Synthesis and antiproliferative activity in vitro of new tricyclic 2-thioxo-1H,3H-imidazo[4,5-b]pyridine derivatives

Article abstract of DOI:10.1080/10426500600892594

A series of 2-arylidenethiazolo[2′,3′:2,3]imidazo[4,5-b] pyridine-2-yl-3-ones were prepared by a reaction of 6-bromo- (4) or 3H-imidazo[4,5-b]pyridin-2-ylthioglycolic acid (3) with appropriate aromatic aldehydes. All new compounds were examined for their

Full text of DOI:10.1080/10426500600892594

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Synthesis and Antiproliferative
Activity in vitro of New
Tricyclic 2-Thioxo-1 H ,3
H -imidazo[4,5-b]pyridine
Derivatives
Hanna Liszkiewicz a , Maria W. Kowalska b & Joanna
Wietrzyk c
a Department of Drugs Technology, Wroclaw Medical
University, Poland
b Faculty of Chemistry, University of Wroclaw,
Poland
c Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, Wroclaw, Poland
Version of record first published: 01 Feb 2007.
To cite this article: Hanna Liszkiewicz, Maria W. Kowalska & Joanna Wietrzyk (2007):
Synthesis and Antiproliferative Activity in vitro of New Tricyclic 2-Thioxo-1 H ,3 H -
imidazo[4,5-b]pyridine Derivatives, Phosphorus, Sulfur, and Silicon and the Related
Elements, 182:1, 199-208
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Phosphorus, Sulfur, and Silicon, 182:199–208, 2007
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500600892594
Synthesis and Antiproliferative Activity in vitro of New
Tricyclic 2-Thioxo-1H,3H-imidazo[4,5-b]pyridine
Derivatives
Hanna Liszkiewicz
Department of Drugs Technology, Wroclaw Medical University, Poland
Maria W. Kowalska
Faculty of Chemistry, University of Wroclaw, Poland
Joanna Wietrzyk
Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, Wroclaw, Poland
A series of 2-arylidenethiazolo[2ꢀ,3ꢀ:2,3]imidazo[4,5-b]pyridine-2-yl-3-ones were
prepared by a reaction of 6-bromo- (4) or 3H-imidazo[4,5-b]pyridin-2-ylthioglycolic
acid (3) with appropriate aromatic aldehydes. All new compounds were examined
for their antiproliferative activity in vitro against cells of human cancer cell lines,
using SRB technique. Preliminary screening data indicated that three of the tested
compounds revealed cytotoxic activity.
Keywords 3H-Imidazo[4,5-b]pyridin-2-yl-thioglycolic acid; 6-bromo-3H-imidazo[4,5-
b]-pyridin-2-yl-thioglycolic acid; 2-(arylidene)-thiazolo[2ꢀ,3ꢀ:2,3]imidazo[4,5-b]-pyridin-3-
ones; antiproliferative activity in vitro
Both thiazole and imidazo[4,5-b]pyridine derivatives have been widely
claimed to have diverse biological properties, some of which are
interesting for the potential treatment of human diseases. Their
bioactivity depends on substituents of the heterocyclic ring system
and includes antiviral,1 antibacterial,2,3 antiulcer,4,5 antituberculytic,6
hypoglycemic,7 anticancer2,8,9 and anxiolitic10 action. Compounds with
a thiazole, imidazothiazole, or imidazopyridine moiety are capable of
generating various reactive intermediates that result in DNA cleavage.
DNA degradation plays an important role in chemotherapy and
carcinogenesis.9,11,12
Received April 29, 2006; accepted June 9, 2006.
Address correspondence to Hanna Liszkiewicz, Department of Drugs Technology,
Wroclaw Medical University, Nankiera 1 Sq., Wroclaw, 50-140 Poland.
199

200
H. Liszkiewicz et al.
Continuing our studies of the chemical and biological properties of
imidazo[4,5-b]pyridine derivatives13 and inspired by the effect of fusing
the thiazole ring to an aromatic skeleton on the antitumor activity,9 we
synthesized a series of novel tricyclic thiazoloimidazopyridines.
Because a change of the biological activity may be a conse-
quence of even small structural manipulations, thus to broaden the
scope of potential cytotoxic activity, a side chain containing sub-
stituted arylidene structure was introduced to the triheterocyclic
framework. On the basis of some recent reports, because fluorinated
analogues improved the biological profile,14 including cytotoxicity,15 it
seemed interesting to incorporate a fluorine atom into the arylidene
structure.
RESULTS AND DISCUSSION
Chemistry
The formation of the fused-ring framework usually takes two or
more steps to complete. Substituted imidazopyridines are versatile
precursors of fused nitrogen heterocycles.6,10 The treatment of 2-
thioxo-1H,3H-imidazo[4,5-b]pyridine (1) or 6-bromo-2-thioxo-1H,3H-
imidazo[4,5-b]pyridine (2) with chloroacetic acid in ethanol and the
presence of NaOH gave 3H-imidazo[4,5-b]pyridin-2-yl-thioglycolic acid
(3) and 6-bromo-3H-imidazo[4,5-b]pyridin-2-yl-thioglycolic acid (4), re-
spectively (Scheme 1). These acids were used as starting compounds for
constructing the tricyclic skeleton of the thiazoloimidazopyridine ring
system. The preparation of the tricyclic framework followed a pathway
previously described for benzoimidazothiazolones.16 It is a straightfor-
ward one-pot approach to furnish the nitrogen heterocyclic framework
with a suitable substituent.
When acid 3 or 4 was refluxed in acetic acid anhydride with an
appropriate aromatic aldehyde and anhydrous sodium acetate, the
reaction resulted in a clean formation of thiazolo[2ꢀ,3ꢀ:2,3]imidazo[4,5-
b]-pyridin-3-ones 5–15. The two-step synthetic route for the substi-
tuted triheterocyclic ring system is outlined in Scheme 1. Substituents
were chosen that are mostly electron-withdrawing groups because they
affect the electron-density distribution pattern what can evoke bio-
logical properties of the molecule. The bioactivity of a molecule is
based mostly on its capability to bind to an active site; thus, the ex-
pected enhancement of biological activity of the obtained compounds
could be attributed to the influence of the electron-withdrawing effect,
which substituents cause on interaction with a biological receptor or
enzyme.17

New Tricyclic 2-Thioxo-1H,3H-imidazo[4,5-b]pyridine Derivatives
201
H
X
X
N
N
ClCH2COOH
S NaOH, EtOH
N
SCH2COOH
N
N
N
H
H
1, 2
3, 4
R-CHO
1, 3; X = H
2, 4; X = Br
CH3COONa
(CH3CO)2O
X
N
R4
N
N
S
H
R3
O
CH -
R2
R1
5–15
No R1 R2
R3
R4
X
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
F
H
H
H
H
H
H
H
H
H
H
CF3
10
11
12
13
14
15
H
H
H
H
H
H
NO2
H
H
H
NO2
OCH3
H
H
H
H
H
H
H
H
Br
H
OCH3 OCH3
OCH3 OCH3 OCH3
H
H
H
SCHEME 1
All new synthesized compounds gave satisfactory elemental analy-
1
ses, and their molecular structure was confirmed by IR and H NMR
spectroscopy. Examination of the IR bands for compounds 5–15 showed
that the characteristic C O vibration was in the range ν = 1740–1710
cm−1. 1H NMR spectra of compounds 5–15 showed a characteristic sin-
glet in the region δ = 8.5–8.0 ppm for the olefinic proton CH-Ar, the po-
sition of which was sensitive to the presence and nature of substituents
in the adjacent phenyl ring. Both steric and electronic effects appear
to be important.18 The introduction of a NO2 group at the orto position
(compound 10) led to a significant deshielding (ꢀδ = 0.47 ppm) of the
olefinic proton as compared to the unsubstituted derivative 5. This ob-
servation supports the opinion that electron-attracting groups at the
orto position in an aromatic ring induce a clear deshielding effect on
the α-proton in the side chain. This effect is probably caused by the

202
H. Liszkiewicz et al.
electron withdrawal of the substituent transmitted to the side chain,
mainly through a p-orbital interaction between the α- and ipso-carbon
atoms.19 A significant deshielding (ꢀδ = 0.38 ppm) was also observed
when two methoxy groups were introduced at the meta and para posi-
tions of the aromatic ring (compound 13). The introduction of a halogen
atom or methoxy group at the para position of the phenyl ring (com-
pounds 6–8, 12) led to an insignificant deshielding of the olefinic proton.
In fact, electronegative atoms and groups exert strong field and induc-
tive effects on a nucleus attached to or near them. This deshielding
effect decreases as the number of bonds between the two centers is in-
creased. However, moieties such as nitro and trifluoromethyl groups
can influence the electronic properties and thus the intermolecular in-
teractions of an entire molecule. This is confirmed in our observation.
The introduction of an NO2 (11) or CF3 (9) group at the para posi-
tion of the phenyl ring causes considerable changes in the electron-
density distribution of the whole molecule. For these two compounds,
changes in shifts of resonance signals were observed not only for the
relatively close olefinic proton but also for remote pyridine protons.
This can lead to the conclusion that the thiazole ring, in particular, the
sulfur atom, participates in the transmission of the substituent effects
from the phenyl ring through the side chain to the imidazopyridine ring
system.
Pharmacology
Antiproliferative Activity In Vitro
The compounds 5–15 were examined for their antiproliferative ac-
tivity in vitro. The following human cancer lines were used: A549 (non-
small cell lung cancer) and SW 707 (rectal adenocarcinoma) (Table I).
Only compounds 5, 10, and 14 revealed weak antiproliferative activity
against the cancer cell lines used for this evaluation.
EXPERIMENTAL
Chemistry
Melting points (uncorrected) were measured with a Boethius melting-
point apparatus. Analyses were performed on a Perkin Elmer 2400 an-
alyzer, and satisfactory results within 0.4% calculated values were
obtained for the new compounds. IR spectra (in KBr) were recorded
with an IR 75 spectrophotometer. 1H NMR spectra were recorded with
a Bruker ARX 300 MHz spectrometer at r.t. using DMSO-d6or CDCl3
as solvents, and chemical shifts refer to the residual solvent signal at

New Tricyclic 2-Thioxo-1H,3H-imidazo[4,5-b]pyridine Derivatives
203
TABLE I Antiproliferative Activity of the
Compounds AL 5, 10 and 14 Against the Cells
of A549 (Nonsmall Cell Lung Cancer) and SW
707 (Rectal Adenocarcinoma)
ID50 [µg/mL SD]
Compound
A 549
SW 707
5
10
14
Cisplatin
60.22 26.14
32.19 12.33
30.17 9,07
0.51 0.03
56.57 35.28
22.81 13.22
27.15 11.12
3.16 0.48
Control
δ 2.50 or 7.24 ppm, respectively. The course of the reaction, and purity
of products were checked by TLC (Kieselgel G, Merck) using diethyl
ether:ethanol = 5:1 as an eluent.
Compounds 1 and 2 were preparated as described.13
Synthesis of 3H-imidazo[4,5-b]pyridin-2-yl-thioglycolic
Acid (3, 4)
0.01 mol 2-thioxo-(3) or 5-bromo-2-thioxoimidazo[4,5-b]pyridine (4)
and 1.2 g (0.03 mol) NaOH in 50 mL ethanol was refluxed for 0.5 h.
After cooling, 0.01 mol chloroacetic acid was added, and the reaction
mixture was refluxed for 2–3 h. The volatile components were removed
in vacuo and water (50 mL) was added. The crude product was filtered
off, washed with water, and dried.
3H-Imidazo[4,5-b]pyridin-2-yl-thioglycolic Acid (3)
Yield 1.9 g (91%), white solid, m.p. 212–215◦C, IR (KBr) ν (cm−1):
3100, 1740, 1580, 1380. 1H NMR (DMSO-d6) δ (ppm): 12.73 (broad 1H,
NH), 8.20 (dd, J= 4.8 Hz, 1.2 Hz, 1H, H-5), 7.82 (dd, J= 7.9 Hz, 1.2 Hz,
1H, H-7), 7.16 (dd, J= 7.9 Hz, 4.8 Hz, 1H, H-6), 4.17 (s, 2H, CH2). Anal.
calcd. for C8H7N3O2S (209.23): C, 45.92; H, 3.37; N, 20.08%. Found: C,
45.80; H, 3.09; N, 19.94%.
6-Bromo-3H-imidazo[4,5-b]pyridin-2-yl-thioglycolic Acid (4)
Yield: 2.5 g (87%), white solid, m.p. 222–225◦C. IR (KBr) ν (cm –1):
1
3120, 1705, 1580, 1290. H NMR (DMSO-d6) δ (ppm): 11.50 (broad,
1H, NH), 8.26 (d, J= 2.0 Hz, 1H, H-5), 8.07 (d, J= 2.0 Hz, 1H, H-7),
4.15 (s, 2H, CH2). Anal. calcd. for C8H6N3BrO2S(288.12): C, 33.35; H,
2.10; N, 14.58%. Found: C, 33.67; H, 1.96; N, 14.34%.

204
H. Liszkiewicz et al.
General Procedure for the Synthesis of
2-(Arylidene)-thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-b]-pyridin-3-ones
(5–15)
A mixture of 0.01 mol imidazo[4,5-b]pyridin-2-yl-thioglycolic acid (3
or 4), 0.01 mol anhydrous sodium acetate, 0.015 mol of the appropriate
aromatic aldehyde, and 15 mL acetic acid anhydride was stirred and
heated at 120◦C (oil bath) for 1.5 h. The precipitate formed was filtered
off and crystallized from dimethylformamide.
2-Benzylidenethiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-b]pyridin-3-one
(5)
Yield: 1.4 g (50%), yellow crystals, m.p. 242–245◦C. IR (KBr) ν (cm−1):
1730, 1610, 1440, 1400. 1H NMR (CDCl3) δ (ppm): 8.57 (dd, J= 4.9 Hz,
1.6 Hz, 1H, H-6), 8.28 (dd, J= 7.9 Hz, 1.6 Hz, 1H, H-8), 8.04 (s, 1H, CH-
Ar), 7.54 (m, 5H, Ar-H), 7.29 (dd, J= 7.9 Hz, 4.9 Hz, 1H, H-7). Anal.
calcd. for C15H9N3OS (279.32): C, 64.50; H, 3.25; N, 15.04%. Found: C,
64.19; H, 2.97; N, 14.97%.
2-(4-Chlorobenzylidene)thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-
b]pyridin-3-one (6)
Yield: 1.9 g (61%), yellow solid, m.p. 291–293◦C. IR (KBr) ν (cm−1):
720, 1605, 1400. 1H NMR (CDCl3) δ (ppm): 8.57 (dd, J= 5.0 Hz, 1.6 Hz,
1H, H-6), 8.28 (dd, J= 7.9 Hz, 1.6 Hz, 1H, H-8), 8.04 (s, 1H, CH-Ar),
7.59–7.49 (m, 4H, Ar-H), 7.30 (dd, J= 7.9 Hz, 5.0 Hz, 1H, H-7). Anal.
calcd. for C15H8N3ClOS (313,76): C, 57.42; H, 2.57; N, 13.39%. Found:
C, 57.78; H, 2.43; N, 13.61%.
2-(4-Bromobenzylidene)thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-
b]pyridin-3-one (7)
Yield: 1.8 g (51%), yellow solid, m.p. 310–312◦C. IR (KBr) ν (cm−1):
1
1720, 1600, 1400. H NMR (CDCl3), δ (ppm): 8.57 (dd, J= 4.0 Hz, 1.6
Hz, 1H, H-6), 8.28 (dd, J= 7.9 Hz, 1.6 Hz, 1H, H-8), 8.03 (s, 1H, CH-
Ar), 7.70–7.66 (m, 2H, Ar-H), 7.50–7.46 (m, 2H, Ar-H), 7.28 (dd, J= 7.9
Hz, 4.9 Hz, 1H, H-7). Anal. Calcd. for C15H8N3BrOS (358.21): C, 50.29;
H, 2.25; N, 11.73%. Found: C, 50.65; H, 2.03; N, 11.76%.
2-(4-Fluorobenzylidene)thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-
b]pyridin-3-one (8)
Yield: 1.8 g (60%), yellow solid, m.p. 288–290◦C. IR (KBr) ν (cm−1):
1720, 1605, 1400. 1H NMR (CDCl3) δ (ppm): 8.57 (dd, J= 5.0 Hz, 1.6 Hz,
1H, H-6), 8.29 (dd, J= 7.9 Hz, 1.6 Hz, 1H, H-8), 8.08 (s, 1H, CH-Ar),
7.65–7.60 (m, 4H, Ar-H), 7.29 (dd, J= 7.9 Hz, 5.0 Hz, 1H, H-7). Anal.

New Tricyclic 2-Thioxo-1H,3H-imidazo[4,5-b]pyridine Derivatives
205
calcd. for C15H8N3FOS (297.31): C, 60.60; H, 2.71; N, 14.13%. Found:
C, 61.02; H, 2.44; N, 14.39%.
2-(4-Trifluorobenzylidene)thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-
b]pyridin-3-one (9)
Yield: 1.8 g (52%), yellow solid, m.p. 324–326◦C, IR (KBr) ν (cm−1):
1
1720, 1605, 1400. H NMR (CDCl3) δ (ppm): 8.45 (dd, J= 4.9 Hz, 1.3
Hz, 1H, H-6), 8.13 (s, 1H, CH-Ar), 7.97 (dd, J= 8.0 Hz, 1.3 Hz, 1H,
H-8), 7.81–7.72 (m, 4H, Ar-H), 7.37 (dd, J= 8.0 Hz, 4.9 Hz, 1H, H-7).
Anal. calcd. for C16H8N3F3OS (347.31): C, 55.33; H, 2.32; N, 12.10%.
Found: C, 55.00; H, 2.19; N, 12.31%.
2-(2-Nitrobenzylidene)thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-
b]pyridin-3-one (10)
Yield: 2.1 g (65%), yellow crystals, m.p. 281–283◦C. IR (KBr) ν (cm−1):
1730, 1620, 1400. 1H NMR (CDCl3) δ (ppm): 8.59 (d, J= 5.0 Hz, 1H, H-
6), 8.51 (s, 1H, CH-Ar), 8.36 (d, J= 8.0 Hz, 1H, H-8), 8.28–8.23 (m,
1H, Ar-H), 7.85–7.64 (m, 3H, Ar-H), 7.36 (dd, J= 8.0 Hz, 5.0 Hz 1H,
H-7). Anal. calcd. for C15H8N4O3S (324.31): C, 55.55; H, 2.49; N, 17.28%.
Found: C, 55.23; H, 2.86; N, 17.01%.
2-(4-Nitrobenzylidene)thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-
b]pyridin-3-one (11)
Yield: 2.3 g (71%), yellow solid, m.p. 347–349◦C. IR (KBr) ν (cm−1):
1740, 1605, 1505, 1050. 1H NMR (CDCl3) δ (ppm): 8.46 (dd, J= 5.0 Hz,
1.4 Hz, 1H, H-6), 8.40–8.36 (m, 2H, Ar-H), 8.32–8.29 (m, 2H, Ar-H), 8.17
(s, 1H, CH), 7.98 (dd, J= 8.1 Hz, 1.4 Hz, 1H, H-8), 7.38 (dd, J= 8.1
Hz, 5.0, Hz, 1H, H-7). Anal. calcd. for C15H8N4SO3 (324.31): C, 55.55,
H, 2.49; N, 17.28%. Found: C, 55.86; H, 2.26; N, 17.27%.
2-(4-Methoxybenzylidene)thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-
b]pyridin-3-one (12)
Yield: 2.9 g (74%), yellow solid, m.p. 268–271◦C. IR (KBr) ν (cm−1):
1
1720, 1600, 1440, 1400. H NMR (CDCl3) δ (ppm): 8.55 (d, J= 4.6 Hz,
1H, H-6), 8.29 (d, J= 7.6 Hz, 1H, H-8), 8.05 (s, 1H, CH-Ar), 7.58 (d,
J= 8.0 Hz, 2H, Ar-H), 7.28 (d, J= 4.6 Hz, 1H, H-7), 7.03 (d, J= 8.0 Hz,
2H, Ar-H), 3.88 (s, 3H, OCH3) Anal. calcd. for C16H11N3O2S (309.34):
C, 62.12; H, 3.58; N, 13.58%. Found: C, 62.35; H, 3.37; N, 13.87%.
2-(3,4-Dimethoxybenzylidene)thiazolo[2 ꢀ,3 ꢀ,2,3]imidazo[4,5-
b]pyridin-3-one (13)
Yield: 2.2 g (65%), yellow solid, m.p. 239–240◦C. IR (KBr) ν (cm−1):
1
1710, 1610, 1400. H NMR (CDCl3) δ (ppm): 8.55 (d, J= 4.6 Hz, 1H,

206
H. Liszkiewicz et al.
H-6), 8.42 (s, 1H, CH-Ar), 8.34 (d, J= 7.6 Hz, 1H, H-8 ), 7.51 (d, J= 8.7
Hz, 1H, Ar-H), 7.29 (dd, J= 7.6 Hz, 4.6 Hz, 1H, H-7), 6.64 (dd, J= 8.7
Hz, 2.2 Hz, 1H, Ar-H), 6.49 (d, J= 2.2 Hz, 1H, Ar-H), 3.92 (s, 3H, OCH3),
3.88 (s, 3H, OCH3). Anal. calcd. for C17H13N3O3S (339.37): C, 60.17; H,
3.86; N, 12.38%. Found: C, 60.51; H, 3.56; N, 12.40%.
2-(3,4,5-Trimethoxybenzylidene)thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,
5-b]pyridin-3-one (14)
Yield: 2.4 g (64%), yellow solid, m.p. 207–209◦C. IR (KBr) ν (cm−1):
1720, 1600, 1465, 1400. 1H NMR (CDCl3) δ (ppm): 8.57 (dd, J= 4.9 Hz,
1.6 Hz, 1H, H-6), 8.29 (dd, J= 7.9 Hz, 4.9 Hz, 1H, H-8), 8.01 (s, 1H, CH-
Ar), 7.28 (dd, J= 7.9 Hz, 4.9 Hz, 1H, H-7), 6.98 (s, 2H, Ar-H), 3.94 (s, 6H,
2 × OCH3), 3.92 (s, 3H, OCH3). Anal. calcd. for C18H15N3O4S (369.39):
C, 58.53; H, 4.09; N, 11.38%. Found: C, 58.17; H, 3.88; N, 11.57%.
2-Benzylidene-7-bromo-thiazolo[2 ꢀ,3 ꢀ:2,3]imidazo[4,5-
b]pyridin-3-one (15)
Yield: 2.1 g (58%), yellow crystals, m.p. 329–332◦C. IR (KBr) ν (cm−1):
1
1730, 1600, 1420. H NMR (CDCl3) δ (ppm): 8.48 (s, 1H, H-6), 8.17 (s,
1H, H-8), 8.08 (s, 1H, = CH-Ar), 7.61–7.51 (m, 5H, Ar-H). Anal. calcd. for
C15H8N3Br1O1S1 (358.21): C, 50,29; H, 2.25; N, 11.73%. Found: 50.24;
H, 2.08; N, 11.90%.
Biology
Antiproliferative Assay in Vitro
Compounds 5–15 were examined in in vitro screening assay. Test
solutions of the compounds (1 mg/mL) were prepared by dissolving
the substance in 100 µL DMSO completed with 900 µL tissue culture
medium. Afterward, the compounds were diluted in the culture medium
to reach the final concentrations of 100, 10, 1, and 0.1 µg/mL.
Cell Lines
The following established in vitro human A549 (nonsmall cell lung
carcinoma) and SW707 (rectal adenocarcinoma) cancer cell lines were
applied. All cancer cell lines were maintained in culture or frozen in
the Cell Culture Collection of the Institute of Immunology and Experi-
mental Therapy, Wrocaw, Poland.
Twentyfour hours before the addition of the tested agents, the cells
were plated in 96-well plates (Sarstedt, USA) at a density of 104 cells
per well in 100 µL culture medium. The cells were cultured in the opti-
MEM+RPMI-1640 (1:1) medium supplemented with 2 mM gluthamine
(Gibco, Warsaw, Poland), streptomycin (50 µg/mL), penicillin (50 U/mL)

New Tricyclic 2-Thioxo-1H,3H-imidazo[4,5-b]pyridine Derivatives
207
(both antibiotics from Polfa, Tarchomin, Poland), and 5% fetal calf
serum (Gibco, Grand Island, USA). The cell cultures were maintained
at 37◦C in a humid atmosphere saturated with 5% CO2. The results of
cytotoxic activity in vitro were expressed as an ID50, which is, the dosis
of compound (in µg/mL) that inhibits the proliferation rate of the tumor
cells by 50% as compared to the controlled untreated cells.
SRB (Sulphorodamine B)
The details of this technique were described by Skehan et al.20 The
cytotoxicity assay was performed after 72 hours of exposure of the cul-
tured cells to varying concentrations (from 0.1 to 100 µg/mL) of the
tested agents. Cells attached to the plastic were fixed by gently layer-
ing cold 50% TCA (trichloroacetic acid, Aldrich) on the top of the culture
medium in each well. The plates were incubated at 4◦C for 1 h and then
washed five times with tap water. The background optical density was
measured in the wells filled with culture medium, without the cells. The
cellular material fixed with TCA was stained with 0.4% sulforhodamine
B (SRB, Sigma) dissolved in 1% acetic acid (POCh, Gliwice, Poland) for
30 min. Unbound dye was removed by rinsing (4x) with 1% acetic acid.
The protein-bound dye was extracted with 10 mM Tris base (POCh,
Gliwice, Poland) for a determination of optical density (at 540 nm) in
a computer-interfaced, 96-well microtiter-plate reader Multiskan RC
photometer (Labsystems, Helsinki, Finland).
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