Syntheses and antiproliferative effects of d-homo- and d-secoestrones

Article abstract of DOI:10.1016/j.steroids.2014.05.015

Substituted and/or heterocyclic d-homoestrone derivatives were synthetized via the intramolecular cyclization of a δ-alkenyl-d-secoaldehyde, -d-secoalcohol or -d-secocarboxylic acid of estrone 3-benzyl ether. The d-secoalcohol was modified at three sites in the molecule. The in vitro antiproliferative activities of the new d-homo- and d-secoestrone derivatives were determined on HeLa, MCF-7, A431 and A2780 cells through use of MTT assay. d-Homoalcohols 3 and 5 displayed cell line-selective cytostatic effects against ovarian and cervical cell lines, respectively. Two d-secoestrones (6 and 12c) proved to be effective, with IC₅₀ values comparable with those of the reference agent cisplatin. A selected compound (6) was tested by tubulin polymerization assay and its cancer specificity was additionally determined by using noncancerous human fibroblast cells.

Full text of DOI:10.1016/j.steroids.2014.05.015

Accepted Manuscript
Syntheses and Antiproliferative Effects of D-homo- and D-secoestrones
Erzsébet Mernyák, Johanna Szabó, Judit Huber, Gyula Schneider, Renáta
Minorics, Noémi Bózsity, István Zupkó, Mónika Varga, Zsolt Bikádi, Eszter
Hazai, János Wölfling
PII:
S0039-128X(14)00128-7
http://dx.doi.org/10.1016/j.steroids.2014.05.015
STE 7577
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 February 2014
13 May 2014
25 May 2014
Please cite this article as: Mernyák, E., Szabó, J., Huber, J., Schneider, G., Minorics, R., Bózsity, N., Zupkó, I.,
Varga, M., Bikádi, Z., Hazai, E., Wölfling, J., Syntheses and Antiproliferative Effects of D-homo- and D-
secoestrones, Steroids (2014), doi: http://dx.doi.org/10.1016/j.steroids.2014.05.015
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Syntheses and Antiproliferative Effects of D-homo- and D-secoestrones
Erzsébet Mernyáka,*, Johanna Szabóa, Judit Hubera, Gyula Schneidera, Renáta Minoricsb,
Noémi Bózsityb, István Zupkób, Mónika Vargac, Zsolt Bikádid, Eszter Hazaid, János Wölflinga
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 Szeged, Hungary
c Cereal Research Non-Profit LTD, P.O.Box 391, H-6701 Szeged, Hungary
d Virtua Drug Ltd, Csalogány u. 4C, H-1015 Budapest, Hungary..
Fax: +36(62)544200
E-mail: bobe@chem.u-szeged.hu

Abstract
Substituted and/or heterocyclic D-homoestrone derivatives were synthetized via the
intramolecular cyclization of
a
δ-alkenyl-D-secoaldehyde, -D-secoalcohol or -D-
secocarboxylic acid of estrone 3-benzyl ether. The D-secoalcohol was modified at three sites
in the molecule. The in vitro antiproliferative activities of the new D-homo- and D-
secoestrone derivatives were determined on HeLa, MCF-7, A431 and A2780 cells through use
of MTT assay. D-Homoalcohols 3 and 5 displayed cell line-selective cytostatic effects against
ovarian and cervical cell lines, respectively. Two D-secoestrones (6 and 12c) proved to be
effective, with IC50 values comparable with those of the reference agent cisplatin. A selected
compound (6) was tested by tubulin polymerization assay and its cancer specificity was
additionally determined by using noncancerous human fibroblast cells.

Keywords
Homoestrone, Secoestrone, Antiproliferative effect, MTT assay, Tubulin polymerization.

1. Introduction
Structural modifications of 17β-estradiol, a proliferation-inducing compound [1], may lead to
antiproliferative estrone derivatives [2,3]. Substitution at position 2 and/or D-ring expansion
usually reduces the estrogeneity, but enhances the antitumor behavior. Hillisch et al. recently
patented antimitotic 2-substituted D-homoestrone-3-sulfamates [4]. We have found that
unsubstituted D-homoestrone exerts a selective cytostatic effect on HeLa cells (IC50 = 5.5
µM), causing cell cycle arrest in the G2/M phase and inducing apoptosis, while its 3-methoxy
counterpart has no impact on the proliferation of human reproductive cancer cell lines (HeLa,
MCF-7 and A2780) [5]. We earlier reported an efficient route for the production of D-
homoestrone from natural estrone, via the key δ-alkenyl-D-secoestrone-3-benzyl ether 17-
carbaldehyde (1) [6]. The latter derivative readily takes part in ring-closure reactions due to
the favorable position of its aldehyde and alkenyl moieties. In a preliminary publication, we
described that reduction or oxidation of the aldehyde function of the 3-methoxy counterpart of
the secoaldehyde leads to a secoalcohol or a secocarboxylic acid, which furnish 16-
halomethyl-17-oxa derivatives in haloetherification or halolactonization processes [7]. In the
3-benzyl ether series only iodoetherification of the 3-benzyl-secoalcohol was carried out in
order to synthetize cytotoxic estrone-talaromycin hybrids [8]. The resulting diastereomeric
mixture of iodomethyl tetrahydropyrans was not separated as the new stereogenic center was
lost in the following reaction step. In in vitro cytotoxicity tests of the natural product hybrids
on A549 human lung cancer cells, the effective dosage for the most potent derivative proved
comparable with that of the well-known anticancer agent cyclophosphamide. In a
continuation of our earlier work, we now set out to produce 3-benzyloxy-D-homoestrone
derivatives by the elaborated synthetic routes [6,7], extending the haloetherification and
halolactonization with selenoetherification and selenolactonization. The 3-benzyl ether
protecting group was preferred because of its easy removal. We planned to examine whether
the synthetized D-homoestrones themselves have antiproliferative properties, without the
formation of steroid-toxin hybrids. The potential antitumor activities of the newly synthetized
D-homo derivatives were tested in vitro on four human reproductive cancer cell lines (HeLa,
MCF-7, A2780 and Ishikawa) and one epidermoid tumor cell line (A431). Examination of the
influence of the protecting group on C-3 and the substituent on C-16 on the antiproliferative
properties was also projected. The tumor specificity of the potent compounds was investigated
on noncancerous human foreskin fibroblast cells.
Besides the synthesis and pharmacological testing of the D-homoestrones, we planned to
investigate the precursors of the cyclization reactions and their derivatives. The 3-benzyloxy
secoalcohol was modified at three sites in the molecule: the alcoholic and/or phenolic hydroxy
moiety and the alkenyl side-chain. The hydroxy groups were esterified under solvent-free
microwave irradiation. The unsaturated side-chain was saturated under the conditions used for
hydrogenolysis of the benzyl ether function.

2. Experimental
2.1. Chemistry
Melting points (mp) were determined with a Kofler hot-stage apparatus and are uncorrected.
Elemental analyses were performed with a Perkin-Elmer CHN analyzer model 2400. Thin-
layer chromatography: silica gel 60 F254; layer thickness 0.2 mm (Merck); eluents: (A)
diisopropyl ether, (B) dichloromethane, (C) 70% dichloromethane/30% hexane, (D) 2% ethyl
acetate/98% dichloromethane; detection with iodine or UV (365 nm) after spraying with 5%
phosphomolybdic acid in 50% aqueous phosphoric acid and heating at 100–120 °C for 10
min. Flash chromatography: silica gel 60, 40–63 µm (Merck). The reactions under microwave
irradiation were carried out with a CEM Corporation focused microwave system, Model
1
Discover SP. H NMR spectra were recorded in CDCl3 solution (if not otherwise stated) with
a Bruker DRX-500 instrument at 500 MHz, with Me4Si as internal standard. 13C NMR spectra
were recorded with the same instrument at 125 MHz under the same conditions. Full scan
mass spectra of the compounds were acquired in the range 50 to 800 m/z with an Agilent
500MS Ion trap mass spectrometer equipped with an electrospray ionization source. Analyses
were performed in positive ion mode. The spectra were collected by continuous infusion of
the steroid solution at 10 ng/µl in 1:1 acetonitrile/5 mM ammonium formate (v/v %) at a flow
rate of 15 µl/min.
2.1.1. General procedure for the preparation of 17a-hydroxy-D-homoestrone derivatives 3 and
5
Compound 2 [6] (372 mg, 1.00 mmol) or 4 [6] (284 mg, 1.00 mmol) was dissolved in
methanol (20 ml), and potassium borohydride (270 mg, 5.00 mmol) was added in small
portions in an ice-water bath. The mixture was allowed to stand at room temperature for 3 h,
then poured onto ice (20 g) and acidified with dilute hydrochloric acid to pH 3. The
precipitate that separated out was filtered off, washed until free from acid and dried. The
crude product was subjected to column chromatography.
2.1.1.1. 3-Benzyloxy-17aβ-hydroxy-D-homoestra-1,3,5(10),16-tetraene (3)
According to section 2.1.1, compound 2 (372 mg, 1.00 mmol) was reacted with potassium
borohydride (270 mg, 5.00 mmol). After work-up, the crude product was subjected to column
chromatography with dichloromethane as eluent. 3 was obtained as a white solid (355 mg,
95%). Mp 202-205 °C, Rf = 0.40A. Anal. Calcd. for C26H30O2: C, 83.38; H, 8.07. Found: C,
83.52; H, 7.95.
1H NMR δ ppm 0.83(s, 3H, 18-H3), 2.83(m, 2H, 6-H2), 3.94(s, 1H, 17a-H), 5.04(s, 2H,
OCH2), 5.52(m, 1H) and 5.74(m, 1H): 16-H and 17-H, 6.71(d, 1H, J = 2.3 Hz, 4-H), 6.78(dd,
1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.23(d, 1H, J = 8.5 Hz, 1-H), 7.24(m, 1H, 4’-H), 7.34(m,
2H, 3’-H and 5’-H), 7.42(m, 2H, 2’-H and 6’-H). 13C NMR δ ppm 11.0(C-18), 26.2(2C), 27.1,
30.1, 37.4(C-13), 37.5, 39.8, 43.0, 43.8, 70.0(OCH2), 78.1(C-17a), 112.4(C-2), 114.5(C-4),
126.4(C-1), 127.4(2C: C-2’,6’), 127.7, 127.8(C-4’), 128.5(2C: C-3’,5’), 130.1, 133.0(C-10),
137.3(C-1’), 137.9(C-5), 156.8(C-3). MS m/z (%): 375 (100), 397 (54), 91 (42).
2.1.1.2. 3,17aβ-Dihydroxy-D-homoestra-1,3,5(10)-triene (5)
According to section 2.1.1, compound 4 (284 mg, 1.00 mmol) was reacted with potassium
borohydride (270 mg, 5.00 mmol). After work-up, the crude product was subjected to column
chromatography with dichloromethane as eluent. 5 was obtained as a white solid (243 mg,
85%). Melting point 208-209 ºC [9]: mp 209–210 ºC. Compound 5 is identical with the
compound described in [9]. Anal. Calcd. for C19H26O2: C, 79.68; H, 9.15. Found: C, 79.92; H,
9.05.
1H NMR (DMSO-d6) δ ppm 0.73(s, 3H, 18-H3), 2.67(m, 2H, 6-H2), 4.29(m, 1H, 17a-H),

6.42(m, 1H, 4-H), 6.50(m, 1H, 2-H), 7.05(m, 1H, 1-H), 8.96(s, 1H, 3-OH).
2.1.2. 3-Benzyloxy-13α-hydroxymethyl-14β-(prop-2-en-yl)-des-D-estra-1,3,5(10)-triene (6)
Compound 1 (374 mg, 1.00 mmol) was dissolved in a 1:1 mixture of dichloromethane and
methanol, and potassium borohydride (270 mg, 5.00 mmol) was added in small portions in an
ice-water bath. The mixture was allowed to stand at room temperature for 0.5 h, then diluted
with water and extracted with dichloromethane. The combined organic phases were washed
with water until neutral and dried over sodium sulfate, and the crude product was subjected to
column chromatography with dichloromethane as eluent. 6 was obtained as a white solid (327
mg, 87%). Oil, Rf = 0.53D. Anal. Calcd. for C26H32O2: C, 82.94; H, 8.57. Found: C, 83.05; H,
1
8.45. H NMR δ ppm 0.80(s, 3H, 18-H3), 2.85(m, 2H, 6-H2), 3.30 and 3.61(2xm, 2x1H, 17-
H2), 5.03(m, 2H, 16a-H2), 5.04(s, 2H, OCH2), 5.93(m, 1H, 16-H), 6.73(d, 1H, J = 2.3 Hz, 4-
H), 6.79(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.22(d, 1H, J =8.5 Hz, 1-H), 7.32(t, 1H, J = 7.3
Hz, 4’-H), 7.38(t, 2H, J = 7.3 Hz, 3’-H and 5’-H), 7.43(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), 13C
NMR δ ppm 16.2(C-18), 26.2, 27.6, 30.4, 32.4, 35.9, 38.9(C-13), 41.2, 43.5, 44.3,
70.0(OCH2), 71.0(C-17), 112.4(C-2), 114.3(C-16a), 114.4(C-4), 126.4(C-1), 127.4(2C: C-
2’,6’), 127.8(C-4’), 128.5(2C: C-3’,5’), 133.1(C-10), 137.3(C-1’), 138.0(C-5), 140.5(C-16),
156.8(C-3). MS m/z (%): 431 (100).
2.1.3. 3-Benzyloxy-14β-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-13α-carboxylic acid (7)
and 3-benzyloxy-14β-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-13α,9α-carbolactone (8)
Compound 1 (374 mg, 1.00 mmol) was dissolved in acetone (3 ml), then cooled in an ice-
water bath and Jones reagent (0.4 ml, 8 N) was added in three portions. The reaction mixture
was allowed to stand at room temperature for 4 h, then diluted with water and extracted with
diethyl ether. The combined organic phases were washed with water until neutral and dried
over sodium sulfate, and the crude product was subjected to column chromatography with
dichloromethane as eluent. The first-eluted 8 was obtained as a white solid (144 mg, 37%).
Mp 110-118 °C, Rf = 0.63D. Anal. Calcd. For C26H28O3: C, 80.38; H, 7.26. Found: C, 80.45;
H, 7.12. 1H NMR δ ppm 1.22(s, 3H, 18-H3), 2.73(m, 2H, 6-H2), 5.05(s, 2H, OCH2), 5.09–
5.16(overlapping multiplets, 2H, 16a-H2), 5.85(m, 1H, 16-H), 6.71(d, 1H, J = 2.3 Hz, 4-H),
6.85(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.26(d, 1H, J = 8.5 Hz, 1-H), 7.34(t, 1H, J = 7.3
Hz, 4’-H), 7.39(t, 2H, J = 7.3 Hz, 3’-H and 5’-H), 7.42(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), 13C
NMR δ ppm 18.6(C-18), 25.3, 26.8, 30.1, 31.9, 34.5, 40.4(C-13), 44.7, 45.2, 69.9(OCH2),
80.6(C-9), 113.5(C-2), 114.3(C-4), 117.0(C-16a), 127.4(2C: C-2’,6’), 127.9(C-4’), 128.5(2C:
C-3’,5’), 129.1(C-1), 130.2 (C-10), 136.2(C-16), 136.9(C-1’), 139.3 (C-5), 158.7(C-3),
178.2(C-17). MS m/z (%): 446 (100), 411 (42).
Continued elution yielded 7, which was obtained as a white solid (223 mg, 57%). Mp 120-122
1
°C. Rf = 0.32D. Anal. Calcd. for C26H30O3: C, 79.97; H, 7.74. Found: C, 80.03; H, 7.68., H
NMR δ ppm 1.19(s, 3H, 18-H3), 2.84(m, 2H, 6-H2), 5.01(m, 2H, 16a-H2), 5.05(s, 2H, OCH2),
5.88(m, 1H, 16-H), 6.73(d, 1H, J = 2.3 Hz, 4-H), 6.80(dd, 1H, J = 8.5 Hz, J= 2.3 Hz, 2-H),
7.20(d, 1H, J = 8.5 Hz, 1-H), 7.32(t, 1H, J = 7.3 Hz, 4’-H), 7.39(t, 2H, J = 7.3 Hz, 3’-H and
13
5’-H), 7.43(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), C NMR δ ppm 15.0(C-18), 25.8, 27.1, 28.7,
30.2, 35.7, 37.4, 40.9, 43.0, 45.4, 47.4(C-13), 70.0(OCH2), 112.5(C-2), 114.5(C-4), 115.3(C-
16a), 126.4(C-1), 127.4(2C: C-2’,6’), 127.9(C-4’), 128.5(2C: C-3’,5’), 132.4(C-10), 137.2(C-
1’), 137.9(C-5), 138.8(C-16), 156.8(C-3), 184.5(C-17). MS m/z (%): 435 (100).
2.1.4. 3-Benzyloxy-16β-bromomethyl-17-oxa-D-homoestra-1,3,5(10)-triene (9a)
Compound 6 (188 mg, 0.50 mmol) was dissolved in dichloromethane (5 ml), then cooled in
an ice-salt bath to -15 ºC, and N-bromosuccinimide (89 mg, 0.50 mmol) was added in three

portions. The mixture was stirred for 30 min, the solvent was evaporated off and the residue
was subjected to column chromatography with 70% dichloromethane/30% hexane, yielding
9a as a white solid (155 mg, 68%). Mp 107–108 °C, Rf = 0.51C. Anal. Calcd. for C26H31BrO2:
1
C, 68.57; H, 6.86. Found: C, 68.82; H, 6.75. H NMR δ ppm 1.01(s, 3H, 18-H3), 2.84(m, 2H,
6-H2), 3.17(m, 1H) and 3.58(overlapping multiplets, 2H): 17-H2 and 16-H, 3.46(m, 2H, 16a-
H2), 5.04(s, 2H, OCH2), 6.73(d, 1H, J = 2.3 Hz, 4-H), 6.79(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-
H), 7.21(d, 1H, J = 8.5 Hz, 1-H), 7.30(t, 1H, J = 7.3 Hz, 4’-H), 7.37(t, 2H, J = 7.3 Hz, 3’-H
and 5’-H), 7.42(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), 13C NMR δ ppm 16.5(C-18), 25.5, 25.7,
28.8, 29.9, 34.0(C-13), 35.1, 36.2, 38.5, 43.7, 47.2, 69.9(OCH2), 77.6(C-16), 79.9(C-17),
112.4(C-2), 114.6(C-4), 126.1(C-1), 127.4(2C: C-2’,6’), 127.8(C-4’), 128.5(2C: C-3’,5’),
132.8(C-10), 137.3(C-1’), 137.8(C-5), 156.8(C-3). MS m/z (%): 457 (63), 455 (100), 453
(37).
2.1.5. 3-Benzyloxy-16β-iodomethyl-17-oxa-D-homoestra-1,3,5(10)-triene (9b) and 3-
benzyloxy-16α-iodomethyl-17-oxa-D-homoestra-1,3,5(10)-triene (10b)
Compound 6 (188 mg, 0.50 mmol) was dissolved in diethyl ether (2 ml), and water (0.7 ml),
sodium hydrogencarbonate (84 mg, 1.00 mmol) and iodine (127 mg, 0.50 mmol) were then
added. The mixture was stirred for 1 h at room temperature, diluted with water and extracted
with diethyl ether. The organic phase was separated and washed with a saturated solution of
sodium thiosulfate and then with water. The organic phase was dried over sodium sulfate and
evaporated, and the residue was subjected to column chromatography with 70%
dichloromethane/30% hexane. 9b eluted first as a white solid (127 mg, 50%). Mp 105–108
°C, Rf = 0.51C. Anal. Calcd. for C26H31IO2: C, 62.15; H, 6.22. Found: C, 62.02; H, 6.35. 1H
NMR δ ppm 1.01(s, 3H, 18-H3), 2.85(m, 2H, 6-H2), 3.18 and 3.58(2xm, 2x1H, 17-H2), 3.26–
3.33(overlapping multiplets, 3H, 16a-H2 and 16-H), 5.04(s, 2H, OCH2), 6.73(d, 1H, J = 2.3
Hz, 4-H), 6.79(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.21(d, 1H, J = 8.5 Hz, 1-H), 7.32(t, 1H,
J = 7.3 Hz, 4’-H), 7.38(t, 2H, J = 7.3 Hz, 3’-H and 5’-H), 7.43(d, 2H, J = 7.3 Hz, 2’-H and 6’-
H), 13C NMR δ ppm 10.6(C-16a), 16.7(C-18), 25.4, 25.7, 29.9, 30.5, 33.9(C-13), 35.0, 38.5,
43.7, 47.2, 69.9(OCH2), 77.4(C-16), 79.9(C-17), 112.4(C-2), 114.6(C-4), 126.1(C-1),
127.4(2C: C-2’,6’), 127.8(C-4’), 128.5(2C: C-3’,5’), 132.8(C-10), 137.3(C-1’), 137.8(C-5),
156.8(C-3). MS m/z (%): 504 (32), 503 (100), 214 (21), 113 (26).
Continued elution yielded 10b as a white solid (64 mg, 25%). Mp 112–113 °C, Rf = 0.47C.
1
Anal. Calcd. for C26H31IO2: C, 62.15; H, 6.22. Found: C, 62.28; H, 6.18. H NMR δ ppm
1.02(s, 3H, 18-H3), 2.84(m, 2H, 6-H2), 3.20(m, 2H, 16a-H2), 3.40 and 3.56(2xm, 2x1H, 17-
H2), 4.23 (m, 1H, 16-H), 5.04(s, 2H, OCH2), 6.72(d, 1H, J = 2.3 Hz, 4-H), 6.79(dd, 1H, J =
8.5 Hz, J = 2.3 Hz, 2-H), 7.24(d, 1H, J = 8.5 Hz, 1-H), 7.32(t, 1H, J = 7.3 Hz, 4’-H), 7.38(t,
2H, J = 7.3 Hz, 3’-H and 5’-H), 7.43(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), 13C NMR δ ppm
6.9(C-16a), 15.9(C-18), 25.3, 25.6, 29.8, 30.5, 34.2(C-13), 35.5, 38.3, 41.7, 43.7,
69.9(OCH2), 72.5(C-17), 73.7(C-16), 112.4(C-2), 114.6(C-4), 126.0(C-1), 127.4(2C: C-2’,6’),
127.8(C-4’), 128.5(2C: C-3’,5’), 132.8(C-10), 137.3(C-1’), 137.8(C-5), 156.8(C-3). MS m/z
(%): 504 (30), 503 (100).
2.1.6. 3-Benzyloxy-16β-phenylselenylmethyl-17-oxa-D-homoestra-1,3,5(10)-triene (9c) and
3-benzyloxy-16α-phenylselenylmethyl-17-oxa-D-homoestra-1,3,5(10)-triene (10c)
Compound 6 (188 mg, 0.50 mmol) was dissolved in acetonitrile (5 ml), and phenylselenyl
bromide (118 mg, 0.50 mmol) was added in three portions. After stirring for 1 h at room
temperature, potassium carbonate (70 mg, 0.50 mmol) was added and the stirring was
continued for 1 h. The solvent was evaporated off and the residue was subjected to column
chromatography with 70% dichloromethane/30% hexane. 9c eluted first as a white solid (192
mg, 36%). Mp 135–137 °C, Rf = 0.47C. Anal. Calcd. for C32H36O2Se: C, 72.30; H, 6.83.

Found: C, 72.44; H, 6.95. 1H NMR δ ppm 1.01(s, 3H, 18-H3), 2.83(m, 2H, 6-H2), 3.06 (m,
1H) and 3.14(overlapping multiplets, 2H) and 3.55(overlapping multiplets, 2H): 16-H and
16a-H2 and 17-H2, 5.04(s, 2H, OCH2), 6.72(d, 1H, J = 2.3 Hz, 4-H), 6.79(dd, 1H, J = 8.5 Hz,
J = 2.3 Hz, 2-H), 7.21(d, 1H, J = 8.5 Hz, 1-H), 7.24–7.28 (overlapping multiplets, 3H) and
7.54 (m, 2H): 2”,3”,4”,5”,6”-H, 7.32(t, 1H, J= 7.3 Hz, 4’-H), 7.38(t, 2H, J = 7.3 Hz, 3’-H and
13
5’-H), 7.43(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), C NMR δ ppm 16.6(C-18), 25.5, 25.7, 29.9,
30.3, 33.8, 34.0(C-13), 35.2, 38.5(C-8), 43.7(C-9), 47.4(C-14), 69.9(OCH2), 77.9(C-16),
80.0(C-17), 112.4(C-2), 114.6(C-4), 126.1(C-1), 126.7(2C: C-2’,6’), 127.4(2C: C-3’,5’),
127.8(C-4’), 128.5(2C: C-3”,5”), 129.0(C-4”), 130.8(C-1”), 132.5(2C: C-2”,6”), 132.9(C-10),
137.3(C-1’), 137.8(C-5), 156.8(C-3). MS m/z (%): 550 (34), 549 (100).
Continued elution yielded 10c (192 mg, 36%). Mp 142–143 °C, Rf = 0.40C. Anal. Calcd. for
C32H36O2Se: C, 72.30; H, 6.83. Found: C, 72.38; H, 6.95. 1H NMR δ ppm 1.00(s, 3H, 18-H3),
2.80(m, 2H, 6-H2), 3.11 and 3.40(2xm, 2x1H, 16a-H2), 3.19 and 3.32(2xd, 2x1H,
J = 11.2 Hz, 17-H2), 4.24(m, 1H, 16-H), 5.03(d, 2H, OCH2), 6.71(d, 1H, J = 2.3 Hz, 4-H),
6.78(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.19(d, 1H, J = 8.5 Hz, 1-H), 7.22–7.26
(overlapping multiplets. 3H): 3”,4”,5”-H, 7.32(t, 1H, J = 7.2 Hz, 4’-H), 7.38(t, 2H, J = 7.2
Hz, 3’-H and 5’-H), 7.43(d. 2H, J = 7.2 Hz, 2’-H and 6’-H), 7.54 (m, 2H, 2”-H and 6”-H), 13C
NMR δ ppm 16.1(C-18), 25.4, 25.5, 25.9, 29.4(C-16a), 29.8(C-6), 34.2, 35.6, 38.4(C-8),
41.8(C-9), 43.7(C-14), 69.9(OCH2), 72.8(C-17), 73.1(C-16), 112.4(C-2), 114.6(C-4),
126.1(C-1), 126.9(2C: C-2’,6’), 127.4(2C: C-3’,5’), 127.8(C-4’), 128.5(2C: C-3”,5”),
12.91(C-4”), 130.1(C-1”), 132.7(2C: C-2”,6”), 132.9(C-10), 137.3(C-1’), 137.9(C-5),
156.8(C-3). MS m/z (%): 549 (100).
2.1.7. 3-Benzyloxy-16β-bromomethyl-17-oxa-D-homoestra-1,3,5(10)-trien-17a-one (11a)
Compound 7 (195 mg, 0.50 mmol) was dissolved in dichloromethane (5 ml), and N-
bromosuccinimide (89 mg, 0.50 mmol) was added. The mixture was stirred at room
temperature for 1 h. The solvent was evaporated off and the residue was subjected to column
chromatography with dichloromethane as eluent. 11a was obtained (193 mg, 82%). Mp 150–
155 °C, Rf = 0.63D. Anal. Calcd. for C26H29BrO3: C, 66.53; H, 6.23. Found: C, 66.72; H, 6.35.
1H NMR δ ppm 1.26(s, 3H, 18-H3), 2.88(m, 2H, 6-H2), 3.56–3.62(overlapping multiplets, 2H,
16a-H2), 4.54(m, 1H, 16-H), 5.04(s, 2H, OCH2), 6.74(d, 1H, J = 2.3 Hz, 4-H), 6.80(dd, 1H, J
= 8.5 Hz, J = 2.3 Hz, 2-H), 7.22(d, 1H, J = 8.5 Hz, 1-H), 7.32(t, 1H, J = 7.3 Hz, 4’-H), 7.38(t,
2H, J = 7.3 Hz, 3’-H and 5’-H), 7.42(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), 13C NMR δ ppm
18.5(C-18), 25.4, 26.2, 26.3, 29.7, 34.2, 35.3, 37.7, 41.3(C-13), 42.6, 43.1, 70.0(OCH2),
78.3(C-16), 112.6(C-2), 114.6(C-4), 126.2 and 127.4(2C) and 127.9 and 128.5(2C): C-
1,2’,3’,4’,5’,6’, 131.9(C-10), 137.2(C-1’), 137.4(C-5), 157.0(C-3), 176.3(C-17). MS m/z (%):
491 (63), 469 (100).
2.1.8. 3-Benzyloxy-16β-iodomethyl-17-oxa-D-homoestra-1,3,5(10)-trien-17a-one (11b)
Compound 7 (195 mg, 0.50 mmol) was dissolved in a 1:1 mixture (5 ml) of dichloromethane
and tetrahydrofuran, and saturated sodium hydrogencarbonate solution (10 ml, pH = 9) and
iodine (127 mg, 0.50 mmol) were added. The mixture was stirred for 1 h at room temperature,
diluted with water and extracted with dichloromethane. The organic phase was separated out
and washed with a saturated solution of sodium thiosulfate and with water. The organic phase
was dried over sodium sulfate and evaporated, and the residue was subjected to column
chromatography with dichloromethane as eluent. 11b was obtained (217 mg, 84%). Mp 158–
160 °C, Rf = 0.66D. Anal. Calcd. for C26H29IO3: C, 60.47; H, 5.66. Found: C, 60.32; H, 5.75.
1H NMR δ ppm 1.26(s, 3H, 18-H3), 2.89(m, 2H, 6-H2), 3.43(m, 2H, 16a-H2), 4.22(m, 1H, 16-
H), 5.04(s, 2H, OCH2), 6.73(d, 1H, J = 2.3 Hz, 4-H), 6.80(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-
H), 7.21(d, 1H, J = 8.5 Hz, 1-H), 7.32(t, 1H, J = 7.3 Hz, 4’-H), 7.38(t, 2H, J = 7.3 Hz, 3’-H

and 5’-H), 7.42(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), 13C NMR δ ppm 9.3(C-16a), 18.7(C-18),
25.4, 26.3, 28.2, 29.7, 34.2, 37.7, 41.2(C-13), 42.7, 43.2, 69.9(OCH2), 78.3(C-16), 112.6(C-
2), 114.6(C-4), 126.2 and 127.4(2C) and 127.9 and 128.5(2C): C-1,2’,3’,4’,5’,6’, 131.9(C-
10), 137.2(C-1’), 137.3(C-5), 156.9(C-3), 176.4(C-17). MS m/z (%): 517 (58), 113 (100).
2.1.9. 3-Benzyloxy-16β-phenylselenylmethyl-17-oxa-D-homoestra-1,3,5(10)-trien-17a-one
(11c)
Compound 7 (195 mg, 0.50 mmol) was dissolved in acetonitrile (5 ml), and phenylselenyl
bromide (118 mg, 0.50 mmol) was added in three portions. After stirring for 1 h at room
temperature, potassium carbonate (70 mg, 0.5 mmol) was added and the stirring was
continued for 1 h. The solvent was evaporated off and the residue was subjected to column
chromatography with dichloromethane as eluent. 11c was obtained (216 mg, 79%). Mp 175–
178 °C, Rf = 0.66D. Anal. Calcd. for C32H34O3Se: C, 70.45; H, 6.28. Found: C, 70.62; H, 6.14.
1H NMR δ ppm 1.26(s, 3H, 18-H3), 2.86(m, 2H, 6-H2), 3.15 and 3.29(2xm, 2x1H, 16a-H2),
4.52(m, 1H, 16-H), 5.04(s, 2H, OCH2), 6.73(d, 1H, J = 2.3 Hz, 4-H), 6.80(dd, 1H, J = 8.5 Hz,
J = 2.3 Hz, 2-H), 7.21(d, 1H, J = 8.5 Hz, 1-H), 7.26–7.34(overlapping multiplets, 4H:
4’,3”,4”,5”-H), 7.39(t, 2H, J = 7.3 Hz, 3’-H and 5’-H), 7.43(d, 2H, J = 7.3 Hz, 2’-H and 6’-
H), 7.56(m, 2H, 2”-H and 6”-H). 13C NMR δ ppm 18.6(C-18), 25.4, 26.2, 27.4, 29.7, 33.3,
34.2, 37.8, 41.3(C-13), 42.7, 43.5, 69.9(OCH2), 79.9(C-16), 112.2(C-2), 114.6(C-4), 126.2
and 127.4(3C) and 127.9 and 128.5(2C) and 129.3(2C): C-1,2’,3’,4’,5’,6’,3”,4”,5”, 129.6(C-
1”), 132.0(C-10), 132.8(2C: C-2”,6”), 137.2(C-1’), 137.4(C-5), 157.0(C-3), 176.8(C-17). MS
m/z (%): 569 (100), 567 (53), 501 (47), 91 (42).
2.1.10. General procedure for the preparation of derivatives 12a and 12dA suspension of 6
(188 mg, 0.50 mmol) or 12c (210 mg, 0.50 mmol) and Pd/C (0.30 g, 10%) in ethyl acetate (20
ml) was subjected to 20 bar of hydrogen pressure at room temperature for 3 h. The catalyst
was then removed by filtration through a short pad of silica gel. After evaporation of the
solvent in vacuo, the crude product was subjected to column chromatography with
dichloromethane as eluent.
2.1.10.1. 3-Hydroxy-13α-hydroxymethyl-14β-propyl-des-D-estra-1,3,5(10)-triene (12a)
According to section 2.1.10, compound 6 (188 mg, 0.50 mmol) was subjected to
hydrogenolysis. The chromatographic purification of the crude product yielded 12a (118 mg,
87%). Mp 60–62 °C, Rf = 0.17D. Anal. Calcd. for C19H28O2: C, 79.12; H, 9.78. Found: C,
1
78.95; H, 9.86. H NMR δ ppm 0.77(s, 3H, 18-H3), 2.82(m, 2H, 6-H2), 3.35 and 3.52(2xd,
2x1H, J = 10.9 Hz, 17-H2), 6.56(d, 1H, J = 2.3 Hz, 4-H), 6.63(dd, 1H, J = 8.5 Hz, J = 2.3 Hz,
2-H), 7.16(d, 1H, J = 8.5 Hz, 1-H), 13C NMR δ ppm 14.7 and 15.9: C-18 and Ac-Me, 25.0,
26.4, 27.4, 30.5, 31.2, 35.6, 38.8(C-13), 41.8, 43.5, 45.3, 71.4(C-17), 112.8(C-2), 114.9(C-4),
126.7(C-1), 132.9(C-10), 138.3(C-5), 153.5(C-3). MS m/z (%): 289 (100).
2.1.10.2. 13α-acetoxymethyl-3-hydroxy-14β-propyl-des-D-estra-1,3,5(10)-triene (12d)
According to section 2.1.10, compound 12c (210 mg, 0.5 mmol, described in section 2.1.11)
was subjected to hydrogenolysis. The chromatographic purification of the crude product
yielded 12d (145 mg, 88%). Mp 84–90 °C, Rf = 0.37D. Anal. Calcd. for C21H30O3: C, 76.33;
1
H, 9.15. Found: C, 76.45; H. 9.22. H NMR δ ppm 0.83(s, 3H, 18-H3), 2.09(s, 3H, Ac-Me),
2.82(m, 2H, 6-H2), 3.86 and 3.92(2xd, 2x1H, J = 11.2 Hz, 17-H2), 4.75(s, 1H, OH), 6.56(d,
1H, J = 2.3 Hz, 4-H), 6.63(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.16(d, 1H, J = 8.5 Hz, 1-H),
13C NMR δ ppm 14.5 and 15.9: C-18 and Ac-Me, 24.8, 26.3, 27.3, 30.5, 31.3, 36.0, 37.4(C-
13), 41.7, 43.4, 45.8, 72.3(C-17), 112.7(C-2), 114.9(C-4), 126.7(C-1), 132.7(C-10), 138.2(C-
5), 153.4(C-3), 170.0(Ac-CO). MS m/z (%): 385 (100).
2.1.11. General procedure for the synthesis of derivatives 12c and 12b Compound 6 (188 mg,

0.50 mmol) or 12a (145 mg, 0.50 mmol) and acetic anhydride (0.1 ml, 1 mmol) were mixed
with silica gel (500 mg), placed into a pressure tube equipped with a stirrer bar and inserted
into the cavity of a Discover SP microwave system apparatus. The mixture was heated at 160
ºC for 5 min, then poured onto water and extracted with diethyl ether. The organic phase was
washed with water until neutral, dried over anhydrous sodium sulfate, filtered and evaporated.
The crude product was subjected to column chromatography.
2.1.11.1. 3-acetoxy-13α-acetoxymethyl-14β-propyl-des-D-estra-1,3,5(10)-triene (12b)
According to section 2.1.11, compound 6 (188 mg, 0.50 mmol) was reacted with acetic
anhydride (0.1 ml, 1 mmol) in a microwave reactor. The crude product was subjected to
column chromatography with dichloromethane as eluent, yielding 12b (160 mg, 86%). Oil, Rf
= 0.69D. Anal. Calcd. for C23H32O4: C, 74.16; H, 8.66. Found: C, 74.25; H, 8.82. 1H NMR
δ ppm 0.83(s, 3H, 18-H3), 2.09 and 2.28(2xs, 2x3H, 2xAc-Me), 2.86(m, 2H, 6-H2), 3.86 and
3.92(2xd, 2x1H, J = 11.3 Hz, 17-H2), 6.79(d, 1H, J = 2.3 Hz, 4-H), 6.83(dd, 1H, J = 8.5 Hz, J
13
= 2.3 Hz, 2-H), 7.27(d, 1H, J = 8.5 Hz, 1-H), C NMR δ ppm 14.1 and 15.5 and 20.7: C-18
and 17-Ac-Me and 3-Ac-Me, 24.4, 25.8, 26.8, 30.0, 30.9, 35.6, 36.9(C-13), 40.9, 43.3, 45.4,
71.7(C-17), 118.2, 120.9, 126.2(C-4), 137.6 and 137.8: C-5 and C-10, 148.0(C-3), 169.4 and
170.9: C-17-Ac-CO and C-3-Ac-CO. MS m/z (%): 427 (100).
2.1.11.2.
13α-acetoxymethyl-3-benzyloxy-14β-(prop-2-en-yl)-des-D-estra-1,3,5(10)-triene
(12c)
According to section 2.1.11, compound 12a (145 mg, 0.50 mmol) was reacted with acetic
anhydride (0.1 ml, 1 mmol) in a microwave reactor. The crude product of 12c was subjected
to column chromatography with 2% ethyl acetate/98% dichloromethane as eluent, yielding
12c (191 mg, 91%). Mp 90–92 °C, Rf = 0.78D. Anal. Calcd. for C28H34O3: C, 80.35; H, 8.19.
1
Found: C, 80.12; H, 8.22. H NMR δ ppm 0.87(s, 3H, 18-H3), 2.09(s, 3H, Ac-Me), 2.85(m,
2H, 6-H2), 3.84 and 4.00(2xd, 2x1H, J = 11.2 Hz, 17-H2), 4.97(m, 2H, 16a-H2), 5.04(s, 2H,
OCH2), 5.84(m, 1H, 16-H), 6.72(d, 1H, J = 2.3 Hz, 4-H), 6.79(dd, 1H, J = 8.5 Hz, J = 2.3 Hz,
2-H), 7.21(d, 1H, J = 8.5 Hz, 1-H), 7.30(t, 1H, J = 7.3 Hz, 4’-H), 7.38(t, 2H, J = 7.3 Hz, 3’-H
and 5’-H), 7.43(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), 13C NMR δ ppm 16.2 and 20.9: C-18 and
Ac-Me, 26.2, 27.5, 30.4, 32.7, 36.3, 37.6(C-13), 41.3, 43.4, 44.8, 69.9(OCH2), 71.9(C-17),
112.4(C-2), 114.4(C-16a), 114.5(C-4), 126.4(C-1), 127.4(2C: C-2’,6’), 127.8(C-4’),
128.5(2C: C-3’,5’), 132.9(C-10), 137.3(C-1’), 137.9(C-5), 139.7(C-16), 156.8(C-3), 171.2(C-
17). MS m/z (%): 431 (100).
2.1.12. Synthesis of 3-benzyloxy-13α-(p-tolylsulfonyloxymethyl)-14β-(prop-2-en-yl)-des-D-
estra-1,3,5(10)-triene (12e)
Compound 6 (188 mg, 0.50 mmol) was dissolved in pyridine (3 ml), then cooled in an ice-
water bath and 286 mg (1.50 mmol) of p-tolylsulfonyl chloride was added in small portions.
After stirring for 3 h at room temperature, the reaction mixture was diluted with water (50 ml)
and extracted with dichloromethane. The organic phase was washed with water until neutral,
dried over anhydrous sodium sulfate, filtered and evaporated. The crude product was
subjected to flash chromatography with dichloromethane as eluent, yielding 12e (252 mg,
95%). Mp 105–110 °C, Rf = 0.82D. Anal. Calcd. for C33H38O4S: C, 74.68; H, 7.22. Found: C,
1
74.83; H, 7.35. H NMR δ ppm 0.81(s, 3H, 18-H3), 2.47(s, 3H, Ts-Me), 2.82(m, 2H, 6-H2),
3.74 and 3.90(2xd, 2x1H, J = 9.4 Hz, 17-H2), 4.92(m, 2H, 16a-H2), 5.03(s, 2H, OCH2),
5.75(m, 1H, 16-H), 6.71(d, 1H, J = 2.3 Hz, 4-H), 6.78 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H),
7.18(d, 1H, J = 8.5 Hz, 1-H), 7.32(t, 1H, J = 7.3 Hz, 4’-H), 7.36–7.39(overlapping multiplets,

4H: 3’,5’,3”,5”-H), 7.43(d, 2H, J = 7.3 Hz, 2’-H and 6’-H), 7.82(d, 2H, J = 8.2 Hz, 2”-H and
13
6”-H), C NMR δ ppm 15.7 and 21.4: C-18 and Ts-Me, 25.7, 27.1, 30.1, 32.4, 35.6. 37.9(C-
13), 40.7, 42.8, 43.8, 69.7(OCH2), 76.5(C-17), 112.2(C-2), 114.2(C-4), 114.5(C-16a), 126.1
and 127.2(2C) and 127.6 and 127.7(2C) and 128.3(2C): C-1,2’,3’,4’,5’,6’,2”,6”, 129.5(2C: C-
3”,5”), 132.4(C-10), 132.7(C-1”), 137.0(C-1’), 137.6(C-5), 138.9(C-16), 144.4(C-4”),
156.5(C-3). MS m/z (%): 530 (100), 552 (45).

2.2. Antiproliferative (MTT) assay
The growth-inhibitory effects of the test compounds were determined on HeLa, MCF-7,
A2780, Ishikawa and A431 cells isolated from cervical, breast, ovarian, endometrial and skin
cancer, respectively. These cell lines were obtained from the European Collection of Cell
Cultures, Salisbury, UK. The cells were cultivated in minimal essential medium (Sigma-
Aldrich, Budapest, Hungary) supplemented with 10% fetal bovine serum, 1% non-essential
amino acids and an antibiotic-antimycotic mixture. All media and supplements were obtained
from Life Technologies (Paisley, Scotland, UK). All cell types were seeded into 96-well plates
at a density of 5000 cells/well and incubated with increasing concentrations (0.1–30 µM) of
the compounds at 37 °C under cell culturing conditions. After a 72-h incubation, cells were
treated with 5.0 mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
solution for 4 h, the precipitated formazan crystals were dissolved in dimethyl sulfoxide, and
the absorbance was read at 545 nm with a microplate reader; wells with untreated cells were
utilized as controls [10]. Sigmoidal concentration–response curves were fitted to the measured
points, and the IC50 values were calculated by means of GraphPad Prism 4 (GraphPad
Software, San Diego, CA, USA). Cisplatin was used as a positive control in the same
concentration range as the test compounds.
To determine the selective cytotoxic effect, compound 6 was subjected to the MTT assay with
intact, noncancerous, human foreskin cells under the same experimental conditions.

2.3. Tubulin polymerization assay
The effect of 3-benzyloxy-D-secoalcohol (6) on tubulin polymerization was tested with the
Tubulin Polymerization Assay Kit (Tebu-bio, Le Perray-en-Yvelines, France) according to the
manufacturer’s recommendations. Briefly, 10 µl of a 0.1 or 0.2 mM solution of the test
compound (6) was placed on a prewarmed (37 °C), UV-transparent microplate. 10 µl 10 µM
paclitaxel and 10 µl General Tubulin Buffer were used as positive and negative control,
respectively. 100 µl 3.0 mg/ml tubulin in 80 mM PIPES pH 6.9, 2 mM MgCl2, 0.5 mM
EGTA, 1 mM GTP, 10.2% glycerol was added to each sample, and the microplate was
immediately placed into a prewarmed (37 °C) UV-spectrophotometer (SpectoStarNano, BMG
Labtech, Ortenberg, Germany) to start the recording reaction. A 60-min kinetic measurement
protocol was applied to determine the absorbance of the reaction solution per minute at
340 nm. For evaluation of the experimental data, a tubulin polymerization curve was used in
which optical density was plotted against time, and the maximum reaction rate (Vmax
:
∆absorbance/min) was calculated. Differences between absorbances determined at two
consecutive timepoints were calculated and the highest difference was taken as the Vmax of the
tested compound in the tubulin polymerization reaction. Each sample was prepared in two
parallels. For statistical evaluation, Vmax data were analyzed by the unpaired Student’s t-test
by using Prism 4 software. The Vmax values of the tested compounds were compared with the
rate of the basal tubulin polymerization (i.e. the untreated control).

2.4. Docking calculations
Docking calculations were carried out according to the DockingServer methodology [11]. The
MMFF94 force field [12] was used for energy minimization of the ligand molecule by using
DockingServer. PM6 semiempirical charges calculated by MOPAC2009 [13] were ascribed to
the ligand atoms. Nonpolar hydrogen atoms were merged, and rotatable bonds were defined.
Docking calculations were carried out on the tubulin structure with the pdb code 1Z2B.
Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were
added with the aid of AutoDock tools [14]. Affinity (grid) maps of 25×25×25 Å grid points
and 0.375 Å spacing were generated by using the Autogrid program [14]. The simulation box
center was set to the center of the crystallized ligand. AutoDock parameter set- and distance-
dependent dielectric functions were used in the calculation of the van der Waals and the
electrostatic terms, respectively.
Docking simulations were performed by using the Lamarckian genetic algorithm and the Solis
& Wets local search method [15]. The initial positions, orientations and torsions of the ligand
molecules were set randomly. Each docking experiment ivolved 50 different runs that were set
to terminate after a maximum of 2.5x106 energy evaluations. The population size was set to
150. During the search, a translational step of 0.2 Å and quaternion and torsion steps of 5
were applied.

3. Results and discussion
3.1. Synthesis of D-homo- and D-secoestrones
The literature reveals several synthetic approaches for the formation of D homologs of
estrone. The total synthesis of D-homoestrone (4) was first reported by Ananchenko et al.
[16]. Semi-synthetic methods, and particularly the ring expansion of five-membered ring D of
estrone analogs, are complex, multistep reactions and therefore exhibit low efficiency [17].
We recently developed an efficient route for the synthesis of D-homoestrone (4) from a δ-
alkenyl-D-secoestrone-3-benzyl ether (1), which is available in several steps from estrone 3-
benzyl ether via a Grob fragmentation as key step [6,18]. In the present work we used the D-
secoaldehyde (1) for the synthesis of D-homoestradiol (5), as the impact of 5 on the
proliferation of the human cancer cell lines HeLa, MCF-7, A2780, Ishikawa and A430 does
not appear to have been described in the literature. The intramolecular Prins cyclization of 1
with 1.1 equivalents of p-toluenesulfonic acid and the subsequent Jones oxidation (and
simultaneous β-elimination of the tosyloxy group) led to the desired α,β-unsaturated ketone
(2, Scheme 1). The hydride reduction of 2 selectively yielded its 17aβ-hydroxy derivative (3).
During the hydrogenation of 2, hydrogenolysis of the 3-benzyl group and saturation of the
16,17-double bond occurred, leading to D-homoestrone (4), hydride reduction of which
selectively yielded 17aβ-hydroxy-D-homoestradiol (5).
Scheme 1.
Hydride reduction of the D-secoaldehyde (1) with potassium borohydride or the oxidation of
1 with Jones reagent (8 N) was carried out in order to obtain the 3-benzyloxy-D-secoalcohol
(6) and the 3-benzyloxy-D-secocarboxylic acid (7), which can be used as starting materials
for electrophile-induced ring-closure reactions (Scheme 2). Jones oxidation of the aldehyde
function in 1 was accompanied by the formation of a bridged lactone 8, similarly as described
earlier [7]. It was previously shown that the secoaldehyde in the 3-methoxy 13β-estrone and
13α-estrone series readily cyclizes and furnishes 17-oxa-D-homo-tetrahydropyrans or 17-oxa-
D-homo-δ-lactones (NBS or I2 were used as electrophile triggers). In the present work, we
carried out halo- or selenoetherification and halo- or selenolactonization reactions to obtain
16-halomethyl- or 16-phenylselenylmethyl-substituted 17-oxa-D-homo-3-benzyl ethers.
Bromoetherification of 6 with an equimolar amount of NBS in MeCN afforded 9a (Scheme 1,
Table 1, Entry 1). The reaction was carried out at -15 °C to avoid the formation of 2-bromo
derivatives by electrophile substitution. A 2:1 diastereomeric ratio was observed in the
iodoetherification with iodine, yielding a mixture of 9b and 10b (Table 1, Entry 2). The
application of phenylselenyl bromide as electrophile trigger led to a different isomeric ratio
(9c + 10c = 1:1, Table 1, Entry 3). The configuration at C-16 in the tetrahydropyrans 10 was
established from NOESY experiments, which demonstrated a crosspeak between the multiplet
of 16-H and the singlet of the angular methyl group. The orientation of the 16a substituent in
the minor diastereomers (10) is therefore the opposite (α) of that of the angular methyl group.
The electrophile-induced cyclizations of the secocarboxylic acid 7 furnished one diastereomer
11 stereoselectively (Table 1, Entries 4‒6). NOESY experiments revealed the β-position for
the 16 substituent. These results are in good agreement with the earlier observations in the 3-
methoxy series [7].
Scheme 2.
Table 1.
For functionalization of the alcoholic and phenolic hydroxy groups in the secoalcohol 6

(Figure 1, Table 2), esterification was carried out by using acetic anhydride under microwave
irradiation on a solid support, since microwave heating is a very effective and non-polluting
method of activation. Irradiation under solvent-free conditions usually leads to higher
conversions and shorter reaction times and has several environmental advantages (green
chemistry) [19]. 6 was subjected to hydrogenolysis in order to remove the benzyl protecting
group and to saturate the alkenyl side-chain (Table 2, Entry 1). The resulting diol 12a was
acetylated with acetic anhydride on a silica gel support in a microwave reactor, yielding 12b
(Table 2, Entry 2). The partially acetylated 12c was synthetized from the secoalcohol 6 by
microwave-induced acetylation (Table 2, Entry 3). Removal of the benzyl ether group from
12c led to 12d (Table 2, Entry 4). Tosylation of 6 furnished 12e (Table 2, Entry 5).
Figure 1.
Table 2.

3.2. Determination of antiproliferative properties of the newly synthetized compounds
(6–12) and the effect of secoalcohol 6 on tubulin polymerization
Microtubuli are polymers of tubulin, which in turn is a dimer involving an α unit and a β unit.
They have a wide range of biological functions, including maintenance of the cytoskeleton
and the organization of intracellular transport, but the formation of mitotic spindles is
generally regarded as the most relevant and is the most intensively investigated aspect [20]. A
variety of cytotoxic agents such as colchicine, vinca alkaloids, taxans, combrestatin and 2-
methoxyestradiol (2ME) influence the equilibrium between the polymerization and
degradation of the microtubuli, and are therefore able to influence cell division in a cell cycle
phase-specific manner [21]. It has recently been established that some agents that interact
with tubulin are also able to target the vascular system of tumors [22], causing vasculature
recession or vasculature ‘normalization’ [23]. 2ME, a well-known nonpolar endogenous
metabolite of 17β-estradiol, is a potent antiproliferative, apoptotic and antiangiogenic agent in
vitro and in vivo at pharmacological concentrations [24]. Investigation of its antitumor activity
on different malignancies in clinical studies revealed that 2ME can be tolerated up to very
high doses (e.g. 1200 mg/day) [25,26]. The most important mechanisms of its action seem to
be the inhibition of neoangiogenesis, microtubule disruption and the upregulation of factors
regulating extrinsic and intrinsic apoptotic pathways [27]. Cushman et al. reported that 2-
ethoxyestradiol is a more potent cytotoxic agent than 2ME in cancer cell cultures, and is also
a tubulin polymerization inhibitor [28]. Hillisch et al. patented the finding that some D-
homoestra-1,3,5(10)-trien-3-yl 2-substituted sulfamates are potential pharmaceuticals for the
treatment of tumorous diseases [4] by acting as tubulin polymerization inhibitors. We recently
characterized the antitumor activities of 13-epimers of D-homoestrone and their 3-methyl
ether counterparts on HeLa, MCF-7 and Ishikawa cells, providing the first evidence that D-
homoestrone selectively suppresses HeLa cell proliferation and modulates cell cycle
progression, leading to the accumulation of cells in the G2/M phase. Moreover, no significant
in vivo estrogenic activity was observed as concerns the weight gain of the uterine tissue of
gonadectomized rats after a 7-day treatment with D-homoestrone. The 3-methyl ether and 13-
epi counterparts did not display any effect on the proliferation of the tested cells [5].
In the present study, we tested D-homoestrone derivatives that are readily available via the D-
homoestrone synthetic pathway [5]. Compounds 2–5 differ only in the functional groups on
C-3 and C-17a, and possess no other substituents. In vitro antiproliferative tests were carried
out on a panel of human adherent cancer cell lines (Table 3). Compounds 3 and 5 proved to
exert substantial cell-line selective antiproliferative activities against ovarian and cervical
cancer cell lines, respectively. 17aβ-D-Homoestradiol (5) behaved similarly to D-homoestrone
(4) [5]. The 3-benzyl ether of the unsaturated ketone 2 was not active. As concerns the results
for 2–5 and D-homoestrone-3-methyl ether [5], three of the five tested 13β-methyl-D-
homoestrone derivatives are potent selective cytotoxic agents, acting in the low micromolar
range. The nature of the protecting group at position 3 has a great influence on the
antiproliferative properties.
Möller et al. found that some 2- and/or 16-halogenated D-homoestrone derivatives are
efficient inhibitors of 17β-hydroxysteroid dehydrogenase type 1, and their binding affinity to
human estrogen receptor α is reduced by 100- to 1000-fold relative to that of estradiol [29].
Accordingly, such modified D-homoestrone derivatives may be used for the treatment of
estradiol-dependent diseases. Those previous results led us to examine the antiproliferative
properties of the synthetized halogen-containing D-homoestrones. The 16-halomethyl-17-oxa-
D-homosteroids (9–11) and their starting compounds (6, 7) were tested in vitro. None of the
16-halomethyl derivatives influenced the proliferation of the tested tumor cells, but the
secoalcohol (6) proved to be effective against all the cell lines, and especially on the estrogen
receptor-positive breast adenocarcinoma (MCF-7), skin epidermoid (A431) and endometrial

(Ishikawa) carcinoma cells (IC50 = 5-8 µM). Structural modifications of secoalcohol 6 at three
sites in the molecule yielded diverse derivatives (12) which displayed different effects on the
proliferation of the cell lines. Acetylation of the 17-OH group led to the 17-acetoxy-3-benzyl
ether (12c) which exhibited similar antiproliferative action to that of its 17-hydroxy
counterpart (6), whereas the 17-tosyl ester (12d) appeared to be inactive. Removal of the 3-
benzyl protecting group and simultaneous saturation of the propenyl side-chain furnished 12a,
which demonstrated complete loss of the growth-inhibitory behavior, and the 16-acetoxy-3-ol
(12d) and the 3,17-diacetate (12b) were also totally inactive. The results of the MTT assays
for the secoestrones 6 and 12 indicate that the benzyl ether protecting group is necessary for
the antiproliferative activity, which is greatly affected by the nature of the 17-functional
group.
Table 3.
We are not aware of literature reports of effective antitumor D-secoestrone derivatives against
the HeLa, MCF-7, A2780, A431 and Ishikawa cell lines. Saloranta et al. described 17-
homoallylic D-secoestrones with low antitumor potentials on these cell lines (IC50 > 30 µM)
[30]. D-Secoestrone nitriles were recently tested in vivo for estrogenic activity. Opening of
ring D resulted in complete loss of the estrogenic activity, whereas some derivatives exerted
moderate antiestrogenic effects [31–33]. As D-secoestrone derivative 6 suppresses the
proliferation of MCF-7, A431 and Ishikawa cells, it was selected for additional in vitro
experiments. Since tumor specificity is one of the greatest challenges facing new anticancer
drug candidates, this feature was additionally investigated for 6. When intact, noncancerous
human foreskin fibroblast cells were incubated with 3.0 or 10 µM 6 for 72 h, a weak
proliferation-inhibiting effect (8.3 and 17.7% inhibition, respectively) was detected (Table 4).
Cisplatin, our positive control molecule, elicited similar inhibitory activity on fibroblast cell
proliferation at these concentrations. Although the tumor specificity of secoestrone 6 does not
differ significantly from that of cisplatin, a compound generally used in the therapy of
cancerous diseases of female reproductive organs, it may be noted that to date there have been
no reports of the tumor specificity of D-secoestrone derivatives with potent antitumor activity.
Table 4.
Various cytotoxic estrones behave as tubulin polymerization inhibitors [3, 29, 34, 35], and
there is evidence relating to a B-homoestrone derivative which enhances tubulin assembly and
stabilizes microtubules similarly to paclitaxel [36]. However, no information was previously
available on the impact of D-secoestrones on tubulin polymerization. We present here data on
the effect of a D-secoalcohol (6) on the kinetics of in vitro tubulin polymerization. Our test
compound (6) significantly enhanced the maximum rate (Vmax) of formation of microtubules
(Figure 2).
Figure 2.
This significant increase in Vmax might be due to the interaction of compound 6 with the
colchicine binding site of tubulin, as previously reported for several estrone analogs [36]. To
determine whether compound 6 interacts with tubulin, it was docked into both the colchicine
and the vinblastine binding sites of tubulin. Molecular docking calculations were carried out
using the available crystal structure of tubulin crystallized with both colchicine and
vinblastine (PDB code: 1Z2B) [37]. The results revealed that both the interaction energy and

the frequency of docking suggest more favorable binding of the ligand at the colchicine
binding site. The interactions of the ligand at the binding site are presented in Figure 3.
Compound 6 is seen to be stabilized mainly by hydrophobic interactions between the
hydrophobic amino acids at the binding site and the hydrophobic ring of the ligand: LEU-248,
ALA-250, LEU-255, ALA-316, VAL-315 and MET-299 in the B chain and VAL-181 in the A
chain are within interacting distance. The alcoholic oxygen of the ligand accepts a hydrogen-
bond from the amide nitrogen of ASN101 in the A chain, whereas the oxygen is donated to
GLN11 in the A chain.
Figure 3.
4. Acknowledgments
The authors thank the Hungarian Scientific Research Fund (OTKA K101659), the New
Hungary Development Plan (TÁMOP-4.2.2.A-11/1/KONV-2012-0047) and the Richter
Gedeon Plc. for financial support. This research was supported by the European Union and
the State of Hungary, co-financed by the European Social Fund in the framework of
TÁMOP 4.2.4. A/2-11-1-2012-0001 ‘National Excellence Program’.

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Footnotes for tables
Table 1 Synthesis of D-homoestrone derivatives (9‒11)
Table 2 Synthesis of D-secoestrone derivatives (12)
Table 3 Antiproliferative properties of the synthetized compounds
* Mean values from two independent determinations with five parallel wells; standard
deviation <15%.
Table 4 Proliferation-inhibitory effects of compound 6 and cisplatin on noncancerous human
foreskin fibroblast cells, determined in the 72-h antiproliferative assay described in section
2.2.
* Mean values from two independent determinations with five parallel wells.

Tables
Table 1.
Entry
Starting
Electrophilic Product
Yield (%)
secosteroid reagent
1
2
3
4
5
6
NBS
NIS
PhSeBr
NBS
NIS
68
6
6
6
7
7
7
9a
9b + 10b
9c + 10c
11a
50 + 25
36 + 36
82
84
79
11b
11c
PhSeBr
Table 2.
Entry
Substrate
Reagent
Reaction
conditions
10 bar, rt
MW, 5 min,
160 ºC
Product
Yield
(%)
87
1
2
H2, Pd/C
Ac2O
6
12a
12a
12b
86
3
Ac2O
MW, 5 min,
160 ºC
91
6
12c
4
5
H2, Pd/C
TsCl
10 bar, rt
3 h, rt
88
95
12c
6
12d
12e
Table 3.
Calculated IC50 values [µM]
HeLa
>30
18.9
3.5
MCF-7
>30
13.9
>30
6.4
A2780
>30
3.0
A431
>30
11.3
>30
5.4
Ishikawa
>30
>30
>30
7.3
2
3
>30
12.0
>30
20.6
>30
>30
>30
>30
>30
19.2
>30
>30
>30
>30
14.4
5
13.2
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
14.9
6
>30
>30
>30
>30
>30
>30
>30
>30
24.1
>30
>30
>30
7.9
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
7.3
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
9.5
7
8
9a
9b
9c
10b
10c
11a
11b
11c
12a
12b
12c

>30
>30
5.3
23.3
>30
7.3
>30
>30
0.7
>30
>30
8.9
>30
>30
3.5
12d
12e
Cisplatin
Table 4.
Compound
Applied concentration
% Inhibition of proliferation
SEM*
[µM]
3.0
8.3 1.2
6
10.0
3.0
17.7 2.1
12.5 1.7
Cisplatin
10.0
22.1 4.6

Scheme legends
Scheme 1. Reagents and conditions: (i) p-TsOH, CH2Cl2, 0 ºC ‒ r.t., 24 h; (ii) Jones reagent (8
N), Me2CO, 0 ºC, 1 h; (iii) KBH4, MeOH, 0 ºC ‒ r.t., 3 h; (iv) H2 (20 bar), Pd/C, EtOAc, 24 h.
Scheme 2. Reagents and conditions: (i) 5 equiv. KBH4, CH2Cl2:MeOH=1:1, 0 ºC ‒ r.t., 0.5 h;
(ii) Jones reagent (8 N), Me2CO, 0 ºC ‒ r.t., 4 h; (iii) NBS, MeCN, -15 ºC, 0.5 h; (iv) I2,
NaHCO3, CH2Cl2/THF, r.t., 1 h; (v) PhSeBr, MeCN, r.t., 1 h.

Schemes
Scheme 1.
O
OH
O
i
H
iii
H
ii
H
H
H
H
2
3
BnO
1
iv
OH
O
iii
H
H
H
H
5
4
HO
Scheme 2.
CH2OH
iii/iv/v
O
O
i
16a
CH2X
H
H
H
9: 16β-CH2X
6
H
10: 16α-CH2X
16a
O
O
H
H
ii
H2CO
1'
O
OH
iii/iv/v
1
6'
5'
2'
3'
CH2X
H
H
11
7
4'
O
+
X
9-11
a
b
c
Br
I
SePh
O
H
H
8

Figure legends
Figure 1. The synthetized D-secoestrone derivatives
Figure 2. Effects of compound 6 on the calculated maximum reaction rate (Vmax) of in vitro
microtubule formation. Kinetic curves of tubulin polymerization in the presence of 100 or 200
µM of 6 and 10 µM paclitaxel (Panel A). Calculated Vmax values of tubulin polymerization
(Panel B). Control: untreated samples, PAC: 10 µM paclitaxel-treated samples. The
experiment was performed in two parallels. Each bar denotes the mean SEM, n = 4. * and
** indicate p < 0.05 and p < 0.01, respectively, compared with the control values.
Figure 3. Interactions of ligand 6 at the colchicine binding site of tubulin

Figures
Figure 1.
CH2OR2
R3
12
R2
R1
R3
H
a
b
c
d
e
H
-CH2-CH3
-CH2-CH3
H
Ac
Ac
Bn
H
H
H
Ac -CH=CH2
Ac
Ts
-CH2-CH3
-CH=CH2
R1O
Bn
12
Figure 2.
Figure 3.