Exploring DNA binding properties and biological activities of dihydropyrimidinones derivatives

Colloids and Surfaces B: Biointerfaces 106 (2013) 28–36

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Exploring DNA binding properties and biological activities of dihydropyrimidinones derivatives Gongke Wang, Changling Yan, Yan Lu ∗ School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, PR China

a r t i c l e

i n f o

Article history: Received 1 December 2012 Accepted 10 January 2013 Available online 18 January 2013 Keywords: DNA binding Spectroscopy Viscosity Isothermal titration calorimetry Structure activity relationship

a b s t r a c t The effects of substituent modifications for three dihydropyrimidinones derivatives on DNA binding properties were investigated using viscometry in combination with spectroscopy and isothermal titration calorimetry (ITC). The results indicated that substitution in 4 rd position of benzene ring has significant effects on DNA binding mode, affinity and energetics. Electron-donating substitution was favorable for intercalating into DNA bases and had higher DNA binding affinity. However, electron-withdrawing substitution was preferable to bind to DNA in partial intercalation mode with relatively weak DNA binding affinity. Simultaneously, electron-donating substitution could result in more favorable binding enthalpy relative to electron-donating substitution and the parent compound. Antitumor activities of these analogs over BEL-7402 and PC-12 cells were studied to explore the structure activity relationships (SARs), which suggested that electron-donating substitution in 4 rd position of benzene ring could greatly enhance the antitumor activities. However, electron-withdrawing substitution has little effect on the antitumor activity. The present results favor the development of potential drugs related with dihydropyrimidinones derivatives in the treatment of some diseases. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Nucleic acids are the biological target for many anticancer and antiviral drugs, hence, DNA and RNA are important targets in drug development. In recent decades, the investigation of the binding interactions between nucleic acids and small-molecule ligands is an active research field, and the latter compounds represent targets for drug design in anticancer and antitumor therapy [1–3]. Along these lines, especially important are the ligands capable of structure and sequence selective binding to DNA, since such compounds may control numerous genetic diseases by selectively inhibiting gene expression in vivo [4]. One of the most successful approaches in sequence-selective targeting of DNA so far is the polyamide approach pioneered by Dervan and coworkers [5], which has led to successful blocking of tumor growth in nude mice [6]. To exploit the full potential of DNA recognition in medicine and biotechnology there is a strong need for new drugs and biosensors that interact with DNA. In view of the complexity of the ligand-DNA recognition process, an investigation on model compounds, which possess only one DNA-binding mode, is desired. Unfortunately, only a few ligands are known that

∗ Corresponding author. Tel.: +86 373 3325249; fax: +86 373 3325249. E-mail addresses: [email protected] (G. Wang), [email protected] (Y. Lu). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.01.019

bind to DNA by the classical intercalative mode exclusively, i.e., by insertion between the neighboring base pair of DNA [7–14]. A great number of ligands, which have an intercalating part endowed with a variety of substituents, bind to DNA by a mixed mode of intercalation accompanied with groove binding or external electrostatic effects [15–22]. Thus, in the case of “classical” intercalator ethidium, the phenyl ring occupies the minor groove, resulting in an overall heterogeneous DNA binding modes [23,24]. In this respect, the binding modes of ethidium derivatives, anthracene derivatives and substituted 9-methylacridinium salts to DNA have been systematically investigated [8,9,25,26]. However, with the occurrence of numerous new diseases resulting from the deterioration of the environment, the candidates of potential new drugs should not have been limited in the common species, such as ethidium derivatives, anthracene derivatives and flavone derivatives, etc. Therefore, the development of heterocyclic drugs offering special effectiveness for the treatment of major diseases is urgent and significant. Several dihydropyrimidinones derivatives (Scheme 1) have been chosen, in the current investigations, for the following reasons. Dihydropyrimidinones and their derivatives have attracted considerable interest in recent years because of their medical importance as calcium channel blockers [27], antihypertensive agents [27], and potent HIVgp120-CD4 inhibitor [28]. Moreover, several alkaloids containing the dihydropyrimidinones as a core unit have been isolated from marine source, which also showed

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Cell lines of human hepatocellular carcinoma (BEL-7402) and rat pheochromocytoma (PC-12) were purchased from American Type Culture Collection. Dihydropyrimidinones derivatives (4a, 4b and 4c) were synthesized with one-pot method in our laboratory. The yields were obviously increased and the reaction time was greatly condensed as compared to the reported investigations [32]. Melting points were obtained on a type of XRC-1 digital melting point apparatus and are uncorrected. NMR spectra were determined using a Bruker Avance-400 spectrometer with TMS as internal standard in DMSO-d6 . Electrospray ionization mass spectra (ESI-MS) were recorded on an AcQuity UPLC-Mass spectrometer (Waters, USA). Other chemicals were of analytical grade and used without further purification. 2.2. Preparation of stock solutions

Scheme 1. Structures of several dihydropyrimidinones derivatives. R represents the C4 substituent on dihydropyrimidinones.

interesting biological properties [29]. Therefore, dihydropyrimidinones are regarded as the potential new drugs targeted on DNA. On the other hand, the pharmacological activities of dihydropyrimidinones and their derivatives have been paid more attention long before, however, dihydropyrimidinones structure activity relationship (SAR) and the assay on the inhibition against tumor or cancer cells in vivo and vitro have few been reported [30]. To uncover its SAR, better understand mechanisms of action and find more potent inhibitors of some tumor cells, we decided to design and synthesize a series of dihydropyrimidinones derivatives and evaluate their DNA-binding properties and pharmacological activities against some tumor cells. Herein, we report a systematic investigation of the DNA binding properties and biological activities of a series of dihydropyrimidinones derivatives, including 5-(ethoxycarbonyl)-6-methyl-4phenyl-3,4-dihydropyrimidin-2(1H)-one (4a), 5-(ethoxycarbonyl) -6-methyl-4-(4-dimethylaminophenyl)-3,4-dihydropyrimidin-2 (1H)-one (4b) and 5-(ethoxycarbonyl)-6-methyl-4-(4-nitrophenyl) -3,4-dihydropyrimidin-2(1H)-one (4c). With this series in hand, we try to establish the influences of (i) the specific binding mode and (ii) the substitution of electron-donating group or electronwithdrawing group on DNA binding properties and biological activities of dihydropyrimidinones derivatives, and further reveal their SARs. We used multiplicate biophysical techniques, such as spectrophotometric titrations, viscosimetric experiments, calorimetric studies and antitumor activity assay to achieve these aims.

ctDNA was dissolved in appropriate buffer solution for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of ctDNA solution was determined spectrophotometrically at 260 nm using molar extinction coefficient of ε260 = 6600 cm−1 M−1 (expressed as molarity of phosphate groups) [33,34]. The final concentration of the stock solution of ctDNA was 2.5 mM in DNA phosphate. The 1.0 × 10−2 M stock solutions of compounds 4a, 4b and 4c were prepared by dissolving appropriate amount of 4a, 4b and 4c in 10 mL ethanol aqueous solution (25%, v/v), respectively, which were diluted to the experimental concentrations with buffer solution. All experiments were carried out in phosphate buffered solution (PBS) of pH 7.40. All stock solutions were stored in the dark at 0–4 ◦ C. 2.3. Synthesis of compound 4a A mixture of benzaldehyde (0.53 g, 5 mM), ethyl acetoacetate (0.98 g, 7.5 mM), urea (0.45 g, 7.5 mM) and FeCl3 ·6H2 O (0.34 g, 1.25 mM) in 10 mL ethanol was refluxed for 6 min under microwave irradiation (200 W). After cooling, the reaction mixture was washed with cold water (2 mL × 20 mL) and residue recrystallized from ethyl acetate:n-hexane (1:3) to afford the pure product (1.24 g, 95%). Mp 202–204 ◦ C; 1 H NMR (DMSO-d6 ): ıH 9.18 (s, 1H), 7.73 (s, 1H), 7.22–7.34 (m, 5H), 5.14 (d, J = 3.2 Hz, 1H), 3.98 (q, J = 7.0 Hz, 2H), 2.25 (s, 3H), 1.09 (t, J = 7.0 Hz, 3H); ESIMS (m/z): 261 [M+H]+ . 2.4. Synthesis of compound 4b A mixture of 4-dimethylaminobenzaldehyde (0.38 g, 2.5 mM), ethyl acetoacetate (0.33 g, 2.5 mM), urea (0.30 g, 5 mM) and FeCl3 ·6H2 O (0.36 g, 1.33 mM) in 10 mL ethanol was refluxed for 5 min under microwave irradiation (250 W). After cooling, the reaction mixture was washed with cold water (2 mL × 25 mL) and residue recrystallized from ethyl acetate:n-hexane (1:3) to afford the pure product (0.34 g, 84%). Mp 255–257 ◦ C; 1 H NMR(CDC13 ): ıH 7.32 (s, 1 H), 7.20 (d, J = 8. 4 Hz, 2H), 6.72 (br, 2H), 5.38 (s,1H), 5.33 (s, 1H), 4.09 (q, J= 7.0 Hz, 2H), 2.95 (s, 6H), 2.32 (s, 3H), 1.20 (t, J = 7.0 Hz, 3H); ESIMS (m/z): 304 [M+H]+ .

2. Materials and methods

2.5. Synthesis of compound 4c

2.1. Materials

A mixture of 4-nitrobenzaldehyde (0.30 g, 2 mM), ethyl acetoacetate (0.26 g, 2 mM), urea (0.27 g, 4.5 mM) and FeCl3 ·6H2 O (0.27 g, 1.0 mM) in 10 mL ethanol was refluxed for 6 min under microwave irradiation (250 W). After cooling, the reaction mixture was washed with cold water (2 mL × 50 mL) and residue recrystallized from ethyl acetate:n-hexane (1:3) to afford the pure product (0.51 g, 83%). Mp 205–207 ◦ C; 1 H NMR (DMSO-d6): ıH 9.33 (s, 1H), 8.20 (d, J=7.2Hz, 2H), 7.87 (s, 1H), 7.50 (d, J = 7.3Hz, 2H), 5.27 (s, 1H), 3.97

Highly polymerized type I calf-thymus DNA (ctDNA) sodium salt was purchased from Sigma Chemical Co. and deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. To check the protein content of ctDNA solution, the absorbance at 260 and 280 nm was recorded. The A260 /A280 ratio was determined as 1.87, indicating that the ctDNA was sufficiently free from protein [31].

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(q, J = 5.4Hz, 2H), 2.26 (s, 3H), 1.07 (t, J = 6.8 Hz, 3H); ESIMS (m/z): 306 [M+H]+ . 2.6. Viscosity measurements Viscosities for the ligand-DNA systems were measured with a suspended level Ubbelohde viscometer that had a flow time of about 200 s for water at 25 ◦ C. Flow time measurements were performed by a Schott AVS 310 photoelectric time unit with a resolution of 0.01 s. At least three time records reproducible to 0.02 s were obtained, and the average value was used in the calculations. Data were presented as (/0 )1/3 versus binding ratio [35], where  is the viscosity of DNA in the presence of the ligand and 0 is the viscosity of DNA in the absence of the ligand. Viscosity values were calculated from the observed flow time of DNA-containing solutions (t > 100 s) corrected for the flow time of buffer alone (t0 ),  = (t − t0 )/t0 . 2.7. Spectroscopy measurements

(1)

where A0 and A are the absorbance of ligand in the absence and presence of ctDNA, εG and εH−G are the absorption coefficients of ligand and its complex with DNA, respectively. Fluorescence measurements were made using a Cary Eclipse fluorescence spectrophotometer with a slit width 5 nm for the excitation and emission beams. Fluorescence quenching studies were carried out with the anionic quencher K4 [Fe(CN)6 ]. Stern–Volmer quenching constant KSV is used to evaluate the fluorescence quenching efficiency. The interaction pattern of the fluorescence probe with DNA can be deduced from the variation of KSV . It is measured according to the Stern–Volmer equation [37]: F0 = 1 + Ksv [Q ] F

(2)

where F0 and F are the fluorescence intensities in the absence and in the presence of quencher (Q), respectively. Fluorescence competitive binding experiment was conducted by adding increasing amounts of dihydropyrimidinones derivatives (0–8 ␮M) directly into the EB (ethidium bromide)–DNA system (cEB = 10 ␮M, cDNA = 100 ␮M). And emission spectra were recorded in the region 550–800 nm using an excitation wavelength of 500 nm. The data were treated according to the classical Stern–Volmer equation [38]: F0 = 1 + Ksq r F

Isothermal titration calorimetry (ITC) was performed using a Model Nano-ITC 2G biocalorimetry instrument (TA, USA) at 25 ◦ C. ctDNA and dihydropyrimidinones derivatives solutions were properly degassed prior to the titrations to avoid the formation of bubbles in the calorimeter cell. The reference cell contained the same volume of ultrapure water. In a standard experiment, the ctDNA (0.172 mM) in the 1.0 mL calorimeter cell was titrated by the dihydropyrimidinones derivatives solutions (2.0 mM) by up to 25 successive automatic injections of 10 ␮L each. The individual injections were programmed at intervals of 400 s. The first injection was ignored in the final data analysis. Integration of peaks corresponding to each injection and correction for the baseline were carried out using NanoAnalyze software provided by the manufacturer. Fitting the data according to the independent binding model resulted in the stoichimetry (n), equilibrium binding constant (Kb ), and enthalpy of complex formation (H0 ). The other thermodynamic parameters were calculated by the following formulas: G0 = −RT ln Kb

UV–vis absorption spectra were measured on a TU-1810 UV spectrophotometer equipped with 1.0 cm quartz cells. A constant concentration of dihydropyrimidinones derivatives (10 ␮M) was titrated with increasing concentration of ctDNA (0–40 ␮M). During the spectra titration, equal amount of DNA was added to both ligand solution and reference solution to eliminate the absorbance of DNA itself. The absorption data were analyzed using the following equation to estimate the binding constant Ka [36]: A0 1 εG εG = + εH−G − εG εH−G − εG Ka [DNA] A − A0

2.8. Isothermal titration calorimetry

(3)

where F0 and F represent the fluorescence intensities in the absence or presence of dihydropyrimidinones derivatives, and r is the concentration ratio of dihydropyrimidinones derivatives to DNA. Ksq is a linear Stern–Volmer quenching constant dependent on the ratio of the bound concentration of EB to the concentration of DNA. The Ksq value is obtained as the slope of F0 /F versus r linear plot. All the spectral measurements were thermostatically controlled by a SHP DC-0515 circulating water thermostat at 25 ◦ C.

0

0

G = H − TS

(4) 0

(5)

2.9. Antitumor activity assays Both human hepatocellular carcinoma (BEL-7402) cells and rat pheochromocytoma (PC-12) cells were used as a model to study the antineoplastic properties of 4a, 4b and 4c. After a routine culture, the cells were released by treatment with 0.25% trypsin when they became almost confluent. Then the cells were counted to 104 cells/cm2 and 200 ␮L of the cells suspension were pipetted into 96-well tissue culture plate. 4 h later, the medium was replaced with the fresh medium containing 0.0008–0.5 mg/ml of 4a, 4b or 4c. And the cells were continued to culture another 48 h. The cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) based on succinic dehydrogenase activity at OD 490 nm (n = 6), with the absorption at 630 nm as the reference wavelength (Multiskan MK3, Thermo Labsystems, USA). The inhibition rate of drugs over tumor cells was calculated as follows: Inhibition rate = (ODc − ODt)/ODc × 100%

(6)

where ODc and ODt are the OD 490 nm values of control group without any treatment and the treatment group, respectively. 3. Results and discussion 3.1. Viscosity studies Hydrodynamic measurements that are sensitive to length change are regarded as the least ambiguous and most critical tests of a binding model in solution in the absence of crystallographic structural data [39,40]. The interaction of the ligands with DNA has an effect on the hydrodynamic properties of the solutions of the biopolymers [35]. At low ligand-to-DNA ratios (r < 0.2), the intercalation of the ligands with the DNA base pairs leads to the elongation of the biomacromolecule and increases the viscosity of the solution. In contrast, a partial or non-classical intercalation of the ligands can bend or kink DNA resulting in a decrease in its effective length with a concomitant decrease in its viscosity [39,41]. The effects of EB, 4a, 4b and 4c on the viscosity of ctDNA are shown in Fig. 1. In the case of EB, which was chosen as a reference compound, a linear dependence of the cubic root of the relative viscosity on the ligand-to-ctDNA ratio was observed with a slope of 0.93, which is consistent with the previous reports [41,42]. The

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Fig. 1. Effects of EB, 4a, 4b and 4c on the relative viscosity of the solution of ctDNA at 25 ◦ C. [ctDNA] = 10 ␮M; [EB] = [4a] = [4b] = [4c] from 0 to 2 ␮M.

compound 4a and 4b behave similarly, leading to an increase in the viscosity of the ctDNA solutions, albeit with slightly smaller slopes (0.75 and 0.85, respectively). At the same time, the increase in the viscosity seems to reach saturation at lower r values as compared to EB. Notably, the addition of 4c leads to a much less pronounced increase in the viscosity of ctDNA solutions (k = 0.61 at r < 0.1 and k = 0.42 at r ≥ 0.1). The experimental results suggest that 4a and 4b bind to ctDNA through an intercalation mode, as demonstrated by comparison with EB. Furthermore, 4b intercalates more strongly than 4a evaluated from the slopes. In contrast, 4c appears to have much less influence on the viscosity of the ctDNA solutions, which indicates 4c may not be a classical intercalator, and an at least partial contribution of the non-classical intercalation mode. In addition, a detailed inspection of the change in the viscosity of ctDNA solutions with an increase in the concentration of 4c reveals two linear parts of the plot (one with a relatively large slope up to r < 0.1 and one part with a smaller slope at r ≥ 0.1). Thus, it may be proposed that at low ligand-to-ctDNA ratios, 4c may be an intercalator, whereas with increasing r values, an obvious contribution of non-classical intercalation to the overall association takes place. The non-classical intercalation dose not result in the lengthening of DNA, and consequently, it dose not lead to an increase in the bulk viscosity of DNA solution, which may explain the much lower slope in the plot of the viscosity of the ctDNA solution versus the ligand-to-ctDNA ratio at r ≥ 0.1. 3.2. Absorption spectroscopic studies The absorption spectra of 4a, 4b and 4c (200–350 nm) underwent significant changes on binding to ctDNA, and these provided a convenient handle to characterize their interactions. Fig. 2 shows the absorption spectrum of 4a, 4b and 4c. In presence of increasing concentration of ctDNA (0–40 ␮M), the visible hypochromism and slight red shift at 286, 287 and 277 nm in the absorbance spectra of 4a, 4b and 4c, respectively, were observed. The extent of hypochromism for 4a and 4b is 26.1% and 42.0%, respectively, which indicates the relative strong binding to ctDNA [43]. Obviously, such hypochromism and red shift occur most likely due to the effective overlap of the ␲ electron cloud of the ligands and base pairs and are suggestive of intercalative binding to ctDNA. As for 4c, the absorption spectrum shows that the addition of ctDNA yields hypochromism about 13.2% under the same experimental conditions, which suggests that 4c has a lower affinity with ctDNA as compared to 4a and 4b. At the same time, these spectral characteristics

Fig. 2. Spectrophotometric titrations of ctDNA to 4a, 4b and 4c, respectively. Arrows indicate the changes of the intensity of the absorption bands upon addition of ctDNA. Inset: plots of A0 /(A − A0 ) vs. 1/[ctDNA] for the titrations of ctDNA to 4a, 4b and 4c, respectively. [ctDNA] from 0 to 40 ␮M; [4a] = [4b] = [4c] = 10 ␮M.

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Table 1 The quenching constants of 4a, 4b and 4c with increasing concentration of quencher [Fe(CN)6 ]4− in the absence and presence of CT-DNA at 25 ◦ C. KSV (×104 L mol−1 )

4a

4b

4c

Without DNA With DNA

2.31 ± 0.06 0.969 ± 0.05

2.14 ± 0.08 0.712 ± 0.04

1.64 ± 0.03 1.03 ± 0.05

seem to be indicated that 4c may be close to the border between a classical intercalation and non-classical intercalation mode. To determine quantitatively the affinity of the three analogs with ctDNA, the absorption titration data were analyzed to estimate the corresponding binding constants by using Eq. (1) described in materials and methods section. Binding isotherms were constructed from these data (inset, Fig. 2), and the best linear fits to the data resulted in binding constants of 2.43 × 104 M−1 , 7.81 × 104 M−1 and 1.42 × 104 M−1 for 4a, 4b and 4c, respectively. The binding affinities determined here are similar to the other intercalators reported [26,43]. Notably, resonance-stabilizing electron-donating substitution in benzene ring at C4 position (4b) was shown to enhance ctDNA binding affinity up to 3-fold relative to 4a. In contrast, electron-withdrawing substitution in benzene ring at C4 position (4c) resulted in a 2-fold decrease in binding affinity relative to 4a. The similar effects of different substitutions on the binding affinity of DNA have been reported in the previous investigation [44]. Although the potential steric hindrance of dimethylamino substituted in benzene ring (4b) seems to be greater than that of nitryl substituted (4c) and the unsubstituted parent (4a), it is still seen that the binding affinity of 4b to ctDNA is larger than that of 4a and 4c. These behaviors may arise from the electron-donating characteristic of dimethylamino, which can improve stacking between the substituted compound and ctDNA bases in the binding sites. Enhancement of base stacking interactions between the ligand chromophore and the ␲-system of the ctDNA bases results from substituent-induced changes in electron density distribution in the dihydropyrimidinones ring. On the contrary, the electron- withdrawing characteristic of nitryl declines stacking between the substituted compound and ctDNA bases in the binding sites, which leads to the weakest binding affinity of 4c with ctDNA in the three compounds. On the other hand, dimethylamino substituted in benzene ring is favorable for hydrophobic effect that accompanies the release of water from the ordered shell surrounding the ligand as the ligand intercalates into the hydrophobic interior of ctDNA, which can also bring about the larger binding affinity of 4b to ctDNA relative to 4a and 4c. 3.3. Fluorescence spectroscopic studies A reliable method of investigating the binding of small molecules to nucleic acids is the fluorescence quenching method [41] where the molecules bound to the surface of the helix will be accessible to the quencher while those buried by intercalation inside the helix will be protected from the quencher. Anionic quencher like [Fe(CN)6 ]4− will not be able to quench the fluorescence of the intercalated molecules. Consequently, the magnitude of the Stern–Volmer quenching constant (Ksv ) of ligands that are bound intercalatively will be lower than that of the free molecules. According to Eq. (2), the Stern–Volmer curves for 4a, 4b and 4c quenched by [Fe(CN)6 ]4− in the absence and presence of ctDNA were constructed respectively (Figs. S1–S3), and the calculated quenching constants (KSV ) were presented in Table 1. From Table 1, we can see that, in the absence of ctDNA, the three compounds are efficiently quenched by [Fe(CN)6 ]4− , resulting in linear Stern–Volmer plots of larger slopes. However, in the presence of ctDNA, the slopes of the plots for 4a and 4b are remarkably decreased. Especially for 4b, this trend becomes more obvious,

Fig. 3. Emission spectra of EB bound to ctDNA in the presence of 4a, 4b and 4c, respectively. Arrows show the intensity changes upon increasing concentrations of 4a. Inset: plots of F0 /F versus r (c4a , 4b or 4c /cctDNA ). [EB] = 10 ␮M, [ctDNA] = 100 ␮M; [4a] = [4b] = [4c] from 0 to 8 ␮M.

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Fig. 4. ITC data for the binding of 4a, 4b and 4c to ctDNA (0.1 M PBS, pH 7.4, 25 ◦ C). Left parts: heat released during the titration of ctDNA by successive additions of a concentrated solution of 4a (top), 4b (midst) and 4c (bottom), respectively; the heat released during the dilution of 4a, 4b and 4c (without DNA) are presented in the top panels, respectively. Right parts: the ligand/DNA binding isotherms after correction of heat for the dilution of 4a, 4b and 4c, respectively; the solid lines represent the best fits according to the independent binding model.

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which indicates the strong protection caused by ctDNA in the binding sites. By contrast, in the presence of ctDNA, the slope of the plot for 4c is slightly decreased, which suggests the relative poor protection. Therefore, the binding affinity of the three compounds with ctDNA is in the order 4b > 4a > 4c. These results obtained here are consistent with the previous viscosity studies and absorption spectral investigations. In order to understand the interaction pattern of the three compounds with ctDNA more clearly, the competitive binding experiments were carried out using EB as a probe. EB is one of the most typical intercalators, emitting intense fluorescence in the presence of DNA. It was previously reported that the enhanced fluorescence of EB–DNA system can be quenched, at least partially by the addition of the second molecule [31,42]. The extent of quenching fluorescence of EB bound to DNA is used to determine the extent of the binding between the second molecule and DNA. The emission spectra of EB bound to ctDNA in the absence and presence of 4a, 4b and 4c are shown in Fig. 3. The fluorescence quenching plots of EB–ctDNA complex by 4a, 4b and 4c (inset, Fig. 3) illustrate that the quenching of EB bound to ctDNA by the three compounds is in good agreement with the classical Stern–Volmer equation (Eq. (3) described in materials and methods section). The linear Stern–Volmer quenching constants (Ksq ) and the quenching extent for EB-DNA system in the presence of 4a, 4b and 4c were, respectively, summarized in Table S1. The larger values of Ksq for 4a and 4b suggest that they have a higher affinity with ctDNA. Furthermore, it is seen that the quenching extent for 4a and 4b has reached up to about 50% when r = 0.8, which indicates that they can easily compete with EB in binding to ctDNA [43,45]. For 4c, much less values of Ksq (0.214) and the quenching extent (13.7%) were observed, which implies that the relatively difficult displacement of EB bound to ctDNA by 4c. Thus, the changes observed here further convince the behaviors of intercalation binding for 4a and 4b and the partial intercalation binding for 4c. 3.4. ITC studies ITC has become an effective tool to thermodynamically characterize the binding of small molecules to macromolecules [45,46]. Furthermore, it can provide significant insight into the energetics in the binding interactions between the ligands and biomacromolecules. Therefore, this technique was used to thermodynamically investigate the formation of the ligand–ctDNA complex. Fig. 4 shows the integrated heats of reaction plotted against the mole ratio of the three compounds to ctDNA after the corrections for dilution effects of 4a, 4b and 4c, respectively. The data points reflect the experimental points while the continuous lines represent the calculated fits of the data. These binding interactions were characterized by exothermic heats. The binding constants, binding stoichiometry and thermodynamic parameters of ligand–ctDNA complexes, evaluated by fitting the integrated heats according to the independent binding model, are provided in Table 2. From Table 2, we can see that the binding constants (Kb ) determined here are in reasonable agreement with the above spectrophotometric titration results. Moreover, the binding affinities determined by ITC are similar to the previous investigations [22,47]. The binding stoichiometry (n) determined here suggests that the binding sites for the three compounds are 5–7 bases length. The binding of each derivative to ctDNA involves an enthalpic stabilization and an entropic destabilization of the ligand–ctDNA complex, which is generally typical for intercalative interaction of small molecules to nucleic acids [48]. Relative to 4a, the significant enhancement for binding enthalpy of 4b resulting from C4 substitution in benzene ring may arise from the improved stacking between the ligand and the DNA bases in the binding site. It is obvious that

the enhancement of base stacking interactions is due to the increase of electron density in dihydropyrimidinones ring resulting from the substitution of electron-donating group (dimethylamino). In addition, the hydrogen bond acceptor sites of dimethylamino group may provide an additional source of ligand–DNA binding enthalpy enhancement. On the other hand, by comparison with 4a, the obvious reduction for binding enthalpy of 4c may originate from the decrease of electron density in dihydropyrimidinones ring resulting from the substitution of electron-withdrawing group (nitrophenyl) in C4 position of benzene ring. In contrast to the changes of binding enthalpies for the three compounds, the changes of binding entropies that result from C4 substitution in benzene ring further confirm their binding mode with ctDNA. The most evident reduction of the binding entropy for 4b may be due to the formation of the most ordered structure in binding to the ctDNA helix. Simultaneity, the most inapparent reduction of the binding entropy for 4c lies in the partial intercalation to ctDNA. The reduction of the binding entropy for 4a lies between 4b and 4c because of the moderate intercalation to ctDNA, forming the relatively ordered complex. In addition to the formation of stable complex, sources of the reduction in binding entropy may include a decrease in rotational entropy brought about by interactions between the C4 substitution in benzene ring and the DNA binding sites. For example, dimethylamino group possesses the potential hydrogen bonding with the DNA binding site; the restrictions in rotational freedom brought about by the potential bonds could contribute to the significant decrease in binding entropy for 4b. 3.5. Antitumor activity studies Antitumor activities of 4a, 4b and 4c were examined in vitro against two tumor cell lines, human hepatocellular carcinoma (BEL7402) cells and rat pheochromocytoma (PC-12) cells using MTT assay method [49,50]. The inhibition rate of the three compounds for 24 h on each cell line was calculated with Eq. (5) (shown in materials and methods section). The bar diagrams of inhibition rate of the three compounds against the two tumor cell lines versus the test sample concentrations are, respectively, presented in Fig. 5. From Fig. 5, we can see that the inhibition rate of 4b over the two cell lines is greater than 4a and 4c, and the inhibition rate of 4a and 4c is nearly equal in the test sample concentrations. At the same time, it is also found that 4a and 4c almost lose their toxicity over the two cell lines when the test sample concentrations are lower than 0.004 mg/ml. These observations suggest that 4b has efficient inhibition to the growth and proliferation of the two tumor cells in a dose-dependent manner. However, 4a and 4c seem to be less effect on the growth of the two tumor cells relative to 4b. With quantitative analysis, we can see that 4b is about 7-fold increase in the inhibition rate over BEL-7402 cell by comparison with 4a and 4c when the sample concentrations are lower than 0.02 mg/ml. In the case of PC-12 cell, it is about 5-fold increase in the same conditions. Additionally, it is noted that the inhibition rate of the three compounds over the two cells, especially for PC-12 cell is almost equivalent when the sample concentration is 0.5 mg/ml. This is likely due to the excessive sample concentration which exceeds the routine therapeutic dose. So the results prove that the cytotoxicity of 4b is much higher than that of 4a and 4c. SAR analysis revealed that the presence of electron-donating substitution in 4 rd position of benzene ring (e.g., 4b) can effectively enhance the antitumor activities of the dihydropyrimidinones derivatives, while the presence of electron-withdrawing substitution in the same position (e.g., 4c) hardly affects their antitumor activities relative to the parent (4a). The evident substituent effect for these analogs studied here may provide an effective approach in improving their biological activities. Therefore, the promising

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35

Table 2 Thermodynamic parameters for the binding of 4a, 4b and 4c to CT-DNA obtained from ITC measurements at 25 ◦ C. Compound

n

Kb (×104 M−1 )

H0 (kJ mol−1 )

S0 (J mol−1 k−1 )

G0 (kJ mol−1 )

4a 4b 4c

0.21 0.15 0.16

1.57 ± 0.14 5.79 ± 0.21 1.03 ± 0.10

−34.74 ± 0.35 −40.82 ± 0.42 −26.85 ± 0.48

−39.19 ± 0.46 −45.75 ± 0.51 −13.24 ± 0.39

−23.95 ± 0.12 −27.18 ± 0.14 −22.90 ± 0.23

SARs studies are another emphasis for this work. The interactions were confirmed with viscosity measurement, UV-absorption spectra, fluorescence spectra and ITC studies. The results indicate that the substituent modifications in 4 rd position of benzene ring for this kind of compounds significantly affect their DNA binding properties. The analogs possessed electron-donating substitute may preferably intercalate into DNA helix bases, enhancing their binding affinities. Whereas, the ones possessed electron-withdrawing substitute tend to bind to DNA with a partial intercalation mode, and show the relative weak binding affinities. The effects of substituent modifications on the DNA binding energies for these analogs were investigated by ITC method in detail, which indicates that electron-donating substitute modifications in 4 rd position of benzene ring are more favorable for DNA binding enthalpy by comparison with electron-withdrawing substitute modifications. The antitumor activity studies reveal that the analogs modificated with electron-donating substitute possess significant biological activities. However electron-withdrawing substitute modifications almost not affect the biological activities. Therefore, the SARs studies may provide an effective approach to further modificate and design new therapeutic agents in some diseases. The results of this study will be taken into account for investigating the modifications of other position in benzene ring on DNA binding behaviors and biological activities for this series of analogs in further experiments. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant no. 21173071, 21273061) and the Research Fund for the Doctoral Program of Higher Education of China (Grant no. 20114104110002). Appendix A. Supplementary data Fig. 5. Inhibition rate of 4a, 4b and 4c over BEL-7402 (top panel) and PC-12 (bottom panel) cells, respectively, in vitro. Each data point is the mean ± standard error obtained from three independent experiments. (Three bars, from left to right: 4a, 4b and 4c, respectively.)

compounds substituted by electron-donating groups, are hopeful for further modification and design to potential antitumor agents. Simultaneously, from the above DNA binding studies and the biological evaluation, it is interesting to note that the DNA binding affinities of dihydropyrimidinones derivatives may correlate with their antitumor activities. For example, the DNA binding affinity of 4b is about 4 to 5-fold increases relative to 4a and 4c, accordingly its inhibition rate over tumor cells is approximately 5-fold increases as compared to 4a and 4c. For 4a and 4c, this correlation is clearer. The less difference of binding affinities between 4a and 4c brings about their almost equivalent inhibition rate over the two tumor cells. The information obtained from this work would be useful to understand the SARs of dihydropyrimidinones derivatives and helpful to develop new potential antitumor agents for some diseases. 4. Conclusions In this study, we used different approaches to explore the DNA binding properties of three dihydropyrimidinones derivatives under simulated physiological conditions. At the same time, the

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.01.019. References [1] T. Biver, M. Venturini, E.A. Jares-Erijman, T.M. Jovin, F. Secco, Biochemistry 48 (2009) 173. [2] M.D. Incalci, C. Sessa, Expert Opin. Investig. Drugs 6 (1997) 875. [3] C.N.N. Soukpoe’-Kossi, A. Ahmed Ouameur, T. Thomas, A. Shirahata, T.J. Thomas, H.A. Tajmir-Riahi, Biomacromolecules 9 (2008) 2712. [4] D.L. Arockiasamy, S. Radhika, R. Parthasarathi, B.U. Nair, Eur. J. Med. Chem. 44 (2009) 2044. [5] L.A. Dickinson, R.J. Gulizia, J.W. Trauger, E.E. Baird, D.E. Mosier, J.M. Gottesfeld, P.B. Dervan, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 12890. [6] L.A. Dickinson, R. Burnett, C. Melander, B.S. Edelson, P.S. Arora, P.B. Dervan, J.M. Gottesfeld, Chem. Biol. 11 (2004) 1583. [7] M.J. Hannon, Chem. Soc. Rev. 36 (2007) 280. [8] C. Bailly, R.K. Arafa, F.A. Tanious, W. Laine, C. Tardy, A. Lansiaux, P. Colson, D.W. Boykin, W.D. Wilson, Biochemistry 44 (2005) 1941. [9] N.C. Garbett, N.B. Hammond, D.E. Graves, Biophys. J. 87 (2004) 3974. [10] J. Joseph, E. Kuruvilla, A.T. Achuthan, D. Ramaiah, G.B. Schuster, Bioconjug. Chem. 15 (2004) 1230. [11] W.D. Wilson, Y.H. Wang, S. Kusuma, S. Chandrasekharan, N.C. Yang, D.W. Boykin, J. Am. Chem. Soc. 107 (1985) 4989. [12] C.V. Kumar, E.H. Asuncion, J. Am. Chem. Soc. 115 (1993) 8547. [13] W.B. Tan, A. Bhambhani, M.R. Duff, A. Rodger, C.V. Kumar, Photochem. Photobiol. 82 (2006) 20. [14] J. Tan, B.C. Wang, L.C. Zhu, Bioorgan. Med. Chem. 17 (2009) 614.

36

G. Wang et al. / Colloids and Surfaces B: Biointerfaces 106 (2013) 28–36

[15] Wilson, F.A. Tanious, H.J. Barton, R.L. Jones, K. Fox, R.L. Wydra, L. Strekowski, Biochemistry 29 (1990) 8452. [16] W.D. Wilson, F.A. Tanious, D. Ding, A. Kumar, D.W. Boykin, P. Colson, C. Houssier, C. Bailly, J. Am. Chem. Soc. 120 (1998) 10310. [17] W.D. Wilson, F.A. Tanious, H.J. Barton, L. Strekowski, D.W. Boykin, J. Am. Chem. Soc. 111 (1989) 5008. [18] T.A. Larsen, D.S. Goodsell, D. Cascio, K. Grzeskowiak, R.D. Dickerson, J. Biomol. Struct. Dyn. 1 (1989) 477. [19] W.D. Wilson, F.A. Tanious, H.J. Barton, R.L. Wydra, R.L. Jones, D.W. Boykin, L. Strekowski, Anti-Cancer Drug Des. 5 (1990) 31. [20] L. Strekowski, W.D. Wilson, J.L. Mokrosz, A. Strekowska, A.E. Koziol, G.J. Palenik, Anti-Cancer Drug Des. 2 (1988) 387. [21] L. Strekowski, S. Chandrasekaran, Y.H. Wang, W.D. Edwards, W.D. Wilson, J. Med. Chem. 29 (1986) 1311. [22] S. Nafisi, F. Manouchehri, H.A. Tajmir-Riahi, M. Varavipour, J. Mol. Struct. 875 (2008) 392. [23] C.C. Tsai, S.C. Jain, H.M. Sobell, Proc. Natl. Acad. Sci. U.S.A. 72 (1975) 628. [24] D.B. Davies, V.I. Pahomov, A.N. Veselkov, Nucleic Acids Res. 25 (1997) 4523. [25] Y. Lu, G.K. Wang, J. Lv, G.S. Zhang, Q.F. Liu, J. Fluoresc. 21 (2011) 409. [26] N.K. Modukuru, K.J. Snow Jr., B.S. Perrin, J. Thota, C.V. Kumar, J. Phys. Chem. B 109 (2005) 11810. [27] C.O. Kappe, W.M. Fabian, M.A. Semones, Tetrahedron 53 (1997) 2803. [28] A.D. Patil, N.V. Kumar, W.C. Kokke, M.F. Bean, A.J. Freyer, C.D. Brosse, S. Mai, A. Truneh, B. Carte, J. Org. Chem. 60 (1995) 1182. [29] N.S. Nandurkar, M.J. Bhanushali, M.D. Bhor, B.M. Bhanage, J. Mol. Catal. A. 271 (2007) 14. [30] D. Nagarathnam, S.W. Miao, B. Lagu, G. Chiu, J. Fang, T.G.M. Dhar, J. Med. Chem. 42 (1999) 4764. [31] J. Marmur, J. Mol. Biol. 3 (1961) 208.

[32] A. Shaabani, A. Bazgir, F. Teimouri, Tetrahedron Lett. 44 (2003) 857. [33] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954) 3047. [34] R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, U.N. Balachandran, Biochem. Biophys. Res. Commun. 271 (2000) 731. [35] G. Cohen, H. Eisenberg, Biopolymers 8 (1969) 45. [36] M. Purcell, J.F. Neault, T. Riahi, Biochim. Biophys. Acta 1478 (2000) 61. [37] S. Das, G.S. Kumar, J. Mol. Struct. 872 (2008) 56. [38] J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4161. [39] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 31 (1992) 9319. [40] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1993) 2573. [41] S. Basili, A. Bergen, F. Dall Acqua, A. Faccio, A. Granzhan, H. Ihmels, S. Moro, G. Viola, Biochemistry 46 (2007) 12721. [42] D. Suh, J.B. Chaires, Bioorg. Med. Chem. 3 (1995) 723. [43] H. Xu, K.C. Zheng, H. Deng, L.J. Lin, Q.L. Zhang, L.N. Ji, New J. Chem. 27 (2003) 1255. [44] R.A. Hutchins, J.M. Crenshaw, D.E. Graves, W.A. Denny, Biochemistry 42 (2003) 13754. [45] R.O. Brien, I. Haq, Biocalorimetry 2: Applications of Calorimetry in the Biological Sciences, John Wiley and Sons, West Sussex, 2004. [46] J.B. Chaires, DNA and RNA Binders: From Small Molecules to Drugs, Wiley-VCH, Weinheim, 2002. [47] S. Nafisi, F. Manouchehri, H.A. Tajmir-Riahi, M. Varavipour, J. Mol. Struct. 827 (2007) 35. [48] J.B. Chaires, Arch. Biochem. Biophys. 453 (2006) 26. [49] T. Mosmann, J. Immunol. Methods 65 (1983) 55. [50] C. Kurumurthy, P.S. Rao, B. Veeraswamy, G.S. Kumar, P.S. Rao, B. Narsaiah, L.R. Velatooru, R. Pamanji, J.V. Rao, Eur. J. Med. Chem. 46 (2011) 3462.