Microwave assisted synthesis and biomedical potency of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives

Article abstract of DOI:10.1016/j.steroids.2014.12.008

A convenient microwave assisted solvent free synthesis as well as conventional synthesis of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives 7-19 from appropriate steroidal precursors 1-6 and methyl salicylate is reported. The microwave assisted synthesis in most cases was more successful regarding reaction time and product yields. It was more environmentally friendly too, compared to the conventional method. The antioxidant activity and cytotoxicity of the synthesized derivatives were evaluated in a series of in vitro tests, as well as their inhibition potency exerted on hydroxysteroid dehydrogenase enzymes (Δ⁵-3βHSD, 17βHSD2 and 17βHSD3). All of the tested compounds were effective in OH radical neutralization, particularly compounds 9, 11 and 14, which exhibited about 100-fold stronger activity than commercial antioxidants BHT and BHA. In DPPH radical scavenging new compounds were effective, but less than reference compounds. 2-Methoxybenzoyl ester 10 exhibited strong cytotoxicity against MDA-MB-231 cells. Most compounds inhibited growth of PC-3 cells, where salicyloyloxy stigmastane derivative 15 showed the best inhibition potency. Compounds 9, 10 and 11 were the best inhibitors of 17βHSD2 enzyme. X-ray structure analysis and molecular mechanics calculations (MMC) were performed for the best cytotoxic agents, compounds 10 and 15. A comparison of crystal and MMC structures of compounds 10 and 15 revealed that their molecules conformations are stable even after releasing of the influence of crystalline field and that the influence of crystal packing on molecular conformation is not predominant.

Full text of DOI:10.1016/j.steroids.2014.12.008

Steroids 94 (2015) 31–40
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Microwave assisted synthesis and biomedical potency of salicyloyloxy
and 2-methoxybenzoyloxy androstane and stigmastane derivatives
a
a
b
b
a
´
Katarina M. Penov Gaši , Evgenija A. Djurendic , Mihály Szécsi , János Gardi , János J. Csanádi ,
c
a
a
a
´
ˇ
´
´
´
´
Olivera R. Klisuric , Sanja V. Dojcinovic-Vujaškovic , Andrea R. Nikolic , Marina P. Savic ,
Jovana J. Ajdukovic , Aleksandar M. Oklješa , Vesna V. Kojic , Marija N. Sakac ,
Suzana S. Jovanovic-Šanta
a
a
d
´
´
´
ˇ a
a,
⇑
a
´
Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia
b First Department of Medicine, University of Szeged, Korányi fasor 8-10, H-6720 Szeged, Hungary
c Department of Physics, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovic´a 4, 21000 Novi Sad, Serbia
d Oncology Institute of Vojvodina, Put Dr Goldmana 4, 21204 Sremska Kamenica, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2014
Received in revised form 19 November 2014
Accepted 12 December 2014
Available online 23 December 2014
A convenient microwave assisted solvent free synthesis as well as conventional synthesis of salicyloyloxy
and 2-methoxybenzoyloxy androstane and stigmastane derivatives 7–19 from appropriate steroidal pre-
cursors 1–6 and methyl salicylate is reported. The microwave assisted synthesis in most cases was more
successful regarding reaction time and product yields. It was more environmentally friendly too, com-
pared to the conventional method. The antioxidant activity and cytotoxicity of the synthesized deriva-
tives were evaluated in a series of in vitro tests, as well as their inhibition potency exerted on
Keywords:
hydroxysteroid dehydrogenase enzymes (D
5-3bHSD, 17bHSD2 and 17bHSD3). All of the tested com-
Steroidal salicylic acid derivatives
Antioxidant activity
Cytotoxicity
pounds were effective in OH radical neutralization, particularly compounds 9, 11 and 14, which exhibited
about 100-fold stronger activity than commercial antioxidants BHT and BHA. In DPPH radical scavenging
new compounds were effective, but less than reference compounds.
D
5-3b-Hydroxysteroid dehydrogenase (D5
-
2-Methoxybenzoyl ester 10 exhibited strong cytotoxicity against MDA-MB-231 cells. Most compounds
inhibited growth of PC-3 cells, where salicyloyloxy stigmastane derivative 15 showed the best inhibition
potency. Compounds 9, 10 and 11 were the best inhibitors of 17bHSD2 enzyme. X-ray structure analysis
and molecular mechanics calculations (MMC) were performed for the best cytotoxic agents, compounds
10 and 15. A comparison of crystal and MMC structures of compounds 10 and 15 revealed that their mol-
ecules conformations are stable even after releasing of the influence of crystalline field and that the influ-
ence of crystal packing on molecular conformation is not predominant.
3bHSD) inhibition
17b-Hydroxysteroid dehydrogenase type 2
and type 3 (17bHSD2 and 3) inhibition
X-ray structural analysis
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
Many phenolic substances of plant and synthetic origin possess
powerful anti-oxidant and cytotoxic properties. Thus, for example,
It is well established that oxidative stress and oxidative damage
are closely associated with carcinogenesis [1]. Thus, the antioxi-
dant properties of androstane steroids which were proven in dif-
ferent cells and tissues [2–4] could potentially reduce cancer
cells formation and proliferation. b-Sitosterol and stigmasterol
expressed antioxidant activity, as well as cytotoxic activity against
human carcinoma cell lines and are used in the prevention of cer-
tain cancers [5–7]. Further, prednisolone and its derivatives,
besides their anti inflammatory effect, exhibited also antioxidant
and cytotoxic activity [8,9].
salicylic acid derivatives are phenolic compounds exhibiting such
activities [10–17].
In addition, some substances could influence on the level of cir-
culating steroids by affecting the steroidogenic enzymes, which is
of high significance for the treatment of different endocrine disor-
ders [18]. For example, some steroid substances expressed them-
selves as good inhibitors of 17b-hydroxysteroid dehydrogenase
type 2 (17bHSD2), so they can be used in the treatment of endo-
crine diseases, i.e., osteoporosis [19–21].
In this study, inspired by recent trends in using microwave
(MW) irradiation methods for preparing steroids [22–24] we
present efficient method for the synthesis of a series of steroidal
salicyloyl and 2-methoxybenzoyl esters by reaction of the
⇑
Corresponding author. Tel.: +381 21 485 2771; fax: +381 21 455 662.
E-mail address: suzana.jovanovic-santa@dh.uns.ac.rs (S.S. Jovanovic´-Šanta).
http://dx.doi.org/10.1016/j.steroids.2014.12.008
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

32
K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
corresponding steroidal compounds with methyl salicylate under
solvent-free MW irradiation conditions. Synthesized compounds
combine the structural characteristics of parent compounds, both
steroidal (testosterone, sitosterol, stigmasterol, 3b-hydroxy-, 11b-
hydroxy- and 3-oxo-steroids) and non-steroidal molecules (salicy-
lates). For comparison purpose, we carried out similar reactions,
using conventional heating in solution (toluene). Analytically pure
steroidal esters were tested in vitro to assess their abilities as free
radical scavengers (anti-oxidants), chemotherapeutic agents, and
was evaporated. The pure compounds 7, 9 and 11–18 were
obtained from crude mixture after chromatography on a silica gel
column (toluene–EtOAc, 10:1 for 7; n-hexane–acetone, 12:1 for
9, 11, 12 and 13; petroleum ether for 14; toluene for 15 and 16;
n-hexane–acetone, 7:1 for 17 and 8:1 for 18).
3-Oxo-androst-4-en-17b-yl salicylate (7). White crystals, mp
265 °C from n-hexane–dichloromethane. IR (KBr): 3110, 2977,
2939, 1671, 1614, 1485, 1324, 1305, 1250, 1230, 1217, 1164,
767. 1H NMR (CDCl3): 0.99 (s, 3H, H-18); 1.21 (s, 3H, H-19); 4.87
(dd, 1H, J1 = 7.7 Hz, J2 = 9.0 Hz, H-17); 5.75 (s, 1H, H-4); 6.86–7.86
(group of signals, 4H, H–Ar); 10.87 (s, 1H, OH from salicyloyl
group). 13C NMR (CDCl3): 12.2 (C-18); 17.4 (C-19); 20.5; 23.5;
27.6; 31.4; 32.7; 33.9; 35.4; 35.6; 36.6; 38.6; 42.9; 50.2; 53.6;
83.4 (C-17); 112.7; 117.6; 119.0; 123.9 (C-4); 129.7; 135.5;
161.6; 170.0; 170.8; 199.4 (C-3). HRMS (TOF) m/z: C26H32O4
[M+H]+ calculated 409.23734, found 409.23684; [M+Na]+ calculated:
431.21928, found: 431.21910.
inhibitors of three hydroxysteroid dehydrogenases (D
5-3bHSD,
17bHSD2 and 17bHSD3), related to the development of endocrine
diseases. Further, comparison of the X-ray crystallographic param-
eters obtained for two products (10 and 15) with the data obtained
by molecular mechanic calculations was carried out to study the
influence of crystalline field.
2. Experimental
(3-Oxo-androst-4-en-17b-yl)-2-methoxybenzoate (8). White
crystals, mp 158 °C from dichloromethane [25].
2.1. Chemical synthesis
3-Oxo-5a-androstan-17b-yl salicylate (9). White crystals, mp
219 °C from n-hexane–dichloromethane. IR (KBr): 3110, 2937,
2853, 1710, 1671, 1614, 1485, 1303, 1250, 1214, 1158, 1091,
765, 702. 1H NMR (CDCl3): 0.95 (s, 3H, H-18); 1.03 (s, 3H, H-19),
4.85 (t, 1H, J = 8.2 Hz, H-17); 6.85–7.86 (group of signals, 4H, H–
Ar); 10.88 (s, 1H, OH from salicyloyl group). 13C NMR (CDCl3):
11.4; 12.3; 20.8; 23.6; 27.6; 28.7; 31.2; 35.1; 35.7; 36.9; 38.0;
38.4; 43.0; 44.6; 46.5; 50.5; 53.6; 83.7 (C-17); 112.8 (C-10); 117.5
(C-30); 119.0 (C-50); 129.7 (C-60); 135.5 (C-40); 161.6 (C-20); 170.1
(C@O from salicyloyl group); 211.74 (C-3). HRMS (TOF) m/z:
2.1.1. General
The infrared spectra (wave numbers in cmꢀ1) were taken on a
Nexus 670 FT-IR spectrometer. Nuclear magnetic resonance
(NMR) spectra were recorded on a Bruker AC 250 apparatus oper-
ating at 250 MHz (proton) and 62.9 MHz (carbon), using standard
Bruker software, with tetramethylsilane as the internal standard.
Chemical shifts are given in ppm (d-scale); coupling constants (J)
are given in Hz. High resolution mass spectra (TOF) were recorded
on a 6210 Time-of-Flight LC/MS Agilent Technologies (ESI+) instru-
ment. GC/MS analyses were performed on an Agilent Technologies
GC 7890A instrument with Mass Selective Detector 5975C. Absor-
bances of the reaction mixtures in free radical scavenging tests
were recorded on a CECIL CE2021 spectrophotometer. The micro-
wave reactor was a monomode system (Microwave Synthesis Sis-
tem – Discover Bench Mate from CEM) with focused waves.
Melting points were determined using a Büchi SMP 20 apparatus
and are uncorrected. Organic solutions were dried over Na2SO4
and evaporated on a rotary evaporator under reduced pressure.
Column chromatography was performed on Merck grade 60 silica
gel (0.063–0.2 mm).
C
26H34O4 [M+H]+ calculated 411.25299, found 411.25272;
[M+Na]+ calculated: 433.23493, found: 433.23503.
(3-Oxo-5 -androstan-17b-yl)-2-methoxybenzoate (10). White
a
crystals, mp 165–167 °C from n-hexane–dichloromethane. IR
(KBr): 3014, 2942, 2851, 1712, 1601, 1582, 1490, 1307, 1253,
1133, 1083, 1025, 756. 1H NMR (CDCl3): 0.91 (s, 3H, H-18); 1.01
(s, 3H, H-19), 3.88 (s, 3H, OCH3); 4.81 (t, 1H, J = 8.7 Hz, H-17);
6.93–7.82 (group of signals, 4H, H–Ar). 13C NMR (CDCl3): 11.4;
12.3; 20.8; 23.6; 27.6; 28.7; 31.1; 35.1; 35.6; 36.8; 38.0; 38.4;
42.8; 44.5; 46.5; 50.5; 53.6; 55.8; 83.0 (C-17); 111.9; 119.9;
120.3; 131.5; 133.3; 159.2; 165.9 (C@O from 2-methoxybenzoyl
group); 211.8 (C-3). HRMS (TOF) m/z: C27H36O4 [M+H]+ calculated
425.26864, found 425.26794; [M+Na]+ calculated: 447.25058,
found: 447.24984.
17b-Hydroxyandrost-5-en-3b-yl salicylate (11). White crystals,
mp 184 °C from n-hexane–acetone. IR (KBr): 3273, 2943, 2905,
1667, 1614, 1485, 1301, 1249, 1211, 1157, 1137, 1086, 755. 1H
NMR (CDCl3): 0.78 (s, 3H, H-18); 1.09 (s, 3H, H-19); 3.67 (t, 1H,
J = 8.4 Hz, H-17); 4.89 (m, 1H, H-3); 5.43 (d, 1H, J = 4.6 Hz, H-6);
6.85–7.87 (group of signals, 4H, H–Ar), 10.92 (s, 1H, OH from sali-
cyloyl group). 13C NMR (CDCl3): 11.0; 19.4; 19.7; 20.6; 23.4; 27.7;
30.5; 31.5; 31.9; 36.5; 37.0; 38.1; 42.7; 50.1; 51.2; 75.2 (C-3); 81.9
(C-17); 112.8; 117.5; 119.0; 122.8 (C-5); 129.9; 135.5; 139.4 (C-4);
161.7; 169.7 (C@O). HRMS (TOF) m/z: C26H34O4 [MꢀH]ꢀ calcu-
lated: 409.23843, found: 409.23834.
2.1.2. General procedure for the preparation of compounds 7–19
2.1.2.1. Microwave irradiation. A mixture consisting of methyl salic-
ylate (18 mmol), the corresponding steroidal compound (1 mmol)
and sodium (3 mmol) was heated to 110 °C. When reaction with
sodium was completed (5–10 min), the mixture was irradiated
for 30 min at temperature 160–200 °C, using a 200 W MW source.
After cooling the reaction mixture to room temperature, water
(100 mL) and HCl (1:1, to pH 7) were added and crude product
was extracted with dichloromethane (3 ꢁ 50 mL). The organic
phase was dried and evaporated, resulted in an oily product.
Chromatographic separation of crude product on silica gel column
(toluene–EtOAc, 10:1 for 7 and 8; toluene–EtOAc, 20:1 for 9 and
10; n-hexane–acetone, 12:1 for 11, 12, 13; petroleum ether for
14; petroleum ether–acetone, 20:1 for 15 and 16; petroleum
ether–acetone, 3.5:1 for 18 and 19) gave the pure products 7–16,
18 and 19.
3b-Hydroxyandrost-5-en-17b-yl salicylate (12). White crystals,
mp 211–212 °C from EtOAc. IR (KBr): 3468, 3134, 2981, 2964,
2936, 2854, 1666, 1611, 1485, 1303, 1248, 1215, 1162, 1092,
760, 733. 1H NMR (CDCl3): 0.96 (s, 3H, H-18); 1.04 (s, 3H, H-19);
3.54 (m, 1H, H-3); 4.86 (dd, 1H, J1 = 7.5, J2 = 9.0 Hz, H-17); 5.38
(d, 1H, = 4.9 Hz, H-6); 6.86–7.87 (group of signals, 4H, H–Ar),
10.90 (s, 1H, OH from salicyloyl group). 13C NMR (CDCl3): 12.2
(C-18); 19.4 (C-19); 20.6; 23.7; 27.7; 31.4; 31.6; 31.7; 36.6; 36.8;
37.2; 42.2; 42.8; 50.0; 51.0; 71.7 (C-3); 83.8 (C-17); 112.9; 117.6;
119.1; 121.2 (C-6); 129.8; 135.5; 140.9 (C-5); 161.6; 170.2
2.1.2.2. Conventional heating. A mixture consisting of methyl salic-
ylate (18 mmol), the corresponding steroidal compound (1 mmol),
sodium (3 mmol) and toluene (3 mL) was refluxed during 1–50 h,
depending of substrate. After the reaction completion, the reaction
mixture was cooled to room temperature, diluted with water
(100 mL), then neutralized with HCl (1:1) and extracted with
dichloromethane (3 ꢁ 50 mL). The extract was dried and solvent
(C@O). HRMS (TOF) m/z:
409.23843, found: 409.23758.
C
26H34O4 [MꢀH]ꢀ calculated:

K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
33
Androst-5-ene-3b, 17b-diyl disalicylate (13). White crystals, mp
212–213 °C from dichloromethane–acetone. IR (KBr): 3423, 3119,
2945, 1667, 1614, 1584, 1486, 1301, 1249, 1217, 1158, 1091,
995, 767, 703. 1H NMR (CDCl3): 0.98 (s, 3H, H-18); 1.11 (s, 3H,
H-19); 4.88 (overlapping, 2H, H-3 and H-17); 5.46 (d, 1H,
J = 4.3 Hz, H-6); 6.85–7.88 (group of signals, 8H, H–Ar); 10.90
and 10,92 (2s, 2H, OH phenolic groups from salicyloyl residues).
13C NMR (CDCl3): 12.2 (C-18); 19.4 (C-19); 20.5; 23.7; 27.6; 27.7;
31.4; 31.6; 36.7; 36.8; 36.9; 38.0; 42.8; 49.9; 50.9; 75.1 (C-3);
83.7 (C-17); 112.8; 117.5; 118.9; 119.0; 122.6 (C-6); 129.8;
129.8; 135.5; 139.4 (C-5); 161.6; 161.7; 169.6 and 170.1 (C@O
from salicyloyl group). MS (m/z, rel.%): 392 (M+–C6H4(2-OH)CO2H,
26.25); 255(M+–2xC6H4(2-OH)CO2H, 25); 194(17.5); 145(28.75);
121(100); 91(60); 55(42.5). For C33H38O6 (530.65) calculated:
74.69% C; 7.22% H; found: 74.72% C; 7.83% H.
(C-3, C@O); 217.1 (C-17, C@O). HRMS (TOF) m/z: C26H28O5
[M+H]+ calculated: 421.20095, found: 421.20111, [M+Na]+ calcu-
lated: 443.18290, found: 443.18378.
(3,17-Dioxo-androsta-1,4-dien-11b-yl)-2-methoxybenzoate (19).
Colorless oil. IR (film): 3054, 2939, 2854, 1738, 1693, 1663, 1601,
1490, 1298, 1252, 1129, 1080, 1071. 1H NMR (CDCl3): 1.12 (s,
3H, H-18); 1.29 (s, 3H, H-19); 3.92 (s, 3H, OCH3); 5.82 (d, 1H,
J = 2.9 Hz, H-11); 6.03 (d, 1H, J = 1.3 Hz, H-4), 6.27 (dd, 1H,
J1 = 1.3 Hz, J2 = 10.1 Hz, H-2); 6.98–7.87 (group of signals, 5H, H–
Ar and H-1). 13C NMR (CDCl3): 15.4; 20.8; 21.8; 31.5; 31.6; 32.5;
35.0; 36.9; 43.1; 46.6; 51.6; 54.8; 55.6; 70.8; 112.0; 120.2;
122.9; 128.4; 131.5; 134.1; 154.6 (C-4); 159.4 (C-20); 164.7 (–O–
C@O); 168.2 (C-5); 186.0 (C-3, C@O); 217.7 (C-17, C@O). HRMS
(TOF) m/z:
C
27H30O5 [M+H]+ calculated: 435.21660, found:
435.21633, [M+Na]+ calculated: 457.19855, found: 457.19797.
Stigmast-5-en-3b-yl salicylate (14). White crystals, mp 168 °C
from EtOAc. IR (KBr): 3141, 2951, 2868, 1670, 1612, 1300, 1249,
1213, 1157, 756, 701. 1H NMR (CDCl3): 0.69 (s, 3H, H-18); 1.07
(s, 3H, H-19); 4.88 (m, 1H, H-3); 5.43 (d, 1H, J = 4.3 Hz; H-6);
6.83–7.87 (group of signals, 4H, H–Ar), 10.93 (s, 1H, OH). 13C
NMR (CDCl3): 11.8 (C-18); 12.0; 18.8; 19.0; 19.3; 19.8; 21.0;
23.0; 24.3; 26.0; 27.8; 28.2; 29.1; 30.3; 31.9; 33.7; 36.1; 36.6;
36.9; 38.1; 39.7; 42.3; 45.8; 50.0; 56.0; 56.7; 75.3 (C-3); 112.9;
117.5; 118.9; 123.1; 129.9; 135.4; 139.3 (C-5); 161.7; 169.6
(C@O). HRMS (TOF) m/z: C36H53O3 Mꢀ calculated: 533.40002,
found: 533.40035.
Stigmasta-5,22-dien-3b-yl salicylate (15). White crystals, mp
180–183 °C from dichloromethane. IR (KBr): 3132, 2950, 2867,
1669, 1612, 1300, 1213, 1157, 1087, 973, 756. 1H NMR (CDCl3):
0.73 (s, 3H, H-18); 1.06 (s, 3H, H-19); 5.05–5.17 (overlapping, 3H,
H-3 and H-22 and H-23); 5.45 (d, 1H, J = 4.3 Hz, H-6); 6.85–7.88
(group of signals, 4H, H–Ar); 10.94 (s, 1H, OH). 13C NMR (CDCl3):
12.0; 12.3; 19.0; 19.3; 21.0; 21.1; 21.2; 22.1; 24.3; 25.4; 27.8;
28.9; 31.8; 31.9; 36.6; 36.9; 38.1; 39.6; 40.5; 42.2; 50.0; 51.2;
55.9; 56.8; 75.2 (C-3); 112.8; 117.5; 118.9; 123.1; 129.3; 129.9;
135.4; 138.3; 139.3 (C-5); 161.7; 169.6 (C@O). MS (m/z, rel.%):
394 (M+–C6H4(2-OH)CO2H, 46.15; 368 (5.76); 351 (8.46); 281
(11.53); 255 (30.76); 207 (16.15); 159 (22.31); 135 (36.15); 105
(51.92); 83 (59.23); 55 (100). For C36H52O3 (532.80) calculated:
81.15% C; 9.84% H; found: 80.93% C; 9.71% H.
2.2. Biological tests
2.2.1. Free radical scavenging assays
Free radical scavenging capacity (RSC) of the synthesized com-
pounds was evaluated by measuring of their ability to neutralize
2,2-diphenyl-1-picrylhydrazyl (DPPH) or hydroxyl (OH) radicals.
Final concentrations of the tested compounds were in the range
of 0.01–8 mM.
2.2.1.1. DPPH assay. The DPPH-assay was performed as described
previously [17]. Different aliquots (0.1–2.0 mL) of 0.01 M sample
solution in dichloromethane were added to DPPH solution in meth-
anol (90 lM, 1 mL; Sigma, St. Louis, MO) and filled with 95 vol.% of
methanol to a final volume of 4 mL. The same reaction mixture with
no tested compounds was used as control. Absorbencies of the reac-
tion mixtures (Asample) and control (Acontrol) were recorded at 515 nm
(CECIL CE2021 spectrophotometer) after 1 h. Commercial synthetic
antioxidants, 3,5-di-tert-butyl-4-hydroxytoluene (BHT) (Aldrich;
Taufkirchen, Germany) and 3-tert-butyl-4-hydroxyanisole (BHA)
(Fluka; Taufkirchen, Germany) were used as positive controls. For
each sample, three replicates were recorded.
The percentage of DPPH radical scavenging capacity (DPPH RSC)
was calculated using the following equation:
(Stigmasta-5,22-dien-3b-yl)-2-methoxybenzoate (16). White
crystals, mp 155–157 °C from EtOAc. IR (KBr): 2954, 2868, 1724,
1601, 1583, 1490, 1463, 1299, 1252, 1132, 1081, 755. 1H NMR
(CDCl3): 0.72 (s, 3H, H-18); 1.06 (s, 3H, H-19); 3.90 (s, 3H, OCH3);
4.87 (m, 1H, H-3); 5.01–5.22 (overlapping, 2H, H-22 and H-23);
5.43 (d, 1H, J = 4.7 Hz, H-6); 6.95–7.79 (group of signals, 4H, H-
Ar). 13C NMR (CDCl3): 12.0; 12.3; 19.0; 19.4; 21.0; 21.1; 21.2;
24.4; 25.4; 27.8; 28.9; 31.9; 31.9; 36.6; 36.9; 37.0; 38.0; 38.2;
39.6; 40.5; 42.2; 50.0; 51.2; 55.9; 55.9; 56.8; 74.3 (C-3); 112.0;
120.0; 120.8; 122.6; 129.2; 131.3; 133.2; 138.3; 139.8; 159.1;
165.5 (C@O). HRMS (TOF) m/z: C37H54O3; [M+Na]+ calculated:
569.39652, found: 569.39639. For C37H54O3 (546,41) calculated:
81.27% C; 9.95% H; found: 80.93% C; 9.65% H.
RSC ð%Þ ¼ ðAcontrol ꢀ Asample=AcontrolÞ ꢁ 100:
IC50 values (the concentration of the tested compound in the
reaction mixture which causes 50% of RSC) were determined by
linear regression analysis from the obtained RSC values.
2.2.1.2. Hydroxyl-radical scavenging assay. Hydroxyl-radicals scav-
enging capacity (OH RSC) of the tested compounds was evaluated
by measuring the degradation of 2-deoxy-
chen, Germany) in the reaction with OH radicals, generated in situ in
Fenton’s reaction [17]. These radicals attack 2-deoxy- -ribose and
D-ribose (Aldrich; Taufkir-
D
degrade it into a series of fragments, some or all of which react with
2-thiobarbituric acid (TBA) (Sigma; St. Louis, MO) at low pH and high
temperature to give a pink chromogen, which can be determined
spectrophotometrically at 532 nm. Different aliquots (0.005–
0.5 mL) of a sample solution in dichloromethane were added to test
tubes (final concentration in the range of 0.01–8 mM, each contain-
ing 0.1 mL of 5 mM H2O2, 0.1 mL of 10 mM FeSO4, 0.1 mL of 0.05 M
11b-Hydroxyandrosta-1,4-diene-3,17-dione (17). White crystals,
mp 185–186 °C from n-hexane–acetone (lit. [26] mp 186.5–
189 °C; lit. [27] mp184–186 °C).
3,17-Dioxo-androsta-1,4-dien-11b-yl salicylate (18) Yellow oil. IR
(film): 3185, 3052, 2940, 2855, 1739, 1665, 1612, 1485, 1299,
1249, 1214, 1157, 1082, 761, 735, 702. 1H NMR (CDCl3): 1.08 (s,
3H, H-18); 1.31 (s, 3H, H-19); 5.84 (d, 1H, J = 2.9 Hz, H-11); 6.05
(s, 1H, H-4); 6.28 (dd,1H, H-2, J1 = 1.8 Hz, J2 = 10.1 Hz); 6.88–7.78
(group of signals, 5H, H–Ar and H-1); 10.85 (s, 1H, OH). 13C NMR
(CDCl3): 15.7 (C-18); 21.2 (C-19); 21.7; 31.3; 31.6; 32.3; 34.9;
36.7; 42.8; 46.2; 51.4; 54.6; 71.8 (C-11); 112.0 (C-10); 118.1 (C-
30); 119.4 (C-50); 123.0 (C-1); 128.6 (C-2); 129.2 (C-60); 136.2 (C-
40); 153.8 (C-4); 162.2 (C-20); 167.8 (O–C@O); 168.7 (C-5); 185.7
2-deoxy-D-ribose, and 0.067 M KH2PO4–K2HPO4 buffer of pH 7.4 to
a final volume of 3 mL). The same reaction mixture without sample
was used as control. After 1 h at 37 °C incubation, 2 mL of TBA
reagent (10.4 mL of 60% (v/v) HClO4, 3 g TBA and 120 g of trichloro-
acetic acid (TCA; Sigma; St. Louis, MO) and 0.2 mL of 0.1 M EDTA
(Sigma; St. Louis, MO) were added to the reaction mixture, and the
tubes were heated at 100 °C for 20 min. After cooling, absorbances
of the reaction mixtures and of the control were recorded at

34
K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
532 nm. Percentage of OH RSC was calculated using the same equa-
tion as for DPPH RSC.
Three replicates were recorded for each sample. BHT and BHA
were used as reference compounds. IC50 values (the concentration
at which 50% of OH radical is neutralized) were determined by lin-
ear regression analysis from the obtained RSC values.
androst-4-ene-3,17-dione and microsomes prepared from liver tis-
sue of adult female rats served as enzyme source in this case. Dur-
ing procedures, tissue preparations suited as enzyme source were
incubated with 1 l
M [14C]-labeled substrate steroid in the presence
of 1 mM coenzyme NAD or NADPH in 0.1 M HEPES buffer (pH = 7.3)
containing 1 mM EDTA and 1 mM dithiothreitol. The appropriate
substrate was added to the incubate in 10
glycol in HEPES buffer solution, whereas test compounds were
applied in 10 L of dimethyl sulfoxide solution. These organic sol-
vent contents of 200 L final volume of the incubation medium
lL of a 25 v/v% propylene
2.2.2. Cytotoxicity assay
The cytotoxicity of newly synthesized compounds was evalu-
ated using previously described method [28]. The chemotherapy
drug doxorubicin (DOX), used as reference compound, was tested
under the same experimental conditions.
l
l
did not reduce the enzyme activity substantially. Incubation was
carried out at 37 °C for 20 min and the enzymatic reaction was
stopped by the addition of ethyl acetate and freezing. After extrac-
tion, unlabeled carriers of the substrate and the product steroids
were added to the samples. Substrates and products were separated
by TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm thick) with
the solvent system diisopropyl ether/CH2Cl2 (50:50 v/v) and UV
spots were used to trace the separated steroids. Spots were cut
out and the radioactivity of the product formed and of the substrate
remaining was measured by means of liquid scintillation counting.
2.2.2.1. Cell lines. Three human tumor and one normal cell lines
were used in this study: estrogen receptor positive human breast
adenocarcinoma (ER+, MCF-7), estrogen receptor negative human
breast adenocarcinoma (ERꢀ, MDA-MB-231), human prostate can-
cer (PC-3) and normal fetal lung fibroblasts (MRC-5). These cells
were grown in Dulbecco’s modified Eagle’s medium with 4.5% of
glucose. Media were supplemented with 10% of fetal calf serum
(FCS, Sigma) and antibiotics (100 IU/mL of penicillin and 100
lg/
Test compounds were applied at 50 lM concentrations and control
mL of streptomycin; ICN Galenika). All cell lines were cultured in
flasks (Costar, 25 cm2) at 37 °C in 100% humidity atmosphere and
5% of CO2 incubator. Only viable cells were used in the assay. Via-
bility was determined by dye exclusion assay with trypan blue.
incubates without test substances were also prepared in every ser-
ies. Two experiments were performed with each test compound
and the standard deviations of the mean enzyme activity results
were within 10%. IC50 values were determined for more potent
inhibitors. In this case, conversion was measured at five or six dif-
ferent concentrations of the test compound between 0.1 and
50 lM. IC50 results were calculated by linear regression analysis fol-
lowing a logit–log transformation of the data, and the standard
deviations were determined from the fitted lines.
2.2.2.2. SRB test. Cytotoxicity was evaluated by colorimetric sulfo-
rhodamine B (SRB) assay [28]. In brief, single cell suspension
(5 ꢁ 103 cells) was plated into 96-well microtiter plates (Costar,
flat bottom). Plates were pre-incubated at 37 °C in a 5% CO2 incu-
bator during 24 h. Solutions of selected substances (at final con-
centrations ranging from 10ꢀ8 M to 10ꢀ4 M) were added to all
wells except the control ones. After incubation period (48 h/
37 °C/5% CO2) cytotoxicity assay was carried out as follows:
2.3. X-ray crystallographic analysis of compounds 10 and 15
X-ray diffraction data for compounds 10 and 15 was collected at
room temperature on an Oxford Diffraction (Agilent Technologies)
50
washed with distilled water, and SRB (75
all wells; 30 min later the plates were washed with citric acid (1%)
and dried at room temperature. Finally, 200 L of 10 mM Tris basis
lL of 80% TCA – was added to all wells; after 1 h the plates were
lL of 0.4%) was added to
Gemini S diffractometer with graphite-monochromated MoK
a
radiation (k = 0.7107 Å). Data reduction for all compounds was per-
formed with the program package CrysAlis RED [32]. Space group
determinations were based on analysis of the Laue class and sys-
tematically absent reflections. Structures were determined by
direct methods using SIR92 [33]. All structures were refined using
full-matrix least-squares. Non-hydrogen atoms were refined aniso-
tropically for all compounds, C–H hydrogen atoms were included
on calculated positions riding on their attached atoms with fixed
distances of 0.93 (CH) or 0.97 Å (CH2), and O–H hydrogen atoms
were identified on difference electron density maps and isotropi-
cally refined. All calculations were performed using SHELXL97
[34], PARST [35] and PLATON [36], as implemented in the WINGX
[37] system of programs. Crystal data and refinement parameters
are summarized in Table S1.
l
(pH 10.5) was added to all wells. Wells containing cells without
tested compounds were used as control. Wells without cells con-
taining only complete medium were used as blank. Absorbances
of samples (Asample) and control (Acontrol) were measured on the
microplate reader. Cytotoxicity was calculated according to the
formula:
CI ð%Þ ¼ ð1 ꢀ Asample=AcontrolÞ ꢁ 100:
Two independent experiments were set out in quadruplicate for
each concentration of the compound. Mean values and standard
deviations (SD) were calculated for each concentration. The IC50
(value that defines the dose of compound that inhibits cell growth
by 50%) for every compound was determined by Median effect
analysis.
3. Results and discussion
2.2.3. Determination of
and their inhibition
D
5-3bHSD, 17bHSD3 and 17bHSD2 activities
3.1. Synthesis of new compounds
Inhibitory effects exerted by the newly synthesized compounds
(7, 9–18) on the rat androgen synthesis were investigated with
in vitro radiosubstrate incubations. Our previously published
The primary objective of this study was the synthesis of salicy-
loyl esters of steroids 1–6 which was achieved by conventional
heating of a mixture of steroidal compound (1–6), sodium and
methyl salicylate in toluene. Otherwise, in solvent-free MW
assisted transesterification reaction, in addition to esterification
of steroidal alcohols, in most cases methylation of the phenol
moiety occurred also, leading to the mixtures of salicyloyl and
2-methoxybenzoyl esters, except in the case of substrates 3 and
4, when only salicyloyl esters were obtained as the reaction prod-
ucts. All mixtures of salicyloyl and 2-methoxybenzoyl esters were
method for
and adapted also to the measurement of 17bHSD3 and 17bHSD2
[31] activities.
5-3bHSD activity was investigated via dehydroepi-
D
5-3bHSD [29,30] was used with certain modifications
D
androsterone–androst-4-ene-3,17-dione conversion, whereas the
17bHSD3 was measured by the androst-4-ene-3,17-dione–testos-
terone conversion. Homogenate of testicular tissue obtained from
adult Wistar rats was used as source of these enzymes. Activity of
the 17bHSD2 was studied via conversion of testosterone to

K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
35
Table 1
The structures of starting and product compounds and reaction conditions for both MW assisted and conventional synthesis.
Substrates
Products
MW irradiation (200 W)
[temp (°C)/time (min)/yield (%)]
Conventional heating
[time (h)/yield (%)]
200/30/34.6
8/19.6
OH
O
O
OH
O
1
O
7
8
200/30/43.4
–
MeO
O
O
O
O
180/30/66
180/30/8.4
13/33.4
OH
O
O
O
O
OH
O
H
2
H
9
–
OMe
O
10
H
160/30/4.2
160/30/6
16/13.4
16/9.6
OH
OH
OH
O
HO
O
11
3
O
O
OH
O
HO
12
160/30/44.4
16/4
O
HO
OH
O
O
13
190/30/53
2/74
OH
O
HO
O
4
14
(continued on next page)

36
K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
Table 1 (continued)
Substrates
Products
MW irradiation (200 W)
[temp (°C)/time (min)/yield (%)]
Conventional heating
[time (h)/yield (%)]
200/30/18
2/43.5
OH
O
HO
O
5
15
200/30/26
2/15
MeO
O
O
16
–
1/32.8
HOH2C
O
O
HO
HO
OH
O
O
17
O
6
200/30/15.2
50/23.3
OH
O
O
O
O
18
200/30/10.6
–
OMe
O
O
O
19
chromatographically separated. We assume that there are two pos-
sible mechanisms of 2-methoxybenzoyl esters formation. First
mechanism supposes that salicyloyl esters could be formed pri-
marily and then methylated in situ under MW irradiation, giving
2-methoxybenzoyl esters. Another mechanism includes reaction
of steroidal alcohols with methyl 2-methoxybenzoate, probably
formed during heating of methyl salicylate at high temperatures
with sodium. However, prednisolone (6) underwent fragmentation
first, and then esterification, giving one fragmentation product and
its salicyloyl ester, when reaction mixture was conventionally
heated, while in the MW assisted reaction salicyloyl and 2-
methoxybenzoyl esters were obtained.
In order to compare the efficiency of both reaction conditions,
we compared the reaction durations, as well as the yields of newly
synthesized compounds (Table 1) for both MW assisted and con-
ventional methods.
Compound 7 was prepared in reaction of testosterone (1) and
methyl salicylate by MW assisted, as well as conventional heating
(Table 1). MW assisted reaction lasted 16 times shorter than con-
ventional one, and compound 7 was obtained in better yield
(34.6% vs. 19.6%). In the MW assisted reaction 2-methoxybenzoyl
ester 8 was obtained as well (43.4%; total yield of compounds 7
and 8 was 78%), which was not obtained by conventional heating.
Compound 8 was prepared before from testosterone and 2-
methoxybenzoyl chloride in the presence of 4-dimethylaminopyr-
idine and triethylamine for 48 h at room temperature [25], which
is almost 100 times slower than in MW assisted reaction. The spec-
troscopic data of compound 8 are in good agreement with earlier
data [25].
Starting from 17b-hydroxy-5a-androstan-3-one (2), MW
assisted reaction resulted in formation of two products: salicyloyl
ester 9 (66%) and 2-methoxybenzoyl ester 10 (8.4%). However,
derivative 9 was the only product formed by conventional heating
(33.4%). The yield of compound 9 was twice higher in the MW
assisted reaction, while the reaction time was 26-fold shorter.
Both MW assisted and conventional heating of 3b,17b-dihy-
droxy-androst-5-ene (3) gave three products: 3b-salicyloyl deriva-
tive 11, 17b-salicyloyl derivative 12 and 3b,17b-bis(salicyloyl)
derivative 13. MW assisted reaction conditions resulted in a
shorter reaction time (32-fold) and 2-fold greater total yield for
all products (11, 12 and 13) (54.6%), with compound 13 (44.4%)
constituting the major product, in comparing to the results of con-
ventional heating where the total yield of all products was 27%.
Transesterification of methyl salicylate with b-sitosterol (4) in
conventional reaction conditions resulted in salicyloyl derivative
14 in 74% yield, while the MW assisted reaction gave the same
product in 53% yield. Otherwise, MW assisted reaction conditions
resulted in a shorter reaction time (4-fold). Similarly, by conven-
tional heating of stigmasterol (5) with methyl salicylate, salicyloyl
derivative 15 and 2-methoxybenzoyl derivative 16 were obtained

K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
37
Table 2
MW assisted reactions, the transesterification reaction gives better
yields in solution (conventional heating) with sterols 4 and 5 pos-
sessing an additional hydrocarbon side chain.
Scavenger activity of the selected steroidal compounds; standard deviations of the
mean results were within 10%.
Compound
DPPH (IC50 mM)
OH (IC50 mM)
It should be noted that at 160 °C and 180 °C under MW irradia-
tion, salicyloylesters were the main or only products, while at higher
temperature (200 °C) 2-methoxybenzoyl esters are, in most cases,
the main reaction products (Table 2). Namely, comparison between
substrates 1 and 2, or 4 and 5 (Table 1), and both pairs with 3 indi-
cated an increasing of the yields of 2-methoxybenzoyl esters with
increasing of temperature. However, the apparent temperature
trend was broken if one compares the product profile of entry 2
(180 °C) and 4 (190 °C). When the reaction was carried out at
160 °C (substrate 3), salicyloyl esters as the only reaction products
were obtained. However, at 180 °C (substrate 2) the salicyloyl ester
was obtained as main product and corresponding 2-methoxyben-
zoyl ester was obtained as side product. By applying 200 °C (sub-
9
10
11
13
14
15
16
17
1.00
1.00
0.65
0.85
0.03
0.15
1.60
0.52
1.64
0.09
0.13
0.04
0.012
0.02
0.60
0.03
0.18
0.02
0.09
0.06
0.09
0.29
0.18
0.19
1.94
2.13
18
b-Sitosterol (4)
Stigmasterol (5)
BHT
BHA
strates 1,
5 and 6) the yields of the corresponding 2-
methoxybenzoyl esters were higher or similar as yields of salicyloyl
esters.
Table 3
In vitro cytotoxicity of the tested compounds: standard deviations of the mean results
were within 10%.
3.2. Biological properties
Compound
IC50 (lM)
3.2.1. Scavenger activity of the tested compounds
MCF-7
MDA-MB-231
PC-3
MRC-5
The antioxidant activities of the synthesized salicylic acid ste-
roidal derivatives were evaluated in a series of in vitro tests and
compared with those of commercial antioxidants BHT and BHA.
In the DPPH assay, the ability of the tested compounds to act as
hydrogen or electron donors in transforming DPPH radical to its
reduced form, DPPH-H, was measured spectrophotometrically
[17]. The hydroxyl radical scavenging activity of the examined
compounds was measured by the deoxyribose assay [17]. The pro-
7
9
>100
>100
68.32
>100
>100
48.25
61.02
45.11
>100
>100
>100
0.75
>100
>100
3.45
16.51
34.79
58.31
26.27
29.31
51.75
86.80
10.89
42.44
18.59
98.79
95.61
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
11
12
13
14
15
16
17
18
DOX
32.91
>100
88.01
>100
19.10
42.02
>100
40.58
0.12
tective effects of the tested compounds on 2-deoxy-D-ribose were
assessed as their ability to remove hydroxyl radicals (formed in
the Fenton reaction) from the test solution and prevent the sugar
degradation. The OH radical scavenging activity of the tested com-
pounds was determined indirectly, by measuring the absorbance of
the pink colored solutions. Scavenger activity of the selected ste-
roidal compounds is given in Table 2.
0.12
in better total yield (58.5%) than in MW assisted reaction condi-
tions (total yield 44%).
MW irradiation and conventional heating of prednisolone (6)
with methyl salicylate and sodium gave androstane derivatives
17, 18 and 19 as a result of primary cleavage of the C17-dihydroxy-
acetone side chain of prednisolone. Here we present degradation of
prednisolone (6) in the highly functionalized 17-ketosteroid 17
(32.8%), achieved with methyl salicylate and sodium in toluene
by conventional heating during 1 h (Table 1). However, the cleav-
age of the C17-dihydroxyacetone side chain of prednisolone under
different reaction conditions was reported before [26,27]. Thus, the
degradation of prednisolone using ethanolic KOH at room temper-
ature during 1.5 h gave 17-ketosteroid 17 in 23% yield [26]. On the
other hand, the reaction of prednisolone with Bi(OTf)3ꢂH2O, in 1,4-
dioxane, at 80 °C, for 14 h [27], afforded 17 in better yield (78%) but
longer time than in our work.
Though all of the tested compounds neutralized DPPH radical,
the highest scavenger activity was displayed by the salicyloyl ester
of b-sitosterol 14, which was slightly more effective than the com-
mercial antioxidant BHT and 3 times more effective than its parent
molecule, b-sitosterol.
All of the tested compounds were more effective in OH radical
neutralization than commercial antioxidants BHT and BHA, partic-
ularly androstane 17b-salicyloyloxy derivative 9, androstane 3b-
salicyloyloxy derivative 11 and salicyloyl ester of b-sitosterol 14,
which exhibited the highest activity with respect to IC50 values,
about 100-fold lower than those observed for BHT and BHA.
3b,17b-Bis-salicyloyl ester 13 was less active than the correspond-
ing mono-3b salicyloyl ester 11. Compound 18 with salicyloyloxy
group at C11 exhibited the lowest activity.
On the other hand, conventional heating of prednisolone (6)
with methyl salicylate for 50 h gave only 11b-salicyloyl ester 18
(23.3%). However, the MW assisted reaction resulted in formation
of two products: compound 18 (15.2%) and 2-methoxybenzoyl
ester 19 (10.6%). The yield of compound 18 was a little bit lower
in the MW assisted reaction, compared to the conventional heat-
ing, but the reaction time was 100-fold shorter.
Based on a comparison of the transesterification reaction of
methyl salicylate with steroidal alcohols 1–6 under solvent-free
MW irradiation conditions versus conventional heating, MW irra-
diation considerably accelerated the reactions (4–100-fold). In
the case of steroidal compounds 1, 2, 3 and 6, MW assisted reaction
resulted in higher total product yields. Although, in general, yields
and reaction times were higher and shorter, respectively, in the
It can be seen that introduction of salicyloyl or 2-methoxyben-
zoyl group in the C3 position of b-sitosterol and stigmasterol led to
an increasing of the OH radical neutralization potential. Namely,
salicyloyl and 2-methoxybenzoyl esters of b-sitosterol and stig-
masterol 14–16 expressed themselves as 2–9-fold better hydroxyl
radical scavengers than their parent compounds, where for b-sitos-
terol antioxidant activity was established before [5]. Salicyloyl
ester of b-sitosterol 14 was more active than salicyloyl ester of
stigmasterol 15.
3.2.2. Cytotoxicity
The synthesized androstane and stigmastane derivatives 7–18
were evaluated for their in vitro cytotoxicity against human breast
adenocarcinoma (MCF-7, ER+ and MDA-MB-231, ERꢀ) and

38
K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
Fig. 1. Cytotoxicity trough the range of investigated concentrations of the tested steroidal compounds against the most affected tumor cell line PC-3 (A: compounds 7, 9 and
10; B: compounds 11–13; C: compounds 14–16 and D: compounds 17 and 18). The corresponding cytotoxicity curves of reference compound doxorubicin is also presented on
each graph (dashed line). All derivatives expressed an expected dose-dependent toxicity.
Fig. 2. Cytotoxicity trough the range of investigated concentrations of the most effective tested steroidal compounds against MDA-MB-231 tumor cell line (A: compounds 10,
11 and 13; B: compounds 15, 16 and 18). The corresponding cytotoxicity curves of reference compound doxorubicin is also presented on both graphs (dashed line). All
derivatives expressed an expected dose-dependent toxicity.
prostate cancer cells (PC-3), as well as normal fetal lung fibroblasts
(MRC-5). Cytotoxicity was determined using the standard SRB
assay, after exposure of cells to the tested compounds for 48 h
[28]. Doxorubicin served as reference compound, used as positive
control for general toxicity. The results of cytotoxicity assay for
the tested compounds are presented in Table 3.
Prostate cancer PC-3 cells were the most sensitive on the tested
steroidal compounds (Table 3). All the tested compounds
decreased this cell line proliferation, where stigmastane 3b-salicy-
loyloxy derivative 15, testosterone salicyloyl ester 7 and com-
pound 17 without salicyloyl group expressed the best cytotoxic
potential (IC50 10.89, 16.51 and 18.59
MDA-MB-231 cell line the most active was 2-metoxybenzoyl ester
10 (IC50 3.45 M), whereas moderate cytotoxicity showed salicy-
loyl ester 15 (IC50 19.10 M).
For both PC-3 and MDA-MB-231 cell line all cytotoxic com-
pounds expressed an expected dose-dependent toxicity (Figs. 1
and 2, respectively). Estrogen receptor positive breast cancer cell
line (MCF-7) was less sensitive on the tested steroidal compounds.
Note that doxorubicin was extremely toxic to normal
noncancerous MRC-5 cells, consistent with its nonspecific cellular
cytotoxicity. In addition, doxorubicin controls showed low
lM, respectively). Against
l
l

K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
39
Table 4
In vitro inhibition of
D
5-3bHSD, 17bHSD3 and 17bHSD2 by selected steroidal compounds; standard deviations of the mean results were within 10%. Non-inhibited control taken
as 100%. S.D.: standard deviation. NI: no inhibition.
Compound
3bHSD inh.
17bHSD3 inh.
17bHSD2 inh.
Rel. conv. at 50
lM (%)
Rel. conv. at 50
lM (%)
Rel. conv. at 50
lM (%)
IC50 (lM)
7
9
78
NI
67
79
69
NI
NI
NI
90
NI
56
84
80
77
NI
NI
76
85
93
NI
95
79
–
–
–
–
74
88
60
78
NI
NI
–
19.0
5.8
3.7
1.8
–
–
–
–
–
10
11
12
13
14
15
16
17
18
–
22.4
Fig. 3. ORTEP drawings of the molecular structures of compounds 10 (a) and 15 (b) with labeling of non-H atoms. Displacement ellipsoids are shown at the 50% probability
level and H atoms are drawn as spheres of arbitrary radii. Intramolecular hydrogen bond is shown as dashed.
cytotoxicity against PC3-cells (IC50 95.61
before [38]. On the contrary, none of the newly synthesized steroi-
dal compounds were toxic against healthy cells (MRC-5).
l
M), which was shown
salicyloyloxy group in C-3 or C-17 position. Similarly, all of the
tested compounds decreased proliferation of prostate cancer PC-
3 cells, where 3- and 17-salicyloyl esters 15 and 7, as well as com-
pound 17 expressed the best cytotoxic effect. 17-(2-Methoxyben-
zoyl) ester 10 showed considerable antiproliferative activity
against MDA-MB-231 cells. Newly synthesized compounds were
no toxic against normal, non-cancerous cells (MRC-5), while doxo-
rubicin was extremely toxic against these cells. Most of the tested
substances effectively inhibited 17bHSD2 enzyme, whereas 17-sal-
icyloyl esters 9 and 11, as well as 17-(2-methoxybenzoyl) ester 10,
were the strongest inhibitors.
3.2.3. Inhibition of
D
5-3bHSD, 17bHSD3 and 17bHSD2 activities
Since hydroxysteroid dehydrogenase enzymes are involved in
the biosynthesis of active steroids, their inhibition constitutes an
interesting approach for treating steroid-dependent cancers. The
inhibitory effects of newly synthesized steroidal compounds
exerted on three enzymes from hydroxysteroid dehydrogenase
family were tested, in order to study potential of these substances
to influence the peripheral conversion of steroid precursors into
biologically active forms.
3.3. Crystallographic study
Inhibitory effects exerted on the rat testicular
D
5-3bHSD and
17bHSD3 activity by the newly synthesized compounds (7, 9–
18), investigated via measurement of steroidal substrate to corre-
sponding product conversion by in vitro radiosubstrate incubation
methods, showed that these compounds do not affect the activity
of these two enzymes (Table 4). However, almost all of the tested
substances expressed the inhibitory effects on the 17bHSD2 activ-
ity, whereas salicyloyl esters 9 and 11, and 2-methoxybenzoyl
ester 10 efficiently inhibited this enzyme (IC50 = 5.8, 1.8 and
To complement our investigation, we defined three-dimensional
structures in the crystalline state of compounds 10 and 15, which
exhibited the best cytotoxic activity against breast i.e. prostate can-
cer cells (Table 3). Representative views of molecules 10 and 15 are
shown in Fig. 3 [39]. Since we investigated behavior of compounds
10 and 15 in biological (live) systems, we were interested in the con-
formation of molecules released from the influence of crystalline
field (the conformations in which the compounds exhibit their bio-
logical activity). After determining three-dimensional structures of
these compounds in crystalline state, next step in our study was
defining the conformation of the molecules 10 and 15 in terms of
energy minima. For this purpose, we performed the MMC using
PCMODEL [40]. The conformation of molecules remains stable at
their energy minima, which is confirmed by comparing selected tor-
sion angles of molecules, obtained from crystal structure analyses
and after MMC (Table S2). As can be seen in Fig. S1, very good
3.7
inhibited it.
Summarizing biological properties of the investigated com-
lM, respectively), while salicyloyl esters 7 and 18 modestly
pounds, it can be concluded that some of androstane and stigmas-
tane salicyloyl and 2-methoxybenzoyl esters have promising
biomedical potential. Namely, all of the tested compounds
were effective in OH radical neutralization, even more than com-
mercial antioxidants, particularly compounds 9, 11 and 14 with

40
K.M. Penov Gaši et al. / Steroids 94 (2015) 31–40
[14] Fletcher S, Singh J, Zhang X, Yue P, Page BDG, Sharmeen S, et al. Disruption of
transcriptionally active Stat3 dimers with nonphosphorylated, salicylic acid-
based small molecules: potent in vitro and tumor cell activities. ChemBioChem
2009;10(12):1959–64.
overlap was observed for the molecular structures of molecules 10
and 15 in the crystalline state and after MMC.
Figs. 3 and S1 illustrate that compound 15 contains intramolec-
ular O–Hꢂ ꢂ ꢂO hydrogen bond (hydrogen bond parameters are given
in Table S3), which remains after MMC. Based on our analyses, the
presence of O–Hꢂ ꢂ ꢂO intramolecular hydrogen bonds appears to
stabilize the conformations of the compound 15. Otherwise, the
absence of intermolecular hydrogen bonding in the crystal packing
of both compounds 10 and 15 (illustrated in Fig. S2) could be one of
the reasons for the stability of their molecular conformations. The
influence of crystal packing molecular conformation is not
predominant.
ˇ
´
´
ˇ
´
[15] Djurendic´ EA, Dojcinovic-Vujaškovic SV, Sakac MN, Jovin EDj, Kojic VV,
Bogdanovic´ GM, et al. X-ray structural analysis, antioxidant and cytotoxic
activity of newly synthesized salicylic acid derivatives. Struct Chem
2010;21:67–78.
´
ˇ
´
´
ˇ
´
´
´
[16] Djurendic E, Dojcinovic-Vujaškovic S, Sakac M, Ajdukovic J, Gakovic A, Kojic V,
et al. Synthesis and biological evaluation of some new 2-oxazoline and
salicylic acid derivatives. ARKIVOC 2011;ii:83–102.
´
ˇ
´
´
´
´
[17] PenovGaši K, Djurendic E, DojcinovicVujaškovic S, Gakovic A, Jovanovic-Šanta
S, Kojic´ V, et al. Synthesis, anti-oxidant activity and cytotoxicity of salicyloyl
derivatives of estra-1,3,5(10)-triene and androst-5-ene. Chem Pap 2012;66:
284–94.
[18] Vihko P, Herrala A, Härkönen P, Isomaa V, Kaija H, Kurkela R, et al. Enzymes as
modulators in malignant transformation. J Steroid Biochem 2005;93:277–83.
[19] Poirier D, Bydal P, Tremblay MR, Sam KM, Luu-The V. Inhibitors of type II 17b-
hydroxysteroid dehydrogenase. Mol Cell Endocrinol 2001;171:119–28.
[20] Tremblay MR, Luu-The V, Leblanc G, Noél P, Breton E, Labrie F, et al.
Acknowledgments
Authors would like to thank the Hungary-Serbia IPA Cross-bor-
der Co-operation Programme (Project No. HUSRB/1002/214/133
RECODAC) and to the Provincial Secretariat for Science and Tech-
nological Development of the Autonomous Province of Vojvodina
(Grant No. 114-451-3600/2013-02).
Spironolactone-related
dehydrogenase: chemical synthesis, receptor binding affinities, and
proliferative/antiproliferative activities. Bioorgan Med Chem 1999;7:1013–23.
inhibitors
of
type
II
17b-hydroxysteroid
´
´
ˇ
´
´
´
[21] PenovGaši KM, Miljkovic DA, MedicMijacevic LjD, Djurendic EA, Stojanovic SZ,
ˇ
Sakac MN, et al. Synthesis, X-ray crystal structures and biological activity of
16-amino-17-substituted-D-homo steroid derivatives. Steroids 2003;68:
667–76.
ˇ
´
[22] Ahonen KV, Lahtinen MK, Valkonen AM, Dracínsky M, Kolehmainen E.
Microwave assisted synthesis and solid-state characterization of lithocholyl
amides of isomeric aminopyridines. Steroids 2011;76:261–8.
[23] Deb S, Wähälä K. Rapid synthesis of long chain fatty acid esters of steroids in
ionic liquids with microwave irradiation: expedient one-pot procedure for
estradiol monoesters. Steroids 2010;75:740–4.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.steroids.2014.
12.008.
[24] Borah JM, Chowdhury P. Expedited Baeyer–Villiger oxidation of steroidal
ketones by microwave irradiation. Steroids 2011;76:1341–5.
[25] Wang J. Androstanes as androgen receptor modulators. WO 2003026568 A3;
2004.
References
[26] Simons SS, Merchlinsky MJ, Johnson DF. 17-Ketosteroids via a base induced
cleavage of C-17-dihydroxy acetone side chain. Steroids 1981;37:281–9.
[27] Pinto RMA, Salvador JAR, Le Roux C, Paixao JA. Bismuth(III) triflate-catalyzed
direct conversion of corticosteroids into highly functionalized 17-ketosteroids
by cleavage of the C17-dihydroxyacetone side chain. J Org Chem 2009;74:
8488–91.
[1] Klaunig JE, Kamendulis LM, Hocevar BA. Oxidative stress and oxidative damage
in carcinogenesis. Toxicol Pathol 2010;38:96–109.
[2] Ahlbom E, Prins GS, Ceccatelli S. Testosterone protects cerebellar granule cells
from oxidative stress-induced cell death through
mechanism. Brain Res 2001;23:255–62.
a receptor mediated
[28] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. New
colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer
Inst 1990;82:1107–12.
[29] Tóth I, Szécsi M, Julesz J, Faredin I. Activity and inhibition of 3-beta-
hydroxysteroid dehydrogenase/delta-5-4-isomerase in human skin. Skin
Pharmacol 1997;10(3):160–8.
[30] Darvas B, Székács A, Fónagy A, Szécsi M, Tóth I. Progesterone in Periplaneta
americana and Neobellieria bullata adults from the procuticle phase until first
progeny production. Gen Comp Endocrinol 1997;107(3):450–60.
[3] Boccuzzi G, Aragno M, Seccia M, Brignardello E, Tamagno E, Albano E, et al.
Protective effect of dehydroepiandrosterone against copper-induced lipid
peroxidation in the rat. Free Radic Biol Med 1997;22:1289–94.
[4] Aragno M, Brignardello E, Tamagno E, Gatto V, Danni O, Boccuzzi G.
Dehydroepiandrosterone administration prevents the oxidative damage
induced by acute hyperglycemia in rats. J Endocrinol 1997;155:233–40.
[5] Paniagua-Pérez R, Madrigal-Bujaidar E, Reyes-Cadena S, Alvarez-González I,
Sánchez-Chapul L, Pérez-Gallaga J, et al. Cell protection induced by beta-
sitosterol: inhibition of genotoxic damage, stimulation of lymphocyte
production, and determination of its antioxidant capacity. Arch Toxicol
2008;82:615–22.
[6] Park C, Moon D-O, Ryu C-H, Choi BT, Lee WH, Kim G-Y, et al. b-Sitosterol
sensitizes MDA-MB-231 cells to TRAIL-induced apoptosis. Acta Pharmacol Sin
2008;29(3):341–8.
[7] Woyengo TA, Ramprasath VR, Jones PJH. Anticancer effects of phytosterols. Eur
J Clin Nutr 2009;63:813–20.
´
´
ˇ
´
[31] Djurendic EA, Klisuric OR, Szécsi M, Sakac MN, Jovanovic-Šanta SS, Ignáth I,
et al. X-ray structural analysis and antitumor activity of new salicylic acid
derivatives. Struct Chem 2014;25:1747–58.
[32] Oxford diffraction CrysAlis CCD and CrysAlis RED. Versions 1.171. Oxford
Diffraction Ltd., Abingdon, Oxfordshire, England; 2008.
[33] Altomare A, Cascarano G, Giacovazzo C, Guagliardi A. Completion and
refinement of crystal structures with SIR92.
343–50.
J Appl Crystallogr 1993;26:
[8] Yildirim A, Sahin YN, Suleyman H, Yilmaz A, Yildirm S. The role of prednisolone
and epinephrine on gastric tissue and erythrocyte antioxidant status in
adrenalectomized rats. J Physiol Pharmacol 2007;58:105–16.
[34] Sheldrick GM. SHELX97, programs for crystal structure analysis. Germany:
University of Göttingen; 1997.
[35] Nardelli MJ. PARST95 – an update to PARST: a system of Fortran routines for
calculating molecular structure parameters from the results of crystal
structure analyses. J Appl Cryst 1995;28:659–62.
[9] Yazar E, Konyalioglu S, Col R, Osman Birdane Y, Levent Bas A, Elmas M. Effects
of vitamin
E and prednisolone on some oxidative stress markers in
endotoxemic rabbits. Rev Med Vet 2004;155:538–42.
[36] Spek AL. PLATON, a multipurpose crystallographic tool. The Netherlands:
University of Utrecht; 1998.
[37] Farrugia LJ. WinGX suite for small-molecule single-crystal crystallography.
J Appl Cryst 1999;32:837–8.
[10] Fujimori T, Yamada S, Yasui H, Sakurai H, Tu Y, Ishida T. Orally active
antioxidative copper(II) aspirinate: synthesis, structure characterization,
superoxide scavenging activity, and in vitro and in vivo antioxidative
evaluations. J Biol Inorg Chem 2005;10:831–41.
[38] David-Beabes GL, Overman MJ, Petrofski JA, Campbell PA, de Marzo AM,
Nelson WG. Int J Oncol 2000;17:1077–86.
[39] Farrugia LJ. Ortep-3 for windows – a version of ORTEP-III with a graphical user
interface (GUI). J Appl Crystallogr 1997;30:565.
[40] Serena software. PCMODEL, version 4.0. Serena Software Bloomington,
Indiana, USA; 1989.
[11] Logrado LPL, Silveira D, Romeiro LAS, de Moraes MO, Cavalcanti BC, Costa-
Lotufo LV, et al. Synthesis and biological evaluation of new salicylate
macrolactones from anacardic acids. J Braz Chem Soc 2005;16:1217–25.
[12] Ott I, Schmidt K, Kircher B, Schumacher P, Wiglenda T, Gust R. Antitumor-
active cobalt–alkyne complexes derived from acetylsalicylic acid: studies on
the mode of drug action. J Med Chem 2005;48:622–9.
[13] Ekinci D, Sentürk M, Küfrevioglu OI. Salicylic acid derivatives: synthesis,
features and usage as therapeutic tools. Expert Opin Ther Pat
2011;21(12):1831–41.