Hybrid molecules of estrone: New compounds with potential antibacterial, antifungal, and antiproliferative activities

Article abstract of DOI:10.1016/j.bmc.2007.02.021

New hybrid molecules of estrone were synthesized as compounds indicating promising biological activity (antibacterial, antimycobacterial, antifungal, and antiproliferative). The prepared molecules contained various heterocyclic units (pyridine, benzylsulf

Full text of DOI:10.1016/j.bmc.2007.02.021

Bioorganic & Medicinal Chemistry 15 (2007) 2898–2906
Hybrid molecules of estrone: New compounds with potential
antibacterial, antifungal, and antiproliferative activities
b,
b
a,
a
J. Adamec,a, R. Beckert, D. Weiß, V. Klimesˇova, K. Waisser,
*
*
*
´
c
d
e
´
U. Mo¨llmann, J. Kaustova and V. Buchta
aDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovske´ho 1203, Hradec Kra´love´, 500 05, Czech Republic
bInstitut fu¨r Organische Chemie und Makromolekulare Chemie der Friedrich-Schiller-Universita¨t Jena,
Lessingstr. 8, D-07743 Jena, Germany
cHans-Kno¨ll-Institut fu¨r Naturstoffforschung, Beutenbergstr. 11, D-07745 Jena, Germany
dNational Reference Laboratory for Mycobacterium kansasii, Regional Institute of Public Health,
Partyza´nske´ na´m. 7, Ostrava 702 00, Czech Republic
eDepartment of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Heyrovske´ho 1203,
Hradec Kra´love´ 500 05, Czech Republic
Received 14 September 2006; accepted 9 February 2007
Available online 13 February 2007
Dedicated to Prof. Roland Mayer (Dresden) on the occasion of his 80th birthday.
Abstract—New hybrid molecules of estrone were synthesized as compounds indicating promising biological activity (antibacterial,
antimycobacterial, antifungal, and antiproliferative). The prepared molecules contained various heterocyclic units (pyridine, ben-
zylsulfanyl derivatives of pyridine or derivatives of tetrazole) linked to estrone by n-heptyl bridges. The compounds with charge
on molecule (the hybrid pyridinium or benzylsulfanylpyridinium salts) exhibited significant biological activity (antibacterial, anti-
mycobacterial, antifungal, and antiproliferative). On the other hand, the compounds not in the form of salts (x-(1-phenyl-5-tetraz-
olylthio)heptylethers of estrone) were inactive. The antimycobacterial activities of three different series of tetrazole derivatives (i.e.,
the hybrid molecules with estrone, tetrazole-5-thiols, and 5-benzylsulfanyl-1-phenyltetrazoles) with the same substituents on phenyl
ring were compared. Amongst them, the 5-benzylsulfanyl-1-phenyltetrazoles were the most potent.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Previously, we have described that alkylsulfanyl group
bound to an electron deficient carbon of heterocyclic
system presents a pharmacophore responsible for a sig-
nificant antibacterial activity (e.g., in some benzylsulfa-
nyl derivatives of pyridine,3 benzimidazole,4 other
benzazoles5 or in some derivatives of tetrazole6–8).
The recent reappearance of tuberculosis and other infec-
tious diseases represents a serious problem worldwide.1
In the past few years, we were trying to find new groups
of compounds active against these diseases. Now it has
been demonstrated that the combination of a steroid
unit, which is able to interact with cell membranes,
and another antimicrobially potent functionality may
result in an increase of activity.2
It has been found before that x-pyridiniumalkylethers
of steroidal phenols2 exhibit good biological activity
(antibacterial). The aim of the present study was to pre-
pare new hybrid molecules where the pyridine was
replaced by a different heterocyclic moiety that itself
exhibited some biological effect. The studied hybrid
molecules contained in their structure a steroidal unit
(3-hydroxy-estra-1,3,5(10)-triene, estrone) and various
heterocyclic compounds (namely: pyridine, benzylsulfa-
nyl derivatives of pyridine or derivatives of tetrazole-5-
thiols) linked by an alkyl bridge. Hybrid molecules (3,
Keywords: Mycobacterium; Steriod; Tetrazole; Benzylsulfanyl pyridine;
Tuberculosis.
*
Corresponding authors. Tel.: +420 495 067 302; fax: +420 495 067
166; e-mail addresses: Jan.Adamec@faf.cuni.cz; C6bera@
uni-jena.de; Vera.Klimesova@faf.cuni.cz
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.021

J. Adamec et al. / Bioorg. Med. Chem. 15 (2007) 2898–2906
2899
Scheme 2) were prepared as ammonium bromides. The
activities of the obtained hybrid molecules were further
compared to the activities of their heterocyclic precursors.
O
Br
N+
N
R
2
O
7
CH3CN, reflux
2. Results and discussion
2.1. Chemistry
R
3a-3d
z
Compound 1, a commercially available pharmaceutical
(estrone), reacted with x,x0-dibromoheptane in the
presence of potassium carbonate to give (x-bromohept-
oxy)-estra-1,3,5(10)-triene-17-one 2 (Scheme 1) as a
key intermediate for the following reactions with (a)
pyridine, (b) 4-benzylsulfanyl pyridine, (c) substituted
4-benzylsulfanyl pyridines, and (d) derivatives of 1-phe-
nyltetrazole-5-thiols (Scheme 2). Reaction of compound
2 with pyridine or benzylsulfanyl derivatives of pyridine
7a–7c (Scheme 3) in boiling acetonitrile resulted in
the desired x-pyridinium salts of the heptylethers of
the 3-hydroxy-estra-1,3,5(10)-trienes (3a–3d, Scheme
2). In the case of the derivative 3b, the reaction was
carried out also in autoclave (3 atm, 100 ꢁC) using meth-
anol as a solvent. Nevertheless, no product was achieved
under these conditions.
O
z
N
N
SH
N
N
N
N
N
S
O
THF, TEA, reflux
N
7
4a-4d
Scheme 2. Syntheses of hybrid molecules of estrone 3a–3d and 4a–4d.
Reagents: R: H (3a), benzylsulfanyl (3b), 4-chlorobenzylsulfanyl (3c),
4-methylbenzylsulfanyl (3d). Z: 4-CH3 (4a), H (4b), 4-Cl (4c), 3,4-Cl2
(4d).
R1
N
R3
N
S
R2
N
S
N
N
Reaction of the intermediate 2 with compounds 5a–5d
(Scheme 3) in boiling anhydrous THF and anhydrous
TEA gave the corresponding derivatives of 3-[70-(1-
phenyl-5-tetrazolylthio)heptyloxy]-estra-1,3,5(10)-triene-
17-one 4a–4d (Scheme 2). Attempts to replace TEA by
sodium ethanolate were unsuccessful; many by-products
were isolated under these drastic conditions.
5a-5d, 6a-6d
7a-7c
Scheme 3. Derivatives of 1-phenyltetrazolyl-5-thiols, 5-benzylsulfanyl-
1-phenyltetrazoles, and 4-benzylsulfanylpyridines. R1, 4-CH3; R2, H
(5a); R1, H; R2, H (5b); R1, 4-Cl; R2, H (5c); R1, 3,4-Cl2; R2, H (5d);
R1, 4-CH3; R2, C6H5CH2 (6a); R1, H; R2, C6H5CH2 (6b); R1, 4-Cl;
R2, C6H5CH2 (6c); R1, 3,4-Cl2; R2, C6H5CH2 (6d); R3, H (7a); R3, 4-
Cl (7b); R3, 4-CH3 (7c).
2.2. Biology
zylsulfanyl-1-phenyltetrazoles 6 and precursor 2 were
evaluated for their antifungal activity.
The prepared compounds 3 and 5 were screened in vitro
for antibacterial, antimycobacterial, antifungal, antipro-
liferative, and cytotoxic activities. For the purpose to
find out which part of the molecule contributes to the
activity, the both parts of the hybrid molecules were
subjected to evaluation assay, that is, the steroid unit 2
and heterocyclic moiety 5 and 7. For the sake of com-
parison of the activity of various tetrazole derivatives,
the previously prepared 5-benzylsulfanyl-1-phenyltet-
razole derivatives6–8 were also included. All tested phe-
nyltetrazole derivatives (4–6) have the same
substituents on the phenyl ring.
2.2.1. Antibacterial and antimycobacterial activities.
Antibacterial activity of some of the studied compounds
was evaluated in vitro against the following strains:
Staphylococcus aureus (SG 511), S. aureus (MRSA 994/
93), S. aureus (MRSA 134/94), Micrococcus luteus (ATCC
10240), Enterococcus faecalis (1528), Mycobacterium
smegmatis (SG 987), M. aurum (SB 66), M. vaccae
(10670), M. fortuitum Borstel. The activities expressed
as minimum inhibitory concentrations (MIC, lg/mL)
are presented in Table 1. The derivatives of tetrazole 4–6
exhibited no activity in the preliminary screening (data
not shown).
It has been reported that some antimycobacterial com-
pounds are often antifungal as well.9,10 Therefore, the
hybrid molecules 3 and 4 together with the 5-ben-
The antimycobacterial activities were studied against
tuberculous and non-tuberculous mycobacterial strains:
Mycobacterium tuberculosis CNCTC My 331/88,
M. kansasii CNCTC My 235/80, M. avium CNCTC
My 330/88, and a clinical isolate of M. kansasii 6 509/
96. The results are presented in Table 2, MICs are
expressed in lmol/L.
O
O
H
i
H
H
HO
Br
O
7
The antibacterial assessment revealed that the prepared
compounds possess mostly only a moderate or slight
activity. Mycobacterial strains were more sensitive to
evaluated compounds. The most active compounds
1
2
Scheme 1. Synthesis of the intermediate 2. Reagents and conditions: (i)
Br(CH2)7Br, K2CO3, anhydrous acetone, reflux, 17 h.

2900
J. Adamec et al. / Bioorg. Med. Chem. 15 (2007) 2898–2906
Table 1. In vitro antibacterial activities; MIC (lg/mL)
Strain/compound
I
II
III
IV
V
VI
VII
VIII
IX
3a
3b
3.12
3.12
3.12
1.56
100
50
6.25
3.12
3.12
3.12
100
25
0.8
0.4
12.5
6.25
1.56
3.12
100
100
100
1.56
100
0.8
3.12
1.56
6.25
6.25
100
25
6.25
3.12
3.12
3.12
25
3.12
1.56
1.56
1.56
50
6.25
6.25
6.25
6.25
50
3.12
6.25
3.12
>100
>100
100
3c
3d
0.8
0.8
7a
7b
100
50
100
100
12.5
25
25
25
12.5
12.5
12.5
100
0.4
7c
CPB
50
0.8
100
0.4
50
1.56
>100
>100
3.12
>100
25
6.25
100
0.78
6.25
100
0.1
1.56
100
0.78
DMSO/MeOH
CIP
>100
0.2
>100
12.5
I, S. aureus (SG 511); II, S. aureus (MRSA 994/93); III, S. aureus (MRSA 134/94); IV, M. luteus (ATCC 10240); V, E. faecalis (1528); VI,
M. smegmatis (SG 987); VII, M. aurum (SB 66); VIII, M. vaccae (10670); IX, M. fortuitum Borstel.
CPB, N-cetylpyridinium bromide; CIP, ciprofloxacin; solvent, DMSO.
Table 2. In vitro antimycobacterial activities; MIC (lmol/L)
Strain
M. tuberculosis
M. avium
M. kansasii
My 331/88
My 330/88
My 235/80
14
6509/96a
14
Days
14
21
14
21
7
21
7
21
2
32
16
4
n
16
32
2
32
32
2
62.5
16
8
n
n
62.5
62.5
32
8
n
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
INH
32
8
32
8
32
8
16
8
32
16
16
16
n
16
4
16
8
8
8
16
16
n
16
16
x
16
16
x
16
16
n
16
16
n
8
8
500
x
x
x
n
x
n
n
n
x
x
n
n
x
n
n
n
n
n
n
n
n
n
n
n
x
n
n
n
x
x
n
n
x
500
x
x
500
x
x
500
x
x
x
500
x
500
x
x
x
x
x
x
x
500
250
32
16
16
16
125
32
62.5
0.5
x
x
x
500
500
16
16
16
8
x
x
250
250
16
16
16
16
62.5
32
32
4
500
500
32
32
16
32
125
62.5
62.5
4
x
250
125
62.5
n
x
x
500
32
32
32
16
125
62.5
62.5
y
500
250
125
n
500
250
125
n
62.5
32
62.5
62.5
125
62.5
62.5
y
n
125
n
32
125
62.5
62.5
1
125
125
62.5
125
y
32
125
62.5
125
y
62.5
125
62.5
125
8
62.5
32
32
y
a Clinical isolate; n, not evaluated because of the precipitation (either growth of bacteria or crystallization of the compound); x, >500; y, >250; INH,
isoniazide.
from evaluated set were the hybrid molecules 3. The hy-
brid molecules 3 showed significant activity in contrast
to the compounds 4 that showed practically no activity.
The antibacterial and antimycobacterial activities are
probably connected with the presence of a charge on
nitrogen atom in the molecule. The comparison of the
antimycobacterial activities of hybrid molecules 3 with
heterocyclic moiety 7 and steroidal moiety 2 confirmed
our hypothesis that steroidal part may result in an in-
crease of the activity. Hydrophobic steroid unit interacts
with cell membranes, leading to an enhancement of the
concentration of the effective molecule in the inside of
the cell. But the way that the steroid unit is connected
to the heterocyclic moiety is very important (see the dif-
ferent activity of compounds 3 and 4).
hybrid molecules 4 were practically inactive. The efficacy
of compounds 6 corresponded to our hypothesis
described previously that alkylsulfanyl group bound to
an electron deficient carbon presents a pharmacophore
responsible for antimycobacterial activity.3–5
The antibacterial activity of the hybrid molecules seems
to be further related to the length of the alkyl chain be-
tween the steroidal unit and the heterocyclic nitrogen.
The antibacterial activity of compound 3a lied between
those of similar homologues with the connective chain
length of 6 and 8 carbons that were prepared previous-
ly.2 This finding corresponded well with the theory
about the relationship between the alkyl chain length
and antibacterial activity of the hybrid molecules.
Regarding the tetrazole derivatives 4–6, 5-benzylsulfanyl
derivatives of 1-phenyltetrazoles 6 exhibited the best
activity; on the other hand, tetrazole-5-thiols 5 and
2.2.2. Antifungal activity. Antifungal activity of com-
pounds 2, 3, 4 and 6 was evaluated against following
fungal strains: Candida albicans ATCC 44859, Candida

J. Adamec et al. / Bioorg. Med. Chem. 15 (2007) 2898–2906
Table 3. In vitro antifungal activities; MIC (lmol/L)
2901
Strain
I
II
III
IV
V
VI
VII
48
>125 >125 >125 >125 >125 >125 >125 >125 >125 >125
VIII
Time (h) 24
48
24
48
24
48
24
48
24
48
24
48
24
72 120
2
3a
>125 >125 >125 >125 >125 >125
3.9
3.9
7.8
n
3.9
3.9
7.8
3.9
15.6
15.6
15.6
n
15.6
15.6
15.6
7.8
15.6
7.8
7.8
n
31.3
7.8
7.8
15.6
15.6
n
15.6
15.6
15.6
7.8
3.9
3.9
7.8
n
7.8
7.8
7.8
7.8
3.9
15.6
15.6
n
62.5
31
>250 >250 31.3
62.5
7.8
3b
3c
31
31
7.8
7.8
n
15.6
7.8
31
7.8
62.5
n
62.5
15.6
15.6
7.8
3d
4a–4d
6a
6b
>250 >250 >250 >250 >250 >250
>250 >250 >250 >250 >250 >250
>250 >250 >250 >250 >250 >250
>250 >250 >250 >250 >250 >250
>250 >250 >250 >250 >250 >250
>250 >250 >250 >250 250
>250 >250 >250 >250 62.5
>250 >250 >250 >250 >250 >250 >250 >250 31.3
>250 >250 >250 >250 >250
250
>250 >250 31.3
62.5
62.5
62.5
125
6c
6d
>250 >250 >250 >250 62.5
62.5
>250 >250 >250 125
>250 >250 >250 >250 26.1
0.27 0.27 1.08 2.16 1.08
>250 >250 31.3
>250 >250 >250 >250 250
FLU
AMB
0.8
0.02
1.6
0.07
1.6
0.07
>250 52.2
0.07 0.14
104.5 13.1
0.14 0.04
52.2
0.14
3.3
1.08
6.5
2.16
52.2
1.08
I, C. albicans (ATCC 44859); II, C. tropicalis (156); III, C. krusei (E28); IV, C. glabrata (20/I); V, T. beigelii (1188); VI, A. fumigatus (231); VII,
Absidia corymbifera (272); VIII, T. mentagrophytes (445).
FLU, fluconazole; AMB, amphotericin B; n, not evaluated.
tropicalis 156, Candida krusei E28, Candida glabrata 20/
I, Trichosporon beigelii 1188, Aspergillus fumigatus 231,
Absidia corymbifera 272, and Trichophyton mentagro-
phytes 445. The MICs (lmol/L) are presented in Table 3.
L-929 for their antiproliferative effects (GI50: concentra-
tion which inhibited cell growth by 50%), and against
HeLa for their cytotoxic effects (CC50 and CC10: cyto-
toxic concentration which contains a specific destructive
action by 50% and 10%, respectively; used particularly
in referring to the lysis of cells), see Table 4. The cells
were incubated with 10 concentrations of the tested
compounds. The antiproliferative and cytotoxic assays
have been described previously.11
As shown in Table 3, significant antifungal activity was
observed only in hybrid compounds carrying the charge
on molecule 3. The hybrid compounds of tetrazole 4
were inactive and the 5-benzyl-1-phenyltetrazoles 6
exhibited only slight activity against T. mentagrophytes.
This finding (the relationship of biological activity and
the charge on the molecule) is well consistent with that
found for the antibacterial activity.
3. Conclusion
A series of new hybrid molecules of estrone with various
heterocycles was prepared. The heterocyclic moiety in-
volved: pyridine, 4-benzylsulfanylpyridine derivatives,
and 1-phenyltetrazole-5-thiol derivatives. Their biologi-
cal activity against various bacterial, mycobacterial,
and fungal strains as well as the antiproliferative and
cytotoxic activities were evaluated. For the comparison,
the precursors of the studied hybrid molecules were
screened too.
2.2.3. Antiproliferative and cytotoxic activities. The com-
pounds 2–7 were assayed against cell lines K-562 and
Table 4. Antiproliferative and cytotoxic effects
Antiproliferative activity
Cytotoxicity
L-929a GI50
K-562b GI50
HeLac CC50
(lg/mL)
(lg/mL)
(lg/mL) CC10 (lg/mL)
2
49.9
16.7
4.0
13.1
5.3
>50 (16.3)
17.5 (5.1)
3.1 (1.2)
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
It was observed that the presence of a charge on the
molecule was important for all the studied activities
(the most active compounds were prepared as salts).
The best potency in the prepared group was found in
the x-pyridinium derivatives of estrone 3.
2.3
1.0
2.8
3.5
4.1 (1.9)
1.9 (1.0)
3.0
>50
>50
>50
>50
>50
>50
>50
33.4
12.5
22.5
16.7
19.1
34.3
21.2
24.3
>50
>50
>50
>50
42.8
>50
42.6
31
>50 (28.7)
>50 (20.3)
>50 (50)
>50 (50)
>50 (33)
>50 (40)
48.9 (26.4)
33.3 (18.1)
19.7 (4)
Comparing the antibacterial, antimycobacterial, anti-
proliferative and cytotoxic activities of three different
series of tetrazole derivatives (i.e., the hybrid molecules
with estrone, tetrazole-5-thiols, and 5-benzylsulfanyl-1-
phenyltetrazoles) with the same substituents it has been
found that the 5-benzylsulfanyl-1-phenyltetrazoles were
the most potent.
24.3
36.1
>50
>50
>50
19.8
37.5
41.6 (8.7)
50 (5)
48.6 (9.9)
23.6 (14.0)
16.3 (8.6)
21.4 (13.3)
4. Experimental
4.1. General methods
One to 10 lg/mL, strong activity; >10 lg/mL, weak activity.
a Mouse fibroblast cells.
b Human leukemia.
All chemicals were purchased from Sigma–Aldrich
(Schnelldorf, Germany). The derivatives of tetrazole
c Human cervical carcinoma; solvent, DMSO.

2902
J. Adamec et al. / Bioorg. Med. Chem. 15 (2007) 2898–2906
5a–5d and 6a–6d were prepared previously in our labora-
tory.6 Melting points were measured on a Koffler appa-
ratus and are uncorrected. Elemental analyses were
performed with a CHNS-932 (LECO) instrument. The
samples for analysis and biological tests were dried over
4.2.2.1.
1-[(17-Oxo-estra-1,3,5(10)-triene-3-yloxy)-
heptyl]-pyridinium bromide (3a). Yellowish crystals
(90% yield), reaction time: 24 h; mp 140–142 ꢁC
(MeOH); H NMR (250 MHz, CDCl3): d 0.88 (s, 3H,
1
18-H), 1.40–2.53 (m, 23H, 3· CH, 10· CH2), 2.83–
2.90 (m, 2H, 6-H), 3.88 (t, 2H, J = 6.3 Hz, –CH2–O–),
5.02 (t, 2H, J = 7.4 Hz, –CH2–N–), 6.60 (d, 1H,
J = 2.9 Hz, 4-H), 6.66 (dd, 1H, J = 2.9 Hz, J = 8.5 Hz,
2-H), 7.16 (d, 1H, J = 8.6 Hz, 1-H), 8.11 (dd, 2H,
J = 6.6 Hz, J = 7.5 Hz, 30-HPy, 50-HPy), 8.45–8.53 (m,
1H, 40-HPy), 9.50 (d, 2H, J = 5.6 Hz, 20-HPy, 60-HPy).
13C NMR (250 MHz, CDCl3): d 220.8, 157.0, 145.1,
145.1, 145.1, 137.7, 131.9, 128.4, 128.4, 128.1, 126.3
114.5, 112.1, 67.6, 62.0, 50.4, 48.0, 44.0, 38.4, 35.8,
31.9, 31.6, 29.6, 29.1, 28.7, 26.5, 25.9, 25.8, 21.5, 13.8.
LMR MS (CI +Q1MS LMR UP LR): 527 (M+Å H,
15), 447 (58), 446 (18), 368 (7), 270 (10), 130 (8), 80
(100). IR (DIAMAND) tmax (CO) 1727 cmꢀ1. Anal.
Calcd for C30H40NO2Br (526.56): C, 68.43; H, 7.66; N,
2.66; Br, 15.17. Found: C, 68.71; H, 7.88; N, 2.47; Br,
15.38.
1
P2O5 at 61 ꢁC and 66 Pa for 24 h. H NMR and 13C
NMR spectra were recorded on a Bruker DRX-400 spec-
trometer either in CDCl3 or DMSO (250 or 400 MHz).
MS spectra were measured on a Finnigan MAT SAQ
710 mass spectrometer. Solvents were purified, dried,
and distilled according to conventional methods. All
reactions were carried out under inert (argon) atmo-
sphere. The reactions were monitored by TLC aluminum
plates, silica gel 60 (Merck, Darmstadt, Germany),
0.2 mm, detection by UV (254 nm) or iodine. For the
column chromatography, silica gel 60 (Lichroprep Si
60, 40–63 lm, Merck) was used; the preparative TLC
was carried out using silica gel F254 (15–40 lm, Merck).
4.2. Syntheses
4.2.1. Procedure for the preparation of 3-(x-bromohept-
oxy)-estra-1,3,5(10)-triene-17-one (2). To a solution of
the phenolic steroid (estrone 1; 1 mmol) in anhydrous
acetone (10 mL) containing freshly dried potassium car-
bonate (2 mmol), x,x0-dibromoheptane (4 mmol) was
added. The reaction mixture was refluxed until comple-
tion (17 h), as determined by TLC. After cooling to
room temperature, the potassium carbonate was filtered
off and washed with acetone. Evaporation resulted in
oily crude product, which was purified by column chro-
matography on silica gel with n-heptane/ethyl acetate
(90:10) and by crystallization in methanol.
4.2.2.2. 4-Benzylsulfanyl-1-[(17-Oxo-estra-1,3,5(10)-
triene-3-yloxy)-heptyl]- pyridinium bromide (3b). Gray
crystals (55% yield), reaction time: 24 h; mp 135–
137 ꢁC; 1H NMR (250 MHz, CDCl3): d 0.89 (s, 3H,
18-H), 1.38–2.54 (m, 23H, 3· CH, 10 · CH2), 2.85–
2.88 (m, 2H, 6-H), 3.88 (t, 2H, J = 6.6 Hz, –CH2–N–),
4.41 (s, 2H, –CH2–S–), 4.74 (t, 2H, J = 7.3 Hz, –CH2–
O–), 6.61 (d, 1H, J = 2.6 Hz, 4-H), 6.69 (dd, 1H,
J = 2.9 Hz, J = 8.6 Hz, 2-H), 7.16 (d, 1H, J = 8.5 Hz,
1H), 7.30–7.44 (m, 5H, HBn), 7.75 (d, 2H, J = 7.2 Hz,
30-HPy,50-HPy), 9.04 (d, 2H, J = 6.9 Hz, 20-HPy, 60-
HPy). 13C NMR (250 MHz, CDCl3): d 220.9, 163.0,
157.0, 149.2, 149.0, 142.7, 137.7, 135.5, 132.7, 131.9,
128.9, 128.8, 128.7, 128.6, 126.3, 123.0, 114.5, 112.1,
67.6, 60.4, 50.4, 48.0, 44.0, 38.4, 36.4, 35.9, 35.7, 31.6,
31.5, 29.6, 29.1, 28.7, 26.5, 25.9, 25.8, 21.6, 13.9. ESI
+VE +HMR BSCAN (LIN) UP PROF NRM: 568.3
(100); IR (DIAMAND) tmax(CO) 1736 cmꢀ1. Anal.
Calcd for C37H46NO2SBr (648.75): C, 68.50; H, 7.15;
N, 2.16; S, 4.94; Br, 12.32. Found: C, 68.56; H, 6.80;
N, 2.38; S, 5.31; Br, 12.02.
4.2.1.1. 3-(x-Bromoheptoxy)-estra-1,3,5(10)-triene-17-
one (2). White crystals (75% yield); mp 72–74 ꢁC (MeOH);
1H NMR (250 MHz, CDCl3): d 0.83 (s, 3H, 18-H), 1.18–
2.41 (m, 23H, 3· CH, 10· CH2), 2.81 (m, 2H, 6-H), 3.33 (t,
2H, J = 6.8 Hz, 70-H), 3.85 (t, 2H, J = 6.5 Hz, 10-H), 6.56
(d, 1H, J = 2.4 Hz, 4-H), 6.63 (dd, 1H, J = 2.9 Hz,
J = 8.5 Hz, 2-H), 7.11 (d, 1H, J = 8.6 Hz, 1-H). 13C
NMR (250 MHz, CDCl3): d 220.8, 157.1, 137.7, 131.9,
126.3, 114.5, 112.1, 67.7, 50.4, 48.0, 44.0, 38.4, 35.9,
33.9, 32.7, 31.6, 30.3, 29.6, 29.2, 28.5, 28.1, 26.6, 25.9,
21.6, 13.8. LMR MS (EI +Q1MS LMR UP LR): 446
(M+Å, 62), 361 (4), 348 (1), 322 (2), 283 (2), 270 (54 ), 242
(9), 226 (9), 213 (17), 199 (7), 185 (52), 172 (32), 146
(46), 133 (25), 115 (20), 97 (29), 91 (16), 81 (10), 69 (15),
55 (100), 41 (48), 30 (2). IR (DIAMAND) tmax (CO)
1734 cmꢀ1. Anal. Calcd for C25H35BrO2 (447.46): C,
67.11; H, 7.88; Br, 17.86. Found: C, 67.32; H, 8.10; Br,
17.66.
4.2.2.3. 4-(4-Chlorobenzylsulfanyl)-1-[(17-oxo-estra-
1,3,5(10)-triene-3-yloxy)-heptyl]-pyridinium bromide (3c).
Yellowish crystals (82% yield), reaction time: 24 h;
1
mp 126–129 ꢁC; H NMR (250 MHz, CDCl3): d 0.88
(s, 3H, 18-H), 1.31–2.54 (m, 23H, 3· CH, 10· CH2),
2.82–2.89 (m, 2H, 6-H), 3.87 (t, 2H, J = 6.5 Hz,
–CH2–N–), 4.42 (s, 2H, –CH2–S–), 4.71 (t, 2H,
J = 7.3 Hz, –CH2–O–), 6.60 (d, 1H, J = 2.7 Hz, 4-H),
6.66 (dd, 1H, J = 2.7 Hz, J = 8.5 Hz, 2-H), 7.15 (d,
1H, J = 8.9 Hz, 1-H), 7.28–7.41 (m, 4H, HBn), 7.80
(d, 2H, J = 6.9 Hz, 30-HPy, 50-HPy), 9.02 (d, .2H,
J = 6.9 Hz, 20-HPy, 60-HPy). 13C NMR (250 MHz,
CDCl3): d 220.9, 162.6, 157.0, 149.3, 142.7, 137.7,
134.5, 134.1, 132.0, 131.5, 130.4, 130.0, 129.4, 129.0,
126.3, 123.2, 114.5, 112.1, 67.6, 60.5, 50.4, 48.0, 44.0,
38.3, 35.9, 35.7, 35.0, 31.6, 31.5, 29.6, 29.1, 28.7, 26.5,
26.0, 25.8, 21.6, 13.9. FAB +Q1MS LMR UP PROF:
603 (40), 600 (5), 547 (6), 505 (7), 480 (10), 479 (16),
447 (10), 420 (8), 398 (12), 360 (10), 348 (14), 334 (17),
4.2.2. General procedure for the preparation of com-
pounds (3a–3d). A solution of compound 2 (0.2 mmol)
and pyridine or benzylsulfanyl derivatives of pyridine
(0.6 mmol) in anhydrous acetonitrile (10 mL) was
refluxed for 24–29 h until complete conversion was
achieved (TLC). The mixture was then concentrated in
vacuo. The precipitate that separated out was filtered
off, washed several times with cold n-heptane, and dried
in vacuo. The compound 3a was further purified by
crystallization in methanol.

J. Adamec et al. / Bioorg. Med. Chem. 15 (2007) 2898–2906
2903
332 (30), 307 (100), 289 (75), 276 (29), 262 (33), 249 (38),
238 (42), 236 (93), 202 (27). IR (DIAMAND) tmax(CO)
1736 cmꢀ1. Anal. Calcd for C37H45ClNO2SBr (683.19):
C, 65.05; H, 6.64; Cl, 5.19; N, 2.05; S, 4.69; Br, 11.70.
Found: C, 64.92; H, 6.71; Cl, 5.29; N, 2.32; S, 4.53;
Br, 11.65.
23H, 3· CH, 10· CH2), 2.86–2.91 (m, 2H, 6-H), 3.40 (t,
2H, J = 7.3 Hz, –CH2–S–), 3.92 (t, 2H, J = 6.4 Hz,
–CH2–O–), 6.63 (d, 1H, J = 2.7 Hz, 4-H), 6.70 (dd, 1H,
J = 2.9 Hz, J = 8.6 Hz, 2-H), 7.18 (d, 1H, J = 8.1 Hz,
1-H), 7.51–7.60 (m, 5H, HPh). 13C NMR (400 MHz,
CDCl3): d 220.8, 157.1, 154.5, 137.7, 131.9, 130.1, 129.8,
129.8, 126.3, 123.9, 123.9, 114.6, 112.1, 67.8, 50.5, 48.0,
44.0, 38.4, 35.9, 33.3, 31.9, 31.6, 29.7, 29.2, 29.0,
28.8, 28.6, 26.6, 25.9, 25.9, 21.6, 13.9. IR (DIAMAND)
4.2.2.4. 4-(4-Methylbenzylsulfanyl)-1-[(17-oxo-estra-
1,3,5(10)-triene-3-yloxy)-heptyl]-pyridinium bromide (3d).
White crystals (42% yield), reaction time: 26 h; mp 108–
t
max(CO) 1731 cmꢀ1. Anal. Calcd for C32H40N4O2S
1
111 ꢁC; H NMR (400 MHz, CDCl3): d 0.88 (s, 3H, 18-
(544.77): C, 70.55; H, 7.40; N, 10.28; S, 5.89. Found: C,
70.79; H, 7.44; N, 10.04; S, 5.69.
H), 1.34–2.53 (m, 26H, 3· CH, 10· CH2, 1· CH3–Bn),
2.84–2.90 (m, 2H, 6-H), 3.88 (t, 2H, J = 6.4 Hz, –CH2–
N–), 4.36 (s, 2H, –CH2–S–), 4.73 (t, 2H, J = 7.3 Hz,
–CH2–O–), 6.61 (d, 1H, J = 2.5 Hz, 4-H), 6.66 (dd, 1H,
J = 2.8 Hz, J = 8.5 Hz, 2-H), 7.15 (d, 1H, J = 7.7 Hz,
1-H), 7.24–7.30 (m, 4H, HBn), 7.73 (d, 2H, J = 7.0 Hz,
30-HPy, 50-HPy), 9.03 (d, 2H, J = 7.0 Hz, 20-HPy, 60-HPy).
13C NMR (400 MHz, CDCl3): d 220.9, 163.2, 157.0,
142.7, 138.5, 137.7, 131.9, 129.9, 129.9, 129.6, 129.5,
129.5, 128.8, 128.6, 126.3, 123.0, 114.6, 112.2, 67.7, 60.4,
50.4, 48.0, 44.0, 38.4, 36.3, 35.9, 35.5, 31.6, 31.5, 29.6,
29.1, 28.7, 26.6, 26.0, 25.8, 21.6, 21.1, 13.9. ESI +VE
+HMR BSCAN (LIN) UP PROF NRM: 582.3. IR
4.2.3.3. 3-(70-[1-(4-Chlorophenyl)-5-tetrazolylthio]hep-
tyloxy)-estra-1,3,5(10)-triene-17-one (4c). Light yellow
crystals (91% yield), reaction time: 15 h; mp 69–
1
71.5 ꢁC. H NMR (400 MHz, CDCl3): d 0.88 (s, 3H,
18-H), 1.28–2.53 (m, 23H, 3· CH, 10· CH2), 2.86–
2.89 (m, 2H, 6-H), 3.40 (t, 2H, J = 7.3 Hz, –CH2–S–),
3.92 (t, 2H, J = 6.4 Hz, –CH2–O–), 6.63 (d, 1H, J = 2.7
Hz, 4-H), 6.70 (dd, 1H, J = 2.9 Hz, J = 8.6 Hz, 2-H),
7.18 (d, 1H, J = 8.1 Hz, 1-H), 7.50–7.59 (m, 4H, HPh).
13C NMR (400 MHz, CDCl3): d 220.8, 157.1, 154.5,
137.7, 136.2, 131.9, 130.0, 130.0, 126.3, 125.1, 125.1,
114.6, 112.1, 67.7, 50.5, 48.0, 44.0, 38.4, 35.9, 33.5,
31.9, 31.6, 29.7, 29.2, 29.0, 28.7, 28.5, 26.6, 26.0, 25.9,
22.7, 13.9. IR (DIAMAND) tmax(CO) 1738 cmꢀ1. Anal.
(KBr)
t
max(CO) 1737 cmꢀ1
.
Anal. Calcd for
C38H48NO2SBr (662.78): C, 68.87; H, 7.30; N, 2.11; S,
4.84; Br, 12.06. Found: C, 69.01; H, 7.12; N, 2.45; S,
5.19; Br, 11.98.
Calcd for C32H39ClN4O S (579.21): C, 66.36; H, 6.79;
2
N, 9.67; Cl, 6.12; S, 5.54. Found: C, 66.76; H, 6.76; N,
9.37; Cl, 5.96; S, 5.17.
4.2.3. General procedure for the preparation of com-
pounds (4a–4d). A solution of 3-(x-bromoheptoxy)-es-
tra-1,3,5(10)-triene-17-one
2
(0.5 mmol) and the
4.2.3.4. 3-(70-[1-(3,4-Dichlorophenyl)-5-tetrazolylthio]hep-
tyloxy)-estra-1,3,5(10)-triene-17-one (4d). White crystals
(97% yield), reaction time: 16 h; mp 108–110 ꢁC. 1H
NMR (400 MHz, CDCl3): d 0.90 (s, 3H, 18-H),
1.24–2.54 (m, 23H, 3· CH, 10· CH2), 2.86–2.91 (m, 2H,
6-H), 3.42 (t, 2H, J = 7.3 Hz, –CH2–S–), 3.92 (t, 2H,
J = 6.4 Hz, –CH2–O–), 6.63 (d, 1H, J = 2.4 Hz, 4-H),
6.69 (dd, 1H, J = 2.6 Hz, J = 8.6 Hz, 2-H), 7.18 (d, 1H,
J = 8.6 Hz, 1-H), 7.48 (dd, 1H, J = 2.5 Hz, J = 8.6 Hz,
5-HPh), 7.64 (d, 1H, J = 8.6 Hz, 6-HPh), 7.76 (d, 1H,
J = 2.5 Hz, 2-HPh). 13C NMR (400 MHz, CDCl3): d
220.8, 157.1, 154.6, 137.7, 134.6, 134.1, 132.8, 131.9,
131.5, 126.3, 125.6, 122.8, 114.6, 112.1, 67.7, 50.5, 48.0,
44.0, 38.4, 35.9, 33.6, 31.9, 31.6, 29.7, 29.2, 29.0, 28.7,
28.5, 26.6, 25.9, 25.9, 13.9. IR (KBr) tmax(CO) 1739
cmꢀ1. Anal. Calcd for C32H38Cl2N4O2S (613.66): C,
62.63; H, 6.24; N, 9.13; Cl, 11.55; S, 5.23. Found: C,
62.66; H, 6.23; N, 9.23; Cl, 11.69; S, 4.91.
corresponding derivative of 1-phenyl-5-tetrazolylthiol
5a–5d (0.75 mmol) in anhydrous THF (6 mL) and anhy-
drous TEA (0.75 mmol) was refluxed for 15–21 h until a
complete conversion was achieved (TLC). Evaporation
resulted in oily crude products, which were purified
by column chromatography on silica gel with
n-heptane/acetone (90:10).
4.2.3.1. 3-(70-[1-(4-Methylphenyl)-5-tetrazolylthio]hep-
tyloxy)-estra-1,3,5(10)-triene-17-one (4a). Yellow crys-
tals (95% yield), reaction time: 21 h; mp 61–63 ꢁC. H
NMR (250 MHz, CDCl3): d 0.90 (s, 3H, 18-H), 1.24-
2.56 (m, 26H, 3· CH, 10· CH2, 1· CH3–Ph), 2.85–
2.93 (m, 2H, 6-H), 3.38 (t, 2H, J = 7.4 Hz, –CH2–S–),
3.91 (t, 2H, J = 6.6 Hz, –CH2–O–), 6.63 (d, 1H,
J = 2.7 Hz, 4-H), 6.70 (dd, 1H, J = 2.8 Hz, J = 8.5 Hz,
2-H), 7.18 (d, 1H, J = 8.4 Hz, 1-H), 7.35 (d, 2H,
1
J = 8.3 Hz, 3-HPh, 5-HPh), 7.41–7.47 (m, 2H, 2-HPh
,
6-HPh). 13C NMR (400 MHz, CDCl3): d 220.9, 157.0,
154.4, 140.4, 137.7, 131.9, 131.2, 130.3, 130.3, 126.3,
123.7, 123.7, 114.5, 112.1, 67.7, 50.4, 48.0, 44.0, 38.4,
35.8, 33.2, 31.6, 29.6, 29.2, 29.0, 28.7, 28.5, 26.5, 25.9,
25.9, 21.6, 21.3, 13.8. IR (DIAMAND) tmax(CO)
1736 cmꢀ1. Anal. Calcd for C33H42N4O2S (558.79): C,
70.93; H, 7.58; N, 10.03; S, 5.74. Found: C, 71.05; H,
7.43; N, 9.90; S, 5.72.
4.2.4. General procedure for the preparation of ben-
zylsulfanyl derivatives of pyridine (7a–7c). Pyridine-4-thi-
ol (5 mmol) in dry N,N-dimethylformamide (8 mL) was
added to a solution of sodium (5 mmol) in dry methanol
(2.5 mL). After 10 min of stirring at room temperature,
the corresponding benzyl halide (5 mmol) was added in
2–3 portions and the resultant suspension was stirred for
3–8 h. The mixture was then poured into an ice bath and
left to stand overnight. The solid was filtered off, washed
with cold water (2· 30 mL), and air-dried. The crude
products were purified by preparative TLC using n-hex-
ane/acetone (2:1–6:1) followed by crystallization in
aqueous ethanol or methanol.
4.2.3.2. 3-(70-[1-Phenyl-5-tetrazolylthio]heptyloxy)-estra-
1,3,5(10)-triene-17-one (4b). White crystals (90% yield),
reaction time: 19 h; mp 70.5–72.5 ꢁC. 1H NMR
(400 MHz, CDCl3): d 0.90 (s, 3H, 18-H), 1.25–2.54 (m,

2904
J. Adamec et al. / Bioorg. Med. Chem. 15 (2007) 2898–2906
4.2.4.1. 4-(Benzylsulfanyl)pyridine (7a). Light yellow
crystals (56% yield); mp 54–57 ꢁC (59–61 ꢁC was
MIC was determined after incubation at 37 ꢁC for
7, 14, and 21 days. MIC (lmol/mL) was the lowest
concentration of an antimycobacterially effective
substance (on the above-stated concentration
scale), at which inhibition of the growth of myco-
bacteria occurred.
reported12).
4.2.4.2. 4-(4-Chlorobenzylsulfanyl)pyridine (7b). Yel-
low crystals (25% yield); mp 70–71 ꢁC (75–76.5 ꢁC was
reported13).
4.3.2. Antifungal activity. The antifungal activity was
determined in the Department of Biological and Medi-
cal Sciences, Faculty of Pharmacy, Charles University,
4.2.4.3. 4-(4-Methylbenzylsulfanyl)pyridine (7c). Light
yellow crystals (40% yield); mp 75–77 ꢁC (82–83 ꢁC was
reported13).
´ ´
Hradec Kralove, Czech Republic.
4.3. Biological assays
4.3.2.1. Test compounds. Before testing, the com-
pounds were dissolved in 100% DMSO so that the final
concentration of DMSO in the test medium did not ex-
ceed 1% of the total solution composition. Twofold seri-
al dilutions ranging from 250 to 0.49 lmol/L were tested
for all compounds providing that a given compound was
soluble in DMSO and no precipitation in the RPMI
medium occurred. All solutions were prepared immedi-
ately before testing.
The prepared steroidal hybrid molecules together with
the heterocyclic precursors were screened in vitro for
antimicrobial, antimycobacterial, and antifungal activi-
ty. Some of the prepared derivatives were tested also
for the antiproliferative and cytotoxic activities.
4.3.1. In vitro antibacterial activity including antimyco-
bacterial. The antibacterial activity was evaluated in two
workplaces:
4.3.2.2. Fungal organisms. A set of eight fungal strains
of potentially pathogenic fungi for humans (C. albicans
ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata
20/I, T. beigelii 1188, A. fumigatus 231, A. corymbifera
272, T. mentagrophytes 445) was used for the antifungal
screening tests by means of modified micro dilution broth
method M27-A (NCCLS 1997).14 They included clinical
isolates from biological materials of patients with suspect-
ed or proven mycosis. The fungal isolates were stored in
Skim Milk Medium (Becton–Dickinson) for 6–10 months
at ꢀ40 ꢁC before use. Prior to testing, each isolate was
subcultured on Sabouraud dextrose agar (SDA, Difco)
to ensure optimal growth characteristics and purity.
(a) The activity against strains included in Table 1 was
evaluated in the Hans-Kno¨ll-Institut, Jena, Germa-
ny. The activity of the studied compounds was
determined as the minimal inhibition concentration
(MIC) according to NCCLS guidelines14 using the
microbroth dilution method. The cells were grown
overnight at 37 ꢁC in Mueller–Hinton broth
(MHB, Difco). Fifty microliters of a compound
solution of 400 lL/mL was serially diluted by factor
two with MHB. The wells were then inoculated
with 50 lL of bacteria to give a final concentration
of 5· 105 CFU/mL. After the microtiter plates were
incubated at 37 ꢁC for 24 h, the MIC (lg/mL) val-
ues were read with a Nepheloscan Ascent 1.4 auto-
matic plate reader (Lab-systems, Vantaa, Finland)
as the lowest dilution of antibiotic allowing no vis-
ible growth.
(b) The antimycobacterial activity against strains
included in Table 2 was evaluated in the National
Reference Laboratory for Mycobacterium kansasii,
Regional Institute of Public Health, Ostrava, Czech
Republic. The following strains, obtained from the
Czech National Collection of Type Cultures
(CNCTC), National Institute of Public Health,
Prague, were used: Mycobacterium tuberculosis
CNCTC My 331/88, M. kansasii CNCTC My
235/80, M. avium CNCTC My 330/88, and a clini-
cal isolate of M. kansasii 6 509/ 96. Antimycobacte-
rial activity of the compounds against these strains
The isolates of yeasts had been grown for 1–4 days, the
isolates of molds for 4–14 days, on SDA at room temper-
ature. Yeast and conidia suspensions were prepared in
sterile water supplemented with 0.01% of Tween 80. Each
suspension of a yeast and a mold was diluted in the ratio of
1:50 in the RPMI 1640 medium to obtain the final inocu-
lum, which ranged from 0.5 · 103–2.5 · 103 CFU/mL and
0.5 · 104–5 · 104 CFU/mL, respectively. The inoculum
size was determined microscopically using the Burker’s
¨
chamber and verified by plating 100 lL of serial dilutions
of each inoculum onto an SDA plate and incubation until
growth became visible.
4.3.2.3. Antifungal assay. A stock solution of each
antifungal compound was prepared in the RPMI 1640
medium with L-glutamine, but without sodium bicar-
bonate (Sevapharma, Prague). The RPMI medium was
buffered to pH 7.0 0.1 with 0.165 M morpholinepro-
panesulfonic acid (MOPS) buffer (Sigma). Each well of
microtiter plate with 200 lL of the medium was inocu-
lated with 10 lL of the inoculum suspension, and the
microtiter plates were incubated at 35 ꢁC in a humid
atmosphere. The MIC was determined visually as the
lowest concentrations that showed 80% and higher
growth inhibition as compared with the growth of the
drug-free control wells. The MIC values were read after
24 and 48 h of incubation without agitation for yeasts,
ˇ
was determined in the Sula semisynthetic medium
ˇ
(Sevapharma, Prague). The Sula liquid medium
(with bovine serum) is routinely used in the Czech
Republic. Each strain was simultaneously inoculat-
ed into a petri dish containing the Lo¨wenstein-
Jensen medium for the control of sterility of the
inoculum and its growth. The compounds were
added to the medium as dimethylsulfoxide (DMSO)
solutions. The final concentrations were 1000, 500,
250, 125, 62.5, 32, 16, 8, 4, 2, and 1 lmol/L. The

J. Adamec et al. / Bioorg. Med. Chem. 15 (2007) 2898–2906
2905
A. fumigatus, A. corymbifera, and 72 and 120 h of incu-
bation for T. mentagrophytes. Drug-free controls and
quality control strains (C. krusei ATCC 6254, C. par-
apsilosis ATCC 22019) were included.
4.3.3.4. Methods of evaluation. Suspension cultures of
K-562 in micro plates were analyzed by an electronic cell
¨
analyzer system CASY 1 (SCHARFE, Reutlingen,
Germany) using an aperture of 150 lm. The software
¨
for data evaluation CASYSTAT (SCHARFE) offers fast
4.3.3. Antiproliferative and cytotoxic effect. Antiprolifer-
ative and cytotoxic activities were evaluated in the
Hans-Kno¨ll-Institut, Jena, Germany.
graphical evaluation of the measurement parameters, for
example, as diagrams of cell diameter distributions, over-
lays of different curves, and cell volume distributions. The
0.2 mL content of each well in the microplate was diluted
1:50 with CASYTON (NaCl, 7.93 g/L; Na2 EDTA,
0.38 g/L; KCl ,0.4 g/L; NaH2PO4 monohydrate,
0.22 g/L; NaH2PO4 dihydrate, 2.45 g/L; NaF, 0.3 g/L;
4.3.3.1. Antiproliferative and cytotoxicity assays.11
The target compounds were assayed against cell lines
K-562 and L-929 for their antiproliferative effects and
against HeLa for their cytotoxic effects. The cells were
incubated with 10 concentrations of the test compounds.
¨
SCHARFE). Every count/milliliter was automatically
calculated from the arithmetic mean of three successive
counts of 0.4 mL each. From the dose response curves
the Gl50 values (concentration which inhibited cell growth
by 50%) were calculated with CASYSTAT. The Gl50 val-
ue was defined as being where the concentration–response
curve intersected the 50% line, determined by means of
the cell counts/milliliter, compared to control.
4.3.3.2. Cells and culture conditions. Cells of estab-
lished suspended cell lines K-562 (DSM ACC 10) and
adherent L-929 (DSM ACC 2) were cultured in RPMI
medium (GIBCO BRL 42402-016), supplemented with
100 U/mL of penicillin G–sodium salt/100 lg/mL of
streptomycin–sulfate (Gibco–BRL 15140-114), 10% of
heat-inactivated fetal bovine serum (Gibco–BRL
10500-064), and L-glutamine (Gibco–BRL 25030-024)
at 37 ꢁC in high density polyethylene flasks (NUNC
156340). HeLa (DSM ACC 57) cells were grown in
RPMI 1640 culture medium (Gibco–BRL 21875-034)
supplemented with 100 U/mL of penicillin G-sodium
salt/100 lg/mL of streptomycin–sulfate (Gibco–BRL
15140-114), 10% of heat-inactivated fetal bovine serum
(Gibco–BRL 10500-064), and 10 mL/L of non-essential
amino acid (Gibco–BRL 11140-035) at 37 ꢁC in high
density polyethylene flasks (NUNC 156340).
The monolayers of the adherent L-929 and HeLa cells
were fixed by glutaraldehyde and stained with a 0.05%
solution of methylene blue for 15 min. After gently
washing, the stain was eluted by 0.2 mL of 0.33 N HCl
in the wells. The optical densities were measured at
630 nm in a DYNATECH MR 7000 micro plate reader.
Comparisons of the different values were performed
with Microsoft EXCEL.
Acknowledgments
The adherent cells of L-929 and HeLa were harvested at
the logarithmic growth phase after soft trypsinization,
using 0.25% trypsin in HBSS containing 0.038% of
EDTA (Gibco–BRL 25200-056).
This work was financially supported by Project No.
MSM 0021620822 of the Ministry of Education of the
Czech Republic, by the Deutsche Forschungsgemeins-
chaft (Sonderforschungsbereich 436), and by the
European Union Erasmus/Socrates II Programme. The
For each antiproliferative assay with L-929 and K-562
approximately 10,000 cells were seeded with 0.1 mL of
RPMI 1640 culture medium (Gibco–BRL 21875-034),
containing 10% of heat-inactivated fetal bovine serum
(Gibco–BRL 10500-064), 100 U/mL of penicillin G–sodi-
um salt/100 lg/mL of streptomycin–sulfate (Gibco–BRL
15140-114), but without HEPES, into 96-well micro
plates. The plates were previously prepared with 10 dilu-
tions of test substances in 0.1 mL RPMI 1640 medium.
ˇ
authors thank Jana Klimesova for the syntheses of
4-(benzylsulfanyl)pyridine derivatives.
´
References and notes
1. Raviglione, M. C. Tuberculosis 2003, 83, 4.
2. Lange, C.; Holzhey, N.; Scho¨necker, B.; Beckert, R.;
Mo¨llmann, U.; Dahse, H.-M. Bioorg. Med. Chem. 2004,
12, 3357.
For cytotoxic assay with HeLa, approximately 10,000
cells were seeded with 0.1 mL RPMI 1640 culture medi-
um per well of the 96-well micro plates. HeLa cells were
preincubated for 48 h without the test substances. The
solutions of the compounds of the corresponding con-
centrations were applied carefully on the monolayers
of HeLa cells after the preincubation time.
ˇ
´
3. Klimesova, V.; Svoboda, M.; Waisser, K.; Pour, M.;
Kaustova, J. Collect. Czech. Chem. Commun. 1999, 64,
´
417.
4. Klimesova, V.; Kocı, J.; Pour, M.; Stachel, J.; Waisser, K.;
ˇ
Kaustova, J. Eur. J. Med. Chem. 2002, 37, 409.
´
ˇ´
´
ˇ´
ˇ
´
´
5. Kocı, J.; Klimesova, V.; Waisser, K.; Kaustova, J.; Dahse,
H.-M.; Mo¨llmann, U. Bioorg. Med. Chem. Lett. 2002, 12,
3275.
ˇ
´
6. Waisser, K.; Adamec, J.; Kunes, J.; Kaustova, J. Chem.
The adherent cells of L-929 and HeLa were seeded into
microplates NUNC 167008 and the suspended cells of
K-562 were seeded into microplates NUNC 163320.
Pap. -Chem. Zvesti 2004, 58, 214.
ˇ
´
7. Adamec, J.; Waisser, K.; Kunes, J.; Kaustova, J. Arch.
Pharm. Chem. Life Sci. 2005, 338, 385.
8. Waisser, K.; Adamec, J.; Dolezˇal, R.; Kaustova, J. Folia
´
4.3.3.3. Incubation conditions. Cells of L-929, K-562,
and HeLa were incubated for 72 h at 37 ꢁC in a humid-
ified atmosphere and 5% CO2.
Microbiol. 2005, 50, 195.
9. Adamec, J.; Waisser, K.; Silva, L.; Buchta, V. Folia
Pharm. Univ. Carol. 2005, 33, 13.

2906
J. Adamec et al. / Bioorg. Med. Chem. 15 (2007) 2898–2906
˚
10. Waisser, K.; Hladuvkova, J.; Hrabalek, A.; Klimesova,
V.; Kubicova, L.; Kunes, J.; Machacek, M.; Palat, K., Jr.;
´
´
ˇ
´
13. Cranham, J. E.; Cummings, W. A. W.; Johnston, A. M.;
Stevenson, H. A. J. Sci. Food Agric. 1958, 9, 147.
14. National Committee for Clinical Laboratory Standards.
Methods for Dilution Antimicrobial Susceptibility
Tests for Bacteria that Grow Aerobically Approved
Standard. NCCLS Document M7-A4, 4th ed.; National
Committee for Clinical Laboratory Standards: Villanova,
PA, 1997.
´
ˇ
´ˇ
´
ˇ
ˇ
´
´
´
Vinsova, J.; Buchta, V.; Jılek, P.; Odlerova, Z.
Folia Pharm. Univ. Carol. 1998, 21–22, 6.
11. Dahse, H.-M.; Schlegel, B.; Gra¨fe, U. Pharmazie 2001, 56,
489.
12. Hassner, A.; Krepski, L. R.; Alexanian, V. Tetrahedron
1978, 34, 2069.