Synthesis, DNA Interaction, and Cytotoxic Activity of a Novel Proflavine

University, Moyzesova 11, SK-04167 Košice, Slovak Republic, and Department of ... Faculty of Chemical and Food Technology, Slovak Technical Universit...
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Bioconjugate Chem. 2007, 18, 93−100

93

Synthesis, DNA Interaction, and Cytotoxic Activity of a Novel Proflavine-Dithiazolidinone Pharmacophore Ladislav Janovec,† Danica Sabolova´,‡ Ma´ria Kozˇurkova´,‡ Helena Paulı´kova´,§ Pavol Kristian,† Ja´n Ungvarsky´,† Erika Moravcˇ´ıkova´,§ Ma´ria Bajdichova´,§ Dusˇan Podhradsky´,‡ and Ja´n Imrich*,† Department of Organic Chemistry, Department of Biochemistry, Institute of Chemistry, Faculty of Science, P.J. Sˇ afa´rik University, Moyzesova 11, SK-04167 Kosˇice, Slovak Republic, and Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak Technical University, Radlinske´ho 9, SK-81237 Bratislava, Slovak Republic. Received June 16, 2006; Revised Manuscript Received September 21, 2006

Five novel proflavine-dithiazolidinone derivatives 4a-4e have been designed and synthesized by the reaction of dialkyl acridin-3,6-diyl dithioureas 3a-3e with methyl bromoacetate. The binding affinity of dithiazolidinone hydrochlorides 5a-5e with calf thymus DNA and plasmid (pUC19) DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence, and CD spectroscopy. The effects of 5a-5e on the thermal denaturation profiles of calf thymus DNA were also studied. From spectrophotometric and spectrofluorimetric titrations, the binding constants for the pUC19 DNA-drug complexes were determined (K ) 6.2-2.2 × 104 M-1). In vitro cytotoxic activities of compounds 5a-5e toward murine leukemia cell line L1210 and human uterus carcinoma HeLa cells were also examined. 2′,2′′-[(Acridin-3,6-diyl)diimino]-3′,3′′-dipropyl-1,3-dithiazolidin4-one hydrochloride (5b) showed the highest activity against these cells with IC50 values of 6.3 µM and 12.9 µM over the course of 72 h.

INTRODUCTION

EXPERIMENTAL PROCEDURES

The interactions of small organic molecules with nucleic acids have been the focus of enhanced interest for DNA complexes over the past three decades (1). The understanding and recognition of the mode of action has allowed considerable progress to be made in the area of anticancer drug design, including acridinyl derivatives (2-8). One of the earliest and most wellknown agents possessing a mutagenic effect toward DNA and inhibition of protein synthesis (9) is 3,6-diaminoacridine (proflavine, 1). As a consequence, simple acridine compounds such as 1 have been used as antibacterial agents (10) and as probes to examine mutagenesis (11). Recently, the interaction of 1 with herring sperm DNA was investigated by cyclic voltametry and UV-vis spectroscopy to determine the binding constant (12). In our previous work, we observed the binding affinities and estimated the binding constants for N-(acridin-9-ylthiocarbamoyl)amino acids with plasmid DNA (pUC 19) (13). These findings inspired us to exploit the considerable intercalation capability of the highly fluorescent proflavine chromophore and prepare novel DNA-binding compounds in combination with the biologically active thiazolidinone skeleton. We wanted to ascertain the influence of structural changes on the intercalation properties, in particular, with reference to alkyl chain elongation. UV-vis spectrophotometry, fluorescence titration, circular dichroism (CD), and quantum chemical calculations (AM1) were all used to study the interaction of the compounds with DNA. The anticancer inhibitory effects were also tested against two tumor cell lines, viz., murine leukemia L1210 and human uterus carcinoma HeLa cells.

Materials and Instrumental Techniques. Plasmid isolation and purification processes have been described in detail previously (14). Solutions of calf thymus DNA (Sigma Chemical Co.) in TE (Tris-EDTA) buffer were sonicated for 5 min and the DNA concentration determined by absorbance at 260 nm. The purity of the DNA was determined by monitoring the value of A260/A280. UV-vis spectra were measured on a Varian Cary 100 UV-vis spectrophotometer in 0.1 M Tris buffer. Fluorescence measurements were made using a Varian Cary Eclipse spectrofluorometer with a slit width of 10 nm for the excitation and emission beams. Fluorescence intensity is expressed in arbitrary units. Thermal denaturation studies were conducted using a Varian Cary 100 UV-vis spectrophotometer supplied with a thermostatic cell holder. The temperature was controlled by a thermostatic bath ((0.1 °C). CD spectra were recorded on a Jasco J-810 spectropolarimeter in 1 cm quartz cuvettes and are the mean result of three scans from which the buffer background had been electronically subtracted. AM1 quantum chemical calculations were performed using the Gamess (version 6) program package (15). The binding affinities were calculated using absorbance spectra according to the method of McGhee and von Hippel using data points from a Scatchard plot (16, 17). The binding data were fitted using Grafit 4 software. 1H (400 MHz) and 13C (100 MHz) NMR spectra were measured on a Varian Mercury Plus NMR spectrometer at room temperature in CDCl3 using TMS as an internal standard (0 ppm for both nuclei). IR spectra were recorded on a Specord 75 IR spectrophotometer (Zeiss, Jena) as KBr discs (0.8 mg/300 mg KBr). Melting points were determined with a Koffler hot-stage apparatus and are uncorrected. Elemental analyses were performed on a Perkin-Elmer CHN 2400 analyzer. Reactions were monitored by thin-layer chromatography (TLC) using Silufol plates with detection at 254 nm. Preparative column chromatography was conducted using Kiesegel Merck 60 column, type

* Corresponding author. Ja´n Imrich, Department of Organic Chemistry, Institute of Chemistry, Faculty of Science, P. J. Sˇafa´rik University, Moyzesova 11, SK-04167 Kosˇice, Slovak Republic. Tel. +421-55905727892. E-mail: [email protected]. † Department of Organic Chemistry, P.J. Sˇ afa´rik University. ‡ Department of Biochemistry, P.J. Sˇ afa´rik University. § Slovak Technical University.

10.1021/bc060168v CCC: $37.00 © 2007 American Chemical Society Published on Web 11/07/2006

94 Bioconjugate Chem., Vol. 18, No. 1, 2007

9385 (grain size 250 nm), or aluminum oxide Merck 90 neutral (grain size 200 nm). Murine leukemia cell line (L1210) and human uterus carcinoma cells (HeLa) were obtained from the American Type Culture Collection (Rockville, MD). The L1210 cell line was kept in a RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (Grand Island Biological Co., Grand Island, NY), penicillin (100 units/mL), and streptomycin (100 µg/mL). HeLa cells were grown as monolayers in DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma) supplemented with 10% FCS and antibiotics penicillin (100 units/mL) and streptomycin (100 µg/ mL). Trypsin (0.25%, Boehringer, Mannheim) was routinely used for subculture. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air. Cell viability in the presence or absence of the experimental agent was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyl tetrazolium bromide; Sigma) microculture tetrazolium assay as described previously (18, 19). Cells harvested in the log phase of growth were counted and seeded (2.5 × 104 cells/100 µL well) in 96-well microtiter plates. After 24 h of incubation at 37 °C and 5% CO2, cultures were treated with varying concentrations of a drug. After 45 h or 69 h exposure to the compound, MTT (50 µL, 1 mg/mL) was added to each well. After 3 h, the cell cultures were centrifuged, the supernatant discarded, and then the resulting pellets thoroughly extracted into 200 µL of DMSO. Absorption at 540 nm was recorded using the MicroPlate Reader (Labsystem Multiscan, Multisoft, Finland). The MTT assay was performed three times for each compound. DNA fragmentation in HeLa and L1210 cells was measured after extraction of the DNA from a constant number of cells. After treatment of both cells lines for 24 h, 2 × 106 cells were collected and washed twice with cold PBS, resuspended in 200 µL TE lytic solution (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5% Triton X-100) and incubated for 10 min with RNase (2.5 mg/ mL) at 50 °C and then with proteinase K (2.5 mg/mL) for 30 min at 50 °C. The DNA was extracted with NaCl (1 mM), EDTA (1 mM), and 2-propanol (200 µL) for 6 h at -20 °C. After centrifugation, the DNA precipitates were washed with ethanol, resuspended in TE (Tris-EDTA) buffer and then separated by electrophoresis over a 1.5% agarose gel containing ethidium bromide (0.5 µg/mL) and the running buffer TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). The gel was run at 6 V/cm for 45 min after which it was photographed under UV transillumination. Synthesis of 3,6-Diisothiocyanatoacridine (2). An aqueous solution (40 mL) of Na2CO3 (400 mg) was added dropwise during 1 h to a well-mixed suspension of 3,6-diaminoacridine hemisulfate hydrate (500 mg, 1.93 mmol) and thiophosgene (0.296 mL, 3.87 mmol) in chloroform (50 mL). Following this, a saturated aqueous solution of NaHCO3 was slowly added until pH 8 was attained (approximately 2 h). The organic layer was then separated and the water layer extracted with chloroform (3 × 15 mL). The combined organic fractions were collected and dried over CaCl2. After filtration, the solution was evaporated in vacuo leaving behind the dark red crude product (approximately 500 mg). The crude product was then taken up in chloroform (100 mL) and purified by flash chromatography over neutral alumina using chloroform (50 mL) as the eluent to provide 300 mg of dark yellow solid product. Yield: 60%, mp 195-197 °C. IR (KBr, cm-1): 2012 (NCS), 1607 (CdC). 1H NMR (CDCl , TMS/ppm): δ 8.73 (s, 1H, CH-9), 7.99 (m, 3 2H, CH-4,5), 7.97 (m, 2H, CH-1,8), 7.38 (dd, 2H, CH-2,7, J ) 1.70 Hz, J ) 8.90 Hz). 13C NMR (CDCl3, TMS/ppm): δ 149.12 (C-4a,10a), 138.57 (NCS), 136.41 (C-9), 133.98 (C-3,6), 130.12 (C-1,8), 124.97 (C-8a,9a), 124.74 (C-2,7), 124.16 (C-4,5). Anal. calculated for C15H7N3S2 (293.37): C, 61.41; H, 2.41; N, 14.32. Found: C, 61.37; H, 2.37; N, 14.13.

Janovec et al.

General Procedure for the Preparation of 1′,1′′-(Acridin3,6-diyl)-3′,3′′-dialkyldithioureas 3a-3e. To a methanol (3 mL) suspension of 3,6-diisothiocyanatoacridine (50 mg, 0.17 mmol), a ten-molar excess of the appropriate amine was added. The reaction mixture was stirred for approximately 1 h while the progress of the reaction was monitored by TLC (toluene/ acetone 3:1) until completion. The solid product was filtered off and dried in vacuo. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-diethyldithiourea (3a). Yield: 65%, mp 228-230 °C. 1H NMR (DMSO, TMS/ppm): δ 9.93 (bs, 2H, 2 × NH), 8.86 (s, 1H, CH-9), 8.27 (s, 2H, CH-4,5), 8.21 (bs, 2H, 2 × NH), 8.02 (d, 2H, CH-1,8, J ) 9.20 Hz), 7.59 (d, 2H, CH-2,7, J ) 9.20 Hz), 3.53-3.60 (m, 4H, 2 × NCH2), 1.18 (t, 6H, 2 × CH3, J ) 7.04 Hz). 13C NMR (DMSO, TMS/ppm): δ 179.78 (CS), 149.27, 141.25, 134.95, 128.56, 122.59, 122.45, 115.91, 48.49 (NCH2), 13.92 (CH3). Anal. calculated for C19H21N5S2 (383.54): C, 59.50; H, 5.52; N, 18.26. Found: C, 59.30; H, 5.25; N, 18.20. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dipropyldithiourea (3b). Yield: 62%, mp 222-223 °C. 1H NMR (DMSO, TMS/ppm): δ 9.93 (bs, 2H, 2 × NH), 8.86 (s, 1H, CH-9), 8.32 (s, 2H, CH-4,5), 8.21 (bs, 2H, 2 × NH), 8.02 (d, 2H, CH-1,8, J ) 8.60 Hz), 7.59 (d, 2H, CH-2,7, J ) 8.60 Hz), 3.46-3.53 (m, 4H, 2 × NCH2), 1.56-1.66 (m, 4H, 2 × CH2), 0.94 (t, 6H, 2 × CH3, J ) 7.81 Hz). 13C NMR (DMSO, TMS/ppm): δ 180.08 (CS), 149.21, 141.40, 135.02, 128.54, 122.57, 122.43, 115.78, 45.62 (NCH2), 21.55 (CH2), 11.37 (CH3). Anal. calculated for C21H25N5S2 (411.59): C, 61.28; H, 6.12; N, 17.02. Found: C, 61.05; H, 5.95; N, 17.38. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dibutyldithiourea (3c). Yield: 61%, mp 218-220 °C. 1H NMR (DMSO, TMS/ppm): δ 9.92 (bs, 2H, 2 × NH), 8.85 (s, 1H, CH-9), 8.30 (s, 2H, CH-4,5), 8.19 (bs, 2H, 2 × NH), 8.01 (d, 2H, CH-1,8, J ) 8.80 Hz), 7.58 (d, 2H, CH-2,7, J ) 8.80 Hz), 3.50-3.57 (m, 4H, 2 × NCH2), 1.55-1.62 (m, 4H, 2 × CH2), 1.32-1.42 (m, 4H, 2 × CH2), 0.93 (t, 6H, 2 × CH3, J ) 7.60 Hz). 13C NMR (DMSO, TMS/ppm): δ 180.00 (CS), 149.31, 141.29, 134.88, 128.51, 122.56, 122.44, 115.84, 43.54 (NCH2), 30.33 (CH2), 19.57 (CH2), 13.65 (CH3). Anal. calculated for C23H29N5S2 (439.65): C, 62.84; H, 6.65; N, 15.93. Found: C, 62.52; H, 6.33; N, 16.18. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dipentyldithiourea (3d). Yield: 60%, mp 232-233 °C. 1H NMR (DMSO, TMS/ppm): δ 9.89 (bs, 2H, 2 × NH), 8.85 (s, 1H, CH-9), 8.29 (s, 2H, CH-4,5), 8.17 (bs, 2H, 2 × NH), 8.01 (d, 2H, CH-1,8, J ) 8.80 Hz), 7.58 (d, 2H, CH-2,7, J ) 8.80 Hz), 3.51-3.56 (m, 4H, 2 × NCH2), 1.57-1.64 (m, 4H, 2 × CH2), 1.32-1.37 (m, 8H, 4 × CH2), 0.91 (t, 6H, 2 × CH3, J ) 6.0 Hz). 13C NMR (DMSO, TMS/ppm): δ 180.03 (CS), 149.31, 141.31, 134.89, 128.49, 122.56, 122.39, 115.86, 43.82 (NCH2), 28.57 (CH2), 27.89 (CH2), 21.80 (CH2), 13.84 (CH3). Anal. calculated for C25H33N5S2 (467.70): C, 64.20; H, 7.11; N, 14.97. Found: C, 64.51; H, 7.36; N, 14.83. 1′,1′′-(Acridin-3,6-diyl)-3′,3′′-dihexyldithiourea (3e). Yield: 60%, mp 212-214 °C. 1H NMR (DMSO, TMS/ppm): δ 9.89 (bs, 2H, 2 × NH), 8.86 (s, 1H, CH-9), 8.29 (s, 2H, CH-4,5), 8.18 (bs, 2H, 2 × NH), 8.01 (d, 2H, CH-1,8, J ) 9.00 Hz), 7.58 (d, 2H, CH-2,7, J ) 9.00 Hz), 3.50-3.55 (m, 4H, 2 × NCH2), 1.56-1.63 (m, 4H, 2 × CH2), 1.30-1.37 (m, 12H, 6 × CH2), 0.89 (t, 6H, 2 × CH3, J ) 6.80 Hz). 13C NMR (DMSO, TMS/ppm): δ 180.01 (CS), 149.27, 141.17, 134.84, 128.39, 122.36, 122.43, 115.70, 43.87 (NCH2), 30.92 (CH2), 28.16 (CH2), 26.06 (CH2), 21.98 (CH2), 13.84 (CH3). Anal. calculated for C27H37N5S2 (495.76): C, 65.42; H, 7.52; N, 14.13. Found: C, 65.19; H, 7.25; N, 14.20. General Procedure for the Preparation of 2′,2′′-[(Acridin3,6-diyl)diimino]-3′,3′′-dialkyl-1,3-dithiazolidin-4-ones 4a4e and the Corresponding Hydrochlorides 5a-5e. To a

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Novel Proflavine−DNA Intercalators

dichloromethane (3 mL) suspension of the appropriate thiourea (0.13 mmol), triethylamine (0.04 mL, 0.286 mmol) was added. The mixture was stirred well for 10 min, followed by the addition of methyl bromoacetate (0.03 mL, 0.316 mmol), and monitored by TLC (toluene-acetone 3:1) until completion, at which time the reaction mixture was homogeneous. The solvent was evaporated in vacuo and the crude product purified over silica gel (toluene-acetone 3:1 as eluent). To produce 5, the product 4 was dissolved in acetone (0.5 mL), and an equimolar solution of HCl in methanol (1 mL of 35% hydrochloric acid in 9 mL of methanol) was added. The mixture was stirred for 1 h followed by the addition of diethyl ether. The resulting precipitant was filtered off and dried in vacuo. 2′,2′′-[(Acridin-3,6-diyl)diimino]-3′,3′′-diethyl-1,3-dithiazolidin-4-one (4a). Yield: 71%; yellow oil. 1H NMR (CDCl3, TMS/ppm): δ 8.68 (s, 1H, CH-9), 7.98 (d, 2H, CH-1,8, J ) 8.80 Hz), 7.70 (s, 2H, CH-4,5), 7.20 (d, 2H, CH-2,7, J ) 8.80 Hz), 3.99 (q, 4H, 2 × NCH2, J ) 7.20 Hz), 3.84 (s, 4H, 2 × SCH2), 1.36 (t, 6H, 2 × CH3, J ) 7.20 Hz). 13C NMR (CDCl3, TMS/ppm): δ 171.53 (CO), 155.03 (CN), 150.14, 150.05, 135.72, 129.58, 124.04, 122.88, 116.96, 38.44 (NCH2), 32.88 (SCH2), 12.55 (CH3). Anal. calculated for 5a, C23H21N5O2S2‚ HCl, yellow solid (500.05), mp 215-216 °C: C, 55.25; H, 4.43; N, 14.01. Found: C, 55.43; H, 4.39; N, 13.89. 2′,2′′-[(Acridin-3,6-diyl)diimino]-3′,3′′-dipropyl-1,3-dithiazolidin-4-one (4b). Yield: 65%; yellow oil. 1H NMR (CDCl3, TMS/ppm): δ 8.69 (s, 1H, CH-9), 7.97 (d, 2H, CH-1,8, J ) 8.80 Hz), 7.74 (s, 2H, CH-4,5), 7.20 (d, 2H, CH-2,7, J ) 8.80 Hz), 3.89 (t, 4H, 2 × NCH2, J ) 7.20 Hz), 3.84 (s, 4H, 2 × SCH2), 1.77-1.86 (m, 4H, 2 × CH2), 1.02 (t, 6H, 2 × CH3, J ) 7.20 Hz). 13C NMR (CDCl3, TMS/ppm): δ 171.72 (CO), 155.12 (CN), 150.25, 149.87, 135.14, 129.84, 123.05, 122.55, 116.35, 43.66 (NCH2), 32.38 (SCH2), 19.79 (CH2), 10.91 (CH3). Anal. calculated for 5b, C25H25N5O2S2‚HCl, yellow solid (528.10), mp 178-180 °C: C, 56.86; H, 4.96; N, 13.26. Found: C, 56.70; H, 4.89; N, 13.29. 2′,2′′-[(Acridin-3,6-diyl)diimino]-3′,3′′-dibutyl-1,3-dithiazolidin-4-one (4c). Yield: 63%; yellow oil. 1H NMR (CDCl3, TMS/ppm): δ 8.73 (s, 1H, CH-9), 7.99 (d, 2H, CH-1,8, J ) 8.80 Hz), 7.78 (s, 2H, CH-4,5), 7.22 (d, 2H, CH-2,7, J ) 8.80 Hz), 3.92 (t, 4H, 2 × NCH2, J ) 7.20 Hz), 3.85 (s, 4H, 2 × SCH2), 1.72-1.80 (m, 4H, 2 × CH2), 1.33-1.48 (m, 4H, 2 × CH2), 0.99 (t, 6H, 2 × CH3, J ) 7.20 Hz). 13C NMR (CDCl3, TMS/ppm): δ 171.76 (CO), 155.31 (CN), 150.33, 149.88, 135.90, 129.60, 124.00, 122.90, 116.78, 43.19 (NCH2), 32.85 (SCH2), 29.36 (CH2), 20.12 (CH2), 13.82 (CH3). Anal. calculated for 5c, C27H29N5O2S2‚HCl, yellow solid (556.15), mp 156158 °C: C, 58.31; H, 5.44; N, 12.59. Found: C, 58.48; H, 5.56; N, 13.22. 2′,2′′-[(Acridin-3,6-diyl)diimino]-3′,3′′-dipentyl-1,3-dithiazolidin-4-one (4d). Yield: 61%, yellow oil. 1H NMR (CDCl3, TMS/ppm): δ 8.67 (s, 1H, CH-9), 7.96 (d, 2H, CH-1,8, J ) 8.80 Hz), 7.69 (s, 2H, CH-4,5), 7.18 (d, 2H, CH-2,7, J ) 8.80 Hz), 3.90 (t, 4H, 2 × NCH2, J ) 7.20 Hz), 3.83 (s, 4H, 2 × SCH2), 1.74-1.81 (m, 4H, 2 × CH2), 1.36-1.43 (m, 8H, 4 × CH2), 0.93 (t, 6H, 2 × CH3, J ) 6.40 Hz). 13C NMR (CDCl3, TMS/ppm): δ 171.71 (CO), 155.24 (CN), 150.25, 149.88, 135.79, 129.57, 123.99, 122.87, 116.86, 43.38 (NCH2), 32.84 (SCH2), 28.93 (CH2), 26.89 (CH2), 22.35 (CH2), 13.98 (CH3). Anal. calculated for 5d, C29H33N5O2S2‚HCl, yellow solid (584.21), mp 175-176 °C: C, 59.62; H, 5.87; N, 11.99. Found: C, 59.33; H, 5.68; N, 12.03. 2′,2′′-[(Acridin-3,6-diyl)diimino]-3,3′′-dihexyl-1,3-dithiazolidin-4-one (4e). Yield: 58%, yellow oil. 1H NMR (CDCl3, TMS/ ppm): δ 8.68 (s, 1H, CH-9), 7.97 (d, 2H, CH-1,8, J ) 8.80 Hz), 7.70 (s, 2H, CH-4,5), 7.19 (d, 2H, H-2,7, J ) 8.80 Hz), 3.91 (t, 4H, 2 × NCH2, J ) 7.20 Hz), 3.83 (s, 4H, 2 × SCH2),

Scheme 1. Synthesis of the Novel Proflavine Derivatives 4a-4ea

a Reagents and conditions: R ) ethyl - a; n-propyl - b; n-butyl - c; n-pentyl - d; n-hexyl - e; (i) CSCl2, CHCl3/H2O, Na2CO3, RT; (ii) RNH2, CH3OH, RT; (iii) BrCH2CO2CH3, CH2Cl2, RT; (iv) RNCS, CH2Cl2, RT.

1.73-1.81 (m, 4H, 2 × CH2), 1.32-1.43 (m, 12H, 6 × CH2), 0.91 (t, 6H, 2 × CH3, J ) 6.80 Hz). 13C NMR (CDCl3, TMS/ ppm): δ 171.71 (CO), 155.27 (CN), 150.31, 149.86, 135.88, 129.58, 123.97, 122.89, 116.86, 43.44 (NCH2), 32.84 (SCH2), 31.45 (CH2), 27.19 (CH2), 26.51 (CH2), 22.56 (CH2), 14.04 (CH3). Anal. calculated for 5e, C31H37N5O2S2‚HCl, yellow solid (612.26), mp 118-120 °C: C, 60.81; H, 6.26; N, 11.44. Found: C, 60.98; H, 6.25; N, 11.59.

RESULTS Synthesis of the Novel Proflavine Derivatives 4a-4e. The reaction course for the synthesis of 2′,2′′-[(acridin-3,6-diyl)diimino]-3′,3′′-dialkyl-1,3-dithiazolidin-4-ones 4a-4e is depicted in Scheme 1. Physicochemical values as well as the methods used for the structure determination are given in the Experimental Procedures section. Spectroscopic Properties. The absorption spectra of proflavine derivatives 5a-5e with pUC19 DNA all exhibited broad absorption bands in the region 250-500 nm and the spectra of the acridine derivatives significant absorption peaks in the range 350-460 nm. Figure 1 provides an example illustrating the characteristic changes in the absorption spectra during titration

Figure 1. Absorption titration of compound 5b (2.6 µM) in 0.1 M Tris buffer (pH 7.4) with increasing concentration of pUC19 DNA (from top to bottom: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 µM bp). Inset: Scatchard plot of 5b complexation.

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Figure 3. Correlation of the chain length (Å) of the alkyl substituents in 5a-5e with the DNA binding constants K obtained from spectrophotometry (k ) -2.135; r ) 0.965; S ) 0.572) and spectrofluorimetry (k ) -1.512; r ) 0.983; S ) 0.159). Figure 2. Changes in emission fluorescence spectra of 5b (5.2 µM) in 0.1 M Tris buffer (pH 7.4) with increasing concentration of pUC19 DNA from top to bottom: 0, 25, 41, 62, 83, 101, 124, 145, and 163 µM bp; λex ) 320 nm. Inset: Scatchard plot of 5b complexation.

of compound 5b with increasing amounts of pUC19 DNA. The binding of the drugs to DNA was characterized by bathochromic shifts and marked hypochromism. The red shift of the drug absorption band was pronounced with 5c and 5d, whereas only mild shifts occurred for 5a, 5b, and 5e (Table 1). The representative fluorescence spectra of 5b with pUC19 DNA were obtained by the addition of the DNA to the drug solution which resulted in a decrease of fluorescence (Figure 2). Similar fluorescence quenching was observed for all investigated derivatives. Binding parameters for the interactions of 5a-5e with DNA obtained from spectrophotometric and spectrofluorimetric analysis are summarized in Table 2. Correlation of the chain length (Å) of the alkyl substituents in 5a-5e with the DNA binding constants K obtained from spectrophotometry (k ) -2.135; r ) 0.965; S ) 0.572) and spectrofluorimetry (k ) -1.512; r ) 0.983; S ) 0.159) are depicted in Figure 3. As seen in Figure 4, the addition of 5b to calf thymus DNA induced a slight increase of the CD signal centered at 275 nm

and the negative peak at 243 nm. Similar CD changes were also observed for the complexes with the other drugs. Figure 5 provides thermal denaturation curves of DNA melting experiments for the calf thymus DNA in the presence of 5a-5e, whose Tm values are presented in Table 1. AM1 Calculations. To establish the steric structure of the intercalators under study, AM1 calculations were performed (Figure 6, Table 3). Cytotoxic Activity in Vitro. The inhibitory effects of compounds 5a-5e against two tumor cell lines (L1210, HeLa) were tested and the resulting concentration-response curve plotted. The concentrations that produced 25% (IC25) or 50% (IC50) inhibition of cell viability were calculated and the results collected in Table 4. Effects of 5b on the viability of HeLa (A) and L1210 cells (B) incubated for 0-72 h without (control) or with 0-100 µM 5b are shown in Figure 7. Changes of cells morphology have been examined using fluorescent and light microscopy (Figure 8). The induction of apoptosis by 5b in L1210 and HeLa cells has been evaluated by electrophoresis of fragmented DNA (Figure 9).

DISCUSSION We report an efficient strategy for the synthesis of novel proflavine-dithiazolidinone derivatives. As the starting com-

Table 1. UV-vis DNA Binding Properties λmax (nm)

hypochromism

Tma

compound

free

bound

∆λ

%

(°C)

5a 5b 5c 5d 5e

381 393 393 396 390

383 397 400 406 392

2 4 7 10 2

23 22.7 29.0 18.2 22.8

80 79 79 78 77

a T measurements were performed in BPE buffer pH 7.1 (6 mM Na HPO , 2 mM NaH PO , 1 mM EDTA) using 10 µM drug and 20 µM bp calf thymus m 2 4 2 4 DNA with a heating rate of 1 °C min-1.

Table 2. Photophysical and Binding Parameters of 5a-5e from Spectrophotometric and Spectrofluorimetric Analysis compd 5a 5b 5c 5d 5e

spectrophotometry K × 104 n

spectrofluorimetry K × 104 n

6.2 ( 0.2 5.3 ( 0.4 4.7 ( 0.2 4.6 ( 0.3 3.9 ( 0.4

5.5 ( 0.3 4.7 ( 0.3 3.9 ( 0.2 3.6 ( 0.4 2.2 ( 0.3

2.1 ( 0.5 2.3 ( 0.4 2.7 ( 0.3 2.6 ( 0.5 3.2 ( 0.8

2.4 ( 0.4 2.8 ( 0.5 3.3 ( 0.3 3.0 ( 0.4 3.9 ( 0.6

λem (nm)

Φfa

∆Gb (kJ mol-1)

420, 460 420, 500 430, 490 420, 490 425, 500

0.75 0.51 0.94 0.89 1

-27.3 -26.9 -26.6 -26.6 -26.2

a The fluorescence quantum yields were calculated using 2′,2′′-[(acridin-3,6-diyl)diimino]-3′,3′′-dihexyl-1,3-dithiazolidin-4-one hydrochloride (5e) as a standard (Φf ) 1). b The standard Gibb’s free energy change (∆G° ) -RT ln K) for derivatives 5a-5e is approximately -26.52 kJ mol-1 at 25 °C, thereby indicating the spontaneity of the binding with DNA.

Bioconjugate Chem., Vol. 18, No. 1, 2007 97

Novel Proflavine−DNA Intercalators

Table 3. Structural and Electronic Features of Proflavine-Thiazolidinone Derivatives 4a-4e structural/electronic parameters

4a

4b

compound 4c

4d

4e

φ (C4-C3-N6′-C2′) 104.57° 104.60° 105.56° 105.08° 105.28° φ (C5-C6-N6′′-C2′′) 104.33° 104.63° 105.51° 104.94° 105.07° alkyl chain length (Å) 1.52 2.48 3.83 4.98 6.29 dipole moment (D) 2.02 1.98 1.92 1.89 1.88

Figure 4. Circular dichroism spectra of the calf thymus DNA (20 µM) (solid line) in the absence and in the presence of 5b (10 µM) (dashed line) in 0.1 M Tris buffer (pH 7.4).

Figure 5. Melting curves of calf thymus DNA (violet ]) with 5a (light blue O), 5b (black B), 5c (red 4), 5d (yellow 3), and 5e (green 0) measured at 260 nm in BPE buffer pH ) 7.1.

Figure 6. Minimum-energy conformation of the propyl-proflavine derivative 4b obtained using Gamess software. Color code for the atoms: dark blue, nitrogen; light blue, carbon; red, oxygen; yellow, sulfur.

pound for the preparation of newly designed intercalators, the commercially available proflavine hemisulfate was used. We attempted to prepare the intermediate thioureas 3a-3e by the treatment of the free base proflavine (1) with aliphatic isothiocyanates to incorporate aliphatic chains into the resulting structures. The free base was obtained by the addition of triethylamine to the solution of proflavine hemisulphate in a

mixture DMSO/methanol, then poured into brine, and the resulting precipitate filtered off and dried overnight in vacuo. Unfortunately, the reaction of 1 did not proceed even with a ten-molar excess of ethylisothiocyanate and 30 h heating. In addition, proflavine (1) as the free base underwent decomposition during the course of the reaction, and an attempt to carry out the reaction with free base 1 released in situ also failed. The reason for these failures is probably the poor nucleophilicity of the amino group in 1 and the low electrophilicity of the NCS carbon of the alkylisothiocyanates. The problem was circumvented by preparing thioureas 3a-3e using a reverse approach via reaction of the corresponding alkyl amines with the novel synthon, 3,6-diisothiocyanatoacridine (2) (20). To obtain a higher yield of 2, the synthesis was reduced to a one-pot procedure by in situ reaction of free base 1 with thiophosgene. Diisothiocyanate 2 reacted readily with the amines in methanol, whereas in chloroform, toluene, and methylene chloride, decomposition of the products was observed. The obtained thioureas 3a-3e were filtered off and dried in vacuo. Because of their low stability, they were immediately employed after drying in the subsequent reaction. Aliphatic nonbranched amines were used to study the structural dependence of the chain length vs DNA intercalation capability of the final products 5a5e. For the preparation of the corresponding thiazolidin-4-ones 5a-5e, our previous experience with the regioselectivity of thiazolidin-4-one formation was exploited (21). The synthesis was performed by the reaction of methyl bromoacetate with thioureas 3a-3e in the presence of triethylamine under anhydrous conditions; the resulting products 4a-4e were obtained pure by column chromatography over silica gel. The regioselective formation of the product 4a was proven by a gHMBC NMR experiment revealing a cross peak between the methylene signal of the ethyl group and the carbonyl carbon. In the case of compounds 4b-4e, the structure was verified by the presence of a SCH2 signal at ca. 3.8 ppm, typical for regioisomers analogous to 4 (21-23). To enhance their water solubility, the products 4a-4e were converted to their corresponding hydrochlorides 5a-5e. The alternative regioisomers 6a-6e were not observed. Electronic absorption spectra of the synthesized derivatives exhibited significant absorptions in the range 350-460 nm, which are typical for transitions between the π-electronic energy levels of the acridine ring. To obtain additional information on the new proflavine derivatives 5a-5e, fluorescence measurements were also performed. The fluorescence spectra of the proflavine derivatives 5a-5e each exhibited a broad absorption band in the range 350-600 nm. Differences were found in the fluorescence intensities of the compounds with the highest intensity displayed by the derivative 5e. Binding constants (K) and neighbor exclusion parameters (n) clearly indicate a direct dependence between the structural changes and the resulting intercalation capability. Because n ranges from 2.1 to 3.9, multiple-site binding is occurring which affects interaction at the neighboring sites. Aslanoglu (12) determined the binding constant (K ) 2.32 × 104 M-1) and the binding site size (n ) 2.07 bp) for the interaction between proflavine and DNA. Our binding constants determined by UVvis measurements ranged above this from 3.9 to 6.2 × 104 M-1

98 Bioconjugate Chem., Vol. 18, No. 1, 2007

Janovec et al.

Table 4. Effect of 5a-5e on the Viabilitya of HeLa and L1210 Cells L1210

HeLa

48 h

72 h

48 h

72 h

IC (µM)

IC25

IC50

IC25

IC50

IC25

IC50

IC25

IC50

5a 5b 5c 5d 5e

2.6 ( 0.4 2.8 ( 0.5 2.8 ( 0.5 10.0 ( 0.8 25.0 ( 1.0

8.2 ( 0.5 6.7 ( 1.6 8.5 ( 0.7 100.0 ( 0.5 100.0 ( 0.5

3.5 ( 1.1 2.8 ( 0.3 4.0 ( 0.9 10.5 ( 1.0 45.0 ( 0.7

11.0 ( 1.7 6.3 ( 1.0 20.5 ( 0.8 100.0 ( 1.0 90.0 ( 1.2

3.0 ( 0.3 2.8 ( 0.5 11.5 ( 0.9 23.0 ( 1.0 27.0 ( 0.5

15.5 ( 1.0 20.3 ( 0.3 ND ND ND

3.0 ( 0.4 5.3 ( 1.1 5.0 ( 0.8 4.0 ( 0.5 3.1 ( 0.5

12.5 ( 0.9 12.9 ( 0.3 25.0 ( 1.0 57.0 ( 0.9 78.0 ( 0.8

a Cell viability was evaluated by MTT assay. Cells were incubated for 0-72 h without (control) or with 0-100 µM of compound. IC 25 and IC50 are concentrations that produced 25% and 50%, respectively, inhibition of cell viability. ND: inhibition was not obtained (IC50 > 100 µM). Results are expressed as a mean ( S.D. (n ) 3).

Figure 8. Morphology of leukemia cells L1210 (A,B) and cervical carcinoma HeLa cells (C,D) treated with 5b (24 h incubation, 10 µM). Magnification 400×. Image A: control L1210 cells stained with Hoechst 44433 and propidium iodide. Images A, B, and C were obtained using a fluorescence microscope and image D using a light microscope. Arrows indicate membrane blebbing.

Figure 7. Effects of 5b on the viability of HeLa (A) and L1210 cells (B). Cells were incubated for 0-72 h without (control) or with 0-100 µM 5b. Cell viability was evaluated by MTT assay. Results are expressed as a percentage of the control.

and by spectrofluorimetry from 2.2 to 5.5 × 104 M-1 while the binding site sizes were similar to the value determined by Aslanoglu. The slight changes are induced by the thiazolidinone moiety on the proflavine skeleton which is supposed to stabilize the double helix DNA structure. This interpretation was also supported by thermal denaturation studies. The melting of DNA is a phenomenon observed when the double-stranded DNA molecules are heated and separate into two single strands; it occurs due to the disruption of the intermolecular forces such as π-π stacking and hydrogenbonding interactions between the DNA base pairs. DNA melting experiments revealed that the Tm of calf thymus DNA (76 °C) increases in the presence of 5a-5e to within the range 77-80 °C. An increase in the helix stabilization is due to intercalation of proflavine-dithiazolidinone derivatives into DNA. To elucidate the drug binding mechanism, CD was also used. It is one of the spectroscopic methods most amenable for

determining a drug-binding mode. Changes in the CD spectrum of DNA as a result of complexation can often be ascribed to alteration of the DNA structure. Agents intercalating into the DNA generally give no induced CD or weakly negative signals in the drug absorption band (24). Our observations suggest that intercalation exists between 5a-5e and calf thymus DNA. To determine the steric effects of the drugs, AM1 calculations were performed. From these studies, it was deduced that the structure depicted in Figure 6 is the most suitable for DNA intercalation. The results indicate that steric influence on the size of the binding constants is negligible, as the values of the analogous dihedral angles of the thiazolidin-4-one and proflavine rings were nearly identical. However, Joseph et al. demonstrated that, by introducing a subtle steric factor, one could alter the DNA binding properties of aryl-substituted acridinium derivatives (25). The values of the dipole moments, which are related to some extent to the polarity, can be considered a measure of lipophilicity of 5a-5e (Table 3). Despite only small differences in the values of the binding constants, we have found that there exists a linear correlation between the chain length (Å) and binding ability. The cells showed different responses to the tested compounds, though the L1210 cells appeared to be more sensitive than the HeLa cells. The results clearly indicate that substances 5a-5c possess strong cytotoxic effects toward cancer cell lines. In the present study, 5b is one of the most effective compounds in

Novel Proflavine−DNA Intercalators

Bioconjugate Chem., Vol. 18, No. 1, 2007 99

whereby the binding affinity decreases with increasing length of the side chain. All of the compounds displayed cytotoxic activity in vitro and inhibited the growth of cancer cells. We can therefore summarize that the newly synthesized molecules afford interesting possibilities to develop novel DNA-targeted anticancer agents.

ACKNOWLEDGMENT This work was supported by the Slovak Grant Agency VEGA, grants no.1/1274/04, 1/1173/04, 1/ 1272/04, and 1/2471/05 and by the State NMR Programme no. 2003SP200280203. Dr. Karel D. Klika is thanked for correction of the language and assistance in manuscript preparation.

LITERATURE CITED

Figure 9. Agarose gel electrophoresis of DNA fragments from cells treated with 5b. HeLa and L1210 cells were incubated with 5b (5 or 10 µM) for 24 h. DNA fragmentation was visualized by ethidium bromide after DNA agarose gel electrophoresis. Lane 1: size marker (10000-250 bp). HeLa cells: lane 2, control; lane 3, treated with 5 µM 5b; lane 4, treated with 10 µM 5b. L1210: lane 5, control; lane 6, treated with 5 µM 5b; lane 7, treated with 10 µM 5b.

terms of its ability to inhibit proliferation in a time- and dosedependent manner (IC50 6.3 and 12.9 µM; 72 h treatment). Incubation of cells in the presence of 5b at concentrations higher than 3 µM resulted in significant growth inhibition. The ability of 5b to inhibit cancer cell proliferation and the aforementioned properties of drug binding into DNA suggests that this compound could cause apoptosis, also known as programmed cell death. Apoptosis is characterized by distinct morphological features such as cell shrinkage, chromatin condensation, plasma membrane blebbing, oligonucleosomal DNA fragmentation, and finally, the breakdown of the cell into smaller units. Changes in cell morphology were examined using fluorescent microscopy to predict mode of death induced by 5b. The fluorescence of 5b is sufficient for intracellular localization of this drug and propidium iodide was utilized for identification of necrotic cells. The altered morphology of L1210 cells was evident after 24 h treatment with 10 µM 5b (20% of necrotic cells), and this compound was accumulated by L1210 cells which clearly exhibited cytoplasmic blebbing (a hallmark of apoptosis) and shape irregularity (Figure 8B). When human cervical carcinoma HeLa cells were treated with 10 µM 5b, the nuclei and the cytoplasms exhibited green fluorescence (Figure 8C), and although necrotic features were not observed, membrane blebbing was evident (Figure 8D). In addition to characteristic changes in morphology, the induction of apoptosis by 5b in L1210 and HeLa cells has been evaluated by electrophoresis of fragmented DNA. L1210 cells cultured with 5b (5 µM or 10 µM) for 24 h exhibited a characteristic DNA ladder pattern of apoptosis. Negligible cleavage of DNA into nucleosomal fragments was seen with 5b-treated HeLa cells. Since drug action usually requires internalization of the drug, a high rate of uptake of the most potent antiproliferative drug 5b by L1210 was confirmed (about 50% of 5b was accumulated after rapid incubation of the cells with the compound). A linear correlation between the binding constants of 5a-5e to DNA and the length of the alkyl chain has been established

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