Synthesis, crystal structure, catecholase activity, DNA ... - NOPR - niscair

Indian Journal of Chemistry Vol. 54A, February 2015, pp. 170-178

Synthesis, crystal structure, catecholase activity, DNA cleavage and anticancer activity of a dinuclear manganese(III)-bipyridine complex Dhananjay Deya, Abhranil Dea, Sukanta Pala, Partha Mitrab, Anandan Ranjanic, Loganathan Gayathric, Saravanan Chandralekad, Dharumadurai Dhanasekaranc, Mohammad Abdulkadhar Akbarshae, Niranjan Kolea & Bhaskar Biswasa, * a

Department of Chemistry, Raghunathpur College, Purulia 723 133, India Email: [email protected]/ [email protected] b Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Kolkata 700 032, India c Department of Microbiology, Bharathidasan University, Tiruchirappalli 620 024, India d Department of Chemistry, Urumu Dhanalakshmi College, Tiruchirappalli 620 019, India e Mahatma Gandhi-Doerenkamp Center, Bharathidasan University, Tiruchirappalli 620 024, India Received 23 April 2014, revised and accepted 19 January 2015 Design and synthesis of a dinuclear manganese(III) complex [Mn2(µ-O)(µ-O2CMe)2(H2O)2(2,2'-bipy)2](NO3)2.5H2O (1) [2,2'-bipy = 2,2'-bipyridine] with mono-µ-oxo and di-µ-acetato bridge has been reported. Single crystal X-ray diffraction study reveals that (1) crystallizes in monoclinic system with P21/n space group. Evaluation of (1) as a model system for the catechol oxidase enzyme by using 3,5-di-tert-butylcatechol (DTBC) as the substrate in methanol medium, reveals that the complex exhibits efficient catalytic activity with kcat value of 8.89×102. (1) cleaves the pBR 322 DNA without addition of an activating agent. Further, the anticancer activity of (1) on human hepatocarcinoma cell line (HepG2) has been examined. The apoptosis in the cell line has been assessed by the changes in cell morphology, which shows the efficacy of (1) to induce 55% of apoptotic for 24 h. Interestingly, the observed IC50 values reveal that (1) effects conformational change on DNA strongly and exhibits remarkable cytotoxicity. Keywords: Coordination chemistry, Manganese, X-ray structure, Catecholase activity, DNA cleavage, Anticancer activity

Design and synthesis of multinuclear manganese assemblies are of interest to synthetic chemists because of their fundamental applications towards metallobiomolecules such as superoxide dismutase1, catalase2, photosystem II3 of green plants, etc. Catechol oxidase is a copper enzyme having a hydroxobridged dicopper(II) center in the active site. This enzyme catalyzes the oxidation of a wide range of o-diphenols (catechols) to the corresponding o-quinones coupled with 2e/2H+ reduction of O2 to H2O, in a process known as catecholase activity4-7. Extensive biomimetic studies have been carried out with dicopper(II) complexes, derived from nitrogen-containing dinucleating ligands, as the model compounds4,5. Some mononuclear, dinuclear, and oligonuclear systems of manganese acting as catalysts for catecholase activity, are known8-10. Structures of several binuclear Mn(III) complexes along with magnetic properties of the [Mn2(µ-RCOO)2(µ-O)]2+ core have been described in the literature11-15. Though the perchlorate salt of the dinuclear cation, [Mn2(µ-CH3COO)2(µ-O)]2+ has been previously reported14,15 but we have synthesized the nitrate salt of the same cationic dinuclear unit using a

different methodology. The examples of the dinuclear MnIIIMnIII catalyst for catechol oxidation are very limited16. Since carboxylate is a suitable leaving group for incoming catecholate moiety to show catecholase activity, it is of interest to explore such catalytic aspect with the dinuclear MnIIIMnIII compounds having carboxylate bridges. The design of small cationic molecules that react at specific sequences of DNA under physiological conditions via oxidative and hydrolytic cleavage has been of great interest in the field of bioinorganic chemistry. The application of octahedral complexes has allowed the targeting of specific DNA sites by matching the shape, symmetry and functionality of the metal complex to that of the DNA target17. Due to the unusual binding properties and general photo-activity, these coordination compounds were suitable candidates as DNA secondary structure probe, photocleavers and antitumor drugs18-20. Herein, we have synthesized and crystallographically characterized a dinuclear acetatooxo-manganese(III) complex containing 2,2'-bipyridine at room temperature. We have investigated the catalytic activity of [Mn2(µ-O)(µ-O2CMe)2(H2O)2(2,2'-

DEY et al.: SYNTHESIS & CHARACTERISATION OF Mn(III)-BIPYRIDINE COMPLEX

bipy)2](NO3)2.5H2O (1) complex towards 3,5-di-tert-butylcatechol as bio-mimetic model for catecholase oxidase in methanolic medium. Literature survey indicates that magnetic property and magnetostructural relationship of the dinuclear 2+ 11-15 [Mn2(µ-RCOO)2(µ -O)] core remain the most fundamental aspect of the investigation. The catalase activity of the identical cationic dinuclear core (without single crystal study) has been investigated by Corbella et al.21 We have also explored the cleavage efficiency on pBR 322 DNA without addition of external agents. Investigation of cytotoxic effect of (1) on human hepatocarcinoma cell line (HepG2) reflects remarkable efficiency to kill the affected DNA molecules. Materials and Methods High purity 2,2'-bipyridine (Lancaster, UK), ammonium ceric nitrate (Aldrich, UK), manganese(II) chloride tetrahydrate (E. Merck, India), glacial acetic acid (E. Merck, India) were purchased and used as received. All the other reagents and solvents were of analytical grade (AR grade) purchased from commercial sources and were used as received. Infrared spectrum (KBr) was recorded with a FTIR-8400S Shimadzu spectrophotometer in the range 400–3600 cm–1. Ground state absorption was measured with a Jasco V-530 UV-vis spectrophotometer. Elemental analyses were performed on a Perkin-Elmer 2400 CHN microanalyser. Electrospray ionization (ESI) mass spectrum was recorded using a Q-tof-micro quadruple mass spectrometer. Synthesis of [Mn2(µ-O)(µ-O2CMe)2(H2O)2(2,2'-bipy)2](NO3)2.5H2O (1)

A 60-40 v/v AcOH-H2O solution (10 mL3) of 2,2'-bipyridine (0.156 g, 1 mmol) was added dropwise to a solution of MnCl2.4H2O (0.197 g, 1 mmol) in the same solvent (10 mL3) and mixed slowly on a magnetic stirrer with slow stirring for 10 minutes. The colour of the solution turned pink. After that solid CH3COONa (0.164 g, 2 mmol) was added to the reaction mixture. Then solid ammonium ceric nitrate (1.10 g, 2 mmol) was added to the above solution when the pink solution turned into a dark brown clear solution. Stirring was continued for another 10 minute. Lastly, the solution was filtered and the supernatant liquid was kept in air for slow evaporation. After a few days, the fine microcrystalline compound that separated out was washed with toluene and dried in vacuo over silica gel indicator. Yield: 0.163 g (82.7% based on metal salt). Anal (%): Calc for C24H36N6O18Mn2 (1): C, 35.74; H, 4.50;

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N, 10.42; Found: C, 35.81; H, 4.38; N, 10.52. IR (KBr pellet, cm-1): 3418, 1604, 1587, 1484, 1426, 1384, 852. UV-vis (λ, nm): 239, 281, 318, 442. Single crystal X-ray diffraction study

Crystal diffraction data were collected using a Bruker Smart Apex CCD diffractometer. The data were collected with graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) at 293 K. The structure was resolved by direct method and refined by full-matrix least-squares procedures using the SHELXL-97 software package22,23. The crystallorgraphic data and bond distance, bond angle table are summarized in Table 1 and Table S1 (Supplementary Data). Catalytic oxidation of 3,5-DTBC

In order to examine the catecholase activity of the complex, a 10−4 M solution of (1) in methanol solvent was treated with 100 equiv. of 3,5-di-tert-butylcatechol (3,5-DTBC) under aerobic conditions at room temperature. Absorbance vs. wavelength (wavelength scans) of the solution was recorded at regular time intervals of 5 min in the wavelength range 300–500 nm up to 60 min. It may be noted here that a blank experiment without catalyst did not show formation of the quinone up to 6 h in MeOH. To determine the dependence of rate on substrate concentration and various kinetic parameters, a 10−4 M solution of the complex was treated with at least Table 1—Crystal data and structure refinement parameters for (1) Parameters

(1)

Empirical formula Formula weight Temperature (K) Crystal system Space group a (Å) b (Å) c (Å) Volume (Å3) Z ρ (g cm–3) µ (mm–1) F (000) θ ranges (º) Rint R (reflections) wR2 (reflections) Final R indices Largest peak and hole (eA˚-3)

C24H22N6O18Mn2 806.08 293 Monoclinic P 21/n 17.9334(11) 9.9386(6) 19.1126(11) 3365.3(3) 4 1.564 0.836 1608 1.5-29.0 0.119 40452 8588 0.0982, 0.3280 2.48, -1.92

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10 equiv. of substrate so as to maintain pseudo first order conditions. The reaction was followed spectrophotometrically by monitoring the increase in the absorbance at 401 nm (Quinone band maxima) as a function of time (time scan) up to 60 minutes.

(measurement) and 630 nm (reference) using a 96-well plate reader (Bio-Rad, Hercules, CA, USA). Data were collected for three replicates each and used to calculate the mean. The percentage inhibition was calculated, from this data, using the formula:

DNA cleavage studies

 Mean OD (control)-Mean OD (treated)  % Inhibition =   × 100 Mean abs (control)  

Cleavage of pBR322 DNA by the dinuclear manganese(III) complex as (1) was monitored in 5 mM Tris–HCl/50 mM NaCl buffer (pH 7.1) medium by agarose gel electrophoresis technique. The complex (25, 50, 75 and 100 µg) solutions were incubated with plasmid DNA for an hour at 37 °C. After incubation, the reaction was quenched by addition of loading buffer (2 µL of bromophenol blue dye). The samples were then carefully electrophoresed for 30 min at 50 V along with the control DNA on 1% agarose gel using Tris-acetic acid EDTA (TAE) buffer. Then the gel was stained using EB and photographed under UV light to determine the extent of DNA cleavage. The results were compared with the control. Anticancer activity of (1)

The human hepatocarcinoma (HepG2) cell line was obtained from the National Center for Cell Science (NCCS), Pune, India. The cells were cultured in DMEM medium (Sigma–Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Gibco) and 100 U/mL of penicillin and 100 µg/mL of streptomycin as antibiotics (Gibco), in 96 well culture plates, at 37 °C, in a humidified atmosphere of 5% CO2, in a CO2 incubator (Forma, Thermo Scientific, USA). All experiments were performed using cells from passage 15 or less. Cytotoxicity assay (MTT assay)

The Mn(III) complex (1) in the concentration range 50–500 µM/mL dissolved in DMSO was added to the wells 24 h after seeding of 5×103 cells per well in 200 µL of fresh culture medium. DMSO solution was used as the solvent control. A miniaturized viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was carried out according to the method described by Mosmann24. After 24 and 48 h, 20 µL of MTT solution (5 mg/mL in phosphatebuffered saline (PBS)) was added to each well and the plates were wrapped with aluminum foil and incubated for 4 h at 37 °C. The purple formazan product was dissolved by addition of 100 µL of DMSO to each well. The absorbance was monitored at 570 nm

From these data, the IC50 values (the concentration at which (1) killed 50% of the cells at the respective durations of treatment) for 24 and 48 h treatment were arrived at. Acridine orange and ethidium bromide staining was performed as described by Spector et al.25 The cell suspension of each sample containing 5×105 cells, was treated with 25 µL of AO and EB solution (3.8 µM of AO and 2.5 µM of EB in PBS) in triplicate and examined under a fluorescent microscope (Carl Zeiss, Germany) using an UV filter (450–490 nm). The morphological changes observed were photographed. The human hepatocarcinoma (HePG2) cells were cultured in 6-well plates and treated with IC50 concentration of the Mn(III) complex. After 24 h incubation, the treated and untreated cells were harvested and stained with Hoechst 33258 (1 mg/mL, aqueous) for 5 min at room temperature. A drop of the cell suspension was placed on a glass slide, and a cover slip was laid over to reduce light diffraction. 300 Cells per samples were counted in duplicate and observed at ×400 with a fluorescent microscope (Carl Zeiss, Jena, Germany). fitted with a 377–355 nm filter. The percentage of cells reflecting pathological changes was calculated. Results and Discussion Synthesis and characterisation

The dinuclear Mn(III) complex was prepared by mixing of 2,2'-bipyridine and sodium acetate to a solution of MnCl2·4H2O followed by the addition of solid ammonium ceric nitrate (CAN) in aqueous acetic acid solution (v/v) at room temperature. The schematic presentation of synthesis is given below: Mn2+ + 2,2'-bipy + NaOAc + CAN → (1) The dinuclear manganese(III)-bipyridine complex containing oxo- and acetate-bridged was prepared in aqueous acetic acid solutions. The air-stable moistureinsensitive compound is soluble in different polar solvents like methanol, ethanol, acetonitrile and water. Here, CAN is used to oxidize the manganese ion at


higher oxidation level, it also supplies heavier nitrate anion to stabilize the complexes. The IR spectrum of (1) contains characteristic strong peak for the NO3– anion at ~1384 cm–1. The peaks at 1604 and 1587 cm–1 are due to presence of imine chromophore of bipyridine ligand. Peaks characteristic for the oxo- and acetato-bridges are clearly observed at ~852 and 1484, 1426 cm–1 respectively26,27. Water molecules in the structure of the complex were detected in the IR spectra by a broad band centered at about 3418 cm–1 (Supplementary Data, Fig. S1). To probe the solution stability of the complex, we performed UV–vis spectral measurements for methanolic solution of (1) at room temperature. Bands at ~239, 281, 318 and 442 nm are observed (Supplementary Data, Fig. S2). Low intensity and broad transitions at 442 nm attributed to ligand field bands in MnIII octahedral field28,29 and remained unaffected for at least 5 days, revealing that the complex is stable in solution at room temperature. Mass spectral analysis of the manganese(III) complex further consolidates this fact by producing molecular ion peak at m/z 592.93 (Calc. 593.06) (Supplementary Data, Fig. S3) in methanol, which corresponds to the existence of the stable cationic species as [Mn2(µ-O)(µ-OAc)2(H2O)2(2,2'-bipy)2]2+ in solution. The single crystal X-ray diffraction analysis reveals that the crystal lattice of (1) consists of dinuclear cationic [Mn2(µ-RCOO)2(µ-O)]2+ core with coordinated and lattice water molecules and nitrate as counter anions. The ORTEP diagram with atom numbering scheme of the (1) is shown in Fig. 1. The cationic complex shows two acetato and one oxo bridges. A chelating 2,2'-bipyridine (bipy) acting as terminal ligand and one water molecule complete the octahedral coordination of each manganese atom.

Fig. 1—ORTEP diagram of (1)2+ (30% ellipsoid probability) with atom numbering scheme.

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Catecholase activity

Catechol oxidases are type III copper proteins which catalyze the oxidation of catechols to quinones in the presence of oxygen30. The catecholase activity of (1) was studied using 3,5-di-tert-butylcatechol (3,5-DTBC) as a convenient model substrate, in air saturated methanol solvent at room temperature (25 °C). For this purpose, a 1×10−4 M solution of this complex was treated with 1×10−2 M (100 equiv.) of 3,5-DTBC and the course of the reaction was followed by recording the UV–vis spectra of the mixture at an interval of 5 min for 1 h (Scheme 1). Spectral bands at 239, 281, 318 and 442 nm appeared in the electronic spectrum of (1) in methanol, whereas 3,5-DTBC showed a single band at 284 nm. As the reaction proceeded, there was a gradual decrease in intensity of the band due to the catechol at 284 nm31 and an initial new band was formed at ~401 nm (Fig. 2). This indicates the formation of the respective quinone derivative; the band maximum was gradually shifted to 401 nm. 3,5-DTBQ, which was purified by column chromatography and isolated in high yield (78.4% for 1) by slow evaporation of the eluant, was identified by 1H NMR spectroscopy

Catalytic oxidation of 3,5-DTBC to 3,5-DTBQ in air-saturated methanol solvent Scheme 1

Fig. 2—Increase of quinone band at 401 nm after addition of 100 equivalents of (3,5-DTBC) to a solution containing (1) (10-4 M) in methanol at 25 °C. [The spectra were recorded after every 5 min].

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(Supplementary Data, Fig. S4). 1H NMR (CDCl3, 400 MHz) δH: 1.15 (s, 9H), 1.20 (s, 9H), 6.15 (d, J = 2.4 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H). Kinetic studies

Kinetic studies were performed to understand the extent of the efficiency of (1). The kinetics of oxidation of 3,5-DTBC was determined by the method of initial rates and involved monitoring the growth of the quinone band at 401 nm as a function of time31. Kinetic experiment was performed with (1) (at a constant concentration of 1×10-4 M) and 3,5-DTBC (varying the concentration from 1×10-3 M to 1×10-2 M) in methanol by UV-vis spectrophotometry. The experimental procedure involved the preparation of stock solutions of the dinuclear MnIII complex and the substrate 3,5-DTBC at higher concentrations in methanol medium. 3,5-DTBC (2 mL) of appropriate concentration obtained by accurate dilution from the stock solution was taken in the UV-vis quartz cell and kept for a while inside the cell holder which was attached with a thermostat to keep the temperature at 25 °C. Then, 0.04 mL of stock solution of the complex was added to achieve the ultimate concentration of the complex as 1×10-4 M. The formation of 3,5-DTBQ was monitored with time at a wavelength of 401 nm. The initial rate method was applied to determine the rate constant value for each concentration of the substrate; each experiment was repeated thrice. The average rate constant values for (1) show that the rate is first order at low concentrations of the 3,5-DTBC substrate, but zero order at its higher concentrations. Figure 3 represents the dependency of initial rate on the concentration of catechol for complex (1) as representative in methanol medium. By applying GraFit32 program for Michaelis-Menten

Fig. 3—Plot of rate versus [substrate] in presence of (1) in MeOH. [Inset: Lineweaver-Burk plot].

enzymatic kinetics, KM and Vmax was calculated where KM is Michaelis–Menten constant for the Mn(III) complex and Vmax is maximum initial rate attained for a particular concentration of the metal complex in the presence of a large excess of the 3,5-DTBC. The turnover number (kcat) value was obtained by dividing the Vmax value with the concentration of the dinuclear Mn-complex. The observed kinetic parameters are found as Vmax (M s-1) = 2.47×10-5 (std. error = 9.39 × 10-7); rate constant KM (M) = 8.19×10-4 (std. error = 5.95 × 10-5 ). We also calculated the turnover number to evaluate the efficiency for the conversion of maximum number of substrate molecules to product molecules per catalyst per time unit. The turnover number for the present dinuclear Mn(III) catalyst was Kcat (h-1) = 8.89 × 102 which reflects good efficiency of (1) for conversion of catechol molecules into quinone. However, we could not isolate the complex after the catalysis reaction, since it was used in 10-4 M concentration. DNA cleavage studies

The incubation of pBR322 plasmid DNA in the presence of the (1) was studied to determine the efficiency with which it sensitizes DNA cleavage by monitoring the transition from the naturally occurring, covalently closed circular form (Form I) to the open circular relaxed form (Form II). When circular plasmid DNA was subjected to electrophoresis, the intact supercoiled DNA (form I) migrates relatively fast. If scission occurs on one strand, the supercoiled DNA will relax to generate a slower moving open circular form (form II). If both strands are cleaved, a linear form (form III) will be generated that migrates between form I and form II32. The ability of the (1) to cleave DNA was assayed by gel electrophoresis on pBR 322 DNA as the substrate in 5 mM Tris–HCl/50 mM NaCl buffer (pH 7.1) medium in the absence of external additives. The DNA was mixed with the different concentration (25, 50, 75 and 100 µg) of Mn(III) complex and was incubated at 37 ºC for 1 h. Figure 4 reveals the cleavage activity of (1) on

Fig. 4—Concentration dependent cleavage of the bacterial genomic DNA by (1). [Lane 1 shows the control DNA. Lane 2 shows DNA + sample (25 µg); lane 3, DNA+ sample (50 µg); lane 4, (75 µg); lane 5, (100 µg)].

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pBR 322 DNA. Lane 1 shows the pBR 322 DNA control. Lane 2 shows the DNA treated with 25 µg of the (1). There was complete cleavage of the DNA with the addition of 25 µg of the complex. Further addition of different concentrations (50, 75 and 100 µg) of the complex in Lane 3, Lane 4, Lane 5 did not show bands for the DNA. This finding clearly suggests that the concentration of the complex needed to cleave the pBR 322 DNA was 25 µg. It is advantageous to perform DNA cleavage studies without addition of activating additives because opening of parallel cytotoxicity is associated with the external addition17,33-38. Reddy et al.34 and Mishra et al.35 showed polynuclear transition metal complexes containing labile ligands coordinated at metal centre can catalyses DNA cleavage by attacking the phosphorous atom in phosphate groups. In the present study, it is the hydrolytic cleavage of DNA which is responsible for slicing DNA at phosphate diester bonds in presence of coordinated water molecules. The half life of a typical phosphate diester bond of DNA in neutral water under ambient conditions is estimated to be in the order of tens to hundred billions of years. This means that a catalyst has to accelerate this reaction in many folds to achieve an effective hydrolysis of the phosphate backbone of DNA within an acceptable timeframe38. General mechanism of this method is that the hydrolysis reaction is facilitated by the presence of metal complexes, which can activate the phosphate group towards nucleophilic attack, activate water or hydroxide as nucleophile or increase the leaving group ability of the departing alcohol. The presence of two coordinated labile water molecules in (1) makes it susceptible to a nucleophilic attack at P-atom of the DNA phosphate backbone, forming a five coordinate intermediate, which is stabilized by the dinuclear Mn(III) catalyst (1). Subsequent cleavage of either the 3′-PO (as seen is most often in enzymatic systems) or the 5′-PO results in a strand scission (Scheme 2).

obtained by plotting the cell viability against the concentration of the complex (Supplementary Data, Fig. S4). The results revealed that the IC50 at 48 h (110±0.3 µM) is lower than that at 24 h (120±0.4), clearly indicating that the complex exhibits cytotoxicity against HepG2 in a dose and duration-dependent manner. Thus, the cytotoxicity exhibited by the complex is consistent with its strong binding with DNA, and its efficiency in cleaving DNA in the absence of an external agent is responsible for its potency to induce cell death through different modes of interaction between the cationic complex and DNA. Apoptotic cell death is known as characterized by different cellular changes such as cell shrinkage, nuclear condensation, DNA fragmentation, membrane blebbing and formation of apoptotic bodies. These apoptotic characteristics as produced in human hepatocarcinoma cell (HepG2) by the (1) were analyzed by AO/EB staining. In this staining method, the fluorescence pattern depends on the viability and membrane integrity of the cells. In general, dead cells are permeable to ethidium bromide and fluoresce orange-red, whereas live cells are permeable to acridine orange only and thus fluoresce green. The cytological changes which were observed in the treated cells are classified into four types based on the fluorescence emission and morphological features of Base O O

The [Mn2(µ-CH3COO)2(µ-O)(bpy)2(H2O)2]2+ cationic unit in (1) has the ability to strongly bind and cleave DNA in the absence of an external agent and DNA cleavage is considered as an important step for a drug to act as an anticancer agent39-41. The cytotoxicity of the (1) dissolved in DMSO was investigated against a human hepatocarcinoma cell line (HepG2) by MTT assay. The IC50 values of (1) were

H

O

H

H

H

O

H

P

Base

O

N O

O

N

O

H

Mn

H

O

O

H

H

O

H

H

O

O O

O

H

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O

Base

H

Mn

Anticancer activity of (1)

H

O

O

O N N

H

H

O

H

H

H

DNA Strand

Proposed mechanism of hydrolytic cleavage of plasmid DNA prompted by (1) Scheme 2

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chromatin condensation in the AO/EB stained nuclei: (i) Viable cells, which are having highly organized nuclei, fluoresce green. (ii) Early apoptotic cells, which show nuclear condensation, emit orange-green fluorescence. (iii) Late apoptotic cells, with highly condensed or fragmented chromatin, the nuclei fluoresce orange to red. (iv) Necrotic cells fluoresce orange to red, with no indication of chromatin fragmentation. All these morphological changes were observed after the treatment of cancer cells with the complex. Figure 5 indicates the apoptotic morphologies induced by (1) at IC50 concentration for 24 h. as well as the efficacy of (1) to induce 55% of apoptotic. There is no indication for necrotic cell death compared with untreated controls by the complex for 24 h. The IC50 values and morphological changes consolidate the potential anticancer activity of the molecule. However, further studies are needed in this direction to confirm the mode of cell death induced by 1. Morphological changes in the nucleus and chromatin were revealed by Hoechst 33528 staining method. The cells treated with IC50 concentration of the complex showed some changes in their morphology of nuclei compared to the control untreated nuclei. In the untreated control cells the nuclei were round with intact chromatin while after treatment with (1) for 24 h changes such as chromatin marginalization, condensation and fragmentation were observed. Figure 6 indicates the apoptotic nuclear

morphology induced at IC50 concentration by the complex for 24 h. It is interesting that 55% of treated cells exhibited abnormal nuclei (Fig. 6). The cytotoxic effect of (1) against tumor cell line (HepG2) was investigated to evaluate the antiproliferative efficiency of (1) in comparison with cisplatin (Table 2). DMSO at 0.5% concentration in the culture medium was used as negative control, with cisplatin as the positive control. Despite the high cytotoxic activity of the dinuclear manganese(III)bipyridine against HepG2 cells, the effectiveness of cytotoxicity was relatively low when compared to cisplatin, the IC50 values of which was 7.2±0.4 µM for 24 h treatment period. However, cytotoxic potential apart, cisplatin has been established to produce toxic side effects42 which is not expected with the manganese complex in the present study. The cytotoxicity of the manganese(III) complex is probably due to the extended planar ring induced by bipyridine43 and presence of labile coordinated water molecules at each of the Mn(III) centres in (1) allows favourable coordination of DNA bases, especially guanine44. Table 2—Inhibitory rates (%) and IC50 of complex (1) and cisplatin against hepatocarcinoma cell line (HepG2) after incubation for 24 h and 48 ha Complex (1) Cisplatin IC50 (µM) IC50 (µM) Inhibitory Inhibitory rates (%) 24 h 48 h rates (%) 24 h 48 h HepG2 50 120±0.4 110±0.3 50 7.2±0.4 6.09±0.4 a

Results represented as ‘mean±SD’ of three independent experiments.

Fig. 5—(a) Representative morphological changes observed for (1) with AO/EB staining against HepG2 at 24 h incubation. (b) The effect of (1) on HepG2 cell as revealed in acridine orange and ethidium bromide staining. [Relative percentage of morphological changes was determined and classified into two categories: viable apoptosis as compared with the control cells after 24 h incubation].

Fig. 6—(a) Representative morphological changes observed for (1) with Hoechst 33258 staining against HepG2 at 24 h incubation. (b) The effect of (1) on HepG2 cell as revealed in Hoechst staining. [Relative percentage of morphological changes was determined and classified into two categories: normal and abnormal nuclei as compared with the control cells after 24 h incubation].


Conclusions Herein, we have synthesized and crystallographically characterized an acetato-oxo-bridged dinuclear manganese(III) complex containing 2,2'-bipyridine. We have investigated the catalytic activity of the Mn(III) complex towards 3,5-di-tert-butylcatechol as biomimetic model for catecholase oxidase in methanolic medium. Turnover number (TON) for our dinuclear Mn(III) catalyst as Kcat (h-1) = 8.89×102 reflects its good efficiency for the conversion of catechol molecules into quinone. We have also explored the cleavage efficiency on pBR 322 DNA without addition of external agents and the complex completely cleaves supercoiled plasmid pBR 322 DNA at a concentration of 25 µg. Investigation into the cytotoxic effect of the dinuclear Mn(III) on human hepatocarcinoma cell line (HepG2) shows its remarkable efficiency to kill the affected DNA molecules. Detailed experimentation related to DNA interaction, cleavage and cytotoxicity on this complex will help to develop clinically relevant information for possible new anticancer agents. Supplementary Data Crystallographic data for 1 are available under CCDC 996461, free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223336033; Email: [email protected] or www: http://www.ccdc.cam.ac.uk). Other supplementary data associated with this article i.e., Figs S1-S4 and Table S1, are available in the electronic form at http://www.niscair.res.in/jinfo/ijca/IJCA_54A(02)170178_SupplData.pdf. Acknowledgement BB gratefully acknowledges the University Grants Commission, New Delhi, India (No. F. PSW-84/1213(ERO) dated 05/02/2013) and Department of Science and Technology, New Delhi, India (SB/FT/CS-088/2013 dated 21/05/2014) for financial support. BB is also thankful to the Indian Association for the Cultivation of Science, Kolkata, India for single crystal X-ray diffraction study, proton NMR and mass spectral studies. References 1 Manganese Redox Enzymes, edited by E J Larson & V L Pecoraro (VCH Publications, New York) 1992. 2 Whittaker M M, Barynin V V, Antonyuk S V & Whittaker J W, Biochemistry, 38 (1999) 9126. 3 Yachandra V K, Sauer K & Klein M P, Chem Rev, 96 (1996) 2927. 4 Koval I A, Gamez P, Belle C, Selmeczi K & Reedijk J, Chem Soc Rev, 35 (2006) 814.

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