DNA binding acridine-thiazolidinone agents affecting intracellular glutathione

Article abstract of DOI:10.1016/j.bmc.2012.09.068

Three new acridine-thiazolidinone derivatives (2a-2c) have been synthesized and their interactions with calf thymus DNA and a number of cell lines (leukemic cells HL-60 and L1210 and human epithelial ovarian cancer cell lines A2780) were studied. The comp

Full text of DOI:10.1016/j.bmc.2012.09.068

Bioorganic & Medicinal Chemistry 20 (2012) 7139–7148
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
DNA binding acridine–thiazolidinone agents affecting intracellular glutathione
Helena Paulíková a, , Zuzana Vantová a,b, L’uba Hunáková , Lydia Cizeková , Mária Carná ,
⇑
e
a
a
ˇ
ˇ
´
ˇ
c
c
d
d
d
ˇ
Mária Kozurková , Danica Sabolová , Pavol Kristian , Slávka Hamul’aková , Ján Imrich
a Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Faculty of Chemical and Food Technology, Radlinského 9, SK-81237 Bratislava,
Slovak Republic
b Department of Chemical and Biochemical Engineering, Faculty of Chemical and Food Technology, Faculty of Chemical and Food Technology, Radlinského 9, SK-81237
Bratislava, Slovak Republic
c Department of Biochemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik University, Moyzesova 11, SK-04167 Košice, Slovak Republic
d Department of Organic Chemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik University, Moyzesova 11, SK-04167 Košice, Slovak Republic
e Cancer Research Institute, Vlárska 7, SK-81237 Bratislava, Slovak Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2012
Revised 21 September 2012
Accepted 26 September 2012
Available online 12 October 2012
Three new acridine–thiazolidinone derivatives (2a–2c) have been synthesized and their interactions with
calf thymus DNA and a number of cell lines (leukemic cells HL-60 and L1210 and human epithelial ovar-
ian cancer cell lines A2780) were studied. The compounds 2a–2c possessed high affinity to calf thymus
DNA and their binding constants determined by spectrofluorimetry were in the range of 1.37 ꢀ 106–
5.89 ꢀ 106 Mꢁ1. All of the tested derivatives displayed strong cytotoxic activity in vitro, the highest activ-
ity in cytotoxic tests was found for 2c with IC50 = 1.3 0.2
7.7 0.5
lM (HL-60), 3.1 0.4 lM (L1210), and
Keywords:
Acridine
DNA-intercalation
Glutathione
Antitumor
l
M (A2780) after 72 h incubation. The cancer cells accumulated acridine derivatives very fast
and the changes of the glutathione level were confirmed. The compounds inhibited proliferation of the
cells and induced an arrest of the cell cycle and cell death. Their influence upon cells was associated with
their reactivity towards thiols and DNA binding activity.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
9-ylimino)-3-substituted-4-oxo-thiazolidin-5-ylidene]acetate
which were synthesized recently by our group from 1-alkyl(acyl)-
Antitumor effects of acridines are associated not only with their
interaction with DNA but also with proteins, especially some key
enzymes important in cell proliferation as topoisomerases or telo-
merases.1–3 Acridines can interact with DNA either via intercala-
tion or groove-binding and may thus block a DNA replication,
transcription, or DNA repair.4 In the meantime, the best known
acridine drugs used in antitumor therapy are amsacrine and DACA.
Damage of DNA by these compounds is usually strengthened by
the oxidative stress.5 The search for novel anticancer drugs encour-
ages also syntheses of new acridine derivatives.6–8 Several series of
novel 9- and 3,6-substituted acridines have recently been prepared
in our laboratory and investigated for their DNA binding properties
and in vitro antineoplastic activity.9–13 Besides antitumor effects
found at micromolar concentrations, all new substances displayed
a potent DNA binding with binding constants in the range of 105–
106 Mꢁ1. This affinity as well as UV–vis, spectrofluorimetric and CD
studies confirmed an intercalation of our ligands into DNA to be a
principal mode of interaction.
4-(acridin-9-yl)thiosemicarbazides and dimethyl acetylenedicar-
boxylate (DMAD).14–17 The present study was designed to expand
our knowledge of the mechanism of cytotoxicity of related acri-
dine–thiourea derivatives which possess also reactivity towards
thiols. Three new acridine–thiazolidinone derivatives were pre-
pared from N-(acridin-9-yl)-N0-substituted thioureas (R = sec-bu-
tyl, tert-butyl, and 4-bromophenyl) and DMAD and their DNA
binding activity was evaluated. Although, anticancer activity of
acridines is mainly related to their capacity to bind to DNA, the
cytotoxicity of these may be enhanced by an increase of their elec-
trophilicity to facilitate the reaction with essential SH groups pres-
ent in living organisms, especially a main low-molecular thiol,
glutathione. Reactivity of acridines with sulfhydryls could modify
SH-proteins and alter intracellular glutathione redox status, which
modulates a lot of cellular events, for example, selective gene
expression, DNA synthesis, or regulation of the cell cycle.18–20 In vi-
tro cytotoxic effects of these compounds on murine leukemic cells
L1210, human promyelocytic leukemic HL-60 cells, and A2780 hu-
man ovarian adenocarcinoma cells were monitored. To investigate
the mechanism of cytotoxicity, a cellular accumulation of acridine–
thiazolidinone derivatives was studied and their influence on the
intracellular glutathione level was evaluated. The cell death and
changes in the cell cycle were observed.
In search for new pharmacophoric structures we were inspired
by acridine–thiazolidinone derivatives such as methyl [2-(acridin-
⇑
Corresponding author.
E-mail address: helena.paulikova@stuba.sk (H. Paulíková).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.068

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H. Paulíková et al. / Bioorg. Med. Chem. 20 (2012) 7139–7148
O
R
4
N 3
S
5
O
2
S
R
HN
N
N
H
N
O
1´
8´
5´
CH3
DMAD
MeOH, 24h
N
4´
2a-c
1a-c
R = sec-Bu (a), tert-Bu (b), p-Br-Ph (c)
Scheme 1. Synthesis of tested compounds 2a–2c.
2. Results and discussion
2.1. Chemistry
0.5
Three new derivatives were prepared from N-(acridin-9-yl)-N0-
substituted thioureas (R = sec-Bu (a), tert-Bu (b), 4-Br-Ph (c)) and
DMAD (Scheme 1).
0.4
0.3
0.2
0.1
0
To a stirred solution of corresponding N-(acridin-9-yl)-N0-
substituted thiourea 1 (5 mmol) in methanol (30 mL), DMAD
(6 mmol) was added and the solution was stirred for 20–24 h at
room temperature.21,22 The reaction course was followed by TLC,
the eluent benzene/acetone, 9:1. The crude product was isolated
by evaporation of the solvent, purified by column chromatography
on silica gel using the same eluent as above and obtained as pale
yellow crystals.
300
350
400
450
500
550
600
2.2. DNA binding studies
Wavelength (nm)
Acridine intercalation into DNA, a strong stacking interaction
between an aromatic chromophore of the ligand and DNA base
pairs, is characteristic of a hypochromism and red shifts in the
UV–vis spectra.23–25 Interaction of ligands 2a–2c with calf thymus
(ct) DNA was therefore examined by UV–vis, fluorimetric titra-
tions, and thermal melting experiments. UV–vis data of com-
pounds 2a–2c are displayed in Table 1 and absorption spectra of
2c in the absence and presence of calf thymus DNA are shown in
Figure 1. The spectra show a significant absorption in the range
Figure 1. UV–vis spectral changes of 2c (25
lM) upon addition of ctDNA (from top
to bottom 0–30 M bp) in 0.01 M Tris (pH 7.4), 24 °C.
l
tures (Tm) in the presence and absence of studied derivatives 2a–
2c are shown in Table 1. The Tm of free ctDNA was 70.5 °C and in-
creased to 74–81 °C upon 2a–2c intercalative binding in accord
with literature.10
In a spectrofluorimetric study, free 2a–2c exhibited a broad
emission band in the range of 420–550 nm. Excitation wavelength
was set to 384 nm and the spectra were monitored at fixed drug
of 300–600 nm, typical for transitions between
p-electron energy
levels of the acridine ring. As the DNA concentration increased,
the bands showed moderate hypochromicities (21.5–35.9%) and
slight bathochromic shifts which indicate an insertion of the com-
pounds into DNA. The overall changes in the UV–vis spectra sug-
gest that intercalation is the dominant binding mode.
DNA binding properties of compounds 2a–2c were also evalu-
ated by thermal melting experiments where duplex DNA was ther-
mally denatured into single-stranded components in the presence
and absence of these ligands. The DNA helix denaturation can be
determined by monitoring a change of the DNA absorbance at
260 nm as a function of temperature. The DNA melting tempera-
concentration 26.7 lM. Titration with increasing concentration of
ctDNA continued until no further changes in the spectra of the
drug–DNA complexes were recorded (Fig. 2). Binding of the acri-
dine probes into DNA helix was found to reduce the fluorescence
of studied acridine derivatives and represents another independent
proof of intercalation.
The binding constants K of 2a–2c with ctDNA determined by
spectrofluorimetry were calculated according to a McGhee and
von Hippel method.26,27 Their large values in the range of
1.37 ꢀ 106–5.89 ꢀ 106 Mꢁ1 increased in the order 2b < 2a < 2c
and are indicative of a high affinity of the acridine chromophore
to DNA-base pairs (see Table 2). The binding affinity is approxi-
mately ten-times higher than K values of related acridine-bis-imi-
Table 1
dazolidinone intercalators (1.9 ꢀ 105–7.1 ꢀ 105 Mꢁ1 9
)
derived
UV–vis absorption data of acridine derivatives 2a–2c
aTm
(°C)
from proflavine (acridine-3,6-diamine), daunomycin (7.09 ꢀ
105 Mꢁ1), or acridine orange (3.19 ꢀ 105 Mꢁ1),28 even two-order
magnitudes higher than those of acridine-bis-thiazolidinones
(2.2–6.2 ꢀ 104 Mꢁ1).12 These data clearly show that less bulky
mono-9-substituted acridine–thiazolidinones studied here are
better suited for intercalative (threading) binding than bis-3,6-
substituted proflavine derivatives. Accordingly, as we have proven
Compound kmax free
kmax bound
(nm)
D
(nm)
k
Hypochromicity
(%)
(nm)
2a
2b
2c
326
324
326
331
333
330
5
9
4
26.3
31.2
24.2
75
74
81
a
Tm was measured in BPE buffer (6 mM Na2HPO4, 2 mM Na2HPO4, 1 mM EDTA),
pH 7.1.

H. Paulíková et al. / Bioorg. Med. Chem. 20 (2012) 7139–7148
7141
When compared with cisplatin, the cytotoxicity of our com-
pounds 2a–2c was lower against L1210 and A2780 and similar
against HL-60 cells (Table 3). All 2a–2c were less cytotoxic against
200
150
100
50
HL-60 cells than amsacrine (IC50 = 0.08 l
M, 72 h).37
The cytotoxicities of the substances 2a–2c were verified by a di-
rect cell counting method (DCC) using a trypan blue exclusion test.
Growth curves of the HL-60 cells were obtained by dye exclusion
tests (Fig. 3) and from these IC50 values were calculated. All deriv-
atives displayed an inhibitory effect with respect to the control.
When longer-term experiments to determine IC50 (72 h) are
employed one should remember that several divisions of cultured
cells have been completed at the end and repair mechanisms low-
ering the therapeutic effect may be running. In our case, luckily,
the IC50 values did not deteriorate upon long-term incubation con-
firming a persisting prolonged cytotoxicity of 2a–2c on the studied
HL-60 cells (Table 4).
410
420
430
440
450
460
470
The cytotoxicity of 2a–2c against NIH-3T3 mouse embryonic
fibroblast cells has been examined. Cytotoxicity of 2c against
NIH-3T3 cells (IC50 = 3.5 lM, 72 h) was lower than toxicity against
Wavelength (nm)
leukemia cells (L1210 or HL-60) but higher than toxicity against
A2780 cells.
Figure 2. Spectrofluorimetric titration of derivative 2c (26.7
buffer (pH 7.4, 24 °C) by increasing concentration of ctDNA (from top to bottom, 0–
32 M bp, at 2 M intervals), kex = 384 nm.
lM) in 0.01 M Tris
l
l
2.4. Accumulation of 2a–2c in the cells
Cells were incubated with 2a–2c at 37 °C for 4 h, washed with
PBS, and monitored by fluorescence microscopy. In the control
experiment, cells were not treated (data not presented). Leukemic
cells accumulated acridine derivatives 2a–2c already after 4 h
incubation. As revealed in Figure 4, HL-60 cells became bright-blue
about 4 h after addition of all tested derivatives. The rapid internal-
ization of 2a–2c was shown also for A2780 (not shown).
An intracellular uptake of 2c was confirmed by HPLC. A well-re-
solved peak that corresponds to 2c was achieved on a C18 column
using the mobile phase consisting of ammonium formate
(150 mM) in 80% methanol with pH adjusted to 4.5 using formic
acid. The detection of 2c was carried out at wavelength 370 nm
(Fig. 5A, 2c (1 nmol) was injected). HL-60 cells were incubated
with the substance for 10 or 60 min, then cells were washed twice
with PBS, extracted with methanol, and the extract was analyzed
(Fig. 5B and C). HPLC chromatograms showed that 2c
(Rt = 32.8 min) was accumulated in cells already after 10 min incu-
bation. The level of 2c did not change after 60 min incubation.
Table 2
Fluorescence data of compounds 2a–2c
Compound
kem (nm)
Ufa
K ꢀ 106 (Mꢁ1
2.21 0.25
1.37 0.13
5.89 0.81
)
2a
2b
2c
421
422
425
0.61
1.00
0.75
a
Relative fluorescence intensity was calculated using the compound 2b as a
standard (Uf0 = 1).
Table 3
Cytotoxicity of derivatives 2a–2c against leukemic and A2780 cells—MTT-assay
Compound
IC50
L1210
72 h
3.4 0.5 2.1 0.3 3.3 0.5 7.5 0.7 20.8 1,3 12.6 1.1
5.4 0.7 3.7 0.6 17.9 2.2 20.9 1.9 >30 23.7 2.8
1.4 0.3 1.3 0.2 1.7 0.4 3.1 0.4 12.5 0.2 7.7 0.5
ND 2.2 0.3 ND 1.3 0.3 ND 2.73 0.6
(lM)
HL-60
72 h
A2780
72 h
48 h
48 h
48 h
2a
2b
2c
Cisplatin
2.5. SH-reactivity of 2a–2c and changes of intracellular level of
glutathione
It is known that high degree of SH-reactivity is the shared fea-
ture of a variety of anticancer agents. Cysyk et al. reported that
amsacrine was able to create GSH-acridine thioether conjugate.30
Accordingly, a similar metabolic transformation of 2a–2c may be
expected also here. Although their cytotoxic effect should be asso-
ciated mainly with their DNA binding activity, these compounds
possessing at least five electrophilic sp2 carbons can also interact
with intracellular thiols to afford several target structures. To con-
firm such an assumption, we studied an interaction with a model
by molecular dynamics calculations of binding energies,9 preferen-
tial mode of DNA binding of proflavine derivatives is a minor-
groove binding, not a threading binding as in this case. The reasons
for that may be looked for in an electrostatic energy change and
entropy decrease accompanying the DNA–ligand binding.9
2.3. Cytotoxicity of 2a–2c
thiolate, N-acetyl-L-cysteine (NAC). UV–vis spectrophotometric
We performed also a basic pre-screening of cytostatic parame-
ters of derivatives 2a–2c. Leukemic cells HL-60 (human) and L1210
(mice) and human epithelial ovarian cancer cell lines A2780 were
treated with derivatives 2a–2c in a concentration range of 0.25–
analysis (Fig. 6) confirmed that 2c reacted with NAC to provide a
NAC-2c conjugate which displayed a distinctly different absor-
bance spectrum compared to that of the 2c. An attempt to render
an NMR proof of the conjugate structure was precluded by a very
different solubility of both reagents not allowing to see sufficient
concentrations of the product(s) with supposed limited stability
in the reaction mixture.
10 lM on the leukemic cells and in the range of 1.0–50 lM on
A2780 cells for 3 days. IC50 values were determined using a MTT-
assay (Table 3). All derivatives displayed inhibitory effect with re-
spect to the control. The most potent cytotoxic derivative was 2c
and the A2780 cell line was less sensitive to cytotoxic action of
2a–2c than leukemic cells.
Moreover, in a following kinetic study, second-order rate con-
stants of the reaction of 2c with N-acetyl-L-cysteine (Table 5) in-
creased in the range of ca. one order of magnitude upon the pH

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H. Paulíková et al. / Bioorg. Med. Chem. 20 (2012) 7139–7148
Figure 3. Inhibitory effects of derivatives 2a–2c on the proliferation of HL-60 cells. Three-day cell growth is shown in the presence of 2a–2c derivatives: 2a—sec-Bu, 2b—tert-
Bu, 2c—p-Br-Ph. Viability of cells was measured by a dye exclusion test using trypan blue. Cells were incubated for 0–72 h without (control) or with 0–10
compound. Results are expressed as a mean S.D. (n = 3).
lM of the
raise from 6.0 to 7.0 confirming thus a kinetic control in the reac-
tion. Such behavior proves that thiol reacts with the acridine deriv-
ative in a thiolate form.
Table 4
Cytotoxicity of derivatives 2a–2c against human leukemic cells HL-60—DCC method
Glutathione (GSH) is a dominant non-protein thiol in mamma-
lian cells whose main role is to detoxificate electrophilic xenobiot-
ics.31 We suppose that compounds 2a–2c could also form
conjugates with intracellular GSH (either spontaneously or enzy-
matically in reactions catalyzed by glutathione-S-transferase),
even though we did not succeed to confirm them by direct chem-
ical synthesis. Moreover, SH groups of proteins may be also as-
sumed to react with 2a–2c. If GSH traps the electrophilic acridine
derivatives, it actually protects SH groups of proteins against bind-
ing of 2a–2c.
Compound
IC50
HL-60
(lM)
48 h
72 h
2a
2b
2c
2.0 0.4
3.5 0.7
1.6 0.6
1.7 0.4
2.8 0.5
0.9 0.1
Cells were incubated for 0–72 h without (control) or with 0–10
pounds 2a–2c. IC50 values are concentrations that produced 50% inhibition of the
cell viability. Results are expressed as a mean S.D. (n = 3).
l
M of the com-
Figure 4. Intracellular accumulation of compounds 2a–2c (5
control was stained with Hoechst 33342.
lM each) in HL-60 cells, visualized by fluorescence microscopy after 4 h incubation (magnification 40 ꢀ 10). The

H. Paulíková et al. / Bioorg. Med. Chem. 20 (2012) 7139–7148
7143
Figure 5. HPLC analysis of intracellular accumulation of 2c. HL-60 cells (2 ꢀ 106/mL) were incubated with 10
lM 2c for 10 min (B) and 60 min (C). Standard of 2c (A) (1 nmol/
20
l
L) Rt = 32.8 min; A = 1366.622 mV s.
We therefore examined the level of intracellular glutathione in
treated cells. When HL-60 cells were incubated with 2a–2c for
short-time (Fig. 7), the intracellular concentration of GSH (total
glutathione) decreased. However, at long-term incubation, the le-
vel of intracellular glutathione was recovered, the most with the
derivative 2b. GSH depletion might trigger the downstream events
of apoptosis by leaving cells unprotected against thiol oxidation
radical production.32 As seen in Figure 7, the concentration of
GSH returned to the control level only in cells incubated with 2b
for 24 h.
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
2c
2c + NAC
Table 5
Second-order rate constants for 2c reaction with NAC at various
pH
250
300
350
400
450
500
pH
k2 [Mꢁ1 sꢁ1
]
Wavelength (nm)
7.0
6.5
6.0
9.9 0.9
3.8 0.7
2.0 0.5
Figure 6. The spectral changes observed for 2c after reaction with NAC. UV–vis
spectra: 2c (solid line) and the spectrum recorded after addition of NAC (dashed
line). Spectra were measured in the solution of 50% DMSO and 100 mM Tris/HCl (pH
7) at 25 °C. The mixture of 2c and NAC was kept at 25 °C for 10 min. The final
The reaction was followed in the solution of 50% DMSO and
100 mM Tris/HCl at 25 °C. The final concentration of 2c and
NAC was 20 lM and 1 mM, respectively.
concentration of 2c and NAC was 20 lM and 1 mM, respectively.

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H. Paulíková et al. / Bioorg. Med. Chem. 20 (2012) 7139–7148
To determine the role of glutathione in cytotoxicity of 2a–2c,
HL-60 cells were pretreated with -buthionine–sulfoximine
(BSO). BSO application at concentration of 10 M prior to 2a–2c
treatment induces a depletion of GSH by inhibition of -glutamyl-
cysteine synthetase, a key enzyme active in glutathione biosynthe-
sis. BSO itself exhibited no significant toxicity on HL-60 cells (data
not shown). The results of trypan blue staining assay showed that
BSO pre-treatment significantly augmented inhibition of the cell
growth induced by three acridine–thiazolidinones (Fig. 8).
20
18
16
14
12
10
8
2 h
24 h
L
l
c
The depletion of intracellular glutathione can be associated
with oxidative stress. Some acridine derivatives are able to induce
oxidative stress. Blasiak et al.5 found that free radicals may be in-
volved in the formation of DNA lesions induced by amsacrine.
We verified induction of oxidative stress in the presence of 2c. Oxi-
dative stress has been determined by DHE assay. The production of
6
4
2
0
control
2a
2b
2c
superoxide in 2c (5
firmed (Fig. 9B), and when cells were pretreated with BSO
(10 M for 6 h), the generation of superoxide was enhanced in
lM, 1 h) treated HL-60 cells has been con-
Figure 7. Glutathione in treated HL-60 cells. 2a and 2b (2
derivatives were incubated with cells for 2 h and for 24 h.
l
M) and 2c (1 lM)
l
the presence of 2c (Fig. 9C).
100
90
80
70
60
50
40
30
20
10
0
2.6. Arrest of cell cycle and cellular death
As interactions of compounds 2a–2c with DNA and cellular thi-
ols can induce a damage of cellular processes, an arrest of the cell
cycle may be expected. We have really found that incubation of
HL-60 cells with 2c resulted in accumulation of cells in the G1-
phase of the cell cycle (Fig. 10), and the percentage of treated cells
(1 lM 2c, 48-h incubation) in the subG0 region (apoptotic cells) in-
creased to 25%. The results show that low concentrations of 2c can
induce apoptosis (identified by a subG0 cell population).
To assess a type of cell death induced by higher concentration of
2a–c, we analyzed their morphological changes after staining with
propidium iodide (PI). Uptake of PI (red fluorescence) indicates a
loss of membrane integrity, a characteristic hallmark of late apop-
totic and necrotic cells. As seen in Figure 11, HL-60 cells treated
2a
2a+BSO
2b
2b+BSO
2c
2c+BSO
Figure 8. Cytotoxicity of 2a–2c in BSO pretreated HL-60 cells. Cells were pretreated
with BSO (10 M) for 6 h and than incubated with 2a (1.5 M), 2b (3.0 M), and 2c
(1.0 M), respectively, for 48 h. The number of viable cells was determined by
trypan blue staining assay and the results are expressed as the percentage of all
cells.
with 5 lM 2a–2b and 2.5 lM 2c reduced their volume (cell shrink-
ing) and were PI-positive after 24 h incubation.
Next studies (by dual FDA/PI fluorescent staining using FACS)
showed that 2c at concentration about 2 lM induced necrosis of
l
l
l
l
cells (Fig. 12).
Figure 9. Oxidative stress induced by 2c in human leukemia cells HL-60. Cells were incubated without 2c (A, control), in the presence of 5
added after pre-treatment with BSO (10 M for 6 h, C). The positive control (D, menadione) and cells treated with BSO (E, 10 M BSO for 6 h) are shown. Oxidative stress was
assessed by the DHE assay, optical (upper) and fluorescence (lower) microphotographs, magnification 40 ꢀ 10.
lM of 2c for 1 h (B) or 2c was
l
l

H. Paulíková et al. / Bioorg. Med. Chem. 20 (2012) 7139–7148
7145
Figure 10. Analysis of cell cycle of HL-60 cells non-treated (A) and treated with 2c (0.5 lM, B) and 2c (1 lM, C) for 48 h. Analyses were performed with CANTO II as described
in Section 4.3.
Figure 11. Microphotographs of HL-60 cells treated with 5
positive marked with an arrow, magnification 40 ꢀ 10).
lM 2a and 2b (B, C) and 2.5 lM 2c (D) for 24 h and non-treated cells (A). HL-60 cells were stained with PI (PI-
Figure 12. Analysis of cell death induced by 2
lM 2c (B) and 4
lM 2c (C) after 24 h incubation with HL-60 cells, nontreated cells (A). Cell death was detected by dual FDA/PI
fluorescent staining using FACS.
order of magnitude greater than the K of daunomycin. All tested
3. Conclusion
derivatives displayed a strong cytotoxic activity in vitro: they
inhibited the growth of cancer cells and induced the cellular death
at micromolar concentrations. The compounds also temporarily
New acridine–thiazolidinone derivatives 2a–2c have shown the
high affinity to calf thymus DNA with the binding constants K one

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H. Paulíková et al. / Bioorg. Med. Chem. 20 (2012) 7139–7148
changed the level of intracellular glutathione. The cytotoxicity of
2a–2c seems to be associated with their DNA binding activity
and the induction of oxidative stress. The new acridine–thiazolid-
inone substances may be inspiring for further development of
DNA-targeted anticancer agents affecting also glutathione system
of cells.
(FBS) were obtained from Grand Island Biological Co., USA, and
all other chemicals from Lachema (Czech Republic).
4.3. Biological studies
4.3.1. UV–vis absorption measurements
UV–vis spectra were measured on a Varian Cary 100 UV–vis
spectrophotometer in 0.01 M Tris buffer (pH 7.4). A solution of
the calf thymus DNA (ctDNA) in TE (Tris–EDTA) buffer was soni-
cated for 5 min and the DNA concentration was determined from
its absorbance at 260 nm. The purity of DNA was determined by
monitoring the value A260/A280. The concentration of DNA at
260 nm was expressed as base pairs ranged from 0 to 30 lM bp.
The derivatives 2a–2c were all dissolved in DMSO from which
working solutions were prepared by dilution using 0.01 M Tris buf-
fer to a final concentration 25 lM. All measurements were per-
4. Experimental
4.1. Synthesis
NMR spectra were measured on a 400 MHz NMR spectrometer
Varian Mercury Plus. IR spectra were obtained on a Specord IR 75
spectrophotometer and CHN analysis was carried out on a CHN
analyzer Perkin–Elmer CHN 2400.
formed at 24 °C.
4.1.1. Methyl 2-[2-(acridin-9-ylimino)-4-oxo-3-sec-butyl-1,3-
thiazolan-5-yliden]acetate (2a)
4.3.2. Fluorescence measurements
Mp 175–177 °C, yield 57%. For C23H21N3O3S (419.50) Calcd:
65.85% C, 5.05% H, 10.02% N. Found: 65.07% C, 4.90% H, 9.72% N;
IR spectrum (CHCl3): 1720 (ester C@O), 1693 (C@N), 1640 (amide
C@O), 1613 (C@C). 1H NMR spectrum (CDCl3, d): 1.15 (t, 3H,
3J = 7.4 Hz, CH3), 1.79 (d, 3H, 3J = 6.9 Hz, CH3), 2.25 (m, 2H, CH2),
3.66 (s, 3H, OCH3), 4.99 (sextet, 1H, 3J = 6.9 Hz, CH), 6.90 (s, 1H,
Fluorescence was scanned on a Varian Cary Eclipse spectrofluo-
rimeter with a 10 nm slit width for excitation and emission beams.
Emission spectra were recorded in the region 410–470 nm using
an excitation wavelength 384 nm. Fluorescence intensities are ex-
pressed in arbitrary units. Fluorescence titrations were conducted
by adding increasing amounts of ctDNA directly into the cell con-
taining the solution of compounds 2a–2c (c = 26.7 ꢀ 10ꢁ6 M,
0.01 M Tris buffer, pH 7.4). The concentration range of DNA was
3
@CH), 7.47 (dd, 2H, J = 8.6, 7.2 Hz, H-20,70), 7.78 (dd, 2H, 3J = 8.4,
3
7.2 Hz, H-30,60), 7.80 (d, 2H, J = 8.6 Hz, H-10,80), 8.23 (d, 2H,
3J = 8.4 Hz, H-40,50).
0ꢁ32
lM bp. All measurements were performed at 24 °C.
4.1.2. Methyl 2-[2-(acridin-9-ylimino)-4-oxo-3-tert-butyl-1,3-
thiazolan-5-yliden]acetate (2b)
4.3.3. Tm measurements
Thermal denaturation studies were conducted using a Varian
Cary Eclipse spectrophotometer equipped with a thermostatic cell
holder. The temperature was controlled by a thermostatic bath
( 0.1 °C). The absorbance at 260 nm was monitored for either
Mp 203–205 °C, yield 64%. For C23H21N3O3S (419.50) Calcd:
65.85% C, 5.05% H, 10.02% N. Found: 65.62% C, 4.96% H, 9.95% N;
IR spectrum (CHCl3): 1726 (ester C@O), 1690 (C@N), 1640 (amide
C@O), 1610 (C@C). 1H NMR spectrum (CDCl3, d): 2.05 (s, 9H,
CH3), 3.64 (s, 3H, OCH3), 6.82 (s, 1H, @CH), 7.49 (dd, 2H, 3J = 8.6,
ctDNA (10 lM) or a mixture of DNA with 2a–2c (5 lM) in BPE buf-
3
7.1 Hz, H-20,70), 7.79 (dd, 2H, J = 8.8, 7.1 Hz, H-30,60), 7.83 (d, 2H,
fer, pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA), with a
heating rate 1 °C/min. Melting temperatures were determined as
a maximum of the first derivative plots of melting curves.
3
3J = 8.6 Hz, H-10,80), 8.27 (d, 2H, J = 8.8 Hz, H-40,50).
4.1.3. Methyl 2-[2-(acridin-9-ylimino)-3-(4-bromophenyl)-4-
oxo-1,3-thiazolan-5-yliden]acetate (2c)
4.3.4. Equilibrium binding titration
Binding affinities were calculated from absorbance spectra
according to the method of McGhee and von Hippel using data
points from the Scatchard plot. The binding data were fitted using
GNU Octave 2.1.73 software.29
Mp 161–163 °C, yield 79%. For C25H16BrN3O3S (518.40) Calcd:
57.92% C, 3.11% H, 8.11% N. Found: 57.49% C, 2.97% H, 7.95% N;
IR spectrum (CHCl3): 1726 (ester C@O), 1700 (C@N), 1643 (amide
C@O), 1613 (C@C). 1H NMR spectrum (CDCl3, d): 3.71 (s, 3H,
OCH3), 7.03 (s, 1H, @CH), 7.50 (ddd, 2H, 3J = 8.4, 7.1 Hz,
4J = 1.0 Hz, H-20,70), 7.55 (d, 2H, 3J = 8.0 Hz, Ph), 7.78 (d, 2H,
4.3.5. Cell culture conditions
3
3J = 8.0 Hz, Ph), 7.79 (dd, 2H, J = 8.2, 7.1 Hz, H-30,60), 7.88 (d, 2H,
Human myeloid leukemia cell line HL-60, mouse leukemia cell
line L1210, human ovarian carcinoma cell line A2780 were ob-
tained from Dr. P. Ujhazy, Roswell Park Cancer Institute, Buffalo.
The cell lines (HL-60, L1210 and A2780) were grown in RPMI
1640 medium supplemented with 10% FBS, penicillin (100 units/
4
3J = 8.4 Hz, H-10,80), 8.26 (dd, 2H, 3J = 8.2 Hz, J = 1.0 Hz, H-40,50).
4.2. Materials
mL), and streptomycin (100 lg/mL). Medium for HL-60 and
Propidium iodide (PI), Triton X-100, reduced form of glutathi-
A2780 cell lines was supplemented also with 2 mM glutamine.
The cells were maintained at 37 °C in a humidified 5% CO2 atmo-
sphere. NIH-3T3 mouse embryonic fibroblast cells were obtained
from the American Type Culture Collection, Rockville, MD (USA).
NIH-3T3 cells were routinely cultured in DMEM supplemented
one (GSH), L-buthionine-sulfoximine (BSO), menadione, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
Hoechst 33342, fluorescein diacetate (FDA), dihydroethidium
(DHE), dimethyl sulfoxide (DMSO), DMEM and RPMI-1640 med-
ium, trypsin, b-nicotinamide adenine dinucleotide 20-phosphate
reduced tetrasodium salt hydrate (NADPH), glutathione reductase
with 10% FBS, 2 mM
L-glutamine, penicillin (100 lg/mL) and strep-
tomycin (100 g/ml) in 5% CO2 at 37 °C.
l
from baker’s yeast, plasmid pUC19 (2761 bp, DH 5a), cisplatin,
agarose (type II No-A-6877), and calf thymus DNA were obtained
from Sigma–Aldrich. EDTA and RNase A were purchased from
Serva, 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) from Merck,
4.3.5.1. MTT-assay.
the presence of 2a–2c (concentration in the range 0–30
The viability of cells was determined in
M) or ab-
l
sence of tested derivatives using a MTT microculture tetrazolium
N-acetyl-
L-cysteine (NAC) and glutathione reductase from
assay as described previously.33
Calbiochem. Penicillin, streptomycin, and fetal bovine serum

H. Paulíková et al. / Bioorg. Med. Chem. 20 (2012) 7139–7148
7147
4.3.5.2. DCC.
The cell proliferation, growth curves, and cyto-
10 mM DTNB, and 500
lL of 150 mM sodium phosphate buffer
toxic potential of compounds were determined by a trypan blue
dye exclusion test (DCC). For three-day experiments, cells were
seeded (1 ꢀ 105 cells/mL) in Petri dishes and tested substances
(or 1% DMSO-control) were added after 24 h; then cell proliferation
was checked after 24, 48, and 72 h. Growth curves were obtained
and inhibitory concentrations, IC50, were determined. All dye
exclusion tests were made three times.
(pH 6.2). The assay was initiated by the addition of 10 lL of gluth-
atione reductase (470 U/mL). The rate of formation of 5-thio-2-
nitrobenzoic acid was monitored spectrophotometrically at
412 nm (Specord 250, Analytic Jena). tGSH concentration values
were calculated from a standard curve and expressed in nmol/mg
prot. Protein concentrations were determined by a method of
Lowry et al. using BSA as standard.35
4.3.6. Fluorescence microscopy
4.3.12. Reactivity of 2c with NAC
HL-60 cells harvested in log phase were counted and seeded
Reactivity of 2c with NAC was investigated in a system contain-
ing 100 mM Tris/HCl buffer (pH 6.0–7.0) containing 50% of DMSO
at 25 °C. The kinetic measurements were performed under condi-
tions for a pseudomonomolecular reaction and the reaction rates
were measured spectrophotometrically on a Specord 250 (Analytic
Jena). The rate constants (k2 [Mꢁ1 sꢁ1]) were calculated
(k0 = k2[NAC], k0–pseudo-first-order rate constant) using the pKa
(0.5 ꢀ 106/mL).
4.3.7. Intracellular accumulation of compounds
HL-60 cells were treated with 2a–2c. After 4 or 24 h incubation
at 37 °C in a humidified atmosphere of 5% CO2 in air, the cells were
washed in PBS and observed by an optical microscope and a fluo-
rescence microscope (Axio Zeiss Imager A1, camera AxioCam
Mrc). The cells without 2a–2c (a control) were stained with
values of N-acetyl-L-cysteine (pKa = 9.5).
10
lM of Hoechst for 10 min than washed in PBS.
4.3.13. RP-HPLC analysis of cellular uptake
ODS columns were used for HPLC analysis of acridine deriva-
4.3.8. Analysis of necrosis
tive.36 The chromatographic separation of 2c was achieved on a
HL-60 cells were treated with 2a–2c for 24 h. After incubation,
the cells were washed with PBS, PI was added to a final concentra-
tion of 1 lg/mL, and cells were incubated for 10 min at room tem-
perature. The samples were re-washed twice with PBS and
observed by an optical microscope and a fluorescence microscope
(Axio Zeiss Imager A1, camera AxioCam Mrc).
C18 column (Watrex, 250 ꢀ 4, Reprosil 100 C18 with 5
lm particle
size) using degassed mobile phase—methanol and ammonium for-
mate (MP: a mixture of methanol and 50 mM ammonium formate
in the ratio of 80:20 (v/v), pH was adjusted to 4.5 with formic acid),
the flow rate was 0.5 mL/min. The column temperature was main-
tained at 25 °C and the detection was carried out at 370 nm. The
standard concentration was 1 nmol/20
was 20 L). The amount of 2c in the cells was quantified using
methanol extraction and RP-HPLC analysis. The cell pellets were
re-suspended in 100 L of methanol and mixed for 10 min. The
lL (the injection volume
4.3.9. Flow cytometry—PI staining for cell cycle analysis
l
The control and drug-treated cells (0.5 ꢀ 106/mL) with 0.5 and
1
l
M 2c for 48 h were harvested by centrifugation and permeabi-
l
lized by detergent treatment (0.1% Triton X-100 in PBS) for 30 min.
After incubation with RNAase (50 g/mL) at 37 °C, DNA was
stained with propidium iodide (50 g/mL). Canto II flow cytometer
mixture was kept for 1 h at 4 °C. The tubes were then centrifuged
l
at 6700ꢀg for 15 min (4 °C) to pellet any solid material. The extract
l
(20 lL) was injected into the HPLC system (LC–10AT pump, SPD–
(Becton-Dickinson Mountain View, CA, USA) was used for mea-
surements according to the manufacturer instructions. Data were
evaluated with FCS Express 4.0 (de Novo) software. Forward/side
light scatter characteristics was used to exclude the cell debris
from the analysis and lin/peak PMT4 for the discrimination of
doublets.
10Ai, UV–vis detector, Shimadzu, Japan).
4.3.14. Fluorescence detection of superoxide
Dihydroethidium (DHE) was used to estimate intracellular
superoxide production in HL-60 cells.38 Cells (4 ꢀ 105) in medium
were incubated for 24 h at 37 °C, then 2c was applied without or in
the presence of BSO (10
tion of 2c). Influence of the compounds on the generation of free
radicals was monitored after 1 h. DHE (20 M) was added and
lM of BSO has been added 6 h before addi-
4.3.10. Flow cytometry—FDA/PI staining for detection of
apoptosis/necrosis
l
Apoptosis of HL-60 cell line treated with 2 and 4
l
M 2c for 24 h
incubated for 20 min at 37 °C. After incubation, the cells were
washed in PBS and fluorescence was monitored with a fluorescent
microscope (Axio Zeiss Imager A1, camera AxioCam Mrc).
was detected with fluorescent staining using flow cytometry. From
2.5 ꢀ 105 to 5 ꢀ 105 cells of each sample were re-washed twice
with PBS and the pellet was stained with fluorescein diacetate
(200
quently with 5
l
L of 10 nmol/L FDA, 30 min, 25 °C in the dark) and subse-
L of 1 mg/mL PI. Forward and side scatter, red
Acknowledgments
l
(PI) and green (FDA) fluorescence were measured by flow cytome-
try (Canto II, Becton-Dickinson) and analyzed by FCS Express 4.0
(de Novo) software.
Financial support from the Slovak Grant Agency VEGA (Grants
1/0097/10, 2/0177/11 and 1/0672/11) and APVV-0282-10 are
gratefully acknowledged.
4.3.11. Determination of cellular glutathione
References and notes
Cellular glutathione was measured as described.34 Briefly,
2 ꢀ 106 cells were incubated with different concentrations of 2a–
2c for the indicated time intervals, then harvested by centrifuga-
tion, and washed with PBS. The cell pellets were re-suspended in
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