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ORIGINAL ARTICLE
Year : 2015  |  Volume : 11  |  Issue : 1  |  Page : 105-113

Cytotoxic potency of self-assembled Ruthenium(II)-NHC complexes with pincer type 2, 6-bis(N-methylimidazolylidene/benzimidazolylidene)pyrazine ligands


1 Department of Basic Science and Humanities, Global Institute of Science and Technology; School of Applied Science, Haldia Institute of Technology, Haldia; Department of Chemistry, Jadavpur University, Kolkata, India
2 School of Applied Science, Haldia Institute of Technology, Haldia; Department of Chemistry, Institute of Technology and Management University, Turari Campus, Gwalior, India
3 School of Applied Science, Haldia Institute of Technology, Haldia, India
4 Department of Chemistry, Jadavpur University, Kolkata, India
5 Department of Chemistry, National Dong Hwa University, Hualion, Taiwan, China
6 Central Research Facility, Indian Institute of Technology Kharagpur, Kharagpur, India

Date of Web Publication16-Apr-2015

Correspondence Address:
Santi M Mandal
Central Research Facility, Indian Institute of Technology Kharagpur, Kharagpur West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.150416

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 > Abstract 

Objective: To study the cytotoxic potency of self-assembled Ruthenium(II)-NHC complexes with 2,6-di-(N-methylimidazolylidene/benzimidazolylidene)pyrazine ligands.
Materials and Methods: Ru(II)-N-heterocyclic (Ru-NHC) complexes, Bis-[2,6-di-(N-methylimidazol-2-ylidene)pyrazine]ruthenium(II) hexaflurophosphate (3), Bis-[2,6-di-(N-methylbenzimidazol-2-ylidene)pyrazine]ruthenium(II) hexaflurophosphate (4) have been synthesized from corresponding ligands 2,6-di-(N-methylimidazolium)pyrazine dichloride (1); 2,6-di-(N-methylbenzimidazolium)pyrazine dichloride (2). Complexes were studied to determine their pro-apoptotic activity against HCT15 and Hep2 cell lines, and antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus epidermidis and Candida albicans.
Results: Both, complex 3 and 4, formed a nanosphere structure in aqueous growth medium. Cytotoxicity study revealed that complex 3 was more effective than complex 4. Complexes mainly target cellular DNA and bacterial cell wall.
Conclusion: This is the first report on the formation of nanoball structure of Ru(II)-NHC complexes. Thus, complex 3 provides a new insight to develop antitumor or antimicrobial drug.

Keywords: Antimicrobial, DNA binding, NHC, Ruthenium (II)-NHC complex, Nanoball, pro-apoptotic


How to cite this article:
Roymahapatra G, Dinda J, Mishra A, Mahapatra A, Hwang WS, Mandal SM. Cytotoxic potency of self-assembled Ruthenium(II)-NHC complexes with pincer type 2, 6-bis(N-methylimidazolylidene/benzimidazolylidene)pyrazine ligands. J Can Res Ther 2015;11:105-13

How to cite this URL:
Roymahapatra G, Dinda J, Mishra A, Mahapatra A, Hwang WS, Mandal SM. Cytotoxic potency of self-assembled Ruthenium(II)-NHC complexes with pincer type 2, 6-bis(N-methylimidazolylidene/benzimidazolylidene)pyrazine ligands. J Can Res Ther [serial online] 2015 [cited 2019 Sep 20];11:105-13. Available from: http://www.cancerjournal.net/text.asp?2015/11/1/105/150416


 > Introduction Top


After the isolation of stable imidazole-2-ylidene by Arduengo in 1991, [1] a series of N-heterocyclic carbenes (NHCs)-based metal complexes have been developed due to their potential application in catalysis and biomedicine. [2],[3],[4],[5],[6] N-heterocyclic carbenes, [1],[2],[3],[4],[5],[6],[7],[8] derived from the deprotonation of 1,3 disubstituted imidazolium or benzimidazolium salts by external base or proper choice of metal salt gives the Metal-NHC (M-NHCs), which are used in metal-mediated catalytic reaction with interesting photophysical properties. [2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12] Beyond catalysis, M-NHCs recently contributed interesting results as promising antimicrobial and anticancer drugs. [10],[11],[12],[13],[14],[15] Though initially the imidazoline NHC ligands comprised N-alkylated derivatives, the file is enriched with N-functionalized NHC by pyridine, pyrimidine, pyrazole, oxazole, thiazole, carbazole, etc. [2],[3],[4],[5],[6],[7],[8],[9],[14],[15] Most of N-alkylated NHCs acts as mono dentate ligands, whereas incorporation of wingtip such as pyridine/pyrimidine/oxazole/thiazole in the backbone offers either mono-, bi-, or tridentate NHC depending upon the designing of the ligand and choice of metal. As NHC ligand is a strong ó-donor and weak ð-acceptor than phosphine, it is used as an alternative of phosphine in catalysis. It is needless to mention that pincer carbene complexes should be much more stable than the other similar types of complexes because of their chelating effect (strong M-carbon bond), which prevents the complex from oxygen, moisture, and heat. [16],[17] In literature, there are several examples of organometallic pincer ligands where donor centers are COS, OCS, CNS, CCN, NNC, CNC, etc., in which pyrazole, thiazole, pyrimidine, and mostly pyridine are used as heterocyclic backbones with other C-donor centers like benzene or substituted benzene. CNC pincer M-NHC complexes are mostly pyridine based. [18] Recently, pyrazine-bridged imidazoline or benzimidazoline ligands have been synthesized and used in this study. [6],[7],[8],[9],[14],[15],[16],[17],[18] A steady growth of interest in Ru(II)-NHC anticancer drugs and photoluminescence study of Ru(II)-NHC complexes over the last 20 years is reflected in the accelerating growth of publications. [13],[19],[20],[21],[22],[23] Interestingly, Ru anticancer drugs have been growing rapidly since NAMI-A (ImH+)[RuIIICl4(Im)(S-dmso)], where Im = imidazole and S-dmso = S-bound dimethylsulfoxide) or KP1019 (IndH+)-[RuIIICl4(Ind)2], where Ind = indazole, has successfully completed phase-I clinical trials and an array of other Ru complexes have shown promises for future development. [21] Several metals, Cu(I), Ag(I), Au(I), Au(III), Pd(II), Pt(II)-NHC are reported as anticancer drugs. [13] Nowadays, the emergence of bacterial or fungal pathogens with enhanced antibiotic resistance is a great threat to infection control. Several pyrazine functionalized M-NHC complexes [14] demonstrated the deleterious effects against multidrug-resistant pathogens, and Hindi et al. [24] also summarized the antimicrobial activities of several M-NHCs whereas the reports are inadequate of Ru(II)-NHC for their antimicrobial properties along with anticancer activity. Interestingly, the secondary infection through human pathogens remains a major complication in patients with malignant disease, [25] which encouraged us to study both the anticancer and antimicrobial properties of Ru(II)-NHC.


 > Experimental Top


Reagents and chemicals

All reactions were carried out under nitrogen using standard Schlenk-type flask. Workup procedures were done in air. All the solvents and chemicals were purchased and used without further purification. The chemicals were purchased from Sigma Aldrich, Lancaster, UK and OMSYS, Kolkata. Silica with 200 nm particle size and 4 nm pore size was purchased from Sigma-Aldrich, USA. 1 HNMR and 13 CNMR spectra were recorded on a Bruker 400 spectrometer. Chemicals shifts, δ in ppm are reported to the internal standard TMS for 1 HNMR and 13 CNMR. Microanalysis were performed on a Perkin-Elmer model 2400 instrument for C, H, N% calculations, electronic absorption measurements were done with Varian Cary 1 UV-Vis spectrophotometer. Fast atom bombardment (FAB) mass spectra were recorded on a VG-7070E instrument and xenon (10 keV) was used for bombarding atoms.

General synthesis

Synthesis of ligand 2,6-di-(N-methylimidazolium)pyrazine dichloride, 1

The ligand 2,6-di-(N-methylimidazolium)pyrazine dichloride, 1, was synthesized according to the reported procedure. [6],[14],[26] 2, 6-dichloropyrazine was added to 1-methylimidazole under neat leads to chloride salt; the fume was observed and the mixture became hot. The wet solid was heated neat at 75°C with stirring for 1.5 h. The crude yellowish solid product was washed first with THF and then with diethyl ether to get the pure white mass (Yield; 86%). 1 HNMR (CD 3 CN, 25°C, 400 Mz): δ 9.44 (s, 2H, H a1 ), 9.21 (s, 2H, H b1 ), 8.21 (d, 2H, J = 8.0 Hz, H c1 ), 7.66 (d, 2H, J = 5.2 Hz, H d1 ), 4.02 (s, 6H, N-CH 3 ). 13 CNMR (CD 3 CN, 400 MHz): 140.7 (C2-Pz), 136.3 (C3-Pz), 135.4 (NCN), 125.34 (C4-imi), 119.3 (C5-imi), 36.7 (N-CH3). Mass M/Z + =313.2[L], 241.8[L] 2+ . Anal. Calcd. For C 12 H 14 N 6 Cl 2 , C, 46.02; H, 4.50; N, 26.83%, Found 45.98; H, 4.48; N, 26.79%.

Synthesis of ligand 2,6-di-(N-methylbenzimidazolium)pyrazine dichloride, 2.

The ligand 2,6-di-(N-methylbenzimidazolium)pyrazine dichloride, 2, was synthesized according to the reported procedure. [6],[26] 2,6-dichloropyrazine was added to 1-methylbenzimidazole and the mixture was heated neat at 125°C with stirring for 4 h and the mixture became solid. The crude yellowish solid product was washed first with THF and then with diethyl ether to get the pure white mass of 2. Yield: 4.44 g (11.72 mmol, 80%). 1 HNMR (CD 3 CN, 25°C, 400 Mz): δ 9.73 (s, 2H, H a ), 9.39 (s, 2H, H b ), 8.36 (d, 2H, 2 J = 8.0 Hz, H c ), 8.05 (d, 2H, 2 J = 7.6 Hz, H f ), 7.88 (m, 4H, 2 J = 7.52 Hz, H d],{ e ), 4.23 (s, 6H, N-CH 3 ). 13 CNMR (CD 3 CN, 400 MHz): 144.3 (2Pz), 144.0 (3Pz), 139.9(NCN), 130.0, 129.4, 118.5, 116.7, 115.9, 115.2 (Ar-C) 34.8(N-CH 3 ). Mass M/Z+ =413.3[L], 341.6[L] 2+ . Anal. Calcd. For C 20 H 18 N 6 Cl 2 , C, 58.12; H, 4.39; N, 20.33, Found C,58.09; H, 4.36; N, 20.29%.

Synthesis Ru(II)-NHC complexes; Bis- [2,6-di-(N-methylimidazol- 2-ylidene)pyrazine]ruthenium(II) hexaflurophosphate, 3, and Bis- [2, 6-di-(N-methylbenzimidazol-2-ylidene)pyrazine]ruthenium(II) hexaflurophosphate, 4

The Ru(II)-NHC complexes, 3 and 4 were synthesized by reported procedure. [26] The relevant ligands were added to ethylene glycol solutions containing RuCl 3·3H 2 O. After the solution was heated at 180°C for 6 h, the reaction mixture was cooled to room temperature. A saturated aqueous solution of KPF 6 was added to the reaction mixture. Immediate brown red precipitate was observed for both cases. The ppt. was filtered, dried, and purified though column chromatography using CH 3 CN: CHCl 3 (2:1) to obtain orange red mass of the complexes 3 and 4. The products were re-crystallized from acetonitrile and diethyl ether. Yields were 58% for complex 3, and 51% for complex 4.

For complex 3, 1 HNMR (CD 3 CN, 25°C, 400 Mz): δ 9.13 (s, 2H, H b1 ), 8.12 (d, 1H, H c1 ), 7.05 (d, 1H, H d1 ), 2.74 (s, 1H, N-CH 3 ). 13 CNMR (CD 3 CN, 400 MHz): 142.79 (C2Pz), 138.34 (C3Pz), 137.67 (NCN), 126.63 (C4-imi), 120.92 (C5-imi), 37.6 (N-CH 3 ). Mass M/Z + =582.2 [Ru (L) 2] +2 , 242.2[L] 2+ . Anal. Calcd. For C 24 H 24 N 12 RuP 2 F 12 , C, 33.03; H, 2.75; N, 19.27. Found 32.99; H, 2.74; N, 19.25.

For complex 4, 1 HNMR (CD 3 CN, 25°C, 400 Mz): δ 9.32 (s, 2H, H b ), 8.28 (d, 2H, 2 J = 8.0 Hz, H c ), 7.46 (d, 2H, 2 J = 7.6 Hz, H f ), 7.35 (m, 4H, 2 J = 7.52 Hz, H d],{ e ), 4.92 (s, 6H, N-CH 3 ). 13 CNMR (CD 3 CN, 400 MHz): 145.94 (2Pz), 145.62 (3Pz), 131.36 (NCN), 131.8, 131.2, 120.2,118.1, 117.2, 116.9 (Ar-C) 36.8 (N-CH 3 ). Mass M/Z + =781.7[Ru (L) 2 } +2 , 341.2[L] 2+ . Anal. Calcd. For C 40 H 32 N 12 RuP 2 F 12 , C, 44.78; H, 2.99; N, 15.67. Found 44.76; H, 2.97; N, 15.66.

Structure determination

Suitable single crystals were formed in case of complex 3. Crystal parameter and crystal data [Table S2 [Additional file 3][INLINE:1]] are reported earlier. [26] In order to understand the structure of synthesized complex 4, the structure was drawn according to the NMR and Mass data found, and was further generated the optimized structure of complexes 4 (correlating with the structure of complex 3) with DFT technic. Geometries of both complexes (3 and 4) were optimized at B3LYP/LANL2DZ level of theory using the Gaussian G-03-E01 program. [27] The number of imaginary frequencies of all the molecules turns out to be zero, implying that they correspond to minimum energy structures on the potential energy surface.

Antimicrobial assays

Pseudomonas aeruginosa ATCC 27853 (Gram-negative bacteria), Staphylococcus epidermidis NCIM2493 (Gram-positive bacteria), and Candida albicans SJ11 (unicellular fungus) were used in this study. Bacterial cultures were maintained in Mueller-Hinton Broth (MHB) and a total inoculum load of ca. 10 5 cells per well-maintained as described earlier. [28],[29],[30] Fungal species [29] were collected from a mature solid medium culture plate (Sabouraud dextrose agar), mixed with liquid RPMI 1640 (Himedia, India), and were incubated for 24 h at 30°C to get the relevant turbidity of 0.5 × 10 4 CFU/mL used as inoculums. Minimum inhibitory concentrations (MIC) of complex 3 and 4 against both Gram-positive and Gram-negative bacteria were determined according to CLSI guidelines. [31] The concentration of each compound used for the assay ranged from 0.48 μM to 1 mM. MIC values were determined where no bacterial growth was observed in naked eye. [14] The complex concentration in first lane was 1 mM and further serial dilution was maintained to consecutive wells. Two wells, where no compounds were added, were used as positive controls, and compounds with the absence of microorganisms were used as negative controls in order to maintain the experimental sterility. In order to determine, the bacterial growth or killing kinetics in the presence of complex 3, bacterial cells were grown in 100 mL of Mueller-Hinton Broth (MHB) supplemented with different doses of complex (4-20 μM), at 37°C. Growth or killing rates that directly correlate to bacterial concentrations were determined by measuring OD at 600 nm. The OD values were converted into concentration of cells measured in CFU per milliliter (1.0 OD corresponded to 2.16 × 10 8 CFU/mL). Similarly, C. albicans was inoculated in 2 mL Sabouraud dextrose broth supplemented with different doses of complex 3 (4-20 μM), and incubated at 30°C at 180 rpm. The in vitro growth rates were measured at OD 600 and using the conversion factor of 3 × 10 7 CFU/mL per 1 U OD 600 . [32] All individual experiments were repeated thrice.

Pro-apoptotic assays

MTT assay

Human colon carcinoma cell lines (HCT15) and human epidermoid cancer cells (Hep2) were obtained from the National Center for Cell Sciences (Pune, India). The cells were cultured as monolayers in MEM medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum and antibiotics, and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 following Mandal et al. 2012. [33] In brief, cells were plated in a 96-well flat-bottomed plate and allowed to attach to the culture surface overnight. On the next day, the broth was aspirated off, washed with PBS (1X) buffer and 200 μL of each complex-containing (0-100 μM) medium were separately added in triplicates. After 72-h incubation, the medium was aspirated off again and washed with PBS (1X) buffer, following the addition of 50 μL of 3- [4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazoli-um bromide (MTT reagent) at a standard concentration of, 3 mg.mL−1 to each well and further incubating for 3 h. After incubation, the MTT-containing medium was aspirated off and once more washed with PBS (1X) buffer and then 200 μL of DMSO solution was added to each well. After 10 min, optical density was determined using a MultiSkan plate reader (LabSystems) at a wavelength of 570 nm. The IC 50 values were calculated from the curves drawn by plotting % of survival cells versus compound concentration (in μM). The IC 50 value indicates the amount of drug needed for 50% growth inhibition of the cancer cells relative to untreated (drug-free cancer cells).

DNA cleavage activity

Supercoiled plasmid DNA, pTZ57R/T (400 ng) was used to check the supercoiled DNA cleavage activity for each set of experiments. Each 20-μL reaction mixture containing 2 μL of 10X Tris-NaCl buffer (pH 7.2), 1 μL of 20 mM DTT (Dithiothreitol), appropriate amount of complex solution and final volume was made with Milli-Q water. The resulting reaction mixture was incubated at 37°C for 2 h. The reaction was stopped at 4°C, followed by the addition of 4 μL of 6X loading buffer (glycerol with bromophenol blue). The reaction mixture was loaded on a 1.0% agarose gel containing 1 μg.mL−1 ethidium bromide. The gels were run at a constant voltage of 80 V for 90 min in Tris-Borate-EDTA (TBE) buffer. The gels were visualized under a UV transilluminator.

Circular dichroism

Circular dichroism spectra were recorded at 37°C using a JASCO (J-810) Spectropolarimeter from JASCO International Company Limited (Hachioji, Tokyo, JAPAN). The scanning rate was 100 nm/min with a response time 1 s. Spectra were recorded at standard sensitivity (100 mdeg) with a data-pitch of 0.5 nmin continuous mode. The scanning range was 350-190 nm and all spectra were the average of three consequent accumulations. The cuvettes used were 2 mm Quartz Suprasil precision cells, and the baseline was corrected using the same solution buffer as a reference. CD spectra were collected from the reaction mixture containing DNA (Calf thymus DNA) and metal compounds in buffer, incubated at 37°C for 12 h and blank CT DNA was used as control.

Cell cycle analysis

Cells were seeded at an equal density per dish and allowed to adhere. After 24 h, cells were synchronized in complete medium supplemented with 2% FBS (for 24 h) according to Kues et al. [34] and further treated with the different complex concentrations along with the control (0.1% DMSO). After treatment, cells were harvested and washed in phosphate buffer saline (PBS) and incubated in 70% ethanol for 45 min at 4°C or kept at −20°C overnight for fixation. Cells were centrifuged, washed and then incubated with Propidium iodide (PI) solution (40 μg.mL -1 PI, 100 μg.ml−1 RNaseA in PBS) at 37°C for 1 h. Apoptotic cells were determined by their hypochromic sub-diploid staining profiles. The distribution of cells in the different cell cycle phases was analyzed from the DNA histogram using Becton-Dickinson FACS Calibur flow cytometer and Cell Quest Pro software.

Scanning electron microscopy

P. aeruginosa and C. albicans cells were harvested from the log phase of their respective growth medium. The cells were then washed three times with 1X PBS buffer and resuspended in the same saline buffer. The cells were treated with complex 3 with their MIC concentration (8 mg. L−1 ) for 30 min. Then, washed thoroughly with PBS buffer and 5-10 μL of cells suspended solution was placed on the lysine-coated glass cover slip as a drop-caste method. The fixed cells were dried in vacuum and kept on desiccators until use. Samples were then fixed onto a graphite stub and kept in an auto sputter coater (E5200, Bio-Rad) under low vacuum up to 120 s for gold coating. Surface morphology was studied by using a scanning electron microscope (JEOL JSM5800) with an accelerated voltage between 5-20 kV.


 > Results and discussion Top


Synthesis and characterization of complexes

The ligands 1 and 2 have been synthesized in 86% and 80% yield, respectively, by reported procedure from the respective precursors. [6],[14],[26] The presence of NCHN proton in 1 HNMR spectrum (in CD 3 CN) confirmed their formation. The NCHN proton in 1 HNMR spectrum appeared at 9.44 ppm for ligand 1 and at 9.73 ppm for ligand 2. The 0.29-ppm downfield shift for ligand 2 than ligand 1 is observed in case of 1 HNMR spectrum and 4.5-ppm downfield shift in 13 CNMR spectrum. Complex 3 was prepared in 58% yield by the complexation of ligand 1 with RuCl 3·3H 2 O in refluxing ethylene glycol solution at 180°C [Scheme 1 [Additional file 1]]. Complex 4 was prepared (in 51% yield) by the complexation of ligand 2 and RuCl 3·3H 2 O with refluxing in ethylene glycol solution at 180°C [Scheme 1]. Absence of imidazolium C2 proton, downfield shifting of pyrazine and other aromatic protons of complex 3 and 4 in the 1 HNMR spectrum confirm the formation of complexes. The crystals of 3 suitable for X-ray data collection were grown by diffusion of diethyl ether into acetonitrile solution. Crystallographic data [CCDC-772881 (for complex 3)] can be obtained free of charge from The Cambridge Crystallographic Data Centre (CCDC) via www.ccdc.cam.ac.uk/data_request/cif. Recently, crystallographic data of complex 3 has been published by Lee et al. [35] We have optimized both the structures of complex 3 and complex 4 [the structure of complex 4 were drawn according to NMR, Mass data and correlated with the structure of complex 3] using Gaussian E03W [27] at the B3LYP/LANL2DZ level of theory. The optimized structures (ORTEP view) of both the complexes 3 and 4 are shown in [Figure 1] and [Figure 2], respectively. Crystallographic data of Complex 3 are given in Table S2. The bond parameters of the complexes 3 and 4 are summerized in [Table S1a and b [Additional file 2]]. The Ru-C carbene (M-C NHC ) bonds are found (2.04754-2.06126Ε for complex 3, 2.08803-2.08805 Ε for complex 4) and Ru-N pyrazine (M-N NHC ) (2.00471-2.00943Ε for complex 3, 2.03512 Ε for complex 4) bond distances are consistence with the reported crystallographic data [28],[32] and in both complexes ligands are nearly perpendicular to each other (88.77°) show octahedral geometry.
Figure 1: ORTEP view of optimized geometry of Ru(II)-NHC complex 3,30% probability) [H atoms and PF6 are removed for clarity]

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Figure 2: ORTEP view of optimized geometry of Ru(II)-NHC complex 4, 30% probability) [H atoms and PF6 are removed for clarity]

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Pro-apoptotic activity

Inhibition of cell proliferation was examined by incubating human cancer cell lines, HCT15 and Hep2 with different concentrations of complex 3 and 4, the cell proliferation was measured by the standard MTT assay. Complex 3 exhibited a clear and significant inhibition of cell proliferation against both HCT15 and Hep2 cell lines [Figure 3]a. Complex 3 was three to four fold more active than complex 4, which might be due to the presence of additional benzyl group that decreased the reactivity of the compound. Interestingly, the obtained IC 50 values of complex 3 was lower than other reported ruthenium complex, gold complex, and some platinum-based compounds against Hep2 cancer cell lines. [8] The supercoiled plasmid DNA used as substrate in order to determine the DNA-cleaving activity of the synthesized ruthenium complex 3. Results showed that two types of DNA scission observed in this system as nicked plasmid (form II), the linear DNA (form III), and the supercoiled plasmid (form I) [Figure 3]b. Therefore, it allows for easy recognition of both single- and double-strand DNA. It was found that 1-μM concentration of complex 3 had no activity whereas increasing the concentration of complex level, the form II of cleaved plasmid increased significantly. Interestingly, at a concentration of 20 μM of complex 3, almost 50% DNA cleavage was observed but at 100 μM concentration, the DNA remained in the lane unable to migrate within the 1% agarose gel. It might be due to the aggregation of DNA that coordinated with the Ru complex. It reveals that complex 3 efficiently nicked the supercoiled DNA and aggregate simultaneously.

DNA-binding propensity of the complexes to calf thymus (CT) DNA was studied using CD spectroscopy. The DNA conformational change induced by complex 3 was investigated. The samples contained CT DNA (50 μg.mL−1 ) in the presence or absence of complex 3 (10 μM) in 10 mM phosphate buffer (pH 7.2). The ionic strength was kept constant and the R value remained constant at 0.1 for all investigated compounds. The changes observed in the native right-handed B-form DNA on interaction with the complex 3 is as shown in [Figure 3]c. The right-handed B-DNA exhibits remarkable changes on its characteristic positive and negative bands at 272 and 248 nm, respectively, on interaction with of ruthenium complex. The complex induces a drastic decreased in intensity for both the positive (272 nm) and negative bands (248 nm). The decrease in positive band intensity indicates destabilization of base-stacking, and the decrease in the negative band intensity points to loss in right-handed helicity. [36] Inhibition of cell proliferation was started from 5 μM concentrations of complexes and gradually increased with increasing the complex concentration. The IC 50 values of complex 3 and 4 against Hep2 cells and HCT15 cells were listed in [Table 1]. Complex 3 seems to induce cell cycle arrest at the G0-G1 phase, as shown in [Figure 3]d (a, b, c). There were 2.81%, 13.54%, and 59.19% relative increases in cell number at the G0-G1 phase in HCT15 cell lines with complex 3 at a concentrations of 0 μM, 5 μM, and 20 μM, respectively, compared with control cultures. Apoptosis induction occurred in a dose-dependent manner following treatment. This induction of programmed cell death was targeted on DNA cleavage and aggregation, confirmed by DNA cleavage and CD spectroscopic study. Our results show that the exposure of complex 3 against HCT15 cells resulted in an improvement of apoptotic process verified from DNA binding/cleavage assay and the assessment of sub-G0/G1 cells by FACS analysis.
Figure 3: (a) Pro-apoptotic activity of complex 3 and 4 against human carcinoma cell lines. MTT assay represents the inhibition of cell proliferation of HCT15 (green and cyan color) and Hep2 (yellow and bluish color) cells after treatment with complex 3 (green and yellow) and complex 4 (Cyan and bluish). (b). Agarose gel electrophoresis image of supercoiled plasmid DNA after treatment with different concentrations (lane 1, 0 μM; lane 2, 1 μM; lane 3, 5 μM; lane 4, 10 μM; lane 5, 20 μM; lane 6, 50 μM; lane 7, 100 μM) of complex 3. (c) CD spectra indicating the conformational change of right-handed DNA (blue color) upon addition of complex 3 (red color) at a concentration of 20 μM. (a) Representative histogram plot of HCT15 cancer cells treated with complex 3 for 24 h. Control (0.1% DMSO) cultures, (b) 5 μM of complex 3 and (c) 20 μM of complex 3 indicating cell cycle arrest at the G0-G1 phase. Inside the histogram, the symbols indicate the different phases (M1, G0-G1 phase; M2, G1 phase; M3, S phase, and M4, G2/M phase) of cell cycle

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Table 1: MIC values (μM) against microbes and IC50 values (μM) against HCT15 and Hep2 cell lines

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Antimicrobial activity

The antibacterial and antifungal activities of complexes 3 and 4 were tested against both Gram-positive and Gram-negative bacteria and fungus. All tested bacteria and fungus were more susceptible to complex 3 than complex 4. The MIC values of complexes are listed in [Table 1]. To kill the fungus taking two fold more complex 3 concentrations than to inhibit the bacterial growth. The killing activity of complex 3 for P. aeruginosa was faster than C. albicans [Figure 4]. The killing activity was concentration dependent, and no cell growth was seen even after 8 and 16 μM concentration of complex 3 for P. aeruginosa and C. albicans, respectively. Further, the interaction of bacteria and fungus with complex 3 was observed using SEM images [Figure 5]. We are representing here only SEM images of Candida cells for better image quality among all obtained images. As seen in these images, the complex 3 effectively interact with the cell wall of Candida and made significant damage; finally, the cytoplasmic materials leached out to surrounding environments. This result suggests that complex 3 directly affecting the cell wall might be the primary cause for cell death. The obtained MIC values are also corroborating with the mechanism of cell wall target where less concentration was required for bacterial inhibition than fungal cell. The differences between the MIC values among bacteria and fungus might be due to the variations of their cell wall compositions. Bacteria have thinner peptidoglycan layer than fungal chitin layer or complex 3 have more affinity to peptidoglycan layer than fungal chitin layer. Thus, complex 3 targets to the cell wall of microbes and resulting slow down the growth rate. Moreover, we have also performed both antimicrobial and pro-apototic activities each one-month interval for 3 months using the same complexes those are stored at room temperature. It was observed that no variation in obtained results which revealed their minimum storage stability.
Figure 4: Growth kinetics of P. aeruginosa (closed circle) with 108 CFU/mL and C. albicans (open circle) with 107 CFU/mL in the presence of different concentrations of complex 3

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Figure 5: SEM micrographs of C. albicans treated with complex 3. Cells were treated with complex 3 (10 µM) (b) and control group was treated with 0.1% DMSO (a)

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Self-assembled nanoball structure

Most interesting observation happened when DMSO solubilized complex 3 and 4 were added to culture medium for antimicrobial or pro-apoptotic assay. Immediately, we observed that a colloidal suspension appeared when complexes were taken into aqueous phase. Then, 10 μL of colloidal suspension was placed on the glass cover slip as a drop-caste method and analyzed with SEM. The SEM images revealed their nanosphere structures in aqueous phase [Figure 6]. The diameter of nanospheres were analyzed during SEM analysis and found to be 311.9 ± 13.2 nm and 704.4 ± 21.7 nm of complex 3 and 4, respectively. The size of complex 4 was almost two-fold greater than that of complex 3. This might be due to the extra benzene moieties in ligand of complex 4. We have considered the nanosphere as nanoball due to their large sizes. It seems to be obvious that the nanoball is formed in aqueous phase due to their self-assembled nature. It is also documented that a hydrogen-bonding interaction occurred between the outward pyrazinyl 'N' and the NHC ring proton of complex 3 (N14···H−C45), which extends along with the b-axis to give a 1D infinite chain in acetonitrile solvent. [32] In this case, both the hydrophobic (PF6 salts are highly insoluble in water) and hydrogen-bonding interaction takes a major part for the formation of spherical structure may result from a closure of the 1D chain along two axes. Earlier, same types of nanostructures have been described from peptide self-assembled mechanism as hydrogen bonds and stacking of aromatic moieties. [37] Recently, a highly soluble, novel hydroxyl functionalized Cu (II) nanoball have been reported. [38] However, the development of self-assembled structures with specific shape and size around the metal centers is of great interest as many applications can be foreseen due to their self-assembly phenomenon. In the light of this finding, complex 3 and 4 needs to be explored further for structure-function activity of their unique nanoball shape and other applications in addition to drug development or drug delivery.
Figure 6: SEM micrographs of complex 3 and 4. Images were captured after addition of DMSO solubilized complex 3 (a) and complex 4 (b) after addition to culture medium

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 > Conclusion Top


In summary, we designed and synthesized two pyrazine functionalized Ru (II)-NHC complexes of methylimidazolylidene and methylbenzimidazolylidene. Both the complexes were evaluated for pro-apoptotic and antimicrobial activities, which revealed that Ru (II)-methylimidazolylidene complex was more active than benzimidazolylidene complex against cancer cell lines, Hep2 and HCT15. DNA binding and cleavage studies revealed a potential candidate of inducing cancer cell apoptosis. Simultaneously, complex 3 showed potential antimicrobial activities against human pathogens, S. epidermidis, P. aeruginosa and C. albicans by targeting their cell wall and DNA or plasmid inside the cell. Always, complex 3 was more active than complex 4. It might be due to their nanosize, which enhanced the higher penetrating ability. Thus, complex 3 may lead to the discovery of potent antitumor or antimicrobial agents.

 
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