Synthesis, anti-oxidant activity, and cytotoxicity of salicyloyl derivatives of estra-1, 3, 5(10)-triene and androst-5-ene

Article abstract of DOI:10.2478/s11696-012-0150-6

Since many estrane and androstane derivatives exhibit cytotoxic, anti-oxidant, or anti-hormone activity, new steroidal derivatives were synthesised from appropriate estrogen or androgen precursors in order to obtain potential therapeutics for the treatment of steroid-dependent diseases. Starting from estradiol (I ), 6-oxo derivatives V and VII were prepared. 17β-Salicyloyl-6-oxo derivatives VI and VIII were synthesised by the reaction of compounds V or VII with methyl salicylate in the presence of sodium. 17β-Salicyloyloxy estradiol IX was prepared from estradiol. Beckmann fragmentation of 16-oxyimino alcohols XII and XIII with methyl salicylate yielded corresponding Dseco derivatives XIV and XV. Simultaneous fragmentation and acylation of compound XII resulted in 3β-salicyloyl-D-seco derivative XVI which was also obtained from compound XIV. Anti-oxidant assays of the newly synthesised compounds V-IX, XIV, and XVI indicated a stronger capacity for hydroxyl radical scavenging, and a weaker capacity for DPPH radical scavenging, compared with the standard anti-oxidants BHA and BHT. Compounds V, XIV, and XVI showed higher or the same activity as BHT. The cytotoxicity of new compounds was evaluated against human breast and prostate carcinoma cells. Compound VI exhibited strong cytotoxicity against MDA-MB-231 cells; compound XIV exhibited strong cytotoxicity against PC-3 cell line, while compound VII moderately inhibited the growth of PC-3 cells.

Full text of DOI:10.2478/s11696-012-0150-6

Chemical Papers 66 (4) 284–294 (2012)
DOI: 10.2478/s11696-012-0150-6
ORIGINAL PAPER
Synthesis, anti-oxidant activity, and cytotoxicity of salicyloyl
derivatives of estra-1,3,5(10)-triene and androst-5-ene
a
a
aKatarina Penov Gaši, Evgenija Djurendi´c, Sanja Dojčinovi´c-Vujaškovi´c,
aAndrea Gakovi´c, Suzana Jovanovi´c-Šanta*, Vesna Koji´c, Marija Sakač
a
b
a
aDepartment of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad,
Trg Dositeja Obradovi´ca 3, 21000 Novi Sad, Serbia
bOncology Institute of Vojvodina, Institutski put 4, 21204 Sremska Kamenica, Serbia
Received 3 June 2011; Revised 15 December 2011; Accepted 21 December 2011
Since many estrane and androstane derivatives exhibit cytotoxic, anti-oxidant, or anti-hormone
activity, new steroidal derivatives were synthesised from appropriate estrogen or androgen precursors
in order to obtain potential therapeutics for the treatment of steroid-dependent diseases. Starting
from estradiol (I ), 6-oxo derivatives V and VII were prepared. 17β-Salicyloyl-6-oxo derivatives
VI and VIII were synthesised by the reaction of compounds V or VII with methyl salicylate in
the presence of sodium. 17β-Salicyloyloxy estradiol IX was prepared from estradiol. Beckmann
fragmentation of 16-oxyimino alcohols XII and XIII with methyl salicylate yielded corresponding D-
seco derivatives XIV and XV. Simultaneous fragmentation and acylation of compound XII resulted
in 3β-salicyloyl-D-seco derivative XVI which was also obtained from compound XIV. Anti-oxidant
assays of the newly synthesised compounds V–IX, XIV, and XVI indicated a stronger capacity for
hydroxyl radical scavenging, and a weaker capacity for DPPH radical scavenging, compared with
the standard anti-oxidants BHA and BHT. Compounds V, XIV, and XVI showed higher or the
same activity as BHT. The cytotoxicity of new compounds was evaluated against human breast
and prostate carcinoma cells. Compound VI exhibited strong cytotoxicity against MDA-MB-231
cells; compound XIV exhibited strong cytotoxicity against PC-3 cell line, while compound VII
moderately inhibited the growth of PC-3 cells.
c
ꢀ 2012 Institute of Chemistry, Slovak Academy of Sciences
Keywords: seco-androstane, 6-oxo-estradiol, androst-5-ene, salicyloyl, anti-oxidant activity, cyto-
toxic activity
Introduction
istry during ageing in female rats was investigated
(de Aguiar et al., 2008). The esterification of estro-
gens with fatty acids at the C-17 position, which in-
creased their hydrophobicity, was reported to enhance
their anti-oxidative potential in LDL oxidation stud-
ies in vitro (Shwaery et al., 1998). The influence of
the 6-oxo function on the anti-oxidant activity of es-
trane derivatives was studied (Badeau et al., 2005).
Estrogen hormones and their derivatives show anti-
oxidative as well as cytotoxic activity. Many estro-
genic compounds with cytotoxic activity were synthe-
Many researchers have claimed that estrogens are
important brain protectors, because of their anti-
oxidant activity in neurons. In vitro (Vedder et al.,
1999) and in vivo studies (Rontu et al., 2004) demon-
strated that high non-physiological doses of estra-
diol were neuro-protective. Possible anti-oxidant prop-
erties of novel estradiol derivatives were examined
(Klinger et al., 2002). Also, the effect of estradiol
valerate on brain oxidative stress and blood biochem-
*Corresponding author, e-mail: suzana.jovanovic-santa@dh.uns.ac.rs

K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
285
al., 2000) which exhibited strong cytotoxicity against
PC-cells (Djurendi´c et al., 2009). A salicyloyl deriva-
tive of compound XIV (compound XVI ) was prepared
in this study, and its cytotoxic and anti-oxidant activ-
ities were tested.
sised by coupling different cytotoxic agents with estra-
diol and their anti-proliferative activity was assessed
(Devraj et al., 1996; Kasiotis et al., 2001; Sadeghi-
Aliabadi & Brown, 2004; Jones et al., 2001; Liu et al.,
2004; Ali et al., 2006).
Many phenolic substances of plant (Al-Dabbas et
al., 2006; Ljubuncic et al., 2005; Cicerale et al., 2010)
and synthetic origin (Fujisawa et al., 2002; Sestili et
al., 2002) possess powerful anti-oxidant and cytotoxic
properties (Al-Dabbas et al., 2006; Fujisawa et al.,
2002). For example, salicylic acid derivatives are phe-
nolic compounds exhibiting anti-oxidant and cytotoxic
activities against different human carcinoma cell lines
(Rotem et al., 2000; Etcheverry et al., 2000; Logrado
et al., 2005; Erickson et al., 1997; Ott et al., 2005;
Fujimori et al., 2005; Djurendi´c et al., 2010, 2011).
In our previous papers, we described the synthe-
sis of some 16,17-seco-estrane and androstane deriva-
tives (Jovanovi´c-Šanta et al., 2003, 2011; Sakač et al.,
2005a, 2008; Penov Gaši et al., 2001, 2003b). Some of
them exhibited anti-estrogen or potent anti-aromatase
effects. We also described the synthesis of some 16,17-
secoandrost-5-ene-16-nitrile derivatives and evaluated
their cytotoxic activity (Djurendi´c et al., 2008, 2009;
Sakač et al., 2008). The results showed that some
16,17-secoandrostane derivatives exhibited a strong
cytotoxic activity against PC-3 cells (Djurendi´c et al.,
2008, 2009; Sakač et al., 2008).
In the present investigation, the synthesis and bio-
logical activity of some new estrane and androstane
derivatives are described. Some of the newly syn-
thesised compounds combine the structural charac-
teristics of the parent compounds, both steroidal
(estradiol) and non-steroidal anti-oxidants (salicy-
lates). By connecting these structures, we expected
to obtain compounds with better anti-oxidant prop-
erties. That was the reason for studying the anti-
oxidant activity of the estrane derivatives with free
or protected phenolic hydroxyl groups in the steroidal
skeleton, as well as with the phenolic hydroxyl
group within the salicyloyl moiety. The cytotoxic-
ity of the synthesised compounds against several hu-
man tumour and normal cell lines (MCF-7, MDA-
MB-231, PC-3, and MRC-5) was evaluated after
48 h treatment. The effect of the salicyloyl group
on the cytotoxicity of the synthesised steroid com-
pounds was also investigated and the influence of
the 6-oxo-function on anti-oxidant and cytotoxic ac-
tivity of estrane derivatives was studied. 3β,17β-
Dihydroxy-16-oxyimino-androst-5-en (XII ) (Miljkovi´c
et al., 1973) served as a starting compound in
preparing several D-secoandrostane derivatives, which
showed cytotoxicity against MCF-7, MDA-MB-231,
PC-3, but not against MRC-5 cells (Djurendi´c et
al., 2008, 2009). In the present paper, compound
XII in the reaction with methyl salicylate unexpect-
edly gave 3β-hydroxy-17-oxo-16,17-secoandrost-5-en-
16-nitrile (XIV ) (Miljkovi´c et al., 1973; Penov Gaši et
Experimental
Melting points were determined using a Bu¨chi SMP
20 apparatus and the results are uncorrected. IR spec-
tra were recorded on a Nexus 670 FT-IR spectrometer.
1H (250 MHz) and 13C NMR spectra were recorded
on a Bruker AC 250 apparatus using tetramethylsilane
as the internal standard. HRMS spectra (TOF) were
recorded on a 6210 Time-of-Flight LC/MS Agilent
Technologies (ESI+) instrument. Absorbance of the
reaction mixtures in free radical scavenging tests was
recorded on a CECIL CE2021 spectrophotometer. Ab-
sorbance in the cytotoxicity test was measured on the
microplate reader (Multiscan MCC340, Labsystems)
at 540/690 nm. Organic solutions were dried with
Na2SO4. Column chromatography was performed on
silica gel 60 (0.063–0.02 mm, Merck).
3,17β–Dihydroxy-estra-1,3,5(10)-trien-6-one
(V)
Methanolic solution (1.5 M) of NaOMe (10 mL,
15 mmol) was added to a solution of III (1.11 g,
2.8 mmol) in anhydrous dichloromethane (15 mL) and
the reaction mixture was stirred at r.t. for 3.5 h. An-
other portion of methanolic NaOMe (5 mL, 7.5 mmol)
was added and the mixture was left overnight. The
solvent was evaporated under reduced pressure, the
residue was poured into aqueous HCl (2 M, 150 mL)
and the resulting precipitate was washed with water
to pH 7 and recrystallised from ethyl acetate to afford
crystalline product V (0.74 g, 92.5 %); m.p. 282–285◦
C
(Pejanovi´c et al. (1995) reported 278–280◦C, Longwell
and Wintersteiner (1940) reported 281–283◦C).
3-Hydroxy-6-oxo-estra-1,3,5(10)-trien-17β-yl
salicylate (VI)
To a mixture of compound V (0.56 g, 1.97 mmol),
methyl salicylate (1.82 g, 12 mmol) and metallic
sodium (0.30 g, 12.9 mmol) heated at 140◦C, dry
toluene (4 mL) was added and the reaction mixture
was heated to reflux for 5.5 h, cooled to r.t., poured
into cold water (50 mL), acidified with diluted HCl
(ϕr = 1 : 1) to pH 5–6, and extracted with chlo-
roform (4 × 30 mL). The combined organic layers
were dried (Na2SO4), filtered and evaporated under
reduced pressure to give a brown oil residue (2.70 g)
which was purified by column chromatography using
toluene/ethyl acetate (ϕr = 9 : 1 and ϕr = 5 : 1)
as the eluent. Recrystallisation from acetone afforded
pure VI (0.25 g, 32 %; m.p. 272◦C).

286
K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
3-Benzyloxy-17β-hydroxy-estra-1,3,5(10)-
trien-6-one (VII)
Penov Gaši et al. (2001) reported 125–128◦C), respec-
tively.
A suspension of V (0.5 g, 1.7 mmol), K2CO3
(0.47 g, 3.4 mmol), benzyl chloride (2.4 mL), acetone
(12 mL), and methanol (12 mL) was heated to reflux
for 8 h, cooled to r.t. and filtered to remove K2CO3.
The solvents and residual benzyl chloride were evapo-
rated under reduced pressure to give light-yellow crys-
tals of crude VII (0.66 g, 89.4 %). Recrystallisation
from methanol afforded pure VII (white crystals, m.p.
176◦C).
17-Oxo-16,17-secoandrost-5-ene-16-nitrile-
3β-yl salicylate (XVI)
A mixture of XII (0.10 g, 0.32 mmol) or XIV (0.12
g, 0.39 mmol), methyl salicylate (0.45 g, 3.0 mmol),
and metallic sodium (0.20 g, 8.6 mmol) was heated to
120◦C followed by the addition of dry toluene (3 mL).
The reaction mixture was heated to reflux (18 h for
XII or 15 h for XIV ) and treated analogously as for IX
to afford an oily crude product which was purified by
column chromatography using toluene/ethyl acetate
(ϕr = 12 : 1). Recrystallisation from hexane/acetone
(ϕr = 2 : 1) or ethanol afforded XVI (24.8 mg, 19 %
starting from XII or 26.7 mg, 16 % starting from XIV,
respectively; m.p. 234◦C).
3-Benzyloxy-6-oxo-estra-1,3,5(10)-trien-17β-yl
salicylate (VIII)
Dry toluene (3 mL) was added to a mixture of
VII (0.12 g, 0.25 mmol), methyl salicylate (0.45 g,
3.0 mmol), and metallic sodium (0.20 g, 8.6 mmol)
heated at 140◦C and the reaction mixture was heated
to reflux for 10 h, cooled to r.t., poured into cold wa-
ter (20 mL), acidified with diluted HCl (ϕr = 1 : 1) to
pH 5–6, and extracted with chloroform (4 × 30 mL).
The combined organic layers were dried (Na2SO4), fil-
tered and evaporated under reduced pressure to af-
ford a brown oily residue (0.90 g) which was purified
by column chromatography using hexane/acetone (ϕr
= 9 : 1) as the eluent. Recrystallisation from hex-
ane/acetone (ϕr = 3 : 1) afforded pure VIII (0.079
g, 56.6 %; m.p. > 330◦C).
Biological tests
Free radical scavenging capacity (RSC) of the syn-
thesised compounds was evaluated by measuring their
ability to neutralise 2,2-diphenyl-1-picrylhydrazyl
.
.
(DPPH ) and HO radicals.
The DPPH-assay was performed as described
previously (Mimica-Dukic et al., 2004). Different
aliquots (0.1–2.0 mL) of 0.01 M sample solution in
.
dichloromethane were added to DPPH in methanol
(90 µM, 1 mL; Sigma, St. Louis, MO) and filled
with 95 vol. % of methanol to a final volume of
4 mL. The same reaction mixture without the com-
pounds under test was used as the control. Ab-
sorbance of the reaction mixtures (Asample) and con-
trol (Acontrol) was recorded at 515 nm after 1 h.
Commercial synthetic anti-oxidants, 3,5-di-tert-butyl-
4-hydroxytoluene (BHT) (Aldrich, Germany) and 3-
tert-butyl-4-hydroxyanisole (BHA) (Fluka, Germany),
estradiol, and methyl salicylate were used as con-
trols. For each sample, three replicates were recorded.
The percentage of DPPH radical scavenging capac-
ity (DPPH RSC) was calculated using the following
equation:
3-Hydroxy-estra-1,3,5(10)-trien-17β-yl
salicylate (IX)
Analogously as for VIII, heating of dry toluene
(4 mL), estradiol (I ) (0.272 g, 1 mmol), methyl sali-
cylate (2.0 g, 12 mmol), and metallic sodium (0.1 g,
4.3 mmol) for 13 h and the same reaction procedure
(except for acidification to pH 1) afforded a brown
oily product which was purified by column chromatog-
raphy using toluene/ethyl acetate (ϕr = 18 : 1) as
the eluent to give light-yellow crystalline IX (0.142 g,
36 %, m.p. 81–84◦C).
Reaction of compounds XII and XIII with
methyl salicylate
RSC(%) = 100 × (Acontrol − Asample/Acontrol) (1)
IC50 values (the concentration of the tested com-
pound in the reaction mixture which causes 50 % of
RSC) were determined by linear regression analysis
from the RSC values obtained.
The hydroxyl-radicals’ scavenging capacity (HO
RSC) of the tested compounds was evaluated by mea-
A mixture of XII or XIII (0.17 mmol), methyl
salicylate (0.47 mmol), and dry toluene (1 mL)
was gradually heated to 110◦C, and metallic sodium
(1.39 mmol) was added. The reaction mixture was
heated to reflux (1 h for XII or 15 min for XIII )
and treated analogously as for IX. Column chromatog-
raphy of the crude product using hexane/acetone
(ϕr = 7 : 1) for XIV or toluene/ethyl acetate (ϕr
= 2 : 1) for XV as the eluents afforded pure XIV
(40 %, m.p. 130–131◦C; Penov Gaši et al. (2000) re-
ported 130–131◦C) and XV (53 %, m.p. 125–128◦C;
.
suring the degradation of 2-deoxy-d-ribose (Aldrich,
.
Germany) in the reaction with OH radicals, gener-
ated in situ in Fenton’s reaction (Mimica-Dukic et al.,
2004). These radicals attack 2-deoxy-d-ribose and de-
grade it to a series of fragments, some or all of which
react upon heating with 2-thiobarbituric acid (TBA)

K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
287
(Sigma, St. Louis, MO) at low pH to give a pink chro-
mogen, which can be determined spectrophotometri-
cally 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 containing 0.1 mL of 5 mM H2O2, 0.1 mL
of 10 mM FeSO4, 0.1 mL of 0.05 M 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 a sample was used as the control. After an in-
cubation period of 1 h at 37◦C, 2 mL of TBA reagent
(10.4 mL of 60 vol. % HClO4, 3 g of TBA, and 120 g
of trichloroacetic acid (Sigma, St. Louis, MO, USA))
and 0.2 mL of 0.1 M EDTA (Sigma, St. Louis, MO,
USA) were added to the reaction mixture and the
tubes were heated at 100◦C for 20 min. After cool-
ing, absorbance of the reaction mixtures and of the
control was recorded at 532 nm. The percentage of
half an hour later the plates were washed with citric
acid (1 %) and dried at room temperature. Finally,
200 µL of 10 mM Tris (pH 10.5) base was added to
all wells. Absorbance was measured on the microplate
reader. The wells without cells, containing the com-
plete medium only, acted as blank. Cytotoxicity was
calculated according to the following equation:
CI(%) = (1 − Asample/Acontrol) × 100
(2)
Two independent experiments were conducted in
quadruplicate for each concentration of the com-
pound. The IC50 (value that defines the dose of a
compound that inhibits cell growth by 50 %) of com-
pounds was determined by median effect analysis.
Results and discussion
.
HO RSC was calculated using Eq. (1).
The synthesis of the estrane salicyloyl derivatives
is outlined in Fig. 1. Starting from estradiol (I ),
the reaction with propanoic acid anhydride in pyri-
dine gave estradiol dipropionate (II ) (Shuvalova et
al., 1983). Oxidation of II with chromium(VI) oxide–
3,5-dimethylpyrazole complex afforded 6-oxo deriva-
tive III as the main product and 6-oxo-9α-hydroxy
derivative IV as a by-product (Sakač et al., 2005b).
De-esterification of compound III to 3,17β-dihydroxy
derivative V was achieved with NaOMe. Previously,
Pejanovi´c et al. (1995) obtained V by hydrolysis of
ethyl ester in III using an ethanolic solution of potas-
sium hydroxide. The transesterification of methyl sal-
icylate with compound V to afford 3-hydroxy-17β-
salicyloyloxy derivative VI was carried out in toluene
in the presence of sodium under reflux. In view of the
planned investigation of the effect of the free phenolic
hydroxyl group in ring A of compound VI (benzy-
loxy derivative) on the biological activity, compound
V was transformed to the benzyl ether derivative VII
by reaction with benzyl chloride in the mixture of ace-
tone and methanol, in the presence of K2CO3 (Fig. 1)
under reflux. The transesterification of methyl salicy-
late with compound VII to afford 3-benzyloxy-17β-
salicyloyloxy derivative VIII was performed in the
presence of sodium in toluene under reflux for 10 h.
The salicyloyl derivative IX was obtained from estra-
diol I under similar reaction conditions within 13 h.
Dehydroepiandrosterone X, the starting compound
in the androstane series (Fig. 2), gave 16-oxyimino
derivative XI following the procedure by Stodola et
al. (1941). The reduction of hydroxyimino ketone XI
with NaBH4 afforded compound XII (Miljkovi´c et al.,
1973). Hydroxy oxime XIII was prepared by a selec-
tive addition of methylmagnesium iodide to the 17-oxo
group of XI (Penov Gaši et al., 2001).
Three replicates were recorded for each sample.
Estradiol, methyl salicylate, BHT, and BHA were used
as reference compounds. IC50 values (the concentra-
tion at which 50 % of HO is neutralised) were de-
termined by linear regression analysis from the RSC
values obtained.
The cytotoxicity of the synthesised compounds
was evaluated using the method described previously
(Djurendi´c et al., 2010). The chemotherapy drug dox-
orubicin (Dox), used as a control, was tested under
the same experimental conditions.
.
Three human tumour and one normal cell lines
were used in this study: estrogen receptor positive
human breast adenocarcinoma (ER+, MCF-7), es-
trogen receptor negative human breast adenocarci-
noma (ER-, MDA-MB-231), human prostate cancer
(PC-3), and normal foetal lung fibroblasts (MRC-5).
These cells were grown in Dulbecco’s modified Eagle’s
medium with 4.5 % of glucose. Media were supple-
mented with 10 % of foetal calf serum and antibiotics
(100 IU mL−1 of penicillin and 100 µg mL−1 of strep-
tomycin; ICN Galenika). All cell lines were cultured
in flasks (Costar, 25 cm2) at 37◦C in an atmosphere
of 100 % humidity and 5 % of CO2 in an incubator.
Only viable cells were used in the assay. Viability was
determined by dye exclusion assay with trypan blue.
Cytotoxicity was evaluated by colourimetric sul-
phorhodamine B (SRB) assay (Skehan et al., 1990).
Briefly, a single-cell suspension (5 × 103 cells) was
plated into 96-well microtitre plates (Costar, flat bot-
tom). The plates were pre-incubated at 37◦C in a 5 %
CO2 incubator for 24 h. The substances tested (final
concentrations ranging from 10−8 M to 10−4 M) were
transferred into all wells except for the control ones.
After the incubation period (48 h/37◦C/5 % CO2), the
cytotoxicity assay was carried out as follows: 50 µL of
80 % trichloroacetic acid (TCA) was added to all wells;
an hour later the plates were washed with distilled wa-
ter, and 75 µL of 0.4 % SRB was added to all wells;
In our previous works, we found that 16-oxyimino-
17β-hydroxy-17α-substituted and 17α-unsubstituted
estrane and androstane derivatives underwent the
Beckmann fragmentation reaction and formation of D-

288
K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
Fig. 1. Synthesis of estrane salicyloyl derivatives. Reaction conditions: i) propionic anhydride, pyridine; ii) CrO3, dimethylpyrazole,
CH2Cl2, –50◦C; iii) NaOMe, CH2Cl2; iv) methyl salicylate, Na, toluene, reflux, 5.5 h; v) BnCl, K2CO3, acetone, CH3OH,
reflux; vi) methyl salicylate, Na, toluene, reflux, 10 h; vii) methyl salicylate, Na, toluene, reflux, 13 h.
seco derivatives in the reaction with different reagents
(Penov Gaši et al., 2000, 2001, 2003a; Miljkovi´c et al.,
1973; Sakač et al., 2005b). In the present work, in
order to obtain salicyloyl derivatives of XII, the reac-
tion with methyl salicylate in the presence of sodium
was performed at the boiling point of toluene during
1 h. However, fragmentation product XIV was formed.
Similarly, heating of the mixture of XIII and methyl
salicylate for 15 min resulted in the formation of com-
pound XV. This represents a new method of fragmen-
tation of compounds XII and XIII, which was earlier
achieved by action of some other reagents (Penov Gaši
et al., 2000, 2001) resulting in 16,17-seco derivatives
XIV (Penov Gaši et al., 2000) and XV (Penov Gaši et
al., 2001) (Fig. 2).
The fragmentation reaction probably proceeded

K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
289
Fig. 2. Synthesis of androstane derivatives and mechanism of fragmentation of compounds XII and XIII with methyl salicylate.
Reaction conditions: i) amyl nitrite, tert-BuOK, tert-BuOH; ii) NaBH4, EtOH; iii) MeMgI, ether, THF; iv) methyl
salicylate, Na, toluene, reflux, 1 h; v) methyl salicylate, Na, toluene, reflux, 15 h; vi) methyl salicylate, Na, toluene, reflux,
18 h.
Fig. 3. Reaction of XII and XIII with acetic anhydride in pyridine. Reaction conditions: i) Ac2O, Py; ii) NaOEt, EtOH.
via salicyloyl derivatives XIIa and XIIIa, intermedi-
ates formed by preferential acylation of the oxyimino
group rather than the alcoholic 3β- and 17β-hydroxy
groups, according to Fig. 2. This reaction scheme ex-
plains the fragmentation reaction in terms of the Grob
fragmentation (the axis of the an electron pair on the
oxygen atom of the 17β-hydroxy group, the bond be-
tween C-16 and C-17 and the N-salicyloyloxy bond are
all parallel, thus fulfilling the stereoelectronic require-
ment for a smooth concerted fragmentation process).

290
K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
Table 1. Spectral data of newly prepared compounds
Compound
Spectral data
—
—
VI
VII
VIII
IR, ν˜/cm−1: 3177, 2924, 1675 (C-6 O), 1650 (C O of ester), 1612, 1577, 1305, 1214, 1157, 1091, 763
—
—
1H NMR (CD3COCD3), δ: 1.03 (s, 3H, H-18), 5.00 (t, 1H, H-17, J = 8.32 Hz), 6.94–7.95 (m, 7H, Ar-H), 8.61 (s,
1H, C-3–OH), 10.85 (s, 1H, OH of salicyloyl)
13C NMR (CD3COCD3), δ: 12.46 (C-18), 23.60, 26.17, 28.16, 37.24, 40.59, 43.26, 43.92, 44.29, 49.99, 84.39 (C-17),
112.91, 112.99, 118.19 (C-3ꢀ), 120.14 (C-5ꢀ), 121.86 (C-1ꢀ), 127.64, 130.75 (C-6ꢀ), 134.38, 136.70, 138.96, 156.70,
—
162.54, 170.09 (O—C O), 197.18 (C-6)
—
HRMS, m/z (found/calc.): 407.18510/407.18530 ([M + H]+, C25H27O5)
IR, ν˜/cm−1: 3540, 2940, 1680, 1633, 1580, 1507, 1433, 1327, 1293, 1153, 1053, 760
1H NMR (CDCl3), δ: 0.80 (s, 3H, H-18), 3.76 (t, 1H, H-17, J = 8.46 Hz), 5.11 (s, 2H, CH2 of benzyl), 6.86–7.87
(m, 8H, Ar-H)
13C NMR (CDCl3), δ: 10.88 (C-18), 22.81, 25.48, 30.39, 36.21, 40.08, 42.93, 43.02, 44.00, 49.86, 70.10 (CH2 of
benzyl), 81.46 (C-17), 110.72, 122.13, 126.64, 127.52, 128.01, 128.56, 133.32, 136.60, 139.82, 157.28, 197.88 (C-6)
HRMS, m/z (found/calc.): 377.21030/377.21112 ([M + H]+, C25H29O3); 399.19152/399.19307 ([M + Na]+
,
C25H28NaO3)
IR, ν˜/cm−1: 3435, 2946, 1683 (C-6 O), 1665 (C O of ester), 1610, 1487, 1304, 1248, 1217, 1158, 1092, 758
—
—
—
—
1H NMR (CDCl3), δ: 0.98 (s, 3H, H-18), 4.96 (t, 1H, H-17, J = 8.30 Hz), 5.10 (s, 2H, CH2 of benzyl), 6.86–7.87
(m, 11H, Ar-H), 10.87 (s, 1H, OH of salicyloyl)
13C NMR (CDCl3), δ: 12.18 (C-18), 23.06, 25.36, 27.51, 36.50, 39.73, 42.73, 43.16, 43.90, 49.55, 70.09 (CH2 of
benzyl), 83.15 (C-17), 110.73, 112.65, 117.61, 119.10, 122.18, 126.69, 127.53, 128.05, 128.59, 129.77, 133.27, 135.65,
—
136.57, 139.51, 157.33, 161.65, 170.06 (O—C O), 197.55 (C-6)
—
HRMS, m/z (found/calc.): 497.23185/497.23225 ([M + H]+, C32H33O5); 519.21407/519.21420 ([M + Na]+
,
C32H32NaO5)
IX
IR, ν˜/cm−1: 3421, 2927, 2872, 1669, 1613, 1584, 1485, 1303, 1249, 1215, 1158, 1092, 756
1H NMR (CD3COCD3), δ: 1.02 (s, 3H, H-18), 4.97 (t, 1H, H-17, J = 8.29 Hz), 6.53–7.94 (m, 7H, Ar-H), 8.02 (s,
1H, OH-3), 10.87 (s, 1H, OH of salicyloyl)
13C NMR (CD3COCD3), δ: 12.67 (C-18), 17.12, 23.92, 26.98, 28.01, 28.28, 30.22, 37.70, 39.61, 44.11, 44.61, 50.30,
84.74 (C-17), 113.61, 115.95, 118.17, 120.12, 127.06, 130.73, 131.65, 136.65, 138.31, 155.89, 155.99, 162.53, 170.97
—
(C O)
—
HRMS, m/z (found/calc.): 391.19078/391.19148 ([M – H]+, C25H27O4)
IR, ν˜/cm−1: 3418, 2942, 2886, 2243 (CN), 1727 (C-17 O), 1665 (C O of ester), 1612, 1581, 1486, 1329, 1297,
—
—
—
—
XVI
1217, 772
1H NMR (CDCl3), δ: 1.11 (s, 3H, H-18), 1.24 (s, 3H, H-19), 4.90 (m, 1H, H-3), 5.46 (m, 1H, H-6), 6.85–7.87 (m,
4H, Ar-H), 9.39 (s, 1H, H-17), 10.88 (s, 1H, OH of salicyloyl)
13C NMR (CDCl3), δ: 13.34 (C-18), 17.52 (C-19), 18.82, 19.22, 27.53, 31.42, 31.48, 32.76, 36.59, 36.80, 37.67, 40.93,
ꢀ
ꢀ
ꢀ
ꢀ
—
48.63, 49.78, 74.71 (C-3), 112.00 (C-1 ), 117.55 (C-3 ), 118.51 (C—N), 119.06 (C-5 ), 121.57 (C-6), 129.88 (C-6 ),
—
ꢀ
ꢀ
—
135.62(C-4 ), 139.02 (C-5), 161.71 (C-2 ), 169.64 (O—C O), 204.93 (C-17)
—
HRMS, m/z (found/calc.): 444.21432/444.21453 ([M + Na]+, C26H31NNaO4)
The proposed mechanism of fragmentation of XII and
XIII with methyl salicylate can be compared with
the mechanism of fragmentation of compound XIII
with acetic anhydride in pyridine, yielding the corre-
sponding 17-methyl-15-cyano-17-oxo derivative XVIII
(Penov Gaši et al., 2001) (Fig. 3).
cylate in the presence of sodium as a result of the
simultaneous acylation in ring A and fragmentation
in ring D.
The spectral data of the prepared compounds are
summarised in Table 1.
Previous investigations revealed that the reac-
tion of compound XII with acetic anhydride in
pyridine afforded 3β,17β-diacetoxy-16-acetoxyimino-
androst-5-ene (XVII ) which did not undergo the frag-
mentation reaction because of acetylation of the 17β-
OH group (Penov Gaši et al., 2000). However, in this
experiment, no acylation of 17β-OH group in XII with
methyl salicylate was observed, probably for steric rea-
sons, which enabled the free 17β-OH group to partici-
pate in the initial fragmentation reaction step (Fig. 3).
Transesterification of methyl salicylate with com-
pound XIV in the presence of sodium gave 3β-
salicyloyl-D-seco derivative XVI (Fig. 2.). This com-
pound was also obtained from XII and methyl sali-
Biological properties
The in vitro anti-oxidant activity of the synthe-
sised estrane and androstane derivatives was evalu-
ated and compared with those of estradiol, methyl sal-
icylate, and commercial anti-oxidants BHT and BHA
(Table 2., Fig. 4). In the DPPH assay, the ability of
the compounds under test to act as donors of hy-
.
drogen atoms or electrons in transforming DPPH
into its reduced form, DPPH-H, was measured by
the spectrophotometric method (Mimica-Dukic et al.,
2004). The compounds under test were able to reduce
the stable purple-coloured radical DPPH into yellow-
coloured DPPH-H form. The hydroxyl radical scav-
.

K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
291
Table 2. Radical scavenging activities of synthesised and ref-
Table 3. In vitro cytotoxicity of synthesised compounds and
erence compounds
doxorubicin
IC50/µM
IC50/mM
Compound
.
.
Compound
Cell line
DPPH
OH
V
VI
VII
VIII
IX
0.099
0.140
0.345
0.400
0.550
0.035
0.039
0.497
0.570
0.040
0.012
0.001
0.015
0.141
0.020
0.200
0.047
0.019
0.004
0.011
1.940
2.13
MCF-7 MDA-MB-231
PC-3
MRC-5
III
IV
V
VI
VII
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.75
> 100
> 100
> 100
10.40
> 100
> 100
> 100
56.00
> 100
0.12
> 100
> 100
> 100
85.24
24.46
> 100
> 100
6.00
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.12
XIV
XVI
VIII
IX
XIV
XVI
Doxorubicin
Estradiol (I)
Methyl salicylate
BHT
> 100
95.61
BHA
DPPH scavengers than estradiol and methyl salicy-
late.
Comparing the OH radical scavenger capacity of
the salicyloyl derivatives (VIII and XVI ) and their
precursors (VII and XIV ), it is obvious that intro-
duction of the salicyloyl function was the reason for
the increase in scavenger activity. In a comparison
of the OH radical scavenger activity of salicyloyloxy
derivatives VI and VIII with that of compound IX,
it could be seen that introduction of the 6-oxo func-
tion increased OH radical scavenger activity. Further,
compound VI (3-hydroxy derivative) showed higher
DPPH radical scavenging activity than compound
VIII (3-benzyl analogue), while both compounds are
less potent DPPH scavengers than commercial anti-
oxidants BHT and BHA. However, both compounds
VI and VIII showed higher activity towards OH rad-
icals than BHT and BHA. Compound VI, showing
higher activity towards OH radicals, was 129 times
more active than BHT and 142 times more active than
BHA; this was attributed to the free hydroxyl group
in ring A. It is also worth noting that the presence of
benzyl ether in ring A slightly reduced the activity of
compound VIII.
Fig. 4. Radical scavenging activities of synthesised and control
compounds.
enging activity of the compounds under test was mea-
sured in the deoxyribose assay (Mimica-Dukic et al.,
2004). The protective effects of the compounds under
test on 2-deoxy-d-ribose were assessed as their abil-
ity to remove hydroxyl radicals (formed in the Fen-
ton reaction) from the test solution and prevent sugar
degradation. The OH radical scavenging activity of
the compounds under test was determined indirectly
by measuring the absorbance of the pink-coloured so-
lutions.
The synthesised compounds V–VIII, XIV, and
XVI expressed higher DPPH scavenger activity when
compared with estradiol and methyl salicylate, where
the highest activity was shown by androstane deriva-
tives XIV and XVI, but lower than commercial anti-
oxidants BHT and BHA (compounds XIV and XVI
showed the same activity as BHT). Compounds V–
IX, XIV, and XVI were more potent OH radical scav-
engers compared with BHT and BHA, where com-
pound V was even 1940 times more active than BHT
and 2130 times more active than BHA. Compared
with estradiol, only compound V was a better OH rad-
ical scavenger (4 times). From all the salicyloyl deriva-
tives examined, estrane salicyloyl derivatives VI and
VIII and androstane derivative XVI were more potent
Cytotoxicity
The cytotoxic effect of the synthesised compounds
against ER+ human breast adenocarcinoma (MCF-7),
ER- human breast adenocarcinoma (MDA-MB-231),
human prostate cancer (PC-3), and normal foetal lung
fibroblasts (MRC-5) was examined by the standard
SRB assay after exposure of the cells to the com-
pounds under test for 48 h (Skehan et al., 1990). The
results are presented in Table 3.
As outlined in Table 3, 17β-salicyloyl derivative VI
showed the highest cytotoxicity against MDA-MB 231
cell line (IC50 value of 10.40 µM). Compound VIII,
3-benzyloxy analogue of VI, was less cytotoxic. On

292
K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
the other hand, compound VII with free 17β-hydroxyl
group, exhibited higher cytotoxicity against the PC-
3 cells than doxorubicin, which was not practically
toxic against these cells. Compound VI was more cy-
totoxic against MDA-MB-231 and PC-3 cells than its
analogue without salicyloyloxy function (compound
V ). On the other hand, salicyloyloxy derivatives VIII
and XVI were less cytotoxic against tumour cells than
their analogues without salicyloyloxy function (com-
pounds VII and XIV ). Comparing the effects of 17-
salicyloyloxy estradiol IX and its 6-oxo analogue VI, it
is obvious that introduction of the 6-oxo function in-
creases cytotoxicity against MDA-MB-231 and PC-3
cells.
Acknowledgements. This work was financially supported by
a research grant from the Provincial Secretariat for Science
and Technological Development of the Autonomous Province
of Vojvodina (Grant No. 114-451-1987/2011-01).
References
Al-Dabbas, M. M., Suganuma, T., Kitahara, K., Hou, D.
X., & Fujii, M. (2006). Cytotoxic, antioxidant and an-
tibacterial activities of Varthemia iphionoides Boiss. ex-
tracts. Journal of Ethnopharmacology, 108, 287–293. DOI:
10.1016/j.jep.2006.05.006.
Ali, H., Ahmed, N., Tessier, G., & van Lier, J. E. (2006). Syn-
thesis and biological activities of nucleoside–estradiol conju-
gates. Bioorganic & Medicinal Chemistry Letters, 16, 317–
319. DOI: 10.1016/j.bmcl.2005.10.004.
None of the newly synthesised compounds exhib-
ited cytotoxicity against healthy MRC-5 cells. By con-
trast, doxorubicin was toxic in very small doses.
Badeau, M., Adlercreutz, H., Kaihovaara, P., & Tikkanen, M. J.
(2005). Estrogen A-ring structure and antioxidative effect on
lipoproteins. Journal of Steroid Biochemistry & Molecular
Biology, 96, 271–278. DOI: 10.1016/j.jsbmb.2005.04.034.
Cicerale, S., Lucas, L., & Keast, R. (2010). Biological activi-
ties of phenolic compounds present in virgin olive oil. Inter-
national Journal of Molecular Sciences, 11, 458–479. DOI:
10.3390/ijms11020458.
de Aguiar, R. B., Dickel, O. E., Cunha, R. W., Monserrat, J. M.,
Barros, D. M., & Martinez, P. E. (2008). Estradiol valerate
and tibolone: effects upon brain oxidative stress and blood
biochemistry during aging in female rats. Biogerontology, 9,
285–298. DOI: 10.1007/s10522-008-9137-7.
Conclusions
In this paper, the synthesis and assaying of anti-
oxidant capacity and cytotoxic activity of new ben-
zyl and salicyloyl analogues of 6-oxo estradiol are de-
scribed. Benzyloxy and salicyloyloxy derivatives of
6-oxo estradiol VI, VII, and VIII expressed higher
DPPH scavenger capacity when compared with estra-
diol and methyl salicylate. Further, 6-oxo estradiol V
was 5 times more active against DPPH radical and
4 times against OH radical than estradiol. Similarly,
6-oxo salicyloyloxy derivative VI was a 4 times more
potent DPPH- and 13 times more potent OH scav-
enger than its analogue IX without the 6-oxo function.
All synthesised compounds showed a much higher OH
scavenger capacity than BHT and BHA. The most po-
tent OH scavenger of all the compounds examined was
6-oxo-estradiol V, which was more effective than the
reference compounds.
Chemical transformation of dehydroepiandroste-
rone afforded 3-hydroxy and 3-salicyloyloxy D-seco
derivatives. The fragmentation reactions of com-
pounds XII and XIII were carried out by a new
method, using methyl salicylate in the presence of
sodium. D-seco derivatives XIV and XV were ob-
tained, which exhibited higher DPPH scavenger ca-
pacity than estradiol and methyl salicylate, but simi-
lar to commercial anti-oxidant BHT.
Devraj, R., Barrett, J. F., Fernandez, J. A., Katzenellen-
bogen, J. A.,
& Cushman, M. (1996). Design, synthe-
sis, and biological evaluation of ellipticine–estradiol conju-
gates. Journal of Medicinal Chemistry, 39, 3367–3374. DOI:
10.1021/jm9602930.
Djurendi´c, E., Dojčinovi´c Vujaškovi´c, S., Sakač, M., Ajdukovi´c
J., Gakovi´c A., Koji´c, V., Bogdanovi´c, G., Klisuri´c, O.,
& Penov Gaši, K. (2011). Synthesis and biological evalua-
tion of some new 2-oxazoline and salicylic acid derivatives.
ARKIVOC, 2011(ii), 83–102.
Djurendi´c, E. A., Dojčinovi´c-Vujaškovi´c, S. V., Sakač, M. N.,
Jovin, E. D., Koji´c, V. V., Bogdanovi´c, G. M., Klisuri´c, O. R.,
Stankovi´c, S. M., Lazar, D. V., Fabian, L., & Penov-Gaši, K.
M. (2010). X-ray structural analysis, antioxidant and cyto-
toxic activity of newly synthesized salicylic acid derivatives.
Structural Chemistry, 21, 67–78. DOI: 10.1007/s11224-009-
9524-y.
Djurendi´c, E. A., Zaviš, M. P., Sakač, M. N., Čanadi, J. J.,
Koji´c, V. V., Bogdanovi´c, G. M., & Penov Gaši, K. M.
(2009). Synthesis and antitumor activity of new D-seco and
D-homo androstane derivatives. Steroids, 74, 983–988. DOI:
10.1016/j.steroids.2009.07.007.
Djurendi´c, E. A., Zaviš, M. P., Sakač, M. N., Koji´c, V. V.,
Bogdanovi´c, G. M., & Penov Gaši, K. M. (2008). Syn-
thesis and biological evaluation of some A,D-ring modified
16,17-secoandrostane derivatives. Collection of Czechoslovak
Chemical Communications, 73, 627–636. DOI: 10.1135/cccc
20080627.
Taking into consideration that most cytotoxic com-
pounds among newly synthesised derivatives have no
specific group that could be attributed to the cyto-
toxic effect, it is not possible to correlate their struc-
tures with their cytotoxicity.
Erickson, K. L., Beutler, J. A., Cardellina, J. H., II,
&
Boyd, M. R. (1997). Salicylihalamides A and B, novel cy-
In comparing the cytotoxicity profiles of the syn-
thesised compounds and doxorubicin, it may be noted
that compound VII was 4 times more cytotoxic
against PC-3 cells than doxorubicin. Compounds VI,
VII, and XIV were cytotoxic against tumour cells, but
not toxic against healthy cells (MRC-5 cell line), while
doxorubicin was very toxic against the healthy cells.
.
totoxic macrolides from the marine sponge Haliclona sp
The Journal of Organic Chemistry, 62, 8188–8192. DOI:
10.1021/jo971556g.
Etcheverry, S. B., Williams, P. A. M., Barrio, D. A., Sálice,
V. C., Ferrer, E. G., & Cortizo, A. M. (2000). Synthesis,
characterization and bioactivity of a new VO2+/aspirin com-
plex. Journal of Inorganic Biochemistry, 80, 169–171. DOI:
10.1016/s0162-0134(00)00026-x.

K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
293
Fujimori, T., Yamada, S., Yasui, H., Sakurai H., In, Y., & Ishida,
T. (2005). Orally active antioxidative copper(II) aspirinate:
synthesis, structure characterization, superoxide scavenging
activity, and in vitro and in vivo antioxidative evaluations.
Journal of Biological Inorganic Chemistry, 10, 931–941.
DOI: 10.1007/s00775-005-0031-3.
Fujisawa, S., Atsumi, T., Kadoma, Y., & Sakagami, H. (2002).
Antioxidant and prooxidant action of eugenol-related com-
pounds and their cytotoxicity. Toxicology, 177, 39–54. DOI:
10.1016/s0300-483x(02)00194-4.
Ott, I., Schmidt, K., Kircher, B., Schumacher, P., Wiglenda,
T., & Gust, R. (2005). Antitumor-active cobalt–alkyne com-
plexes derived from acetylsalicylic acid: Studies on the mode
of drug action. Journal of Medicinal Chemistry, 48, 622–629.
DOI: 10.1021/jm049326z.
Pejanovi´c, V. M., Petrovi´c, J. A., Csanádi, J. J., Stankovi´c
S. M., & Miljkovi´c, D. A. (1995). Synthesis and unusual
Beckmann fragmentation reaction of syn-3-methoxy-6α,l7β-
dihydroxyestra-1,3,5(10)-trien-7-one oxime. Tetrahedron, 51,
13379–13384. DOI: 10.1016/0040-4020(95)00871-5.
Jones, G. B., Hynd, G., Wright, J. M., Purohit, A., Plourde, G.
W., II, Huber, R. S., Mathews, J. E., Li, A., Kilgore, M. W.,
Bubley, G. J., Yancisin, M., & Brown, M. A. (2001). Target-
directed enediynes: Designed estramycins. The Journal of
Organic Chemistry, 66, 3688–3695. DOI: 10.1021/jo0055842.
Jovanovi´c-Šanta, S. S., Andri´c, S., Andri´c, N., Bogdanovi´c, G.,
& Petrovi´c, J. A. (2011). Evaluation of biological activity
of new hemiesters of 17-hydroxy-16,17-secoestra-1,3,5(10)-
triene-16-nitrile. Medicinal Chemistry Research, 20, 1102–
1110. DOI: 10.1007/s00044-010-9442-y.
Jovanovi´c-Šanta, S., Petrovi´c, J., Andri´c, S., Kovačevi´c, R., Đu-
rendi´c, E., Sakač, M., Lazar, D., & Stankovi´c, S. (2003). Syn-
thesis, structure, and screening of estrogenic and antiestro-
genic activity of new 3,17-substituted-16,17-seco-estratriene
derivatives. Bioorganic Chemistry, 31, 475–484. DOI: 10.
1016/s0045-2068(03)00101-9.
Kasiotis, K. M., Magiatis, P., Pratsinis, H., Skaltsounis, A. L.,
Abadji, V., Charalambous, A., Moutsatsou, P., & Haroutou-
nian, S. A. (2001). Synthesis and biological evaluation of
novel daunorubicin-estrogen conjugates. Steroids, 66, 785–
791. DOI: 10.1016/s0039-128x(01)00110-6.
Klinger, W., Lupp, A., Karge, E., Baumbach, H., Eichhorn, F.,
Feix, A., Fu¨ldner, F., Gernhardt, S., Knels, L., Kost, B.,
Mertens, G., Werner, F., Oettel, M., R¨omer, W., Schwarz,
S., Elger, W., & Schneider, B. (2002). Estradiol, testosterone,
dehydroepiandrosterone and androstenedione: novel deriva-
tives and enantiomers. Interactions with rat liver micro-
somal cytochrome P450 and antioxidant/radical scavenger
activities in vitro. Toxicology Letters, 128, 129–144. DOI:
10.1016/s0378-4274(01)00540-9.
Liu, C., Strobl, J. S., Bane, S., Schilling, J. K., McCracken,
M., Chatterjee, S. K., Rahim-Bata, R., & Kingston, D. G. I.
(2004). Design, synthesis, and bioactivities of steroid-linked
taxol analogues as potential targeted drugs for prostate and
breast cancer. Journal of Natural Products, 67, 152–159.
DOI: 10.1021/np030296x.
Ljubuncic, P., Azaizeh, H., Portnaya, I., Cogan, U., Said, O.,
Abu Saleh, K., & Bomzon, A. (2005). Antioxidant activ-
ity and cytotoxicity of eight plants used in traditional Arab
medicine in Israel. Journal of Ethnopharmacology, 99, 43–47.
DOI: 10.1016/j.jep.2005.01.060.
Logrado, L. P. L., Silveira, D., Romeiro, L. A. S., de Moraes, M.
O., Cavalcanti, B. C., Costa-Lotufo, L. V., do Ó Pessoa, C.,
& dos Santos, M. L. (2005). Synthesis and biological evalu-
ation of new salicylate macrolactones from anacardic acids.
Journal of the Brazilian Chemical Society, 16, 1217–1225.
DOI: 10.1590/s0103-50532005000700020.
Longwell, B., & Wintersteiner, O. (1940). Estrogens with oxy-
gen in ring B. III. 6-keto-α-estradiol. The Journal of Biolog-
ical Chemistry, 133, 219–229.
Penov Gaši, K., Cvjeti´canin, S., Stojanovi´c, S., Kuhajda,
K., Stupavský, L., Čanadi, J., Molnar-Gabor, D., Medi´c-
Mijačevi´c, L., & Sakač, M. (2000). The chemical transfor-
mation of 3β,17β-dihydroxy-16-oximino-5-androstene. Acta
Periodica Technologica, 31, 675–683.
Penov Gaši, K. M., Miljkovi´c, D. A., Medi´c-Mijačevi´c, L.
D., Djurendi´c, E. A., Stojanovi´c, S. Z., Sakač, M. N,
Djurendi´c, M. D., Stankovi´c, S. M., Lazar, D., Andri´c, S.,
& Kovačevi´c, R. (2003a). Synthesis, X-ray crystal struc-
tures and biological activity of 16-amino-17-substituted-
D-homo steroid derivatives. Steroids, 68, 667–676. DOI:
10.1016/s0039-128x(03)00097-7.
Penov Gaši, K. M., Stankovi´c, S. M., Csanádi, J. J., Djurendi´c,
E. A., Sakač, M. N., Medi´c Mijačevi´c, L., Arcson, O.
N., Stojanovi´c, S. Z., Andri´c, S., Molnar Gabor, D., &
Kovačevi´c, R. (2001). New D-modified androstane deriva-
tives as aromatase inhibitors. Steroids, 66, 645–653. DOI:
10.1016/s0039-128x(01)00096-4.
Penov-Gaši, K., Stojanovi´c, S., Sakač, M., Djurendi´c, E., Jo-
vanovi´c-Šanta, S., Stankovi´c, S., Andri´c, N., & Popsavin, M.
(2003b). Synthesis, crystal structure and antiaromatase ac-
tivity of 17-halo-16,17-seco-5-androstene derivatives. Journal
of Serbian Chemical Society, 68, 707–714.
Rontu, R., Solakivi, T., Teisala, K., Lehtimäki, T., Punnonen,
R., & Jokela, H. (2004). Impact of long-term hormone
replacement therapy on in vivo and in vitro markers of
lipid oxidation. Free Radical Research, 38, 129–137. DOI:
10.1080/10715760310001641169.
Rotem, R., Tzivony, Y., & Flescher, E. (2000). Contrasting ef-
fects of aspirin on prostate cancer cells: Suppression of pro-
liferation and induction of drug resistance. The Prostate, 42,
172–180. DOI: 10.1002/(SICI)1097-0045(20000215)42:3<172
::AID-PROS2>3.0.CO;2-R.
Sadeghi-Aliabadi, H., & Brown, J. (2004). Synthesis, analysis
and biological evaluation of novel steroidal estrogenic pro-
drugs for the treatment of breast cancer. Pharmaceutical Bi-
ology, 42, 367–373. DOI: 10.1080/13880200490519686.
Sakač, M., Gakovi´c, A., Stojanovi´c, S., Djurendi´c, E., Koji´c,
V., Bogdanovi´c, G., & Penov Gaši, K. (2008). Synthesis and
biological evaluation of a series of A,B-ring modified 16,17-
secoandrostane derivatives. Bioorganic Chemisty, 36, 128–
32. DOI: 10.1016/j.bioorg.2008.01.002.
Sakač, M. N., Miljkovi´c, D. A., Penov Gaši, K. M., Popsavin, M.,
Klisuri´c, O. R., Stankovi´c, S. M., Andri´c, S., & Kovačevi´c, R.
(2005a). Synthesis, X-ray crystal structure and antiestrogenic
activity of 17-methyl-16,17-secoestra-1,3,5(10)-triene deriva-
tives. Collection of Czechoslovak Chemical Communications,
70, 63–71. DOI: 10.1135/cccc20050063.
Sakač, M. N., Penov Gaši, K. M., Popsavin, M., Djurendi´c, E.
A., Andri´c, S., & Kovačevi´c, R. M. (2005b). Synthesis and es-
trogenic activity screening of some 6,9-disubstituted estradiol
derivatives. Collection of Czechoslovak Chemical Communi-
cations, 70, 479–486. DOI: 10.1135/cccc20050479.
Sestili, P., Diamantini, G., Bedini, A., Cerioni, L., Tommasini,
I., Tarzia, G., & Cantoni, O. (2002). Plant-derived pheno-
lic compounds prevent the DNA single-strand breakage and
cytotoxicity induced by tert-butylhydroperoxide via an iron-
chelating mechanism. Biochemical Journal, 364, 121–128.
Miljkovi´c, D., Petrovi´c, J., Staji´c, M., & Miljkovi´c, M. (1973).
The Beckmann fragmentation reaction of some α-hydroxy ke-
toximes. The Journal of Organic Chemistry, 38, 3585–3588.
DOI: 10.1021/jo00960a031.
Mimica-Dukic, N., Bozin, B., Sokovic, M., & Simin, N. (2004).
Antimicrobial and antioxidant activities of Melissa officinalis
L. (Lamiaceae) essential oil. Journal of Agricultural and Food
Chemistry, 52, 2485–2489. DOI: 10.1021/jf030698a.

294
K. Penov Gaši et al./Chemical Papers 66 (4) 284–294 (2012)
Shuvalova, S. D., Men’shova, N. I., & Grinenko, G. S. (1983).
Synthesis of estradiol esters from estrone. Pharmaceutical
Chemistry Journal, 17, 663–664. DOI: 10.1007/bf00764184.
Shwaery, G. T., Vita, J. A., & Keaney, J. F., Jr. (1998). An-
tioxidant protection of LDL by physiologic concentrations
of estrogens is specific for 17-beta-estradiol. Atherosclerosis,
138, 255–262. DOI: 10.1016/s0021-9150(98)00020-3.
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon,
J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., &
Boyd, M. R. (1990). New colorimetric cytotoxicity assay for
anticancer-drug screening. Journal of the National Cancer
Institute, 82, 1107–1112. DOI: 10.1093/jnci/82.13.1107.
Stodola, F. H., Kendall, E. C., & McKenzie, B. F. (1941).
Studies on steroid α-ketols. II. A new partial synthesis of 5-
androstene-3,16,17-triol: an intermediate in the preparation
of 16-hydroxytestosterone. The Journal of Organic Chem-
istry, 6, 841–844. DOI: 10.1021/jo01206a008.
Vedder, H., Anthes, N., Stumm, G., Wu¨rz, C., Behl, C., &
Krieg, J. C. (1999). Estrogen hormones reduce lipid peroxida-
tion in cells and tissues of the central nervous system. Jour-
nal of Neurochemistry, 72, 2531–2538. DOI: 10.1046/j.1471-
4159.1999.0722531.x.