Synthesis and cytotoxic activity of new acridine-thiazolidine derivatives

Article abstract of DOI:10.1016/j.bmc.2012.04.007

Although their exact role in controlling tumour growth and apoptosis in humans remains undefined, acridine and thiazolidine compounds have been shown to act as tumour suppressors in most cancers. Based on this finding, a series of novel hybrid 5-acridin-9

Full text of DOI:10.1016/j.bmc.2012.04.007

Bioorganic & Medicinal Chemistry 20 (2012) 3533–3539
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cytotoxic activity of new acridine-thiazolidine derivatives
Francisco W.A. Barros a, Teresinha Gonçalves Silva b, Marina Galdino da Rocha Pitta b, Daniel P. Bezerra c,
Letícia V. Costa-Lotufo a, Manoel Odorico de Moraes a, Cláudia Pessoa a, Maria Aline F.B. de Moura d,
Fabiane C. de Abreu d, Maria do Carmo Alves de Lima b, Suely Lins Galdino b, Ivan da Rocha Pitta b,
,
⇑
Marilia O.F. Goulart d,
⇑
a Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil
b Laboratório de Planejamento e Síntese de Fármacos – LPSF, Grupo de Pesquisa em Inovação Terapêutica – GPIT, Universidade Federal de Pernambuco, Avenida Moraes Rego 1235,
Cidade Universitária, CEP 50670-901 Recife, Pernambuco, Brazil
c Departamento de Fisiologia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil
d Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Maceió, Alagoas, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Although their exact role in controlling tumour growth and apoptosis in humans remains undefined, acri-
dine and thiazolidine compounds have been shown to act as tumour suppressors in most cancers. Based
on this finding, a series of novel hybrid 5-acridin-9-ylmethylene-3-benzyl-thiazolidine-2,4-diones were
synthesised via N-alkylation and Michael reaction. The cell viability was analysed using a 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and DNA interaction assays were per-
formed using electrochemical techniques.
Received 7 January 2012
Revised 27 March 2012
Accepted 4 April 2012
Available online 10 April 2012
Keywords:
Acridine
Ó 2012 Elsevier Ltd. All rights reserved.
Thiazolidines
Cytotoxicity
DNA sensors
1. Introduction
of acute leukaemias and lymphomas but is ineffective in solid tu-
mours. Widespread clinical use of this compound has been limited
Acridine derivatives have been used for commercial purposes for
more than a century. They are capable of interacting with nuclear
DNA in a sequence-specific manner and with biological targets,
such as topoisomerase I and II (topo I and II) and telomerase. These
compounds play an important role in a variety of diseases, and they
have been used clinically in past decades as antiviral,1,2 antiprion,3
antiprotozoal,4 anti-inflammatory, antineoplasic5 and as analgesic
compounds.6
The biological activity of acridines has been attributed to the
planarity of these aromatic structures, which can intercalate with-
in double-stranded DNA, thus interfering with cellular functions.7
Indeed, the mode of action of acridines has been the focus of con-
tinuous and exciting research.8 In cancer chemotherapy, the bio-
logical targets of acridines include DNA topoisomerases I and/or
II, telomerase/telomeres and protein-kinases.9–15
by problems, such as side effects, drug resistance and poor bioavail-
ability, which have encouraged the further structural modification
of this compound.16
In contrast, thiazolidine compounds have emerged as antineo-
plastic agents with a broad spectrum of antitumour activity against
many human cancer cells.17–23 These molecules are agonists of per-
oxisome proliferator-activated receptor
c (PPARc), which is ex-
pressed in many human tumours, including lung, breast, colon,
prostate and bladder,24 and they modulate the proliferation and
apoptosis of many cancer cell types. Arylidene-thiazolidine-2,4-
diones were also synthesised and screened as anti-inflammatory
compounds, showing considerable biological efficacy, when com-
pared to rosiglitazone, agonist of PPAR
drug.25 As cytotoxic compounds, they were shown to be moderately
active in a range higher than 30
M.26 Troglitazone and derivatives
c and used as a reference
l
Amsacrine is the best-known acridine, and it exhibits potent
cytotoxic activity and has been found to be clinically useful. It
was also one of the first DNA-intercalating agents to be considered
as a topoisomerase II inhibitor. Amsacrine is active in the treatment
were assayed as anticancer against PC-3 and androgen dependent
LNCaP cells. The respective IC50 values for troglitazone and its
D
2-dehydro derivative were 30 2 and 20
2 lM in PC-3 cells
and 22 3 and 14
1
l
M in LNCaP cells.27
Considering these facts, our strategy was to couple acridine and
thiazolidine nucleus to obtain a new class of compounds, the thiaz-
acridine derivatives. By assaying their biological activities using di-
verse techniques based on various mechanisms of action, we found
⇑
Corresponding authors.
E-mail address: mofg@qui.ufal.br (M.O.F. Goulart).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.007

3534
F. W. A. Barros et al. / Bioorg. Med. Chem. 20 (2012) 3533–3539
these derivatives to be a new class of drugs that are effective in
cancer therapy.
from diphenylamine with zinc dichloride in acetic acid according
to Tsuge et al. (1963).32 Subsequently, the oxidation of 1 with
pyridinium chlorochromate, according to Mosher and Natale
(1995),33 gave the 9-acridinaldehyde 2. The synthetic pathways
are illustrated in Scheme 1.
It is clearly of fundamental importance to explore the factors
that determine the affinity and selectivity of DNA-binding com-
pounds to ascertain the nature and potency of such molecules,
particularly with respect to their potential to cause DNA damage.
In this context, the need for stable, low cost, and readily adaptable
analytical tools for the detection of DNA damage has been the driv-
ing force in the development of DNA-biosensor technology.28,29 An
electrochemical DNA-biosensor is a receptor-transducer that em-
ploys double-stranded DNA (dsDNA) immobilised onto the surface
of an electrochemical transducer as a molecular recognition ele-
ment through which specific DNA-binding processes may be as-
sessed.28,30 The interaction of an analyte (i.e., a drug, pro-drug or
in situ-generated intermediate) with dsDNA may lead to the rup-
ture of hydrogen bonds and the consequential opening of the dou-
ble helix, resulting in increased accessibility to the constituent
bases. The extent of DNA damage may be determined by monitor-
ing the oxidation of the exposed bases by voltammetric methods.
The electrochemical characteristics of such dsDNA-biosensors have
been evaluated, and it is clear that this approach can provide a
greater understanding of the mechanism of interaction between
drugs and DNA and can offer new insights in rational drug
design.30,31
The acridinylidene-thiazolidine 9 was isolated as a single isomer.
X-ray crystallographic studies and 13C NMR have demonstrated a
preferred Z configuration for 5-benzylidene-thiazolidinones and
3
5-benzylidene-imidazolinones.34,35 The JCH-coupling constant va-
lue between the ethylene hydrogen and the carbon atom located
at the 4-position of the heterocyclic ring is of special interest in this
structural elucidation.36 In contrast, compounds 10 and 12 were iso-
lated as isomeric mixtures. The Z isomer was the major stereoiso-
mer formed, and the isomers were readily identified by 1H NMR,
as the ethylene hydrogen is more deshielded in the Z isomer than
it is in the E isomer. The Z isomer was isolated for the derivative 11.
2.2. Biological assays
2.2.1. Cytotoxicity assay
The cytotoxicity of the acridine-thiazolidine derivatives and of
some of the precursors: 3 (3-acridin-9-yl-2-cyano-acrylic acid ethyl
ester), 5 (3-(4-methyl-benzyl)-thiazolidine-2,4-dione) and 6 (3-(4-
bromo-benzyl)-thiazolidine-2,4-dione) was evaluated against tu-
mour cell lines of different histotypes using a previously described
MTT assay.37 Amsacrine was used as a positive control. Table 1
summarises the IC50 data for cytotoxic activity. The results indi-
cated that the precursor thiazolidine-2,4-diones 5 and 6 were
Herein, we describe the synthesis of the following acridine-
thiazolidine derivatives: 5-(acridin-9-ylmethylene)-3-(4-methyl-
benzyl)-thiazolidine-2,4-dione (9), 5-(acridin-9-ylmethylene)-
3-(4-bromo-benzyl)-thiazolidine-2,4-dione (10), 5-(acridin-9-
ylmethylene)-3-(4-chloro-benzyl)-thiazolidine-2,4-dione (11),
inactive (IC50 >25
3-acridin-9-yl derivative was significantly active against HL-60
(4.07 M). The thiazacridines exhibit relatively high cytotoxicity
lg/mL) (not shown in Table 1) and that the
and
5-(acridin-9-ylmethylene)-3-(4-fluoro-benzyl)-thiazolidine-
2,4-dione (12). Moreover, the cytotoxic properties of these
derivatives and of some of their precursors were examined in differ-
ent histotype cancer cell lines, and they were also tested for DNA
interaction, using electrochemical methods and DNA biosensors.
l
against colon carcinoma and glioblastoma tumour cell lines, pos-
sessing IC50 values in the range of 7.4–46.4, 7.2–35.5, 5.8–29.0,
and 5.6–58.0
amsacrine was most active showing IC50 values ranging from 0.08
to 3.3 M.
lM for (9, 10, 11) and (12), respectively. However,
2. Results and discussion
2.1. Chemistry
l
The cytotoxicity of the quimeric thiazacridines was also evalu-
ated against normal cells (PBMC and V79). The results presented
in Table 1 show that the cytotoxic effects of these acridine-thiazol-
idine derivatives were less pronounced in normal cells, with a
selectivity index (SI) for glioblastoma (SF-295) of 3.7, 3.1, 3.1 and
5.9 for 9–12, respectively. In addition, these compounds were not
found to be active in leukaemia, breast carcinoma or normal lym-
phoblast cells.
The thiazacridine derivatives 9–12 were synthesised by the
nucleophilic addition of substituted 3-benzyl-thiazolidine-diones
5–8 on 3-acridin-9-yl-2-cyano-acrylic acid ethyl ester 3 (Scheme
1). Indeed, the direct condensation of 9-acridinaldehyde 2, with
the substituted thiazolidines 5–8, did not lead to the expected
acridinylidene-thiazolidines. 9-Methyl-acridine 1 was prepared
CH3
CHO
N
O
PCC
NH
S
N
O
4
2
1
NCCH2COOCH2CH3
RC6H4CH2Cl
COOCH2CH3
CN
O
O
NC5H5
N
N
+
N
S
S
N
R
R
O
O
5 - 8
Scheme 1. Synthetic route involved in the preparation of acridine-thiazolidine derivatives.
3
9 R = CH3 10 R = Br 11 R = Cl 12 R = F

Table 1
Cytotoxic activity of acridine-thiazolidine derivatives on cancer and normal cells, in
lg/mL (lM)
Cell lineb
Histotype
Acridine derivativesa
9
10
11
12
3
Amsacrinec
Tumour cell
HL-60
K-562
CEM
HCT-8
HCT-15
SW-620
COLO-205
MDA-MB-231
HS-578-T
MX-1
PC-3
DU-145
SF-295
Promyelocytic leukaemia
Myeloblastoid leukaemia
Lymphoblastic leukaemia
Colon carcinoma
Colon carcinoma
Colon carcinoma
Colon carcinoma
Breast carcinoma
Breast carcinoma
Breast carcinoma
Prostate carcinoma
Prostate carcinoma
Glioblastoma
>25 (60.9)
>25 (60.9)
>25 (60.9)
3.1 (7.4) 2.4–3.8
11.9 (29.2) 7.5–19.0
11.0 (26.8) 6.7–18.1
19.1 (46.4) 8.6–42.3
>25 (60.9)
>25 (52.6)
>25 (52.6)
>25 (52.6)
5.3 (11.2) 4.5–6.3
9.4 (19.8) 4.9–18.0
16.9 (35.5) 11.2–23.3
>25 (52.6)
Nd
Nd
>25 (58.0)
>25 (58.0)
>25 (58.0)
3.6 (8.3) 3.0–4.3
9.3 (21.5) 6.6–13.1
8.7 (20.1) 5.8–13.1
2.7 (6.3) 1.8–4.1
>25 (58.0)
>25 (58.0)
>25 (58.0)
5.6 (13.0) 3.7–8.5
4.1 (9.5) 3.5–4.8
2.5 (5.8) 1.8–2.8
2.5 (5.7) 1.8–3.5
12.5 (29.0) 8.4–18.7
9.6 (22.2) 7.7–11.8
>25 (60.3)
>25 (60.3)
>25 (60.3)
2.3 (5.6) 1.8–3.0
24.0 (58.0) 13.4–43.2
12.9 (31.3) 7.8–19.4
9.5 (23.0) 5.5–16.4
Nd
1.23 (4.07) 0.98–1.55
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
0.03 (0.08) 0.01–0.09
Nd
Nd
0.1 (0.3) 0.03–0.3
0.1 (0.3) 0.1–1.7
0.1 (0.3) 0.1–0.2
0.4 (1.1) 0.4–2.0
Nd
>25 (60.9)
>25 (60.9)
Nd
Nd
Nd
Nd
Nd
7.3 (17.7) 5.5–9.5
>25 (60.9)
3.2 (7.8) 2.6–4.0
8.1 (19.7) 5.6–11.7
8.6 (21.1) 5.3–14.11
6.4 (15.6) 4.9–8.2
4.1 (8.6) 2.4–6.8
Nd
3.4 (7.2) 2.8–4.1
17.7 (37.2) 13.4–23.4
24.1 (50.7) 17.8–32.6
12.3 (25.9) 10.2–14.9
11.2 (27.1) 6.1–20.8
Nd
2.3 (5.6) 1.8–2.9
3.9 (9.3) 2.6–5.8
3.9 (9.5) 2.7–5.6
5.8 (14.1) 4.9–7.0
1.3 (3.3) 0.5–3.2
0.10 (0.3) 0.06–0.18
0.18 (0.5) 0.08–0.43
0.5 (1.4) 0.3–0.9
Nd
16.52 (54.68) 11.60–23.52
7.04 (23.30) 5.73–8.66
Nd
Nd
OVCAR-8
UACC-62
MDA-MB-435
Normal cells
PBMCd
Ovarian carcinoma
Melanoma
Melanoma
Nd
Peripheral lymphoblast
Lung fibroblastse
>25 (60.9)
11.7 (28.5) 7.8–18.5
>25 (52.6)
10.7 (22.6) 6.4–18.1
>25 (58.0)
7.7 (17.9) 5.7–10.5
>25 (60.3)
13.5 (32.7) 9.1–20.1
Nd
Nd
8.5 (21.7) 6.8–9.9
2.3 (5.8) 1.7–3.6
V79
a
b
c
Data are presented as IC50 values in lg/mL (lM) and as the 95% confidence interval obtained by nonlinear regression for all of the cell lines from two independent experiments, performed in duplicate, after 72 h of incubation.
Cell survival was evaluated by the MTT assay, as reported in the Section 4.
Amsacrine was used as a positive control.
Cell survival was evaluated by the Alamar blue assay, as reported in the Section 4.
Chinese hamster cell line. Nd: not determined.
d
e

3536
F. W. A. Barros et al. / Bioorg. Med. Chem. 20 (2012) 3533–3539
The selectivity index of amsacrine to glioblastoma was of 11.6
we propose that the modified acridine-thiazolidines could be
promising key structures in anticancer drug development.
showing a greater selectivity for tumour cells compared with
thiazacridines. The SI was calculated with the following formula:
SI = IC50(V79)/IC50(SF-295).
4. Experimental part
4.1. Chemistry
Although the thiazacridines were shown to be less active and less
selective than amsacrine, the selectivity for solid tumour cell lines is
interesting and deserves attention as a target for study. In fact, some
compounds, such as bisannulated acridines, have been shown to
have solid tumour-selective cytotoxicity.38 Some aminoderivatives
of azapyranoxanthenone have shown greater cytotoxic potential
against colon tumour cells than against leukaemia cells.39 However,
the basis for this solid tumour selectivity remains unclear.
None of the compounds described here were able to cause
haemolysis in mouse erythrocytes, even at the highest concentra-
The melting points were measured in capillary tubes on a Buchi
(or Quimis) apparatus. Thin layer chromatography was performed
on silica gel plates from Merck (60F254). The infrared spectra of 1%
KBr pellets were recorded on a Bruker IFS66 spectrometer. 1H NMR
spectra were recorded on a Bruker AC 300 P spectrophotometer
using DMSO-d6 as the solvent, with tetramethylsilane as an inter-
nal standard. Electronic impact mass spectra were measured at
70 eV on a Finnigan GCQ Mat Quadrupole Ion-Trap. The MS data
fully agreed with the proposed structures.
The chemical data on 5-acridin-9-ylmethylene-3-(4-methyl-
benzyl)-thiazolidine-2,4-dione 9, 5-acridin-9-ylmethylene-3-(4-
bromo-benzyl)-thiazolidine-2,4-dione 1042 and 5-(acridin-9-
ylmethylene)-3-(4-fluoro-benzyl)-1,3-thiazolidine-2,4-dione 1243
are reported elsewhere.42,43 Thiazolidine-2,4-dione was N-(3)-
alkylated in the presence of potassium hydroxide, which enabled
the thiazolidine potassium salt to react with substituted benzyl
halide in a hot alcohol medium.
tion (200 lg/mL) (data not shown). The absence of lytic effects sug-
gests that the cytotoxicity of these compounds is not related to
membrane disruption and is likely related to more specific cellular
pathways. The target could be DNA, as has been observed for acri-
dine compounds.40,41
2.2.2. DNA interaction assay
Results from the dsDNA biosensor show positive interactions
for all of the acridines, as represented by the appearance of guano-
sine (Ep = 0.8 V) and adenosine (Ep = 1.0 V) peaks (Figs. 1A–4A).
This clearly demonstrates that damage from the compounds
caused a distortion of the double helix and exposure of the bases
to oxidation. In the experiments using ssDNA in solution, a de-
crease of the oxidation waves of the nucleobases, guanosine and
adenosine, with eventual anodic potential shifts (mainly for 11),
were observed, confirming the interaction with DNA (Figs. 1B–4B).
4.1.1. (5Z)-5-(Acridin-9-ylmethylene)-3-(4-chloro-benzyl)-1,3-
thiazolidine-2,4-dione 11
4-Chloro-benzylthiazolidine (0.9 mmol) and 3-acridin-9-yl-2-
cyano-acrylic acid ethyl ester (0.9 mmol) were dissolved in abso-
lute ethanol (8 mL). The solution was refluxed for 4 h in the
presence of a small amount of piperidine as a catalyst. The precip-
itate obtained was filtered and washed with water. C24H15ClN2O2S.
Yield 51%. Mp 204–206 °C. TLC Rf 0.73, (n-hexane/ethyl acetate 6:4).
3. Conclusion
IR cmÀ1 (KBr)
m
1748, 1695, 1624, 1379, 1334, 1149, and 760. 1H
The acridine-thiazolidines derivatives herein investigated
showed promising cytotoxic activity. The synthesis of the quimeric
compounds is worthy once there was an increase of cytotoxic
activity compared to precursors. Although less active and selective
than the positive control amsacrine, all of them exhibited relatively
high cytotoxicity, predominantly on colon carcinoma and glioblas-
toma tumour cell lines, whereas no activity on leukaemia, breast
carcinoma, or normal lymphoblast cells was observed. Taking into
account the selectivity for cells of solid tumours, as well as, the po-
sitive interaction with the DNA shown in the electrochemical tests,
NMR (d ppm, DMSO-d6) 4.88 (s, CH2); 7.46 (s, 4H benzyl), 7.69
(dt, 2H 2,7-acridin, J = 7.8 and 1.2 Hz), 7.92 (dt, 2H 3,6-acridin,
J = 7.8 and 1.2 Hz), 8.15 (d, 2H 1,8-acridin, J = 8.4 Hz), 8.24 (d, 2H
4,5-acridin, J = 8.4 Hz), 8.79 (s, 1H, ethylene). 13C NMR (DMSO-d6,
DEPT): d 44(CH2), 122.3(2C), 125.6 (2CH), 126.9 (2CH), 128.5
(2CH), 129 (C), 129.7 (C), 129.9 (2CH), 130.7 (2CH), 131.9 (C),
132.5 (C), 134.2 (C), 138.0 (2C), 148 (2CH), 164 (CO), 166.8 (CO).
Ms EI 70 eV, m/z (%) 430 (M+ 2.5), 305(20.3), 235(100), 234(75.1),
231(80.1), 203(16.8), 190(16.9), and 125(28.8).
24
16
A
B
9 + dsDNA
9 and ssDNA solution
ssDNA solution
14
dsDNA
20
16
12
8
O
O
N
12
N
S
N
S
CH3
O
N
CH3
O
10
8
6
4
4
2
0
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.2
0.4
0.6
0.8
1.0
1.2
1.4
(V) vs Ag/AgCl/Cl- (0.1 mol L-1)
E
(V) vs Ag/AgCl/Cl- (0.1 M)
E
Figure 1. (A) Differential pulse voltammogram (DPV) at pH 4.5 for the oxidation of 9 attached to the surface of carbon paste electrode (CPE) in the presence of a ssDNA
solution (1.0 Â 10À4 M). (B) Differential pulse voltammogram, at pH 4.5, of the dsDNA biosensor in the absence and in the presence of 9 in solution (1.0 Â 10À4 M).
Equilibration time = 5 s,
DEs = 2 mV, DEdpv = 50 mV, m .
= 5 mV sÀ1

F. W. A. Barros et al. / Bioorg. Med. Chem. 20 (2012) 3533–3539
3537
24
20
16
12
8
16
B
10 + ssDNA solution
A
10 + dsDNA
dsDNA
ssDNA solution
14
O
O
N
12
N
S
N
Br
O
S
N
Br
10
8
O
6
4
4
2
0
0
0.2
0.4
E
0.6
0.8
1.0
1.2
1.4
0.2
0.4
0.6
0.8
1.0
1.2
1.4
(V) vs Ag/AgCl/Cl- (0.1 M)
E
(V) vs Ag/AgCl/Cl- (0.1 M)
Figure 2. (A) Differential pulse voltammogram at pH 4.5 for the oxidation of 10 attached to the surface of CPE in the presence of a ssDNA solution (1.0 Â 10À4 M). (B)
Differential pulse voltammogram at pH 4.5 of the dsDNA biosensor in the absence and in the presence of 10 in solution (1.0 Â 10À4 M). Equilibration time = 5 s,
DEs = 2 mV,
D
Edpv = 50 mV,
m
= 5 mV sÀ1
.
11 + dsDNA
dsDNA
16
14
12
10
8
24
B
11 + ssDNA solution
ssDNA solution
A
20
16
12
8
O
O
N
N
S
N
Cl
S
N
O
Cl
O
6
4
4
2
0
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.2
0.4
0.6
0.8
1.0
1.2
1.4
(V) vs Ag/AgCl/Cl- (0.1 M)
E
(V) vs Ag/AgCl/Cl- (0.1 M)
E
Figure 3. (A) Differential pulse voltammograms at pH 4.5 for the oxidation of 11 attached to the surface of CPE in the presence of a ssDNA solution (1.0 Â 10À4 M). (B)
Differential pulse voltammogram at pH 4.5 of the dsDNA biosensor in the absence and in the presence of 11 solution (1.0 Â 10À4 M). Equilibration time = 5 s,
DEs = 2 mV,
D
Edpv = 50 mV,
m
= 5 mV sÀ1
.
12 + dsDNA
dsDNA
16
14
12
10
8
A
B
12 + ssDNA
ssDNA
24
20
16
12
8
O
O
N
N
S
N
F
O
S
N
F
O
6
4
4
2
0
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.2
0.4
0.6
0.8
1.0
1.2
1.4
-
E
(V) vs Ag/AgCl/Cl (0.1 mol L-1)
-
E
(V) vs Ag/AgCl/Cl (0.1 M)
Figure 4. (A) Differential pulse voltammogram at pH 4.5 for the oxidation of 12 attached to the surface of CPE in the presence of a ssDNA solution (1.0 Â 10À4 M). (B)
Differential pulse voltammogram at pH 4.5 of the dsDNA biosensor in the absence and in the presence of 12 in solution (1.0 Â 10À4 M). Equilibration time = 5 s,
DEs = 2 mV,
D
Edpv = 50 mV, m
= 5 mV sÀ1

3538
F. W. A. Barros et al. / Bioorg. Med. Chem. 20 (2012) 3533–3539
4.2. Biological
lowing the method described by Costa-Lotufo et al. (2002).45 The
diluted compounds were tested at concentrations ranging from
1.5 to 200 mg/mL. Triton X-100 (1%) was used as a positive control.
After incubation at room temperature for 30 min followed by cen-
trifugation, the supernatant was removed, and the liberated hae-
moglobin was measured spectrophotometrically at 540 nm.
4.2.1. MTT assay
The cytotoxic effects of the synthesised compounds were eval-
uated against the following human cancer cell lines, all obtained
from the National Cancer Institute, Bethesda, MD, USA: HL-60 (pro-
myelocytic leukaemia), K-562 (myeloblastoid leukaemia), CEM
(lymphoblastic leukaemia), HCT-8 (colon carcinoma), HCT-15 (co-
lon carcinoma), SW-620 (colon carcinoma), COLO-205 (colon carci-
noma), MDA-MB-231 (breast carcinoma), HS-578-T (breast
carcinoma), MX-1 (breast carcinoma), PC-3 (prostate carcinoma),
DU-145 (prostate carcinoma), SF-295 (glioblastoma), OVCAR-8
(ovarian carcinoma), UACC-62 (melanoma), and MDA-MB-435
(melanoma). Chinese hamster lung fibroblasts (V79 – normal
cells), kindly provided by Dr. J.A.P. Henriques (Federal University
of Rio Grande do Sul, Porto Alegre, Brazil), were also used. The cell
lines were maintained in RPMI-1640 medium (cancer cells) or
MEM with Earle’s salts (V79 cells) supplemented with 10% foetal
4.3. DNA interaction assay
4.3.1. Electrochemical approaches
The following experiments were conducted to evaluate the inter-
action of acridine-thiazolidine derivatives with DNA by electroche-
mical methods. Electrochemical experiments including differential
pulse voltammetry (DPV) were performed using an Autolab (Echo-
Chemie, Utrecht, Netherlands) PGSTAT 20 or PGSTAT-30. The work-
ing electrodes were a BAS (Bioanalytical Systems, West. Lafayette,
IN, USA) GC electrode of 3-mm diameter or a carbon paste electrode
and a carbon paste electrode modified with acridine-thiazolidine
derivatives. The counter electrode was a platinum coil, and the re-
ference electrode was Ag|AgCl, ClÀ (0.1 M); all electrodes were con-
tained in a single-compartment electrochemical cell with a 10 mL
capacity. The optimised differential pulse voltammetry parameters
bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 lg/
mL streptomycin at 37 °C with 5% CO2.
Cell growth was quantified by the ability of living cells to reduce
a yellow dye, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-
zolium bromide (MTT), to a purple formazan product.37 For all of
the experiments, the cells were seeded in 96-well plates
(0.7 Â 105 cells/well for adherent cells and 0.3 Â 106 cells/mL for
were as follows: pulse amplitude (
D
Esw) of 50 mV, pulse width of
Es] of
70 ms and a scan rate of 5 mV sÀ1 (using a step potential [
D
2 mV). The glassy carbon electrode was polished with alumina on
a polishing felt (BAS polishing kit). After mechanical cleaning, the
electrochemical pretreatment of the glassy carbon electrode in-
volved a sequence of five cyclic potential scans from 0 to +1.4 V in
acetate buffer at pH 4.5. All of the experiments were carried out at
room temperature (25 1 °C).
suspended cells). After 24 h, the compounds (0.048–25 lg/mL),
dissolved in DMSO, were added to each well (using an HTS
(high-throughput screening) Biomek 3000, Beckman Coulter, Inc.
Fullerton, California, USA) and incubated for 72 h. Amsacrine (Sig-
ma Aldrich Co., St. Louis, MO, USA) was used as a positive control.
At the end of the incubation, the plates were centrifuged, and the
medium was replaced by fresh medium (150
0.5 mg/mL MTT. After 3 h, the formazan product was dissolved in
150 L DMSO, and the absorbance was measured using a multi-
plate reader (DTX 880 Multimode Detector, Beckman Coulter,
Inc., Fullerton, California, USA). The substance effect was quantified
as the percentage of the control absorbance at 595 nm.
l
L) containing
4.3.1.1. Preparation of the dsDNA-GC biosensor and its interac-
tion with acridine-thiazolidine derivatives.
The electroche-
l
mical procedure for the investigation of the acridine-thiazolidine
derivatives and dsDNA interaction involved three steps: prepara-
tion of the electrode surface, immobilisation of the dsDNA gel
and voltammetric transduction. The GC electrode was first po-
lished with alumina, using a Metrohm felt-polishing pad, until
the surface displayed a mirror-like appearance. The electrode
was then electrochemically pretreated with a sequence of five cyc-
lic potential scans from 0 to +1.4 V versus Ag|AgCl, ClÀ (0.1 M) in
acetate buffer,28 washed thoroughly with distilled/deionised
water, dried and placed in an upright position in the cell.
To immobilise the dsDNA (calf thymus, type 1), the surface of
the electrode was coated with 10 lL of calf thymus DNA solution
(containing 12.0 mg of dsDNA in 1.0 mL of acetate buffer). The
quantity (0.36 mg) of dsDNA employed was estimated to be suffi-
cient to cover the entire surface of the GC electrode.28 The dsDNA
was allowed to dry at room temperature under a stream of nitro-
4.2.2. Alamar blue assay
The cytotoxic effects of the synthesised compounds were eval-
uated against PBMC (peripheral blood mononuclear cells) from
healthy donors using the Alamar blue assay.44 Heparinised blood
(from healthy, non-smoker donors who had not taken any drug
for at least 15 days prior to sampling) was collected, and the PBMC
were isolated by a standard method of density-gradient centrifuga-
tion using Ficoll-Hypaque. PBMC were cultivated in RPMI-1640
medium supplemented with 20% foetal bovine serum, 2 mM gluta-
mine, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C
with 5% CO2. Phytohaemagglutinin (2%) was added at the begin-
ning of the culture period.
gen. Immediately after drying, 20 lL of an ethanolic solution of
Briefly, the PBMC were plated in 96-well plates (3 Â 105 cells/mL
the acridine-thiazolidine derivatives (10À4 M) was added with se-
quential drying. The prepared biosensor was then put into the ap-
propriate cell, covered with 5 mL of aqueous acetate buffer and
analysed at E = 0 to 1.4 V.28 For each series of experiments, an iden-
tical dsDNA-GC electrode was prepared as a reference blank as a
control; this electrode was not treated with substrate but received
the same pre- and post-treatments as the test electrode.
in 100 mL of medium). After 24 h, the compounds (0.048–25 lg/
mL), dissolved in DMSO, were added to each well (using an HTS
Biomek 3000, Beckman Coulter, Inc., Fullerton, California, USA)
and incubated for 72 h. Amsacrine (Sigma Aldrich Co., St. Louis,
MO, USA) was used as a positive control. At 24 h before the end of
the incubation, 10 mL of a stock solution (0.312 mg/mL) of Alamar
blue (resazurin, Sigma Aldrich Co., St. Louis, MO, USA) was added to
each well. The absorbance was measured using a multiplate reader
(DTX 880 Multimode Detector, Beckman Coulter, Inc., Fullerton,
California, USA), and the effect of the drug was quantified as the
percentage of the control absorbance at 570 and 595 nm.
The procedure produced a thick-layer dsDNA-modified elec-
trode. Because uniform coverage of the electrode surface had been
achieved, any new peaks observed in the presence of the additive
were due solely to the interaction of the analyte with the DNA film
not from the diffusion process in solution.29
4.2.3. Haemolysis assay
4.3.1.2. Preparation of the carbon paste electrode modified
The test was performed in 96-well plates using a 2% mouse er-
ythrocyte suspension in 0.85% NaCl containing 10 mM CaCl2 fol-
with acridine-thiazolidine derivatives.
interaction between test substances and ssDNA are generally
Analyses of the

F. W. A. Barros et al. / Bioorg. Med. Chem. 20 (2012) 3533–3539
3539
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dine samples (10À4 mol LÀ1
) and stirred for approximately
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complete evaporation, the mixture was subjected to a nitrogen
atmosphere. Next, 20 lL of commercial mineral oil was added
and mixed strongly to achieve a uniform texture. In this case, a
blank (control) was also produced by the addition of ethanol only
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4.3.1.3. Preparation of ssDNA and its interaction with acridine-
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Single-stranded DNA (ssDNA) was
prepared by dissolving 3.0 mg of dsDNA in 1.0 mL of hydrochloric
acid (1 M) and heating for 1 h until complete dissolution. This
treatment was followed by neutralising the solution with 1.0 mL
of sodium hydroxide (1 M) and 9 mL of acetate buffer was then
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Freshly prepared ssDNA solution was added to the cell, and sin-
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+1.4 V versus Ag|AgCl, ClÀ (0.1 M). Two peaks corresponding to
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potentials of +0.815 and +1.131 V, respectively. To ensure reprodu-
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oxidation current and potential of the bases were very similar (rsd
of 5%). After this process, the carbon paste electrode modified with
acridine-thiazolidine derivatives was inserted into the ssDNA
solution, and the DPV experiment was performed. An unmodified
carbon paste electrode was also employed in the DPV experiments
involving the ssDNA solution and was used for comparison.
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This study was supported by the Brazilian National Research
Council and National Institute of Science and Technology for
Pharmaceutical Innovation (CNPq/RENORBIO/INCT_IF), as well as
the INCT-Bioanalítica. The authors wish to thank Professor José
Dias de Souza Filho (Universidade Federal de Minas Gerais, Belo
Horizonte, MG, Brazil) for the obtention of the 13C NMR.
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Supplementary data associated with this article can be found, in
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