DNA binding, anti-tumour activity and reactivity toward cell thiols of acridin-9-ylalkenoic derivatives

Article abstract of DOI:10.1007/s12039-015-0851-9

Abstract In this paper, we describe the synthesis, biochemical properties and biological activity of a series of new 9-substituted acridine derivatives with a reactive alkene moiety: 9-[(E)-2-phenylethenyl] acridine (1) and methyl (2E)-3-(acridin-9-yl)-pr

Full text of DOI:10.1007/s12039-015-0851-9

c
J. Chem. Sci. Vol. 127, No. 5, May 2015, pp. 931–940. ꢀ Indian Academy of Sciences.
DOI 10.1007/s12039-015-0851-9
DNA binding, anti-tumour activity and reactivity toward cell thiols of
acridin-9-ylalkenoic derivatives
O SALEMa, M VILKOVAb, J PLSIKOVAa, A GROLMUSOVAc, M BURIKOVAc,
M PROKAIOVAb, H PAULIKOVAc, J IMRICHb and M KOZURKOVAa,∗
aDepartment of Biochemistry, P J Šafárik University in Kosice, Faculty of Science, Moyzesova 11,
SK-04001 Kosice
bDepartment of Organic Chemistry, P J Šafárik University in Kosice, Faculty of Science, Moyzesova 11,
SK-04001 Kosice
cDepartment of Biochemistry and Microbiology, Faculty of Chemical and Food Technology,
Slovak Technical University, Radlinského 9, SK-81237 Bratislava, Slovak Republic
e-mail: maria.kozurkova@upjs.sk
MS received 10 October 2014; revised 15 January 2015; accepted 24 January 2015
Abstract. In this paper, we describe the synthesis, biochemical properties and biological activity of a series
of new 9-substituted acridine derivatives with a reactive alkene moiety: 9-[(E)-2-phenylethenyl] acridine (1)
and methyl (2E)-3-(acridin-9-yl)-prop-2-enoate (2). The interaction of derivatives 1 and 2 with calf thymus
DNA was investigated using UV-Vis, fluorescence and circular dichroism spectroscopy. The binding constants
K were estimated as being in the range of 1.9 to 7.1 × 105 M−1, and the percentage of hypochromism was
found to be 40–57% (from spectral titration). UV-Vis, fluorescence, and CD measurements indicate that the
compounds were effective DNA-intercalating agents. Electrophoretic separation proved that ligands 1 and 2
relaxed topoisomerase I at a concentration of 5 μM. Ester 2 was shown to have a stronger cytostatic effect on
leukemia cell line L1210 than alkene 1. The incubation of ligands 1 and 2 with the ovarian carcinoma cell line
A2780 confirmed their extensive cytotoxic effects, an effect which was particularly pronounced in the case of
ligand 2. Cytotoxicity tests against A2780 cells demonstrate that a conjugate of compound 2 with L-cysteine
(3) is less cytotoxic than compound 2, especially at concentrations greater than 10 μM.
Keywords. DNA binding; anti-tumour activity; acridin-9-ylalkenoic derivatives, glutathione.
(m-AMSA), 4ꢁ-(acridin-9-ylamino)-methanesulfon-m-
anisidide, has been in clinical use since the 1970s. The
effect of amsacrine and its second-generation analogues
is closely associated with the ability of these DNA
intercalators to interfere adversely with DNA strand
cleavage.1,8 The cytotoxic effect of most acridine-based
drugs is based on their ability to suppress topoisomerase
activity. Some acridine derivatives can act as multi-
target anti-cancer drugs. In addition to demonstrating
a high affinity to DNA, acridine derivatives with reac-
tivity towards thiols are able to modulate the level of
cellular thiols. The reactivity of such substances with
sulfhydryls can alter intracellular glutathione redox
status (GSSG/GSH), which modulates a wide range
of cellular events such as selective gene expression,
DNA synthesis, and the regulation of the cell cycle
Moreover a reduction in the GSH level decreases the
detoxification potency of the cells and GSH depletion
might trigger subsequent apoptosis by leaving the cells
unprotected against the production of thiol oxidation
radicals.9–11
1. Introduction
Acridine is one of the most commonly used DNA
intercalating scaffolds for anti-cancer, anti-viral, anti-
malarial, and anti-bacterial agents and has been the focus
of extensive studies.1–4 DNA is generally the primary
intracellular target of anti-cancer drugs, so any interac-
tion between small molecules and DNA can cause DNA
damage in cancer cells thereby inhibiting the growth
of cancer and resulting in cell death or apoptosis.5–7
The intercalation of planar molecules between base
pairs causes the DNA helix to extend and decreases
its winding angle. The archetypal DNA-intercalating
agents are 9-aminoacridine and its derivatives. These
compounds bind reversibly to DNA by intercalation,
but they do not usually interact with it covalently.
The intercalation of the acridines is facilitated by
cation formation and sufficient levels of molecular
planarity. The 9-anilinoacridine derivative amsacrine
∗For correspondence
931

932
O Salem et al.
(DMSO, Serva), and calf thymus DNA were obtained
In our previous work12 we studied new acridine–
from Sigma-Aldrich Chemie (Germany). EDTA, RNase
A, and proteinase K were purchased from Serva
(Germany). Plasmid pUC 19 (2761 bp, DH 5α),
agarose (type II No-A-6877) (Sigma), 5,5ꢁ-dithio-bis(2-
nitrobenzoic acid) (DTNB) were obtained from Merck
(Germany). All other chemicals were purchased from
Lachema (Czech Republic).
thiazolidinone derivatives and their interactions with
calf thymus DNA and HL60, L1210 and A2780 cell
lines. All of the tested derivatives displayed strong cyto-
toxic activity in vitro. The cancer cells accumulated
acridine derivatives very rapidly and changes in the glu-
tathione level were confirmed. The compounds inhib-
ited the proliferation of the cancer cells and induced an
arrest in the cell cycle and cell death. Their influence
on the cells was associated with their reactivity towards
thiols and DNA binding activity.
2.2 Chemistry
In this paper we describe the synthesis, biochemical
properties, and biological activity of novel 9-substituted
acridine derivatives with a reactive alkene moiety: 9-
[(E)-2-phenylethenyl]acridine (1) and methyl (2E)-3-
(acridin-9-yl)-prop-2-enoate (2). These compounds may
show potential as anti-cancer agents due to their sensi-
tivity toward nucleophilic groups present in biomole-
cules. Our study focuses on the capability of both
compounds to bind to DNA and interfere with human
topoisomerase I, and also examines the in vitro anti-
proliferative activity of the derivatives against mouse
lymphocytic leukemia cells (L1210) and human ovarian
cancer cell lines (A2780). The high level of reactivity
between the thiol group and the double bond is well-
known, and therefore the new derivatives were also
tested for their ability to reduce the level of glutathione
in cells. For this purpose, a conjugate of ester 2 with
cysteine 3-{[1-(acridin-9-yl)-3-methoxy-3-oxopropyl]
sulphanyl}-2-aminopropanoic acid 3 was prepared and
the effect of the ester on cell viability was evaluated.
2.2a Synthesis of 9-[(E)-2-phenylethenyl]acridine (trivial
9-(2-styryl)acridine) (1): Sodium hydride (60% purity,
145 mg, 3.63 mmol) was added to a solution of benzyl-
triphenyl-phosphonium chloride (1000 mg, 2.57 mmol)
in anhydrous tetrahydrofuran (5 mL). The solution was
cooled to −10◦C and stirred for 30 min. An equimolar
amount of acridin-9-carbaldehyde13 (500 mg, 2.41 mmol)
was added and the solution was stirred for 1 h at −10◦C.
The resulting mixture was allowed to reach room tem-
perature and the stirring was continued for 12 h. The
solvent was evaporated under reduced pressure and the
residue was purified with column chromatography (SiO2,
9:1 v/v cyclohexane/acetone) to produce derivative 1 as
a pure Z isomer.
Yield: 320 mg (47%), M.p. 184–185◦C (ethyl
acetate). 1H NMR (400 MHz, 25◦C, CDCl3): δ 8.32 (dd,
J = 8.4, 1.0 Hz, 2H, H-1ꢁ,8ꢁ), 8.25 (br d, J = 8.4
Hz, 2H, H-4ꢁ,5ꢁ), 7.88 (d, J = 16.4 Hz, 1H, H-β), 7.78
(ddd, J = 8.4, 6.4, 1.0 Hz, 2H, H-3ꢁ,6ꢁ), 7.68 (dd, J =
8.0, 1.6 Hz, 2H, H-2ꢁꢁ,6ꢁꢁ), 7.51 (ddd, J = 8.4, 6.4, 0.8
Hz, 2H, H-2ꢁ,7ꢁ), 7.46 (t, J = 8.0 Hz, 2H, H-3ꢁꢁ,5ꢁꢁ),
7.40 (m, 1H, H-4ꢁꢁ), 7.04 (d, J = 16.4 Hz, 1H, H-α)
ppm. 13C NMR (100 MHz, 25ꢁC, CDCl3): δ 148.7 (C-
4ꢁa,10ꢁa), 143.2 (C-9ꢁ), 139.5 (C-α), 136.5 (C-1ꢁꢁ), 130.0
(C-3ꢁ,6ꢁ), 129.9 (C-4ꢁ,5ꢁ), 129.0 (C-3ꢁꢁ,5ꢁꢁ), 128.8 (C-4ꢁꢁ),
126.9 (C-2ꢁꢁ,6ꢁꢁ), 125.9 (C-1ꢁ,8ꢁ), 125.6 (C-2ꢁ,7ꢁ), 124.5
(C-8ꢁa,9ꢁa), 122.1 (C-β) ppm. For C21H15N (281.36):
calcd. C 89.65, H 5.37, N 4.98; found C 89.48, H 5.44,
N 4.83.
2. Experimental
2.1 Materials and chemicals
The studied analogues (table 1) were prepared prior
to the experiment and were dissolved in DMSO to a
final concentration of 2×104 μM. All chemicals and
reagents were of reagent grade and were used with-
out further purification. Propidium iodide (PI), Hoechst
33342, ethidium bromide, Triton X–100, reduced form 2.2b Synthesis of methyl (2E)-3-(acridin-9-yl)-prop-
of glutathione (GSH), 3-(4,5-dimethylthiazol-2-yl)-2,5- 2-enoate (2): A mixture of methyl (triphenylphospho-
diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide ranylidene)acetate (1.66 g, 4.96 mmol) and acridin-9-
Table 1. UV-Vis absorption data of acridine derivatives 1–3.
Ligand λmax free λmax bound
ꢀλ
(nm)
K
Hypochromicity
(%)
(nm)
(nm)
[M−1
]
1
2
3
368
361
361
369
362
363
1
1
2
7.1 × 105
1.9 × 105
3.4 × 106
57
40
18

Anti-tumour activity of acridine derivatives
933
carbaldehyde (1.0 g, 4.83 mmol) in dry chloroform buffer was sonicated for 5 min and the DNA concentra-
(5 mL) was stirred overnight at room temperature. The tion was determined from its absorbance at 260 nm.
solvent was evaporated under reduced pressure and The purity of DNA was determined by monitoring the
the residue was purified with column chromatography value A260/A280. DNA concentration measured at 260 nm
(SiO2, 9:1 v/v cyclohexane/acetone) to produce deriva- and expressed as micromolar equivalents of the base
tive 2 as yellow needles of pure Z isomer.
pairs ranging from 0 to 110 μM bp for 9-[(E)-2-pheny-
Yield: 570 mg (45%), M.p. 150–152◦C (ethyl lethenyl]acridine (1) and from 0 to 95 μM bp for methyl
1
acetate). H NMR (400 MHz, 25◦C, CDCl3): δ 8.54 (2E)-3-(acridin-9-yl)-prop-2-enoate (2) and 3-{[1-(acridin-
(d, J = 16.4 Hz, 1H, H-β), 8.25 (dd, J = 9.0, 1.0 9-yl)-3-methoxy-3-oxopropyl]sulfanyl}-2-aminopropa-
Hz, 2H, H-4ꢁ,5ꢁ), 8.17 (dd, J = 8.4, 1.0 Hz, 2H, H- noic acid (3). The derivatives 1–3 were dissolved in
1ꢁ,8ꢁ) 7.80 (ddd, J = 9.0, 6.6, 1.0 Hz, 2H, H-3ꢁ,6ꢁ), DMSO, from which working solutions were prepared
7.56 (ddd, J = 8.4, 6.6, 1.0 Hz, 2H, H-2ꢁ,7ꢁ), 6.48 (d, through dilution to a concentration of 25 μM using a
J = 16.4 Hz, 1H, H-α), 3.93 (s, 3H, CH3) ppm. 13C 0.01 M Tris-HCl buffer. All measurements were perfor-
NMR (100 MHz, 25◦C, CDCl3): δ = 166.3 (C=O), med at 24◦C.
148.8 (C-4ꢁa,10ꢁa), 139.7 (C-β), 139.6 (C-9ꢁ), 130.4
(C-4ꢁ,5ꢁ), 130.0 (C-3ꢁ,6ꢁ), 129.1 (C-α), 126.7 (C-2ꢁ,7ꢁ),
2.3b Equilibrium binding titration: The binding
125.4 (C-1ꢁ,8ꢁ), 124.0 (C-8ꢁa,9ꢁa), 52.4 (CH3) ppm. For
affinities were calculated from absorbance spectra
C17H13NO2(263.30): calcd. C 77.55, H 4.98, N 5.32;
according to the method of McGhee and von Hippel
using data points from a Scatchard plot.14 The binding
data were fitted using GNU Octave 2.1.73 software.15
found C 77.33, H 4.82, N 5.21.
2.2c Synthesis of conjugate 3-{[1-(acridin-9-yl)-3-
methoxy-3-oxopropyl]sulfanyl}-2-aminopropanoic acid
(3): A methanolic solution (0.5 mL) of derivative 2
(0.02 g, 0.076 mmol, 1.0 equiv.) was added dropwise to
a stirred solution of L-cystein (0.02 g, 0.114 mmol, 1.5
equiv.) in a mixture of methanol (1.5 mL) and Tris.HCl
(pH=7.4, 1 mL). The reaction mixture was stirred at
room temperature for ca. 1 h until the initial quantity
of derivative 2 had disappeared (TLC). The precipitate
which had formed was filtered off and washed with dry
methanol.
2.3c Fluorescence
measurements: Fluorescence
measurements of free derivatives 1 and 2 were per-
formed on a Varian Cary Eclipse spectrofluorimeter
with a 5 nm slit width for excitation and emission beams
at a concentration of 20 μM in a 0.01 M Tris-HCl
buffer at pH=7.4. Emission spectra were recorded in
the region of 400–700 nm using excitation wavelengths
of λ1 =368 & λ2 =361 nm. Fluorescence intensities
are expressed in arbitrary units. Fluorescence titra-
tions were carried out by adding increasing amounts
of ctDNA directly into the cell containing solutions of
ligands 1 and 2 (c=20 μM, 0.01 M Tris-HCl, pH=7.4).
The concentration range of DNA for derivative 1 was
1–12 μM bp and 0–70 μM bp for derivative 2. All
measurements were performed at 24◦C.
Yield: 15 mg (52%), M.p. 188–190◦C. 1H NMR (600
MHz, DMSO-D6, 25◦C): δ 8.37 (dd, J = 9.0, 1.2 Hz,
2H, H-1ꢁ,8ꢁ), 8.14 (dd, J = 9.0, 1.2 Hz, 2H, H-4ꢁ,5ꢁ),
7.84 (ddd, J = 9.0, 6.6, 1.2 Hz, 2H, H-3ꢁ,6ꢁ), 7.66 (ddd,
J = 9.0, 6.6, 1.2 Hz, 2H, H-2ꢁ,7ꢁ), 4.27 (dd, J = 14.4,
9.0 Hz, 1H, COCHa), 4.08 (dd, 1H, J = 14.4, 6.6 Hz,
1H, COCHb), 4.01 (dd, J = 9.0, 6.6 Hz, 1H, SCH), 3.97
(m, 1H, NCH), 3.38 (s, 3H, OCH3), 3.08 (d, J = 5.4
Hz, 2H, SCH2) ppm. 13C NMR (150 MHz, DMSO-D6,
25◦C): δ 171.5 (COOCH3), 169.5 (COOH), 148.5 (C-
4ꢁa,10ꢁa), 141.7 (C-9ꢁ), 130.6 (C-3ꢁ,6ꢁ), 130.3 (C-4ꢁ,5ꢁ),
126.7 (C-2ꢁ,7ꢁ), 125.3 (C-1ꢁ,8ꢁ), 125.1 (C-8ꢁa,9ꢁa), 52.7
(OCH3), 52.4 (NCH), 48.1 (SCH), 31.8 (SCH2), 29.5
(C-CO) ppm.
2.3d Circular dichroism: CD spectra of complexes
of ctDNA (1.03× 10−3 M bp) with ligands 1 and 2 (28
μM) were recorded 10 min after mixing on a J-810
Jasco spectropolarimeter at 24◦C. All measurements
were performed in 0.01 M Tris-HCl buffer (pH=7.4).
A rectangular quartz cell with a 1 cm path length was
used to obtain spectra from 200 to 350 nm. The results
are presented as a mean of three scans from which the
buffer background was electronically subtracted.
2.3 Biological activity
2.3e Topoisomerase I relaxation assay: Calf thymus
2.3a Spectral and DNA binding studies: UV-vis topoisomerase I (Takara, Japan) was used to determine
spectra were measured on a Varian Cary 100 UV-Vis topoisomerase I inhibition activity. pUC 19 DNA (1.4
spectrophotometer in 0.01 M Tris-HCl buffer (pH= μg) was used as the substrate in a reaction volume of
7.3). The solution of calf thymus DNA (ctDNA) in TE 20 μL containing the reaction buffer and 0.1% bovine

934
O Salem et al.
serum albumin. The tested inhibitors were added as nmol/20 μL (the injection volume was 20 μL). The
indicated, and the reaction was initiated by the addition amount of derivative 2 in the cells was measured using
of 3 units of topoisomerase I. Reactions were carried methanol extraction and RP-HPLC analysis. The cell
out at 37◦C for 45 min.
pellets were re-suspended in 100 μL of methanol and
mixed for 10 min. The mixture was kept at 4◦C for 1 h.
The tubes were then centrifuged at 6700 g for 15 min
(4◦C) in order to produce pellets from the solid mate-
rial and the extract (20 μL) was injected into the HPLC
system (LC-10AT pump, SPD-10Ai, UV-vis detector,
Shimadzu, Japan).
2.3f Gel electrophoresis: Gel electrophoresis was
performed at 7 V/cm for 2 h in a TBE buffer on a 0.8%
agarose gel. The gel was stained with ethidium bromide
(1 mg/mL) and photographed under UV light.
2.3g Cell culture conditions: Mouse leukemia cell
line L1210 and human ovarian carcinoma cell line
A2780 were obtained from Dr. Ujhazy, Roswell Park
Cancer Institute, Buffalo, USA. The cell lines were
grown in a RPMI 1640 medium supplemented with
10% FBS, penicillin (100 U/mL), and streptomycin
(100 μg/mL). The medium for the A2780 cell lines
was also supplemented with 2 mM glutamine. The
cells were maintained at 37◦C in a humidified 5% CO2
atmosphere.
2.3k HPLC analysis of GSH: GSH concentration
was measured using Chen’s method which utilizes the
measurement of a GSH DTNB adduct (GSH-TNB;
TNB-5-thionitrobenzoic acid) with HPLC.16 Cells (2
million A2780 cells detached by trypsinization) were
washed with PBS three times before ice-cold methanol
(0.2 mL) was added to the mixture of cells. The mix-
ture was homogenized and centrifuged at 4000 rpm for
15 min in order to remove the protein, the cell lysate
supernatant (50 μL) was vortex-mixed with a solution
of 0.15 M Tris-HCl (40 μL; pH=7.5, containing 1 mM
EDTA) and a solution of DTNB (10 mM, 10 μL) was
added. The mixture was allowed to stand at room tem-
perature for 15 min and subjected to HPLC analysis.
The calibration solutions of GSH (10–100 μM) were
prepared by the reaction with DTNB in the same condi-
tions as those of the cell lysate supernatant. HPLC anal-
ysis was carried out on a Shimadzu HPLC system (LC-
10AT pump, SPD-10Ai, UV-vis detector). An aliquot
of 20 μL of sample solution was injected (Rheodyne,
7725i) into a C18 column (Watrex, 250× 4, Reprosil 100
C18 with 5 μm particle size) and the mixed disulfide (of
GSH with thionitrobenzoate) was eluted at a flow rate
of 0.5 mL/min with isocratic elution. Prior to analysis,
the C18 column was pre-equilibrated for 30 min with a
mobile phase consisting of methanol–ammonium for-
mate (23 mM, pH=5.8) (10:90) at room temperature.
The injection volume was 20 μL and a wavelength of
356 nm was used for detection.
2.3h DCC: Cell proliferation, growth curves, and
cytotoxic potential of compounds were determined
using a trypan blue dye exclusion test (DCC). For
the three-day experiments, cells were seeded (1× 105
cells/mL) in Petri dishes and the tested substances (or
1% DMSO-control) were added after 24 h. Cell prolif-
eration was then checked after 24 h and 48 h. All dye
exclusion tests were performed in triplicate.
2.3i Intracellular accumulation of acridines 1 and
2 evaluated with fluorescence microscopy: The cells
harvested in the log phase were counted and seeded
(0.5× 106/mL). After 24 h incubation at 37◦C in a
humidified atmosphere of 5% CO2 in air, acridines
1 and 2 were added for 2 h, and the cells were
then washed in PBS and observed using an optical
microscope and a fluorescence microscope (Axio Zeiss
Imager A1, camera AxioCam Mrc).
2.3j Intracellular accumulation of acridine evaluated 2.3l Fluorescence detection of superoxide: Fluores-
with HPLC: ODS columns were used for HPLC anal- cence detection of superoxide dihydroethidium (DHE)
ysis of the acridine derivatives. The chromatographic was used to estimate intracellular superoxide produc-
separation of ester 2 was performed on a C18 column tion in the cells.17 Cells (4× 105) in medium were incu-
(Watrex, 250 × 4, Reprosil 100 C18 with 5 μm par- bated for 24 h at 37◦C, before derivatives 1 and 2 were
ticle size) using a degassed mobile phase (a mixture added. The influence of the substances on the genera-
of methanol and 50 mM ammonium formate at a ratio tion of free radicals was monitored after 1 h. DHE (20
of 80:20 (v/v), pH adjusted to 4.5 with formic acid) μM) was added and incubated for 20 min at 37◦C. After
at a flow rate of 0.5 mL/min. The column tempera- incubation, the cells were washed in PBS and fluores-
ture was maintained at 25◦C and the detection was car- cence was monitored with a fluorescence microscope
ried out at 370 nm. The standard concentration was 1 (Axio Zeiss Imager A1, camera AxioCam Mrc).

Anti-tumour activity of acridine derivatives
935
3. Results and Discussion
was improved through the use of a technique previously
described by Märkl and Merz21 which requires neither
a catalyst nor an anhydrous medium. In this way, the
yield of alkene 2 was increased to 45% (scheme 1).
3.1 Chemistry
9-[(E)-2-Phenylethenyl]acridine (1) was prepared from
benzyltriphenylphosphonium chloride which was first
dehydrohalogenated with sodium hydride at low tem- 3.2 Spectral and DNA binding studies
perature to produce a corresponding yield as an orange
suspension.18,19 9-acridinecarbaldehyde13 was added to
this mixture thereby resulting in the formation of
alkene 1 at a yield of 47% after 12 h. The same car-
baldehyde was transformed in a Wittig reaction in
order to target methyl 3-(acridin-9-yl)-prop-2-enoate
(2) with methyl (triphenylphosphoranylidene)acetate.20
The typically unsatisfactory yield from this reaction
The interaction of acridine compounds 1, 2, and 3 with
calf thymus DNA (ctDNA) was monitored using spec-
trophotometric titrations in an aqueous buffer. The UV-
vis spectra showed significant absorption at the range
of 300–450 nm, typical for transitions between elec-
tron energy levels of the acridine ring.22–24 Represen-
tative titrations are shown in figure 1. In addition to
the significant hypochromism of samples 1 and 2 (40–
57%), the smaller hypochromism of sample 3 (18%),
and a partial loss of the fine structure of the absorp-
tion bands, the absorption maxima of all DNA-ligand
complexes exhibited very small bathochromic shifts of
approximately 1 nm (sample 3 - 2 nm) (table 1). The
absence of tight isosbestic points in the titration experi-
ments indicates that more than one type of DNA-ligand
complex was formed. The values of hypochromism in
this work (40–57%) are comparable to those revealed in
our previous work (34–54%).25
R
H
O
N
N
R = Ph (1) , COOCH3 (2)
Scheme 1. Synthesis of acridin-9-ylalkenoic derivatives 1
and 2. Reagents and conditions: Ph3P+CH2Ph Cl−, NaH,
THF, −10◦C → rt, 12 h and Ph3P=CHCOOCH3, CHCl3, rt,
12 h, resp.
0,4
1,2
2
1
1
0,3
0,8
0,6
0,4
0,2
0
0,2
0,1
0
300
350
400
450
500
300
400
500
600
Wavelenght (nm)
Wavelength (nm)
1,2
1
3
0,8
0,6
0,4
0,2
0
300
350
400
450
500
Wavelength
Figure 1. UV-vis spectrophotometric titration of 9-[(E)-2-phenylethenyl]acridine
(compound1), acridine ester (compound 2), 3-{[1-(acridin-9-yl)-3-methoxy-3-
oxopropyl]sulfanyl}-2-aminopropanoic acid (compound 3), 25 μM) in 0.01 M Tris-
HCl buffer (pH=7.4, 24◦C) at increasing concentrations of ctDNA (from top to
bottom, 0–110 μM bp, at 10 μM intervals).

936
O Salem et al.
Table 2. Fluorescence data of acridine ligands 1 and 2 with
ctDNA.
Compound
λex
(nm)
λem
(nm)
F/Fa0
1
2
368
361
526
500
1.0
0.95
aRelative fluorescence intensity of free derivatives (9.7 μM
in 0.01 M Tris-HCl buffer, pH=7.4, 24◦C).
Figure 3. Circular dichroism spectra of ctDNA (1.03×
10−4 M bp) in both the presence and the absence of deriva-
tives 1 and 2 (28 μM) in 0.01 M Tris-HCl buffer (pH=7.4,
24◦C).
UV-Vis titration data were then used in combination
with McGhee and von Hippel plots in order to deter-
mine the binding constants of ligands 1 and 2 with
ctDNA. 14, 15 The binding parameters obtained from the
spectrophotometric analysis are summarized in table 1.
The binding constants determined by spectrophotome-
try were in the range of 1.9 × 105–3.4× 106 M−1. The
binding affinity values of the studied derivatives with
calf thymus DNA are comparable with the K values
of other intercalators, e.g., acridine bis-imidazolinones
(1.9–7.1×105 M−1), daunomycin (7.09×105 M−1), and
acridine orange (3.19×105 M−1).12
The spectrofluorimetric results show that the free
acridine ligands 1 and 2 exhibited a distinct fluores-
cence (table 2). The binding of alkenes 1 and 2 with the
DNA helix was corroborated by the significant reduc-
tion in fluorescence after consecutive additions of DNA
to the ligand solution. This can be seen in figure 2 which
shows the representative fluorescence spectra of 1 and
2 (figure 2).
Circular dichroism was used in order to monitor con-
formational changes after the addition of ligands 1 and2
to DNA. The complexes produced a CD spectrum typ-
ical of B-form DNA with positive and negative Cot-
ton effects at 278 and 245 nm, respectively, (figure 3);
these results are in accordance with previous biophys-
ical studies of similar compounds.12 Changes to the
negative bands showing non-significant red shifts were
observed for both compounds. The positive band at
278 nm showed an increase of molar ellipticity and a
mild red-shift of the band maxima in combination with
an increase in intensity following the addition of com-
pounds 1 and 2 to the DNA sample. These findings
would suggest that the intercalation has stabilized the
right-handed B-form of the DNA.
a
150
100
50
0
450
500
550
600
650
700
Wavelength (nm)
2
600
400
200
0
b
3.3 Topoisomerase I relaxation assay
Topoisomerases are essential DNA-targeting enzymes
which induce DNA strand cleavage, a process which
in turn leads to the reorganization and reconnection
of the damaged DNA strand. The resultant relaxation
of supercoiled DNA is necessary during the transcrip-
tion or replication process. DNA intercalators have been
shown to interfere significantly with this physiological
process;26–31 the ligand that occupies the topoisomerase
binding site can suppress the association of topoiso-
merase with DNA, thereby influencing topoisomerase
activity. DNA intercalators which inhibit topoisomerase
activity or which form stabilized ternary complexes
450
500
550
600
Wavelength (nm)
Figure 2. Spectrofluorimetric titration of 9-[(E)-2-pheny-
lethenyl]acridine-compound 1 (a) and acridine ester – com-
pound 2 (b) (20 μM) in 0.01 M Tris-HCl buffer (pH=7.4,
24◦C) at increasing concentrations of ctDNA (from top to
bottom, 0–12 μM bp, at 1 μM and 0–70 μM bp, at 5 μM
intervals, respectively).

Anti-tumour activity of acridine derivatives
937
3.4 Effects of compounds 1 and 2 on mouse leukemia
cell line L1210 and human ovarian carcinoma cells
A2780
The biological effects of acridine derivatives 1 and 2
were studied by analyzing changes in viability of L1210
and A2780 cancer cell lines in vitro.
Figure 4. Inhibition of topoisomerase I-induced DNA
relaxation by acridine ligands compound 1 and 2. Native
supercoiled pUC 19 (1.4 μg, lane DNA) was incubated for
45 min at 37◦C with 3 U of topoisomerase I in both the
absence (lane TopoI) and presence of ligands (lane 1a, 2a
- 5 μM, lane 1b, 2b - 30 μM, lane 1c, 2c - 60 μM). The
DNA samples were run on agarose gel followed by ethidium
bromide staining; sc - supercoiled plasmid, oc - open-circular
plasmid.
3.4a Cytotoxicity of the 9-[(E)-2-phenylethenyl]acri-
dine (1): 9-[(E)-2-phenylethenyl] acridine 1 showed
a higher level of cytotoxicity towards A2780 cells than
towards L1210 cells. While the compound suppressed
the proliferation of the leukemic L1210 cells over the
entire range of concentrations (5–50 μM), the inhibi-
tion level was considerably higher at the highest con-
centration of 50 μM; the number of A2780 cells was
reduced to approximately one third of the original num-
ber after 48 h (figure S1). The IC50 values (48 h) for
L1210 and A2780 cells were 18.90 μM and 5.45 μM,
respectively.
The cells incubated with 9-[(E)-2-phenylethenyl]
acridine 1 for 2 h had visibly accumulated this deriva-
tive. The microphotographs (figure 5) confirm that the
compound was distributed differently in the A2780 and
L1210 cells. Figure 6A shows that microgranules of 1
were formed in the L1210 cells.
with DNA and topoisomerase therefore show potential
use in the development of DNA-targeting anticancer
drugs.30
In order to study the effect of our ligands on the
DNA relaxation, supercoiled plasmid pUC 19 was incu-
bated with topoisomerase I in the presence of com-
pounds 1 and 2 at concentrations of 5, 30 and 60 μM.
As is shown in figure 4, the supercoiled DNA was fully
relaxed by the enzyme in the absence of compounds
1 and 2 (line TopoI). Similarly, a greater proportion
of the relaxed form of DNA was observed when lig-
ands 1 and 2 were added at the lower concentration of
5 μM (line 1a, 1b). However, as the concentration of
ligands increased to 30 and 60 μM, the proportions of 3.4b Cytotoxicity of ester 2: The inhibitory effect of
supercoiled plasmid form exceeded those of the relaxed ester 2 on the proliferation of L1210 and A2780 cells
form (lines 1b, 2b). It is possible to suggest that this was found to be even stronger than that of 9-[(E)-
may be a result of topoisomerase I inhibition through 2-phenylethenyl]acridine 1 (figure S2) and the A2780
a concurrent intercalation of ligands 1 and 2 into the cells were again seen to be more sensitive to compound
topoisomerase binding site. It is also assumed that a 2 than the L1210 cells. A mild suppression of A2780
ternary complex consisting of topoisomerase, DNA, cells was observed the relatively low concentration of
and ligand could be stabilized significantly under these 5 μM concentration, while the highest concentration of
conditions.
50 μM resulted in a rapid and strong inhibition after
Figure 5. Intracellular uptake of 9-[(E)-2-phenylethenyl]acridine compound 1 in cells L1210 and A2780.
Fluorescence of compound 1 (50 μM) in the cells was monitored with a fluorescence microscope after 2 h
incubation; magnification: 400×, scale 20 μm.

938
O Salem et al.
Figure 6. Intracellular uptake of ester 2 into L1210 and A2780 cells. The cells were incubated without
(control) and with compound 2 (50 μM) for 2 h; magnification: 400×, scale bar, 20 μm.
Table 3. Levels of GSH in L1210 and A2780 cells after
treatment with ester 2.
24 h which also continued after 48 h. The IC50 values
(48 h) for L1210 and A2789 cells were 7.86 μM and
4.60 μM, respectively.
The different levels of toxicity which ester 2 demon-
strated against L1210 and A2780 cells could be asso-
ciated with different levels of accumulation of ester 2
within these cell lines.
Microphotographs taken after incubation showed that
compound 2 had accumulated in both types of cells.
Compound 2 was not evenly localized in leukemic
L1210 cells and that it was likely to be preferentially
accumulated in the nucleus (figure 6). However, A2780
cells after incubation with derivative 2 showed a visible
fluorescence in all cell structures.
nM GSH per 1× 106 cells
L1210 control
0.6 0.2
0
2.0 0.2
1.3 0.1
L1210 + 2 (15 μM)
A2780 control
A2780 + 2 (15 μM)
is possible to suggest that ester 2 is present in the cells
in a ‘free’ form or as a conjugate with glutathione (and
other thiols, as well). Only cca. 1% of the ‘free’ form
of ester 2 was found in cells. Thus it can be assumed
that a conjugate of compound 2 with a reduced form of
glutathione is the predominant form of conjugation in
the cells.
As the depletion of intracellular glutathione can
induce oxidative stress in cells, it was also possible to
verify the presence of free radicals in the cells after
incubation with compound 2 for 1 h. As is shown in
figure 7, the DHE assay confirmed the presence of
strong oxidative stress in the cells along with a higher
rate of free radical generation than was found in cells in
the presence of menadione.
3.4c Changes of intracellular level of glutathione:
The acridin-9-ylalkenoic derivatives contain either
deactivated alkene 1, and ester 2 includes the neigh-
bouring ester group polarized C=C double bond, there-
fore it would be expected that the studied compounds
demonstrate reactivity towards nucleophilic HS-group-
containing biomolecules including glutathione (GSH),
the main intracellular low-molecular thiol. This form
of interaction would also be expected to produce some
new conjugated molecule(s) and change the level of
glutathione in cells. Therefore the changes of GSH in 3.4d Interaction of conjugate 3-{[1-(acridin-9-yl)-3-
the cells treated with ester 2 were examined and the methoxy-3-oxopropyl]sulfanyl}-2-aminopropanoic acid
concentration of the compound in the cells measured. with thiols: In order to examine the possible forma-
After treatment with ester 2, a full depletion of GSH tion of conjugate(s) of ester 2 with thiols and to ver-
in L1210 cells and a 35%-decrease of the GSH level ify the cytotoxicity of these conjugates, a reaction prod-
in A2780 cells was observed following treatment with uct of compound 2 with L-cysteine was synthesized in
ester 2 (table 3). In contrast, no significant changes to methanol/Tris.HCl buffer (2:1) at room temperature in
the GSH levels of the cells were observed after the addi- order to produce a precipitate of 3-{[1-(acridin-9-yl)-3-
tion of 9-[(E)-2-phenylethenyl]acridine 1 practically methoxy-3-oxopropyl]sulfanyl}-2-aminopropanoic acid
(data not shown) and therefore its reactivity towards (3) which would be pure enough for spectroscopic iden-
thiols was not studied.
tification. Because of its low solubility, 1D and 2D NMR
The intracellular concentration of compound 2 was analysis (1H, 13C, gCOSY, TOCSY, gHSQC, gHMBC,
determined using HPLC analysis (figure S3). The cells NOESY) was performed in DMSO-D6 and confirmed
were incubated with compound 2 (15 μM) for 2 h. It the structure of conjugate 3 as shown in scheme 2. The

Anti-tumour activity of acridine derivatives
939
Figure 7. Oxidative stress in A2780 cells induced by compounds 1 and 2. The cells were incubated with compound 1 or 2
(10 μM) for 1 h and oxidative stress was determined using a DHE assay. A – negative control, B – positive control (20 μM
menadione), C– compound 2, D – compound 1 (magnification 400×); scale 20 μm.
results show that conjugate 3 had only limited stability sides of the acridine ring, thereby enhancing the cyto-
in DMSO-D6 and underwent a slow partial decomposi- toxic properties of the acridine skeleton. The saturation
tion into the starting compounds and some other decom- of this double bond into a single C–C bond which inter-
position/cyclization product over the course of a few rupts extended electron conjugation within compound 3
hours.
may be seen as one possible reason for the decrease in
The creation of the GSH–xenobiotics conjugate acts the originally considerable cytotoxicity of the studied
as a method of detoxifying the xenobiotic drug, and acridinyl-alkenoic compounds. The reduced cytotoxic-
therefore we decided to compare the cytotoxicity of ity of 9-[(E)-2-phenylethenyl]acridine 1 towards both
compound 3 with that of the pure ester 2. As can be seen cell lines may be due to a series of factors such as the
in figure S4, conjugate 3 is less cytotoxic than the pure less polar character of the compound, higher hydropho-
ester 2, especially at concentrations higher than 10 μM. bicity, increased volume, lower flexibility, and also the
Although the reaction of compound 2 with thiol did not expected coplanarity of the acridine and phenyl sub-
eliminate its cytotoxicity entirely, a trend toward dimin- stituents at the double bond. All these factors can con-
ishing it was noted after the formation of the conjugate. tribute to the reduced ability of compound 1 to penetrate
These results suggest that the double CH=CH bond and the intracellular membranes and incorporate itself into
the methyl ester group in ester 2 conjugated at opposite the nucleus.
O
OH
*
O
*
O
O
O
H
H2N
CH3
CH3
S
H
NH2 HCl
MeOH, Tris.HCl
rt
HS
O
1´
4´
8´
5´
9´
+
8´a
9´a
4´a
OH
10´a
N
N
3
2
Scheme 2. Synthesis of conjugate 3-{[1-(acridin-9-yl)-3-methoxy-3-oxopropyl]sulfanyl}-2-aminopropanoic acid (3).

940
O Salem et al.
7. Plsikova J, Janovec L, Koval J, Ungvarsky J, Mikes
4. Conclusions
J, Jendzelovsky R, Fedorocko P, Imrich J, Kristian P,
Kasparkova J, Brabec V and Kozurkova M 2012 Eur. J.
Med. Chem. 57 283
Our studies show that ester 2 demonstrates a stronger
cytostatic effect on leukemia cell line L1210 than
9-[(E)-2-phenylethenyl]acridine 1. The incubation of
compounds 1 and 2 with ovarian carcinoma cell line
A2780 showed extensive cytotoxic effects which were
especially marked in the case of compound 2. The
mechanism of the cytotoxicity of studied acridine
derivatives can be related to their affinity towards DNA
and their capability to inhibit topoisomerase I. An addi-
tional factor may be the different characters of com-
pounds 1 and 2; the former is more hydrophobic, more
bulky and less flexible, whereas the latter is more polar
and flexible during transportation through intracellu-
lar membranes and more reactive towards nucleophilic
low-weight cell metabolites. In addition, compounds
1 and 2 are both able to induce oxidative stress in
cells. The fact that ester 2 shows a higher reactiv-
ity towards thiols than 9-[(E)-2-phenylethenyl]acridine
1 can be taken as further proof of the crucial role
of polarity in the interaction of the studied molecules
with detoxification agents such as glutathione or
cysteine.
8. Su T L, Lin Y-W, Chou T-C, Zhang X, Bacherikov V A,
Chen C-H, Liu L F and Tsai T-J 2006 J. Med. Chem. 49
3710
9. Schafer F Q and Buettner G R 2001 Free Radic. Biol.
Med. 30 1191
10. Go Y M and Jones D P 2010 Antioxid. Redox Signal. 13
489
11. Kern J C and Kehrer J P 2005 Front. Biosci. 10 1727
12. Paulikova H, Vantova Z, Hunakova L, Cizekova L,
Carna M, Kozurkova M, Sabolova D, Kristian P,
Hamulakova S and Imrich 2012 J. Bioorg. Med. Chem.
20 7139
13. Bedlovicova Z, Imrich J, Kristian P, Danihel I, Sabolova
D, Kozurkova M, Böhm S, Paulikova H and Klika KD
2010 Heterocycles 80 1047
14. McGhee J D and von Hippel P H 1974 J. Mol. Biol. 86
469
15. Busa J, Octave TechnicalUniversity in Kosice, Kosice
2006
16. Chen W, Zhao Y, Seefeldt T and Guan X 2008 J. Pharm.
Biomed. Anal. 48 1375
17. Zhao H, Joseph J, Fales H M, Sokoloski E A, Levine
R L, Vasquez-Vivar J and Kalyanaraman B 2005 Proc.
Natl. Acad. Sci. U.S.A. 102 5727
18. Vedejs E, Marth C F and Ruggeri R 1988 J. Am. Chem.
Soc. 110 3940
19. Vedejs E and Marth C F 1988 J. Am. Chem. Soc. 110
Supplementary Information
3948
20. Vlassa M, Molnariu C and Silberg J A 1994 Rev. Roum.
Chemie 39 315
21. Märkl G and Merz A 1973 Synthesis 295
22. Carlson C B and Beal P A 2000 Bioorg. Med. Chem.
Lett. 10 1979
Figures S1 to S4 are provided in the supplementary
information and is available at www.ias.ac.in/chemsci.
Acknowledgement
23. Delcros J G, Tomasi S, Carrington S, Martin B, Renault
J, Blagbrough I S and Uriac P 2002 J. Med. Chem. 45
5098
24. Baranovsky S F, Bolotin P A, Evstigneev M P and
Chenyshev D N 2009 J. Appl. Spectrosc. 76 132
25. Janovec L, Kozurkova M, Sabolova D, Ungvarsky J,
Paulikova H, Plsikova J, Vantova Z and Imrich J 2011
Bioorg. Med. Chem. 19 1790
This study was supported by Slovak Research and
Development Agency VEGA grants 1/0001/13 and
1/0790/14.
References
26. Pommier Y 2006 Nat. Rev. Cancer 6 789
27. Chaudhuri P, Majumder H K and Bhattacharya S 2007
J. Med. Chem. 50 2536
28. Leppard J B and Champoux J J 2005 Chromosoma 114
75
1. Lang X, Li L, Chen Y, Sun Q, Wu Q, Liu F, Tan C, Liu
H, Gao C and Jiang Y 2013 Bioorg. Med. Chem. 21 4170
2. Chaires J B 1998 Curr. Opin. Struct. Biol. 8 314
3. Graves D E and Velea L M 2000 Curr. Org. Chem. 4
915
29. McClendon A K and Osheroff N 2007 Mutat. Res. 623
4. Ihmels H and Otto D 2005 Top. Curr. Chem. 258
83
161
30. Kathiravan M K, Khilare M M, Nikoomanesh K, Chothe
A S and Jain K S 2013 J. Enz. Inhib. Med. Chem. 28 419
31. Basili S, Basso G, Faccio A, Granzhan A, Ihmels H,
Moro S and Viola G 2008 Chem. Med. Chem. 3 1671
5. Vantova Z, Paulikova H, Sabolova D, Kozurkova M,
Suchanova M, Janovec L, Kristian P and Imrich J 2009
Int. J. Biol. Macromol. 45 174
6. Lord C J and Ashworth A 2012 Nature 481 287