3,6-Bis(3-alkylguanidino)acridines as DNA-intercalating antitumor agents

European Journal of Medicinal Chemistry 57 (2012) 283e295

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Original article

3,6-Bis(3-alkylguanidino)acridines as DNA-intercalating antitumor agents Jana Plsikova a, Ladislav Janovec b, Jan Koval c, Jan Ungvarsky b, Jaromir Mikes c, Rastislav Jendzelovsky c, Peter Fedorocko c, Jan Imrich b, Pavol Kristian b, Jana Kasparkova d, Viktor Brabec d, Maria Kozurkova a, * a

Department of Biochemistry, P. J. Safarik University in Kosice, Faculty of Science, Moyzesova 11, 04001 Kosice, Slovak Republic Department of Organic Chemistry, P. J. Safarik University in Kosice, Faculty of Science, Moyzesova 11, 04001 Kosice, Slovak Republic c Department of Cellular Biology, P. J. Safarik University in Kosice, Faculty of Science, Moyzesova 11, 04001 Kosice, Slovak Republic d Department of Molecular Biophysics and Pharmacology, Institute of Biophysics, Academy of Sciences of the Check Republic, Kralovopolska 135, 612 65 Brno, Czech Republic b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 January 2012 Received in revised form 12 September 2012 Accepted 13 September 2012 Available online 23 September 2012

A series of 3,6-bis(3-alkylguanidino) acridines was prepared and the interaction of these novel compounds with calf thymus DNA was investigated with UVevis, fluorescence and circular dichroism spectroscopy, in addition to DNA melting techniques. The binding constants K were estimated to range from 1.25 to 5.26
105 M1, and the percentage of hypochromism was found to be 17e42% (from spectral titration). UVevis, fluorescence and circular dichroism measurements indicated that the compounds act as effective DNA-intercalating agents. Electrophoretic separation proved that ligands 6aee relaxed topoisomerase I at a concentration of 60 mM, although only those with longer alkyl chains were able to penetrate cell membranes and suppress cell proliferation effectively. The biological activity of novel compounds was assessed using different techniques (cell cycle distribution, phosphatidylserine externalization, caspase-3 activation, changes in mitochondrial membrane potential) and demonstrated mostly transient cytostatic action of the ethyl 6c and pentyl 6d derivatives. The hexyl derivative 6e proved to be the most cytotoxic. Different patterns of cell penetration were also observed for individual derivatives. Principles of molecular dynamics were applied to explore DNAeligand interactions at the molecular level. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Acridine Intercalator Topoisomerase I Cytotoxic Antitumor activity Caspase-3 Molecular modeling

1. Introduction Interaction of small molecules with DNA has been studied for almost half of a century in hope of learning design principles for the targeting of specific DNA sequences in order to control gene expression [1e3]. The unique structural features of DNA due to the relative planar stacking of aromatic bases along the helix sugar phosphate backbone make it a particularly interesting target for drug design. Considerable efforts have been invested to develop sequence specific DNA binding ligands. Single dye molecules usually bind to DNA by two main interactions e by intercalation or groove-binding. Most dye aggregates are too large to fit into the corresponding binding area and bind to the phosphate backbone [4,5]. Dervan et al. have developed series of polyamines [6,7] which have expanded the sequenceselective DNA binding ligands [8,9]. In recent years, a number of carbohydrate-containing molecules interacting with DNA have been developed [10,11]. Arya’s research group developed a class of carbohydrates that bind to DNA within the major groove and * Corresponding author. Tel.: þ421 55 6223582; fax: þ421 556222124. E-mail addresses: [email protected], [email protected] (M. Kozurkova). 0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2012.09.020

include aminoglycoside (neomycin) conjugates [12e16]. Because aminoglycosides do not have a chromophore for spectrophotometric analysis, authors presented their findings from competition dialysis of three acridines with increasing positive charge against 14 different nucleic acids. 9-aminoacridine and quinacrine showed a clear preference for DNA triplex, whereas neomycineeacridine conjugate binding to RNA triplex [17]. Acridine and its derivatives are a dye class whose interaction with DNA has been extensively studied [18e23]. The archetypal DNA intercalating agents are 9-aminoacridine and its derivatives, which bind reversibly to DNA by intercalation, but usually do not covalently interact with it. The intercalation of the acridines is facilitated by cationic ionization and sufficient molecular planarity. Two acridine compounds, Acranil and Atabrine (quinacrine hydrochloride), are drugs with a number of different medical applications [24e26]. One of the interesting groups of acridine derivatives are guanidines. Guanidines are important molecules with a wide range of interesting biochemical and pharmaceutical properties. The isolation, structural identification and synthesis of naturally occurring guanidines have been reviewed by Berlinck [27]. Guanidines are found widely in natural products, pharmaceutically active compounds and in molecules used for supramolecular studies [28e30]. More recently, complex guanidine-containing natural

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products have been isolated and subjected to synthetic studies. For example, the tricyclic guanidine ptilomycalin A [31] was isolated from the sponges harvested from both the Caribbean and the Red Sea, and was found to have cytotoxic, antifungal, antimicrobial, and antiviral effects [31,32]. Recent studies of synthetic biologically-active guanidines developed by Berlinck have found evidence of antimicrobial activity, thrombin and Naþ/Hþ exchange inhibition, as well as inhibition of NO synthase, antithrombotic or antidiabetic activity and the effective delivery of anticancer agents [33]. In this paper we describe the synthesis and report the biochemical and biological activities of new guanidine derivatives bearing acridine moiety which have been designed as potential anticancer agents. In our study we focus on the capacity of the derivatives to bind to DNA and to interfere with human topoisomerase I, in addition to the derivatives’ in vitro antiproliferative activity on human promyelocytic leukemia cells (HL-60). Molecular dynamics was also employed in the study to reveal modes of interaction with DNA.

4aee, which produced guanidines 5aee in a yield of 29e42% in the reaction with ammonium hydroxide. Guanidines 5aee were then purified by column chromatography on silica gel. Finally, the derivatives 6aee were obtained as hydrochlorides salts. 2.2. Biological activities 2.2.1. Spectral and DNA binding studies The absorption spectra of the newly synthesized guanidine derivatives 6aee in the presence of ctDNA show that all studied derivatives displayed absorption bands in the region of 300e 450 nm. The different substituents were found to have a slight effect on UVevis spectra, with maximal absorptions of around 371 nm. It is widely accepted that if the compounds could intercalate DNA, the UVevis curve of their complex would demonstrate bathochromic shifts and hypochromicity [35e37]. The absorption spectra of 6e (12.8 mM) in both the absence and presence of calfthymus DNA (0e240 mM) are given in Fig. 1. As the concentration of DNA increases, the curve shows significant hypochromicities (17e42%) and a slight bathochromic shift indicating that compound 6e forms a complex with DNA. UVevis data for compounds 6aee are displayed in Table 1. The absence of tight isosbestic points in titration experiment indicates that more than one type of DNAeligand complex could be formed. Comparing the value of hypochromism in this work (17e42%), with our previous work (34e54%) [34], we suggest the less proximity of the proflavine chromophore to DNA for our compounds which can be caused by weaker interaction between the electronic states of the

2. Results and discussion 2.1. Chemistry In the preparation of the target bis(guanidino)acridines 6aee, the initial 3,6-bis(thiourea)acridines 3aee were synthesized from commercial proflavine 1 via 3,6-diisothiocyanatoacridine 2 by our recent procedure (Scheme 1) [34]. The thioureas 3aee obtained during this process were used for the preparation of carbodiimides

i H 2N

N

SCN

NH2

NCS

N 2

1

ii

R

N=C=N

N

N=C=N

iii

R

R

4a-4e

S

S N H

N H

N

N H

N H

R

3a-3e

iv

R

NH

NH N H

N H

N 5a-5e

N H

N H

R

v

R

NH

NH N H

N H

N 2HCl. 4H2O

N H

N H

R

6a-6e

Scheme 1. R ¼ a e ethyl; b e n-propyl; c e n-butyl; d e n-pentyl; e e n-hexyl; (i) CSCl2, CHCl3/H2O, Na2CO3, RT. (ii) R-NH2, CH3OH, RT. (iii): HgO, Na2SO4 (dry), THF (dry), 55  C. (iv) NH4OH, THF. (v) Solution of 36% HCl in acetone (1:9).

J. Plsikova et al. / European Journal of Medicinal Chemistry 57 (2012) 283e295

285

0,4

Absorbance

0,3

0,2

0,1

0 360

400

Fig. 2. First derivative of the helix denaturation curves of ctDNA (black line) with compound 6a (blue line), 6b (green line), 6c (violet line), 6d (red line) and 6e (yellow line) measured at 260 nm in BPE buffer, pH 7.1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

440

Wavelength (nm) Fig. 1. UVevis spectrophotometric titration of derivative 6e (12.8 mM) in 0.01 M Tris buffer (pH 7.4, 24  C) by increasing concentration of ctDNA (from top to bottom, 0e240 mM bp, at 2 mM intervals).

chromophore and those of the DNA bases (different substituent on proflavine chromophore). Thermal denaturation studies were used for the additional evidence of intercalation into the DNA. Duplex DNA is thermally denatured into single-strand components in the presence and absence of compounds. Compounds that physically associate with DNA typically stabilize the duplex, hence increasing the midpoint denaturation temperature (Tm) [38]. Thus, the helix denaturation of DNA can be determined by monitoring the absorbance of DNA bases at 260 nm as a function of temperature (Tm). However, the Tm will increase slightly (Tm < 0.6  C) when interaction of small molecules with DNA through nonspecific electrostatic interactions with the phosphate backbone of DNA occurs. DNA melting curves in the presence and absence of studied derivatives 6aee are presented in Fig. 2. The Tm of the ctDNA was 70.5  C in the absence of derivatives and in presence of 6aee increased from 84 to 89  C (Table 1). Therefore, it can be concluded that proflavine derivatives 6aee interact with DNA through the intercalative mode. During spectrofluorimetric study, acridine ligands 6aee exhibited an emission band in the range of 400e600 nm. Excitation wavelength was 384 nm, and spectra were monitored at a fixed concentration of 9.6 mM. Titration with increasing concentrations of ctDNA continued until no further changes in the spectra of the drugeDNA complexes were recorded (Fig. 3). Binding of the proflavine probes to DNA helix was found to reduce the florescence of the probes [39] and that represents further independent proof of intercalation. Fluorescence titration data were used to determine the binding constants of ligands 6aee with calf thymus DNA using McGhee and von Hippel plots [40,41]. The constants were derived from Table 1 UVevis absorption data of guanidine derivatives 6aee. Compound 6a 6b 6c 6d 6e a

lmax free

lmax bound

(nm)

(nm)

370 370 371 370 372

372 373 372 372 376

Dl (nm)

Hypochromicity (%)

Tma

2 3 1 1 5

23 32 17 31 42

84 85 86 88 89

nonlinear curve fitting, a method which has been described previously [42]. The binding parameters from spectrophotometric analysis are summarized in Table 2. Typical binding constant for intercalation complexes between organic dyes and DNA range from 104 to 106 M1 and are usually significantly smaller than the binding constant of groove binders (105e109 M1) [23]. The calculated binding constants, K, and the neighbor exclusion parameters, n, clearly indicate a direct link between the intercalation capability and structural changes of proflavine derivatives 6aee. The high values of binding constants K, determined by fluorescence titration in the range of 5.26 to 1.25
105 M1, prove the high affinity of the proflavine ligands to DNA-base pairs and correspond to typical binding constants for intercalation complexes [43,44]. The binding constants for our studied derivatives 6aee are also of the same order of magnitude as those found in our previous study [42,45]. Estimates for n lay in the range 2.1e4.1 bp. The relative binding constants K increased as follows: 6e < 6d < 6c < 6b < 6a. Interestingly, a linear inverse relationship was found between the drug binding affinities and the chain length ( A) of the alkyl substituents (r ¼ þ0.971, k ¼ 1.06, S ¼ 0.15) (Fig. 4); it was discovered that the binding decreased with the increasing substituent length. Depending on the nature of alkyl substituents the binding constants decreased with a growing bulk of alkyls in the order: ethyl > propyl > butyl > pentyl > hexyl. The same trend has been observed in all of our former studies [34,43]. Clearly,

700 600

Florescence (a.u.)

320

500 400 300 200 100

Tm measurements were performed in BPE buffer, pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA) using 12.4 mM drug and 7.64 mM bp ctDNA with a heating rate of 1  C min1. Tm of ctDNA is 70.5  C.

0 400

450

500

550

600

Wavelength (nm) Fig. 3. Spectrofluorimetric titration of derivative 6e (9.6 mM) in 0.01 M Tris buffer (pH 7.4, 24  C) by increasing the concentration of ctDNA (from top to bottom, 0e257 mM bp, at 2 mM intervals), lex ¼ 384 nm.

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Table 2 DNA binding constants, K, neighbor exclusion parameters, n of proflavine ligands 6aee with ctDNA, determined by fluorescence spectrophotometric titration.

lex (nm)

lem (nm)

F/F0a

K (M1)

6a 6b 6c 6d 6e

371 371 372 373 371

472 471 472 472 473

0.96 0.68 1.00 0.66 0.71

5.26 5.01 4.25 2.36 1.25








105 105 105 105 105

n

DGb (kJ mol1)

2.1 2.9 3.5 3.9 4.1

32.7 32.5 32.1 30.7 29.1

Elipticity (mdeg-1)

Compound

6

a

Fluorescence quantum yields were calculated using 6c as standard (F/F0 ¼ 1). b The standard Gibb’s free-energy change (DG ¼ RTlnK) for derivatives 6aee is approximately 31.2 kJ/mol at 25  C and indicates the spontaneous binding of the compounds to DNA.

4 2 0 -2 -4 -6 240

280

300

Wavelength (nm) Fig. 5. CD spectra of the ctDNA (76.8 mM) in the absence and presence of 6aee (6.5 mM) in 0.01 M Tris buffer (pH 7.4).

Compounds 6a and 6e are too small to be orientated and thus show no intrinsic LD signal. Any LD signals that arise in the spectroscopic regions of complexes 6a and 6e in the presence of ctDNA, therefore, indicate binding of the complex to the ctDNA in a specific orientation(s). The LD signal yielded by the sample of ctDNA in the presence of 6a and 6e is decreased in the region above 300 nm (in the range of 330e430 nm) so that the LD spectra (Fig. 6) show that both 6a and 6e bind to DNA in a specific orientation(s), not randomly. Moreover, negative sign of the LD signal that arises in the 330e430 nm regions suggests that the angle of the long axis of 6a and 6e to the axis of the DNA double helix is more than 54 as expected for an intercalator [49].

0 -0,02

LD

increasing the hydrophobicity of alkyl substituents, which are symmetrically located in the 3, 6-positions of the acridine skeleton, impedes the DNA binding and renders the binding energy more positive. Circular dichroism (CD) is a powerful and reliable tool which is used to determine the conformation of biomacromolecules. CD spectra monitor the asymmetric environment of the compounds when bound to DNA and therefore can be used to obtain information on the binding mode. The free compounds do not have CD spectra, but they have induced CD when bound to DNA. The CD spectrum of free ctDNA has a negative band at 245 nm due to helicity, and a positive band at 279 nm due to base stacking which is characteristic of DNA in the right-handed B form [46e48]. When our compounds were incubated with ctDNA, the CD spectra displayed changes in both positive and negative CD bands (Fig. 5). A decrease of negative bands was observed for compounds 6aee. The positive band at 279 nm showed an increase of molar ellipticity following the addition of all proflavine derivatives to the DNA. This phenomenon could be due to the stabilization of a right-handed Bform of DNA by intercalation. LD, which can be used to probe the orientation of molecules, was also exploited to characterize further DNA binding mode of 6a and 6e. Long molecules such as DNA (minimum length of w250 base pairs) can, in a flow Couette cell, be orientated through viscous drug [49]. Small unbound molecules are not orientated in the experiment and show no signal. Similarly, the molecules that bound randomly to ctDNA show no signal. However, molecules bound in a specific orientation with respect to the ctDNA show a LD signal.

260

-0,04 -0,06 -0,08 -0,1 250

300

350

400

450

500

550

500

550

Wavelength (nm)

0

LD

-0,02 -0,04 -0,06 -0,08 -0,1 250

300

350

400

450

Wavelength (nm) Fig. 4. Correlation of chain lengths ( A) of alkyl substituents in proflavine derivatives 6aee with DNA binding constants (K
105 M1) obtained from spectrophotometry (k ¼ 1.06, r ¼ 0.971, S ¼ 0.15).

Fig. 6. Linear dichroism spectra of ctDNA (3.11
104 M, black line) in the presence of increasing amounts of 6a (up) and 6e (bottom) (11e58
106 M) in 10 mM Tris solution, pH ¼ 7.4.

J. Plsikova et al. / European Journal of Medicinal Chemistry 57 (2012) 283e295

1,18

n/no

1,16 1,14

6a

1,12

6b

1,1

6c

1,08

6d

1,06

6e

1,04 1,02 1 0

0,04

0,08

0,12

0,16

r Fig. 7. Effect of increasing amounts of 6aee (12e60
106 M) on the relative viscosity of ctDNA (3.11
104 M) in 10 mM Tris buffer, pH ¼ 7.4.

The DNA LD bands (220e300 nm) confirm that the DNA remains in the presence of 6a and 6e in the B-DNA conformation, however, some structural changes in DNA are suggested by the increase in the amplitude of DNA negative LD band at 260 nm upon drug addition (Fig. 6). An increase in the amplitude of the negative 260 nm LD band of DNA is usually associated with DNA stiffening [49e51] so that the effect of 6a and 6e on this DNA LD signal is consistent with an intercalative mode of interaction of both drugs. The intercalators dramatically increase the length of DNA, resulting in an increased viscosity. In contrast, the groove binders, do not lengthen the DNA helix, and do not increase the viscosity of DNA solutions. Hence, viscosity provides a simple, theoretically sound means of distinguishing DNA binding mode [52]. Our viscosity data (Fig. 7) demonstrate that compounds 6aee cause a significant increase in viscosity of DNA solution, 6e being most effective. This increase in viscosity is due to the increase in separation of base pairs at intercalation sites, which results in an increase in overall DNA contour length [52e54]. Thus, the results illustrate the lengthening of DNA indicating the intercalation of 6aee. 2.2.2. DNA mobility shifts No ethidium bromide was added to the agarose gel during the electrophoretic stage of the mobility shift experiments in order to test the effect of the binding of the 6aee compounds to bulk pUC 19 plasmid DNA. The bands that had migrated further from the well were identified as containing the supercoiled plasmid DNA (SC). The relaxed open circular DNA (OC) traveled the shortest distance due to its shape. When compounds 6aec were added to the plasmid DNA, a progressive reduction in DNA mobility of the band representing the supercoils was observed (Fig. 8). The band assigned to the supercoiled plasmid DNA was not observed in 6a, 6b

287

samples at concentration 60 mM (lane 1d, 2d) and 80 mM (lane 1e, 2e) and in 6c at 80 mM (lane 3e). Instead, a broad smeared band was observed at the position of the relaxed circular band. It is obvious that no gel shift was observed for samples 6dee (concentrations 5, 20, 40, 60, 80 mM) under the experimental conditions applied. A reduction of the electrophoretic mobility of DNA was observed upon an increase in the concentration of the sample. Interestingly, at low concentrations the bands were sharp but started to become diffuse and less intense at higher concentrations, which indicate partially denatured or degraded DNA. DNA degradation may be based on photocleavage. Photocleavage of DNA by 6aee has been shown to occur mainly via single strand cleavage, which is rather unlikely to lead to the observed diffuse bands. 2.2.3. Topoisomerase I relaxation activity Topoisomerase-targeting drugs can be divided into two broad classes that vary widely in their mechanisms of action [55,56]. The class I drugs include anthracyclines, actinomycins, ellipticines, alkaloids etc. These drugs act by stabilizing covalent topoisomerase-DNA complexes. Class I drugs are also referred to as “topoisomerase poisons” because they transform the enzyme into a potent cellular toxin. Class II drugs, by contrast, interfere with the catalytic function of enzyme without trapping the covalent complex. The drugs in this class are referred to as “topoisomerase inhibitors”. The main topoisomerase inhibitors are coumarine antibiotics, fostriecin analogs, benzimidazole and anthraquinoline derivatives, anthraimidazole derivatives etc. [57e64]. The ligand that occupies the topoisomerase binding site may suppress the association of topoisomerase with DNA, thus influencing the topoisomerase activity. DNA intercalators that inhibit topoisomerase activity or form stabilized ternary complexes with DNA and topoisomerase have a high potential for development as DNAtargeting anticancer drugs [64]. Five proflavine derivatives 6ae6e were evaluated for their Topo I relaxation activity. Fig. 9a illustrates the human DNA Topo I relaxation activity of these derivatives. As is shown, supercoiled pBR322 was fully relaxed by the enzyme in the absence of the studied samples. Compounds 6aee showed Topo I relaxation activity at very high ligands concentrations 60 mM. Campothecine (10 mM) and ethidium bromide (5 mM) were used as controls. We employed also a DNA unwinding assay using relaxed plasmid DNA (pBR322) and human topoisomerase I in the presence of two concentrations (5 and 60 mM) of acridine derivatives (6aee). Campothecine (10 mM), topotecane (30 mM) and ethidium bromide (5 mM) were used as controls. Representative results of such assays in the presence of acridine drugs are shown in Fig. 9b. In the presence of (6aee) relaxed plasmid DNA was converted into fully supercoiled DNA. These results showed that all five acridine derivatives could intercalate into DNA and induced constrained negative supercoils.

Fig. 8. Native gel electrophoresis of supercoiled plasmid DNA (pUC 19) in the presence and absence of increasing 6aee compounds. (Lane 1ae5a: 5 mM, lane 1be5b: 20 mM, 1ce5c: 40 mM, 1de5d: 60 mM, 1ee5e: 80 mM).

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J. Plsikova et al. / European Journal of Medicinal Chemistry 57 (2012) 283e295

a

1

2

6a 3a 3b

6b 4a

6a

6b

6c 5a

4b

6d 5b

6a

6b

6e 7a

7b

CPT EtBr

7b

CPT

TOPO EtBr

R

S

b

1

2

3a

3b

4a

6c 4b

5a

5b

6d 6a

6e 6b

7a

Fig. 9. a) Effects of acridine ligands 6aee on the relaxation of pBR322 plasmid DNA by topoisomerase I. pBR322 (1.4 mg, lane 1) was incubated for 45 min at 37  C with 3 units of topoisomerase I in the absence (lane 2) or presence of ligands (lane 3ae7a: 5 mM, lane 3be7b: 60 mM). Line CPT  pBR322 þ Topo I þ campothecine (10 mM), line EtBr  pBR322 þ Topo I þ ethidium bromide (5 mM). The DNA samples were run on agarose gel followed by ethidium bromide staining. S ¼ supercoiled plasmid, R ¼ relaxed, opencircular plasmid. b) effects of acridine ligands 6aee on the relaxation of relaxed pBR322 plasmid DNA by topoisomerase I. Relaxed pBR322 (1.4 mg, lane 1) was incubated for 45 min at 37  C with 3 units of topoisomerase I in the absence (lane 2) or presence of ligands (lane 3ae7a: 5 mM, lane 3be7b: 60 mM). Line CPT  relax pBR322 þ Topo I þ campothecine (10 mM), line TOPO  relax pBR322 þ Topo I þ topotecane (30 mM), line EtBr  relax pBR322 þ Topo I þ ethidium bromide (5 mM). The DNA samples were run on agarose gel followed by ethidium bromide staining.

2.2.4. Biological studies To determine whether the tested compounds 6aee demonstrate any biologically relevant effects, an elementary evaluation of HL-60 cell number was initially carried out with different concentrations and incubation periods. Three different concentrations (5, 15 and 25 mM) of the tested compounds were evaluated for biological action after 24, 48 and 72 h treatment. The analysis of variations in total cell number demonstrates the core ability of the tested compounds to affect cancer cell proliferation and cell cycle distribution. The 6a derivative was capable to suppress cell numbers comparably to the other two ones already 24 h after addition and even at the lowest tested concentration (Table 3). However, these initial effects vanished latter on and the surviving populations were proliferating further. The ability of derivatives 6aee to impair cell proliferation was tested by flow cytometric analysis of the cell cycle. The HL-60 cells were exposed to different concentrations of 6a, 6d and 6e, but similarly to the results of variations in cell numbers, derivatives 6aec did not affect the cell cycle distribution significantly (data not shown). The other two derivatives, 6d and 6e, demonstrated more considerable effects that were both concentration- and timedependent, but which were more pronounced in populations treated with derivative 6e. In the case of derivative 6d, we did not record any significant changes in cell cycle progression (data not shown) despite the significant suppression of cell numbers. But in contrast, the 6e derivative induced cell cycle block in G0/G1-phase at the expense of both the S-and the G2/M-phase (Table 4). As with the changes in total cell number, the percentage of cells with dissipated mitochondrial membrane potential did not increase greatly after treatment with derivative 6a (data not shown). Likewise, the treatment with derivative 6d showed significant results only at the highest tested concentration and only after 72 h treatment. In contrast, but again in accordance with the changes in total cell number, the activity of derivative 6e affected the

mitochondrial physiology and caused mitochondrial membrane potential dissipation in up to 70% of cells (Fig. 10). In order to prove the stimulation of programmed cell death, the activation of typical apoptotic execution caspase-3 was monitored by a flow cytometric analysis. The changes in caspase-3 activation showed trend very similar to the changes observed in MMP analyses. Neither 6a nor 6d derivatives had significant effects on caspase-3 activation (except for the highest concentration of 6d/ 72 h treatment where 46.8% of cells showed signs of activated caspase-3). Nevertheless, the derivative 6e stimulated caspase-3 activation in a very similar manner to MMP (Fig. 11), indicating the specific activation of apoptosis in target cells. In order to support these results demonstrating the onset of programmed cell death (MMP dissipation and caspase-3 activation), analysis using Annexin/PI double staining was also performed. Once again, derivates 6a and 6d were ineffective and failed to have any significant effect on the onset of apoptosis or necrosis (data not shown), while derivate 6e significantly increased the induction of necrosis 48 and 72 h after the treatment (Fig. 12).

Table 3 Percentage of total cell number. 24 h Control 6a 5 mM 15 mM 25 mM 6d 5 mM 15 mM 25 mM 6e 5 mM 15 mM 25 mM

100.00 71.70 78.95 71.98 76.19 75.20 78.32 73.70 74.49 76.71

48 h          

3.09 8.56* 5.01 5.25* 3.29* 3.57* 3.84* 3.76* 4.80* 3.78*

100.00 78.56 80.03 86.85 81.90 77.14 67.98 72.18 52.70 40.67

72 h          

5.48 6.74 6.66 2.20 4.12 5.18 4.73* 2.86* 8.00* 6.76*

100.00 79.32 84.97 94.84 83.30 54.40 48.67 57.54 33.13 20.46

         

5.21 3.70 2.58 6.75 4.01 3.10* 4.45* 3.92* 1.71* 2.21*

The results are presented as a mean  SD, statistical significance: p < 0.05 (*) for particular experimental group compared to untreated control.

J. Plsikova et al. / European Journal of Medicinal Chemistry 57 (2012) 283e295

289

Table 4 Cell cycle distribution of 6e. S

G1 24 h

48 h

72 h

Control 5 mM 15 mM 25 mM Control 5 mM 15 mM 25 mM Control 5 mM 15 mM 25 mM

35.96 36.31 41.77 51.52 38.29 45.10 64.37 65.72 47.02 48.93 70.31 73.59

           

1.63 0.63 6.56 6.67* 0.22 10.62 10.70* 5.39* 6.19 7.86 6.85* 1.31*

44.41 47.31 46.58 38.11 45.29 43.54 27.52 27.77 40.30 43.60 23.84 22.50

G2/M            

0.77 1.69 6.31 5.22 1.23 7.26 8.68* 4.03* 3.15 7.31 6.29* 0.93*

19.62 16.37 11.65 10.37 16.42 11.36 8.11 6.51 12.68 7.47 5.85 3.91

           

0.96 1.07 0.28* 1.45* 1.03 3.41 2.26* 1.38* 3.21 1.53 0.58* 0.62*

The results are presented as a mean  SD, statistical significance: p < 0.05 (*) for particular experimental group compared to untreated control.

Since action of the drugs generally requires interaction with the cells or even internalization, we verified the activity of the studied derivatives using a fluorescence microscopy. Their fluorescence was visualized by an inverted fluorescence microscope Leica DMI6000 B and merged together with a brightfield image. The data clearly demonstrates vast differences in the abilities of derivatives to enter the tumor cells. Whereas derivative 6a enters only cells with typical necrotic morphology and avoids living cells, the 6d and 6e derivatives enter all cells regardless of their physiological status (Fig. 13). In summary, we may suggest that among the tested compounds the derivative 6e inhibits the cell proliferation associated with changes in the cell cycle progression, and induces apoptosis of HL60 cells through a caspase-3 dependent pathway. All these results are in agreement with studies of resveratrol [65], which has been shown to activate caspase-3, and inhibit the proliferation and/or induce apoptotic cell death in a number of tumor cells, including HL-60. There are various approaches to testing of cytotoxicity. Reporting IC50 (or possibly other IC-values) based on for example MTT-test is one of the ways. It is advantageous as it is easy and straightforward as well as it is also easy to evaluate. However, according to literature as well as our own experience, the MTT assay (or other similar ones) is giving us only a one number that represents result of multiple processes on cellular and cell population level. For testing of new drugs we have compared various approaches (including comparison using IC50 values) and found that it might be quite misleading to compare various drugs only at IC50

Fig. 10. The effect of the 6e on changes in mitochondrial membrane potential (MMP). The results are presented as a mean  SD, statistical significance: p < 0.05 (*) for particular experimental group compared to untreated control.

Fig. 11. The effect of the 6e on changes in caspase-3 activation. The results are presented as a mean  SD, statistical significance: p < 0.05 (*) for particular experimental group compared to untreated control.

concentrations (unpublished results). Therefore we are using approach based on limited number of fixed concentrations that are used to compare toxicity of the drugs e to get more information at lower number of tested concentrations. It is always a trade-off whether to simply test extensive number of drugs/drug concentrations or to gather more information on limited matrix of drugs vs. concentrations. As the MTT assay measures metabolic activity of the cells, decrease in signal might be a result of changes in cell proliferation and/or cell viability and/or cell metabolism. Therefore these assays are giving only one number that is a sum of these processes, but much more could be hidden behind. Sometimes, as it was presented for example in our previous and above mentioned study of Janovec et al. [34], the IC50 values determined by MTT assay (presented as cytotoxicity) and IC50 values determined by DCC test (presented as viability) are showing high level of similarity, although some differences can be found. This is by our own experience a very rare case. Other analyses such as cell counts, viability, mitochondrial membrane potential dissipation, caspase-3 activation or others are giving a glance on processes that are involved in cytotoxic action of the drug and more precise information. For example high toxic rate can be result of necrosis as well as apoptosis. Percentage of cells with activated caspase-3 and its comparison to overall toxicity can help to answer this question. Our recent results (partially presented only those which are significant) proved, that derivative 6e causes mitochondrial membrane potential dissipation together with intensive activation of caspase-3 and significant drop in cell viability. Some differences, like IC50 for 6e established by DCC test [34] at 2.44 mM and insignificant changes to untreated control established in our recent manuscript by e.g. MMP dissipation method at 5 mM concentration can be explained by differences in experimental design, cell cultivation conditions (including cell passage number, density of seeding, etc.), different drug preparation and methods that were used. The trend is, however, similar as 6e already at 15 mM concentration induces massive elevation in percentage of cells with dissipated MMP. The cells themselves are therefore sensitive to tested drug at concentrations that are if not equal but still in similar range. When considering difference in methods used, we may notice that even when the analyses in recent manuscript were executed from the same pool of cells, the methods do not consider the same attribute of the cell. Therefore even when percentage of cells with dissipated MMP and activated caspase-3 is approx. 40% (15 mM/ 48 h), the viability is approx. 80%. Even though the results make sense as MMP dissipation and caspase-3 activation are part of the

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Fig. 12. The effect of the 6e on changes of cell death induction. The results are presented as a mean  SD, statistical significance: p < 0.05 (*) for particular experimental group compared to untreated control.

apoptotic machinery and the loss of membrane integrity/viability is mostly a final stage of cell demise. 2.3. Molecular modeling Electrophoretic gel mobility shift assay provides an evidence of the reduction of the bands mobility upon decreasing of alkyl chain of ligands 6aee of pre-stained dsDNA sample (Fig. 8). Of note, the bands were sharp for long alkyl chains derivatives 6d, 6e but

started to become diffuse and less intense at higher concentrations for short arms compounds 6aec. This may indicate partial denaturation or degradation of dsDNA [66]. To shed more light on these experimental results we applied principles of molecular dynamics. We explored all possible DNAeligand interactions from the point of the intercalation modes, including major, minor and threading modes. We decided to study a dynamic of the putative interaction complexes. The idea behind this study was to explore possible DNA structural changes adopted upon ligand binding. Decamers

Fig. 13. Internalization of all three derivatives into the HL-60 cells.

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dAdT and dCdG were chosen as the DNA models. Only border ligands 6a, 6e which best fit the extremes in the bands mobility were chosen for the experiment in order to save time (data not show). We would like to mention here that a deal was not to propose the ligand interaction pose in its complex with DNA, but to assume changes and circumstances that might help to understand DNA degradation observed in the experiments. Thus, the results presented here must be considered as a crude assumption. Molecular dynamics simulations were performed using the same procedure described previously [34]. Briefly, entry coordinates were gained by docking study in Autodock 4.2. The water-box as solvent was used and periodic boundary conditions were applied within NAMD 2.6 software. Finally, 1 ns production run was executed to achieve the equilibrium of the solvated complexes and entire structures were evaluated visually. As depicted in Fig. 14, a certain level of distortion of the phosphodiester backbone is visible in the complex with ethyl derivative 6a having minor extent for the complex with 6e. In order to rationalize this observation we extended the period of the simulation for the complex [dCdG]10-6a to 7 ns. During in simulation course the acridine chromophore was expelled from its intercalation cavity and thus released ligand 6a formed minor groove binding like positions (Fig. S1eS3). From the structure obtained in the simulation it is possible to assume that when the guanidine core is close to the phosphoribose backbone at the point of maximal DNA distortion (Fig. S1), a direct attack on DNA strands could occur [66]. Such an attack could be made by hydroxyl species generated by the guanidine core at physiological pH. This assumption is in accordance with the fact that hexyl derivate 6e, which offered minimal backbone distortion during the simulation, does not

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provide shift in band mobility. Although in the view of the energetic terms the point of the maximal distortion with the energy 3403 kcal mol1 seems to be structurally meaningless in comparison to the intercalation (3562 kcal mol1) and the minor groove binding pose (3544 kcal mol1), it might be still considered as the highly probable structure where disruption of DNA strand could be generated. Though all above mentioned findings should be considered as preliminary results, we regard them sufficiently interesting to be presented here. Further studies that are beyond the scope of this work are carried out now to discover the mechanisms underlying the observed diffuse bands. 3. Conclusion A new series of 3,6-bis(3-alkylguanidino)acridine 6aee had been synthesized. These compounds showed strong DNA binding activity (K ¼ 1.25e5.26
105 M1) which decreased as the length of side alkyl substituents increased. UVevis, fluorescence, circular and linear dichroism spectroscopy, DNA melting and viscosimetric techniques indicated that the studied compounds act as effective DNA-intercalating agents. In summing up the biological results, we may conclude that derivatives 6a and 6d showed a mostly transient cytostatic action at the concentrations used in the experiment. An initial decrease in total cell numbers (after 24 h) induced by derivatives 6a and 6d, the minimal occurrence of cells with dissipated mitochondrial potential, and the insignificant changes in cell cycle progression indicate the initial transient suppression of cell proliferation without induction of programmed cell death (no activation of caspase-3). On the other hand, derivative 6e initially proved to be both cytostatic and cytotoxic when incubated with cells for longer periods of time. These findings are supported by the insignificant changes in programmed cell death hallmarks such as MMP dissipation, caspase-3 activation (with the exception of derivative 6e at 25 mM) or phosphatidylserine externalization and cell death/ survival. Generally, 3,6-bis(3-alkylguanidine)acridines had better DNA binding than structurally resemble 3,6-bis(3-alkylurea)acridines. This is probably due to presence of positively charged guanidine cores that might increase electrostatic attractive forces between guanidine derivatives and DNA. This fact might advance guanidine derivatives in an attraction to negatively charged DNA than urea derivatives, neutral at physiological pH. Conversely, 3,6-bis(3alkylthiourea)acridines have in one magnitude lower binding constants what might be prescribed to thiourea group obtaining sulfur atom disable to form strong hydrogen bonds with nucleic bases or phosphodiester backbone of DNA. Interestingly, 3,6bis(imidazolidinones)acridines obtained slight higher DNA binding capability than 3,6-bis(-3-alkylguanidine)acridines, although it is hardly to clarify the reason of these differences. 4. Experimental section 4.1. Chemistry

Fig. 14. Superposition of the phosphodiester backbone of the complex [dCdG]10-6a (red) and the complex [dCdG]10-6e (white) on atoms C5, N3, N1 of the guanidine residue no. 6. For clarity ligands 6a, 6e and all hydrogens were omitted. Picture was prepared using Chimera software [78]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4.1.1. General method for the synthesis of (acridine-3,6-diyldiimino) bis[(alkylamino) methaniminium]dichloride tetrahydrates (6aee) Bis-thiourea 3aee (0.17 mM) was suspended in anhydrous THF (3 mL), anhydrous Na2SO4 (13 mM), Et3N (25 mM), and a small amount of dried CaCl2 was added. The suspension was stirred at room temperature until it turned red, at which point HgO (7 mmol) was added and intensive stirring continued at 55  C until all thiourea was spent (cca. 1 h, TLC monitoring, eluent acetone/MeOH/ Et3N 3:1:1). The precipitate was then filtered off, and the excess of ammonium hydroxide was added to the crude THF solution of

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carbodiimides 5aee. At the end of the reaction (TLC), the reaction mixture was filtered off, the product was purified by flash chromatography on silica gel, eluent acetone/MeOH/Et3N (3:1:1) and arose in the form of oil. To obtain corresponding hydrochlorides 6aee, oil residues were dissolved in acetone and 5 equivalents of the solution of 36% HCl in acetone (1:9) was added. The resulting orange precipitate was collected by filtration, washed with acetone and diethyl ether, and dried in vacuo. NMR spectra were measured on a Varian Mercury Plus 400 MHz NMR spectrometer in CD3OD at ambient temperature. The d chemical shifts were referenced to tetramethylsilane (0 ppm), the magnitudes of coupling constants J are expressed in Hz. Melting points were determined using a Boetius hot-stage apparatus and are uncorrected. Elemental analyses were performed on a PerkineElmer analyzer CHN 2400. Reactions were monitored by a thin-layer chromatography (TLC) using Silufol plates with detection at 254 nm. Preparative column chromatography was conducted using Kiesegel Merck 60 column, type 9385, grain size 250 nm. All tested compounds had a purity of at least 95%. 4.1.2. Synthesis of (acridine-3,6-diyldiimino)bis[(ethylamino) methaniminium]dichloride tetrahydrate (6a) Yield ¼ 34%, orange solid, m.p. ¼ 210e212  C. For C19H23N7$2HCl$4H2O (494.43) calculated: 46.16% C, 6.73% H, 19.83% N; found: 45.85% C, 6.52% H, 20.06% N. 1 H NMR (400 MHz, CD3OD): 9.67 (s, 1H, H-9), 8.46 (d, 2H, H-1,8, J ¼ 8.8), 8.04 (s, 2H, H-4,5), 7.74 (d, 2H, H-2,7, J ¼ 8.8), 3.49 (q, 4H, 2
NeCH2, J ¼ 7.2), 1.38 (t, 6H, 2
CH3, J ¼ 7.2). 13 C NMR (100 MHz, CD3OD): 155.51 (2
C-NH), 148.66 (C9), 147.76 (C3, C6), 142.99 (C4a, C10a), 133.69 (C1, C8), 124.23 (C8a, C9a), 123.42 (C2, C7), 107.08 (C4, C5), 38.74 (2
NeCH2), 14.00 (2
CH3). 4.1.3. Synthesis of (acridine-3,6-diyldiimino)bis[(n-propylamino) methaniminium]dichloride tetrahydrate (6b) Yield ¼ 37%, orange solid, m.p. ¼ 195e197  C. For C21H27N7$2HCl$4H2O (522.48) calculated: 48.28% C, 7.14% H, 18.77% N; found: 48.55% C, 6.35% H, 18.82% N. 1 H NMR (400 MHz, CD3OD): 9.64 (s, 1H, H-9), 8.44 (d, 2H, H-1,8, J ¼ 8.8), 8.03 (s, 2H, H-4,5), 7.73 (d, 2H, H-2,7, J ¼ 8.8), 3.41 (t, 4H, 2
NeCH2, J ¼ 7.2), 1.78 (sext, 4H, 2
CH2, J ¼ 7.2), 1.08 (t, 6H, 2
CH3, J ¼ 7.2). 13 C NMR (100 MHz, CD3OD): 155.88 (2
CeNH), 148.31 (C9), 147.47 (C3, C6), 143.46 (C4a, C10a), 133.73 (C1, C8), 124.47 (C8a, C9a), 123.65 (C2, C7), 107.95 (C4, C5), 45.52 (2
NeCH2), 23.03 (2
CH2), 11.71 (2
CH3). 4.1.4. Synthesis of (acridine-3,6-diyldiimino)bis[(n-butylamino) methaniminium]dichloride tetrahydrate (6c) Yield ¼ 29%, orange solid, m.p. ¼ 200e202  C. For C23H31N7$2HCl$4H2O (550.53) calculated: 50.18% C, 7.51% H, 17.81% N; found: 50.26% C, 7.68% H, 18.01% N. 1 H NMR (400 MHz, CD3OD): 9.64 (s, 1H, H-9), 8.44 (d, 2H, H-1,8, J ¼ 8.8), 8.03 (s, 2H, H-4,5), 7.73 (d, 2H, H-2,7, J ¼ 8.8), 3.45 (t, 4H, 2
NeCH2, J ¼ 6.8), 1.75 (m, 4H, 2
CH2), 1.51 (m, 4H, 2
CH2), 1.03 (t, 6H, 2
CH3, J ¼ 7.2). 13 C NMR (100 MHz, CD3OD): 155.64 (2
CeNH), 148.32 (C9), 147.48 (C3, C6), 143.15 (C4a, C10a), 133.62 (C1, C8), 124.26 (C8a, C9a), 123.46 (C2, C7), 107.53 (C4, C5), 43.58 (2
NeCH2), 31.57 (2
CH2), 21.06 (2
CH2), 14.09 (2
CH3). 4.1.5. Synthesis of (acridine-3,6-diyldiimino)bis[(n-pentylamino) methaniminium]dichloride tetrahydrate (6d) Yield ¼ 42%, orange solid, m.p. ¼ 205e207  C. For C25H35N7$2HCl$4H2O (578.59) calculated: 51.90% C, 7.84% H, 16.95% N; found: 52.03% C, 7.52% H, 16.75% N.

1 H NMR (400 MHz, CD3OD): 9.67 (s, 1H, H-9), 8.46 (d, 2H, H-1,8, J ¼ 8.8), 8.03 (s, 2H, H-4,5), 7.74 (d, 2H, H-2,7, J ¼ 8.8), 3.43 (t, 4H, 2
NeCH2, J ¼ 7.2), 1.77 (m, 4H, 2
CH2), 1.45 (m, 8H, 4
CH2), 0.98 (t, 6H, 2
CH3, J ¼ 6.8). 13 C NMR (100 MHz, CD3OD): 155.63 (2
CeNH), 148.63 (C9), 147.70 (C3, C6), 143.02 (C4a, C10a), 133.68 (C1, C8), 124.21 (C8a, C9a), 123.43 (C2, C7), 107.16 (C4, C5), 43.81 (2
NeCH2), 30.05 (2
CH2), 29.24 (2
CH2), 23.43 (2
CH2), 14.38 (2
CH3).

4.1.6. Synthesis of (acridine-3,6-diyldiimino)bis[(n-hexylamino) methaniminium]dichloride tetrahydrate (6e) Yield ¼ 34%, orange solid, m.p. ¼ 235e237  C. For C27H39N7$2HCl$4H2O (606.64) calculated: 53.46% C, 8.14% H, 16.16% N; found: 53.51% C, 7.93% H, 16.39% N. 1 H NMR (400 MHz, CD3OD): 9.66 (s, 1H, H-9), 8.45 (d, 2H, H-1,8, J ¼ 8.8), 8.03 (s, 2H, H-4,5), 7.73 (d, 2H, H-2,7, J ¼ 8.8), 3.43 (t, 4H, 2
NeCH2, J ¼ 7.2), 1.76 (m, 4H, 2
CH2), 1.51e1.36 (m, 12H, 6
CH2), 0.95 (t, 6H, 2
CH3, J ¼ 7.2). 13 C NMR (100 MHz, CD3OD): 155.64 (2
CeNH), 148.57 (C9), 147.68 (C3, C6), 143.10 (C4a, C10a), 133.67 (C1, C8), 124.27 (C8a, C9a), 123.43 (C2, C7), 107.24 (C4, C5), 43.85 (2
NeCH2), 32.62 (2
CH2), 29.51 (2
CH2), 27.59 (2
CH2), 23.68 (2
CH2), 14.42 (2
CH3). 4.2. Materials All chemicals and reagents were purchased of reagent grade and used without further purification. Propidium iodide (PI), Hoechst 33342, ethidium bromide, Triton X-100, reduced form of glutathione (GSH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and calf thymus DNA were obtained from SigmaeAldrich Chemie (Germany). EDTA, RNase A and proteinase K were purchased from Serva (Germany), plasmid pUC 19 (2761 bp, DH 5a), agarose (type II No-A-6877) (Sigma) and 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) were purchased from Merck (Germany) and all other chemicals were purchased from Lachema (Czech Republic). 4.3. Biological studies 4.3.1. UVevis absorption measurements UVevis spectra were measured on a Varian Cary 100 UVevis spectrophotometer in 0.01 M Tris buffer (pH 7.4). The concentration of ctDNA (Sigma Chemical Co.) ranged from 0 to 2.44
104 M bp. The guanidine derivatives were all dissolved in DMSO from which working solutions were prepared by dilution using 0.01 M Tris buffer to a concentration of 1.28
105 M. All measurements were performed at 25  C. 4.3.2. Fluorescence measurements Fluorescence measurements were scanned on a Varian Cary Eclipse spectrofluorimeter with a 5 nm slit width for excitation and emission beams. Emission spectra were recorded in the region 400e600 nm using an excitation wavelength of 384 nm. Fluorescence intensities are expressed in arbitrary units (au). Fluorescence titrations were conducted by adding increasing amounts of ctDNA directly into the cell containing the solution of 6aee (c ¼ 9.58
106 M, 0.01 M Tris buffer, pH 7.4). The concentration range of DNA was 0e2.57
104 M bp. All measurements were performed at 25  C. 4.3.3. CD and LD spectroscopy CD spectra were recorded on a Jasco J-810 spectropolarimeter in 1 mm quartz cuvettes and are the mean result of three scans from which the buffer background had been electronically subtracted.

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All measurements were performed in 0.01 M Tris buffer (pH 7.4), the concentration of ctDNA was 1.95
101 mM and the concentration of 6aee was 6.52 mM. Flow LD spectra were collected by using a flow Couette cell in a Jasco J-720 spectropolarimeter adapted for LD measurements. Long molecules, such as DNA (minimum length of w250 bp), can be orientated in a flow Couette cell. The flow cell consists of a fixed outer cylinder and a rotating solid quartz inner cylinder, separated by a gap of 0.5 mm, giving a total pathlength of 1 mm. LD spectra of ctDNA at the concentration of 3.11
108 M modified by guanidine derivatives (11e58
106 M) were recorded at 25  C in 10 mM Tris buffer in the range of 210e550 nm. 4.3.4. Viscosimetric titration The relative viscosity of ctDNA solutions at the concentration of 3.11
108 M in the presence of 6aee (12e60
106 M) was measured by microviscometry (AMVn Automated Micro Viscometer, Anton Paar GmbH, Austria) using a 1.6-mm capillary tube at 25  C. Density of the solutions was measured by Density Meter DMA 4500 (Anton Paar GmbH, Austria). 4.3.5. 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 ctDNA (7.64 mM) or a mixture of ctDNA with 6aee (12.4
106 M) in BPE buffer, (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA, pH 7.1), with a heating rate of 1  C min1. The melting temperatures were determined as the maximum of the first derivative plots of melting curves. 4.3.6. Equilibrium binding titration The absorption and fluorescence data were used to construct binding plots according to the method of McGhee and von Hippel using data points from a Scatchard plot [40,41]. The binding data were obtained using GNU Octave 2.1.73 software [42] to extract the K and n values. 4.3.7. DNA unwinding assay A DNA unwinding assay was used to assess the ability of the acridine-based compounds to intercalate into plasmid DNA. This assay monitors the extent of intercalation of a drug as a function of the conversion of relaxed DNA into negatively supercoiled DNA. pBR322 or relaxed pBR322 (1.4 mg) was applied as the substrate in a reaction volume of 20 mL containing the reaction buffer (10
) and a 0.1% bovine serum albumin (BSA). The appropriate unwinding assay was added as indicated, and the reaction was initiated by the addition of 3 units of topoisomerase I (Takara, Japan). Reactions were carried out at 37  C for 45 min. Gel electrophoresis was performed at 7 V/cm for 2 h in TBE buffer (45 mM Tris base, 45 mM boric acid, 1 mM EDTA, pH 8.0) on a 0.8% agarose gel. Gel was stained with ethidium bromide (1 mg/mL) and photographed under UV light. 4.3.8. Cell culture and experimental design HL-60 cells (human promyelocytic leukemia, ATCC, USA) were grown in complete RPMI medium (Gibco, USA) supplemented with 7.5% NaHCO3 (10 mL L1), penicillin (100 U mL1), streptomycin (100 mg mL1), amphotericin (25 mg mL1, Invitrogen, USA) and 10% heat-inactivated fetal calf serum (FCS, PAA Laboratories GmbH, Austria) and maintained at 37  C, 95% humidity and in a 5% CO2 atmosphere. For the experiments, cells were seeded in Ø 60 mm Petri dishes (TPP, Switzerland), to which the studied chemical compounds were added. Results were analyzed 24, 48, and 72 h after compound addition.

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4.3.9. Quantitation of total cell number Absolute numbers of cells within individual groups were counted with Coulter Counter (Model ZF, Coulter Electronics Ltd., Luton, Bedfordshire, UK). The total cell numbers were expressed as a percentage of the untreated control. 4.3.10. Analysis of cell cycle parameters Cells were treated for 24, 48, or 72 h and subsequently harvested by centrifugation, washed in cold phosphate-buffered saline (PBS), fixed in cold 70% ethanol and kept at 4  C overnight. Prior to analysis, cells were washed twice in PBS, resuspended in staining solution (0.1% Triton X-100, 0.137 mg mL1 of ribonuclease A and 0.02 mg mL1 of propidium iodide or PI), incubated in darkness at RT for 30 min and analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The ModFit 3.0 (Verity Software House, Topsham, ME, USA) software was used to generate DNA content frequency histograms and to quantify the number of cells in the individual cell cycle phases. 4.3.11. Detection of mitochondrial membrane potential (MMP) Cells (5
105) were treated for 24, 48, or 72 h and subsequently harvested by centrifugation, washed once with Hank’s balanced salt solution (HBSS), resuspended in HBSS supplemented with 0.1 mM tetramethylrhodamine ethyl ester perchlorate (TMRE; SigmaeAldrich, St. Louis, MO, USA) and incubated for 20 min at 37  C in darkness and analyzed using a BD FACSCalibur flow cytometer. The cells (1
104 per sample) were gated according to FSC
SSC dot blot and the TMRE fluorescence was analyzed in FL2 channel. The results are presented as the percentage of cells with decreased fluorescence indicating dissipated MMP. 4.3.12. Caspase-3 activation Activation of caspase-3 was analyzed using a FITC Active Caspase-3 Apoptosis Kit (BD Pharmingen, Franklin Lanes, NJ, USA, cat.# 550480) according to the manufacturer’s instructions. Cells were harvested together, washed in cold PBS, fixed and permeabilized for 20 min on ice, washed again twice in cold PBS, incubated with antibody for 30 min at RT, washed and finally analyzed using the BD FACSCalibur flow cytometer. Results were evaluated as percentages of positively stained cells. 4.3.13. Phosphatidylserine externalization analysis Phosphatidylserine externalization and cell survival were analyzed using Annexin V-FITC (BD Pharmingen) and propidium iodide (PI; SigmaeAldrich) double-staining. Cells (1.5
105) were treated for 24, 48, or 72 h and subsequently harvested by centrifugation, stained with Annexin V-FITC (according to manufacturer’s instructions) in binding buffer (10 mM HEPES, 2.5 mM CaCl2, 140 mM NaCl) for 10 min, washed, shortly stained with PI (1 mg mL1) and analyzed using the BD FACSCalibur flow cytometer. Results were analyzed using a CellQuest Pro software. 4.3.14. Uptake of tested derivatives Cells were treated with all three derivatives at 50 mM for 18 h, washed with PBS, resuspended in HBSS, mounted as native slides and visualized with inverted fluorescent Leica DMI6000 B microscope (Leica Microsystems, Wetzlar, Germany). To visualize all cells, the fluorescence channel was merged with brightfield picture. The microscope setup was as follows: Objective: HCX APO U-V-I 40.0
0.75 DRY/Camera: DFC 420 C (8 bit, 3
3 binning, pixel size ¼ 0.3 mm)/Filter cube: A4 (Ex ¼ 360/40; DM ¼ 400; Em ¼ 470/40). 4.3.15. Molecular modeling Chemaxon was used to determine the charge of ligands 6a and 6e at physiological pH [67]. Thus, two additional hydrogen atoms

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were added on the guanidine moieties of ligands to set charge to þ2. Molecular models and coordinates of acridine ligands 6a, 6e were built through building options of a GABEDIT package and then minimized using a GAMESS software (Ver.: June 6, 1999) at a RHF/ AM1 semi-empirical level of theory [68,69]. Docking simulations were carried out using an AUTODOCK program ver. 4.2, while a MGL TOOLS 1.4.3 was used to prepare the input files [70e72]. All calculations were carried out in a NAMD 2.6 using a TIP3P potential for waters, a parm99.dat parameters set for nucleic acids, and GAFF atom types for the ligand [73e76]. ANTECHAMBER and XLEAP modules as a part of AMBERTOOLS 1.0 software package were applied to extrapolate missing ligand forceefield parameters and derive charges using an AM1-BCC method. Trajectories were examined visually using a VMD 1.8.6 software package [77] and Chimera software [78]. Precise descriptions of each step of molecular dynamics simulations and initial docking simulations have been published previously [79]. Shortly, we built pseudo-intercalation cavities in accordance with the published work [79] using our own building protocol that was successfully applied for related acridine derivatives [34]. In the first step, docking runs were performed to obtain the initial input coordinates for molecular dynamics simulations. Input structures were chosen in the view of the energetic terms. Thus, the solvated intercalation models were minimized and equilibrated. During these runs DNAeligands complexes were kept constrained. Subsequently, constrains were removed and entire solvated complexes were fully relaxed without additional restrains to allow exploration of a possible oligonucleotide structure distortion. 4.3.16. Statistical analysis Results were calculated as mean  standard deviation (S.D.) of at least three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc tests and results were deemed significant if p < 0.05. Acknowledgment This study was supported by the Slovak Research and Development Agency under contract VVCE-0001-07, VEGA grant No. 1/  0672/11, and Internal Grant Program of the P.J. Safárik University in Kosice (VVGS40/12e13, VVGS-PF-2012e16). Molecular graphics images were produced using a UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR01081). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2012.09.020. References [1] J.B. Chaires, Drug-DNA interactions, Curr. Opin. Struct. Biol. 8 (1998) 314e320. [2] D.E. Graves, L.M. Velea, Intercalative binding of small molecules to nucleic acids, Curr. Org. Chem. 4 (2000) 915e929. [3] B. Wang, J. Tan, L. Zhu, Selective binding of small molecules to DNA: application and perspectives, Colloids Surf. B 79 (2010) 1e4. [4] L. Strekowski, B. Wilson, Noncovalent interaction with DNA: an overview, Mut. Res. 623 (2007) 3e13. [5] G.M. Blackburn, M.J. Gait (Eds.), Nucleic Acids in Chemistry and Biology, IRL, Oxford, UK, 1996, p. 375. [6] W.S. Wade, M. Mrksich, P.B. Dervan, Design of peptides that bind in the minor groove of DNA at 50 -(A, T)5G(A, T)C(A, T) sequences by a dimeric side-by-side motif, J. Am. Chem. Soc. 114 (1992) 8783e8794. [7] S. White, E.E. Baird, P.B. Dervan, Effects of the A$T/T$A degeneracy of pyrroleimidazole polyamide recognition in the minor groove of DNA, Biochemistry 35 (1992) 12532e12537.

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