Stereoselective synthesis of the four 16-hydroxymethyl-3-methoxy- and 16-hydroxymethyl-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-ol isomers and their antiproliferative activities

Article abstract of DOI:10.1016/j.steroids.2018.02.008

The reduction of 16-hydroxymethylene-3-methoxy-13α-estra-1,3,5(10)-trien-17-one (14) and 16-hydroxymethylene-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-one (16) yielded a mixture of two diastereomeric diols, the 16α-hydroxymethyl,17β-hydroxy and 16β-hydroxymethyl,17α-hydroxy isomers (17a-20a) in a ratio of 6:1. We describe a straightforward synthetic route to transform the isomers with trans functional groups attached to ring D (17a-20a) into isomers with cis functional groups (25a-28a). We determined the in vitro antiproliferative activities of compounds 17a–20a and 25a–28a by means of MTT assays against a panel of human adherent cancer cell lines HeLa, A2780, MCF-7, T47D, MDA-MB-231 and MDA-MB-361.

Full text of DOI:10.1016/j.steroids.2018.02.008

Accepted Manuscript
Stereoselective synthesis of the four 16-hydroxymethyl-3-methoxy- and 16-hy-
droxymethyl-3-benzyloxy-13 α-estra-1,3,5(10)-trien-17-ol isomers and their
antiproliferative activities
Anita Kiss, Erzsébet Mernyák, János Wölfling, Izabella Sinka, István Zupkó,
Gyula Schneider
PII:
S0039-128X(18)30043-6
https://doi.org/10.1016/j.steroids.2018.02.008
STE 8235
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 December 2017
12 February 2018
22 February 2018
Please cite this article as: Kiss, A., Mernyák, E., Wölfling, J., Sinka, I., Zupkó, I., Schneider, G., Stereoselective
synthesis of the four 16-hydroxymethyl-3-methoxy- and 16-hydroxymethyl-3-benzyloxy-13 α-estra-1,3,5(10)-
trien-17-ol isomers and their antiproliferative activities, Steroids (2018), doi: https://doi.org/10.1016/j.steroids.
2018.02.008
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Stereoselective synthesis of the four 16-hydroxymethyl-3-methoxy-
and 16-hydroxymethyl-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-ol isomers
and their antiproliferative activities
Anita Kissa, Erzsébet Mernyáka, János Wölflinga, Izabella Sinkab, István Zupkób,
Gyula Schneidera*
aDepartment of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
bDepartment of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720
Hungary
Abstract
The reduction of 16-hydroxymethylene-3-methoxy-13α-estra-1,3,5(10)-trien-17-one (14)
and 16-hydroxymethylene-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-one (16) yielded a
mixture of two diastereomeric diols, the 16α-hydroxymethyl,17β-hydroxy and 16β-
hydroxymethyl,17α-hydroxy isomers (17a20a) in a ratio of 6:1. We describe a
straightforward synthetic route to transform the isomers with trans functional groups attached
to ring D (17a20a) into isomers with cis functional groups (25a-28a).
We determined the in vitro antiproliferative activities of compounds 17a–20a and 25a–28a
by means of MTT assays against a panel of human adherent cancer cell lines HeLa, A2780,
MCF-7, T47D, MDA-MB-231 and MDA-MB-361).
Keywords: 13α-Estrone, Stereocontrolled synthesis, Stereoisomers, Antiproliferative activity.
1. Introduction
Breast cancer is the most frequently diagnosed cancer in women worldwide [1]. Since
estrogens are known to play a role in the development of many breast cancers, a logical
approach to the treatment of estrogen-sensitive breast cancer is the use of anti-estrogens that
block the interaction of estrogens with their specific receptor. The literature provides evidence
that inversion of the configuration of estra-1,3,5(10)-trien-17-one at C-13 may lead to the loss
of estrogenic activity [2, 3]. These investigations revealed that the C-13 epimers of estradiols
1

possess low affinity for the estrogen receptor, with no uterotropic activity when tested on
mice. The probable explanation of the negligible receptor binding ability of the 13-epi-
estradiols of ring C and D [4]. 13α-Estrone is readily available from natural estrone by the
epimerization method developed by Yaramenko and Khvat, using 1,2-phenylenediamine and
acetic acid [5]. As 13α-estradiols possess low affinity for the estrogen receptor and are readily
available, we have chosen these compounds as scaffolds for the design of 13α-estrone
derivatives as potencial antitumor agents.
Hydrophilic functional groups attached to ring D in the steroid skeleton have strong effect on
biological properties of these compounds. Earlier we reported preparation and configuration
determinations of the four possible isomers of 16-hydroxymethyl-estra-1,3,5(10)-trien-3,17-
diol [6], and their 3-methyl [7, 8] and 3-benzyl ethers [6].
We describe here the preparation of four possible isomers of 16-hydroxymethyl-3-methoxy-
and 16-hydroxymethyl-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-ols (17a20a and 25a28a).
The reduction of 16-hydroxymethylene-3-methoxy-13α-estra-1,3,5(10)-trien-17-one (14)
and 16-hydroxymethylene-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-one (16) with NaBH4 in
ethanol yielded a mixture of two diastereomeric diols 17a20a in a 6:1 ratio. The
simultaneous development of the two chiral centers permits formation of the four isomers
(17a20a and 25a28a). However, only two isomers (17a20a) were detected by thin-layer
chromatography [9, 10].
Here, we describe a straightforward synthetic route to transform the isomers with trans
functional groups attached to ring D (17a20a) into isomers with cis functional groups
(25a28a).
We set out to obtain answers to the following questions: (1) how the antiproliferative
activities in the 16-hydroxymethyl-13α-estrane-3-methoxy- and 16-hydroxymethyl-3-
benzyloxy series differ, and (2) how the antiproliferative activities are influenced by the
relative steric positions of the 16-hydroxymethyl and 17-hydroxy groups.
2

2. Experimental
2.1. General
Melting points (mp) were determined on Kofler block and are uncorrected. Specific rotations
о
were measured in CHCl3 (c 1 in CHCl3) at 20 C with a POLAMAT-A (Zeiss-Jena)
polarimeter and are given in units of 10-1 deg cm2 g-1. Elementary analysis data were
determined with PerkinElmer CHN analyzer model 2400. The reactions were monitored by
TLC on Kieselgel (Merck Si 254 F) layers (0.25 mm thick); solvent system: (A)
acetone/toluene/hexane (30:35:35 v/v), (B) diisopropyl ether. The spots were detected by
spraying with 5% phosphomolybdic acid in 50% aqueous H3PO4. The Rf values were
determined for the spots observed by illumination at 254 and 365 nm. Flash chromatography:
silica gel 60, 40-63 μm. All solvents were freshly distilled prior to use. NMR spectra were
recorded on a Bruker DRX 500 instrument at 500 (1H NMR) or 125 MHz (13C NMR).
Chemical shifts are reported in ppm (δ scale), and coupling constants (J) are in Hz. For the
determination of multiplicities, the J-MOD pulse sequence was used.
2.2. 3-Methoxy-13-estra-1,3,5(10)-trien-17-one (13)
A mixture of compound 1 (25 g, 87.9 mmol) in acetic acid (250 ml) and o-phenylenediamine
(13.5 g, 125 mmol) was treated at reflux temperature for 3 h. After cooling, the mixture was
slowly poured onto ice and the resulting precipitate was filtered off, washed with water and
dried. The solid was dissolved in CH2Cl2 (300 ml), the solution was washed with water (3 ×
250 mL), dried (Na2SO4) and the solvent evaporated. The crude product was dissolved in
methanol (200 ml) and Girard-P reagent (18.8 g, 100 mmol) and acetic acid (20 ml) were
added to the solution. The reaction mixture was heated at reflux for 3 h, and then allowed to
stand at room temperature overnight. After white crystals formed, the mixture was neutralized
with a dilute Na2CO3 solution. The precipitate was filtered off and dissolved in CH2Cl2 (250
ml), the solution was washed with water (3 × 250 mL), dried (Na2SO4) and the solvent
evaporated Yield of pure 13: 23.7 g (95%). It is identical with the compound described in ref.
[9].
3

2.3. 16-Hydroxymethylidene-3-methoxy-13-estra-1,3,5(10)-trien-17-one (14)
Compound 13 (25 g, 88 mmol) was dissolved in anhydrous toluene (200 ml), then NaOMe
(9.5 g, 0.18 mmol) and freshly distilled ethyl formate (100 ml, 1.3 mol) were added to the
solution. The mixture was stirred for 6 h at 50 °C and allowed to stand overnight at room
temperature. It was then diluted with water (1000 ml) and carefully poured onto a mixture of
ice (100 g) and concentrated HCl (25 ml). The white precipitate separating out was filtered
off, washed thoroughly with water and dried. Recrystallization from CH2Cl2/hexane gave 14
(25.2 g, 90%). Mp: 103107 °C; Rf = 0.70 (ss A); [α] 25 = + 72 (c 1 in CHCl3). (Found: C,
D
76.93; H, 7.52. C20H24O3 (312.17) requires: C, 76.89; H, 7.74%).1H NMR (δ, ppm, CDCl3):
1.12 (s, 3H, 18-H3), 2.82 (m, 2H, 6-H2), 3.76 (s, 3H, 3-OCH3), 6.60 (s, 1H, 4-H), 6.70 (d, 1H,
J = 8.5 Hz, 2-H), 7.10 (s, 1H, 16-CH), 7.19 (d, 1H, J = 8.5 Hz, 1-H). 13C NMR (δ, ppm,
CDCl3): 25.3 (C-18), 25.7, 28.2, 28.6, 30.4, 31.6, 41.2, 41.9, 48.9, 50.8 (C-13), 55.2 (3-
OCH3), 62.1, 111.1 (C-16), 111.7 (C-2), 113.5 (C-4), 126.8 (C-1), 131.9 (C-10), 138.0 (C-
5),157.5 (C-3), 159.3 (16-CH), 213.9 (C-17).
2.4. 16-Hydroxymethyl-3-methoxy-13-estra-1,3,5(10)-trien-17-ol (17a) and 16-
hydroxymethyl-3-methoxy-13-estra-1,3,5(10)-trien-17-ol (18a)
Compound 14 (15 g, 48 mmol) was suspended in MeOH (300 ml) and then KBH4 (13 g,
240 mmol) was added in small portions at room temperature. The mixture was allowed to
stand for 6 h, then poured onto ice (1000 g) and acidified with dilute HCl to pH 3. The
precipitate separating out was filtered off, washed until free from acid and dried. The mixture
obtained consisted of diols 17a and 18a in a ratio of 6:1 (yield 15 g, 98%) [9].
2.5. 17β-Hydroxy-3-methoxy-13α-estra-1,3,5(10)-trien-16α-ylmethyl acetate (17b) and 17α-
hydroxy-3-methoxy-13α-estra-1,3,5(10)-trien-16β-ylmethyl acetate (18b)
A mixture of 17a and 18a (10 g, 31.6 mmol) was dissolved in pyridine (40 ml) and a
solution of Ac2O (3.2 ml, 32 mmol) in pyridine (60 ml) was added dropwise with cooling in
salt-ice bath. The mixture was allowed to stand for 5 h at room temperature, and then poured
onto a mixture of ice (400 g) and concentrated H2SO4 (40 ml). The precipitate separating out
was filtered off and dissolved in CH2Cl2 (60 ml). The solution was washed with water, dried
over Na2SO4 and evaporated in vacuo. The residue obtained was subjected to
chromatographic separation (silica gel, hexane/CH2Cl2 (75:25, v/v)) to give 17c and 18c as oil
4

(2.4 g, 18.8%). Continued elution with CH2Cl2 and CH2Cl2/ethyl acetate (95:5, v/v) resulted in
17b as a white powder (5.6 g, 50%), 18b as an oil (1.1 g, 10%), and a residual mixture of 17a
and 18a (2.0 g, 20%). After alkaline methanolysis of the mixture of diacetates (17c and 18c),
the resulting mixture of diols (17a and 18a) was recycled.
17b: Mp: 104105 °C; Rf = 0.45 (ss A); [α]D25= + 91 (c 1 in CHCl3). (Found: C, 73.86; H,
1
8.32. C22H30O4 (358.48) requires: C, 73.71; H, 8.44%). H NMR (δ, ppm, CDCl3): 1.08 (s,
3H, 18-H3), 2.08 (s, 3H, Ac-H3), 2.78 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OCH3), 4.17 (m, 2H, 16a-
13
H2), 6.60 (s, 1H, 4H); 6.72 (t, 1H, J = 6.0 Hz, 2-H), 7.16 (d, 1H, J = 8.5 Hz, 1-H). C NMR
(δ, ppm, CDCl3): 21.0 (C-18), 27.9, 28.8, 29.0, 29.3 (Ac-CH3), 30.4, 38.5, 40.8, 43.5 (C-13),
44.9, 50.2, 55.2 (3-OCH3), 66.9 (C-16a), 84.6 (C-17), 112.0 (C-2), 113.2 (C-4), 127.5 (C-1),
133.6 (C-10), 137.9 (C-5), 157.2 (C-3), 171.3 (Ac-C=O). 18b: Oil; Rf = 0.40 (ss A); [α] 25 = +
D
19 (c 1 in CHCl3). (Found: C, 73.92; H, 8.25. C22H30O4 (358.48) requires: C, 73.71; H,
8.44%). 1H NMR (δ, ppm, CDCl3): 0.98 (s, 3 H, 18-H3), 2.03 (s, 3H, Ac-H3), 2.79 (m, 2 H, 6-
H2), 3.78 (s, 3H, 3-OCH3), 3.94 (m, 1H, 17-H), 4.10 (dd, 1H, J = 10.9 Hz, J = 6.1 Hz, 16a-
H2), 4.20 (dd, 1H, J = 10.9 Hz, J = 5.5 Hz, 16-H2), 6.62 (d, 1H, J = 2.7 Hz, 4-H), 6.73 (dd,
13
1H, J = 8.6 Hz, J = 2.7 Hz, 2-H), 7.23 (d, 1H, J = 8.6 Hz, 1-H). C NMR (δ, ppm, CDCl3):
20.8 (Ac-CH3), 22.9 (C-18), 26.5, 28.1, 28.3, 30.4, 33.0, 42.1, 42.3, 43.2, 43.8 (C-13). 48.7,
55.0 (3-OCH3), 67.5 (C-16a), 75.8 (C-17), 111.7 (C-2), 113.5 (C-4), 126.8 (C-1), 132.1 (C-
10), 137.9 (C-5), 171.1 (Ac-C=O).
2.6. 3-Methoxy-17-oxo-13α-estra-1,3,5(10)-trien-16α-ylmethyl acetate (21)
Compound 17b (2 g, 5.6 mmol) was dissolved in acetone (50 ml), and Jones reagent (2 ml)
was added dropwise with cooling in ice. The reaction mixture was poured onto ice and the
precipitate separating out was filtered off and dried. The substance obtained was crystallized
from acetone/hexane to give pure 21 (1.1 g, 57%). Mp: 117119 °C; Rf = 0.60 ( ss A); [α] 25
D
=  21 (c 1 in CHCl3). (Found: C, 74.35; H, 8.04. C22H28O4 (356.48) requires: C, 74.13; H,
1
7.92%). H NMR (ppm, CDCl3): 1.04 (s, 3H, 18-H3), 2.04 (s, 3H, Ac-H3), 2.84 (s, 2H, 6-
H2), 3.76 (s, 3H, 3-OCH3), 4.29 (d, 2H, J = 3.0 Hz, 16a-H2), 6.61 (s, 1H, 4-H), 6.70 (d, 1H, J
13
= 6.5 Hz, 2-H), 7.18 (d, 1H, J = 8.0 Hz, 1-H). C NMR (, ppm, CDCl3): 20.8 (C-18), 24.5
(Ac-CH3), 25.5, 28.4, 28.5, 30.3; 32.8, 41.4 (C-8), 41.9 (C-9), 45.2 (C-14), 47.6 (C-16), 50.2
(C-13), 55.2 (3-OCH3), 63.3 (C-16a), 111.7 (C-2), 113.6 (C-4), 126.9 (C-1), 131.7 (C-10),
137.9 (C-5), 157.5 (C-3), 170.8 (Ac-C=O), 219.7 (C-17 C=O).
5

2.7. 3-Methoxy-17-oxo-13α-estra-1,3,5(10)-trien-16β-ylmethyl acetate (22)
Compound 18b (2 g, 5.6 mmol) was dissolved in acetone (100 ml) and a solution of Jones
reagent (2 ml) was added dropwise with cooling in ice. The mixture was poured onto ice. The
oily substance separating out was extracted with CH2Cl2, dried over Na2SO4 and evaporated
in vacuo, giving product 22 (1.2 g, 60%). Mp: 144145 оC; Rf = 0.60 (ss A); [α]D25 =  18 (c
1
1 in CHCl3). (Found: C, 74.35; H, 8.04. C22H28O4 (356.48) requires: C, 74.13; H, 7.92%). H
NMR (δ, ppm, CDCl3): 0.90 (s, 3H, 18-H3), 2.04 (s, 3H, Ac-H3), 2.90 (m, 2H, 6-H2), 3.78 (s,
3H, 3-OCH3), 4.33 (s, 2H, 16a-H2), 6.65 (s, 1H, 4-H), 6.72 (d, 1H, J = 7.5 Hz, 2-H), 7.20 (d,
1H, J = 8.5 Hz, 1-H). 13C NMR (δ, ppm, CDCl3): 13.1 (Ac-CH3), 20.8 (C-18), 25.7, 25.9,
26.7, 29.6, 31.9, 37.8, 44.1, 48.2 (C-13), 48.8, 48.9, 55.2 (3-OCH3), 63.1 (C-16a), 111.6 (C-
2), 113.9 (C-4), 126.2 (C-1), 131.9 (C-10), 137.6 (C-5), 157.6 (C-3), 170.7 (Ac-C=O), 218.8
(C-17 C=O).
2.8. 16-Hydroxymethyl-3-methoxy-13-estra-1,3,5(10)-trien-17-ol (25a)
LiAlH4 (1.2 g, 32 mmol) was suspended in anhydrous diethyl ether (100 ml), then t-BuOH
(9 ml, 96 mmol) was added carefully during stirring and cooling with salt-ice. Compound 21
(2.3 g, 6.4 mmol) dissolved in diethyl ether (100 ml) was added dropwise to the suspension.
The reaction mixture was stirred for 3 h and was decomposed by the careful addition of water
(75 ml) during stirring and cooling. Next, it was acidified with dilute HCl to pH 5. The
organic phase was separated and the aqueous phase was extracted with diethyl ether. The
substance obtained on washing, drying and evaporation of the solvent was subjected to
chromatographic separation on silica gel with CH2Cl2/ethyl acetate (99:1, v/v) to give pure
compound 25a (630 mg, 31%). Mp: 124125 °C; Rf = 0.35 (ss A); [α] 25 = + 22 (c 1 in
D
1
CHCl3). (Found: 76.03; H, 8.74. C20H28O3 (316.44) requires: C, 75.91; H, 8.92%). H NMR
(δ, ppm, CDCl3): 1.03 (s, 3H, 18-H3), 2.81 (d, 2H, J = 5.0 Hz, 6-H2), 3.78 (s, 3H, 3-OCH3),
13
6.62 (s, 1H, 4-H), 6.72 (d, 1H, J = 7.0 Hz, 2-H), 7.22 (d, 1H, J = 8.5 Hz, 1-H). C NMR (δ,
ppm, CDCl3): 23.7 (C-18), 26.9, 28.7, 30.4, 34.1, 41.7, 40.7, 40.7, 44.7 (C-13), 50.2 (C-16),
55.2 (3-OCH3), 64.4 (C-16a), 76.2 (C-17), 111.7 (C-2), 113.5 (C-4), 126.9 (C-1), 132.5 (C-
10), 138.2 (C-5), 157.4 (C-3). Further elution with CH2Cl2/ethyl acetate (95:5) gave 17a (1.35
g, 67%).
6

2.9. 16-Hydroxymethyl-3-methoxy-13-estra-1,3,5(10)-trien-17-ol acetonide (25c)
Compound 25a (663 mg, 2 mmol) was dissolved in acetone (10 ml) in the presence of a
catalytic amount of p-toluenesulfonic acid. The reaction mixture was treated at reflux
temperature for 1 h. It was then neutralized with morpholine, diluted with CH2Cl2 (50 ml)
and washed with water. The organic layer was dried over Na2SO4 and evaporated in vacuo.
Recrystallization from methanol gave compound 25c (680 mg, 95%). Mp: 138140 °C; Rf =
0.85 (ss B); [α]D25 = + 18 (c 1 in CHCl3). (Found: C, 77.68; H, 8.92. C23H32O3 (356.50)
1
requires: C, 77.49; H, 9.05%). H NMR (δ, ppm, CDCl3): 1.08 (s, 3H, 18-H3), 1.35 and 2.80
(two s, 6H, (CH3)2C), 2.80 (m, 2H, 6-H2), 3.78 (s, 3H, 3-OCH3), 6.61 (s, 1H, 4-H), 6.73 (d,
1H, J = 8.5 Hz, 2-H), 7.20 (d, 1H, J = 8.5 Hz, 1-H). 13C NMR (δ, ppm, CDCl3): 22.7 and 24.2
(two C, (CH3)2C), 27.0, 27.8 (C-18), 29.0, 30.1, 30.4, 33.2, 37.1, 40.1, 40.8, 45.1 (C-13),
50.7, 55.2 (3-OCH3), 75.1 (C-17), 98.2 (C-20), 111.9 (C-2), 113.4 (C-4), 127.1 (C-1), 133.2
(C-10), 138.0 (C-5), 157.3 (C-3).
2.10. 16α-Hydroxymethyl-3-methoxy-13α-estra-1,3,5(10)-trien-17α-ol phenylboronate (25d)
Compound 25a (95 mg, 0.3 mmol) was dissolved in ethanol (5 ml) and phenylboronic acid
(45 mg, 0.3 mmol) was added. The precipitate formed in 15 min was filtered off and dried
(25d: 116 mg, 86%). Mp: 128130 °C; Rf = 0.70 (ss B); [α] 25 = + 8.5 (c 1 in CHCl3). (Found:
D
1
C, 77.86; H, 7.98. C26H31BO3 (402.35) requires: C, 77.62; H, 7.77%). H NMR (δ, ppm,
CDCl3): 1.36 (s, 3H, 18-H3), 2.77 (m, 2H, 6-H2), 3.64 (d, 1H, J = 10.5 Hz, 16a-H2), 3.78 (s,
3H, 3-OCH3), 3.90 (t, 1H, J = 10.5 Hz, 16a-H2), 4.33 (dd, 1H, J = 10.5 Hz, J = 5.0 Hz, 17-H),
6.59 (s, 1H, 4-H), 6.74 (dd, 1H, J = 8.5 Hz, J = 2.0 Hz, 2-H), 7.15 (d, 1H, J = 8.5 Hz, 1-H),
7.36 (t, 2H, J = 7.0 Hz, 3’- and 5’-H), 7.41 (t, 1H, J = 7.5 Hz, 1’-H), 7.82 (d, 2H, J = 7.5 Hz,
2’-H and 6’-H).
2.11.
17α-Hydroxy-3-methoxy-13α-estra-1,3,5(10)-trien-16α-ylmethyl p-toluenesulfonate
(29)
Compound 25a (316 mg, 1 mmol) was dissolved in anhydrous pyridine (10 ml) and then a
solution of p-toluenesulfonyl chloride (285 mg, 1.5 mmol) in anhydrous pyridine (5 ml) was
added dropwise during cooling with ice. The reaction mixture was allowed to stand for 24 h
and then poured onto a mixture of ice (100 g) and sulfuric acid (5 ml). The precipitate
separating out was filtered off and recrystallized from a mixture of acetone/hexane to give 29
о
(415 mg, 88%). Mp: 120123 C; Rf = 0.55 (ss A). (Found: C, 69.05; H, 7.36. C27H34O5S
7

1
(470.63) requires: C, 68.90; H, 7.28%). H NMR (δ, ppm, CDCl3): 0.96 (s, 3H, 18-H3), 2.45
(s, 3H, Ts-H3), 2.78 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OCH3), 4.06 (dd, 1H, J = 9.0 Hz, J = 7.0
Hz, 16a-H2), 4.12 (d, 1 H, J = 9.0 Hz, 17-H), 4.28 (dd, 1H, J = 9.0 Hz, J =7.0 Hz, 16a-H2),
6.60 (s, 1H, 4-H), 6.71 (dd, 1H, J = 9.0 Hz, J = 2.5 Hz, 2-H), 7.18 (d, 1H, J = 8.5 Hz, 1-H),
7.35 (d, 2H, J = 8.0 Hz, 3’-H and 5’- H), 7.80 (d, 2H, J = 8.0 Hz, 2’-H and 6’-H). 13C NMR (δ,
ppm, CDCl3): 21.6 (C-18), 23.3 (Ts-CH3), 26.8, 28.8, 30.2, 30.3, 33.1, 39.2, 40.5, 40.7, 44.8
(C-13), 49.7 (C-14), 55.2,71.9 (C-16a), 75.4 (C-17), 111.9 (C-2), 113.4 (C-4), 127.0 (C-1),
129.8 (C-2’ and C-6’), 132.6 (C-3’ and C-5’), 132.6 (C-16), 133.1 (C-1’), 138.0 (C-5), 144.7
(C-4’), 157.4 (C-3).
2.12. 17,16-Epoxymethano-3-methoxy-13-estra-1,3,5(10)-triene (33)
Compound 29 (380 mg, 1 mmol) was dissolved in methanol (15 ml) and heated at reflux
with NaOCH3 (162 mg, 3 mmol) for 2 h. The reaction mixture was diluted with water, the
precipitate separating out was filtered off, dried and crystallized from a mixture of
acetone/hexane to give 33 (265 mg, 88%). Mp: 115116.5 °C; Rf = 0.75 (ss A); [α]D25 = + 19
(c 1 in CHCl3). (Found: C, 80.76; H, 8.92. C20H26O2 (298.42) requires: C, 80.50; H, 8.78%).
1H NMR (ppm, CDCl3): 1.25 (s, 3H, 18-H3), 2.80 (m, 2H, 6-H2), 3.19 (m, 1H, 16-H), 3.77
(s, 3H, 3-OCH3), 4.15 (t, 1H, J = 6.0 Hz, 16a-H2), 4.77 (t, 1H, J = 6.0 Hz, 16a-H2), 4.84 (d,
1H, J = 6.0 Hz, 17-H), 6.60 (d, 1H, J = 2.0 Hz, 4-H); 6.72 (dd, 1H, J =8.5 Hz, J = 2.5 Hz, 2-
13
H); 7.19 (d, 1H, J = 8.5 Hz, 1-H). C NMR (δ, ppm, CDCl3): 23.3 (C-18), 27.2, 29.1, 30.5,
32.1, 35.8, 37.3, 39.2, 42.4, 45.2 (C-13), 53.4, 55.2, (3-OCH3), 75.8 (C-16a), 94.2 (C-17),
111.8 (C-2), 113.4 (C-4), 127.2 (C-1), 133.0 (C-10), 137.9 (C-5), 157.3 (C-3).
2.13. 16β-Hydroxymethyl-3-methoxy-13α-estra-1,3,5(10)-trien-17β-ol (26a)
Compound 22 (356 mg, 1 mmol) was dissolved in methanol (15 ml) and NaBH4 (378 mg, 10
mmol) was added in small portions at room temperature. The reaction mixture was allowed to
stand for 24 h, then it was diluted with water and the precipitate was collected by filtration
and recrystallized from acetone/hexane to obtain 26a (260 mg, 82%). Mp: 104106 °C; Rf =
0.30 (ss A); D25 = + 24 (c 1). (Found: C, 75.83; H, 8.77. C20H28O3 (316.44) requires: C,
75.91; H, 8.92%). 1H NMR (δ, ppm, CDCl3): 0.95 (s, 3H, 18-H3), 2.79 (m, 2H, 6-H2), 3.72 (t,
1H, J = 7.5 Hz, 17-H), 3.77 (s, 3H, 3-OCH3), 3.85 (d, 1H, J = 3.0 Hz, 16a-H2), 3.88 (d, 1H, J
= 4.5 Hz, 16a-H2), 6.60 (s, 1H, 4-H), 6.71 (d, 1H, J = 7.0 Hz, 2-H), 7.19 (d, 1H, J = 8.5 Hz, 1-
8

H). 13C NMR (δ, ppm, CDCl3): 28.9, 29.3, 30.0 (C-18), 30.4, 30.5, 32.1, 40.2, 43.3, 43.3, 45.3
(C-13), 51.2, 55.1 (3-OCH3), 63.1 (C-16a), 85.4 (C-17), 111.8 (C-2), 113.3 (C-4), 127.4 (C-
1), 133.2 (C-10), 138.3 (C-5), 157.2 (C-3).
2.14. 16-Hydroxymethyl-3-methoxy-13-estra-1,3,5(10)-trien-17-ol acetonide (26c)
Compound 26a (663 mg, 2 mmol) was dissolved in acetone (10 ml) in the presence of a
catalytic amount of p-toluenesulfonic acid. The reaction mixture was treated at reflux
temperature for 1 h. It was then neutralized with morpholine, the solution was diluted with
CH2Cl2 (50 ml) and washed with water. The organic layer was dried over Na2SO4 and
evaporated in vacuo. Recrystallization from methanol gave compound 26c (665 mg, 93%).
Mp: 137139 °C; Rf = 0.80 (ss B); [α] 25 = + 15.5 (c 1 in CHCl3). (Found: C, 77.58; H, 9.23.
D
1
C23H32O3 (356.50) requires: C, 77.49; H, 9.05%). H NMR (δ, ppm, CDCl3): 1.23 (s, 3H, 18-
H3), 1.36 and 2.17 (two s, 6H, (CH3)2C), 3.14 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OCH3), 6.60 (d,
1H, J = 3.0 Hz, 4-H), 6.71 (dd, 1H, J = 9.0 Hz, J = 3.0 Hz, 2-H), 7.19 (dd, 1H, J = 17.0 Hz, J
= 9.0 Hz, 1-H). 13C NMR (δ, ppm, CDCl3): 8.6, 19.8 (C-18), 28.5, 29.7, 29.8, 30.8, 32.5, 33.7,
36.4, 41.2, 44.5, 46.2, 51.5, 55.1, 55.2 (3-OCH3), 62.1 (C-16a), 81.7 (C-17), 98.1 (C-20),
111.7 (C-2), 113.3 (C-4), 127.5 (C-1), 133.2 (C-10), 138.5 (C-5), 157.1 (C-3).
2.15. 16β-Hydroxymethyl-3-methoxy-13α-estra-1,3,5(10)-trien-17β-ol phenylboronate (26d)
Compound 26a (79 mg, 0,25 mmol) was dissolved in ethanol (5 ml) and phenlyboronic acid
(38 mg, 0.25 mmol) was added. The precipitate formed in 15 min was filtered off and dried
(26d: 125 mg, 93%). Mp: 87 89 °C; Rf = 0.75 (ss B); [α] 25 = + 6 (c 1 in CHCl3). (Found: C,
D
1
77.83; H, 7.96. C26H31BO3 (402.35) requires: C, 77.62; H, 7.77%). H NMR (δ, ppm,
CDCl3,): 1.16 (s, 3H, 18-H3), 2.81 (m, 2H, 6-H2), 3.79 (s, 3H, 3-OCH3), 3. 87 (dd, 1H, J =
11.0 Hz, J = 7.0 Hz, 16a-H2), 4.23 (d, 1H, J = 7.0 Hz, 17-H), 4.27 (dd, 1H, J = 6.0 Hz, J =
11.0 Hz, 16a-H2), 6.62 (s, 1H, 4-H), 6.75 (dd, 1H, J = 1.5 Hz, J = 8.0 Hz, 2-H), 7. 21 (d, 1H, J
= 8.5 Hz, 1-H), 7. 37 (t, 2H, J = 7.5 Hz, 3’-H and 5’-H), 7. 43 (d, 1H, J = 7.5 Hz, 4’-H), 7.86
(d, 2H, J = 7.5 Hz, 2’-H and 6’-H). 13C NMR (δ, ppm, CDCl3): 23.8 (C-18), 26.7, 29.1, 30.4,
30.5, 32.4, 35.9, 39.3, 41.4, 45.2 (C-13), 49.5, 55.2 (3-OCH3), 63.9 (C-16a), 78.5 (C-17),
112.0 (C-2), 113.3 (C-4), 127.4 (C-1), 127.5 (C-2’ and C-6’), 130.6 (C-4’), 133.2 (C-10),
133.9 (C-3’ and C-5’), 137.8 (C-5), 157.3 (C-3).
9

2.16. 17β-Hydroxy-3-methoxy-13α-estra-1,3,5(10)-trien-16β-ylmethyl p-tolenesulfonate (30)
Compound 26a (316 mg, 1 mmol) was dissolved in anhydrous pyridine (10 ml) and then a
solution of p-toluenesulfonyl chloride (285 mg, 1.5 mmol) in anhydrous pyridine (5 ml) was
added dropwise during cooling with ice. The reaction mixture was allowed to stand for 24 h
and then poured onto a mixture of ice (100 g) and sulfuric acid (5 ml). The precipitate was
filtered, dissolved in dichloromethane and chromatographed on silica gel with a mixture of
о
dichloromethane/hexane (1:1, v/v) to obtain 30 (340 mg, 72%). Mp: 138140 C; Rf = 0.50
1
(ss A). (Found: 68.98; H, 7.41. C27H34O5S (470.63) requires: C, 68.90; H, 7.28%). H NMR
(δ, ppm, CDCl3): 0.96 (s, 3H, 18-H3), 2.45 (s, 3H, Ts-H3), 2.78 (m, 2H, 6-H2), 3.77 (s, 3H, 3-
OCH3), 4.07 (dd, 1H, J = 12.0 Hz, J = 8.0 Hz, 16a-H2), 4.12 (d, 1H, J = 11.0 Hz, 17-H), 4.28
(dd, 1H, J = 12.0 Hz, J = 8.0 Hz, 16a-H2), 6.60 (d, 1H, J = 3.5 Hz, 4-H), 6.71 (dd, 1H, J =
10.5 Hz, J = 3.5 Hz, 2-H), 7.18 (d, 1H, J = 10.5 Hz, 1-H), 7.35 (d, 2H, J = 10.5 Hz, 3’-H and
5’-H), 7.80 (d, 2H, J = 10.5 Hz, 2’-H and 6’-H). 13C NMR (δ, ppm, CDCl3): 21.6 (C-18), 23.3
(Ts-CH3), 26.8, 28.8, 29.7, 30.3, 30.3, 33.1, 39.2, 40.5, 40.7, 44.8 (C-13), 49.8 (C-14), 55.2,
71.9 (C-16a), 75.5 (C-17), 111.9 (C-2), 113.4, (C-4), 127.0 (C-1), 127.9 (C-2’ and C-6’), 129.8
(C-3’ and C-5’), 132.6 (C-10), 133.2 (C-1), 138.0 (C-5), 144.7 (C-4’), 157.4 (C-3).
2.17. 17β,16β-Epoxymethano-3-methoxy-13α-estra-1,3,5(10)-triene (34)
Compound 30 (570 mg, 1.5 mmol) was dissolved in methanol (15 ml) and heated at reflux
with NaOCH3 (243 mg, 4.5 mmol) for 2 h. The reaction mixture was diluted with water, the
precipitate separating out was filtered off, dried and crystallized from a mixture of
acetone/hexane to give 34 (390 mg, 87%). Mp: 105106 °C; Rf = 0.80 (ss A); [α] 25 = + 28 (c
D
1
1 in CHCl3). (Found: C, 8.31; H, 8.96. C20H26O2 (298.42) requires: C, 80.50; H, 8.78%). H
NMR (δ, ppm, CDCl3): 1.25 (s, 3H, 18-H3), 2.80 (m, 2H, 6-H2), 4.15 (t, 1H, J = 5.5 Hz, 16a-
H2), 4.77 (t, 1H, J = 6.5 Hz, 16a-H2), 4.84 (d, 1H, J = 6.5 Hz, 17-H), 6.60 (d, 1H, J = 3.0 Hz,
4-H), 6.72 (dd, 1H, J = 9.0 Hz, J = 3.0, Hz 2-H), 7.19 (d, 1H, J = 9.0 Hz, 1-H). 13C NMR (δ,
ppm, CDCl3): 23.3 (C-18), 27.2, 29.2, 30.5, 32.1, 35.9, 37.3, 39.2, 42.4, 45.2 (C-13), 53.4,
55.2 (3-OCH3), 75.8 (C-16a), 94.3 (C-17), 111.8 (C-2), 113.4 (C-4), 124.2 (C-1), 133.0 (C-
10), 137.9 (C-5), 157.3 (C-3).
2.18. 16α-Acetoxymethyl-3-methoxy-13α-estra-1,3,5(10)-trien-17β-acetate (17c), 16β-
acetoxymethyl-3-methoxy-13α-estra-1,3,5(10)-trien-17α-acetate (18c), 16α-acetoxymethyl-3-
10

methoxy-13α-estra-1,3,5(10)-trien-17α-acetate (25b) and 16β-acetoxymethyl-3-methoxy-13α-
estra-1,3,5(10)-trien-17β-acetate (26b)
General procedure
Compound 17a, 18a, 25a or 26a (150 mg, 0.47 mmol) was dissolved in a mixture of
pyridine (3 ml) and acetic anhydride (3 ml) and the solution was allowed to stand at room
temperature for 12 h Then it was diluted with water and the oil separating out was extracted
with CH2Cl2 (3 × 15 ml). The organic phase was washed with NaHCO3 solution, and then
with water, dried and evaporated in vacuo. The residual oil was chromatographed on silica gel
with a mixture of CH2Cl2/hexane (1:3, v/v). 17c: Oil (186 mg, 98%); Rf = 0.65 (ss B);  25 =
D
(c 1 in CHCl3). (Found: 72.14; H, 8.27. C24H32O5 (400.51) requires: C, 71.97; H,
8.05%). 1H NMR (δ, ppm, CDCl3): 1.08 (s, 3H, 18-H3), 2.00 (s, 3H, Ac-H3), 2.05 (s, 3H, Ac-
H3), 2.84 (d, 2H, J = 3.0 Hz, 6-H2), 3.79 (s, 3H, 3-OCH3), 4.15 (d, 2H, J = 6.0 Hz, 16a-H2),
4.81 (d, 1H J = 5.0 Hz, 17-H), 6.63 (s, 1H, 4-H), 6.73 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, 2-H),
13
7.20 (d, 1H, J = 8.5 Hz, 1-H). C NMR (δ, ppm, CDCl3): 20.9 (Ac-CH3); 21.30 (Ac-CH3),
28.4, 29.1, 29.6, 29.8 (C-18), 30.4, 33.1, 40.4 (C-8), 40.8 (C-9), 44.1 (C-13), 44.7 (C-14),
51.9 (C-16), 55.2 (3-OCH3), 66.4 (C-16a), 84.6 (C-17), 111.9 (C-2), 113.4 (C-4), 127.3 (C-1),
132.7 (C-10), 138.2 (C-5), 157.4 (C-3), 170.6 (C=O), 171.0 (C=O). 18c: Oil (179 mg, 95%);
Rf = 0.65 (ss B); [α] 25 = 0.0 (c 1 in CHCl3). (Found: C, 71.63; H, 8.24. C24H32O5 (400.51)
D
1
requires: C, 71.97; H, 8.05%). H NMR (δ, ppm, CDCl3): 1.02 (s, 3H, 18-H3), 2.00 (s, 3H,
Ac-H3), 2.07 (s, 3H, Ac-H3), 2.82 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OCH3), 4.05 (m, 2H, 16a-H2),
5.30 (d, 1H, J = 9.0 Hz, 17-H), 6.61 (d, 1H, J = 3.5 Hz, 4-H), 6.71 (dd, 1H, J = 8.5 Hz, J = 2.5
Hz, 2-H), 7.22 (d, 1H, J = 8.5 Hz, 1-H). 13C NMR (δ, ppm, CDCl3): 20.9 (Ac-CH3), 21.0 (Ac-
CH3), 23.8 (C-18), 26.2; 28.3, 30.5, 33.1, 39.7, 42.1, 42.9, 44.1 (C-13), 48.8, 55.2 (3-OCH3),
66.9 (C-16a), 76.5 (C-17), 111.7 (C-2), 113.6 (C-4), 127.0 (C-1), 132.1 (C-10), 137.9 (C-5),
170.7 (C=O), 170.8 (C=O). 25b: Oil (90 mg, 95%); Rf = 0.72 (ss B); [α] 25 =  5.5 (c 1 in
D
1
CHCl3). (Found: C, 72.08; H, 8.28. C24H32O5 (400.51) requires: C, 71.97; H, 8.05%). H
NMR (δ, ppm, CDCl3): 1.05 (s, 3H, 18-H3), 2.02 (s, 3H, Ac-H3), 2.06 (s, 3H, Ac-H3), 2.81 (t,
2H, J = 5.0 Hz, 6-H2), 3.77 (s, 3H, 3-OCH3), 4.12 (m, 2H, 16a-H2), 5.34 (d, 1H, J = 9.5 Hz,
17-H ), 6.61 (s, 1H, 4-H), 6.71 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, 2-H), 7.19 (d, 1H, J = 8.5 Hz,
1-H). 13C NMR (δ, ppm, CDCl3): 20.9 (18-CH3), 20.9 (Ac-CH3), 24.2 (C-18), 26.6, 28.7 (Ac-
CH3), 29.3, 30.3, 30.5, 33.4, 36.9 (C-8), 40.5 (C-9), 41.1 (C-14), 44.3 (C-13), 49.7 (C-16),
55.2 (3-OCH3), 65.1 (C-16a), 111.8 (C-2), 113.5 (C-4), 126.9 (C-1), 132.4 (C-10), 137.9 (C-
11

5), 157.4 (C-3), 170.8 (C=O), 170.9 (C=O). 26b: Oil (109 mg, 96%); Rf = 0.70 (ss B); [α] 25
=
D
+ 26 (c 1 in CHCl3). (Found: C, 72.15; H, 8.27. C24H32O5 (400.51) requires: C, 71.97; H,
8.05%). 1H NMR (δ, ppm, CDCl3): 1.08 (s, 3H, 18-H3), 1.98 (s, 3H, Ac-H3), 2.02 (s, 3H, Ac-
H3), 2.81 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OCH3), 4.01 (dd, 1H, J = 11.0 Hz, J = 6.5 Hz, 16a-
H2); 4.10 (t, 1H, J = 11.0 Hz, 16a-H2), 5.12 (d, 1H, J = 5.0 Hz, 17-H), 6.61 (d, 1H, J = 3.0 Hz,
4-H), 6.72 (dd, 1H, J = 8.5 Hz, J = 3.0 Hz, 2-H), 7.17 (d, 1H, J = 8.5 Hz, 1-H).13C NMR (δ,
ppm, CDCl3): 20.8 (Ac-CH3), 21.2 (Ac-CH3), 28.6, 29.7, 30.0 (C-18), 30.4, 31.3, 32.4, 39.8,
40.2, 43.5, 44.9 (C-13), 55.1 (3-OCH3), 63.9 (C-16a), 83.4 (C-17), 111.9 (C-2), 113.0 (C-4),
127.5 (C-1), 132.8 (C-10), 138.0 (C-5), 157.3 (C-3), 170.4 (C=O), 171.0 (C=O).
2.19. 3-Benzyloxy-13-estra-1,3,5(10)-trien-17-one (15)
A mixture of compound 3 (25 g, 70 mmol) in acetic acid (200 ml) and o-phenylenediamine
(20 g, 183 mmol) was treated at reflux temperature for 3 h. After cooling, the mixture was
carefully poured onto ice, the precipitate formed was filtered off, washed with water and
dried. The solid was dissolved in CH2Cl2 (300 ml) and the solution was washed with water (3
× 250 ml), dried over Na2SO4 and evaporated in vacuo. The crude product was dissolved in
methanol (250 ml) and Girard-P reagent (18.8 g, 100 mmol) and acetic acid (20 ml) were
added to the solution. The reaction mixture was heated at reflux for 3 h and then allowed to
stand overnight at room temperature. After neutralization with dilute Na2CO3, the precipitate
was filtered off. The precipitate was dissolved in CH2Cl2 (250 ml) and the solution was
washed with water (3 × 250 ml) to remove the water-soluble derivative of residual normal
estrone-3-benzyl ether. After drying over Na2SO4, the solvent was evaporated in vacuo to give
15 (24.4 g, 97%). 15 is identical with the compound described in ref. [10].
2.20. (Z)-3-Benzyloxy-16-hydroxymethylidene-13-estra-1,3,5(10)-trien-17-one (16)
Compound 15 (28 g, 77.7 mmol) was dissolved in anhydrous toluene (100 ml) and then
NaOMe (10.6 g, 0.2 mmol) and freshly distilled ethyl formate (100 ml, 1.3 mmol) were
added. The mixture was stirred for 6 h at 50 °C and allowed to stand overnight at room
temperature. It was then diluted with water (1 L) and carefully poured onto a mixture of ice
(100 g) and concentrated HCl (22 ml). The white precipitate separating out was filtered off,
washed thoroughly with water and dried. Recrystallization from CH2Cl2/hexane gave 16 (27.5
g, 91%), as white crystals. Mp: 180182 °C; Rf = 0.75 (ss A); [α] 25 =  34 (c 1 in CHCl3).
D
12

(Found: C, 80.56; H, 7.12. C26H28O3 (388.51) requires: C, 80.38; H, 7.26%. 1H NMR (δ, ppm,
CDCl3): 1.15 (s, 3H, 18-H3), 2.81 (m, 2H, 6-H2), 5.02 (s, 2H, Bn-H2), 6.68 (d, 1H, J = 2.4 Hz,
4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.4 Hz, 2-H), 7.09 (s, 1H, =CH), 7.18 (d, 1H, J = 8.6 Hz,
1-H), 7.30 (t, 1H, J = 7.3 Hz, 4’-H), 7.37 (t, 2H, J = 7.3 Hz, 3’- and 5’-H), 7.41 (d, 2H, J = 7.3
Hz, 2’-H and 6’-H). 13C NMR (ppmCDCl3): 24.7, 25.9 (C-18), 27.7, 27.9, 30.4, 30.9, 41.1,
43.6, 44.6, 47.8, 51.2 (C-13), 62.1, 64.4, 69.9, 112.7 (C-2), 114.6 (C-4), 127.0 (C-1), 127.4
(C-2’ and -6’), 127.8 (C-4’), 128.5 (C-3’ and -5’), 132.1 (C-10), 137.2 (C-1’), 138.0 (C-5),
156.8 (C-3), 223.4 (C=O). 16 is identical with the compound described in ref. [10].
2.21. 3-Benzyloxy-16-hydroxymethyl-13-estra-1,3,5(10)-trien-17-ol (19a) and 3-
benzyloxy-16-hydroxymethyl-13-estra-1,3,5(10)-trien-17-ol (20a)
Compound 16a (9 g, 23.2 mmol) was suspended in MeOH (300 ml), and KBH4 (6.2 g, 116
mmol) was added in small portions at room temperature. The mixture was allowed to stand
for 5 h, then poured onto ice (750 g) and acidified with dilute HCl to pH 5. The precipitate
separating out was filtered off, washed until free from acid and dried. The mixture of 19a and
20a (8.0 g, 98%) was obtained in a ratio of 6:1. 20a is identical with the compound described
in ref. [10].
2.22. 17β-Hydroxy-3-benzyloxy-13-estra-1,3,5(10)-trien-16α-ylmethyl acetate (19b) and
17-hydroxy-3-benzyloxy-13-estra-1,3,5(10)-trien-16-ylmethyl acetate (20b)
A mixture of 19a and 20a (10 g, 25.5 mmol) was dissolved in pyridine (40 ml) and a
solution of Ac2O (2.6 ml) in pyridine (55 ml) was added dropwise with cooling in salt-ice.
The mixture was allowed to stand for 5 h at room temperature, and then poured onto a
mixture of ice (400 g) and concentrated H2SO4 (40 ml). The precipitate separating out was
filtered off and dissolved in CH2Cl2 (60 ml), and the solution was washed with water (3 × 60
ml), dried over Na2SO4 and evaporated in vacuo. The mixture of products was subjected to
chromatographic separation (silica gel, CH2Cl2/hexane 25:75, v/v) to give 19c and 20c as oil
(2.1 g, 17%). Continued elution with CH2Cl2 and CH2Cl2/ethyl acetate (99:1, v/v) resulted in
19b as an oil (5.6 g, 51%), 20b as a white powdery compound (1.0 g, 9%), and a residual
mixture of 19a and 20a (2.0 g, 20%). After alkaline methanolysis of the mixture of diacetates
(19c and 20c) and diols (19a and 20a), the products formed were recycled. 19b: Oil; Rf =
0.45 (ss A); [α]D25 = + 75 (c 1 in CHCl3). (Found: C, 77.46; H, 7.96. C28H34O4 (434.58)
13

1
requires: C, 77.39; H, 7.89 %). H NMR (ppm, CDCl3): 1.08 (s, 3H, 18-H3), 2.08 (s, 3H,
Ac-H3), 2.79 (m, 2H, 6-H2), 3.51 (d, 1H, J = 7.5 Hz, 17-H), 4.12 (dd, 1H, , J = 11.0 Hz, J =
6.5 Hz, 16a-H2), 4.23 (dd, 1H, J = 11.0 Hz, J = 6.0 Hz, 16a-H2), 5.03 (s, 2H, Bn-H2), 6.68 (s,
1H, 4-H), 6.79 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, 2-H), 7.16 (d, 1H, J = 8.5 Hz, 1-H), 7.32 (t,
1H, J = 7.5 Hz, 4’-H), 7.37 (t, 2H, J = 7.5 Hz, 3’-H and 5’-H), 7.42 (d, 2H, J = 7.5 Hz, 2’-H
and 6’-H). 20b: Mp: 6668 °C; Rf = 0.35 (ss A); [α] 25 = + 17 (c 1 in CHCl3). (Found: C,
D
77.52; H, 8.02. C28H34O4 (434.58) requires: 77.39; H, 7.89%). 1H NMR (ppm, CDCl3): 0.99
(s, 3H, 18-H3), 2.08 (s, 3H, Ac-H3), 2.79 (m, 2H, 6-H2), 3.65 (d, 1H, J = 7.5 Hz, 17-H), 4.05
(dd, 1H, J = 11.0 Hz, J = 5.0 Hz, 16a-H2), 4.37 (m, 1H, 16a-H2), 5.03 (s, 2H, Bn-H2), 6.68 (d,
1H, J = 2.2 Hz, 4-H), 6.77 (dd, 1H, J = 8.5 Hz, J = 2.2 Hz, 2-H ), 7.19 (d, 1H, J = 8.5 Hz, 1-
H), 7.29 (t, 1H, J = 7.2 Hz, 4’-H), 7.35 (t, 2H, J = 7.2 Hz, 3’- and 5’-H), 7.40 (d, 2H, J = 7.2
Hz, 2’-H and 6’-H). 13C NMR (, ppm, CDCl3): 21.0 (Ac-CH3), 28.9, 29.0 30.3 (C-18), 30.4,
31.8, 32.0, 39.9, 42.3, 43.8, 44.9 (C-13), 51.9, 64.5 (C-16a), 69.9 (Bn-CH2), 82.2 (C-17),
112.7 (C-2), 114.3 (C-4), 127.4 (C-1), 127.6 (C-2’ and -6’), 127.8 (C-4’), 128.5 (C-3’ and -
5’), 133.7 (C-10), 137.4 (C-1’), 138.1 (C-5), 156.4 (C-3), 171.8 (Ac-C=O).
2.23. 3-Benzyloxy-17-oxo-13-estra-1,3,5(10)-trien-16α-ylmethyl acetate (23)
Compound 19b (3 g, 8.4 mmol) was dissolved in acetone (100 ml) and Jones reagent was
added dropwise during cooling with ice. The reaction mixture was poured onto ice (300 g)
and extracted with CH2Cl2. The CH2Cl2 solution was washed with water, dried and
evaporated. The substance obtained was crystallized from acetone/hexane to give 13 (1.1 g,
60%) as white crystals. Mp: 153155 °C; Rf = 0.55 (ss A); [α] 25 =  32.8 (c 1 in CHCl3).
D
1
(Found: C, 77.81; H, 7.65. C28H32O4 (432.56) requires: C, 77.75; H, 7.46%). H NMR (,
ppm, CDCl3): 1.05 (s, 3H, 18-H3), 2.05 (s, 3H, Ac-H3), 2.84 (m, 2H, 6-H2), 4.30 (d, 2H, J =
4.0 Hz, 16a-H2), 5.02 (s, 2H, Bn-H2), 6.70 (d, 1H, J = 2.0 Hz, 4-H), 6.78 (dd, 1H, J = 2.0 Hz,
J = 8.5 Hz, 2-H), 7.18 (d, 1H, J = 8.5 Hz, 1-H), 7.31 (t, 1H, J = 7.0 Hz, 4’-H), 7.38 (t, 2H, J =
7.5 Hz, 3’-H and 5’-H), 7.42 (d, 2H, J = 7.5 Hz, 2’-H and 6’-H). 13C NMR (ppm, CDCl3):
20.8 (C-18), 24.5 (Ac-CH3), 25.6, 28.5, 30.3, 32.8, 41.4, 41.8, 45.2, 47.6, 50.2 (C-13), 63.3
(C-16a), 69.9 (Bn-CH2), 112.6 (C-2), 114.6 (C-4), 126.9 (C-1), 127.4 (C-2’ and C-6’), 127.8
(C-4’), 128.5(C-3’ and C-5’), 132.0 (C-10), 137.2 (C-1’), 137.9 (C-5), 156.8 (C-3), 170.8
(Ac-C=O), 219.6 (C-17 C=O).
14

2.24. 3-Benzyloxy-17-oxo-13-estra-1,3,5(10)-trien-16-ylmethyl acetate (24)
Compound 20b (2 g, 5.6 mmol) was dissolved in acetone (80 ml) and Jones reagent was
added dropwise during cooling in ice. The reaction mixture was poured onto ice (300 g) and
extracted with CH2Cl2. The organic solution was washed with water, dried and evaporated.
The residue is oily product 24 (1.45 g, 60%); Rf = 0.50 (ss A); [α] 25 = 8.5 (c 1 in CHCl3).
D
1
(Found: 77.63; H, 7.67. C28H32O4 (432.56) requires: C, 77.75; H, 7.46%). H NMR (ppm,
CDCl3): 1.14 (s, 3H, 18-H3), 1.96 (s, 3H, Ac-H3), 2.83 (m, 2H, 6-H2), 4.18 (dd, 1H, J = 11.0
Hz, J = 6.5 Hz, 16a-H2), 4.30 (dd, 1H, J = 11.0 Hz, J = 4.5 Hz, 16a-H2), 5.02 (s, 2H, Bn-H2),
6.69 (s, 1H, 4-H), 6.78 (d, 1H, J = 8.5 Hz, 2-H), 7.18 (d, 1H, J = 9.0 Hz, 1-H), 7.32 (t, 1H, J =
7.0 Hz, 4’-H), 7.38 (t, 2H, J = 7.5 Hz, 3’-H and 5’-H), 7.42 (d, 2H, J = 7.5 Hz, 2’- H and 6’-
H). 13C NMR (δ, ppm, CDCl3): 20.9 (C-18), 25.2, 26.1 (Ac-CH3), 27.8, 27.9, 29.7, 30.3, 31.0,
41.0, 42.6, 42.9, 47.6, 50.4 (C-13), 64.3 (C-16a), 69.9 (Bn-CH2), 112.6 (C-2), 114.5 (C-4),
127.0 (C-1), 127.4 (C-2’ and C-6’), 127.8 (C-4’), 128.5 (C-3’ and C-5’), 132.2 (C-10), 137.2
(C-1’), 137.8 (C-5), 156.7 (C-3), 170.8 (Ac-C=O), 218.5 (C-17 C=O).
2.25. 3-Benzyloxy-16-hydroxymethyl-13-estra-1,3,5(10)-trien-17-ol (27a)
LiAlH4 (850 mg, 22.5 mmol) was carefully suspended in anhydrous diethyl ether (70 ml),
then t-BuOH (6.3 ml, 67.5 mmol) was added dropwise during stirring and cooling with salt-
ice. The mixture was allowed to warm up to room temperature, then the solution of compound
23 (2.3 g, 4.5 mmol) in a mixture of diethyl ether/THF (35ml, 15 ml) was added slowly
dropwise to the suspension. The reaction mixture was stirred for 3 h and then decomposed by
the careful addition of water (50 ml) during stirring and cooling with salt-ice. Next, it was
acidified with dilute HCl to pH 5 and then extracted with diethyl ether. The substance
obtained on washing, drying and evaporation of the solvent was purified by column
chromatography on silica gel. Elution with CH2Cl2/ethyl acetate (95:5, v/v) gave pure
compound 27a (530 mg, 30%). Mp: 124126 °C; Rf = 0.55 (ss A); [α] 25 = + 25.8 (c 1 in
D
1
CHCl3). (Found: C, 79.67; H, 8.34. C26H32O3 (392.54) requires: C, 79.56; H, 8.22%). H
NMR (δ, ppm, DMSO): 0.88 (s, 3H, 18-H3), 2.71 (d, 2H, J = 5.5 Hz, 6-H2), 5.03 (s, 2H, Bn-
H2), 6.68 (s, 1H, 4-H), 6.75 (d, 1H, J = 8.5 Hz, 2-H), 7.18 (d, 1H, J = 8.5 Hz, 1-H), 7.31 (t,
1H, J = 5.5 Hz, 4’-H), 7.37 (t, 2H, J = 7.5 Hz, 3’-H and 5’-H), 7.41 (d, 2H, J = 7.5 Hz, 2’-H
13
and 6’-H). C NMR (δ, ppm, DMSO): 24.2 (C-18), 26.7, 28.5, 30.0, 30.5, 33.4, 40.3, 41.1,
41.1, 44.4 (C-13), 49.6, 63.4 (C-16a), 69.05 (Bn-CH2), 73.8 (C-17), 112.5 (C-2), 114.3 (C-4),
15

126.9 (C-1), 127.5 (C-2’ and C-6’), 127.7 (C-4), 128.4 (C-3’ and C-5’), 132.7 (C-10), 137.4
(C-1’), 137.8 (C-5), 156.1 (C-3). Further elution with CH2Cl2/ethyl acetate (95:5, v/v) gave
19a (1.0 g, 60%).
2.26. 3-Benzyloxy-16-hydroxymethyl-13-estra-1,3,5(10)-trien-17-ol acetonide (27c)
Compound 27a (393 mg, 1 mmol) was dissolved in acetone (3 ml) and a catalytic amount of
p-toluenesulfonic acid was added. The reaction mixture was treated at reflux temperature for
1 h. It was then neutralized with morpholine, diluted with CH2Cl2 and washed with water.
The organic layer was dried over Na2SO4 and evaporated in vacuo. Purification by column
chromatography on silica gel with CH2Cl2/hexane (1:3, v/v) gives 27c (389 mg, 90%). Mp:
134136 °C; Rf = 0.80 (ss B); [α] 25 = - 2 (c 1 in CHCl3). (Found: C, 80.67; H, 8.41. C29H36O3
D
1
(432.67) requires: C, 80.52; H, 8.39%). H NMR (ppm, CDCl3): 0.93 (s, 3H, 18-H3), 1.25
and 1.39 (two s, 6H (CH3)2C), 2.80 (m, 2H, 6-H2), 3.74 (d, 1H, J = 3.0 Hz, 16a-H2), 4.05 (dd,
1H, J = 11.5 Hz, J = 4.0 Hz, 16a-H2), 5.04 (s, 2H, Bn-H2), 6.71 (s, 1H, 4-H), 6.80 (dd, 1H, J =
8.5 Hz, J = 2.5 Hz, 2-H), 7.23 (d, 1H, J = 8.5 Hz, 1-H), 7.33 (t, 1H, J = 7.5 Hz, 4’-H), 7. 39 (t,
13
2H, J = 7.5 Hz, 3’-H and 5’-H), 7.45 (d, 2H, J = 7.5 Hz, 2’-H and 6’-H). C NMR (ppm,
CDCl3): 19.8 (C-18), 28.6 and 29.0 (two C, (CH3)2C), 29.8, 29.9, 30.8, 32.5, 33.7, 36.4, 41.2,
44.5, 45.0 (C-13), 51.6, 62.1 (C-16’), 69.9 (Bn-CH2), 81.7 (C-17), 97.4 (CH3)2C), 112.43 (C-
2), 114.3 (C-4), 127.4 (C-2’ and C-6’), 127.5 (C-1), 127.8 (C-4), 127.8 (C-3’ and C-5’), 133.5
(C-10), 137.4 (C-1), 138.6 (C-5), 156.4 (C-3).
2.27.
3-Benzyloxy-16-hydroxymethyl-13-estra-1,3,5(10)-trien-17-ol phenylboronate
(27d)
Compound 27a (130 mg, 0.3 mmol) was dissolved in ethanol (6 ml) and phenylboronic acid
(45 mg, 0.3 mmol) was added. The precipitate formed in 15 min was filtered off and dried
(27d: 132 mg, 92%). Mp: 111113 °C; Rf = 0.70 (ss B); [α] 25 =  14.0 (c 1 in CHCl3).
D
1
(Found: C, 80.47; H, 7.23. C32H35BO3 (478.45) requires: C, 80.33; H, 7.37%.). H NMR (δ,
ppm, CDCl3): 1.16 (s, 3H, 18-H3), 2.80 (m, 2H, 6-H2), 3.87 (dd, 1H, J= 11.5 Hz, J=7.0 Hz,
16a-H2), 5.04 (s, 2H, Bn-CH2); 6.70 (d, 1H, J = 2.0 Hz, 4-H); 6.82 (dd, 1H, J = 8.5 Hz, J =
2.5 Hz, 2-H), 7.21 (d, 1H, J = 8.5 Hz, 1-H), 7.32 (t, 1H, J = 7.5 Hz, 4’-H), 7.38 (dd, 4H, J =
16.0 Hz, J = 8.5 Hz, 3’- H and 5’-H, 3”-H and 5”-H), 7.44 (d, 3H, J = 7.5 Hz, 2’-H and 6’-H,
4”-H), 7.86 (d, 2H, J = 7.0 Hz, 2”-H and 6”-H). 13C NMR (δ, ppm, CDCl3): 23.8 (C-18), 26.7;
16

29.1, 30.4, 32.4, 35.9, 39.3, 41.4, 45.2 (C-13), 49.5, 63.7 (C-1”), 63.9 (C-16a), 70.0 (Bn-CH2),
78.5 (C-17), 112.8 (C-2), 114.4 (C-4), 127.4 (C-2” and C-6”), 127.5 (C-3” and C-5”) 127.8
(C-1), 128.5 (C-2’ and C-6’), 133.5 (C-4”), 133.5 (C-10), 133.9 (C-3’ and C-5’), 137.3 (C-1’),
137.9 (C-5), 156.6 (C-3).
2.28. 3-Benzyl-16α-p-toluenesulfonyloxymethyl-13α-estra-1,3,5(10)-trien-17α-ol (31)
Compound 27a (393 mg, 1 mmol) was dissolved in anhydrous pyridine (15 ml) and a
solution of p-toluenesulfonyl chloride (285 mg, 1.5 mmol) in anhydrous pyridine (10 ml) was
added dropwise while cooled in an ice-bath. The reaction mixture was allowed to stand for 24
h and then poured onto a mixture of ice (100 g) and sulfuric acid (10 ml). The precipitate was
о
filtered off and crystallized from methanol to give 31 (490 mg, 89%). Mp: 115116 C; Rf =
1
0.85 (ss A). (Found: C, 72.67; H, 6.95. C33H38O5S (546.73) requires: C, 72.50; H, 7.01%). H
NMR (δ, ppm, CDCl3): 0.97 (s, 3H, 18-H3), 2.45 (s, 3H, Ts-H3), 2.78 (m, 2H, 6-H2), 4.07 (dd,
1H, J = 9.5 Hz, J = 7.0 Hz, 16a-H2), 4.12 (d, 1H, J = 8.5 Hz, 17-H), 4.28 (dd. 1H, J = 9.5 Hz,
J = 7.0 Hz, 16a-H2), 5.03 (s, 2H, Bn-H2), 6.69 (d, 1H, J = 2.0 Hz, 4-H), 6.79 (dd, 1H, J = 8.5
Hz, J = 2.0 Hz, 2-H), 7.18 (d, 1H, J = 9.0 Hz, 1-H), 7.35 (m, 5H, 2’-H, 3’-H, 4’-H, 5’-H and
13
6-H), 7.42 (d, 2H, J = 7.0 Hz, 3’’-H and 5’’-H), 7.81 (d, 2H, J = 8.0 Hz, 2’’-H and 6’’). C
NMR (δ, ppm, CDCl3): 21.6 (C-18), 23.3 (Ts-CH3) 26.8, 28.8, 30.2, 30.3, 33.1, 39.2, 40.5,
40.7, 44.8 (C-13), 49.7 (C-14), 69.9 (C-16a), 71.9 (Bn-CH2), 75.5 (C-17), 112.7 (C-2), 114.5
(C-4), 127.0 (C-1), 127.4 (C-2’ and C-6’), 127.8 (C-4’), 127.9 (C-2” and C-6’’), 128.5 (C-3’ and
C-5), 129.8 (C-3’’ and C-5’’), 132.9 (C-10), 133.1 (C-1’’), 137.3 (C-1’), 138.0 (C-5), 144.7 (C-
4’’), 156.7 (C-3).
2.29. 16,17-Epoxymethano-3-benzyloxy-13-estra-1,3,5(10)-triene (35)
Compound 31 (547 mg, 1 mmol) was dissolved in methanol (15 ml) and heated at reflux
with NaOCH3 (162 mg, 3 mmol) for 2 h. The reaction mixture was diluted with water, the
precipitate was filtered off, dried, and crystallized from a mixture of acetone/hexane to give
35 (325 mg, 86%). Mp: 9193 °C; Rf = 0.75 (ss B); [α] 25 = + 19.2 (c 1 in CHCl3). (Found: C,
D
1
83.62; H, 7.92. C26H30O2 (374.53) requires: C, 83.38; H, 8.07%. H NMR (δ, ppm, CDCl3):
1.26 (s, 3H, 18-H3), 2.80 (m, 2H, 6-H2), 3.20 (m, 1H, 16-H), 4.15 (t, 1H, J = 5.5 Hz, 16a-H2),
4.78 (t, 1H, J = 7.0 Hz, 16a-H2), 4.85 (d, 1H, J = 7.0 Hz, 17-H), 5.04 (s, 2H, Bn-H2), 6.70 (d,
1H, J = 2.0 Hz, 4-H), 6.80 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, 2-H), 7.19 (d, 1H, J = 8.5 Hz, 1-
17

H), 7.32 (t, 1H, J = 7.0 Hz, 4’-H), 7.39 (t, 2H, J = 7.5 Hz, 3’-H and 5’-H), 7.43 (d, 2H, J = 7.5
Hz, 2’-H and 6’-H). 13C NMR (δ, ppm, CDCl3): 23.3 (C-18), 27.2, 29.2, 30.4, 32.1, 35.9,
37.3, 39.2, 42.4, 45.2 (C-13), 53.4, 69.9 (Bn-CH2), 75.8 (C-16a), 94.2 (C-17), 112.6 (C-2),
114.5 (C-4), 127.2 (C-1), 127.4 (C-2’ and C-6’), 127.8 (C-4’), 128.5 (C-3’ and C-5’), 133.3
(C-10), 137.3 (C-1’), 137.9 (C-5), 156.6 (C-3).
2.30. 3-Benzyloxy-16-hydroxymethyl-13-estra-1,3,5(10)-trien-17-ol (28a)
Compound 24 (2.2 g, 5 mmol) was dissolved in methanol (50 ml) and NaBH4 (570 mg, 15
mmol) was added in small portions at room temperature. The reaction mixture was allowed to
stand for 12 h, then it was diluted with water and the precipitate was collected by filtration
and recrystallized from acetone/hexane to obtain 28a (890 mg, 45%). Mp: 119121 °C; Rf =
0.50 (ss A); [α]D25 = + 31.9 (c 1 in CHCl3). (Found: C, 79.33; H, 8.37. C26H32O3 (392.54)
requires: C, 79.56; H, 8.22%). 1H NMR (δ, ppm, DMSO): 0.85 (s, 3H, 18-H3), 2.71 (s, 2H, 6-
H2), 4.38 (m, 2H, 16a-H2), 5.03 (s, 2H, Bn-H2), 6.68 (s, 1H, 4-H), 6.75 (d, 1H, J = 8.0 Hz, 2-
H), 7.19 (d, 1H, J = 8.5 Hz, 1-H), 7.31 (t, 1H, J = 6.5 Hz, 4’-H), 7.37 (t, 2H, J = 7.5 Hz, 3’-H
and 5’-H), 7.41 (d, 2H, J = 7.0 Hz, 2’-H and 6’-H). 13C NMR (δ, ppm, DMSO): 23.4 (C-18),
26.1; 27.8, 28.1, 30.0, 32.8, 41.9, 42.1, 43.4 (C-13), 45.0, 46.6, 63.6 ( C-16a), 68.9 (Bn-CH2),
72.9 (C-17), 112.3 (C-2), 114.2 (C-4), 126.7 (C-1), 127.4 (C-3’ and C-5’), 127.6 (C-4’), 128.3
(C-2’ and C-6’), 132.4 (C-10), 137.3 (C-1’), 137.8 (C-5), 156.0 (C-3).
2.31. 3-Benzyloxy-16-hydroxymethyl-13-estra-1,3,5(10)-trien-17-ol acetonide (28c)
Compound 28a (250 mg, 0.64 mmol) was dissolved in acetone (25 ml) and a catalytic
amount of p-toluenesulfonic acid was added. The reaction mixture was treated at reflux
temperature for 1 h, neutralized with morpholine, diluted with CH2Cl2 and then washed with
water. The organic layer was dried over Na2SO4 and evaporated in vacuo. The crude product
was recrystallized from methanol to give 28c (240 mg, 86%). Mp: 118120 °C; Rf = 0.70 (ss
B); [α]D25 = + 2.0 (c 1 in CHCl3). (Found: C, 80.36; H, 8.51. C29H36O3 (432.67) requires: C,
1
80.52; H, 8.39%). H NMR (δ, ppm, CDCl3): 0.93 (s, 3H, 18-H3), 1.25 and 1.39 (2 s, 6H, 2
(CH3)2C), 2.80 (m, 2H, 6-H2), 3.74 (d, 1H, J = 4.0 Hz, 16a-H2), 4.05 (dd, 1H, J = 11.5 Hz, J =
4.0 Hz, 16a-H2), 6.72 (s, 1H, 4-H), 6.80 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, 2-H), 7.22 (d, 1H, J =
8.5 Hz, 1-H), 7.31 (m, 1H, 4’-H), 7.45 (t, 2H, J = 7.5 Hz, 3’- and 5’-H), 7.40 (d, 2H, J = 7.5
Hz, 2’- and 6’-H). 13C NMR (δ, ppm, CDCl3): 19.8 (C-18), 28.6 ((CH3)2C), 29.0, 29.7
18

((CH3)2C), 29.9, 30.8, 32.5, 33.7, 36.4, 41.2, 44.5, 45.0 (C-13), 51.6, 62.1 (C-16a), 69.9 (Bn-
CH2), 81.7 (C-17), 97.4 ((CH3)2C), 112.4 (C-2), 114.3 (C-4), 127.4 (C-2’ and -6’), 127.5 (C-
1), 127.6 (C-4’), 128.5 (C-3’ and -5’), 133.5 (C-10), 137.4 (C-1’), 138.5 (C-5), 156.4 (C-3).
2.32.
3-Benzyloxy-16-hydroxymethyl-13-estra-1,3,5(10)-trien-17-ol phenylboronate
(28d)
Compound 28a (130 mg, 0.33 mmol) was dissolved in ethanol (2 ml) and phenlyboronic
acid (45 mg, 0.3 mmol) was added. The precipitate formed in 20 min was filtered off and
dried affording 28d (147 mg, 93%). Mp: 112114 °C; Rf = 0.68 (ss B); [α] 25 =  12 (c 1 in
D
1
CHCl3). (Found C, 80.57; H, 7.62. C32H35BO3 (478.45) requires: C, 80.33; H, 7.37%). H
NMR (δ, ppm, CDCl3): 1.16 (s, 3H, 18-H3), 2.80 (m, 2H, 6-H2), 3.87 (dd, 1H, J = 11.5 Hz, J
= 7.0 Hz, 16a-H2), 4.23 (d, 1H, J = 7.5 Hz, 17-H), 4.26 (dd, 1H, J = 11.5 Hz, J = 6.0 Hz, 16a-
H2), 5.04 (s, 2H, Bn-H2), 6.70 (d, 1H, J = 2.0 Hz, 4-H), 6.82 (dd, 1H, J = 8.5 Hz, J = 2.0 Hz,
2-H), 7.21 (d, 1H, J = 8.5 Hz, 1H), 7.33 (t, 1H, J = 7.0 Hz, 4’-H), 7.38 (dd, 4H, J = 16.0 Hz, J
= 8.5 Hz, 3’-H and 5’-H, 3”-H and 5”-H), 7.44 (d, 3H, J = 7.0 Hz, 2’-H and 6’-H, 4”-H), 7.86
(d, 2H, J = 7.0 Hz, 2”-H and 6”-H). 13C NMR (δ, ppm, CDCl3): 23.8 (C-18), 26.7, 29.1, 30.4,
30.5, 32.4; 35.9, 39.3, 41.4, 45.2 (C-13), 49.5, 63.7 (C-1”), 63.9 (C-16a), 70.0 (Bn-CH2), 78.5
(C-17), 112.8 (C-2), 114.4 (C-4), 127.4 (C-2” and C-6”), 127.5 (C-3” and C-5”), 127.8 (C-1),
128.5 (C-2’ and C-6’), 130.6 (C-4”), 133.5 (C-10), 133.9 (C-3’ and C-5’), 137.7 (C-1’), 137.9
(C-5), 156.6 (C-3).
2.33. 17β-Hydroxy-3-Benzyloxy-13α-estra-1,3,5,(10)-trien-16β-ylmethyl-p-toluenesulfonate
(32)
Compound 28a (393 mg, 1 mmol) was dissolved in anhydrous pyridine (15 ml) and a
solution of p-toluenesulfonyl chloride (285 mg, 1.5 mmol) in anhydrous pyridine (10 ml) was
added dropwise during cooling with ice. The reaction mixture was allowed to stand for 24 h
and then poured onto a mixture of ice (100 g) and sulfuric acid (10 ml). The resulting
precipitate was filtered off, dissolved in CH2Cl2 and chromatographed on silica gel.
CH2Cl2/hexane (1:1, v/v) eluted pure 32. Mp: 157159 оC; Rf = 0.80 (ss A). (Found: C, 72.32;
1
H, 6.87. C33H38O5S (546.73) requires: C, 72.50; H, 7.01%). H NMR (δ, ppm, CDCl3): 0.94
(s, 3H, 18-H3), 2.45 (s, 3H, Ts-H3), 2.73 (s, 2H, 6-H2), 3.78 (d, 1H, J = 3.5 Hz, 17-H), 4.02
(dd, 1H, J =8.5 Hz, J = 6.5 Hz, 16-H2), 4.25 (t, 1H, J = 9.0 Hz, 16a-H2), 5.02 (s, 2H, Bn-H2),
19

6.67 (s, 1H, 4-H), 6.78 (d, 1H, J = 8.0 Hz, 2-H), 7.17 (d, 1H, J = 8.5 Hz, 1-H), 7.35 (m, 5H,
2’-H, 3’-H, 4’-H, 5’-H and 6’-H), 7.42 (d, 2H, J = 7.5 Hz, 3’’-H and 5’’-H), 7.80 (d, 2H, J = 7.5
Hz, 2’’-H and 6’’-H). 13C NMR (δ, ppm, CDCl3): 21. 6 (C-18), 28.8, 29.0, 30.0 (Ts-CH3),
30.4, 31.5, 31.9, 39.9, 41.8, 43.7, 45.0 (C-13), 50.93 (C-14), 69.9 (C-16a), 70.9 (Bn-CH2),
82.2 (C-17), 112.7 (C-2), 114.4 (C-4), 127.4 (C-2’ and C-6’), 127.6 (C-1), 127.8 (C-4’), 127.9
(C-2’’ and C-6’’), 128.5 (C-3’ and C-5’), 129.9 (C-3’’ and C-5’’), 133.1 (C-1’’), 133.4 (C-10),
137.3 (C-1’), 138.1 (C-5), 144.7 (C-4’’), 156.5 (C-3).
2.34. 16,17-Epoxymethano-3-benzyloxy-13-estra-1,3,5(10)-triene (36)
Compound 32 (136 mg, 0.3 mmol) was dissolved in methanol (15 ml) and heated at reflux
with NaOCH3 (54 mg, 1 mmol) until full conversion (2.5 h). The solution was poured into
water and mixed with NH4Cl. The precipitate formed was filtered off, dried, and crystallized
from a mixture of acetone/hexane to give 36 (95 mg, 84%). Mp: 118120 °C; Rf = 0.70 (ss B);
[α]D25 = + 17.0 (c 1 in CHCl3). (Found: C, 83.56; H, 7.95. C26H30O2 (374.53) requires: C,
1
83.38; H, 8.07%). H NMR (δ, ppm, CDCl3): 0.70 (s, 3H, 18-H3), 2.90 (m, 2H, 6-H2), 3.99
(dd, 1H, J = 4.0 Hz, J = 6.0 Hz, 16a-H2), 4.77 (t, 1H, J = 7.0 Hz, 16a-H2), 4.80 (d, 1H, J = 5.0
Hz, 17-H), 5.04 (s, 2H, Bn-H2), 6.73 (d, 1H, J = 1.5 Hz, 4-H), 6.79 (dd, 1H, J = 8.5 Hz, J =
1.5 Hz, 2-H), 7.24 (s, 1H, 1-H), 7.32 (t, 1H, J = 7.5 Hz, 4’-H), 7.38 (t, 2H, J = 7.5 Hz, 3’-H
13
and 5’-H), 7.44 (d, 2H, J = 7.5 Hz, 2’-H and 6’-H). C NMR (δ, ppm, CDCl3): 26.1 (C-18),
29.0, 29.5, 30.7, 32.7, 34.0, 38.0, 42.3, 42.5, 45 (C-13), 52.9, 69.9 (C-16a), 75.4 (Bn-CH2),
75.8 , 98.1 (C-17), 112.4 (C-2), 114.5 (C-4), 127.2 (C-1), 127.4 (C-2’ and C-6’), 127.8 (C-4’),
128.5 (C-3’ and C-5’), 133.2 (C-10), 137.4 (C-1’), 138.4 (C-5), 156.6 (C-3).
2.35. 16α-Acetoxymethyl-3-benzyloxy-13α-estra-1,3,5(10)-trien-17β-acetate (19c), 16β-
acetoxymethyl-3-benzyloxy-13α-estra-1,3,5(10)-trien-17α-acetate (20c), 16α-acetoxymethyl-
3-benzyloxy-13α-estra-1,3,5(10)-trien-17α-acetate (27b) and 16β-acetoxymethyl-3-benzyloxy-
13α-estra-1,3,5(10)-trien-17β-acetate (28b)
General procedure
Compound 19a, 20a, 27a or 28a (150 mg, 0.38 mmol) was dissolved in a mixture of
pyridine (3 ml) and acetic anhydride (3 ml) and the solution was allowed to stand at room
temperature for 12 h. It was then diluted with water and the oil separating out was extracted
with CH2Cl2 (3 × 15 ml). The organic phase was washed with NaHCO3 solution and then with
20

water, dried and evaporated. The residual oil was chromatographed on silica gel with a
mixture of CH2Cl2/hexane (1:3, v/v). Product 19c (178 mg, 98%) was crystallized from
methanol. Mp: 109111 °C; Rf = 0.62 (ss B); [α] 25 = + 8.5 (c 1 in CHCl3). (Found: 75.78; H,
D
7.85. C30H36O5 (476.61) requires: C, 75.60; H, 7.61%). 1H NMR (δ, ppm, CDCl3): 1.08 (s,
3H, 18-H3), 2.00 (s, 3H, Ac-H3), 2.05 (s, 3H, Ac-H3), 2.82 (m, 2H, 6-H2), 4.15 (d, 2H, J = 6.0
Hz, 16a-H2), 4.82 (d, 1H, J = 5.0 Hz, 17-H), 5.04 (s, 2H, Bn-H2), 6.72 (d, 1H, J = 2.0 Hz, 4-
H), 6.80 (dd, 1H, J = 8.5 Hz, J = 2.0 Hz, 2-H), 7.21 (d, 1H, J = 8.5 Hz, 1-H), 7.32 (t, 1H, J =
7.0 Hz, 4’-H), 7.38 (t, 2H, J = 7.0 Hz, 3’-H and 5’-H), 7.43 (d, 2H, J = 7.0 Hz, 2’-H and 6’-
H). 13C NMR (δ, ppm, CDCl3): 20.9 (Ac-CH3), 21.3 (Ac-CH3), 28.4, 29.1, 29.6; 29.8 (C-18),
30.4, 33.1, 40.4, 40.8, 44.1 (C-13), 44.7, 51.9, 66.9 (C-16a), 69.9 (Bn-CH2), 84.6 (C-17),
112.6 (C-2), 114.4 (C-4), 127.3 (C-1), 127.5 (C-2’ and C-6’), 127.8 (C-4’), 128.5 (C-3’ and
C-5’), 132.9 (C-10), 137.3 (C-1’), 138.5 (C-5), 156.6 (C-3), 170.6 (C=O); 171.0 (C=O). 20c:
Mp: 7172 °C; Rf = 0.60 (ss B); [α] 25 = + 11.5 (c 1 in CHCl3). (Found: C, 75.38; H, 7,71.
D
1
C30H36O5 (476.61) requires: C, 75.60; H, 7.61%). H NMR (δ, ppm, CDCl3): 1.02 (s, 3H, 18-
H3); 2.00 (s, 3H, Ac-H3), 2.07 (s, 3H, Ac-H3), 2.82 (m, 2H, 6-H2), 4.05 (m, 2H, 16a-H2), 5.03
(s, 2H, Bn-H2), 5.31 (d, 1H, J = 9.0 Hz, 17-H), 6.70 (s, 1H, 4-H); 6.79 (dd, 1H, J = 9.0 Hz, J =
2.0 Hz, 2-H), 7.22 (d, 1H, J = 9.0 Hz, 1-H); 7.31 (t, 1H, J = 7.0 Hz, 4’-H), 7.38 (t, 2H, J = 7.0
Hz, 3’-H and 5’-H), 7.43 (d, 2H, J = 7.0 Hz, 2’-H and 6’-H). 13C NMR (δ, ppm, CDCl3): 20.9
(Ac-CH3), 21.0 (Ac-CH3), 23.8 (18-CH3), 26.2, 28.3, 30.5, 33.1, 39.7, 42.1, 42.8, 44.1 (C-13),
48.8, 66.9 (C-16a), 70.0 (Bn-CH2), 76.5 (C-17), 112.6 (C-2), 114.6 (C-4), 126.9 (C-1), 127.4
(C-2’ and C-6’), 127.8 (C-4’), 128.5 (C-3’ and C-5’), 132.4 (C-10), 137.3 (C-1’), 138.0 (C-5),
156.7 (C-3), 170.7 (C=O), 171.0 (C=O). 27b: Mp: 106108 °C; Rf = 0.62 (ss B); [α] 25 = +
D
15.6 (c 1 in CHCl3). (Found: C, 75.42; H, 7.96. C30H36O5 (476.61) requires: C, 75.60; H,
1
7.61%). H NMR (δ, ppm, CDCl3): 1.05 (s, 3H, 18-H3); 2.03 (s, 3H, Ac-H3), 2.07 (s, 3H, Ac-
H3), 2.80 (m, 2H 6-H2), 4.02 (dd, 1H, J = 11.0 Hz, J = 7.0 Hz, 16a-H2), 5.03 (s, 2H, Bn-H2),
5.34 (d, 1H, J = 9.0 Hz, 17-H), 6.70 (d, 1H, J = 2.0 Hz, 4-H), 6.78 (dd, 1H, J = 8.5 Hz, J = 2.0
Hz, 2-H), 7.20 (d, 1H, J = 8.5 Hz, 1-H), 7.32 (t, 1H, J = 7.5 Hz, 4’-H), 7.38 (t, 2H, J = 7.5 Hz,
3’-H and 5’-H), 7.38 (t, 2H, J = 7.5 Hz, 3’-H and 5’-H), 7.43 (d, 2H, J = 7.5 Hz, 2’-H and 6’-
H). 13C NMR (δ, ppm, CDCl3): 20.9 (Ac-CH3), 20.9 (Ac-CH3), 24.2 (C-18), 26.6, 28.7, 30.3,
30.5, 33.4; 36.9, 40.5, 41.2; 44.2 (C-13), 49.7, 65.1 (C-16a), 70.0 (Bn-CH2), 76.0 (C-17),
112.6 (C-2), 114.5 (C-4), 126.9 (C-1), 127.4 (C-3’ and C-5’), 132.7 (C-10), 137.3 (C-1’),
138.0 (C-5), 156.7 (C-3), 170.8 (C=O), 170.9 (C=O). 28b: Mp: 104106 °C; Rf = 0.62 (ss B);
[α]D25 = + 9.0 (c 1 in CHCl3). (Found: 75.44; H, 7.82. C30H36O5 (476.61) requires: C, 75.60; H,
21

7.61%). 1H NMR (δ, ppm, CDCl3): 1.26 (s, 3H, 18-H3), 2.00 (s, 3H, Ac-H3), 2.02 (s, 3H, Ac-
H3), 2.80 (m, 2H, 6-H2), 4.01 (m, 16a-H2), 4.11 (m, 1H, 16a-H2), 5.03 (s, 2H, Bn-H2), 5.12 (d,
1H, J = 5.0 Hz, 17-H), 6.70 (s, 1H, 4-H), 6.80 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, 2-H), 7.17 (d,
1H, J = 8.5 Hz, 1-H), 7.32 (t, 1H, J = 7.5 Hz, 4’-H), 7.38 (t, 1H, J = 7.5 Hz, 3’-H and 5’-H),
7.32 (d, 2H, J = 7.5 Hz, 4’-H), 7.38 (t, 2H, J = 7.5 Hz, 3’-H and 5’-H), 7.43 (d, 2H, J = 7.5
13
Hz, 2’-H and 6’-H). C NMR (δ, ppm, CDCl3): 20.9 (Ac-CH3), 21.2 (Ac-CH3), 28.6, 29.7,
30.2 (C-18), 30.4, 31.3, 32.4, 39.9, 40.2, 43.5, 44.9 (C-13), 51.4, 64.0, (C-16a), 69.9 (Bn-
CH2); 83.4 (C-17), 112.7 (C-2), 114.4 (C-4), 127.4 (C-2’ and C-6’), 127.6 (C-1), 127.8 (C-4’),
128.5 (C-3’ and C-5’), 133.1 (C-10), 137.3 (C-1’), 138.1 (C-5), 156.6 (C-3), 170. 4 (C=O),
170.9 (C=O).
2.36. Cell cultures and antiproliferative assays
The growth-inhibitory effects of the compounds were tested in vitro by means of the MTT
assay on HeLa, A2780, MCF-7, T47D, MDA-MB-231 and MDA-MB-361 cells, isolated
from cervical, ovarian and breast cancer, and on noncancerous human fibroblasts (MRC-5 cell
line). The growth-inhibitory effects of the compounds were tested in vitro by means of the
MTT assay on a panel containing four breast cancer cell lines (MCF-7, T47D, MDA-MB-231
and MDA-MB-361), a cervical and an ovarian cancer cell line (HeLa and A2780,
respectively).
The cell lines were obtained from the European Collection of Cell Cultures (Salisbury, UK).
The cells were maintained in minimal essential medium supplemented with 10% fetal bovine
serum (FBS), 1% non-essential aminoacids and an antibiotic-antimycotic mixture (AAM). All
chemicals, if otherwise not specified, were purchased from Sigma-Aldrich Ltd. (Budapest,
o
Hungary). All cell lines were grown in a humidified atmosphere of 5% CO2 at 37 C. For
pharmacological investigations, 10 mM stock solutions of the tested compounds were
prepared with dimethyl sulfoxide (DMSO). The highest applied DMSO concentration of the
medium (0.3%) did not have any substantial effect on the determined cellular functions. All
cell types were seeded into 96-well plates at a density of 5000 cells/well and allowed to stand
overnight under cell culturing conditions, and the medium containing the tested compound
was then added. After a 72 h incubation with two concentrations (10 and 30 µM) of the
compounds, viability was determined by the addition of 20 µl 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml) for 4 h. The precipitated
formazan crystals were solubilized in DMSO and the absorbance was determined at 545 nm
22

with an ELISA reader [11]; wells with untreated cells were utilized as controls. Two
independent experiments were performed with 5 parallel wells; cisplatin, an agent
administered clinically in the treatment of certain gynecological malignancies, was used as
positive control in the same concentration range as the test compounds. For the most effective
compounds, the assays were repeated with a set of dilutions (0.03−30 µM), and sigmoidal
dose-response curves were fitted to the measured data in order to determine the IC50 values by
means of Graphpad Prism 4.0 (Graphpad Software; San Diego, CA, US).
3. Results and discussion
3.1. Synthetic studies
We have previously reported the preparation and determination of the configurations of the
four possible isomers of the 3-methoxy-and 3-benzyloxy-16-hydroxymethyl-estra-1,3,5(10)-
trien-17-ols (5a8a and 9a12a) of 13β-estrone derivatives. Treatment of 3-methoxy- and 3-
benzyloxyestra-1,3,6(10)-trienes (1 and 3) with NaOMe and ethyl formate gave 3-methoxy-
and 3-benzyloxy-16-hydroxymethylidene-estra-1,3,5(10)-trien-17-ones (2 and 4). The 16-
formyl compounds were reduced with KBH4 in methanol yielding a mixture of three (5a7a
and 9a11a) of the four possible isomers of 3-methoxy- and 3-benzyloxy-16-
hydroxymethylestra-1,3,5(10)-trienes in a ratio of 50:45:5 in 94% yield. Fourth isomers 8a
and 12a were prepared from 16α-acetoxymethyl-17β-p-toluenesulfonate mixed esters (6c and
10c) by neighboring group participation during solvolysis in aqueous AcOH. The structures of
1
the isomers were confirmed unambiguously by their IR, H and 13C NMR spectra [6, 7, 8]
(Scheme 1).
Scheme 1
In contrast to the intensive research on 13β-estrone derivatives, reactions of 13α-estrones
are much less studied [2, 9, 10, 12]. Thus, due to our ongoing interest in 16-
hydroxymethyl,17-hydroxy steroids, we extended the earlier investigations to 13α-estrone
derivatives. In this publication we describe an extension of the C-16 hydroxymethylation to 3-
methoxy- and 3-benzyloxy-13α-estra-1,3,5(10)-trien-17-ones (13 and 15). The 3-methoxy-
and 3-benzyloxy-estra-1,3,5(10)-trien-17-ones (1 and 3) were epimerized by the method of
Yaremenko and Khvat [5], resulting in 3-methoxy- and 3-benzyloxy-13α-estra-1,3,5(10)-
23

trien-17-ones (13 and 15) in good yield [9, 10]. Formylation of 13 and 15 with ethyl formate
in the presence of NaOMe gave the 16-hydroxymethylidene-17-ketones (14 and 16) in high
yield.
16-Hydroxymethylidene-17-ketones 14 and 16 were reduced with KBH4 in methanol to give
selectively 17, 18 and 19, 20. Two new chiral centers are formed in this reaction, but only two
of the possible four isomers could be obtained in a 6:1 ratio. These compounds could not be
separated by flash chromatography on silica gel. Selective acetylation of the primary hydroxyl
groups was therefore carried out and the resulting acetates (17b, 18b and 19b, 20b) were
separated on a silica gel column.
Compounds 17b, 19b and 18b, 20b were oxidized by the Jones method to 3-methoxy- and
3-benzyloxy-17-oxo-13α-estra-1,3,5(10)-trien-16α-ylmethyl acetates (21, 23), and 3-
methoxy- and 3-benzyloxy-17-oxo-13α-estra-1,3,5(10)-trien-16β-ylmethyl acetates (22, 24).
Reduction of 21 and 23 with lithium tri-tert-butoxyaluminum hydride afforded isomeric 16α-
hydroxymethyl-3-methoxy- and 3-benzyloxy-16α-hydroxymethyl-13α-estra-1,3,5(10)-trien-
17α-ols (25a and 27a) and 16α-hydroxymethyl-3-methoxy- and 3-benzyloxy-16α-
hydroxymethyl-13α-estra-1,3,5(10)-trien-17β-ols (17a and 19a) in a ratio of 1:2. These
isomers (25a, 17a and 27a, 19a) are separable by flash chromatography. Repeated Jones
oxidation of 17b yields 21, whereas the oxidation of 19b gives 23; both products can be
reused in the reduction process.
Since reduction of the 17-ketone function in steroids normally yields a 17β group (for a few
exceptions, see ref. [13]), the reduction of 22 and 24 with KBH4 in methanol gave the isomers
16β-hydroxymethyl-3-methoxy- and l6β-hydroxymethyl-3-benzyloxy-13α-estra-1,3,5(10)-
trien-17β-ols (26a and 28a) (Scheme 2).
Scheme 2
For confirmation of the configurations assumed for 25a, 27a and 26a, 28a, transformations
were carried out with the individual isomers. Treatment of 25a, 27a and 26a, 28a with
acetone in the presence of a catalytic amount of p-toluenesulfonic acid afforded the
corresponding cyclic acetonide derivatives 25c, 27c and 26c, 28c. It is known from our earlier
observations that only the cis-diols participate in acetonide building reaction [6]. Furthermore,
24

they were reacted with phenylboronic acid to give the corresponding cyclic phenylboronic
esters 25d, 27d and 26d, 28d in very good yields.
When treated with p-toluenesulfonyl chloride in pyridine at low temperature, the primary
hydroxymethyl groups were tosylated yielding 17α-hydroxy-3-methoxy- and 17α-hydroxy-3-
benzyloxy-13α-estra-1,3,5(10)-trien-16α-ylmethyl p-toluenesulfonates (29 and 31) and 17β-
hydroxy-3-methoxy- and 17β-hydroxy-3-benzyloxy-13α-estra-1,3,5(10)-trien-16β-ylmethyl
p-toluenesulfonates (30 and 32).
The alkaline reaction of these 1,3-tosylhydrines leads to the formation of oxetanes. The
process can be regarded as a neighboring group participation charachterized by the general
symbol (O-4). Oxethane formation takes place readily when the participating groups are in a
favourable steric configuration [14].
On alkaline methanolysis of 29 and 31, α oxethanes 33 and 35 were formed, condensed to
ring D. Alkaline methanolysis of 30 and 32, in turn, gave β oxethanes 34 and 36 (Scheme 3).
Scheme 3
In conclusion, we have synthesized two new cis 16-hydroxymethyl,17-hydroxy isomer pairs
25, 27 and 26, 28 in the 3-methoxy- and 3-benzyloxy-estra-13-epi-1,3,5(10)-triene-17-one
series, respectively. These compounds were transformed quantitatively into their cyclic
acetonides and phenylboronic esters. Furthermore, alkaline methanolysis of their 16-p-
toluensulfonyloxymethyl-17-hydroxy derivatives afforded the corresponding α and β oxetanes
condensed to ring D ring in the sterane skeleton.
3.2.Determination of the antiproliferative properties of the 16-hydroxymethyl-3-methoxy- and
16-hydroxymethyl-3-benzyloxy-13α-estra-1,3,5(10)-trien-17-ol isomers
The antiproliferative activities of compounds 17a–20a and 25a–28a were determined in
vitro by means of MTT assays against HeLa (cervical carcinoma), A2780 (ovarian carcinoma)
and a set of breast cancer cell lines differing in receptor status [16]. These cell lines included
MCF-7 (expressing estrogen and progesterone receptors), T47D (expressing estrogen,
progesterone and androgen receptors), MDA-MB-361 (expressing estrogen and progesterone
receptors and HER2), and a triple-negative cell line MDA-MB-231.
The antiproliferative activities of the compounds depended on the nature of the protecting
group at the 3-hydroxy function and on the steric orientation of the substituents at C-16 and
25

C-17. The 3-methyl ethers (17a, 18a, 25a, 26a) did not influence the proliferation of the
breast cancer cell lines markedly. Nevertheless, HeLa and A2780 seemed to be more sensitive
to these compounds. 17α-Hydroxy derivatives (18a and 25a) displayed outstanding cell
growth-inhibitory properties against HeLa with IC50 values in the low micromolar range. The
16α,17β-diastereomer (17a) seemed to be the least potent in the methyl ether series, exerting
remarkable antiproliferative action on HeLa and A2780 with IC50 values over 20 µM. This is
in good agreement with the findings of Poirier et al. showing that in the 13-epimeric 3,17-
estradiol series the 13α-methyl-3,17α-diol inhibits the proliferation of certain cancer cell lines
mostly [15].
The 3-benzyl ethers (19a, 20a, 27a, 28a), on the whole, proved to be more potent in
comparison with their 3-methyl ether counterparts (17a, 18a, 25a, 26a). These compounds
(19a, 20a, 27a, 28a) displayed moderate inhibitory properties with IC50 values in the range of
10–20 µM against the panel of breast cancer cell lines. The receptor status of the cells seems
irrelevant, since no considerable cell line dependent differences between the IC50 values were
observed. The 16β,17β-isomer (28a) may be concerned as the most active on the breast panel,
because it displayed similar good potency against all four cell lines. All 3-benzyl ethers (19a,
20a, 27a, 28a) inhibited the proliferation of HeLa and A2780 cell lines markedly. The
16β,17α-diastereomer (20a) was found to be the most potent and selective compound with an
IC50 value of 3.36 µM against HeLa and 7.74 µM against A2780.
The two most potent 3-benzyl ethers (19a and 28a) were selected for cancer selectivity
investigations by means of the MTT assay, using the non-cancerous human fibroblast cell line
MRC-5. Compounds 19a and 28a inhibited the proliferation of MRC-5 cell line dose-
dependently. The 16β,17β-isomer (28a) proved to be tumor-selective by displaying IC50
values at least two times higher on healthy fibroblast cells than on cancer cell lines. The
16β,17β-isomer (28a) exerted a similar feature on fibroblasts in comparison with breast
cancer cell lines but only a limited cancer selectivity could be identified when compared its
action on HeLa or A2780 cells with that on MRC-5 cell line.
Table 1
Acknowledgments
The work of Anita Kiss was supported by a PhD Fellowship of the Talentum Fund of Richter
Gedeon Plc. (Budapest). Financial support from the Economic Development and Innovation
26

Operative Programme of Hungary (GINOP-2.3.2-15-2016-00038) is gratefully
acknowledged. This research was supported by the Hungarian Scientific Research Fund
(OTKA K113150).
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Legends for Schemes and Tables
Scheme 1. Reagents and conditions: (i) NaOMe, HCOOEt, anhydrous toluene, 50 оC; (ii) KBH4,
MeOH; (iii) TsCl, pyridine; (iv) KOAc, AcOH
Scheme 2. Reagents and conditions: (i) 1,2-phenylenediamine, AcOH, 110 оC; (ii) NaOMe, HCOOEt,
anhydrous toluene, 50 оC; (iii) KBH4, MeOH; (iv) Ac2O, pyridine; (v) CrO3, acetone; (vi) Li(OtBu)3H,
diethyl ether; (vii) NaBH4, MeOH; (viii) acetone, TsOH; (ix) EtOH, PhB(OH)2.
Scheme 3. Reagents and conditions: (i) TsCl, pyridine; (ii) NaOMe, MeOH.
Table 1. Antiproliferative activities of compounds 17a20a and 25a28a.
29