Design and synthesis of 21-alkynylaryl pregnenolone derivatives and evaluation of their anticancer activity

Article abstract of DOI:10.1016/j.bmc.2014.10.012

A series of novel C21-alkynylaryl derivatives of pregnenolone were synthesized and screened for anticancer activity against a panel of seven human cancer cell lines (LNCaP, A549, MCF7, HeLa, A431, HepG2, HT29). The data revealed that these compounds can be potential antitumour agents against the specific cell models. Compound 6f bearing a 2-trifluoromethylphenyl group displayed improved cytotoxicity towards all cancer cell lines used. A431 cells were the most sensitive with derivatives 6e-6h bearing electron withdrawing substituents exhibiting high potency with IC₅₀ values ranging between 2.18 and 0.54 μM and drastic inhibition of the prosurvival PI3K-Akt/PKB pathway.

Full text of DOI:10.1016/j.bmc.2014.10.012

Bioorganic & Medicinal Chemistry 22 (2014) 6980–6988
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 21-alkynylaryl pregnenolone derivatives and
evaluation of their anticancer activity
György Szalóki a, Athanasia Pantzou a, Kyriakos C. Prousis a, Olga Mavrofrydi b, Panagiota Papazafiri b,
Theodora Calogeropoulou a,
⇑
a Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece
b University of Athens, Department of Biology, Division of Animal and Human Physiology, Panepistimiopolis, Athens 15784, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel C21-alkynylaryl derivatives of pregnenolone were synthesized and screened for antican-
cer activity against a panel of seven human cancer cell lines (LNCaP, A549, MCF7, HeLa, A431, HepG2,
HT29). The data revealed that these compounds can be potential antitumour agents against the specific
cell models. Compound 6f bearing a 2-trifluoromethylphenyl group displayed improved cytotoxicity
towards all cancer cell lines used. A431 cells were the most sensitive with derivatives 6e–6h bearing elec-
tron withdrawing substituents exhibiting high potency with IC50 values ranging between 2.18 and
Received 22 June 2014
Revised 4 October 2014
Accepted 9 October 2014
Available online 25 October 2014
Keywords:
Alkynylaryl derivatives
Pregnenolone
0.54
l
M and drastic inhibition of the prosurvival PI3K-Akt/PKB pathway.
Ó 2014 Elsevier Ltd. All rights reserved.
Anticancer activity
1. Introduction
carcinoma (H460) cell lines.6 In addition, pregnenolone derivatives
bearing heterocyclic moieties were found to exhibit anticancer
activity. More specifically, 17-pyrazolinyl derivatives of pregneno-
lone possess interesting activity in vitro against a panel of five
human cancer cell lines and especially against HT-29, HCT-15
and 502713 cell lines.7 In addition, 21-triazolyl derivatives of preg-
nenolone were found to possess anticancer activity.8 A more
detailed study of heterocylic analogues of pregnenolone, involved
pyrazolines, hydrazones, pyrazoles and oximes which were
screened against HepG2 and MDA-MB-231 cancer cell lines.9
17b-Chalconyl derivatives of pregnenolone and their correspond-
ing pyrazoline epimers were evaluated against a panel of four
human cancer cell lines.10
Cancer is one of the leading causes of death affecting millions of
people worldwide. Even though there is an arsenal of available
drugs there is an urgent need to develop new agents to overcome
the limitations of the current therapies.
Steroids represent an important class of naturally occurring
biomolecules which exhibit an astonishing range of biological
activities. The rigid tetracyclic skeleton, the presence of several
functional groups and the easy modification of the steroids have
made them attractive substrates for many different targets. An
extensive focus of research activities have been directed towards
the rational modification of steroids. In particular, a number of ste-
roid derivatives have been reported to possess anticancer activity
while, their excellent ability to penetrate cell membranes is bene-
ficial to their bioavailability.1,2 The majority of structural studies
towards new anticancer steroids involved A and D rings of the
steroid skeleton and to a lesser extent B ring modifications.3–5 In
particular, the presence of heterocyclic moieties in D-ring modified
steroids has been reported to enhance their biological activities.
Pregnenolone, a naturally occurring steroid has been utilized as
a template for the synthesis of steroid derivatives with improved
anticancer profile. Thus, the nitrochlorambucil ester of pregneno-
lone exhibited a significant in vitro cytotoxic activity in brain
posterior fossa, medulloblastoma (Daoy), and lung large cell
Recently, benzylidine pregnenolones and their oxime deriva-
tives were tested for their cytotoxic activities against a panel of
six human cancer cell lines and three of them were very potent
especially against HCT-15 and MCF-7 cell lines.11
We have been involved in the area of steroid research for the
past few years and we have successfully synthesized various cong-
eners with neuroprotective,12 GABAA modulating13 and anticancer
activities.14 Thus, in continuation of our research on steroid based
medicinal chemistry we set out to explore the anticancer proper-
ties of alkynylaryl pregnenolones. We were inspired by the prom-
ising results of benzylidine pregnenolones11 and we incorporated
in our design the substitution of the double bond spacer between
the aromatic moiety and the C17-carbonyl by an alkyne function-
ality. Thus, we maintained the same number of carbons (two) how-
ever, we introduced a nonflexible spacer of high electron density
⇑
Corresponding author. Tel.: +30 2107273833; fax: +30 2107273831.
E-mail address: tcalog@eie.gr (T. Calogeropoulou).
http://dx.doi.org/10.1016/j.bmc.2014.10.012
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

G. Szalóki et al. / Bioorg. Med. Chem. 22 (2014) 6980–6988
6981
R
R
O
O
HO
HO
21-alkynylaryl pregnenolone derivatives
benzylidene pregnenolone derivatives
Figure 1. Design of new derivatives.
(Fig. 1). Furthermore, to the best of our knowledge this class of
compounds has not been previously investigated.
Therefore, in this study, we describe the synthesis and SAR of
C21-alkynylaryl pregnenolone derivatives and their cytotoxic
activity against a panel of seven human cancer cell lines.
treatment with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,
and N,O-dimethylhydroxyl amine hydrochloride in the presence
of DMAP. Subsequent reaction of compound 4 with the n-BuLi
and the appropriate arylalkyne in the presence or absence of TME-
DA at ꢀ40 °C afforded compounds 5a–5e and 5f–5i, respectively.
Finally, deprotection of the 3b-hydroxyl group using HF.pyridine
complex afforded the final compounds 6a–6i.
2. Results and discussion
2.1. Chemistry
2.2. Evaluation of cytotoxic activity
The synthesis of the compounds of the present study 6a–i is
depicted in Scheme 1. Thus, protection of the 3-hydroxyl group
of pregnenolone (1) as the tert-butyldiphenylsilyl ether afforded
compound 2, which was treated with sodium hypobromide in
dioxane/water to give the corresponding 17b-carboxylic acid 3,
which in turn was transformed to the Weinreb amide 4 by
Compounds 6a–6i were assayed for in vitro cytotoxicity against
a panel of human cancer cell lines namely, LNCaP (prostate
carcinoma), A549 (lung adenocarcinoma), MCF7 (breast adenocar-
cinoma), HeLa (cervix adenocarcinoma), A431 (epithelial carci-
noma), HepG2 (hepatocellular carcinoma) and HT29 (colon
carcinoma) using the MTT assay. The IC50 values (lM) for 24 h
O
O
H3CO
O
.
OH
NH HCl
NaOBr
Dioxane-water
H3C
TBDPSCl, DMF
500C
r.t.
DMAP, EDCI, DCM
r.t.
TBDPSO
TBDPSO
O
HO
1
2
3
Ar
Ar
n-BuLi, TMEDA, THF
O
O
N
Ar
-40 0C
HF-pyridine,
DCM
O
TBDPSO
r.t.
5a-5e
5f-5i
O
Ar
HO
TBDPSO
6a-i
4
Ar
TBDPSO
n-BuLi, THF, -40 0
C
F
g
h
a
b
d
e
OCH3
CF3
F
Ar =
F
c
f
OCH3
i
F3C
Scheme 1. Synthesis of compounds 6a–i.

6982
G. Szalóki et al. / Bioorg. Med. Chem. 22 (2014) 6980–6988
and 48 h incubation are reported in Table 1. Our design involves
the variation of the substitution pattern of the aromatic ring of
the 21-alkynylaryl moiety in order to obtain information on the
structure activity relations of this class of compounds. Thus, we
introduced electron donating groups (methoxy, methyl) or elec-
tron withdrawing groups (fluorine, trifluoromethyl, ethynyl) or
combinations thereof. A number of correlations can be drawn by
reviewing the data in Table 1. In general, all the compounds exhibit
increased in vitro cytotoxicity after 48 h incubation when com-
pared with the 24 h experiment. Therefore, we opted to target
the discussion of our results using the IC50 values of the longer
experiment.
8.34 1.26
IC50 = 26.45 2.3
most active derivative against A431 (IC50 = 0.54 0.08
fluoro substituted compounds 6a and 6h are both very active
against LNCaP cells exhibiting IC50 = 9.48 0.75 and
IC50 = 10.78 1.42 M, respectively. However, there is a marked
difference in activity of 6a and 6h with respect to A549 and
HT29 cells which is even more pronounced against MCF7 and HeLa
cells. Specifically, the 3-fluoro derivative 6a possesses an
l
M, for HepG2 IC50 = 16.45 2.55
M. Surprisingly however, compound 6g is the
M). The
lM and for HT29
l
l
lM
l
IC50 = 24.95 3.70
for HT29 cells, an IC50 = 14.70 2.48
IC50 = 6.44 0.82 for HeLa cells. In contrast the 4-fluoro
analogue 6h is slightly active against A549 cells (IC50
40.57 5.76 M) and HT29 cells (IC50 = 44.70 5.92 M) and inac-
tive against MCF7 and HepG2 cells (IC50 >50 M). It is worth noting
however, that this order is reversed for A431 cells with compound
6h to possess an IC50 = 2.18 0.34 M while, 6a an IC50 = 45.32
3.15 M. Furthermore, the combination of a 3-methyl and a 4-flu-
oro substituent on the phenyl ring, compound 6b, leads to an
increase in activity against MCF7 (IC50 = 11.30 0.42 M), HT29
M) and A549 cells (IC50 = 28.2 2.55
lM for A549 cells, an IC50 = 28.58 3.20
lM
l
M for MCF7 cells and an
lM
Thus, the 2-trifluoromethyl substituted derivative 6f is the most
=
potent against LNCaP cells (IC50 = 5.44 0.40
(IC50 = 6.30 0.84 M), MCF7 cells (IC50 = 4.42 0.76
cells (IC50 = 3.85 0.60 M), HepG2 cells (IC50 = 12.64 2.17
and HT29 cells (IC50 = 9.87 1.55 M), while it exhibits excellent
activity against A431 cells (IC50 = 1.78 0.22 M). Conversely, the
l
M), A549
M), HeLa
M),
l
l
l
l
l
l
l
l
l
l
l
3-trifluoromethylphenyl substituted analogue 6g is less active
than the 2-substituted congener with IC50 values for LNCaP
l
IC50 = 17.40 2.81
l
M, for A549 cells IC50 = 20.74 6.70
lM,
cells (IC50 = 26.70 4.18
l
for MCF7 cells IC50 = 18.56 2.11
l
M, for HeLa cells IC50
=
lM), while, it decreases activity against LNCaP cells (IC50 =
Table 1
Cytotoxic activity of compounds 6a–6i
O
Ar
HO
Compound
Ar
(h)
LNCaP
IC50
A549
MCF7
IC50
HeLa
A431
IC50
HepG2
IC50
HT29
IC50
(lM)
a
a
a
a
a
a
a
IC50
M)
IC50
M)
(l
M)
(
l
(lM)
(l
(l
M)
(l
M)
24
48
14.70 1.38
9.48 0.75
>50
24.95 3.70
28.40 2.27
14.70 2.48
21.38 2.81
6.44 0.82
>50
45.32 3.15
>50
>50
>50
28.58 3.20
F
6a
6b
24
48
>50
38.40 2.80
40.05 5.45
28.2 2.55
17.60 1.98
11.30 0.42
>50
>50
>50
>50
>50
>50
>50
26.70 4.18
F
24
48
>50
46.2 4.80
>50
>50
39.00 2.00
7.78 1.72
>50
32.4 2.80
39.31 3.20
12.46 1.34
>50
48.56 5.20
>50
>50
6c
6d
6e
24
48
41.70 4.66
30.80 2.32
44.30 0.99
30.47 4.40
44.15 3.04
18.8 3.96
>50
23.57 3.18
44.12 5.30
13.67 0.88
>50
45.70 4.94
48.35 5.30
22.68 4.60
OCH3
24
48
19.10 0.99
16.85 1.22
>50
20.20 0.60
11.25 0.35
5.15 0.35
>50
>50
4.85 0.78
1.52 0.24
42.57 4.80
17.85 2.35
>50
>50
24
48
7.90 1.84
5.44 0.40
15.77 4.06
6.30 0.84
13.48 5.54
4.42 0.76
13.75 2.18
3.85 0.60
4.21.05 0.45
1.78 0.22
38.42 4.13
12.64 2.17
24.56 3.75
9.87 1.55
6f
F3C
24
48
26.20 1.30
17.40 2.81
29.31 21.9
20.74 6.70
31.45 4.76
18.56 2.11
25.10 3.19
8.34 1.26
2.28 0.42
0.54 0.08
45.68 5.36
16.45 2.55
>50
26.45 2.93
6g
6h
CF3
24
48
12.40 2.80
10.78 1.42
>50
40.57 5.76
>50
>50
>50
43.70 5.16
7.16 1.04
2.18 0.34
>50
>50
>50
44.70 5.92
F
24
48
>50
47.60 6.41
>50
>50
>50
>50
>50
26.70 3.45
>50
>50
>50
>50
>50
>50
6i
OCH3
a
IC50 values in lM are reported as the mean SD from 3 to 4 independent experiments.

G. Szalóki et al. / Bioorg. Med. Chem. 22 (2014) 6980–6988
6983
38.4 2.8
l
M), HeLa and A431 cells (IC50 = >50
l
M) with respect to
the IC50 values in Table 1, and the viability was determined after
24 h. (Table 2).
analogue 6h. In contrast the 3-methyl,4-methoxyphenyl derivative
6d exhibits weak activity against LNCaP, A549 and HepG2 cells and
is more potent against MCF7 cells (IC50 = 18.8 3.96
cells (IC50 = 13.67 0.88 M), while the 4-methoxyphenyl deriva-
tive 6i is inactive against A549, MCF7, A431, HepG2 and HT29
cells (IC50 = >50 M), slightly active against LNCaP cells
The results obtained by trypan blue were similar to those
obtained by the MTT test and reveal a clear cytotoxic effect of these
compounds. Morphological alterations, such as rounded cells were
also visible as soon as after 8 h of incubation (data not shown).
Interestingly, the extent of the cytotoxic effects of the compounds
depended on the tumour cell line investigated with A431, the epi-
dermoid carcinoma, being the most sensitive. These cells are highly
sensitive to mitogenic stimuli because overexpress the epidermal
growth factor receptor. The best-characterized pathway known
to transduce growth factor activation and promote cell growth or
survival in cancer cells, is the Akt/protein kinase B (Akt), a key
effector of phosphatidylinositol 30-kinase (PI3K).15 Phosphoryla-
tion of Akt at Thr308 and Ser473 is required for activation and—
in this form—is found consistently hyperactivated in many
tumours. Based on the above, we evaluated the effects of the most
lM) and A431
l
l
(IC50 = 47.60 6.41
(IC50 = 26.70 3.45
l
l
M) and more potent against HeLa cells
M). The 2,4,5-trimethylphenyl analogue 6c
exhibits high potency against MCF7 cells (IC50 = 7.78 1.72
and A431 cells (IC50 = 12.46 1.34 M), lower activity against HeLa
cells (IC50 = 32.4 2.80 M) which is decreased even more against
LNCaP cells (IC50 = 46.2 4.80 M) and HepG2 cells (IC50
48.56 5.20 M), and the activity is abolished for A549 and HT29
cells. Finally, introduction of a 3-ethynylphenyl substituent, com-
pound 6e, results in good activity against LNCaP, A549 cells and
HepG2 cells (IC50 = 16.85 1.22
IC50 = 17.85 2.35 M, respectively), and excellent against A431
cells (IC50 = 1.52 0.24 M) and MCF7 cells (IC50 = 5.15 0.35 M).
lM)
l
l
l
=
l
lM, IC50 = 20.20 0.60 lM and
l
active compounds on the levels of phosphorylated Akt (Ser473
)
(Fig. 3). Cells were treated with compounds 6e–6g at a concentra-
l
l
In general, electron withdrawing substituents favour activity
against LNCaP cells (compounds 6a, 6e, 6f, 6g, 6h) while their com-
bination with electron donating groups is detrimental to activity
against this cell line (6b versus 6e). Concerning A549 cells no clear
conclusion can be drawn, however the trend observed for LNCaP
cells is observed also for A549. The majority of the alkynylaryl
pregnenolone derivatives of the present study are very potent
against MCF7 cells and the activity is very much dependent on
the electronegativity and the position of the substituents on the
phenyl ring. Compounds 6a, 6f, 6g and 6h bearing electron with-
drawing substituents are very potent against HeLa cells, while,
derivatives 6e, 6f, 6g and 6h exhibit excellent activity against
A431 cells. Compounds 6e, 6f and 6g and compound 6f are the
most potent against HepG2 and HT29 cells, respectively.
tion corresponding to their IC50 value. Cell lysates were prepared
24 h later and 50 lg each, were loaded into each well and fraction-
ated by 10% SDS–PAGE. Immunoblotting was performed as
described in ‘Materials and Methods’. Equal loading was verified
by blotting the membranes with an antibody against b-actin.
Indeed, western blot analysis demonstrated that cytotoxic
compounds 6e–6g interfere with the PI3K-Akt/PKB pathway which
is a crucial survival signalling pathway for a variety of human
cancers (Fig. 3).
Finally, we also examined the effect of dehydroepiandrosterone
(DHEA) against the cell lines of the present study in order to get an
insight as to whether the biological activity we observe for com-
pounds 6a–i is related to the substitution on position C17. DHEA
is inactive against all the cell lines (IC50 >50
l
M) and marginally
Even though an incubation period of 48 h is most frequently
used for the evaluation of antiproliferative action, we also exam-
ined the effects of our compounds at 24 h incubation in order to
get an insight on their acute cytotoxic activity. As we observe in
Table 1 the potent compounds (i.e., 6a, 6b, 6e, 6f, 6g, 6h) exhibit
good activity even after a short incubation period (24 h) which of
course improves after 48 h. This is also depicted in Figure 2 which
includes the dose response curves of the most potent compound 6f
against LNCaP, A549 and MCF7 cell lines.
active against A549 cells (IC50 = 47.8 3.80 lM) and thus, we can
Table 2
Cell viability—expressed as percentage of viable cells over the total number of cells—
after 24 h of exposure to compounds 6e–6h
Compound
HeLa
(10
LNCaP
(10 M)
A431
MCF7
l
M)
l
(5
lM)
(10
lM)
6e
6f
6g
6h
93.2 4.5
70.8 10.3
92.5 6.2
90.3 7.6
0
92.1 6.8
25.6 4.7
0
0
0
0
18.9 2.2
94.2 7.3
90.4 7.7
The acute cytotoxic activity of the potent derivatives was also
corroborated by determination of cell viability using the trypan
blue exclusion test. HeLa, MCF7 and LNCaP cells were treated with
98.4
1
28.6 3.2
10 lM concentration of compounds 6e, 6f, 6g and 6h whereas
A431 cells were treated with 5
lM concentration, in line with
LNCaP HeLa
6f 6f
MCF7 MCF7
6e 6f
C
C
C
C
A
B
C
125
100
75
50
25
0
pAkt
acꢀn
LNCaP
A549
MCF-7
pAkt
acꢀn
C
6e 6f 6g 6h
pAkt
acꢀn
0
2
4
24h 6g
0
10
20
30
40
50
μΜ
Figure 3. Effect of selected compounds on Akt phosphorylation in cancer cell lines
The phosphorylation of Akt (pAkt-Ser473) is shown in the top panels, and total levels
of actin are shown in the bottom panels. (A) effects of 6e, 6f on pAkt levels in LNCaP,
HeLa and MCF7 cells; (B) effects of 6e, 6f, 6g and 6h on pAkt in A431 cells; (C) time
dependent effect of 6g on pAkt in A431 cells. C: untreated cells.
Figure 2. Dose response curves of the cytotoxic activity of compound 6f on LNCaP,
A549 and MCF7 cell lines. Values are the mean standard deviation of four
independent experiments.

6984
G. Szalóki et al. / Bioorg. Med. Chem. 22 (2014) 6980–6988
conclude that the 20-ketoalkynylaryl moiety is responsible for
imparting anticancer activity to the corresponding pregnenolone
derivatives.
4.1.2. 3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-carboxylic
acid (3)
To a solution of compound 2 (3.2 g, 5.83 mmol) in dioxane
(77 mL) and water (23 mL) was added at 0 °C a solution of NaOBr
(158 mL) (prepared by mixing a solution of 15.3 g NaOH in
131 mL water with 84.6 mL dioxane and 5.08 mL Br2 at 0 °C). The
resulting mixture was stirred at ambient temperature overnight,
and the mixture was cooled to 0 °C and was quenched with satu-
rated Na2SO3, followed by an additional 10 min stirring at the same
temperature. The mixture was extracted with ethyl acetate and the
organic phase was washed with water and brine, was dried over
anhydrous Na2SO4 and was evaporated in vacuo. Compound 3
was obtained as white crystals (3.22 g, 99%) and was pure enough
3. Conclusions
A series of novel 21-alkynylaryl derivatives of pregnenolone
were synthesized and evaluated for their anticancer activity
against seven human cancer cell lines. The majority of the com-
pounds exhibited anticancer activity and the 2-trifluoromethyl-
phenyl derivative 6f was found to posses promising activity
against all seven cell lines. A431 cells were the most sensitive
against the compounds of the present study, with derivatives 6e–
6h bearing electron withdrawing substituents exhibiting high
to be employed in the next step without any further purification.
20
mp 195–198 °C; [
a
]
D
ꢀ19.33° (ca. 0.00119 g/mL, CHCl3); IR (CHCl3,
cmꢀ1): 3237, 2935, 1720; Rf: 0.46, cyclohexane/ethyl acetate 9:1;
1H NMR (CDCl3): d 0.76 (s, 3H), 0.85–0.96 (m, 3H), 1.02 (s, 3H),
1.10 (m, 9H), 1.18–2.21 (m, 16H), 2.38 (t, 1H, J = 9.3 Hz), 3.52–
3.63 (m, 1H), 5.15 (d, 1H, J = 4.9 Hz), 7.36–7.47 (m, 6H), 7.70–
7.74 (m, 4H); 13C NMR (CDCl3): d 13.1, 19.1, 19.4, 20.6, 23.3,
24.5, 26.9, 27.0, 30.2, 31.7, 31.8, 31.9, 36.5, 37.2, 37.9, 42.4, 44.1,
49.9, 55.1, 56.3, 73.2, 120.8, 127.42, 127.44, 129.41, 129.43,
134.74, 134.75, 135.73, 135.74, 141.3, 180.5; Anal. Calcd for C36-
H48O3Si: C, 77.65; H, 8.69. Found C, 77.34; H, 9.07.
potency with IC50 values ranging between 2.18 and 0.54 lM. Fur-
thermore, these compounds were found to interfere with the
PI3K-Akt/PKB pathway. These results could further support future
design of pregnenolone-based anticancer drugs.
4. Experimental section
4.1. Chemistry
1H NMR spectra were recorded at 600 or 300 MHz, 13C NMR
spectra at 150.9 or 75.5 MHz and 19F NMR spectra at 564.8 MHz.
1H and 13C NMR spectra are internally referenced to residual sol-
vent signals (CDCl3). 19F NMR spectra are referenced to CCl3F. Data
for 1H NMR are reported as follows: chemical shift (d ppm), multi-
plicity (s = singlet, d = doublet, dd = doublet of doublet, ddd = dou-
blet of doublet of doublet, m = multiplet), coupling constant and
integration. Data for 13C NMR are reported in terms of chemical
shift (d ppm). Melting points (°C) are uncorrected. IR spectra were
obtained on an ATR-IR spectrometer. HR-mass spectra were
recorded on UHPLC LC-MSn Orbitrap Velos-Thermo instrument.
Elemental analyses were obtained using a Perkin Elmer Series II
CHNS/O 2400 instrument. THF was distilled over sodium in the
presence of benzophenone, DCM was distilled over calcium
hydride and DMF was dried over 4 Å molecular sieves. Thin-layer
chromatography (TLC) was performed on glass plates coated with
silica gel (0.2 mm, 60 F254) and flash chromatography using silica
gel (200–400 mesh).
4.1.3. N-Methoxy,N-methyl-3b-(t-butyldiphenylsilyloxy)-
androst-5-en-17b-carboxamide (4)17
To a stirred solution of 3 (1.2 g, 2.16 mmol) in dry CH2Cl2
(12 mL) were sequentially added N,O-dimethylhydroxylamine
hydrochloride (316 mg, 3.23 mmol), 4-dimethylaminopyridine
(395 mg, 3.23 mmol) and EDCI (620 mg, 3.23 mmol) at room tem-
perature and the reaction mixture was stirred overnight. When the
reaction was completed, brine was added and the mixture was
diluted with ethyl acetate. The organic phase was washed with
5% HCl solution and brine, it was dried over anhydrous Na2SO4
and was evaporated in vacuo. The residue was purified by flash col-
umn chromatography (petroleum ether–acetone, 95:5) to afford
compound 4 (1.21 g, 94%) as a white crystalline solid. mp 143–
20
46 °C; [a]
ꢀ45.73° (ca. 0.00164 g/mL, CHCl3); IR (CHCl3, cmꢀ1):
D
2932, 1652; Rf: 0.26, petroleum ether/acetone 9:1; 1H NMR
(CDCl3): d 0.73 (s, 3H), 0.77–0.88 (m, 3H), 0.98 (s, 3H), 1.05 (s,
9H), 1.14–2.38 (m, 16H), 2.72–2.75 (m, 1H), 3.18 (s, 3H), 3.48–
3.58 (m, 1H), 3.63 (s, 3H), 5.13 (d, 1H, J = 5.0 Hz), 7.32–7.44 (m,
6H), 7.66–7.69 (m, 4H); 13C NMR (CDCl3): d 13.8, 19.1, 19.4, 20.9,
24.8, 24.9, 27.0, 31.8, 31.9, 32.0, 36.5, 37.2, 38.9, 42.3, 42.4, 45.1,
50.1, 50.7, 56.7, 60.9, 73.2, 120.9, 127.4, 127.5, 129.3 129.4,
134.7, 134.8, 135.5, 135.6, 141.3, 174.7; MS (ESI+), m/z 600.39
[M+H]+; Anal. Calcd for C38H53NO3Si: C, 76.08; H, 8.90; N, 2.33.
Found C, 76.24; H, 9.07; N, 2.63.
4.1.1. 3b-(t-Butyldiphenylsilyloxy)-5-pregnen-20-one (2)16
To a solution of pregnenolone (1) (2.0 g, 6.32 mmol) in DMF
(40 mL) imidazole (1.1 g, 15.8 mmol) was added at 0 °C and
the reaction was stirred at that temperature for 30 min, and
TBDPSCl (4.7 mL, 15.8 mmol) was added. The reaction was
warmed 50 °C and was stirred overnight. Subsequently, the reac-
tion was cooled to room temperature, was quenched with satu-
rated NH4Cl, and the solvent was evaporated under reduced
pressure. The residue was diluted with H2O and was extracted
with ethyl acetate. The organic phase was washed with brine,
was dried over anhydrous Na2SO4 and was evaporated in vacuo.
4.1.4. General procedures for the synthesis of the alkynylaryl
pregnenolone derivatives 5a–i
Method A. To a solution of n-BuLi (1.6 M solution in hexanes) in
anhydrous THF (0.33 M, 2 equiv) at ꢀ40 °C was added TMEDA
(2 equiv) and the resulting solution was stirred at that temperature
for 15 min. Subsequently, the appropriate alkyne (3 equiv) was
added at ꢀ40 °C and the reaction was stirred at that temperature
for 1 h after which a solution of the Weinreb amide 4 (1 equiv)
in THF (0.055 M) was added and the temperature was allowed to
rise to ꢀ10 °C over 3 h and the reaction was stirred at ꢀ10 °C until
completion. The reaction was quenched with saturated NH4Cl solu-
tion (3 mL) and was diluted with diethyl ether. The organic layer
was washed with brine, was dried over anhydrous Na2SO4 and
the solvent was evaporated in vacuo. The residue was purified by
flash column chromatography to afford the desired compounds
5a–e.
The residue was recrystallized from ethanol to afford 2 in quan-
20
titative yield (3.5 g, 100%). mp 125–129 °C;
[a
]
6.04° (ca.
D
0.00149 g/mL, CHCl3); Rf: 0.68 (cyclohexane/ethyl acetate 8:2);
1H NMR (CDCl3): d 0.60 (s, 3H), 0.81–0.91 (m, 3H), 0.98 (s,
3H), 1.06 (s, 9H), 1.15–2.03 (m, 14H), 2.10 (s, 3H), 2.14–2.38
(m, 2H), 2.49 (t, 1H, J = 9.0 Hz), 3.48–3.58 (m, 1H), 5.12 (d, 1H,
J = 4.9 Hz), 7.33–7.44 (m, 6H), 7.66–7.72 (m, 4H); 13C NMR
(CDCl3): d 13.4, 19.3, 19.6, 21.2, 23.0, 24.6, 27.2, 31.7, 31.9,
32.0, 32.1, 36.7, 37.4, 39.1, 42.6, 44.2, 50.1, 57.1, 63.9, 73.3,
121.0, 127.63, 127.65, 129.61, 129.64, 134.94, 134.98, 135.95,
135.96, 141.4, 209.8; Anal. Calcd for C37H50O2Si: C, 80.09; H,
9.08. Found C, 79.68; H, 8.79.

G. Szalóki et al. / Bioorg. Med. Chem. 22 (2014) 6980–6988
6985
Method B. To a solution of n-BuLi (1.6 M solution in hexanes) in
anhydrous THF (0.33 M, 2 equiv) was added at ꢀ40 °C the appro-
priate alkyne (3 equiv) and the reaction was stirred at that temper-
ature for 1 h. Subsequently a solution of the Weinreb amide 4
(1 equiv) in THF (0.055 M) was added and the temperature was
allowed to rise to ꢀ10 °C over 3 h and the reaction was stirred at
ꢀ10 °C until completion. The reaction was quenched with satu-
rated NH4Cl solution (3 mL) and was diluted with diethyl ether.
The organic phase was washed with brine, was dried over anhy-
drous Na2SO4 and the solvent was evaporated in vacuo. The residue
was purified by flash column chromatography to afford the desired
compounds 5f–i.
1.13–2.16 (m, 14H), 2.22 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.30–2.36
(m, 2H), 2.42 (s, 3H, CH3), 2.71 (t, J = 8.7 Hz, 1H), 3.51–3.61 (m,
1H), 5.15 (d, J = 4.3 Hz, 1H), 7.02 (s, 1H), 7.31 (s, 1H), 7.36–7.45
(m, 6H), 7.69–7.71 (m, 4H); 3C NMR (CDCl3): d 13.3, 19.0, 19.1,
19.4, 19.8, 20.1, 20.9, 22.6, 24.5, 26.9, 31.7, 31.8, 31.9, 36.5, 37.2,
38.7, 42.4, 45.1, 50.0, 56.9, 64.8, 73.2, 91.0, 92.6, 117.1, 120.7,
127.41, 127.43, 129.40, 129.42, 131.1, 134.2, 134.4, 134.7, 134.8,
135.72, 135.73, 139.4, 139.9, 141.3, 189.0; HR-MS (ESI+): [M+H]+
found 683.4289. C47H59O2Si requires 683.4279; [M+Na]+ found
705.4108. C47H58O2SiNa requires 705.4098.
4.1.4.4. 1-[3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-yl]-3-
(2-methyl-4-methoxyphenyl)-2-propyn-1-one (5d).
Com-
4.1.4.1. 1-[3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-yl]-3-
pound 5d was prepared according to Method A from Weinreb
amide 4 (0.2 g, 0.33 mmol) and 1-ethynyl-2-methyl-4-methoxy-
benzene (146 mg, 1.0 mmol) as a crystalline solid after purification
by flash column chromatography (petroleum ether–acetone, 98:2)
(207 mg, 91% yield). mp 66–69 °C; IR: 2932, 2183, 1654 cmꢀ1; Rf:
0.43, petroleum ether 40–60 °C/acetone 9:1; 1H NMR (CDCl3): d
0.76 (s, 3H), 0.88–0.90 (m, 3H), 1.02 (s, 3H), 1.09 (s, 9H), 1.16–
2.38 (m, 16H), 2.49 (s, 3H), 2.70–2.72 (m, 1H), 3.58 (br s, 1H),
3.81 (s, 3H), 5.17 (br s, 1H), 6.72–6.78 (m, 2H), 7.41–7.51 (m,
7H), 7.71 (br s, 4H); 13C NMR (CDCl3): d 13.3, 19.0, 19.3, 20.9,
21.0, 22.5, 24.4, 26.9, 31.7, 31.8, 36.5, 37.2, 38.6, 42.4, 45.0, 49.9,
55.2, 56.8, 64.8, 73.1, 91.2, 92.9, 111.6, 112.0, 115.3, 120.7, 127.3,
127.4, 129.3, 129.4, 134.6, 134.62, 134.7, 135.4, 135.7, 141.2,
144.3, 161.4, 188.9; HR-MS (ESI+): [M+H]+ found 685.4096. C46H57-
O3Si requires 685.4071; [M+Na]+ found 707.3916. C46H56O3SiNa
requires 707.3891.
(3-fluorophenyl)-2-propyn-1-one (5a).
Compound 5a was
prepared according to Method A from Weinreb amide 4 (0.4 g,
0.66 mmol) and 1-ethynyl-3-fluorobenzene (0.24 mL, 2.00 mmol)
as an oil after purification by flash column chromatography (petro-
leum ether–acetone, 95:5) (300 mg, 68%). IR: 2932, 2198,
1663 cmꢀ1; Rf: 0.47, petroleum ether/acetone 95:5; 1H NMR
(CDCl3): d 0.71 (s, 3H), 0.83–0.92 (m, 3H), 0.98 (s, 3H), 1.06 (s,
9H), 1.12–2.38 (m, 16H), 2.69 (t, J = 8.6 Hz, 1H), 3.50–3.58 (m,
1H), 5.13 (d, J = 4.4 Hz, 1H), 7.07–7.23 (m, 2H), 7.28–7.42 (m,
8H), 7.66–7.69 (m, 4H); 13C NMR (CDCl3): d 13.2, 19.1, 19.3, 20.9,
22.2, 24.3, 26.9, 31.6, 31.7, 36.4, 37.2, 38.4, 42.4, 45.1, 49.9, 56.9,
64.8, 73.1, 88.9 (d, JC–F = 3.5 Hz), 89.4, 117.9 (d, JC–F = 21.1 Hz),
119.3 (d, JC–F = 23.1 Hz), 120.7, 122.0 (d, JC–F = 9.2 Hz), 127.3,
127.4, 128.6 (d,
JC–F = 3.2 Hz), 129.37, 129.39, 130.2 (d,
JC–F = 8.5 Hz), 134.6, 134.7, 135.6, 135.7, 141.1, 162.1 (d,
J
C–F = 248.2 Hz), 188.5; 19F NMR (CDCl3): d (ꢀ111.94)–(ꢀ111.86)
(m); HR-MS (ESI+): [M+H]+, found 659.3709. C44H52FO2Si requires
659.3715; [M+Na]+, found 681.3523. C44H51FO2SiNa requires
681.3535.
4.1.4.5. 1-[3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-yl]-3-
(3-ethynylphenyl)-2-propyn-1-one (5e).
Compound 5e was
prepared according to Method A from Weinreb amide 4 (0.2 g,
0.33 mmol) and 1,3-diethynylbenzene (0.14 mL, 1.0 mmol) as an
oil after purification by flash column chromatography (petroleum
ether 40–60 °C/ethyl acetate, 95:5) (104 mg, 47% yield). IR: 2932,
2194, 1661 cmꢀ1; Rf: 0.58, cyclohexane/ethyl acetate 9:1; 1H
NMR (CDCl3): d 0.73 (s, 3H), 0.85–0.92 (m, 3H), 1.00 (s, 3H), 1.08
(s, 9H), 1.14–2.40 (m, 16H), 2.71 (t, 1H, J = 8.6 Hz), 3.13 (s, 1H),
3.51–3.61 (m, 1H), 5.15 (d, 1H, J = 4.7 Hz), 7.32–7.72 (m, 14H);
13C NMR (CDCl3): d 13.3, 19.1, 19.3, 20.9, 22.3, 24.3, 27.0, 31.7,
31.80, 31.82, 36.4, 37.1, 38.5, 42.4, 45.1, 49.9, 56.9, 64.9, 73.1,
78.6, 82.1, 89.4, 89.5, 120.7, 120.8, 122.9, 127.4, 127.5, 128.7,
129.4, 129.41, 132.9, 133.9, 134.7, 134.8, 135.7, 135.8, 136.0,
141.2, 188.8; HR-MS (ESI+): [M+H]+ found 665.3824. C46H53O2Si
requires 665.3809; [M+Na]+ found 687.3641. C46H52O2SiNa
requires 687.3629.
4.1.4.2. 1-[3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-yl]-3-
(3-methyl-4-fluorophenyl)-2-propyn-1-one
(5b).
Com-
pound 5b was prepared according to Method A from Weinreb
amide 4 (0.1 g, 0.16 mmol) and 4-ethynyl-1-fluoro-2-methylben-
zene (0.06 mL, 0.50 mmol) as yellow crystals after purification by
flash column chromatography (petroleum ether 40–60 °C/acetone,
95:5) (80 mg, 71%). mp 143–145 °C; IR: 2932, 2190, 1651 cmꢀ1; Rf:
0.26, petroleum ether 40–60 °C/acetone 9:1; 1H NMR (CDCl3): d
0.72 (s, 3H), 0.84–0.95 (m, 3H), 1.00 (s, 3H), 1.07 (s, 9H), 1.14–
2.19 (m, 16H), 2.28 (d, J = 1.9 Hz, 3H), 2.69 (t, J = 8.8 Hz, 1H),
3.50–3.60 (m, 1H), 5.14 (d, J = 5.1 Hz, 1H), 7.01 (dd, J = 8.5, 9.1 Hz,
1H), 7.34–7.46 (m, 8H), 7.68–7.71 (m, 4H); 13C NMR (CDCl3): d
13.2, 14.3 (d, JC–F = 3.7 Hz), 19.1, 19.4, 20.9, 22.3, 24.3, 26.9, 31.7,
31.82, 31.85, 36.5, 37.2, 38.4, 42.4, 45.1, 49.9, 56.9, 64.9, 73.1,
88.9 (d,
JC–F = 23.4 Hz), 115.9 (d, JC–F = 3.9 Hz), 120.7, 125.8 (d,
C–F = 18.3 Hz), 127.4, 127.5, 129.4, 129.5, 132.5 (d, JC–F = 8.8 Hz),
J
C–F = 1.4 Hz), 90.4 (d, JC–F = 0.9 Hz), 115.7 (d,
4.1.4.6. 1-[3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-yl]-3-
(2-trifluoromethylphenyl)-2-propyn-1-one (5f).
Compound
J
5f was prepared according to Method B from Weinreb amide 4
(0.1 g, 0.16 mmol) and 1-ethynyl-2-trifluoromethylbenzene
(0.07 mL, 0.50 mmol) as an oil after purification by flash column
chromatography (cyclohexane–ethyl acetate, 98:2) (112 mg, 95%
yield). IR: 2931, 2206, 1664 cmꢀ1; Rf: 0.44, petroleum ether 40–
60 °C/acetone 9:1; 1H NMR (CDCl3): d 0.75 (s, 3H), 0.86–0.92 (m,
3H), 1.00 (s, 3H), 1.09 (s, 9H), 1.15–2.72 (m, 16H), 2.73 (t,
J = 8.8 Hz, 1H), 3.52–3.62 (m, 1H), 5.15 (d, J = 4.4 Hz, 1H), 7.35–
7.45 (m, 6H), 7.51–7.59 (m, 2H), 7.69–7.73 (m, 6H); 13C NMR
(CDCl3): d 13.6, 19.3, 19.7, 21.1, 22.9, 24.8, 27.2, 32.0, 32.11,
32.15, 36.8, 37.4, 38.6, 42.7, 45.7, 50.2, 57.1, 65.1, 73.4, 85.9, 93.1
(q, JC–F = 0.9 Hz), 118.4 (q, JC–F = 2.1 Hz), 120.7, 123.1 (q,
JC–F = 272.5 Hz), 126.1 (q, JC–F = 5.0 Hz), 127.4, 127.5, 129.4, 129.5,
130.1, 131.6 (q, JC–F = 0.9 Hz), 132.5 (q, JC–F = 31.1 Hz), 134.7,
134.8, 135.4, 135.7, 135.8, 141.3, 188.9; 19F NMR (CDCl3): d
ꢀ62.1 (s); HR-MS (ESI+): [M+H]+ found 709.3689. C45H52F3O2Si
134.7, 134.8, 135.7, 135.8, 136.2 (d, JC–F = 6.0 Hz), 141.2, 162.5 (d,
J
C–F = 252.1 Hz), 188.9; 19F NMR (CDCl3): d (ꢀ111.43)–(ꢀ111.34)
(m).; HR-MS (ESI+): [M+H]+, found 673.3878. C45H54FO2Si requires
673.3972; [M+Na]+, found 695.3696. C45H53FO2SiNa requires
695.3691.
4.1.4.3. 1-[3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-yl]-3-
(2,4,5-trimethylphenyl)-2-propyn-1-one (5c).
Compound 5c
was prepared according to Method A from Weinreb amide 4 (0.2 g,
0.33 mmol) and 1-ethynyl-2,4,5-trimethylbenzene (0.148 g,
1.0 mmol) as a crystalline solid after purification by flash column
chromatography (petroleum ether 40–60 °C/acetone, 95:5)
(103 mg, 45% yield). mp 74–76 °C; IR: 2932, 2184, 1656 cmꢀ1; Rf:
0.60, petroleum ether 40–60 °C/acetone 95:5; 1H NMR (CDCl3): d
0.75 (s, 3H), 0.85–0.93 (m, 3H), 1.01 (s, 3H), 1.09 (s, 9H),

6986
G. Szalóki et al. / Bioorg. Med. Chem. 22 (2014) 6980–6988
requires 709.3683; [M+Na]+ found 731.3510. C45H51F3O2SiNa
requires 731.3503.
CH2Cl2. The organic layer was washed with brine, was dried over
anhydrous Na2SO4 and was evaporated in vacuo. The residue was
purified by flash column chromatography to obtain the desired
products 6a–i.
4.1.4.7. 1-[3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-yl]-3-
(3-trifluoromethylphenyl)-2-propyn-1-one
(5g).
Com-
pound 5g was prepared according to Method B from Weinreb
amide 4 (0.1 g, 0.16 mmol) and 1-ethynyl-3-trifluoromethylben-
zene (0.07 mL, 0.50 mmol) as an oil after purification by flash col-
umn chromatography (cyclohexane/ethyl acetate, 98:2) (70 mg,
59% yield). IR 2931, 2206, 1663 cmꢀ1; Rf: 0.61, petroleum ether/
acetone 9:1; 1H NMR (CDCl3): d 0.74 (s, 3H), 0.84–0.94 (m, 3H),
1.00 (s, 3H), 1.07 (m, 9H), 1.15–2.40 (m, 16H), 2.72 (t, J = 8.7 Hz,
1H), 3.50–3.60 (m, 1H), 5.14 (d, J = 4.8 Hz, 1H), 7.34–7.45 (m,
6H), 7.52 (t, J = 7.8 Hz, 1H), 7.68–7.74 (m, 6H), 7.80 (br s, 1H);
13C NMR (CDCl3): d 13.6, 19.4, 19.6, 21.2, 22.6, 24.6, 27.2, 31.99,
32.08, 32.1, 36.8, 37.4, 38.8, 42.7, 45.5, 50.2, 57.2, 65.2, 73.4,
88.6, 90.1, 120.9, 121.5, 123.6 (q, JC–F = 272.6 Hz), 127.3 (q,
JC–F = 3.7 Hz), 127.4, 127.5, 129.2, 129.4 (q, JC–F = 3.9 Hz), 129.42,
129.5, 131.6 (q, JC–F = 33.0 Hz), 134.7, 134.8, 135.74, 135.75,
135.8 (q, JC–F = 1.2 Hz), 141.6, 188.9; 19F NMR (CDCl3): d ꢀ63.4
(br s); HR-MS (ESI+): [M+H]+ found 709.3702. C45H52F3O2Si
709.3683; [M+Na]+ found 731.3525. C45H51F3O2SiNa 731.3503.
4.1.5.1. 17b-[1-Oxo-3-(3-fluorophenyl)-2-propynyl]-androst-5-
en-3b-ol (6a).
Compound 6a was prepared according to the
general method described above from compound 5a (293 mg,
0.45 mmol) as a crystalline solid after purification by flash column
chromatography (petroleum ether 40–60 °C/acetone, 9:1) as crys-
20
talline solid (75 mg, 40% yield). mp 131–132 °C; [
a
]
ꢀ29.21°
(c = 0.09583, CHCl3); IR: 3460, 2933, 2202, 1650 cmꢀ1D; Rf: 0.33,
petroleum ether 40–60 °C/acetone 8:2; 1H NMR (CDCl3): d 0.73
(s, 3H), 0.99 (s, 3H), 1.05–2.38 (m, 19H), 2.71 (t, J = 8.6 Hz, 1H),
3.46–3.53 (m, 1H), 5.33 (d, J = 4.8 Hz, 1H), 7.10–7.38 (m, 4H); 13C
NMR (CDCl3): d 13.3, 19.3, 20.9, 22.3, 24.3, 31.5, 31.7, 31.8, 36.5,
37.2, 38.4, 42.1, 45.1, 49.9, 56.9, 64.9, 71.5, 89.0 (d, JC–F = 3.4 Hz),
89.3, 118.0 (d, JC–F = 21.2 Hz), 119.4 (d, JC–F = 23.0 Hz), 121.2,
121.9 (d, JC–F = 9.3 Hz), 128.6 (d, JC–F = 3.3 Hz), 130.3 (d,
JC–F = 8.4 Hz), 140.7, 162.2 (d, JC–F = 248.1 Hz), 188.8; 19F NMR
(CDCl3): d (ꢀ112.15)–(ꢀ112.09) (m); HR-MS (ESI+): [M+H]+ found
421.2525. C28H34FO2 requires 421.2537.
4.1.4.8. 1-[3b-(t-Butyldiphenylsilyloxy)-androst-5-en-17b-yl]-3-
4.1.5.2. 17b-[1-Oxo-3-(3-methyl-4-fluorophenyl)-2-propynyl]-
(4-fluorophenyl)-2-propyn-1-one (5h).
Compound 5h was
androst-5-en-3b-ol (6b).
Compound 6b was prepared
prepared according to Method B from Weinreb amide 4 (0.1 g,
0.16 mmol) and 1-ethynyl-4-trifluoromethylbenzene (60 mg,
0.50 mmol) as an oil after purification by flash column chromatog-
raphy (cyclohexane–ethyl acetate, 98:2) (72 mg, 66% yield). IR:
2933, 2203, 1658 cmꢀ1; Rf: 0.68, cyclohexane/ethyl acetate 9:1;
1H NMR (CDCl3): d 0.73 (s, 3H), 0.85–0.94 (m, 3H), 1.00 (s, 3H),
1.06 (m, 9H), 1.14–2.39 (m, 16H), 2.70 (t, J = 8.7 Hz, 1H), 3.50–
3.60 (m, 1H), 5.14 (d, J = 4.8 Hz, 1H), 7.07 (t, J = 8.6 Hz, 2H), 7.35–
7.45 (m, 6H), 7.53–7.58 (m, 2H), 7.68–7.71 (m, 4H); 13C NMR
(CDCl3): d 13.2, 19.1, 19.4, 21.0, 22.3, 24.3, 26.9, 27.0, 31.7, 31.8,
36.5, 37.2, 38.5, 42.4, 45.2, 49.9, 57.0, 64.8, 73.1, 89.1 (d,
according to the general method described above from compound
5b (36 mg, 0.05 mmol) as a crystalline solid after purification by
flash column chromatography (cyclohexane-ethyl acetate, 95:5)
20
(11 mg, 47% yield). mp 138–140 °C; [
a
]
D
ꢀ57.61° (c = 0.04166,
CHCl3); IR: 3307, 2928, 2193, 1643 cmꢀ1; Rf = 0.57, cyclohexane/
ethyl acetate 95:5; 1H NMR (CDCl3): d 0.74 (s, 3H), 1.01 (s, 3H),
1.05–2.04 (m, 16H), 2.18–2.23 (m, 1H), 2.28 (s, 3H), 2.32–2.40
(m, 2H), 2.71 (t, J = 8.6 Hz, 1H), 3.48–3.58 (m, 1H), 5.36 (d,
J = 4.2 Hz, 1H), 7.00 (t, J = 8.8 Hz, 1H,), 7.36–7.41 (m, 2H); 13C
NMR (CDCl3): d 13.3, 14.3 (d, JC–F = 3.4 Hz), 19.4, 21.0, 22.4, 24.4,
31.6, 31.8, 31.9, 36.5, 37.3, 38.5, 42.2, 45.1, 50.1, 57.0, 64.8, 71.7,
JC–F = 1.4 Hz), 89.8, 116.1 (d, JC–F = 22.3 Hz), 116.3 (d, JC–F
=
88.8 (d, JC–F = 1.4 Hz), 90.5, 115.7 (d, JC–F = 23.4 Hz), 115.9 (d, JC–F =
3.5 Hz), 120.8, 127.4, 127.5, 129.4, 129.5, 134.7, 134.8, 135.1 (d,
JC–F = 8.8 Hz), 135.7, 135.8, 141.2, 163.8 (d, JC–F = 253.6 Hz), 188.8;
19F NMR (CDCl3): d (ꢀ106.92)–(ꢀ106.83) (m).
3.9 Hz), 121.3, 125.8 (d, JC–F = 18.3 Hz), 132.5 (d, JC–F = 8.8 Hz),
136.2 (d, JC–F = 6.0 Hz), 140.7, 162.6 (d, JC–F = 252.1 Hz), 189.0; 19F
NMR (CDCl3):
d
(ꢀ111.34)–(ꢀ111.30) (m); HR-MS (APCI+):
[M+H]+ found 435.2683. C29H36FO2 requires 435.2694.
4.1.4.9. 1-[3b-(t-Butyldiphenylsilyoxy)-androst-5-en-17b-yl]-3-
(4-methoxyphenyl)-2-propyn-1-one (5i).
prepared according to Method B from Weinreb amide 4 (0.1 g,
0.16 mmol) and 1-ethynyl-4-methoxybenzene (0.12 mL,
Compound 5i was
4.1.5.3. 17b-[1-Oxo-3-(2,4,5-trimethylphenyl)-2-propynyl]- and-
rost-5-en-3b-ol (6c).
Compound 6c was prepared according
to the general method described above from compound 5c
0.83 mmol) as a crystalline solid after purification by flash column
chromatography (cyclohexane–ethyl acetate, 95:5) (99 mg, 89%
yield). mp 159–160 °C; IR 2932, 2180, 1636 cmꢀ1; Rf: 0.29, cyclo-
hexane/ethyl acetate 95:5; 1H NMR (CDCl3): d 0.71 (s, 3H), 0.82–
0.92 (m, 3H), 0.98 (s, 3H), 1.06 (s, 9H), 1.12–2.38 (m, 16H), 2.68
(t, J = 8.7 Hz, 1H), 3.48–3.59 (m, 1H), 3.84 (s, 3H), 5.13 (d,
J = 4.7 Hz, 1H,), 6.88 (d, J = 8.8 Hz, 2H), 7.34–7.44 (m, 6H), 7.51 (d,
J = 8.8 Hz, 2H), 7.67–7.69 (m, 4H).; 13C NMR (CDCl3): d 13.2, 19.1,
19.4, 20.9, 22.4, 24.4, 27.0, 31.78, 31.85, 31.88, 36.5, 37.2, 38.5,
42.4, 45.1, 50.0, 55.4, 56.9, 64.8, 73.1, 89.2, 92.1, 112.1, 114.3,
120.8, 127.4, 127.5, 129.4, 129.5, 134.7, 134.8, 134.9, 135.7,
135.8, 141.3, 161.5, 189.0; HR-MS (ESI+): [M+H]+, found
(99 mg, 0.15 mmol) as a crystalline solid after purification by flash
column chromatography (petroleum ether 40–60 °C/acetone, 9:1)
20
(41 mg, 64% yield). mp 101–102 °C; [
a]
ꢀ79.59° (c = 0.07916,
D
CHCl3); IR: 3506, 2931, 2184, 1644 cmꢀ1; Rf: 0.13, petroleum ether
40–60 °C/acetone 9:1; 1H NMR (CDCl3): d 0.75 (s, 3H), 1.00 (s, 3H),
1.05–2.04 (m, 16H), 2.20 (s, 3H), 2.25 (s, 3H), 2.28–2.36 (m, 3H),
2.41 (s, 3H), 2.72 (t, J = 8.6 Hz, 1H), 3.48–3.56 (m, 1H), 5.35 (d,
J = 3.9 Hz, 1H), 7.01 (br s, 1H), 7.29 (br s, 1H); 13C NMR (CDCl3): d
13.3, 19.0, 19.4, 19.8, 20.1, 21.0, 22.6, 24.4, 31.5, 31.7, 31.9, 36.5,
37.2, 38.6, 42.2, 45.1, 50.0, 56.8, 64.9, 71.7, 91.1, 92.7, 117.0,
121.3, 131.1, 134.2, 134.4, 139.5, 140.0, 140.7, 189.1; HR-MS
(ESI+): [M+Na]+ found 467.2903. C31H40O2Na requires 467.2921.
671.3913.
C
45H55O3Si requires 670.3915.; [M+Na]+, found
693.3731 C45H54O3SiNa requires 693.3734.
4.1.5.4.
17b-[1-Oxo-3-(2-methyl-4-methoxyphenyl)-2-propy-
Compound 6d was prepared
nyl]-androst-5-en-3b-ol (6d).
4.1.5. General procedure for the synthesis of compounds 6a–i
To a solution of the appropriate steroid 5a–i (1 mmol) in dry
CH2Cl2 (0.06 M) was added dropwise at 0 °C HF-pyridine (10 equiv)
and the mixture was stirred at ambient temperature for 12 h. After
completion the reaction was cooled to 0 °C, was quenched by addi-
tion of water (3 mL), was stirred for 5 min and was extracted with
according to the general method above from 5d (200 mg,
0.29 mmol) as a crystalline solid after purification by flash column
chromatography (petroleum ether 40–60 °C/acetone, 9:1) (48 mg,
20
37%). mp 63–64 °C; [
a
]
D
ꢀ25.07° (c = 0.05583, CHCl3); IR: 3374,
2932, 2179, 1650 cmꢀ1; Rf: 0.11, petroleum ether 40–60 °C/ace-
tone 9:1; 1H NMR (CDCl3): d 0.73 (s, 3H), 0.84–0.90 (m, 1H), 0.99

G. Szalóki et al. / Bioorg. Med. Chem. 22 (2014) 6980–6988
6987
(s, 3H), 1.03–2.41 (m, 18H), 2.46 (s, 3H), 2.72 (t, J = 8.6 Hz, 1H),
3.49–3.55 (m, 1H), 3.81 (s, 3H), 5.35 (d, J = 3.9 Hz, 1H), 6.70–6.76
(m, 2H), 7.47 (d, J = 8.4 Hz, 1H); 13C NMR (CDCl3): d 13.3, 19.3,
20.9, 21.0, 22.6, 24.5, 31.5, 31.7, 31.8, 36.5, 37.2, 38.6, 42.1, 45.1,
50.0, 55.3, 56.8, 64.7, 71.6, 91.3, 92.8, 111.7, 112.0, 115.3, 121.3,
135.5, 140.7, 144.4, 161.4, 189.0; HR-MS (ESI+): [M+H]+ found
447.2878. C30H39O3 requires 447.2894; [M+Na]+ found 469.2694.
1.01 (s, 3H), 1.05–2.33 (m, 19H), 2.72 (t, J = 8.6 Hz, 1H), 3.49–3.55
(m, 1H), 5.36 (d, J = 5.1 Hz, 1H), 7.05–7.11 (m, 2H), 7.54–7.59 (m,
2H); 13C NMR (CDCl3): d 13.6, 19.6, 21.3, 22.6, 24.7, 31.9, 32.0,
32.1, 36.7, 37.5, 38.8, 42.4, 45.4, 50.3, 57.2, 65.1, 71.9, 89.3 (d,
JC–F = 1.42 Hz), 90.2, 116.4 (d, JC–F = 22.3 Hz), 116.6 (d, JC–F
3.51 Hz), 121.5, 135.3 (d, JC–F = 8.85 Hz), 141.1, 164.1 (d, JC–F
=
=
253.6 Hz), 189.1; 19F NMR (CDCl3): d ꢀ106.9 (m); HR-MS (ESI+):
C30H38O3Na requires 469.2713.
[M+Na]+ found 443.2339. C28H33FNaO2 requires 443.2357.
4.1.5.5. 17b-[1-Oxo-3-(3-ethynylphenyl)-2-propynyl]-androst-5-
en-3b-ol (6e). Compound 6i was prepared according to the
4.1.5.9. 17b-[1-Oxo-3-(4-methoxyphenyl)-2-propynyl]-androst-
5-en-3b-ol (6i).
Compound 6i was prepared according to the
general method above from 5i (99 mg, 0.14 mmol) as an oil after
purification by flash column chromatography (petroleum ether–
general method described above from compound 5i (76 mg,
0.11 mmol) as an oil after purification by flash column chromatog-
raphy (cyclohexane–ethyl acetate, 95:5) as oil (11 mg, 23% yield).
IR: 3395, 2928, 2180, 1654 cmꢀ1: Rf: 0.40, petroleum ether 40–
60 °C/acetone 9:1; 1H NMR (CDCl3): d 0.75 (s, 3H), 1.01 (s, 3H),
1.05–2.41 (m, 19H), 2.71 (t, J = 8.8 Hz, 1H,), 3.48–3.59 (m, 1H),
3.84 (s, 3H), 5.36 (d, J = 5.0 Hz, 1H,), 6.90 (d, J = 8.8 Hz, 2H), 7.52
(d, J = 8.8 Hz, 2H,); 13C NMR (CDCl3): d 13.3, 19.4, 21.0, 22.4, 24.4,
31.6, 31.8, 31.9, 36.6, 37.2, 38.5, 42.2, 45.1, 50.1, 55.4, 57.0, 64.8,
71.7, 89.2, 92.2, 112.0, 114.3, 121.4, 134.9, 140.8, 161.6, 188.9;
HR-MS (ESI+): [M+H]+ found 433.2734. C29H37O3 requires
433.2664.
20
acetone, 9:1) (33 mg, 52%). [
a]
ꢀ41.67° (c = 0.13916, CHCl3); IR:
D
3299, 2933, 2194, 1658 cmꢀ1; Rf: 0.10, petroleum ether/acetone
9:1; 1H NMR (CDCl3): d 0.74 (s, 3H), 0.84–0.91 (m, 1H), 1.01 (s,
3H), 1.04–2.40 (m, 18H), 2.72 (t, J = 8.7 Hz, 1H), 3.13 (s, 1H),
3.48–3.58 (m, 1H), 5.35 (d, J = 5.0 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H),
7.51–7.56 (m, 2H), 7.66 (br s, 1H); 13C NMR (CDCl3): d 13.3, 19.3,
21.0, 22.3, 24.4, 31.5, 31.8, 31.9, 36.5, 37.2, 38.4, 42.2, 45.2, 50.0,
56.9, 64.9, 71.7, 78.5, 82.1, 89.3, 89.5, 120.7, 121.3, 122.9, 128.7,
132.9, 134.0, 136.0, 140.8, 188.8; HR-MS (APCI+): [M+H]+ found
427.2622. C30H35O2 requires 427.2632.
4.1.5.6. 17b-[1-Oxo-3-(2-trifluoromethylphenyl)-2-propynyl]-
4.2. Pharmacology
androst-5-en-3b-ol (6f).
Compound 6f was prepared accord-
ing to the general method described above from 0.14 compound 5f
(100 mg, 014 mmol) as a crystalline solid after purification by flash
4.2.1. Cells and culture conditions
Seven human cancer cell lines were used for the evaluation of
the anticancer activity of the new analogues: A549 (alveolar
adenocarcinoma), A431 (epidermoid carcinoma), HeLa (cervical
adenocarcinoma), HepG2 (hepatocellular carcinoma), HT29 (colo-
rectal adenocarcinoma), LNCaP (prostatic carcinoma), and MCF7
(breast adenocarcinoma). Cells were cultured as monolayers in
DMEM medium supplemented with 10% (v/v) foetal bovine serum
column chromatography (cyclohexane–ethyl acetate, 75:25)
20
(35 mg, 53%). mp 61–62 °C; [
a
]
ꢀ38.18° (c = 0.01833, CHCl3);
IR: 3364, 2932, 2205, 1661 cmꢀ1D; Rf: 0.13, petroleum ether 40–
60 °C/acetone 9:1; 1H NMR (CDCl3): d 0.75 (s, 3H), 1.00 (s, 3H),
1.04–2.41 (m, 19H), 2.74 (t, J = 8.8 Hz, 1H), 3.47–3.57 (m, 1H),
5.35 (d, J = 5.1 Hz, 1H), 7.51–7.59 (m, 2H), 7.70–7.73 (m, 2H); 13C
NMR (CDCl3): d 13.5, 19.6, 21.2, 22.8, 24.8, 31.8, 32.0, 32.1, 36.8,
(FBS), 100 U/ml penicillin and 100 lg/ml streptomycin and were
37.4, 38.5, 42.4, 45.8, 50.2, 57.1, 65.0, 71.9, 85.9, 93.3 (q, JC–F
=
grown in 100 mm or 60 mm tissue culture dishes in a humidified
5% CO2 atmosphere at 37 °C. When 80–90% confluence was
attained, the monolayers were subcultured by means of 0.25%
trypsin-1 mM EDTA or used for experiments.
0.9 Hz), 118.8 (q, JC–F = 2.1 Hz), 121.5, 123.4 (q, JC–F = 273.6 Hz),
126.4 (q, JC–F = 5.1 Hz), 130.4, 131.9 (q, JC–F = 0.9 Hz), 132.5 (q,
JC–F = 31.2 Hz), 135.6, 141.0, 189.3; 19F NMR (CDCl3): d ꢀ62.2
(m); HR-MS (APCI+): [M+H]+ found 471.2497. C29H34F3O2 requires
471.2505.
4.2.2. Cell viability assay
The cytotoxic potential of the C21-modified steroidal ana-
logues was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Cells in their log phase
of growth were harvested, counted and seeded (104 cells/well in
4.1.5.7. 17b-[1-Oxo-3-(3-trifluoromethylphenyl)-2-propynyl]-
androst-5-en-3b-ol (6g).
Compound 6g was prepared accord-
ing to the general method above from 5g (60 mg, 0.08 mmol) as a
crystalline solid after purification by flash column chromatography
100
incubation at 37 °C and 5% CO2 to allow cell attachment, cultures
were treated with varying concentrations (0.1–100 M) of test
lL DMEM) in 96-well tissue culture dishes. After 24 h of
(cyclohexane–ethyl acetate, 75:25) (18 mg, 47%). mp 175–178 °C;
20
[
a]
D
ꢀ29.19° (c = 0.03083, CHCl3); IR: 3489, 2922, 2206,
1648 cmꢀ1
;
Rf = 0.13, petroleum ether/acetone 9:1; 1H NMR
l
samples (steroidal derivatives) made with 1:2 serial dilutions.
The different steroidal derivatives were dissolved in a mixture
of DMSO/water (1:1) and then introduced into the medium con-
taining the cancer cell lines. Four replicates were set up for each
experimental condition. Test samples were left in contact with
the cells for 24 or 48 h. Untreated cells served as a control group.
MTT solution (at a final concentration of 0.5 mg/ml) was added in
each well 4 h before the end of incubation period and the assay
was stopped by replacement of the MTT-containing culture med-
(CDCl3): d 0.75 (s, 3H), 1.01 (s, 3H), 1.07–2.41 (m, 19H), 2.74
(t, J = 8.7 Hz, 1H), 3.48–3.58 (m, 1H), 5.35 (d, J = 4.9 Hz, 1H), 7.53
(t, J = 7.8 Hz, 1H), 7.68–7.75 (m, 2H), 7.80 (br s, 1H); 13C NMR
(CDCl3): d 13.3, 19.4, 21.0, 22.4, 24.4, 31.6, 31.7, 31.9, 36.5, 37.2,
38.5, 42.2, 45.3, 50.0, 56.9, 64.9, 71.7, 88.4, 89.7, 121.3, 123.4 (q,
JC–F = 272.7 Hz), 127.0 (q, JC–F = 3.7 Hz), 129.3, 129.4 (q, JC–F
=
3.9 Hz), 131.3 (q, JC–F = 33.1 Hz), 135.8 (q, JC–F = 1.1 Hz), 140.8,
188.7; 19F NMR (CDCl3): d ꢀ63.5 (m); HR-MS (APCI+): [M+H]+
found 471.2496. C29H34F3O2 requires 471.2505.
ium with 100 ll of 0.1 N HCl in anhydrous isopropanol. Cell via-
bility was determined by measuring absorbance at 545 nm using
a microplate reader. The mean OD value of respective blank was
subtracted from the mean OD value of each experimental set. Per-
cent growth in the presence of test material was calculated con-
sidering the growth in the absence of any test material as 100%
and, in turn, percent growth inhibition in the presence of test
material was calculated. Finally the IC50 values (Table 1) were
4.1.5.8. 17b-[1-Oxo-3-(4-fluorophenyl)-2-propynyl]-androst-5-
en-3b-ol (6h).
Compound 6h was prepared according to the
general method above from 5h (65 mg, 0.09 mmol) as a viscous
oil after purification by column chromatography (cyclohexane–
20
ethyl acetate, 75:25) (24 mg, 58%). [
a]
D
ꢀ48.57° (c = 0.03500,
CHCl3); IR (CHCl3,): 3256, 2935, 2213, 1638 cmꢀ1; Rf: 0.16, petro-
leum ether 40–60 °C/acetone 9:1; 1H NMR (CDCl3): d 0.75 (s, 3H),

6988
G. Szalóki et al. / Bioorg. Med. Chem. 22 (2014) 6980–6988
calculated from three to four independent experiments, using
Microsoft Office Excel software.
Supplementary data
Supplementary data (Copies of 1H and 13C NMR spectra of com-
pounds 6a–6i and 5a–5i. Copies of HR-MS spectra of compounds
6a–6i.) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmc.2014.10.012.
4.2.3. Trypan blue exclusion test
Cells were incubated for 24 h with the most active compounds
(6e, 6f, 6g, 6h) at a concentration of 10 lM or 5 lM. Following
treatments, the medium in each plate was collected. Cells attached
to wells were removed by trypsinization and pooled with their cor-
responding medium. Cells were centrifuged at 1200 rpm for 5 min
and the pellet was resuspended in PBS containing 0.2% trypan blue.
A total of 200–300 cells were counted in each out of three separate
experiments and the mean was used for calculations. Viability was
determined as the percentage of viable cells over the total number
of cells.
References and notes
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Cells were washed twice with ice-cold PBS, harvested by scrap-
ing from dishes, lysed in ice-cold Buffer G [20 mM b-glycerophos-
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(v/v) Triton X-100 and a mixture of protease inhibitors [200
lM
leupeptin, 5 mM DTT, 300 M PMSF and 10 M E64]; and incu-
l
l
bated for 30 min at ꢀ80 °C. Lysates were then centrifuged
(10,000 rpm, 10 min, 4 °C) and the supernatant (total protein
extract) was collected. Protein concentration was determined by
the Bradford assay (BioRad Laboratories). After quantification the
samples were supplemented with 0.33 volumes of SDS–PAGE sam-
ple buffer [330 mM Tris–HCl, pH6.8, 10% (w/v) SDS, 13% (v/v) glyc-
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SDS–PAGE on 10% (w/v) polyacrylamide gels and electrotransfered
onto nitrocellulose membranes. The membranes were then
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[20 mM Tris–HCl, pH 7.5, 137 mM NaCl, 0.05% (v/v) Tween-20]
for 1 h at room temperature and probed overnight at 4 °C with pri-
mary antibodies against pAkt (1:1000, cell signalling) or b-actin
(1:2500, Sigma-Aldrich). After washing with TBS-T (4 ꢁ 5 min),
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milk] for 1 h at room temperature. After washing the blots in
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minescence (ECL; Amersham Biosciences) reagent, exposed to
super RX film.
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Acknowledgement
This work was supported in part by the European Union under
grant agreement MEST-CT-2005-0020575 (2006–2010).