Synthesis and cytotoxic activity of some 17-picolyl and 17-picolinylidene androstane derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.06.030

New 17-picolyl and 17-picolinylidene androstane derivatives, 3-10, 15, 18, 19, 22 and 23, were synthesized starting from 17α-picolyl-androst-5-en- 3β,17β-diol (1) and 17(Z)-picolinylidene-androst-5-en-3β-ol (2). Reaction of 1 with m-chloroperoxybenzoic ac

Full text of DOI:10.1016/j.ejmech.2012.06.030

European Journal of Medicinal Chemistry 54 (2012) 784e792
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxic activity of some 17-picolyl and 17-picolinylidene
androstane derivatives
,
a
ꢀ a
ꢀ a
ꢁ a
ꢀ b
*
Evgenija A. Djurendic , Jovana J. Ajdukovic , Marija N. Sakac , János J. Csanádi , Vesna V. Kojic ,
a
ꢀ b
ꢁ
Gordana M. Bogdanovic , Katarina M. Penov Gasi
Department of Chemistry, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia
b Oncology Institute of Vojvodina, Institutski put 4, 21204 Sremska Kamenica, Serbia
a
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
New 17-picolyl and 17-picolinylidene androstane derivatives, 3e10, 15, 18, 19, 22 and 23, were synthe-
Received 21 September 2011
Received in revised form
14 June 2012
Accepted 15 June 2012
Available online 23 June 2012
sized starting from 17
-ol (2). Reaction of 1 with m-chloroperoxybenzoic acid gives 5
with Jones reagent, 3,6-dione derivative 4; while 17 -picolyl-androst-5-en-3
,4 ,17 -triol (6) derivatives are obtainable from 1 using SeO2 in dioxane. Base-catalyzed tosyl group
elimination from 7 or 9 affords AB conjugated derivatives 8 and 10. Oppenauer oxidation of 1 and 2 yields
4-en-3-one derivatives 11 and 12, which, with H2O2 in 4 M NaOH, affords 4 ,5 and 4 ,5 -epoxides 13,
a
-picolyl-androst-5-en-3
b,17
b
-diol (1) and 17(Z)-picolinylidene-androst-5-en-
,6 -epoxy N-oxide derivative 3, or,
,4 ,17 -triol (5) or
3b
a a
a
b
a
b
3b
b
b
a
a
b b
Keywords:
14, 16 and 17. New 4-methoxy-3-keto derivatives 15 and 18 were obtained from 13 and 14, or, with
methanol in 4 M NaOH, from 16 and 17. Reduction of 11 with NaBH4 gives 22, which was then acetylated
to obtain 23. All new derivatives were screened for antitumor activity against human breast adenocar-
cinoma ERþ, MCF-7; human breast adenocarcinoma ER-, MDA-MB-231; prostate cancer AR-, PC-3;
human cervix carcinoma, HeLa; and colon cancer, HT-29 cells; as well as one human non-tumor cell
line, MRC-5. Compounds 3, 5, 6, 8, 10, 18, 19 and 22 exhibited significant antitumor activity against MDA-
MB-231 breast cancer cells; while 5, 6 and 10 also showed strong cytotoxicity against HT-29. Only
compound 19 exhibited significant activity against MCF-7 breast cancer cells. No compounds displayed
cytotoxicity against non-tumor MRC-5 cells.
Androst-5-ene derivatives
17a-Picolyl
17-Picolinylidene derivatives
Cytotoxicity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
biologically active compounds [6,7]. Similarly, in our own work
[8e10], we described the synthesis and characterization of several
Steroids are a fundamental class of natural biological signaling
molecules, which have demonstrated potential for development
into potent drugs for the treatment of a large number of diseases,
including: brain tumors, breast cancer, prostate cancer, cardiovas-
cular disease or autoimmune disorders [1,2].
Androgens are male steroid hormones which have a wide-range
of important physiological functions, including central involvement
in the development of male sex characteristics and other pheno-
types [3e5]. Anti-androgens are a diverse class of drugs which can
compete with circulating androgens for binding sites on androgen
receptors within prostate cells, leading to targeted apoptosis and
inhibition of androgen-dependent prostate cancer growth [5].
Several steroidal derivatives with substitutions at the C-17
position have been previously described in the literature as potent
17a-picolyl and 17-picolinylidene-androst-5-ene derivatives, with
anti-aromatase (CYP19A1) and/or anti-tumor activity against
a range of tumor cell lines. Several of these tested androstane
derivatives displayed significant aromatase inhibitory activity, and/
or strong activity against 3 common tumor cell lines (human cervix
carcinoma, HeLa; human melanoma, FemX; and human myeloge-
nous leukemia, K562), with IC50 values between 4 and 10 mM [8].
Because it is well known [11] that the presence of nitrogen
heteroatoms can interfere with steroidal hydroxylation by directly
interacting with the cytochrome P450 heme iron in aromatase;
these androstane compounds were specifically designed to incor-
porate nitrogen in the steroidal structure. In the present study, we
continue our investigation of C-17 substitutions, and report
a simple synthetic route for the preparation of 17-picolyl and
17-picolinylidene androst-5-ene derivatives. The effect of these
new androstane derivatives on cell proliferation was tested against
a wide-range of common tumor cell lines, including: human breast
* Corresponding author. Tel.: þ381 21 485 27 33; fax: þ381 21 454 065.
ꢀ
E-mail address: jovana.ajdukovic@dh.uns.ac.rs (J.J. Ajdukovic).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.030

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785
adenocarcinoma ERþ, MCF-7; human breast adenocarcinoma ER-,
MDA-MB-231; prostate cancer AR-, PC-3; human cervix carcinoma,
HeLa; and colon cancer, HT-29.
resulted in NOE enhancements at H-3
a
(3.57 ppm) and H-6
(5.71 ppm). In contrast, for compound 5, selective irradiation of the
H-4 proton signal at 3.89 ppm yielded no such NOE enhancement of
the H-3 signal, indicating that the C4-OH proton is directed toward
2. Results and discussion
the
a-position, consistent with our assignment of an a-configura-
tion for the 4-hydroxy group in compound 5.
2.1. Chemistry
Base-catalyzed tosyl group elimination from 7 or 9 was used to
obtain AB conjugated derivatives 8 and 10. Specifically, compound
The initial steps in the preparation of 17-picolyl and
17-picolinylidene androstane derivatives consist of a series of
8, which contains
compound 1 via 7 (in which the 3
a
3,5-diene system, was obtained from
-hydroxy group is tosylated). The
b
different reactions of 17
and 17-picolinylidene-androst-5-en-3
Specifically, compound 3 (5 ,6 -epoxy N-oxide) was obtained from
compound
a
-picolyl-androst-5-en-3
b
-ol (1) [12,13]
tosyl group was then eliminated by reaction of 7 with bis(2-ami-
noethyl)amine under reflux for 1.5 h, yielding only compound 8.
b-ol (2) [12,13] (see Fig. 1).
a
a
A similar scheme for formation of a D
3 double bond was previously
1
by reaction with m-chloroperoxybenzoic acid
reported by King and Bigelow [16], by reaction of epicholesteryl p-
toluenesulfonate with pyridine; and by Numazawa at al. [17], via
elimination of the tosyl group at C-3 in basic media with N-meth-
ylpirrolidone. The structure of compound 8 was identified by the
presence of an 1H NMR multiplet at 5.63 ppm, and a doublet at
5.94 ppm (J ¼ 9.9 Hz), corresponding to H-3 and H-4 protons,
respectively. Consistent with this, 13C NMR signals were also
recorded for C-3 (128.90 ppm) and C-4 (125.14 ppm).
(MCPBA) at 0 ꢀC,1 h / 10 ꢀC, 3 h / and room temperature, 24 h, at
a molar ratio of 1 : MCPBA ¼ 1:3 (Scheme 1). The presence of
5a,6a C
-epoxy function in compound 3 was confirmed by 1H and 13
NMR spectroscopy: the doublet at 2.93 ppm was attributed to H-6
and 13C NMR signals for C-5 and C-6 atoms were identified at 65.98
and 59.15 ppm, respectively. In addition, a signal for the 17 -OH
b
b
proton was visible at 6.65 ppm for compound 3 (but not compound
1), suggesting a lack of rapid proton exchange; while significant
chemical shift changes were also observed for pyridine C-atoms in
compound 3 (but not compound 1). Together, these results strongly
suggest the presence of an intramolecular hydrogen bond between
Similar reactions were performed on the 17-picolinylidene-
substituted derivative 2 (Scheme 1), to obtained compound 10 via
3b tosyl derivative 9. In this case, the tosyloxy group was also
eliminated with bis(2-aminoethyl)amine at reflux for 1 h, and
compound 10 was isolated from the resulting complex reaction
mixture. The identity of compound 10 was also confirmed by NMR.
1H NMR spectra of 10 shows a multiplet at 5.42 ppm for the H-3
proton, and a doublet at 5.95 ppm (J ¼ 9.7 Hz) for the H-4 proton;
while another multiplet signal at 5.61 ppm was assigned to the H-6
proton. Similarly, 13C NMR spectra contain characteristic signals for
C-3, C-4, C-5 and C-6 atoms, at 128.89, 125.16, 141.57 and
124.72 ppm, respectively.
17b-OH and the N-oxide function on the pyridine core in compound
3, which is absent in compound 1.
In contrast with the above described synthesis, reaction of
compound 1 with Jones reagent [14] at 0 ꢀC for 1.5 h affords
17b-hydroxy-17a-picolyl-androst-4-en-3,6-dione (4). Consistent
with this product, 1H NMR data for compound 4 shows the expected
singlet for the H-4 proton at 6.19 ppm, in place of the H-6 doublet at
5.36 ppm visible in 1H NMR spectra of compound 1. In addition,
signals for C-3 and C-6 carbonyl carbons are visible in 13C NMR
spectra of compound 4 at 199.46 and 201.95 ppm, respectively.
Interestingly, reaction of compound 1 with selenium-dioxide in
In addition to the above, we also investigated the effects of
applying Oppenauer oxidation [8] to our starting compounds 1 and
2. Oppenauer oxidation of 1 and 2 yields 4-en-3-one derivatives 11
dioxane was found to give a mixture of 4
a
and 4
b
hydroxylated
and 12, which, with H2O2 in 4 M NaOH, affords 4
a,5a and
derivatives (5 and 6). Although a 4 -hydroxylation mechanism was
b
4b,5b-epoxides 13, 14, 16 and 17 (Scheme 2), respectively.
previously reported for steroidal 5-en-3-ols via selenium-dioxide
oxidation by Ma and Choi [15], these authors obtained only
Compounds 15 and 18, which contain a 4-methoxy group, were
then obtained from 17-picolyl epoxy derivatives 13 and 14 [9] and
17-picolinylidene epoxy derivatives 16 and 17 [9], in reaction with
methanol and sodium-hydroxide at reflux for 3 h. Analysis of
compounds 15 and 18 by 1H NMR confirmed the expected
4-methoxy group signal at 3.60 and 3.59 ppm, respectively. Simi-
larly, 13C NMR signals were observed for C-4 atoms at 147.23 ppm
(15) and at 154.96 ppm (18), respectively.
a 4
the 17
b
-hydroxy group. After separation by column chromatography,
-picolyl-androst-5-ene-3 ,4 -diol (5) product was identi-
a
b a
fied by the appearance of a new 1H NMR triplet signal at 3.89 ppm
(J ¼ 3.8 Hz) corresponding to the H-4 proton. In addition, a C-4
carbon signal was also visible for compound 5 at 65.06 ppm by 13C
NMR. Analogously, for compound 6, a new 1H NMR doublet
appeared at 4.16 ppm (J ¼ 3.2 Hz), corresponding to the H-4 proton,
and a C-4 carbon signal was observed at 77.24 ppm by 13C NMR. The
We next attempted a series of synthetic schemes involving
compounds 11 and 12. Reaction of compounds 11 and 12 with
potassium-tert-butoxide in tert-butanol at 45 ꢀC for 3 h was
b
-configuration of the 4-hydroxy group in compound 6 was
assigned by 1H-NOE NMR: irradiation of the H-4 proton at 4.16 ppm
found to afford 4,17b-dihydroxy-17a-picolyl-androsta-4,6-dien-
Fig. 1. 17a-Picolyl-androst-5-en-3b,17b-diol (1) and 17(Z)-picolinylidene-androst-5-en-3b-ol (2).

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E.A. Djurendic et al. / European Journal of Medicinal Chemistry 54 (2012) 784e792
786
Scheme 1. Reagents and reaction conditions: a) MCPBA, CH2Cl2, 5% NaHCO3, 0 ꢀC, 1 h / 10 ꢀC, 3 h / r.t., 24 h; b) Jones reagent, acetone, 0 ꢀC, 1.5 h; c) SeO2, dioxane, reflux, 25.5 h;
d) p-TsCl, Py, r.t., 45 h (for 1) or 19 h (for 2); e) bis(2-aminoethyl)amine, reflux, 1 h.
3-one (19), accompanied by compound 4, and 4-hydroxy-17-
picolinylidene-androsta-4,6-dien-3-one (21) [10] accompanied by
compound 20 (Scheme 2). 1H NMR analysis of compound 19
showed a double doublet at 6.06 ppm (J6,7 ¼ 9.4 Hz, J7,8 ¼ 2.0 Hz)
and 6.65 ppm (J6,7 ¼ 9.4 Hz, J6,8 ¼ 2.8 Hz) for the H-7 and H-6
protons, as expected. Similarly, 13C NMR spectral signals were
assigned for double bond carbons at 124.71 (C-6), 134.70 (C-5),
137.10 (C-7) and 140.21 (C-4) ppm. The molecular formula of
compound 19 was confirmed by high resolution mass spectra
(HRMS) (TOF); and by comparison with spectroscopic data previ-
ously reported by our group for compound 21 [10].
2.2. Cytotoxicity
As a first step toward testing the potential of the above
synthesized compounds for the development of novel steroidal
therapeutics, we performed a series of cytotoxicity assays. The
cytotoxic activity of selected synthesized compounds was eval-
uated against several common human tumor cell lines, origi-
nating from 4 different solid tumor types. As a control, the
selected compounds were also tested against normal fetal lung
fibroblasts, MRC-5. Cytotoxic activity was evaluated in vitro after
48 h treatment using the SRB assay, as previously described
[18].
Based on the above, we further investigated possible synthetic
routes based on compounds 11 and 12. Interestingly, reduction of
compound 11 with NaBH4 in ethanol at room temperature for 2.5 h
Interestingly, all of the synthesized compounds with a 17a-pi-
colyl substituent (3, 5, 6, 8, 19 and 22) appear to have stronger
cytotoxicity against ER- human breast adenocarcinoma cells (MDA-
MB-231) (see Table 1). Of these, compound 19, which contains
a 4-hydroxy-4,6-dien-3-on system, displayed the strongest cyto-
was found to yield a 3
anistically, attack of the hydride anion in the axial direction of the
carbonyl group could result in generation of the 3 -hydroxy
b-hydroxy derivative, compound 22. Mech-
b
configuration observed for compound 22. Analogously, reduction of
compound 12 with NaBH4 in absolute ethanol at room temperature
for 2 h, affords compound 24 [10]. The 1H NMR spectrum of
compound 22 was characterized by a multiplet at 4.15 ppm,
toxicity (IC50 1.2
system, also displayed significant cytotoxicity (IC50 2.3
comparison, the cytotoxicity for the 17-picolynilidene compound
21 (IC50 9.3 M) [10] was approximately 8-fold less than that of the
17 -picolyl derivative 19 (IC50 1.2 M); while compound 10
(17-picolynilidene derivate) was 2-fold less cytotoxic (IC50 4.6 M)
than compound 8 (IC50 2.3 M). One possibly interesting exception
m
M); while compound 8, which contains a 3,5-dien
m
M). In
m
attributed to the H-3
a
proton; while 13C NMR spectroscopy
a
m
revealed the corresponding C-3 atom signal at 67.93 ppm. The
stereochemistry of compound 22 was confirmed using selective
NOE difference experiments. Because selective irradiation of the
C-10 methyl protons did not result in NOE enhancement of the H-3
proton signal, the H-3 and C-10 angular methyl group are assumed
to be in trans; and the OH group at C-3 is thus directed toward the
m
m
to this rule was compound 18, a 17-picolynilidene derivate with a 4-
methoxy group: compound 18 was measured to have 10-fold
stronger cytotoxicity (IC50 4.1
42.6 M), which also has a 4-methoxy group, but contains
a 17 -picolyl substituent.
mM) than compound 15 (IC50
m
b
position.
a
Finally, NaBH4 reduction of compound 11 to 22, followed by
acetylation with acetic anhydride in absolute pyridine at room
temperature for 24 h was found to give compound 23.
Compounds 5 and 6, which contain an additional 4-hydroxy
group, both displayed significantly stronger cytotoxicity against
ER- human breast adenocarcinoma cells (MDA-MB-231) (17-fold

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E.A. Djurendic et al. / European Journal of Medicinal Chemistry 54 (2012) 784e792
787
Scheme 2. Reagents and reaction conditions: a) 30% H2O2, 4M NaOH, MeOH, 0 ꢀC, 1 h / 10 ꢀC, 24 h; b) 4M NaOH, MeOH, reflux, 3.5 h (or 3 h); c) 1M t-BuOK, t-BuOH, 45 ꢀC, 3 h (or
r.t., 45 h); d) NaBH4, EtOH, r.t., 24 h (or 2 h); e) Ac2O, Py, r.t., 24 h.
and 10-fold, respectively) vs. compound 1 [9] (IC50 96.1
m
M).
in cytotoxicity (IC50 10.2
tively). Similarly, compound 25, which also contains a 3
group [10], showed low cytotoxicity (IC50 39.3 M) against the same
cell line. However, compound 24 [10] (17-picolinylidene), with
a 3 -hydroxy group, appears to be inactive (IC50 > 100 M) against
MDA-MB-231 cells.
m
M vs. IC50 3.8
m
M for 23 vs. 22, respec-
Similarly, compound 3, which contains a 5 ,6 -epoxy and N-oxide
a
a
b-acetoxy
function, also had significantly stronger cytotoxicity (IC50 7.5
than compound 1 against MDA-MB-231 cells.
m
M)
m
Intriguingly, replacement of the 3
b
-hydroxy group in compound
b
m
22 with a 3
b-acetoxy group (compound 23), resulted in a reduction
In contrast with our results from ER- breast adenocarcinoma cell
Table 1
lines, only compound 19 strong cytotoxicity against ER þ human
In vitro cytotoxic activity of the tested compounds e IC50 values.
breast adenocarcinoma cells (MCF-7 cells) (IC50 3.6 mM); although
Compounds
IC50
(
mM)
compound
8 did display moderate MCF-7 cytotoxicity (IC50
17.4 M). Interstingly, compound 19 also displayed the
m
MCF-7
MDA-MB-231
PC-3
HeLa
HT-29
MRC-5
strongest cytotoxicity against ER- breast cancer cells, followed by
compound 8.
With respect to prostate cancer, PC-3 cells were the most
sensitive to compound 1 [9] (IC50 6.3 mM), although the majority
of the tested compounds (3, 5, 8, 10, 12 [9], 17 [9], 18, 19, 22 and
24) showed moderate PC-3 cytotoxicity: ranging from
a maximum IC50 of 10.1 mM (24) to a minimum IC50 of 34.1 mM
(18). Of these, 5, 8, 10, 18 and 22 also displayed moderate cyto-
toxicity against HeLa cancer cells (along with 6). Unlike our results
from ER- breast cancer cells, compound 24 [10] had 2-fold higher
1 [9]
3
4
5
6
8
10
15
18
19
21 [10]
22
23
24 [10]
Doxorubicin
Formestane
>100
>100
>100
>100
>100
17.4
96.1
7.5
>100
5.6
9.0
2.3
4.6
42.6
4.1
1.2
9.3
6.3
33.6
4.3
e
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.12
33.5
>100
21.3
26.6
27.5
23.3
19.1
15.3
36.6
e
25.8
>100
2.6
8.0
11.7
1.8
>100
56.0
>100
e
21.3
52.1
24.7
19.9
54.8
34.1
25.6
>100
22.3
31.3
10.1
95.61
48.36
55.8
>100
>100
3.6
>100
>100
>100
>100
0.75
3.8
26.9
>100
e
>100
10.6
e
cytotoxicity (IC50 10.1
against PC-3 cells.
mM) than compound 22 (IC50 22.3 mM)
10.2
>100
0.12
55.5
1.17
5.55
0.32
>100
Cytotoxicity results against the colon cancer-derived cell line
(HT-29) revealed a different set a compounds with significantly
>100
>100

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788
strong cytotoxic activity: according to IC50 values, 5 and 10 showed
strong cytotoxicity (IC50 2.6 M and 1.8 M, respectively) against
HT-29 cells; followed by compound 6 (IC50 8.0 M).
3. Conclusion
m
m
m
Novel androstane derivatives 3e10, 15, 18, 19, 22 and 23 were
Overall, the ER- breast cancer cell line (MDA-MB-231) appears
tobe the most sensitive to the majority of tested compounds (Fig. 2).
In fact, all of the tested compounds induced a dose-dependent
cytotoxic response against MDA-MB-231 cancer cells: with some
compounds displaying more toxicity than others. For example,
compounds 5, 18 and 19 showed stronger cytotoxic activity for the
whole range of concentrations vs. Formestan (a CYP19A1 aroma-
tase inhibitor in clinical use); whereas compounds 8 and 22
synthesized from 17
17(Z)-picolinylidene-androst-5-en-3
a
-picolyl-androst-5-en-3
b,17b-diol (1) and
b-ol (2), respectively. These
compounds were evaluated for cytotoxic activity against five
common tumor cell lines, and one normal cell line. Synthesized
compounds with a 17-picolyl substituent (3, 5, 6, 8, 10, 18, 19 and
22) showed markedly strong cytotoxic activity against ER- breast
cancer cells (MDA-MB-231 cells); while compound 19 (followed by
8) also displayed the strongest cytotoxicity against ER þ breast
cancer cells (MCF-7); and compounds 5, 6 and 10 were the most
cytotoxic to HT-29 colon cancer cells.
only induced higher cytotoxicity at concentrations > 0.1 mM; and
compounds 3, 4, 6, 15, and 23 showed either similar or less cyto-
toxicity than Formestan.
As an indication of their potential potency, the cytotoxic activ-
ities of compounds 19, 8 and 5 were found to be similar to that of
4. Experimental
Doxorubicin at concentrations ꢁ 0.1
18 displayed higher cytotoxicity than Doxorubicin (up to 10% at
concentrations <1 M against MDA-MB-231 cells).
As a control, all of the newly synthesized compounds were
confirmed to be non-toxic to normal MRC-5 cells, whereas Doxo-
rubicin was very toxic to these cells.
mM. However, only compound
4.1. Chemistry
m
All melting points were determined using a Büchi SMP 20
apparatus and are reported uncorrected. IR spectra were recorded
on a NEXUS 670 SP-IR spectrometer (wave numbers in cmꢂ1).
NMR spectra were recorded on a Bruker AC 250E spectrometer
Fig. 2. Cytotoxic activity of selected synthesized compounds against MD-MB-231 cells. For- formestan; DOX- doxorubicin.

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789
operating at 250 MHz (1H) and 62.5 MHz (13C), and are reported
in ppm (symbol 100 \f “Symbol” \s 11d-scale) downfield from
a tetramethylsilane internal standard; coupling constants (J) are
given in Hz. GC/MS analyses were performed on an Agilent
Technologies GC 6890N instrument with Mass Selective Detector
5973; the first number denotes the m/z value, while ion abun-
dances are given in parentheses. High resolution mass spectra
(TOF) were recorded on a 6210 Time-of-Flight LC/MS Agilent
Technologies (ESIþ) instrument. Chromatographic separations
were performed on silica gel columns (Kieselgel 60,
0.063e0.20 mm, Merck). All reagents used were of analytical
reagent grade. All solutions were dried over anhydrous sodium
sulfate.
4.1.3. 17
androst-5-en-3
a
-Picolyl-androst-5-en-3
b,4a,17b-triol (5) and 17a-picolyl-
b
,4 ,17 -triol (6)
b
b
Selene-dioxide (0.089 g, 0.81 mmol) was added to a solution of
compound 1 (0.200 g, 0.52 mmol) dissolved in dioxane (4 ml), and
the reaction mixture was stirred under reflux for 25.5 h. The
resulting mixture was then filtered, and the precipitate washed
with dichloromethane (10 ml) and ethyl acetate (10 ml). Water
(10 ml) was then added, and the mother liquor was extracted with
dichloromethane (5 ꢃ 10 ml). After drying and solvent removal,
crude products were separated by column chromatography (10 g
silica gel, hexane-ethyl acetate 2:1 and 1:2), affording pure
compound 6 (0.045 g, 22%) and compound 5 (0.058 g, 28%), both in
the form of a colorless oil.
Compound 5: IR (film, cmꢂ1): 3353, 2926, 2854, 1665, 1596,
4.1.1. 5a,6a-Epoxy-17a-picolyl-androstane-N-oxide-3b,17b-diol (3)
1570, 1438, 1377, 1260, 1216, 1053, 1080, 955, 757; 1H NMR
A 5% solution of NaHCO3 (2.5 ml) was added to a solution of
(250 MHz, CDCl3): d 0.98 (s, 3H, H-18),1.04 (s, 3H, H-19), 2.88 (d,1H,
compound 1 (0.305 g, 0.799 mmol) in dichloromethane (10 ml) at
Jgem ¼ 14.5 Hz, CH2Py), 3.10 (d, 1H, Jgem ¼ 14.5 Hz, CH2Py), 3.60
0
ꢀC, to pH 8. Then, MCPBA (0.414 g, 2.38 mmol) was added and
(m, 1H, H-3), 3.89 (t, 1H, J ¼ 3.8 Hz, H-4), 5.64 (m, 1H, H-6), 7.18
the reaction mixture was stirred for 1 h at 0 ꢀC, 3 h at 10 ꢀC and
24 h at room temperature in the dark. The resulting organic layer
was then washed successively with a solution of 10% Na2S2O3
(2 ꢃ 10 ml), 5% NaHCO3 (2 ꢃ 10 ml) and water (2 ꢃ 10 ml). After
drying and removal of dichloromethane, the mixture of reaction
products was separated by column chromatography (15 g silica
gel, chloroform-acetone 1:1), yielding pure compound 3 (0.138 g,
42%, m.p. 248e250 ꢀC after recrystallization from dichloro-
methane-hexane).
(m, 2H, H-30 and H-50, Py), 7.63 (td, 1H, J3 ,4 ¼ J4 ,5 ¼ 7.7 Hz,
0
0
0
0
J4 ,6 ¼ 1.8 Hz, H-4 , Py), 8.46 (d, 1H, J5 ,6 ¼ 4.1 Hz, H-6 , Py); 13C NMR
0
0
0
0
0
0
(62.5 MHz, CDCl3):
d 13.90 (C-18), 18.27 (C-19), 20.47, 21.47, 23.99,
29.69, 31.38, 31.78, 36.07, 37.09, 38.45, 42.00, 42.47, 43.01, 46.09,
65.06 (C-4), 71.35 (C-3), 83.52 (C-17), 121.35 (C-50, Py), 123.81 (C-30,
Py), 124.86 (C-6), 136.79 (C-40, Py), 146.44 (C-5), 147.90þ(C-60, Py),
160.83 (C-20, Py); GC/MS (m/e, %): 271 ([M-CH2Py-2OH] , 41), 260
(100), 253 (58); HRMS (TOF), m/z: for C25H36NO3 [M þ H]þ calcd.
398.26897, found 398.26828.
IR (KBr, cmꢂ1): 3416, 2940, 2868, 1637, 1560, 1489, 1438, 1377,
Compound 6: IR (film, cmꢂ1): 3362, 2926, 2855,1661,1597,1438,
1215, 1060, 1034, 771; 1H NMR (250 MHz, CDCl3):
d
0.87 (s, 3H,
H-18), 1.09 (s, 3H, H-19), 2.56 (d, 1H, Jgem ¼ 13.4 Hz, CH2Py), 2.93
(d, 1H, J6,7 ¼ 4.3 Hz, H-6 ), 3.79 (d, 1H, Jgem ¼ 13.4 Hz, CH2Py), 3.89
1379, 1250, 1052, 1027, 967, 757, 666; 1H NMR (250 MHz, CDCl3):
d
0.98 (s, 3H, H-18), 1.22 (s, 3H, H-19), 2.80 (d, 1H, Jgem ¼ 14.5 Hz,
b
CH2Py), 3.08 (d, 1H, Jgem ¼ 14.5 Hz, CH2Py), 3.57 (m, 1H, H-3), 4.16
(m,1H, H-3), 6.65 (s,1H,17b-OH$$$$O)N-Py), 7.22e7.39 (m, 3H, H-
(d, 1H, J ¼ 3.2 Hz, H-4), 5.71 (m, 1H, H-6), 7.16 (m, 2H, H-30 and H-50,
30, H-40, H-50, Py), 8.28 (d, 1H, J5 ,6 ¼ 6.1 Hz, H-60, Py); 13C NMR
Py), 7.64 (td, 1H, J3 ,4 ¼ J4 ,5 ¼ 7.7 Hz, J4 ,6 ¼ 1.8 Hz, H-40, Py), 8.46
0
0
0
0
0
0
0
0
(62.5 MHz, CDCl3):
d
14.12 (C-18), 15.96 (C-19), 20.42, 23.04, 28.47,
(m, 1H, H-60, Py); 13C NMR (62.5 MHz, CDCl3):
d 14.11 (C-18), 20.27,
30.82 (CH), 31.05, 32.52, 33.74, 34.93, 38.44, 39.74, 42.87 (CH),
47.02, 51.45 (CH), 59.15 (CH), 65.98 (C-5), 68.42 (C-6), 69.16 (C-3),
85.18 (C-17), 124.28 (CH), 127.74 (CH), 128.97 (CH), 139.95 (CH),
150.76 (C-20, Py); GC/MS (m/e, %): 304 ([M-CH2Py-O]þ, 100), 286
([M-CH2Py-O-H2O]þ, 29), 271 (24), 253 (28), 227 (16); HRMS (TOF),
m/z: for C25H35NO4 [M þ H]þ calcd. 414.26389, found 414.26441.
21.05 (C-19), 23.96, 25.41, 29.69, 32.13, 32.64, 36.13, 37.01, 38.14,
43.19, 46.34, 50.33, 51.31, 72.48 (C-3), 77.24 (C-4), 83.48 (C-17),
121.35 (C-50, Py), 124.74 (C-30, Py), 128.46 (C-6), 136.75 (C-40, Py),
142.94 (C-5), 148.01 (C-60, Py), 160.80 (C-20, Py); GC/MS (m/e, %):
304 ([M-CH3Py]þ, 23), 286 ([M-CH3Py-H2O]þ, 45), 260 (100), 247
(75); HRMS (TOF), m/z: for C25H36NO3 [M þ H]þ calcd. 398.26897,
found 398.26769.
4.1.2. 17b-Hydroxy-17a-picolyl-androst-4-ene-3,6-dione (4)
Jones reagent [14] (2 ml) was added dropwise (w 1 drop/10 s) to
a solution of compound 1 (0.352 g, 0.923 mmol) in acetone (35 ml)
at 0 ꢀC, with vigorous stirring, and the reaction mixture was stirred
for 1.5 h. The resulting reaction mixture was then quenched with
methanol (15 ml, to eliminate any excess reagent) and solvent was
removed in vacuo. Water (20 ml) and NaHCO3 (to pH 8) were then
added, and crude product was extracted with dichloromethane
(5 ꢃ 10 ml). Finally, purification by column chromatography (15 g
silica gel, toluene-ethyl acetate 7:1) afforded pure compound 4
(0.044 g, 12%, m.p. 243e245 ꢀC after recrystallization from
dichloromethane-hexane).
4.1.4. General procedure for 17
b
-hydroxy-17
-yl p-toluenesulfonylate (7) and 17-picolinyliden-androst-5-
-yl p-toluenesulfonylate (9)
a-picolyl-androst-5-
en-3b
en-3b
A solution of p-toluenesulfonylchloride (1.028 g, 5.41 mmol for
compound 1 or 0.511 g, 2.69 mmol for compound 2) was added to 1
(0.506 g, 1.33 mmol) or 2 (0.200 g, 0.55 mmol) dissolved in absolute
pyridine (8 ml or 4 ml) at 0 ꢀC; and the resulting mixture was
stirred 45 h (for 1) or 19 h (for 2) at room temperature. Reactions
were stopped by addition of ice, and poured into water (20 or
40 ml). Crude product (7) was precipitated and purified by column
chromatography (30 g silica gel, toluene-ethyl acetate 20:1 and
15:1), giving pure compound 7 (0.140 g, 20%, m.p. 163e165 ꢀC after
recrystallization from dichloromethane-hexane). The reaction
mixture for 9 was extracted with dichloromethane (8 ꢃ 10 ml), and,
after drying and solvent removal, crude product (9) was recrystal-
lized from dichloromethane-hexane, giving pure compound 9
(0.145 g, 51%, m.p. 176e178 ꢀC).
IR (KBr, cmꢂ1): 3299, 3063, 2923, 2857, 1726, 1693, 1596, 1569,
1440, 1378, 1264, 1119, 1055, 1022, 869; 1H NMR (250 MHz, CDCl3):
d
1.01 (s, 3H, H-18), 1.20 (s, 3H, H-19), 2.80 (d, 1H, Jgem ¼ 14.5 Hz,
CH2Py), 3.09 (d, 1H, Jgem ¼ 14.5 Hz, CH2Py), 6.19 (s, 1H, H-4), 7.18
(m, 2H, H-30 and H-50, Py), 7.65 (td, 1H, J3 ,4 ¼ J4 ,5 ¼ 7.7 Hz,
0
0
0
0
J4 ,6 ¼ 1.7 Hz, H-4 , Py), 8.47 (d, 1H, J6 ,5 ¼ 4.4 Hz, H-6 , Py); 13C NMR
0
0
0
0
0
0
(62.5 MHz, CDCl3):
d
14.08 (C-18), 17.50 (C-19), 18.72, 20.56, 23.58,
Compound 7: IR (KBr, cmꢂ1): 3323, 2951, 2870, 1597, 1569, 1473,
31.64, 33.95, 34.84, 35.55, 35.73, 39.75, 42.94, 46.55, 50.94, 50.99,
83.06 (C-17), 121.53 (C-50, Py), 124.68 (C-30, Py), 125.51 (C-4), 136.93
(C-40, Py), 148.02 (C-60, Py), 160.27 (C-20, Py), 160.82 (C-5), 199.46
(C-3), 201.95 (C-6); HRMS (TOF), m/z: for C25H32NO3 [M þ H]þ
calcd. 394.23767, found 394.23581.
1438, 1361, 1188, 1175, 1098, 938, 866, 755, 667; 1H NMR (250 MHz,
CDCl3): d 0.94 (s, 3H, H-18), 1.00 (s, 3H, H-19), 2.45 (s, 3H, CH3 from
OTs group), 2.79 (d, 1H, Jgem ¼ 14.5 Hz, CH2Py), 3.06 (d, 1H,
Jgem ¼ 14.5 Hz, CH2Py), 4.36 (m,1H, H-3), 5.32 (d,1H, J ¼ 4.9 Hz, H-6),
7.16 (m, 2H, H-30 and H-50, Py), 7.34 (d, 2H from OTs group), 7.63 (td,

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E.A. Djurendic et al. / European Journal of Medicinal Chemistry 54 (2012) 784e792
0
0
0
0
0
0
0
1H, J3 ,4 ¼ J4 ,5 ¼ 7.8 Hz, J4 ,6 ¼ 1.7 Hz, H-4 , Py), 7.80 (d, 2H from OTs
0.44 mmol) or compounds 16 and 17 [9] (0.056, 0.14 mmol) in
methanol (22 ml or 7 ml), and the reaction mixture was stirred
under reflux for 3.5 h (or 3 h). After reaction completion, the
resulting reaction mixture was extracted with dichloromethane
(5 ꢃ 10 ml or 5 ꢃ 5 ml), solvent was dried and removed, and
crude product was separated by column chromatography (5 g or
1 g silica gel). Elution with hexane-ethyl acetate 3:1 (for 15) or
toluene-ethyl acetate 9:1 (for 18) afforded pure compound 15
(0.007 g, 4%), or pure compound 18 (0.006 g, 10%), both in the
form of a colorless oil.
group), 8.46 (d, 1H, J5 ,6 ¼ 5.6 Hz, H-60, Py); 13C NMR (62.5 MHz,
0
0
CDCl3):
d 14.05 (C-18), 19.13 (C-19), 20.67, 21.62 (CH3, OTs), 23.93,
28.59, 31.75, 32.05, 32.55, 35.94, 36.45, 36.93, 38.84, 43.11, 46.26,
49.99, 50.94, 82.27 (C-3), 83.42 (C-17),121.34 (C-50, Py),123.23 (C-6),
124.73 (C-30, Py), 127.57 (C-20 and C-60, OTs), 129.72 (C-30 and C-50,
OTs), 134.71 (C-40, OTs), 136.76 (C-40, Py), 138.95 (C-5), 144.38 (C-10,
OTs), 147.95 (C-60, Py), 160.72 (C-20, Py); HRMS (TOF), m/z: for
C32H42NSO4 [M þ H]þ calcd. 536.28291, found 536.28358.
Compound 9: IR (film, cmꢂ1): 2945, 1653, 1586, 1562, 1467, 1432,
1362, 1175, 1098, 938, 865, 753. 1H NMR (250 MHz, CDCl3):
d
0.88
Compound 15: IR (film, cmꢂ1): 3391, 2929, 1681, 1597, 1570,
(s, 3H, H-18), 1.01 (s, 3H, H-19), 2.44 (s, 3H, CH3 from OTs), 4.32
(m, 1H, H-3), 5.32 (d, 1H, J ¼ 5.0 Hz, H-6), 6.26 (s, 1H, H-20), 7.07
(m, 2H, H-30 and H-50, Py), 7.33 (d, 2H from OTs group), 7.65 (t, 1H,
1439, 1379, 1317, 1260, 1214, 1086, 1022, 927, 891, 800, 756; 1H
NMR (250 MHz, CDCl3):
d 1.00 (s, 3H, H-18), 1.22 (s, 3H, H-19), 2.80
(d, 1H, Jgem ¼ 14.3 Hz, CH2Py), 3.07 (d, 1H, Jgem ¼ 14.3 Hz, CH2Py),
J4 ,3 ¼ J4 ,5 ¼ 9.2 Hz, H-40, Py), 7.79 (d, 2H from OTs group), 8.58
3.60 (s, 3H, from 4-OCH3), 4.72 (bs, 1H, 17b
-OH), 7.18 (m, 2H, H-30
0
0
0
0
(m, 1H, H-60, Py); 13C NMR (62.5 MHz, CDCl3):
d
18.50 (C-18), 19.14
and H-5’, Py), 7.63 (td, 1H, J4’,5’ ¼ J4’,3’ ¼ 7.6 Hz, J4’,6’ ¼ 1.7 Hz, H-4’,
(C-19), 20.94, 21.58 (CH3, OTs), 24.94, 28.51, 29.81, 31.48, 31.61,
35.54, 36.40, 36.81 (C-10), 38.81, 45.79 (C-13), 50.09, 53.74 (C-9),
82.17 (C-3), 117.33 (C-20), 120.39 (C-50, Py), 123.16 (C-6), 126.01
(C-30, Py), 127.57 (C-20 and C-60, OTs), 129.70 (C-30 and C-50, OTs),
134.53 (C-40, OTs), 136.55 (C-40, Py), 138.93 (C-5), 144.38 (C-10, OTs),
148.39 (C-60, Py), 156.93 (C-17), 161.24 (C-20, Py).
Py), 8.46 (d, 1H, J6’,5’ ¼ 5.1 Hz, H-6’, Py); 13C NMR (62.5 MHz,
CDCl3):
d 14.15 (C-18), 17.50 (C-19), 20.69, 23.41, 23.79, 29.66,
31.90, 34.25, 34.84, 35.91 (C-10), 36.01, 38.78, 43.02, 44.23 (C-13),
50.20 (C-14), 54.33 (C-9), 60.23 (OeCH3), 83.34 (C-17), 121.43 (C-5’,
Py), 124.75 (C-3’, Py), 136.88 (C-4’, Py), 146.21 (C-5), 147.23 (C-4),
147.91 (C-6’, Py), 160.57 (C-2’, Py), 191.39 (C-3); GC/MS (m/e, %):
316 ([M-CH3Py] þ, 59), 284 ([M-CH3Py-CH3OH] þ, 26), 273 (100);
HRMS (TOF), m/z: for C26H36NO3 [M þ H]þ calcd. 410.26897, found
410.26718.
4.1.5. General procedure for 17
a-picolyl-androsta-3,5-dien-17b-ol
(8) and 17-picolinyliden-androsta-3,5-diene (10)
Compound 7 (0.080 g, 0.149 mmol) or compound 9 (0.379 g,
0.733 mmol) were stirred with bis(2-aminoethyl)amine (4.5 ml, or
20 ml) under reflux for 1 h, cooled to room temperature, poured into
water, and extracted with dichloromethane (5 ꢃ10 ml). The organic
phase was dried, filtered and evaporated. Crude product was then
purified by column chromatography (1 g silica gel, toluene-ethyl
acetate 30:1 for 8, or 6 g silica gel, toluene-ethyl acetate 30:1 for
10), affording pure compound 8 (0.008 g, 15%) and pure compound
10 (0.018 g, 7%), both in the form of a colorless oil.
Compound 18: IR (film, cmꢂ1): 2924, 1682, 1607, 1586, 1561,
1470, 1435, 1359, 1334, 1314, 1207, 1150, 1095, 1066, 1035, 776, 732.
1H NMR (250 MHz, CDCl3):
d 0.93 (s, 3H, H-18), 1.25 (s, 3H, H-19),
3.59 (s, 3H, from 4-OCH3), 6.23 (s, 1H, H-20), 7.04 (m, 1H, H-5’, Py),
7.27 (m, 1H, H-3’, Py), 7.62 (td, 1H, J4’,5’ ¼ J4’,3’ ¼ 7.8 Hz, J4’,6’ ¼ 1.8 Hz,
H-4’, Py), 8.57 (d, 1H, J6’,5’ ¼ 4.7 Hz, H-6’, Py); 13C NMR (62.5 MHz,
CDCl3): d 17.52 (C-18), 18.80 (C-19), 21.06, 23.40, 24.92, 29.74, 31.40,
34.24, 34.81, 35.14, 35.70 (C-10), 38.80, 45.72 (C-13), 53.15 (C-14),
54.43 (C-9), 60.32 (OeCH3), 118.14 (C-20), 120.30 (C-5’, Py), 122.82
(C-3’, Py), 135.89 (C-4’, Py), 146.66 (C-5), 149.16 (C-6’, Py), 154.96
(C-4), 157.46 (C-17), 159.82 (C-2’, Py), 194.14 (C-3); GC/MS (m/e, %):
391 (Mþ, 100), 377 (59), 348 (6), 170 (7), 130 (20), 93 (15); HRMS
(TOF), m/z: for C26H34NO2 [M þ H]þ calcd. 392.25841, found
392.25724.
Compound 8: IR (film, cmꢂ1): 3328, 3017, 2926, 2855, 1652,
1596, 1570, 1438, 1377, 1024, 846, 753; 1H NMR (250 MHz, CDCl3):
d
0.99 (s, 3H, H-18), 1.26 (s, 3H, H-19), 2.82 (d, 1H, Jgem ¼ 14.6 Hz,
CH2Py), 3.09 (d, 1H, Jgem ¼ 14.6 Hz, CH2Py), 5.41 (m, 1H, H-3), 5.63
(m, 1H, H-6), 5.94 (d, 1H, J ¼ 9.9 Hz, H-4), 7.16 (m, 2H, H-30 and
H-50, Py), 7.63 (td, 1H, J4 ,5 ¼ J4 ,3 ¼ 7.7 Hz, J4 ,6 ¼ 1.8 Hz, H-40, Py),
0
0
0
0
0
0
8.47 (m, 1H, H-60, Py); 13C NMR (62.5 MHz, CDCl3):
d
14.21 (C-18),
4.1.7. 4,17
b-Dihydroxy-17
a-picolyl-androsta-4,6-dien-3-one (19)
18.77 (C-19), 20.66, 23.03, 23.89, 29.68, 31.68, 32.20, 32.56, 33.83,
36.04, 43.22, 46.46, 48.53, 51.33, 83.48 (C-17), 121.31 (C-50, Py),
122.80 (C-6), 124.74 (C-30, Py), 125.14 (C-4), 128.90 (C-3), 136.72
(C-40, Py), 141.56 (C-5), 147.99 (C-60, Py), 160.88 (C-20, Py); HRMS
(TOF), m/z: for C25H34NO [M þ H]þ calcd. 364.26349, found
364.26255.
and 17 -hydroxy-17a
b
-picolyl-androst-4-en-3,6-dione (4)
1 M potassium tert-butoxide (2.5 ml) was added dropwise to
a solution of compound 11 (0.202 g, 0.54 mmol) dissolved in tert-
butyl alcohol (6 ml) at room temperature, and the reaction mixture
was stirred at 45 ꢀC for 3 h (protected from light). When the
reaction was complete, the resulting mixture was poured into
water (10 ml) and extracted with ethyl acetate (5 ꢃ10 ml), followed
by dichloromethane (5 ꢃ 10 ml). Combined extracts were then
dried and solvent removed. The resulting crude product was
separated and purified by column chromatography (20 g silica gel,
toluene-ethyl acetate 8:1 and 5:1), giving compound 19 (0.052 g,
25%, m.p. 147e149 ꢀC after recrystallization from methanol) and
compound 4 (0.028 g, 13%, m.p. 243e245 ꢀC after recrystallization
from methanol).
Compound 10: IR (film, cmꢂ1): 3016, 2963, 2851, 1652, 1586,
1560, 1470, 1428, 1372, 1149, 857, 756; 1H NMR (250 MHz, CDCl3):
d
0.94 (s, 3H, H-18), 1.01 (s, 3H, H-19), 5.42 (m, 1H, H-3), 5.61 (m, 1H,
H-6), 5.95 (d,1H, J4,3 ¼ 9.7 Hz, H-4), 6.25 (t,1H, J ¼ 2.4 Hz, H-20), 7.03
(m, 1H, H-50, Py), 7.29 (m, 1H, H-30, Py), 7.61 (td, 1H,
0
0
0
0
0
0
0
0
0
J4 ,5 ¼ J4 ,3 ¼ 7.8 Hz, J4 ,6 ¼ 1.8 Hz, H-4 , Py), 8.57 (d,1H, J6 ,5 ¼ 4.8 Hz,
H-60, Py); 13C NMR (62.5 MHz, CDCl3):
d
18.79 (C-18), 18.83 (C-19),
21.01, 23.03, 24.98, 29.87, 31.62, 31.66, 33.77, 35.32, 35.82, 45.90,
48.67, 54.17, 118.02 (C-20), 120.17 (C-50, Py), 122.77 (C-30, Py), 124.72
(C-6), 125.16 (C-4), 128.89 (C-3), 135.80 (C-40, Py), 141.57 (C-5),
149.16 (C-60, Py), 157.65 (C-17), 160.44 (C-20, Py); HRMS (TOF), m/z:
for C25H32N [M þ H]þ calcd. 346.25293, found 346.25184.
Compound 19: IR (KBr, cmꢂ1): 3423, 3054, 2941, 2872, 1655,
1616, 1597, 1570, 1380, 1265, 894; 1H NMR (250 MHz, CDCl3):
d 1.04
(s, 3H, H-18), 1.12 (s, 3H, H-19), 2.80 (d, 1H, Jgem ¼ 14.5 Hz, CH2Py),
3.10 (d, 1H, Jgem ¼ 14.5 Hz, CH2Py), 6.06 (dd, 1H, J6,7 ¼ 9.4 Hz,
J7,8 ¼ 2.0 Hz, H-7), 6.65 (dd, 1H, J6,7 ¼ 9.4 Hz, J6,8 ¼ 2.8 Hz, H-6), 7.18
(m, 2H, H-30 and H-50, Py), 7.66 (td, 1H, J3 ,4 ¼ J4 ,5 ¼ 7.6 Hz,
0
0
0
0
4.1.6. General procedure for 17b-hydroxy-4-methoxy-17a-picolyl-
androst-4-en-3-one (15) and 4-methoxy-17-picolinyliden-androst-
4-en-3-one (18)
J4 ,6 ¼ 1.8 Hz, H-4 , Py), 8.47 (d, 1H, J5 ,6 ¼ 4.0 Hz, H-6 , Py); 13C NMR
0
0
0
0
0
0
(62.5 MHz, CDCl3):
d 14.08 (C-18), 16.44 (C-19), 20.27, 23.43, 29.70,
A solution of 4 M NaOH (2 ml or 0.6 ml) was added to
a solution of a mixture of compounds 13 and 14 [9] (0.175 g,
31.93, 33.29, 35.75, 36.48, 37.93, 42.98, 46.87, 48.04, 51.44, 83.17
(C-17), 121.55 (C-50, Py), 123.94 (C-30, Py), 124.71 (C-6), 134.70 (C-5),

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E.A. Djurendic et al. / European Journal of Medicinal Chemistry 54 (2012) 784e792
791
136.76 (C-40, Py), 137.10 (C-7), 140.21 (C-4), 147.83 (C-60, Py), 160.39
(C-20, Py), 193.77 (C-3); HRMS (TOF), m/z: for C25H32NO3 [M þ H]þ
calcd. 394.23767, found 394.24972.
were used in the assay. Viability was determined by the dye
exclusion assay with trypan blue.
Cytotoxicity assay. Cytotoxic activity was evaluated using the
colorimetric sulforhodamine B (SRB) assay, following the method of
Skehan et al. [18]. Briefly, a single cell suspension was plated onto
96-well microtitar plates (Costar, flat bottom): 5 ꢃ 103 cells/well in
4.1.8. 17a-Picolyl-androst-4-en-3b,17b-diol (22)
NaBH4 (0.142 g, 3.74 mmol) was added to a solution of
compound 11 (0.203 g, 0.535 mmol) dissolved in absolute ethanol
(15 ml), and the reaction mixture was stirred at room tempera-
ture for 2.5 h. When the reaction was complete, the resulting
mixture was poured into water (20 ml), and crude product was
filtered and recrystallized from dichloromethane-methanol, to
give pure compound 22 (0.191 g, 94%, m.p. 96e98 ꢀC) as white
crystals.
180 m
l of medium. Plates were pre-incubated 24 h at 37 ꢀC, 5% CO2.
Tested substances were then added to all wells (except controls),
at concentrations ranging from 10ꢂ8 to 10ꢂ4 M. After incubation
(48 h/37 ꢀC/5% CO2), the SRB assay was carried out as follows: 50
ml
of 50% trichloroacetic acid (TCA) was added to all wells and plates
were incubated for 1 h; plates were washed with distilled water,
75
incubated for 30 min; plates were then washed with citric acid (1%)
and dried at room temperature. Finally, 200 l of 10 mmol TRIS (pH
ml of 0.4% SRB was then added to all wells and plates were
Compound 22: IR (KBr, cmꢂ1): 3417, 2934, 2853, 1634, 1597,
1570, 1440, 1380, 1327, 1230, 1193, 1101, 1056, 1031, 860, 760, 657,
m
588; 1H NMR (250 MHz, CDCl3):
d
0.97 (s, 3H, H-18), 1.08 (s, 3H,
10.5) was added to all wells, and absorbance (A) was measured at
540/690 nm on a microplate reader (Multiscan MCC340, Labsys-
tems). Wells without cells, but containing complete medium only,
acted as blanks. Cytotoxic activity was calculated according to the
formula:
H-19), 2.78 (d,1H, Jgem ¼ 14.5 Hz, CH2Py), 3.07 (d,1H, Jgem ¼ 14.5 Hz,
CH2Py), 4.15 (m, 1H, H-3), 5.30 (s, 1H, H-4), 7.17 (m, 2H, H-30 and
H-50, Py), 7.64 (td, 1H, J4’,3’ ¼ J4’,5’ ¼ 7.7 Hz, J4’,6’ ¼ 1.8 Hz, H-40, Py),
8.45 (d, 1H, J6’,5’ ¼ 4.4 Hz, H-60, Py); 13C NMR (62.5 MHz, CDCl3):
d
14.16 (C-18), 18.92 (C-19), 20.68 (C-11), 23.84, 29.50 (C-2), 32.14
1 ꢂ ATEST=ACONTROL ꢃ 100
and expressed as a percent of cytotoxic activity (CI %).
Data analysis. Two independent experiments were conducted in
quadruplicate for each concentration of tested compound. Mean
values and standard deviations (SD) was calculated for each
concentration. IC50 value defines the dose of compound that
inhibits cell growth by 50%. The IC50 of each tested compound was
determined by median effect analysis [19].
(C-7), 32.17, 32.92 (C-1), 35.43, 35.88, 36.65 (C-8), 37.42 (C-10),
43.11 (C-20), 46.28 (C-13), 50.37 (C-14), 54.60 (C-9), 67.93 (C-3),
83.41 (C-17), 121.32 (C-50, Py), 123.41 (C-4), 124.71 (C-30, Py), 136.74
(C-40, Py), 147.90 (C-5), 147.96 (C-60, Py), 160.77 (C-20, Py); HRMS
(TOF), m/z: for C25H36NO2 [M þ H]þ calcd. 382.27406, found
382.27769.
4.1.9. 17b-Hydroxy-17a-picolyl-androst-4-en-3b-yl acetate (23)
Acetic anhydride (1.5 ml) was added to a solution of compound
22 (0.049 g, 0.13 mmol) in absolute pyridine (1.5 ml) and the
reaction mixture was stirred at room temperature for 24 h. The
resulting reaction mixture was slowly poured into cold water
(10 ml), yielding a precipitate, which was filtered and recrystallized
from dichloromethane-hexane, affording pure compound 23
(0.031 g, 56%, m.p. 179e181 ꢀC) as white crystals.
Acknowledgments
We would like to thank the Ministry of Education and Science of
the Republic of Serbia for financial support (Grant No. 172021).
Appendix A. Supplementary material
Compound 23: IR (KBr, cmꢂ1): 3437, 2942, 2852, 1730, 1598,
1570, 1441, 1371, 1240, 1097, 1023, 970, 927, 901, 760, 675, 612; 1H
NMR (250 MHz, CDCl3): d 0.97 (s, 3H, H-18), 1.09 (s, 3H, H-19), 2.06
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.06.
030.
(s, 3H, CH3 from OAc), 2.78 (d, 1H, Jgem ¼ 14.6 Hz, CH2Py), 3.05
(d, 1H, Jgem ¼ 14.6 Hz, CH2Py), 5.24 (m, 1H, H-3), 5.30 (m, 1H, H-4),
6.65 (bs, 1H, 17b
-OH$$$$N-Py), 7.15 (m, 2H, H-30 and H-50, Py), 7.63
References
(td, 1H, J4’,3’ ¼ J4’,5’ ¼ 7.8 Hz, J4’,6’ ¼ 1.6 Hz, H-40, Py), 8.45 (d, 1H,
J6’,5’ ¼ 5.2 Hz, H-60, Py); 13C NMR (62.5 MHz, CDCl3):
d 14.15 (C-18),
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