Pd-catalyzed Suzuki–Miyaura couplings and evaluation of 13α-estrone derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.steroids.2020.108731

13α-Estrones are of great value owing to their potent multiple bioactivity, including anticancer activity. 3-OH or 3-OBn derivatives of 2- or 4-[(subst.) phenyl]-13α-estrone as potential antiproliferative agents have been synthesized via facile, microwave

Full text of DOI:10.1016/j.steroids.2020.108731

Steroids 164 (2020) 108731
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Pd-catalyzed Suzuki–Miyaura couplings and evaluation of 13α-estrone
derivatives as potential anticancer agents
a
,
,
1
a
c
b,*
´
Rebeka Jojart 1, Hazhmat Alib , Gergely Horvath , Zoltan Kele , Istvan Zupko
,
´ ´
´
´
´
a,*
´
´
Erzsebet Mernyak
a
´
´
Department of Organic Chemistry, University of Szeged, Dom ter 8, H-6720 Szeged, Hungary
b
c
¨
¨
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eotvos u. 6., H-6720 Szeged, Hungary
´
´
Department of Medicinal Chemistry, University of Szeged, Dom ter 8, H-6720 Szeged, Hungary
A R T I C L E I N F O
A B S T R A C T
Keywords:
13
3-OBn derivatives of 2- or 4-[(subst.) phenyl]-13
thesized via facile, microwave-induced, Pd-catalyzed Suzuki–Miyaura coupling. 2- or 4-Halogenated 13
α
-Estrones are of great value owing to their potent multiple bioactivity, including anticancer activity. 3-OH or
-estrone as potential antiproliferative agents have been syn-
-estrone
Suzuki-Miyaura coupling
α
13α-Estrone
α
Phenylboronic acid
Anticancer
MCF-7
derivatives have been reacted with (4-subst.)phenylboronic acids using Pd(PPh3)4 as catalyst. The nature of para
substituents at the introduced phenyl group did not influence the outcome of couplings. Certain newly synthe-
sized compounds displayed substantial antiproliferative action against human adherent cancer cell lines of gy-
necological origin. Important structure–activity relationships were revealed, which might be helpful in the
Hela
design of potent and selective anticancer derivatives based on the hormonally inactive 13α-estrane core.
1. Introduction
certain structurally different enzymes involved in estrogen biosynthesis
[11,12] can be inhibited by 2- and/or 4-substitued 13α-estrone de-
The primary function of female sex hormones (estrogens) is to
facilitate the development of female secondary sexual characteristics
and to control the reproductive functions [1]. However, certain estro-
gens possess other biological activities, including anti-angiogenic, neu-
roprotective, anticancer effects and others [2]. Despite the known
property of 17β-estradiol promoting cell proliferation, its derivatives
might act as antiproliferative agents [3]. The major risk in the design of
anticancer agents based on the estrane core is the potential hormonal
side effect of the lead compound. One possibility for minimizing the
undesirable estrogenic behavior is the transformation of natural estro-
rivatives (Fig. 2, compound I). The most potent inhibitors displayed IC50
values in ranges similar to those of their 13β counterparts.
It should be highlighted that organic anion transporter protein
OATP2B1, one of the key players in intestinal drug absorption and drug
transport, can also be inhibited by 13α-estrone compounds (Fig. 2,
compound 3a) [13]. The observed inhibitory potencies are comparable
to those of the best OATP2B1 inhibitors ever published.
Not only enzyme inhibitory behavior, but the direct antiproliferative
potential of certain 13α-estrones have also been published by our group
[14–20]. Certain C-3-O and C-16 modified derivatives (Fig. 2, compound
II [14]) displayed outstanding cell growth-inhibitory action against a
range of human adherent cancer cell lines. Investigations of the mech-
anism of action revealed that compound II indicated cell cycle blockade
at the G2–M transition. Caspase activity determinations proved that the
compound induced apoptosis via the intrinsic pathway.
gens into their core-modified analogs [4–6]. 13
epimer of natural estrone (Fig. 1) [4]. The inversion of the configuration
at C-13 results in a modified conformation, and that is why 13 -estrone
and its 17-ol derivatives do not possess estrogenic behavior [7].
α-Estrone (1a) is an
α
Considering that 13α-estrone (1a) is readily available in a single step
from its natural counterpart, it might serve as a promising candidate for
the design of anticancer compounds lacking hormonal action. We have
demonstrated recently that this group of synthetic compounds offers
great possibilities concerning bioactivity [8–10]. It was shown that
The above-mentioned literature data suggest that 13α-estrones are of
great value owing to their potent multiple bioactivity without estrogenic
side effects.
The Suzuki–Miyaura reaction is a palladium-catalyzed cross-
* Corresponding authors.
´
E-mail address: bobe@chem.u-szeged.hu (E. Mernyak).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.steroids.2020.108731
Received 7 July 2020; Received in revised form 3 September 2020; Accepted 11 September 2020
Available online 16 September 2020
0039-128X/© 2020 Elsevier Inc. All rights reserved.

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R. Jojart et al.
Steroids 164 (2020) 108731
coupling between organic boron compounds and organic halides
[21,22]. It requires the addition of a base for activation of a boron re-
agent. The C(sp2)–C(sp2) coupling proceeds under mild conditions and
tolerates a wide range of functional groups. This methodology is a very
powerful tool for diaryl formation, including the synthesis of pharma-
ceuticals and complex natural products.
Finnigan TSQ-7000 triple quadrupole mass spectrometer (Finnigan-
MAT, San Jose, CA) equipped with a Finnigan electrospray ionization
source. Analyses were performed in positive ion mode using flow in-
jection mass spectrometry with a mobile phase of 50% aqueous aceto-
nitrile containing 0.1 v/v% formic acid. The flow rate was 0.3 ml/min.
Five µl aliquot of the samples were loaded into the flow. The ESI
capillary was adjusted to 4.5 kV and N2 was used as a nebulizer gas.
Literature reveals the application of Suzuki–Miyaura coupling in the
–
synthesis of estrone derivatives with diverse biological activities. C
C
couplings on the estrane core were carried out mainly at C-2, C-3, C-4 or
C-17 [23–29]. Estrone derivatives such as aryl halides (at C-2 or C-4) or
aryl or enol triflates (at C-3 or C-17) have been used. However, Ciana
2.1.1. Reaction conditions for Suzuki-Miyaura coupling in 3-OH series
2-Bromo-3-hydroxy-13α-estra-1,3,5(10)-trien-17-one (3a, 175 mg,
0.50 mmol) or 4-bromo-3-hydroxy-13α-estra-1,3,5(10)-trien-17-one
–
et al. developed a copper-catalyzed, site-selective C H arylation
(6a, 175 mg, 0.50 mmol), Pd(PPh3)4 (58 mg, 0.050 mmol, 0.1 equiv),
K2CO3 (276 mg, 2.0 mmol, 4 equiv.), (4-subst.)phenylboronic acid (2
equiv.) and toluene (4 ml) were added under nitrogen atmosphere, in 10
ml Pyrex pressure vessels (CEM, Part #: 908035) with silicone cap
(CEM, Part #: 909210) and the mixture was heated in a CEM microwave
reactor at 100 ◦C for 30 min under stirring. The solvent was evaporated
in vacuo. The residue was purified by flash chromatography with 10%
ethyl acetate/ 90% hexane as eluent.
methodology for the synthesis of 2-phenyl estrone derivatives [30]. Sato
et al. synthesized estrone–isoquinoline hybrids as cortistatin analogs
with substantial anti-angiogenic properties [23]. 17-Pyridyl estrones
have been prepared as effective AKR1C3 inhibitors, which might be
promising drug candidates in the treatment of endometriosis. Suzuki
couplings at C-3 resulted in estrone derivatives as covalent 17β-HSD1
inhibitors [24]. Langer et al. published recently the synthesis of 2- and/
–
or 4-arylestrones via C C coupling. Certain 4-aryl regioisomers proved
to be potent pancreatic lipase inhibitors [25]. Poirier et al. demonstrated
that estrone derivatives bearing 2-(subst.)phenyl ring possess substan-
tial cytochrome P450 (CYP) 1B1 inhibitory activity [29].
2.1.1.1. 3-Hydroxy-2-phenyl-13α-estra-1,3,5(10)-trien-17-one (11a).
Compound 11a was isolated as white crystals (165 mg, 95%). Mp.:
197–205 ◦C. Rf = 0.46. Anal calcd. for C24H26O2: C, 83.20; H, 7.56.
Found: 83.28; H, 7.61. 1H NMR (DMSO‑d6) δ ppm: 0.96 (s, 3H, H-18),
2.72 (m, 2H, H-6), 6.59 (s, 1H, H-4), 7.08 (s, 1H, H-1), 7.25 (t, J = 7.5
Hz, 1H, H-4′), 7.34 (t, J = 7.5 Hz, 2H, H-3′ and H-5′), 7.49 (d, J = 7.1 Hz,
2H, H-2′ and H-6′), 9.17 (s, 1H, 3-OH). 13C NMR (DMSO‑d6) δ ppm: 20.4
(CH2), 24.5 (C-18), 27.7 (CH2), 28.0 (CH2), 29.2 (CH2), 31.6 (CH2), 32.8
(CH2), 40.6 (CH), 40.8 (CH), 48.5 (CH), 49.4 (C-13), 115.4 (C-4), 125.2
(C-2), 126.1 (C-4′), 127.6 (C-1), 127.7 (2C, 2 × CH), 128.9 (2C, 2 × CH),
130.3 (C), 136.8 (C), 138.8 (C), 151.8 (C-3), 220.6 (C-17). MSI m/z (%):
345 (100, [Mꢀ H]-).
Based on the above-mentioned literature results, here we intended to
perform Suzuki–Miyaura couplings on the hormonally inactive 13α-
estrane core. Regarding the promising biological properties of de-
rivatives modified on ring A, C-2 and C-4 have been chosen as subjects
for modifications. Our aim was to synthesize 3-hydroxy and 3-benzyloxy
2- or 4-(subst.)phenyl compounds. The determination of anti-
proliferative properties of the newly synthesized 13α-estrone derivatives
against a panel of five human adherent gynecological cancer cell lines
(MCF-7, MDA-MB-231, Hela, SiHa, A2780) was also performed.
2.1.1.2. 3-Hydroxy-2-(4-chlorophenyl)-13α-estra-1,3,5(10)-trien-17-one
2. Experimental
(12a). Compound 12a was isolated as white crystals (180 mg, 94%).
◦
Mp.: 256–257 C. Rf = 0.43. Anal calcd. for C24H25ClO2: C, 75.68; H,
2.1. Chemistry
6.62. Found: 75.75; H, 6.53. 1H NMR (DMSO‑d6) δ ppm: 0.97 (s, 3H, H-
18), 2.72 (m, 2H, H-6), 6.60 (s, 1H, H-4), 7.10 (s, 1H, H-1), 7.40 (d, J =
8.6 Hz, 2H, H-3′ and H-5′), 7.52 (d, J = 8.6 Hz, 2H, H-2′ and H-6′), 9.31
(s, 1H, 3-OH). 13C NMR (DMSO‑d6) δ ppm: 20.4 (CH2), 24.5 (C-18), 27.7
(CH2), 28.0 (CH2), 29.2 (CH2), 31.5 (CH2), 32.8 (CH2), 40.6 (CH), 40.8
(CH), 48.5 (CH), 49.4 (C-13), 115.5 (C-4), 123.8 (C-2), 127.4 (C-1),
127.7 (2C, 2 × CH), 130.5 (C), 130.6 (2C, 2 × CH), 130.7 (C), 137.3(C),
137.6 (C), 151.8 (C-3), 220.6 (C-17). MSI m/z (%): 379 (100, [Mꢀ H]-).
Melting points (Mp) were determined with a Kofler hot-stage appa-
ratus 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 (ss): 30%
ethyl acetate/70% hexane, detection with I2 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). Reactions under microwave irradiation were
carried out with a CEM Corporation focused microwave system, Model
Discover SP. The maximum power of irradiation was 200 W. 1H NMR
spectra were recorded in DMSO‑d6, CDCl3 solution 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. Mass spectrometry: full scan mass spectra of the
compounds were acquired in the range of 50 to 1000 m/z with a
2.1.1.3. 2-(4-tert-Butylphenyl)-3-hydroxy-13α-estra-1,3,5(10)-trien-17-
one (13a). Compound 13a was isolated as white crystals (187 mg,
93%). Mp.: 238–240 ◦C. Rf = 0.54. Anal calcd. for C28H34O2: C, 83.54; H,
8.51. Found: 83.61; H, 8.42. 1H NMR (DMSO‑d6) δ ppm: 0.96 (s, 3H, H-
18), 1.29 (s, 9H, 4′-C(CH3)3), 2.71 (m, 2H, H-6), 6.58 (s, 1H, H-4), 7.07
(s, 1H, H-1), 7.25 (t, J = 7.5 Hz, 1H, H-4′), 7.40 (2xd, J = 8.5 Hz, 2x2H,
Fig. 1. The structure of 13
α
-estrone (1a) and its 3-benzyl ether (1b).
2

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R. Jojart et al.
Steroids 164 (2020) 108731
H-2′,H-3′, H-5′ and H-6′), 9.14 (s, 1H, 3-OH). 13C NMR (DMSO‑d6) δ
ppm: 20.4 (CH2), 24.5 (C-18), 27.7 (CH2), 28.0 (CH2), 29.2 (CH2), 31.0
(3C, 4′-C(CH3)3), 31.6 (CH2), 32.8 (CH2), 34.0 (4′-C(CH3)3, 40.6 (CH),
40.8 (CH), 48.5 (CH), 49.4 (C-13), 115.4 (C-4), 124.5 (2C, 2 × CH),
125.1 (C-2), 127.6 (C-1), 128.6 (2C, 2 × CH), 130.3 (C), 135.9 (C), 136.5
(C), 148.3 (C), 151.8 (C-3), 220.6 (C-17). MSI m/z (%): 401 (100,
[Mꢀ H]ꢀ ).
(CH3)3), 32.2 (CH2), 33.3 (CH2), 34.6 (4′-C(CH3)3, 40.9 (CH), 41.6 (CH),
49.0 (CH), 49.9 (C-13), 113,3 (C-2), 125.1 (2C, 2 × CH), 125.8 (C-1),
128.0 (C), 130.1 (2C, 2 × CH), 130.7 (C), 135.4 (C), 136.3 (C), 148.7 (C),
152.7(C-3), 221.2 (C-17). MSI m/z (%): 403 (100, [M + H]+).
2.1.2. Reaction conditions for Suzuki-Miyaura coupling in 3-OBn series
3-Benzyloxy-2-bromo-13α-estra-1,3,5(10)-trien-17-one (3b, 220 mg,
0.50 mmol) or 3-Benzyloxy-2-bromo-13α-estra-1,3,5(10)-trien-17-one
(6b, 220 mg, 0.50 mmol), Pd(PPh3)4 (58 mg, 0.050 mmol, 0.1 equiv),
K2CO3 (276 mg, 2.0 mmol, 4 equiv.), (4-subst.)phenylboronic acid (2
equiv.) and toluene (4 ml) were added under nitrogen atmosphere, in 10
ml Pyrex pressure vessels (CEM, Part #: 908035) with silicone cap
(CEM, Part #: 909210) and the mixture was heated in a CEM microwave
reactor at 150 ◦C for 30 min under stirring. The solvent was evaporated
in vacuo. The residue was purified by flash chromatography with 5%
ethyl acetate/ 95% hexane as eluent.
2.1.1.4. 3-Hydroxy-4-phenyl-13α-estra-1,3,5(10)-trien-17-one (14a).
Compound 14a was isolated as white crystals (163 mg, 94%). Mp.:
125–129 ◦C. Rf = 0.56. Anal calcd. for C24H26O2: C, 83.20; H, 7.56.
Found: 83.29; H, 7.50. 1H NMR (DMSO‑d6) δ ppm: 0.96 (s, 3H, H-18),
6.71 (d, J = 8.5 Hz, 1H, H-2), 7.08 (d, J = 8.5 Hz, 1H, H-1), 7.13 (t, J =
7.4 Hz, 2H, H-3′ and H-5′), 7.27 (t, J = 7.4 Hz, 1H, H-4′), 7.36 (over-
lapping multiplets, 2H, H-2′ and H-6′), 8.82 (s, 1H, 3-OH). 13C NMR
(DMSO‑d6) δ ppm: 20.9 (CH2), 24.9 (C-18), 28.3 (CH2), 28.7 (CH2), 29.3
(CH2), 32.2 (CH2), 33.3 (CH2), 40.8 (CH), 41.6 (CH), 49.0 (CH), 49.9 (C-
13), 112.8 (C-2), 125.4 (CH), 126.2 (CH), 127.6(C), 127.8 (2C, 2 × CH),
129.9 (2C, 2 × CH), 130.2 (C), 135.5 (C), 137.9 (C), 152.0 (C-3), 220.6
(C-17). MSI m/z (%): 345 (100, [Mꢀ H]ꢀ ).
2.1.2.1. 3-Benzyloxy-2-phenyl-13α-estra-1,3,5(10)-trien-17-one (11b).
Compound 11b was isolated as white crystals (203 mg, 92%). Mp170 ꢀ
176 ◦C. Rf = 0.70. Anal calcd. for C31H32O2: C, 85.28; H, 7.39. Found:
1
85.36; H, 7.30. H NMR (C6D6) δ ppm: 0.79 (s, 3H, H-18), 2.55–2.66
(overlapping multiplets, 2H, H-6), 4.79 (s, 2H, 3-OCH2), 6.63 (s, 1H, H-
2.1.1.5. 3-Hydroxy-4-(4-chlorophenyl)-13α-estra-1,3,5(10)-trien-17-one
4), 7.01–7.32 (overlapping multiplets, 9H), 7.21 (d, J = 7.4 Hz, 2H), 13
C
(15a). Compound 12a was isolated as white crystals (175 mg, 92%).
Mp.: 71–78 ◦C. Rf = 0.59. Anal calcd. for C24H25ClO2: C, 75.68; H, 6.62.
Found: 75.74; H, 6.56. 1H NMR (DMSO‑d6) δ ppm: 0.96 (s, 3H, H-18),
6.71 (d, J = 8.5 Hz, 1H, H-2), 7.08 (d, J = 8.5 Hz, 1H, H-1), 7.10 (d, J =
8.5 Hz, 1H, H-1), 7.13 (m, 2H, H-3′ and H-5′), 7.41 (m, 2H, H-2′ and H-
6′), 8.94 (s, 1H, 3-OH). 13C NMR (DMSO‑d6) δ ppm: 20.3 (CH2), 24.4 (C-
18), 27.7 (CH2), 28.2 (CH2), 28.7 (CH2), 31.6 (CH2), 32.8 (CH2), 40.2
(CH), 41.0 (CH), 48.4 (CH), 49.3 (C-13), 112.8 (C-2), 125.8 (CH), 126.3
(C), 127.8(2C, 2 × CH), 130.2 (C), 131.0 (C), 131.8(CH), 131.9 (CH),
135.4 (C), 136.7 (C), 152.0 (C-3), 220.6 (C-17). MSI m/z (%): 381 (100,
[M + H]+).
NMR (CDCl3) δ ppm: 21.1 (CH2), 25.2 (C-18), 28.3 (CH2), 28.4 (CH2),
30.3 (CH2), 32.1 (CH2), 33.5 (CH2), 41.5 (CH), 41.6 (CH), 49.4 (CH),
50.1 (C-13), 70.7 (OCH2), 113.6 (C-4), 126.7 (CH), 126.9 (2 × CH),
127.5 (CH), 127.9 (2 × CH), 128.4 (2 × CH), 128.7 (CH), 129.2 (2 ×
CH), 129.6 (C), 132.6 (C), 137.2 (C), 137.5 (C), 138.8 (C), 153.7 (C),
221.4 (C-17). MSI m/z (%): 437 (100, [M + H]+).
2.1.2.2. 3-Benzyloxy-2-(4-chlorophenyl)-13α-estra-1,3,5(10)-trien-17-
one (12b). Compound 12b was isolated as white crystals (221 mg,
94%). Mp.: 149–157 ◦C. Rf = 0.67. Anal calcd. for C31H31ClO2: C, 79.05;
H, 6.63. Found: 79.12; H, 6.54. 1H NMR (CDCl3) δ ppm: 1.06 (s, 3H, H-
18), 2.86 (m, 2H, H-6), 5.02 (s, 2H, 3-OCH2), 6.74 (s, 1H, H-4), 7.21 (s,
1H, H-1),7.28–7.34 (overlapping multiplets, 7H), 7.47 (m, 2H). 13C
NMR (CDCl3) δ ppm: 21.0 (CH2), 25.1 (C-18), 28.2 (CH2), 28.3 (CH2),
30.3 (CH2), 32.0 (CH2), 33.4 (CH2), 41.4 (CH), 41.5 (CH), 49.2 (CH),
50.1 (C-13), 70.5 (3-OCH2), 113.3 (C-4), 126.8 (2C, 2 × CH), 127.6 (C-
2), 127.7 (CH), 128.0 (2C, 2 × CH), 128.4 (3C, 3 × CH), 130.8 (2C, 2 ×
CH), 132.5 (2C, 2 × C), 137.1 (2C, C), 137.6 (C), 153.4 (C-3), 221.5(C-
2.1.1.6. 4-(4-tert-Butylphenyl)-3-hydroxy-13α-estra-1,3,5(10)-trien-17-
one (16a). Compound 13a was isolated as white crystals (183 mg,
91%). Mp.: 211–219 ◦C. Rf = 0.72. Anal calcd. for C28H34O2: C, 83.54; H,
8.51. Found: 83.60; H, 8.42. 1H NMR (DMSO‑d6) δ ppm: 0.96 (s, 3H, H-
18), 1.31 (s, 9H, 4′-C(CH3)3), 6.69 (d, J = 8.5 Hz, 1H, H-2), 7.05 (m, 3H),
7.37 (m, 2H), 8.80 (s, 1H, 3-OH). 13C NMR (DMSO‑d6) δ ppm: 20.9
(CH2), 24.9 (C-18), 28.3 (CH2), 28.8 (CH2), 29.3 (CH2), 31.7 (3C, 4′-C
Fig. 2. Potent, biologically active 13
α
-estrone derivatives (I, 3a and II).
3

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R. Jojart et al.
Steroids 164 (2020) 108731
17). MSI m/z (%): 472 (100, [M + H]+).
cancer cell lines positive for HPV-18 and HPV-16, respectively. The
cancer selectivity of compounds was tested on the non-cancerous mouse
embryo fibroblast cell line NIH/3T3. All cell lines were purchased from
European Collection of Cell Cultures (ECCAC, Salisbury, UK) exception
for Siha (American Tissue Culture Collection, Manassas, VA, USA). Cells
were cultivated in minimal essential medium supplemented with 10%
fetal bovine serum, 1% non-essential amino acids and an anti-
biotic–antimycotic mixture. All media and supplements were obtained
from Lonza Group Ltd., Basel, Switzerland. Near-confluent cancer cells
were seeded onto a 96-well microplate (5000 cells/well) and, after
2.1.2.3. 3-Benzyloxy-2-(4-tert-butylphenyl)-13α-estra-1,3,5(10)-trien-17-
one (13b). Compound 13b was isolated as white crystals (227 mg,
92%). Mp.: 163–166 ◦C. Rf = 0. Anal calcd. for C35H40O2: C, 85.32; H,
8.18. Found: 85.40; H, 8.09. 1H NMR (C6D6) δ ppm: 0.79 (s, 3H, H-18),
1.27 (s, 9H, 4′-C(CH3)3), 2.63 (m, 2H, H-6), 4.82 (s, 2H, 3-OCH2), 6.66
(s, 1H, H-4), 7.03 (t, J = 7.3 Hz, 1H, H-4′′), 7.11 (t, J = 7.6 Hz, 2H, H-3′′
and H-5′′), 7.23 (d, J = 7.5 Hz, 2H, H-2′′ and H-6′′), 7.35(s, 1H, H-1),
7.41 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3) δ
ppm: 21.0 (CH2), 25.1 (C-18), 28.2 (CH2), 28.3 (CH2), 30.2 (CH2), 31.4
(3C, 4′-C(CH3)3), 32.1 (CH2), 33.4 (CH2), 34.5 (4′-C(CH3)3), 41.5 (2C, 2
× CH), 49.2 (CH), 50.1 (C-13), 70.6 (3-OCH2), 113.5 (C-4), 124.8 (2C, 2
× CH), 126.8 (2C, 2 × CH), 127.4 (CH), 128.3 (2C, 2 × CH), 128.6 (CH),
128.9 (C), 129.2 (2C, 2 × CH), 132.4 (C), 135.6 (C), 136.8 (C), 137.5 (C),
149.4 (C), 153.7 (C-3), 221.6 (C-17). MSI m/z (%): 493 (100, [M + H]+).
overnight standing, 200 μL new medium, containing the tested com-
pounds at 10 and 30 µM, was added. After incubation for 72 h at 37 ◦C in
humidified air containing 5% CO2, the living cells were assayed by the
addition of 20 μL of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) solution. MTT was converted by intact
mitochondrial reductase and precipitated as purple crystals during a 4-h
contact period. The medium was next removed and the precipitated
formazan crystals were dissolved in 100
μL of DMSO during a 60-min
2.1.2.4. 3-Benzyloxy-4-phenyl-13α-estra-1,3,5(10)-trien-17-one (14b).
period of shaking at 37 ◦C.
Compound 14b was isolated as white crystals (196 mg, 90%). Mp.:
58–60 ◦C. Rf = 0.72. Anal calcd. for C31H32O2: C, 85.28; H, 7.39. Found:
85.35; H, 7.31. 1H NMR (CDCl3) δ ppm: 1.05 (s, 3H, H-18), 4.95 (d, J =
3.9 Hz, 2H, 3-OCH2), 6.85 (d, J = 8.7 Hz, 1H, H-2), 7.08 (s, 1H, H-1),
7.11 (m, 2H), 7.19–7.25 (overlapping multiplets, 6H), 7.33 (m, 1H),
7.41 (m, 1H). 13C NMR (CDCl3) δ ppm: 21.1 (CH2), 25.1 (C-18), 28.4
(CH2), 28.5 (CH2), 28.9 (CH2), 32.1 (CH2), 33.4 (CH2), 41.0 (CH), 41.8
(CH), 49.5 (CH), 50.1 (C-13), 70.5 (3-OCH2), 111.1 (C-2), 125.7 (CH),
126.5 (2C, CH), 126.6 (CH), 127.3 (CH), 128.0 (2C, 2 × CH), 128.2 (2C,
2 × CH), 129.9 (CH), 130.1 (CH), 131.2 (C), 133.1 (C), 136.6 (C), 137.6
(C), 137.8 (C), 153.9 (C-3), 221.4 (C-17). MSI m/z (%): 437 (100, [M +
H]+).
Finally, the reduced MTT was assayed at 545 nm, using a microplate
reader utilizing wells with untreated cells serving as control [31]. In the
case of the most active compounds (i.e. higher than 50% growth inhi-
bition at 30 µM), the assays were repeated with a set of dilutions,
sigmoidal dose–response curves were fitted to the determined data and
the IC50 values (the concentration at which the extent of cell prolifera-
tion was half that of the untreated control) were calculated by means of
GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA). All in
vitro experiments were carried out on two microplates with at least five
parallel wells. Stock solutions of the tested substances (10 mM) were
prepared in DMSO. The highest DMSO content of the medium (0.3%) did
not have any substantial effect on cell proliferation. Cisplatin (Ebewe
Pharma GmbH, Unterach, Austria) was used as positive control.
2.1.2.5. 3-Benzyloxy-4-(4-chlorophenyl)-13α-estra-1,3,5(10)-trien-17-
one (15b). Compound 15b was isolated as white crystals (224 mg,
95%). Mp.: 67–69 ◦C. Rf = 0.74. Anal calcd. for C31H31ClO2: C, 79.05; H,
6.63. Found: 79.11; H, 6.56. 1H NMR (CDCl3) δ ppm: 1.04 (s, 3H, H-18),
4.96 (d, J = 4.5 Hz, 2H, 3-OCH2), 6.83 (d, J = 8.7 Hz, 1H, H-2), 7.12 (m,
3H), 7.11–7.25 (overlapping multiplets, 4H), 7.28 (m, 1H), 7.37 (m,
2H). 13C NMR (CDCl3) δ ppm: 21.0 (CH2), 25.0 (C-18), 28.3 (CH2), 28.4
(CH2), 29.0 (CH2), 32.1 (CH2), 33.4 (CH2), 40.9 (CH), 41.7 (CH), 49.3
(CH), 50.1 (C-13), 70.2 (3-OCH2), 110.8 (C-2), 126.1 (CH), 126.5 (2C, 2
× CH), 127.4 (CH), 128.2 (2C, 2 × CH), 128.3 (2C, 2 × CH), 129.6 (C),
131.3 (CH), 131.5 (CH), 132.5 (C), 133.0 (C), 136.2 (C), 136.5(C), 137.4
(C), 153.7 (C-3), 221.6 (C-17). MSI m/z (%): 494 (100, [M + Na]+).
3. Results and discussion
The aromatic ring of 13α-estrone might readily be halogenated using
different types of halogenating agents (Scheme 1, [8]). N-Hal-
osuccinimides are suitable for the non-selective transformations of the
phenolic moiety. Due to the activated behavior of ring A, and the ortho-
directing ability of the 3-OH group, a mixture of mono (2- or 4-
substituted) and bis (2,4-disubstituted) derivatives is formed. We
demonstrated recently that the chemo- and regioselectivity of haloge-
nations depend on the reagent and solvent applied [8]. Additionally, the
nature and the size of the functional group at C-3 also influence the
outcome of the reactions. Bis-2,4-derivates (beside the monosubstituted
compounds) were formed exclusively when the starting compound
possessed a phenolic OH group. In the case of methyl or benzyl ethers,
2.1.2.6. 3-Benzyloxy-4-(4-tert-butylphenyl)-13α-estra-1,3,5(10)-trien-17-
one (16b). Compound 16b was isolated as white crystals (222 mg,
90%). Mp.: 66–70 ◦C. Rf = 0.76. Anal calcd. for C35H40O2: C, 85.32; H,
8.18. Found: 85.41; H, 8.10. 1H NMR (CDCl3) δ ppm: 1.05 (s, 3H, H-18),
1.38 (s, 9H, 4′-C(CH3)3), 4.93 (d, J = 6.5 Hz, 2H, 3-OCH2), 6.85 (d, J =
8.6 Hz, 1H, H-2), 7.06 (m, 2H), 7.14 (m, 1H), 7.21 (m, 5H), 7.43 (m,
2H). 13C NMR (CDCl3) δ ppm: 21.0 (CH2), 25.0 (C-18), 28.4 (CH2), 28.5
(CH2), 29.0 (CH2), 31.4 (3C, 4′-C(CH3)3), 32.1 (CH2), 33.4 (CH2), 34.5
(4′-C(CH3)3), 40.9 (CH), 41.8 (CH), 49.4 (CH), 50.1 (C-13), 70.5 (3-
OCH2), 111.3 (C-2), 124.8 (2C, 2 × CH), 125.5 (CH), 126.5 (2C, 2 × CH),
127.2 (CH), 128.1 (3C, 3 × CH), 129.4 (CH), 129.6 (CH), 131.3 (C),
134.6 (CH), 136.8 (C), 137.6 (C), 149.3 (C), 154.1 (C-3), 221.7 (C-17).
MSI m/z (%): 493 (100, [M + H]+).
only monosubstitutions occurred. We showed recently that 13α-estrone
derivatives halogenated at ring A represent a very promising compound
class with diverse biological activities. Enzymes 17β-hydroxysteroid
dehydrogenase 1 (17β-HSD1 and steroid sulfatase (STS) involved in
estradiol biosynthesis could effectively be inhibited by certain halo de-
rivatives. The 3-hydroxy-2,4-bis-iodo derivative proved to be a dual
inhibitor. The 2-bromo-3-hydroxy derivative should also be highlighted
as it also displayed dual inhibitory properties against 17β-HSD1 enzyme
and OATP2B1 transporter [8,13]. These favorable biological properties
of compounds halogenated at ring A inspired us to perform further
synthetic transformations using the compounds as aryl halide substrates.
First, C(sp2)–C(sp) couplings were carried out in order to introduce
large, but apolar groups onto the C-2 and C-4 positions. Sonogashira
couplings with phenylacetylenes in the 3-OH series resulted in new
compounds with substantial 17β-HSD1 inhibitory potentials [9]. Prod-
ucts in the C-2 series, in turn, exhibited exclusive inhibitory potentials.
These results suggest that the nature of the substituents at C-2, C-3 and/
2.2. Determination of antiproliferative activities
The antiproliferative properties of the newly synthesized compounds
(3, 6 and 11–16) were determined on a panel of human adherent cancer
cell lines of gynecological origin. MCF-7 and MDA-MB-231 were isolated
from breast cancers differing in biochemical background, while A2780
cells were isolated from ovarian cancer. Hela and SiHa are cervical
or C-4 exerts a great impact on the biological activity of 13
derivatives.
α-estrone
4

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R. Jojart et al.
Steroids 164 (2020) 108731
Scheme 1. Synthesis of ring A halogenated 13α-estrone derivatives.
13
α
-Estrone was diversified at several sites, including different
as earlier established [15]. The reaction conditions applied here have
been chosen according to our coupling methods described recently. Pd
(PPh3)4 was used as catalyst, K2CO3 as base, and transformations have
modifications at C-3 and/or in ring D with the aim of getting new
compounds with potential antiproliferative behavior. It was shown that
certain derivatives possess substantial cell growth-inhibitory potential
against a number of human cancer cell lines of reproductive origin. We
proved in many cases, that the presence of the apolar, bulky benzyl ether
function at position C-3 is markedly beneficial concerning the anti-
proliferative potential [14,15,17].
–
been carried out under microwave irradiation. First we performed C
C
couplings of substrate 2a,b-7a,b with phenylboronic acid as a model
reagent (Table 1). 2-Substituted regioisomers in the 3-OH series (3a, 4a)
could efficiently be transformed (Table 1, Entries 2 and 3), except for the
2-chloro derivative (2a, Table 1, Entry 1). The latter could only be
coupled using KOt-Bu as base at elevated temperature (150 ◦C) and
longer microwave irradiation (60 min) (Table 1, Entry 13). These con-
ditions proved to be applicable for the transformations of 4-chloro-3-hy-
droxy (5a) and 3-benzyloxy-2- or 4-chloro derivatives (2b and 5b), too
(Table 1, Entries 14–16). Concerning the reactions of bromo or iodo
compounds (3, 4, 6, and 7), these substrates behaved in a similar way in
these reactions (irrespective of the regioisomerism) (Table 1, Entries 2,
3, 5, 6, 8, 9, 11, 12). However, pronounced dehalogenation were
observed in the case of iodo starting compounds (Table 1, Entries 3, 6, 9,
It follows that 3-OH compounds might have increased enzyme
inhibitory properties, whereas 3-OBn derivatives might be promising
cytostatic candidates. Nevertheless, it is worth evaluating new com-
pounds in both series in order to get an insight into the correlation be-
tween activity and structure.
Based on these findings, we selected 13α-estrone and its 3-O-benzyl
counterpart as starting materials. The latter was synthesized in one step
from estrone 3-benzyl ether in reaction with ortho-phenylenediamine in
acetic acid. The Pd/C-catalyzed hydrogenolysis of the 3-benzyl-13α-
estrone product resulted in 13
α
-estrone in high yield. The halogenation
12). The nature of the substituent at C-3 of 13α-estrane core influenced
of 3-OH and 3-OBn substrates was achieved using N-halosuccinimides as
described earlier [8]. Three different electrophile triggers (NCS, NBS or
NIS) were used for the formation of aryl halides in order to investigate
the outcome of couplings as established earlier in Hirao reactions [10].
Benzyl ethers needed harsher conditions (higher reaction temperature)
than their hydroxy counterparts, due to stronger steric hindrance
(Table 1, Entries 8, 9, 11, 12).
–
C
the influence of the nature of the halogen substituent on the C
coupling reaction. The monosubstituted regioisomers (2a,b-7a,b)
served as starting compounds in Suzuki–Miyaura couplings (Scheme 2).
Phenylboronic acid and its two 4-substituted derivatives were chosen
as coupling partners. 4-Chlorophenylboronic acid seemed to be inter-
esting due to the dual (negative inductive but positive mesomeric)
electronic properties of chlorine. The large alkyl substituent in 4-tert-
butylphenylboronic acid might greatly influence the biological behavior
After establishing the appropriate reaction conditions (Table 1, En-
tries 2 and 8), 2- or 4-bromo-3-OH and ꢀ 3-OBn derivatives (3a,b or 6a,
b) have been chosen for further transformations (Scheme 3). Suzu-
ki–Miyaura couplings with subst.-phenylboronic acids afforded the
desired products (11–16) in excellent yields. 12 new arylated com-
pounds have been synthesized. The structure of the newly synthesized
13α
-estrone derivatives (11–16) have been deduced from 1H and 13C
Scheme 2. Suzuki-Miyaura couplings with phenylboronic acid as model reagent.
5

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R. Jojart et al.
Steroids 164 (2020) 108731
means of MTT assay (Table 2). The two breast cancer cell lines involved
in this study have different receptorial status [32–34]. In contrast with
the estrogen receptor positive MCF-7 cell line, MDA-MB-231 is a triple-
negative cell line lacking all current clinically utilized pharmacological
targets (receptors for estrogens, progesterone, and growth factor HER2/
neu) for hormonal treatment. The triple-negative subtype of breast
cancer, therefore, represents the portion of poorest prognosis among
breast cancer cases [35]. The majority of cervical cancers is associated
with human papilloma virus (HPV) infection [36,37]. Despite vaccina-
tion and screening, cervical cancer still causes high mortality rate in less
developed countries. The utilized cell lines Hela and Siha represent HPV-
18 and ꢀ 16 positive cases, respectively. These two types of HPV are
responsible for nearly 70% of cervical cancerous cases. Ovarian cancer is
another common type of gynecological malignancies [38]. Its poor
prognosis and high mortality rates arise from its high recurrence and
acquired resistance to drug treatment. In order to investigate the anti-
proliferative properties of the synthesized compounds, the ovarian
cancer cell line A2780 was also included. Evaluation of the promising
antiproliferative compounds on the mentioned cell lines might initiate
the development of new anticancer agents acting with better tolerance
and higher effectiveness.
Table 1
–
Effect of reaction conditions on C C coupling of halogenated 13α-estrones (2a,
b-7a,b) with phenylboronic acid in toluene catalyzed by Pd(PPh3)4.1
Entry
Substrate
Base
Temp
Reaction time
(min)
Products2 (%)
(◦C)
1
2
2a
3a
K2CO3
K2CO3
100
100
30
30
0
11a + 1a (95 +
2)
3
4a
K2CO3
100
30
11a + 1a (83 +
12)
4
5
5a
6a
K2CO3
K2CO3
100
100
30
30
0
14a + 1a (94 +
3)
6
7a
K2CO3
100
30
14a + 1a (80 +
15)
7
8
2b
3b
K2CO3
K2CO3
150
150
30
30
0
11b + 1b (92 +
4)
9
4b
K2CO3
150
30
11b + 1b (82 +
7)
10
11
5b
6b
K2CO3
K2CO3
150
150
30
30
0
14b + 1b (90 +
4)
12
13
14
15
16
7b
2a
5a
2b
5b
K2CO3
150
150
150
150
150
30
60
60
60
60
14b + 1b (81 +
10)
Our data reveal conclusive structure–activity correlations concern-
ing the bromo derivatives (3a,b and 6a,b, Table 2). The 3-OH com-
pounds (3a and 6a) did not influence the cell growth of the tested tumor
cells markedly. However, the 4-regioisomer 6b of benzyl ethers exerted
outstanding antiproliferative effect against all investigated cell lines.
MCF-7 cells were the most sensitive to 6b with IC50 value in a sub-
micromolar range. Compound 6b exerted somewhat lower inhibition
against the cervical cell lines Hela and SiHa and triple negative breast
cancer MDA-MB-231 with IC50 values in the low micromolar range. It is
worth mentioning that 6b made a marked distinction between the two
breast cancer cell lines, but not between the two cervical ones. The other
3-OBn regioisomer (3b) exhibited weak antiproliferative effect on the
tested cell lines. As concerns compounds 3b and 6b, it should be high-
lighted, that substantial dependence of the regioselectivity on the anti-
proliferative potential was observed (in favor of the 4-regioisomer). The
most relevant compounds were additionally tested against a mouse
fibroblast cell line (NIH-3 T3) in order to obtain preliminary data
KOt-
Bu
11a (43)
KOt-
Bu
14a (51)
11b (41)
14b (40)
KOt-
Bu
KOt-
Bu
1
Aryl halide (0.50 mmol), Pd(PPh3)4 (0.1 equiv), K2CO3 (4 equiv.), (4-subst.)
phenylboronic acid (2 equiv.), toluene, N2 atmosphere.
2
Flash chromatography yield obtained under microwave irradiation.
NMR spectra.
The antiproliferative properties of the phenylated compounds (11a,
b–16a,b) and their direct precursors (3a,b and 6a,b) were determined in
vitro on a panel of human adherent breast (MCF-7 and MDA-MB-231),
cervical (HeLa and SiHa), and ovarian (A2780) cancer cell lines by
Scheme 3. Suzuki–Miyaura couplings of 2- or 4-bromo-13α-estrones. Reactions were performed on a 0.50 mmol scale with 2 equiv of subst.-phenylboronic acid, 0.1
equiv. Pd(PPh3)4, 4 equiv. K2CO3, at 100 ◦C, 30 min under microwave irradiation.
6

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R. Jojart et al.
Steroids 164 (2020) 108731
Table 2
Antiproliferative properties of the synthesized compounds.
1
Comp.
Comp.
Conc.
M)
Inhibition (%) ± SEM [Calculated IC50]
number
(
μ
MCF-7
MDA-MB-231
HeLa
SiHa
A2780
NIH-3T3
n.t.
3a
6a
10
30
–
–
–
–
–
–
24.26 ± 0.76
22.85 ± 1.97
32.26 ± 0.52
20.72 ± 3.56
10
30
–
–
–
–
–
–
n.t.
51.92 ± 1.61
42.09 ± 1.31
54.81 ± 1.14
44.21 ± 1.09
3b
6b
10
30
–
–
–
–
–
–
–
–
–
n.t.
48.57 ± 1.40
10
30
76.67 ± 0.30
79.45 ± 0.85
(0.52)
54.41 ± 0.88
62.36 ± 0.67
(3.91)
60.23 ± 0.42
68.16 ± 0.62
(2.85)
59.04 ± 1.51
63.04 ± 0.41
(2.74)
47.78 ± 2.10
54.75 ± 1.06
30.59 ± 0.47
29.55 ± 0.36
11a
14a
10
30
44.72 ± 0.73
71.54 ± 0.40
(13.12)
31.50 ± 1.77
48.97 ± 0.42
36.82 ± 0.98
63.67 ± 0.30
(17.06)
33.49 ± 0.90
54.36 ± 0.37
35.73 ± 1.21
45.89 ± 0.96
35.48 ± 1.33
40.04 ± 1.48
10
30
–
–
–
–
–
–
–
–
–
n.t.
27.29 ± 1.02
11b
14b
10
30
32.11 ± 1.90
74.50 ± 1.16
(16.87)
–
51.26 ± 0.20
75.53 ± 0.18
(8.98)
25.45 ± 2.09
70.06 ± 1.48
(18.56)
25.44 ± 1.06
73.42 ± 0.41
(18.62)
–
–
76.16 ± 1.05
(18.92)
10
30
–
–
–
–
–
–
–
–
n.t.
27.35 ± 1.36
27.14 ± 0.94
13a
16a
10
30
47.97 ± 0.98
66.68 ± 0.71
(12.82)
42.24 ± 1.02
53.23 ± 0.35
66.29 ± 0.87
77.67 ± 0.42
(6.69)
44.03 ± 1.15
51.95 ± 0.78
52.46 ± 0.20
68.13 ± 0.55
(9.41)
45.30 ± 2.71
51.68 ± 1.96
10
30
–
–
38.70 ± 1.67
97.96 ± 0.15
(11.19)
–
35.83 ± 1.92
97.93 ± 0.17
(10.38)
–
–
96.93 ± 0.33
91.02 ± 0.59
92.43 ± 0.88
(15.73)
(17.19)
(16.90)
13b
16b
10
30
–
–
24.51 ± 1.62
52.36 ± 1.25
–
–
n.t.
n.t.
28.21 ± 0.85
34.85 ± 0.52
20.00 ± 0.79
64.77 ± 1.82
10
30
–
–
–
–
–
–
–
–
32.81 ± 1.33
24.65 ± 2.81
(continued on next page)
7

´ ´
R. Jojart et al.
Steroids 164 (2020) 108731
Table 2 (continued)
Comp.
1
Comp.
Conc.
M)
Inhibition (%) ± SEM [Calculated IC50]
number
(
μ
MCF-7
MDA-MB-231
HeLa
SiHa
A2780
NIH-3T3
12a
15a
10
30
53.41 ± 0.23
59.51 ± 0.22
(5.33)
25.95 ± 0.43
61.92 ± 0.67
(18.32)
73.65 ± 0.22
75.46 ± 0.81
(3.33)
50.85 ± 0.30
53.45 ± 0.95
24.97 ± 0.52
33.61 ± 0.74
26.47 ± 1.33
28.17 ± 0.25
10
30
–
–
–
–
20.20 ± 1.13
82.92 ± 0.45
(14.97)
–
88.41 ± 1.35
52.83 ± 1.31
80.24 ± 0.21
72.66 ± 1.09
20.9 ± 0.42
(19.76)
(17.91)
(21.19)
12b
15b
10
30
–
–
–
–
–
n.t.
36.29 ± 1.21
22.91 ± 0.55
56.17 ± 1.12
47.26 ± 0.35
34.63 ± 0.48
10
30
48.56 ± 0.32
77.13 ± 0.40
(9.95)
–
47.73 ± 1.65
64.12 ± 2.00
–
–
–
–
33.67 ± 1.39
62.32 ± 0.94
55.04 ± 0.61
I
10
30
23.51 ± 1.95
69.00 ± 0.32
(21.51)
–
–
–
–
n.t.
29.66 ± 0.59
59.89 ± 1.16
46.18 ± 0.14
78.34 ± 0.12
(20.82)
Cisplatin
10
30
53.03 ± 2.29
86.90 ± 1.22
(5.78)
–
42.61 ± 2.33
99.93 ± 0.26
(12.43)
88.64 ± 0.50
90.18 ± 1.78
(7.84)
83.57 ± 1.21
95.02 ± 0.28
(1.30)
91.80 ± 0.39
93.68 ± 0.20
(2.70)
71.47 ± 1.20
(19.13)
1Mean value from two independent measurements with five parallel wells; standard deviation < 20%. n.t.: not tested.
2Inhibition values < 20% are not presented.
concerning the cancer selectivity of the elicited action. Compound 6b
greatly influence cytostatic properties.
exerted only a limited (<50% inhibition even at 30
μM) action on this
In general, compounds 13a and 16a bearing the 4′′-t-Bu function
seemed to be more potent than 11a and 14a. The 3-OBn-4′′-t-Bu de-
rivatives (13b and 16b) displayed very weak growth inhibition, irre-
spective of the substitution pattern of ring A.
cell line, though the cells are highly sensitive to reference agent
cisplatin.
A comparison of the data for phenylated 3-OH (11a and 14a) and
brominated 3-OH derivatives (3a and 6a) reveals that the presence of
the phenyl ring instead of a bromine improved the antiproliferative
activities only for the 2-regioisomer. The calculated IC50 values for
Substantial enhancements in cytostatic potential were detected for
the 2-(4′′-chlorophenyl) 3-OH compound (12a), except for A2780 cell
line. 12a exerted pronounced effect on Hela cells with a 3.33 μM IC50
ˆ
compound 11a were over 10 Aµmol against MCF-7 and Hela, and the
value, which is comparable with that of the most potent compound 6b.
The next lowest IC50 value was obtained on MCF-7 for compound 12a,
but it is far behind the corresponding data of 6b. It is important to note
that essential differences arose here in the inhibitory values for the
breast and cervical cell line pairs. This was not the case against Hela and
SiHa concerning the results of 6b. The activity dependence of regioi-
somerism here was also significant, but the tendency was reversed to
that in the case of 3b and 6b. Here the 2-isomer (12a) was far more
potent than its 4-counterpart (15a). The latter does not seem to be true
effects were weaker on the other cell lines including mouse fibroblasts.
11a proved to be more effective than 14a its 4-counterpart. A moderate
activity enhancement was observed in the case of 11b, which might be
attributed to the presence of the 3-OBn function. Strikingly, the phe-
nylated 4-regioisomer (14b) was less effective than its 2-counterpart
(11b), and far less potent than its bromo derivative 6b.
Setting against the results for phenyl and (subst.)phenyl derivatives
demonstrates that the nature and the size of the ring A substituents
8

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R. Jojart et al.
Steroids 164 (2020) 108731
for the (4′′-chlorophenyl)-3-OBn compounds (12b and 15b), since 15b
exerted more pronounced cell growth inhibitory effect against all cell
lines. This is in a good agreement with the data obtained for 3b and 6b,
but the potency of 15b is appreciably lower.
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5487–5497, https://doi.org/10.1021/jo000108x.
Our 2-phenylethynyl derivative (I) synthesized recently was addi-
tionally included in the investigations. We were interested in compari-
son of the antiproliferative properties of 2-phenyl (11a) and 2-
phenylethynyl (I) derivatives. The data in Table 2 reflect that the
moderate activity of 11a decreased substantially, when a short linear
´
´
´
´
[5] K.M. Penov Gasi, D.A. Miljkovic, L.D. Medic Mijacevic, E.A. Djurendic, S.
´
´
´
Z. Stojanovic, M.N. Sakac, M. Dj, S.M. Djurendic, D. Stankovic, S. Lazar, R.
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–
linker (C C) is inserted between the two phenyl groups. Accordingly,
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4. Conclusion
In conclusion, 2- or 4-[(subst.) phenyl]-13
been synthesized via efficient, microwave-induced, Pd-catalyzed Suzu-
ki–Miyaura coupling. Reactions of 2- or 4-halogenated 13 -estrone de-
α-estrone derivatives have
´ ´
¨
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¨
¨
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C. Tomboly, T.L. Rizner, M. Szecsi, E. Mernyak, Synthesis and structure-activity
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afforded the desired phenylated products in high yields. Certain effec-
tive submicromolar or low micromolar cell growth inhibitors (6b, 12a,
and 13a) have been identified against human cancer cell lines of gy-
necological origin. The estrogen receptor positive MCF-7 and the HPV-
18 positive Hela cancer cell lines were the most sensitive to the tested
compounds. None of the most promising agents exerted substantial
growth inhibitory action against fibroblasts indicating the cancer
selectivity of the action. The most potent compound (6b) exerted
outstanding submicromolar antitumoral activity against MCF-7 cell line
and lower calculated IC50 values against all cell lines in comparison to
those of reference agent cisplatin. The most effective phenylated com-
pound (12a) exerted selective low micromolar activities against two cell
lines (MCF-7 and Hela). Compounds 6b and 12a behaved similarly as
concerns breast cell lines MCF-7 and MDA-MB-231. However, 12a
differentiated between the two cervical cell lines Hela and SiHa.
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¨
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Accordingly, ring A substitution of 13α-estrone derivatives leads to
´
´
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effective antitumoral compounds. The substitution pattern of the aro-
matic ring influences cell growth inhibition markedly. The promising
inhibitory data motivate further structural modifications in ring A of
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13α-estrone, and additional investigations on the mechanism of anti-
proliferative action.
´
¨
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Acknowledgements
potential antiproliferative agents, Steroids 113 (2016) 14–21, https://doi.org/
10.1016/j.steroids.2016.05.010.
´
´
´
¨
[16] N. Bozsity, R. Minorics, J. Szabo, E. Mernyak, G. Schneider, J. Wolfling, H.C. Wang,
´
´
The work of Erzsebet Mernyak in this project was supported by the
´
C.C. Wu, I. Ocsovszki, I. Zupko, Mechanism of antiproliferative action of a new d-
´
Janos Bolyai Research Scholarship of the Hungarian Academy of Sci-
secoestrone-triazole derivative in cervical cancer cells and its effect on cancer cell
motility, J. Steroid Biochem. Mol. Biol. 165 (2017) 247–257, https://doi.org/
10.1016/j.jsbmb.2016.06.013.
´
´
ences. The work of Erzsebet Mernyak in this project was supported by
the ÚNKP-19-4-SZTE-71, New National Excellence Program of The
Ministry of Human CapacitieS”. This work was supported by National
Research, Development and Innovation Office-NKFIH through project
OTKA SNN 124329. Support from Ministry of Human Capacities,
Hungary grant 20391-3/2018/FEKUSTRAT is acknowledged.
´
¨
´
´
[17] J. Szabo, N. Jerkovics, G. Schneider, J. Wolfling, N. Bozsity, R. Minorics, I. Zupko,
´
E. Mernyak, Synthesis and in vitro antiproliferative evaluation of C-13 epimers of
triazolyl-d-secoestrone alcohols: the first potent 13α-D-secoestrone derivative,
Molecules 21 (2016) 611, https://doi.org/10.3390/molecules21050611.
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[18] E. Mernyak, J. Szabo, I. Bacsa, J. Huber, G. Schneider, R. Minorics, N. Bozsity,
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I. Zupko, M. Varga, Z. Bikadi, E. Hazai, J. Wolfling, Syntheses and antiproliferative
effects of D-homo- and D-secoestrones, Steroids 87 (2014) 128–136, https://doi.
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Appendix A. Supplementary data
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I. Zupko, J. Wolfling, Synthesis and in vitro antiproliferative evaluation of d-
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α
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