.). Upon completion saturated
aqueous NH4Cl (2 M, 20 mL) was added, and the mixture was extracted with ethyl
acetate (3 x 30 mL). The combined organic phases were washed with brine, dried
(MgSO4), and evaporated in vacuo. The product was purified by flash
chromatography (20% ethylacetate in hexane Rf =0.57) to give the pure product in 88%
yield. 1H NMR (400 MHz, CDCl3) δ 6.79 (s, 1H), 6.56 (s, 1H), 3.78 (s, 3H), 2.75 (dd,
J = 11.1, 5.7 Hz, 2H), 2.60 (s, 1H), 2.39 – 2.30 (m, 2H), 2.28 – 2.16 (m, 1H), 2.09 –
1.68 (m, 7H), 1.56 – 1.24 (m, 4H), 1.00 (s, 9H), 0.91 (s, 3H), 0.17 (s, 6H). 13C NMR
(101 MHz, CDCl3) δ 148.7, 143.0, 133.1, 129.0, 121.0, 110.1, 87.6, 79.9, 74.0, 55.9,
49.5, 47.2, 43.9, 39.4, 39.1, 32.8, 28.9, 27.4, 26.6, 25.8, 22.8, 18.5, 12.8, -4.5, -4.6.
2.5. General procedure for the Cu(I) mediated 1,3-dipolar cycloaddition
TBS protected ethynylestradiol 6 (0.3 – 0.6 mmol) was placed in a 25 mL round-
bottomed flask and dissolved in t-BuOH (4 – 6 mL) and water (2 – 3 mL). Copper(II)
sulfate (0.1 equiv.), sodium ascorbate (0.2 equiv.) and the azide (7a-7k) or (8a-8b)
(1.2 equiv.) were added. The reaction mixture was heated to 50 °C and stirred for 24
hours. Saturated aqueous NH4Cl (20 mL) was added, and the mixture was extracted
with ethyl acetate (3 x 20 mL). The combined organic extract were washed with brine
(10 mL) and dried (MgSO4). The solvent was removed in vacuo. The crude product
was purified by flash chromatography (silica gel, 20% ethyl acetate in hexane) to give
the pure product in 47 - 86% yield.
2.6.
yl)diol (9a)
3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-phenyl-1H-1,2,3-triazol-4-
1
Light yellow oil (231 mg, 83%), eluent: 20% EtOAc in hexane, Rf = 0.27. H NMR
(400 MHz, CDCl3) δ 7.92 (s, 1H), 7.76 (d, J = 7.7 Hz, 2H), 7.52 (t, J = 7.8 Hz, 2H),
7.44 (d, J = 7.4 Hz, 1H), 6.68 (s, 1H), 6.53 (s, 1H), 3.72 (s, 3H), 2.94 (s, 1H), 2.80 –
2.64 (m, 2H), 2.60 – 2.41 (m, 1H), 2.24 – 2.09 (m, 2H), 2.06 – 1.85 (m, 3H), 1.78 –
1.41 (m, 5H), 1.42 – 1.24 (m, 1H), 1.10 (s, 3H), 0.99 (s, 9H), 0.86 – 0.74 (m, 1H),

0.14 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 154.6, 148.7, 143.0, 137.2, 133.2, 129.8,
129.1, 128.6, 121.0, 120.5, 119.5, 110.1, 82.6, 56.0, 48.7, 47.6, 43.8, 39.5, 38.2, 33.2,
29.0, 27.6, 26.5, 25.9, 23.6, 18.5, 14.5, -4.5, -4.5.
2.7.
triazol-4-yl)diol (9b)
3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-methylphenyl)-1H-1,2,3-
1
Light yellow oil (103 mg, 61%), eluent: 20% EtOAc in hexane, Rf = 0.27. H NMR
(400 MHz, CDCl3) δ 7.84 (s, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H),
6.68 (s, 1H), 6.53 (s, 1H), 3.72 (s, 3H), 2.82 – 2.69 (m, 2H), 2.43 (s, 4H), 2.24 – 2.10
(m, 2H), 2.09 – 1.82 (m, 3H), 1.76 – 1.27 (m, 6H), 1.09 (s, 3H), 0.98 (s, 9H), 0.84 –
0.73 (m, 1H), 0.14 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 154.3, 148.8, 143.1, 139.0,
134.9, 133.2, 130.4, 129.2, 121.1, 120.5, 119.5, 110.1, 82.6, 56.0, 48.8, 47.6, 43.9,
39.6, 38.2, 33.2, 29.1, 27.7, 26.6, 25.9, 23.6, 21.3, 18.6, 14.5, -4.4, -4.4.
2.8. General procedure for 7c and 7d
Sodium azide (1.2 mmol, 76 mg) and copper(II) sulfate (0.1 mmol, 16 mg) were
added to a 25 mL round-bottomed flask and dissolved in methanol (3 mL). Then 4-
tert-butylphenylboronicacid (1 mmol, 0.178 g) or 4-hydroxyphenylboronicacid (1
mmol, 138 mg) was added. The reaction mixture was heated to 40 °C, and stirred
until no sign of starting material (TLC), ca. 2 h. Upon completion the reaction mixture
was cooled to room temperature, and the solvent was evaporated and the crude
product was used in the corresponding cycloaddition reactions, with no further
purification.
2.9. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-(tert-butyl)phenyl)-1H-1,2,3-
triazol-4-yl)diol (9c)
Light yellow oil (181 mg, 49% over two steps), eluent: 20% EtOAc in hexane, Rf =
1
0.30. H NMR (600 MHz, CDCl3) δ 7.87 (s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.53 (d, J
= 8.8 Hz, 2H), 6.68 (s, 1H), 6.53 (s, 1H), 3.72 (s, 3H), 2.81 – 2.65 (m, 2H), 2.56 –
2.44 (m, 1H), 2.23 – 2.10 (m, 2H), 2.04 – 1.95 (m, 2H), 1.95 – 1.87 (m, 1H), 1.72 –
1.43 (m, 5H), 1.36 (s, 9H), 1.10 (s, 3H), 0.98 (s, 9H), 0.89 (t, J = 7.0 Hz, 1H), 0.80 (td,
13
J = 12.9, 4.0 Hz, 1H), 0.14 (d, J = 2.0 Hz, 6H). C NMR (151 MHz, CDCl3) δ 154.3,
152.1, 148.7, 143.0, 134.8, 133.2, 129.1, 126.7, 121.0, 120.3, 119.5, 110.0, 82.6, 56.0,
47.5, 43.8, 39.5, 38.2, 34.9, 33.2, 31.4, 29.0, 27.6, 26.5, 25.9, 23.6, 22.8, 18.5, 14.5, -
4.4, -4.5.
2.10. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-hydroxyphenyl)-1H-1,2,3-
triazol-4-yl)diol (9d)
Red oil (162 mg, 47% over two steps), eluent: 20% EtOAc in hexane, Rf = 0.30. H
1
NMR (600 MHz, CDCl3) δ 7.83 (s, 1H), 7.49 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.7 Hz,
2H), 6.66 (s, 1H), 6.52 (s, 1H), 3.71 (s, 3H), 2.82 – 2.60 (m, 2H), 2.56 – 2.39 (m, 1H),
2.24 – 2.16 (m, 1H), 2.18 – 2.10 (m, 1H), 2.02 – 1.93 (m, 2H), 1.92 – 1.85 (m, 1H),
1.72 (d, J = 12.4 Hz, 1H), 1.64 – 1.55 (m, 2H), 1.54 – 1.41 (m, 2H), 1.34 – 1.24 (m,
1H), 1.10 (s, 3H), 0.97 (s, 9H), 0.84 – 0.70 (m, 1H), 0.13 (s, 6H). 13C NMR (151 MHz,
CDCl3) δ 157.2, 153.9, 148.6, 142.9, 133.0, 129.7, 129.0, 122.3, 121.0, 120.2, 116.6,
110.0, 82.6, 56.0, 48.7, 47.5, 43.7, 39.3, 38.0, 33.1, 28.9, 27.4, 26.4, 25.8, 18.4, 14.4, -
4.6.
2.11. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-flourophenyl)-1H-1,2,3-
triazol-4-yl)diol (9e)

1
Light yellow oil (237 mg, 81%), eluent: 20% EtOAc in hexane, Rf = 0.28. H NMR
(400 MHz, CDCl3) δ 7.87 (s, 1H), 7.80 – 7.63 (m, 2H), 7.25 – 7.13 (m, 2H), 6.68 (s,
1H), 6.53 (s, 1H), 3.72 (s, 3H), 2.85 – 2.66 (m, 3H), 2.57 – 2.43 (m, 1H), 2.23 – 2.10
(m, 2H), 2.05 – 1.86 (m, 3H), 1.75 – 1.27 (m, 6H), 1.09 (s, 3H), 0.98 (s, 9H), 0.86 –
0.71 (m, 1H), 0.14 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 163.7, 161.2, 154.8, 148.7,
143.0, 133.5 (d, J = 3.1 Hz), 133.2, 129.1, 122.5, 122.4, 121.1, 119.7, 116.9, 116.7,
110.1, 82.6, 56.0, 48.7, 47.5, 43.8, 39.5, 38.2, 33.2, 29.0, 27.6, 26.5, 25.9, 23.6, 18.5,
14.4, -4.5, -4.5.
2.13.
triazol-4-yl)diol (9f)
3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-chlorophenyl)-1H-1,2,3-
1
Light yellow oil (233 mg, 78%), eluent: 20% EtOAc in hexane, Rf = 0.28. H NMR
(400 MHz, CDCl3) δ 7.88 (s, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.49 (d, J = 8.9 Hz, 2H),
6.67 (s, 1H), 6.53 (s, 1H), 3.72 (s, 3H), 2.81 – 2.66 (m, 2H), 2.65 – 2.54 (m, 1H), 2.50
(ddd, J = 14.5, 9.5, 5.6 Hz, 1H), 2.23 – 2.08 (m, 2H), 2.04 – 1.95 (m, 2H), 1.96 – 1.86
(m, 1H), 1.72 – 1.24 (m, 6H), 1.09 (s, 3H), 0.98 (s, 9H), 0.84 – 0.73 (m, 1H), 0.14 (d,
J = 1.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 154.9, 148.8, 143.1, 135.7, 134.6,
133.2, 130.0, 129.2, 121.7, 121.1, 119.4, 110.1, 82.6, 56.0, 48.8, 47.6, 43.8, 39.5, 38.3,
33.2, 29.0, 27.6, 26.5, 25.9, 23.6, 18.5, 14.5, -4.4, -4.5.
2.14.
triazol-4-yl)diol (9g)
3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-bromophenyl)-1H-1,2,3-
1
Light yellow oil (254 mg, 80%), eluent: 20% EtOAc in hexane, Rf = 0.28. H NMR
(300 MHz, CDCl3) δ 7.89 (s, 1H), 7.64 (s, 4H), 6.67 (s, 1H), 6.53 (s, 1H), 3.72 (s, 3H),
2.83 – 2.58 (m, 2H), 2.53 – 2.38 (m, 1H), 2.26 – 2.07 (m, 3H), 2.08 – 1.84 (m, 3H),
1.76 – 1.23 (m, 8H), 1.09 (s, 3H), 0.98 (s, 9H), 0.14 (s, 6H). 13C NMR (75 MHz,
CDCl3) δ 154.9, 148.7, 143.0, 136.1, 133.1, 133.00, 129.1, 122.3, 121.9, 121.1, 119.3,
110.0, 82.6, 56.0, 48.7, 47.6, 43.8, 39.5, 38.2, 33.2, 29.0, 27.6, 26.5, 25.9, 23.6, 22.8,
18.5, 14.4, -4.5, -4.5. Eluent: 20% EtOAc in hexane, Rf=0.28, yield (254 mg, 80%).
2.15. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-methoxyphenyl)-1H-1,2,3-
triazol-4-yl)diol (9h)
Light yellow oil (239 mg, 81%), eluent: 20% EtOAc in hexane, Rf = 0.23. H NMR
1
(400 MHz, CDCl3) δ 7.80 (s, 1H), 7.65 (d, J = 9.0 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H),
6.67 (s, 1H), 6.52 (s, 1H), 3.86 (s, 3H), 3.72 (s, 3H), 2.86 – 2.66 (m, 2H), 2.56 – 2.39
(m, 2H), 2.31 – 2.07 (m, 2H), 2.04 – 1.84 (m, 3H), 1.74 – 1.22 (m, 6H), 1.09 (s, 3H),
0.98 (s, 9H), 0.81 (td, J = 12.7, 3.8 Hz, 1H), 0.13 (s, 6H). 13C NMR (101 MHz, CDCl3)
δ 159.9, 154.3, 148.8, 143.0, 133.2, 130.7, 129.2, 122.2, 121.1, 119.6, 114.9, 110.1,
82.6, 56.0, 55.8, 48.8, 47.6, 43.8, 39.6, 38.2, 33.2, 29.0, 27.6, 26.6, 25.9, 23.6, 18.5,
14.5, -4.4, -4.5.
2.16. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazol-4-yl)diol (9i)
Light yellow oil (224 mg, 69%), eluent: 20% EtOAc in hexane, Rf = 0.17. H NMR
1
(400 MHz, CDCl3) δ 7.84 (s, 1H), 6.97 (s, 2H), 6.67 (s, 1H), 6.53 (s, 1H), 3.93 (s, 6H),
3.89 (s, 3H), 3.72 (s, 3H), 2.81 – 2.63 (m, 2H), 2.61 – 2.40 (m, 2H), 2.25 – 2.09 (m,
2H), 2.09 – 1.86 (m, 3H), 1.78 – 1.20 (m, 6H), 1.09 (s, 3H), 0.98 (s, 9H), 0.83 (dt, J =
21.7, 10.4 Hz, 1H), 0.14 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 154.5, 154.1, 148.8,
143.1, 138.5, 133.2, 133.1, 129.2, 121.1, 119.8, 110.1, 98.6, 82.7, 61.2, 56.6, 56.1,
48.8, 47.6, 43.8, 39.6, 38.3, 33.2, 29.0, 27.7, 26.6, 25.9, 23.7, 18.6, 14.5, -4.4, -4.5.

2.17. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-(trifluoromethyl)phenyl)-
1H-1,2,3-triazol-4-yl)diol (9j)
Light yellow oil (228 mg, 73%), eluent: 20% EtOAc in hexane, Rf = 0.26. H NMR
1
(300 MHz, CDCl3) δ 7.97 (s, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H),
6.67 (s, 1H), 6.53 (s, 1H), 3.72 (s, 3H), 2.82 – 2.67 (m, 2H), 2.61 – 2.42 (m, 1H), 2.24
– 2.07 (m, 3H), 2.06 – 1.83 (m, 3H), 1.78 – 1.23 (m, 8H), 1.10 (s, 3H), 0.98 (s, 9H),
13
0.14 (s, 6H). C NMR (75 MHz, CDCl3) δ 155.2, 148.8, 143.1, 133.1, 129.2, 127.3,
127.2, 121.1, 120.4, 119.3, 110.1, 82.7, 56.0, 48.8, 47.6, 43.8, 39.6, 38.4, 33.2, 31.7,
29.0, 27.6, 26.5, 25.9, 23.7, 22.8, 18.56, 14.5, -4.4, -4.4.
2.18. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-(trifluoromethoxyl)phenyl)-
1H-1,2,3-triazol-4-yl)diol (9k)
Light yellow oil (216 mg, 67%), eluent: 20% EtOAc in hexane, Rf = 0.23. H NMR
1
(300 MHz, CDCl3) δ 7.96 (s, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.81 (d, J = 8.5 Hz, 2H),
6.67 (s, 1H), 6.53 (s, 1H), 3.72 (s, 3H), 2.81 – 2.66 (m, 2H), 2.60 – 2.43 (m, 1H), 2.25
– 2.07 (m, 2H), 2.07 – 1.87 (m, 3H), 1.78 – 1.22 (m, 8H), 1.10 (s, 3H), 0.98 (s, 9H),
13
0.22 – 0.03 (m, 6H). C NMR (75 MHz, CDCl3) δ 155.2, 148.8, 143.1, 133.1, 129.2,
127.3, 127.2, 121.1, 120.5, 119.3, 110.1, 82.7, 56.1, 48.8, 47.6, 43.8, 39.6, 38.4, 33.2,
29.0, 27.6, 26.5, 26.4, 25.9, 23.7, 22.8, 18.6, 14.5, -4.4, -4.4.
2.19. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(4-methoxybenzyl)-1H-1,2,3-
triazol-4-yl)diol (10a)
Light yellow oil (184 mg, 61%), eluent: 20% EtOAc in hexane, Rf = 0.19. H NMR
1
(600 MHz, CDCl3) δ 7.30 (s, 1H), 7.22 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.6 Hz, 2H),
6.68 (s, 1H), 6.52 (s, 1H), 5.49 (d, J = 14.8 Hz, 1H), 5.44 (d, J = 14.8 Hz, 1H), 3.80 (s,
3H), 3.73 (s, 3H), 2.80 – 2.63 (m, 2H), 2.37 – 2.28 (m, 1H), 2.15 – 2.05 (m, 2H), 1.98
– 1.82 (m, 3H), 1.61 (dt, J = 12.4, 2.9 Hz, 1H), 1.55 – 1.38 (m, 4H), 1.33 – 1.23 (m,
1H), 1.03 (s, 3H), 0.98 (s, 9H), 0.65 (td, J = 12.9, 4.0 Hz, 1H), 0.14 (d, J = 1.2 Hz,
6H).13C NMR (151 MHz, CDCl3) δ 160.0, 154.0, 148.8, 143.0, 133.2, 129.7, 129.1,
126.7, 121.1, 121.0, 114.6, 110.0, 82.4, 56.0, 55.5, 53.9, 48.7, 47.5, 43.9, 39.5, 38.0,
33.2, 29.0, 27.6, 26.5, 25.9, 23.5, 18.6, 14.4, -4.4, -4.5.
2.20. 3-Tert-butyldimethylsiloxy-2-methoxyestra-17-(1-(3,4,5-trimethoxybenzyl)-1H-
1,2,3-triazol-4-yl)diol (10b)
Light yellow oil (214 mg, 64%), eluent: 20% EtOAc in hexane, Rf = 0.16. H NMR
1
(600 MHz, CDCl3) δ 7.38 (s, 1H), 6.65 (s, 1H), 6.52 (s, 1H), 6.46 (s, 2H), 5.49 (d, J =
14.9 Hz, 1H), 5.41 (d, J = 14.9 Hz, 1H), 3.82 (s, 3H), 3.81 (s, 6H), 3.72 (s, 3H), 2.80
– 2.65 (m, 2H), 2.42 – 2.33 (m, 1H), 2.12 – 2.02 (m, 2H), 1.96 – 1.81 (m, 3H), 1.63 –
1.58 (m, 2H), 1.57 – 1.38 (m, 4H), 1.33 – 1.24 (m, 1H), 1.04 (s, 3H), 0.97 (s, 9H),
0.66 (td, J = 12.8, 3.9 Hz, 1H), 0.13 (d, J = 1.4 Hz, 6H). 13C NMR (151 MHz, CDCl3)
δ 154.2, 153.8, 148.7, 143.0, 138.3, 133.1, 130.4, 129.1, 121.3, 121.1, 110.0, 105.1,
82.5, 61.0, 56.3, 56.0, 54.5, 48.7, 47.5, 43.9, 39.5, 38.1, 33.2, 29.0, 27.6, 26.5, 25.9,
23.5, 18.5, 14.4, -4.5, -4.5
2.21. General procedure for the deprotection of the TBS group
The TBS protected steroid phenol (9a-9k or 10a-10b) (0.3-0.6 mmol, 1 equiv.) was
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 dropvise.
The reaction mixture was stirred at room temperature (16-18 h.). Upon completion the
reaction mixture was poured into saturated aqueous NaHCO3 (10 mL) and extracted
with ethyl acetate (4 x 5 mL). The combined organic extract was dried (MgSO4) and

the solvent evaporated in vacuo. The residue was purified by chromatography (silica
gel, 20 – 70% ethyl acetate in hexane) to give the pure product.
2.22. 2-Methoxyestra-17-(1-phenyl-1H-1,2,3-triazol-4-yl)diol (11a)
Light yellow solid (151 mg, 83%), mp. 141-145 °C, eluent: 20% - 50% EtOAc in
1
hexane, Rf = 0.16. H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 7.69 – 7.50 (m, 2H),
7.36 (t, J = 7.7 Hz, 2H), 7.27 (t, J = 7.4 Hz, 1H), 6.53 (s, 1H), 6.46 (s, 1H), 3.64 (s,
3H), 2.74 – 2.51 (m, 2H), 2.43 – 2.22 (m, 1H), 2.12 – 1.92 (m, 2H), 1.95 – 1.68 (m,
3H), 1.59 – 1.08 (m, 6H), 0.94 (s, 3H), 0.71 – 0.51 (m, 1H). 13C NMR (101 MHz,
CDCl3) δ 154.5, 144.7, 143.5, 137.2, 131.8, 129.9, 129.6, 128.8, 120.5, 119.5, 114.7,
108.2, 82.5, 56.2, 48.7, 47.6, 43.7, 39.6, 38.2, 33.1, 29.1, 27.5, 26.7, 23.6, 14.4.
HRMS (EI+) calcd. for C27H31N3O3 [M]•+: 445.2365. Found 445.2362.
2.23. 2-Methoxyestra-17-(1-(4-methylphenyl)-1H-1,2,3-triazol-4-yl)diol (11b)
Light yellow solid (59 mg, 72%), mp. 142-144 °C, eluent: 20% - 50% EtOAc in
1
hexane, Rf = 0.16. H NMR (600 MHz, CDCl3) δ 7.78 (s, 1H), 7.56 (d, J = 8.4 Hz,
2H), 7.23 (d, J = 8.1 Hz, 2H), 6.61 (s, 1H), 6.54 (s, 1H), 3.72 (s, 3H), 2.76 – 2.58 (m,
2H), 2.44 – 2.37 (m, 1H), 2.34 (s, 3H), 2.14 – 2.01 (m, 2H), 1.94 – 1.87 (m, 2H), 1.86
– 1.79 (m, 1H), 1.64 – 1.35 (m, 5H), 1.29 – 1.21 (m, 1H), 1.01 (s, 3H), 0.74 (td, J =
12.9, 4.0 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 154.2, 144.6, 143.4, 138.8, 134.8,
131.7, 130.2, 129.5, 120.3, 119.4, 114.6, 108.1, 82.4, 56.0, 48.6, 47.4, 43.6, 39.5, 38.0,
33.0, 29.0, 27.4, 26.6, 23.5, 21.1, 14.3. HRMS (EI+) calcd. for C28H33N3O3 [M]•+:
459.2522. Found 459.2531.
2.24. 2-Methoxyestra-17-(1-(4-(tert-butyl)phenyl)-1H-1,2,3-triazol-4-yl)diol (11c)
Light yellow solid (110 mg, 76%), mp. 131-133 °C, eluent: 20% - 50% EtOAc in
hexane, Rf = 0.12.1H NMR (600 MHz, CDCl3) δ 7.87 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H),
7.54 (d, J = 8.8 Hz, 2H), 6.69 (s, 1H), 6.62 (s, 1H), 3.81 (s, 3H), 2.85 – 2.69 (m, 2H),
2.54 – 2.40 (m, 1H), 2.24 – 2.07 (m, 2H), 2.02 – 1.94 (m, 2H), 1.95 – 1.86 (m, 1H),
1.75 – 1.67 (m, 1H), 1.68 – 1.54 (m, 2H), 1.55 – 1.41 (m, 2H), 1.36 (s, 9H), 1.34 –
1.25 (m, 2H), 1.09 (s, 2H), 0.80 (td, J = 12.9, 4.0 Hz, 1H). 13C NMR (151 MHz,
CDCl3) δ 154.2, 152.3, 144.7, 143.6, 134.7, 131.8, 129.6, 126.8, 120.3, 119.5, 114.7,
108.2, 82.5, 56.2, 48.8, 47.6, 43.7, 39.6, 38.2, 34.9, 33.2, 31.4, 29.2, 27.6, 26.7, 23.6,
14.4. HRMS (EI+) calcd. for C31H39N3O3 [M]•+: 501.2991. Found 501.2989.
2.25. 2-Methoxyestra-17-(1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)diol (11d)
Red solid (144 mg, 87%), mp. 167-169 °C, eluent: 20% - 50% EtOAc in hexane, Rf =
0.16.1H NMR (600 MHz, CDCl3) δ 7.63 (s, 1H), 7.40 (d, J = 8.8 Hz, 2H), 6.84 (d, J =
8.8 Hz, 2H), 6.52 (s, 1H), 6.45 (s, 1H), 5.29 (s, 1H), 3.63 (s, 3H), 2.72 – 2.51 (m, 2H),
2.40 – 2.24 (m, 1H), 2.08 – 1.96 (m, 2H), 1.87 – 1.77 (m, 2H), 1.78 – 1.65 (m, 1H),
1.53 (d, J = 12.6 Hz, 1H), 1.49 – 1.40 (m, 2H), 1.34 – 1.28 (m, 2H), 1.19 – 1.12 (m,
1H), 0.92 (s, 3H), 0.65 (td, J = 12.8, 3.8 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ
156.8, 154.0, 144.5, 143.4, 131.7, 130.1, 129.5, 122.3, 119.8, 116.5, 114.6, 108.0,
82.5, 56.0, 48.6, 47.4, 43.6, 39.4, 38.0, 33.0, 29.0, 27.4, 26.5, 23.4, 14.3. HRMS (EI+)
calcd. for C28H33N3O3 [M]•+: 461.2315. Found 461.2319.
2.26. 2-Methoxyestra-17-(1-(4-flourophenyl)-1H-1,2,3-triazol-4-yl)diol (11e)
Light yellow solid (144 mg, 75%), mp. 119-124 °C, eluent: 20% - 50% EtOAc in
hexane, Rf = 0.17.1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H), 7.74 (dd, J = 9.0, 4.6
Hz, 2H), 7.24 – 7.15 (m, 2H), 6.69 (s, 1H), 6.62 (s, 1H), 3.80 (s, 3H), 2.85 – 2.65 (m,
2H), 2.58 – 2.39 (m, 1H), 2.24 – 2.09 (m, 2H), 2.03 – 1.86 (m, 3H), 1.73 – 1.20 (m,

6H), 1.09 (s, 3H), 0.81 (td, J = 12.9, 4.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ
163.6, 161.1, 154.6, 144.6, 143.5, 133.4 (d, J = 3.1 Hz), 131.7, 129.5, 122.4, 122.3,
119.6, 116.8, 116.6, 114.7, 108.1, 82.5, 56.1, 48.6, 47.5, 43.6, 39.5, 38.1, 33.1, 29.0,
27.4, 26.6, 23.5, 14.3. HRMS (EI+) calcd. for C27H30FN3O3 [M]•+: 463.2271. Found
463.2268.
2.27. 2-Methoxyestra-17-(1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)diol (11f)
Light yellow solid (149 mg, 79%), mp. 125-124 °C, eluent: 20% - 50% EtOAc in
hexane, Rf = 0.15.1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 1.2 Hz, 1H), 7.83 – 7.63
(m, 2H), 7.58 – 7.41 (m, 2H), 6.69 (s, 1H), 6.62 (s, 1H), 3.80 (s, 3H), 2.89 – 2.65 (m,
2H), 2.59 – 2.37 (m, 1H), 2.25 – 2.09 (m, 2H), 2.09 – 1.85 (m, 3H), 1.79 – 1.24 (m,
6H), 1.09 (s, 3H), 0.81 (td, J = 12.7, 3.5 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ
154.7, 144.6, 143.5, 135.6, 134.5, 131.6, 129.9, 129.5, 121.6, 119.3, 114.6, 108.0,
82.5, 56.0, 48.6, 47.5, 43.6, 39.5, 38.1, 33.0, 29.0, 27.4, 26.6, 23.5, 14.3. HRMS (EI+)
calcd. for C27H30ClN3O3 [M]•+: 479.1976. Found 479.1967.
2.28. 2-Methoxyestra-17-(1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)diol (11g)
Light yellow solid (152 mg, 74%), mp. 137-140 °C, eluent: 20% - 50% EtOAc in
1
hexane, Rf = 0.15. H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H), 7.67 (s, 4H), 6.69 (s,
1H), 6.62 (s, 1H), 3.81 (s, 3H), 2.85 – 2.68 (m, 2H), 2.57 – 2.36 (m, 1H), 2.21 – 2.08
(m, 2H), 2.07 – 1.87 (m, 3H), 1.80 – 1.27 (m, 6H), 1.09 (s, 3H), 0.87 – 0.72 (m, 1H).
13C NMR (101 MHz, CDCl3) δ 154.9, 144.7, 143.6, 136.2, 133.1, 131.8, 129.7, 122.5,
121.9, 119.3, 114.7, 108.2, 82.7, 56.2, 48.8, 47.6, 43.8, 39.6, 38.3, 33.2, 29.2, 27.6,
26.7, 23.6, 14.4. HRMS (EI+) calcd. for C27H30BrN3O3 [M]•+: 523.1461. Found
523.1461.
2.29. 2-Methoxyestra-17-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)diol (11h)
Light yellow solid (161 mg, 82%), mp. 121-127 °C, eluent: 20% - 50% EtOAc in
1
hexane, Rf = 0.14. H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.66 (d, J = 8.9 Hz,
2H), 7.02 (d, J = 8.9 Hz, 2H), 6.69 (s, 1H), 6.62 (s, 1H), 3.87 (s, 3H), 3.81 (s, 3H),
2.77 (dd, J = 14.9, 4.8 Hz, 2H), 2.54 – 2.41 (m, 1H), 2.27 – 2.10 (m, 2H), 2.04 – 1.86
(m, 3H), 1.78 – 1.18 (m, 6H), 1.09 (s, 3H), 0.82 (td, J = 12.9, 3.8 Hz, 1H). C NMR
13
(101 MHz, CDCl3) δ 160.0, 154.2, 144.7, 143.6, 131.8, 130.6, 129.7, 122.2, 119.7,
114.9, 114.7, 108.2, 82.6, 56.2, 55.8, 48.8, 47.6, 43.8, 39.6, 38.2, 33.2, 29.2, 27.6,
26.7, 23.6, 14.4. HRMS (EI+) calcd. for C28H33N3O4 [M]•+: 475.2471. Found
475.2455.
2.30. 2-Methoxyestra-17-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4-yl)diol (11i)
Light yellow solid (133 mg, 71%), mp. 113-116 °C, eluent: 20% - 50% EtOAc in
hexane, Rf = 0.11.1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H), 7.26 (s, 1H), 6.67 (s,
1H), 6.59 (s, 1H), 3.90 (s, 6H), 3.86 (s, 3H), 3.78 (s, 3H), 2.73 (dd, J = 15.9, 5.1 Hz,
4H), 2.50 (ddd, J = 14.6, 9.5, 5.8 Hz, 2H), 2.14 (ddq, J = 13.2, 9.1, 5.6, 4.5 Hz, 3H),
2.01 – 1.93 (m, 3H), 1.92 – 1.85 (m, 2H), 1.75 – 1.19 (m, 8H), 1.07 (s, 3H), 0.81 (td, J
= 12.9, 3.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 154.5, 153.9, 144.6, 143.5, 138.3,
133.0, 131.7, 129.5, 119.8, 114.7, 108.2, 98.4, 82.5, 61.1, 56.5, 56.1, 48.5, 47.4, 43.6,
39.5, 38.1, 33.1, 29.0, 27.4, 26.6, 23.6, 14.3. HRMS (EI+)calcd. for C30H37N3O6
[M]•+: 535.2682. Found 535.2670.
2.31.
(11j)
2-Methoxyestra-17-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)diol

Light yellow solid (108 mg, 59%), mp. 146-149 °C, eluent: 20% - 50% EtOAc in
hexane, Rf = 0.18.1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H), 7.93 (d, J = 8.1 Hz, 2H),
7.80 (d, J = 8.1 Hz, 2H), 6.69 (s, 1H), 6.62 (s, 1H), 3.80 (s, 3H), 2.82 – 2.69 (m, 2H),
2.58 – 2.46 (m, 1H), 2.17 (t, J = 14.0 Hz, 2H), 2.08 – 1.88 (m, 3H), 1.81 – 1.28 (m,
6H), 1.10 (s, 3H), 0.81 (t, J = 12.5 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 155.0,
144.6, 143.5, 139.5, 131.6, 130.6 (q, J = 33.2 Hz), 129.5, 127.1, 127.1, 124.9, 122.2,
120.3, 119.2, 114.6, 108.0, 82.6, 56.0, 48.6, 47.5, 43.6, 39.5, 38.2, 33.0, 29.0, 27.4,
26.6, 23.5, 14.2. HRMS (EI+) calcd. for C28H30F3N3O3 [M]•+: 513.2239. Found
535.2235.
2.32. 2-Methoxyestra-17-(1-(4-(trifluoromethoxyl)phenyl)-1H-1,2,3-triazol-4-yl)diol
(11k)
Light yellow solid (116 mg, 65%), mp. 128-130 °C, eluent: 20% - 50% EtOAc in
hexane, Rf = 0.17. 1H NMR (400 MHz, CDCl3) δ 8.18 – 7.89 (m, 3H), 7.81 (d, J = 8.2
Hz, 2H), 6.69 (s, 1H), 6.63 (s, 1H), 5.42 (s, 1H), 3.81 (s, 3H), 2.84 – 2.71 (m, 2H),
2.58 – 2.46 (m, 1H), 2.26 – 2.07 (m, 2H), 2.09 – 1.89 (m, 3H), 1.75 – 1.32 (m, 6H),
13
1.10 (s, 3H), 0.81 (t, J = 12.5 Hz, 1H). C NMR (101 MHz, CDCl3) δ 155.0, 144.5,
143.5, 139.5, 131.6, 130.8, 130.5, 129.5, 127.1, 127.1 (q, J = 3.6 Hz), 120.3, 119.2,
114.6, 108.0, 82.6, 56.0, 48.6, 47.5, 43.6, 39.5, 38.2, 33.0, 29.0, 27.4, 26.6, 23.5, 14.3.
HRMS (EI+) calcd. for C28H30F3N3O4 [M]•+: 529.2188. Found 529.2183.
2.33. 2 Methoxyestra-17-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)diol (12a)
Light yellow solid (118 mg, 81%), mp. 106-108 °C, eluent: 20% - 50% EtOAc in
1
hexane, Rf = 0.13. H NMR (400 MHz, CDCl3) δ 7.33 (s, 1H), 7.21 (d, J = 8.6 Hz,
2H), 6.88 (d, J = 8.6 Hz, 2H), 6.70 (s, 1H), 6.61 (s, 1H), 5.48 (d, J = 14.7 Hz, 1H),
5.42 (d, J = 14.8 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 2.80 – 2.66 (m, 2H), 2.38 – 2.27
(m, 1H), 2.15 – 2.05 (m, 2H), 1.98 – 1.80 (m, 3H), 1.65 – 1.57 (m, 1H), 1.56 – 1.37
13
(m, 4H), 1.32 – 1.22 (m, 1H), 1.03 (s, 3H), 0.67 (td, J = 12.8, 3.9 Hz, 1H). C NMR
(101 MHz, CDCl3) δ 159.9, 154.0, 144.6, 143.5, 131.7, 129.5, 126.7, 121.0, 114.7,
114.5, 108.2, 105.2, 82.3, 56.1, 55.3, 53.7, 48.5, 47.3, 43.7, 39.4, 37.9, 33.0, 29.0,
27.5, 26.6, 23.4, 14.3. HRMS (EI+) calcd. for C29H35N3O4 [M]•+: 489.2628. Found
489.2617.
2.34. 2-Methoxyestra-17-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)diol (12b)
Light yellow solid (137 mg, 78%), mp. 111-113 °C, eluent: 20 % - 50 % EtOAc in
1
hexane, Rf = 0.12. H NMR (400 MHz, CDCl3) δ 7.39 (s, 1H), 6.67 (s, 1H), 6.60 (s,
1H), 6.47 (s, 2H), 5.49 (d, J = 14.9 Hz, 1H), 5.41 (d, J = 14.9 Hz, 1H), 3.82 (s, 3H),
3.81 (s, 9H), 2.83 – 2.63 (m, 2H), 2.47 – 2.32 (m, 1H), 2.19 – 2.02 (m, 2H), 2.00 –
1.80 (m, 3H), 1.62 – 1.27 (m, 6H), 1.03 (s, 3H), 0.67 (td, J = 12.8, 4.0 Hz, 1H). 13C
NMR (101 MHz, CDCl3) δ 154.2, 153.8, 144.7, 143.6, 138.3, 131.6, 130.4, 129.6,
121.3, 114.7, 108.1, 105.1, 82.5, 61.0, 56.3, 56.2, 54.4, 48.6, 47.4, 43.8, 39.5, 38.1,
33.1, 29.1, 27.5, 26.6, 23.5, 14.4. HRMS (EI+) calcd. for C31H39N3O6 [M]•+: 549.2839.
Found 549.2826.
2.35 Cancer Cell Growth Inhibition
The method applied was that described by Edmondson and coworkers [17] and is
exemplified for the K562 cell line. K562 human chronic myelogeneous leukemia cells
were cultivated in RMPI medium, free of antibiotics and containing 2-
mercaptoethanol (2 µM) and L-glutamine (2 mM), supplemented with foetal calf
serum (FCS) (10% v/v). The cells were adjusted to a concentration depending on their

observed doubling time, (ca. 40000 cells/ml), in RPMI medium supplemented with
FCS (10% v/v). The candidate drug was dissolved in DMSO. A drug solution of 100
l in medium was added to 100 µl of cell solution (40000 cells/ml) in a 96-well
microtitre testplate (4 µl of the compound solution diluted in medium in order to
reach decreasing concentrations). This series of dilutions was continued to afford
samples at different concentrations 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 was
then removed from the incubator and 50 µl of a solution of MTT (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 µl DMSO. The optical density of each well was read at two
wavelengths (λ 540 and 690 nm) using a Titretek Multiscan MCC/340 plate reader.
After processing and analysis through the application 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.
2.36 Inhibition of Tubulin Assembly
The method applied was that described by Lawrence and coworkers [18]. Tubulin was
isolated from porcine brain and stored at –78 °C. Samples were prepared directly in a
96-well microtitre testplate that was preincubated at 4 ºC 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), tubulin (50 µl, 11 mg/ml in Mes
buffer) and the candidate drug (20 µl, Csample in DMSO). The tubulin/drug samples
were immediately placed in a 96-well plate reader, alongside blank samples
containing Mes buffer (198 µl) and the analogs (10 µl, same concentration). The
absorbance (λ 350 nm) was recorded at 25 °C temperature for a period of 60 min, and
the results were compared to untreated controls to evaluate the relative degree of
change in optical density.
3. Results
3.1 Chemistry
The synthesis of the analogs started with the preparation of the steroid 6 as outlined in
Scheme 1. After chromatography, only one stereoisomer was detected by H NMR
1
and HPLC analyses. The depicted stereochemistry at the C-17 position is in accord
with literature [19a], [19b]. Steroid 6 was obtained in 64% yield over the three-step
protocol. Then, under Cu(I) catalysis, different commercially available azides were
reacted with the alkyne 6 affording the 1,2,3-triazoles 9a-9k and 10a-10b in 47-83%
yield. Then deprotection of the TBS-group with excess tetra-n-butyl ammonium
fluoride in THF [20] afforded the desired 1,4-disubstituted 1,2,3-triazoles 11a-11k
and 12a-12b. Deprotection prior to the cycloaddition proved troublesome. The
spectral data of all new products were in accord with their assigned structures.
[INSERT SCHEME 1 HERE]
Scheme 1. Synthesis of triazole analogs of 2-ME (1).

3.2 Biological evaluations
The target triazoles were submitted to the MTT assay for the evaluation of their
cytotoxic effects against the K562 human myeloid leukemia cancer cell line. These
results showed that in the K562 cancer cell line, the two most potent compounds were
2-ME (1) and analog 11k with IC50-values of 0.8 and 0.7 µM, respectively. Almost as
potent with IC50 = 1.0 µM was triazole 11j, while 11e and 12a were slightly less
potent than 1 and 11k (IC50 = 1.8 and IC50 = 1.4 µM). The other analogs that also
exhibited cytotoxic effects in the K562 cell line had IC50-values between 2.4 and 3.5
µM. The data has been compiled in Table 1.
Table 1. Biological evaluations of 2-ME (1) and triazoles 11a-11k, 12a-12b
K562
Cell assay
IC50 (µM )a
OVCAR-3
Cell assay
IC50 (µM )a
WM35
Cell assay
Tubulin
inhibition
Compound
IC50 (µM )a IC50(µM)b
2-ME (1)
11a
11b
11c
0.8
2.8
1.2
2.6
2.1
5.8
2.2
n.d.c
n.d.
n.d.
n.d.
> 10
n.d.
n.d.
> 10
n.d.
8.1
2.4
2.2
6.1
3.5
5.5
7.3
> 10
1.8
> 10
1.6
>10
2.2
11d
11e
3.5
3.4
5.0
11f
3.2
3.4
4.9
11g
11h
11i
2.1
2.6
4.0
> 10
1.0
> 10
1.4
> 10
1.9
11j
0.7
0.9
2.9
5.9
11k
12a
12b
1.4
2.2
3.8
> 10
n.d.
> 10
> 10
> 10
a Results of three experiments performed as triplicates
b Results of three experiments performed as duplicates
cn.d. = not determined
Basically, the same trends were observed in the ovarian cancer cell line
OVCAR-3. Analog 11k was the most potent compound (IC50 = 0.9 µM) also in this
assay. Again, this analog was slightly more potent than the lead compound 1 (IC50 =
1.2 µM). The two analogs 11e and 11j, with the 4-trifluoromethyl and the 4-fluoro-
substituent, respectively, were potent inhibitors with IC50-values of 1.6 and 1.4 µM in
this cell line. In the WM36 melanoma cell line compound 11j was the most potent
(IC50 = 1.9 µM) with slightly higher cytotoxic effects than 2-ME (1) (IC50 = 2.1 µM)
and compound 11e (IC50 = 2.2 µM) In general, the above-mentioned trends were also
observed in this cell line; however, all compounds were slightly less potent with IC50-
values between 2.9-7.3 µM. The two analogs 11i and 12b with a 3,4,5-
trimethoxyphenyl and a 3,4,5-trimethoxybenzyl substituent, respectively, on the
1,2,3-triazole did not show any anti-proliferative effects in any of the three cancer
cell lines. The same result was observed for triazole 11d with a 4-hydroxyphenyl
substituent.

2-ME (1) and the five most cytotoxic triazoles from the MTT-assay were
subjected to a tubulin assembly inhibition assay; in this assay the most potent
compound was 2-ME (1) with IC50 = 2.2 µM, similar to literature values [2]. Among
the five 1,2,3-triazoles tested as tubulin inhibitors, the most potent inhibitor was 11k
with the 4-trifluoromethoxy substituent with a IC50-value of 5.9 µM. Also triazole 11j
showed activity as a tubulin inhibitor (IC50 = 8.1 µM), but the three analogs 11e, 11h
and 12a were all inactive (IC50 > 10 µM).
Microtubules are cylindrical protein polymers composed of α- and β-tubulin
heterodimers. Microtubules are essential for many cellular processes, i.e. maintenance
of cellular shape, intracellular transport and mitotic spindle assembly during cell
division [21]. Inhibition of microtubule formation leads to mitotic arrest and promotes
vascular disruption leading to cell death. Hence, tubulin is one of the most common
biological targets for the development of new anticancer drugs [22].
The most potent 1,4-disubstituted 1,2,3-triazole analogs in the cytotoxicity
assays and in the tubulin polymerization inhibition assays, were compounds 11j and
11k. Apparently, the presence of a trifluoro group is important for both tubulin
polymerization inhibition and cytotoxic activity. Moreover, this functional group also
enhances the lipophilicity of 11j and 11k. The presence of a fluorine atom in the 4-
position, as in compound 11e, contributed to a higher cell death than the other
prepared triazole analogs of the steroid 1. However, this analog did not inhibit the
assembly of tubulin. Similar effects were also observed for the two analogs with a
trimethoxy group in the 4-position, both compound 11h and 12a, the latter derived
from 4-methoxybenzyl azide, showed cytotoxic activities in all cancer cell lines.
However, both compounds were inactive in the tubulin polymerization inhibition
assay. Since 2-ME (1) was the most potent tubulin inhibitor, the antimitotic effects of
the triazoles 11j and 11k were not investigated further.
4. Conclusions
Structure-activity relationship (SAR) studies have shown that 2-ME (1) is amendable
for changes in the C-17 position. The introduction of a 1,4-disubstituted 1,2,3-triazole
ring in this position gave a few analogs that were slightly more cytotoxic than 2-ME
(1). Compound 11k exhibited potent cytotoxic activity in all three cancer cell lines as
well as moderate inhibition of tubulin polymerization. However, this analog was a
less potent tubulin inhibitor than lead compound 1. Hence, this analog only in part
exhibits its cytotoxic effects due to inhibition of tubulin. The steroid 1 has also been
reported to display other mechanisms of actions [5], [6]. Determination if such effects
are mediated by the triazoles reported herein will be investigated.
Acknowledgements
The School of Pharmacy, University of Oslo, is gratefully acknowledged for Ph.D.-
scholarship to E. J. S.
Appendix A. Supplementary Data
Supplementary data associated with this article can be found, in the online version, at
[to be inserted by Editorial office]

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OH
OH
OH
H
H
H
H
MeO
H2N
MeO
HO
H
H
H
H
H
H
H
H
HO
HO
(CH2)2CH3
O
2-Methoxyestradiol (1)
ENMD-1198 (2)
3
4
Fig. 1. Some examples of steroids exhibiting anti-cancer effects.
Scheme 1. Synthesis of triazole analogs of 2-ME (1).

Highlights
• Analogs of the steroid metabolite 2-methoxyestradiol were prepared as
potential new anti-cancer agents
• Cytotoxic effects in the low micromolar range were observed for some
analogs
• The most cytotoxic and interesting analogs also exhibited tubulin
inhibition
• The 1,4-disubstitued-1,2,3-analogs were obtained with excellent
diastero- and regioselectivity