Original Article 317
Novel Ru(III) Complexes with Some Benzothiazole Derivatives: Synthesis, Physicochemical and Pharmacological Investigations
Affiliations
Key words
▶ cytotoxicity ● ▶ ruthenium(III) complexes ● ▶ benzothiazole ● ▶ synthesis ●
A. Nikolova1, G. Momekov2, A. Bakalova1, K. Nikolova1, D. Ivanov1 1 2
Department of Chemistry, Faculty of Pharmacy, Medical University – Sofia, Sofia, Bulgaria Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University – Sofia, Sofia, Bulgaria
Abstract
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In this work we present 3 new complexes of Ruthenium (III) with a general formula HL[Ru(L)2Cl4], where L = benzothiazole, 2-methylbenzothiazole and 2-mercaptobenzothiazole. The syntheses were carried out in polar medium under argon. The compounds obtained were characterised by IR-, 1H-NMR- 13C-NMR-, UVVIS-spectroscopy and conductivity measurements. The ligands behaved as monodentate, bounding Ru(III) through the nitrogen atoms from the heterocycle. The cytotoxicity of the new complexes was tested against 2 human leukemic
Introduction
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received 07.02.2014 accepted 29.05.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382052 Published online: July 3, 2014 Drug Res 2015; 65: 317–322 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence A. Nikolova Department of Chemistry Faculty of Pharmacy Medical University – Sofia 2 Dunav Street 1000 Sofia Bulgaria Tel.: + 359/2/9236 595 Fax: + 359/2/9879 874 [email protected]
Since the discovery of Rosenberg et al. as that Cisplatin has ability to inhibit tumors [1] many different types of coordination compounds have been investigated like potential anticancer agents. Despite that, cisplatin and other platinum drugs have high antitumor as activity their clinical application is limited from several side effects [2]. In recent years, Ru(III) complexes have emerged as a new class of effective anticancer agents against tumors that proved to be resistant to all other chemotherapeutic drugs currently in clinical use. Furthermore, ruthenium complexes generally have less toxic side effects than platinum – based drugs [3]. Some ruthenium (III) complexes [ImH][trans-RuCl4(Im)2] (ICR), [IndH] [trans-RuCl4(Ind)2] (KP1019), and [ImH][transRuCl4(DMSO)Im] (NAMI-A) have been tested as drug candidates in the clinic [4–6]. In the search for novel ruthenium-based complexes with better pharmacological properties, the thiazole analogues of antitumor ICR and NAMI-A were synthesized [7]. Replacement of imidazole with less basic thiazole ligands results in kinetic properties of potential antitumor Ru(III) complexes [8, 9]. Although the molecular
cell lines (K-562 and KE-37), using the MTT-dye reduction assay. The Ru(III) coordination compound with 2-methylbenzothiazole displayed superior activity compared to the other novel complexes. Its IC50 values were comparable to that of the reference cytotoxic drug cisplatin. In general, the ligands displayed only marginal inhibitory effects on the human leukemic cell lines. Moreover, the ability of the complexes to trigger apoptosis was evaluated using a commercially available DNA-fragmentation ELISA kit and the obtained data indicated that their proapoptotic effects well correlate to the MTT-bioassay data.
mechanism of action of the ruthenium complexes is not completely investigated it is presumed that it is similar to that of cisplatin. The complexes of Ru(III) are often fairly inert to ligand substitution due to strong ligand-field stabilization. We expected that the complexes with a less basic ligands such as derivatives of the benzothiazole would have less value of the stability constants, higher rate of hydrolysis, and higher rate of binding to human serum albumin (HAS) and transferrin. We assume that the modified kinetic properties will have as result increased cytotoxicity of ruthenium complexes [3]. On the other hand, compounds containing the benzothiazole (bt) moiety constantly attract the interest of chemists and pharmacists due to their versatile biological activity and industrial applications. Most of them exhibit anticancer [10], antitumor [11], antimicrobial [12], antifungal [13], anti-inflammatory [14] and anti-allergic [15] activities. Furthermore, possessing 2 endocyclic heteroatoms with pronounced donor abilities, e. g., S and N, they are of significant interest in coordination chemistry, binding to the metal centres in a monodentate way or forming supramolecular arrangements [16] and [17].
Nikolova A et al. Ru(III) Complexes with Benzothiazoles … Drug Res 2015; 65: 317–322
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Authors
318 Original Article
Materials and Methods
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All chemicals were purchased from Sigma-Aldrich, reagent grade and were used without further purification. The synthesis of initial solutions of Ru(III) chloride and the metal complexes were carried out under argon atmosphere. The melting points were determined on an electro thermal apparatus (Büchi 535 in an open capillary tube and are not corrected. The chemical analyses were performed on a Euro EA 3000 – Single, Euro Vector SpA (for C, H, N, S and Cl). The content of Ru(III) was determined spectrophotometrically [18]. The IR spectra in the region 4 500–400 cm − 1 (KI palettes) were recorded on a Thermo scientific Nicolet in the region 400–150 cm − 1 (CsI palettes) on a Bruker IFS 113v. Only significant bands are cited in the text. The 1 H NMR and 13C NMR spectra were registered on a Bruker 250 (250 MHz) spectrometer in DMSO-d6. The chemical shifts were expressed as δ values in ppm against TMS as an internal standard. The UV-VIS spectra of 10 − 4 mol/l solutions in DMSO were obtained on a Hewlett Packard-8452 spectrophotometer. The molar conductivity of 10 − 3 mol/l solutions in DMSO was measured by a Metrohm conductomether 660 (cell constant – 0.82 − 1). The cells lines used in this study, namely KE-37 and K-562 were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig, Germany). The cells were grown as suspension type cultures in controlled environment – cell culture flasks at 37 °C in an incubator ̔BB 16-Function Line̛ Heraeus (Kendro, Hanau, Germany) with humidified atmosphere and 5 % CO2. The growth medium was 90 % RPMI1640 supplemented with 10 % FBS and L-glutamine. Cells were maintained in log phase by supplementation with fresh medium, 2 or 3 times a week. The cytotoxicity of the compounds was assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye reduction assay as described by Mossman [19] with some modifications [20].
General procedure for synthesis of the complexes (1–3) The starting solution of RuCl3 was prepared according to a method given in literature [21]. The 3 new complexes of Ru (III) with benzothiazole (L1), 2-methylbenzothiazole (L2) and 2-mercaptobenzothiazole (L3) are synthesized by a modified procedure in regard to the method reported by Keppler [21]: 95 % ethanol (20 ml) was added to the corresponding ligand (L1, L2 or L3, 15 284 mmol) and the solution was stirred for 10 min at room temperature. To this solution of the ligand, starting solution of RuCl3 (8 ml, 2 549 mmol) was added. The molar ratio of Ru (III): L is 1:6. The reaction mixture was stirred at room temperature and evaporated to half of the volume. The initial brown coloured solution slowly changed. The solid products were formed after a week. The residues were filtered off, washed with several small amounts of distilled water and ethanol. The crystals are dried in a desiccator over P2O5. The new complexes exhibited low solubility in water, ethanol, methanol (M < 1.10–3) and were readily soluble in DMSO.
Benzothiazoliumbis(bisbenzothiazole)tetrachlororutenate(III) (1): Light brown powder: 78 % yield, m. p. 169–171 °C. IR (CsI, ν,cm − 1): 311 (Ru-N), IR (nujol, ν,cm − 1): 924 and 1 273 (CNS),
1 480 (C = N), 3 438 (NH). 1H-NMR (DMSO-d6, Me4Si, δ, ppm): 9.92 (s,1H, N = CH), 8.48 (d, 1H, J = 8.1Hz, H-7), 8.38 (d, 1H, J = 8.8Hz, H-4), 7.58 (t, 1H, J = .7Hz, H-5), 7.46 (t, 1H, J = 8.7Hz, H-6), 6.63 (bs,1H, HN). 13C-NMR (DMSO-d6, Me4Si, δ, ppm): 161.8 (C-2), 159.1 (C-9), 137.6 (C-8), 128.0 (C-5), 127.2 (C-6), 125.3 (C-4), 125.3 (C-7). UV-Vis in DMSO λmax/nm (ε/dm3 M − 1 cm − 1): 218 (11629), 254 (6931), 280 (2231), 406 (very broad band 701). Molar conductance, ΛM (DMSO): 42.35 Ω − 1cm2mol − 1. Anal. calcd. for C21H16N3S3Cl4Ru (649.44 g/mol): C, 38.80; H, 2.46; N, 6.47; S, 14.80; Cl, 21.83; Ru, 15.56. Found: C, 38.43; H, 2.31; N, 6.02; S, 14.73; Cl, 22.12; Ru, 14.79 %.
2-methylbenzothiazoliumbis(bis-2-methylbenzothiazole) tetrachlororutenate(III) (2): Dark brown powder: 66 % yield, m. p. 216–218 °C. IR (CsI, ν,cm − 1) 293 (Ru-N). IR (nujol, ν,cm − 1): 928 and 1 277 (CNS), 1 478 (C = N), 3 445 (NH). 1H-NMR (DMSOd6, Me4Si, δ, ppm): 8.12, (d, 1H, J = 8.3Hz, H-7), 8.06 (d, 1H, J = 8.7Hz, H-4), 7.51(t, 1H, J = 8.8Hz, H-5), 7.42 (t, 1H, J = 8.8Hz, H-6), 6.63 (bs, 1H, HN), 3.01 (s, 3H, CH3). 13C-NMR (DMSO-d6, Me4Si, δ, ppm): 172.8 (C-2), 159.2 (C-9), 139.5 (C-8), 127.8 (C-5), 126.6 (C-6), 123.3 (C-4), 123.2 (C-7), 27.1 (C-10). UV-Vis in DMSO λmax/nm (ε/M − 1 cm − 1): 219 (11500), 262 (6800), 282 (2340), 410 (very broad band 680). Molar conductance, ΛM (DMSO): 40.05 Ω − 1 cm2 mol − 1; Anal. calcd. for C24H22N3S3Cl4Ru (691.44 g/mol): C, 41.65; H, 3.18; N, 6.07; S, 13.91; Cl, 20.51; Ru, 14.62. Found: C, 41.95; H, 3.25; N, 6.2; S, 13.56; Cl, 20.73; Ru, 14.95 %. 2-mercaptobenzothiazoliumbis(bis-2-mercaptobenzothiazole) tetrachlororutenate(III) (3): Dark green powder: 72 % yield, m. p. 245–247 °C. IR (CsI, ν,cm − 1): 282 (Ru-N). IR (nujol, ν,cm − 1): 960 and 1 280 (CNS), 1 485 (C = N), 3 438 (NH). 1H-NMR (DMSO-d6, Me4Si, δ, ppm): 8.08 (d, 1H, J = 8.2Hz, H-7), 7.93 (d, 1H, J = 8.7Hz, H-4), 7.58 (t, 1H, J = 8.8Hz, H-5), 7.26 (t, 1H, J = 8.7Hz, H-6), 13.76 (bs,1H, HN), 3.72(bs,1H, SH). 13C-NMR (DMSO-d6, Me4Si, δ, ppm): 196.1 (C-2), 147.3 (C-9), 133.5 (C-8), 129.2 (C-5), 126.0 (C-6) 123.2 (C-7), 117.1 (C-4). UV-Vis in DMSO λmax/nm (ε/M − 1 cm − 1): 208 (5150), 240 (6320), 286 (9391), 418 (very broad band 902). Molar conductance, ΛM (DMSO): 38.06 Ω − 1 cm2 mol − 1. Anal. calcd. for C21H16N3S6Cl4Ru (745.62 g/mol ): C, 33.80; H, 2.15; N, 5.63; S, 25.80; Cl, 19.02; Ru,13.56. Found: C, 34.20; H, 2.23; N, 5.71; S, 26.02; Cl, 18.48; Ru, 13.12 %.
Cell studies In brief, exponentially growing cells were seeded in 96-well microplates (100 μl/well at a density of 3.5 × 105 cell/ml for the adherent and 1 × 105 cells/ml for the suspension cell lines) and allowed to grow for 24 h prior the exposure to the studied compounds. Stock solutions of the ruthenium complexes, the ligands and the reference anticancer drug (cisplatin) were freshly prepared in DMSO and then diluted with RPMI-1640 growth medium. At the final dilutions the solvent concentration never exceeded 0.5 %. Cells were exposed to the tested agents for 72 h, whereby for each concentration a set of 8 separate wells were used, and each MTT-bioassay was run in triplicate. After incubation with the tested compounds MTT solution (10 mg/ml in PBS) aliquots were added to each well. The plates were further incubated for 4 h at 37 °C and the formazan crystals formed were dissolved by adding 110 μl of 5 % HCOOH in 2-propanol. Absorption of the samples was measured using DTX-880 multimode microplate reader. The survival fractions were calculated as percentage of the untreated control, set as 100 % viable. The experimental data were processed
Nikolova A et al. Ru(III) Complexes with Benzothiazoles … Drug Res 2015; 65: 317–322
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The aim of this study was to synthesize and characterize new ruthenium compounds and to test their in vitro cytotoxicity.
using GraphPad Prizm software and were fitted to sigmoidal concentration/response curves, using non-linear regression. The DNA fragmentation as a quantitative merit of the ability of tested compounds to induce apoptosis was detected using a commercially available “Cell Death Detection” ELISA kit (Roche Applied Science), according to the manufacturer’s instructions. Each test was run in triplicate.
Results and Discussion
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Chemistry The new 3 complexes with general formula HL[Ru(L)2Cl4] where L1 = benzothiazole, L2 = 2-methylbenzothiazole and L3 = 2-mer▶ Fig. 1. captobenzothiazole were synthesized, as shown in ● These reactions were performed under argon atmosphere. As a distinct from the described procedures till now, where the ratio of Ru (III): ligand is from 1:10 to 1:19 [7, 21, 29], the new complexes are synthesized in ratio 1:6. Furthermore, instead of a suspension of the ligand in conc. HCl (6 N, 8 N) our ligands were dissolved in ethanol. We think that lower concentration of hydrochloric acid facilitates the substitution of the chloride ions with benzothiazole moiety and because of that the excess of ligand is smaller. The evaporation half of the volume of the solution is in order to increase the yield. Unlike the red brown initial solution of Ru(III) chloride the complex (1) is light brown, (2) is dark brown and (3) is dark green. The schematic structures of ▶ Fig. 2. The comthe complexes obtained are presented in ●
pounds are soluble in DMSO, partially soluble in alcoholic solution and slightly soluble in water. The data from the elemental analysis for the new complexes and the calculated data are in a good agreement. The coordination with Ru(III) ion led to the appearance of new IR-bands in the spectra of the complexes in comparison to the spectra of the free ligands. A new weak broad band at 3 440 cm − 1 due to ν(NH) appeared which was an evidence that the nitrogen atom in the complexes is protonated. In each of the spectra of the free ligands in the range 1 450–1 590 cm − 1, 2 bands that are characteristic for stretching vibrations ν(C = C) and ν(C = N) were observed. In the spectra of the complexes only one peak was observed, which is with lower intensity in comparison to the free ligands. Moreover, in the spectra of each of the free ligands, 2 peaks were observed around 1 010 m and 1 330 s cm − 1, characteristic to stretching vibrations of ν(CNS). In the spectra of the complexes they have shifted with more than 50 cm − 1 to lower frequencies. The formation of the bond between the ruthenium ion and the ligands is confirmed by the appearance of new band in the far IR-spectra of each complex, which can be assigned to ν(Ru-N) in the range 237–260 cm − 1 when it was from the heterocyclic ring. Also in the spectra of the complexes were seen many changes in the range 400–600 cm − 1. Bands in this range are characteristic of ν(C-S) and δ(C-N). These spectral changes probably were due to the formation of a bond between the ruthenium ion and the ligands [22–28]. Certainly, this fact is confirmed by the appearance of a new band in the far IR-spectra
Fig. 1 Synthetic scheme of Ruthenium complexes.
Fig. 2 Schematic structures of the complexes obtained: [1-] benzothiazoliumbis(bisbenzothiazole) tetrachlororutenate(III); [2-] 2-methylbenzothiazoliumbis(bis-2-methylbenzothiazole)tetrachlororutenate(III); [3-] 2-mercaptobenzothiazoliumbis(bis-2-mercaptobenzothiazole)tetrachlororutenate(III).
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320 Original Article
Table 1 Cytotoxic effects of the tested complexes, the corresponding ligands and the reference cytotoxic drug cisplatin after 72 h treatment (MTT-dye reduction assay). Compounds Complex 1 Complex 2 Complex 3 Ligand 1 Ligand 2 Ligand 3 Cisplatin
IC50 (μM) KE-37
K-562
22.34 ± 0.98* 7.74 ± 2.50* 20.97 ± 3.71* > 200 > 200 > 200 4.43 ± 1.41
32.80 ± 5.09* 16.21 ± 2.33* 29.40 ± 4.29* > 200 > 200 > 200 8.61 ± 2.41
Values were the means of 8 replicates ± SD, run in triplicate. The asterisk (*) denotes statistical significance vs. cisplatin (at p ≤ 0.05 set as significance level; paired t-test)
The molar conductivities of freshly prepared 10 − 3 M solutions in DMSO were measured at room temperature. The values of 42.35 ΛM Ω − 1 mol − 1 cm2 for 1, 40.05 for 2 and 37.06 for 3 indicated the ionic nature of these complexes [41]. Based on the results of the physicochemical studies the following structures for Ru (III) ▶ Fig. 2). complexes have been proposed (●
Cell studies The cytotoxicity of the newly described ruthenium(III) coordination compounds and the corresponding ligands were assessed against 2 human leukemic cell lines, namely KE-37 (T-cell leukemia) and K-562 (chronic myeloid leukemia), using the MTT-dye reduction assay. Following a 72 h exposure period the tested complex compounds displayed prominent cytotoxic effects upon the used cell lines, in a concentration-dependent manner, causing 50 % inhibition of the cellular viability at low micromolar concentrations and almost total eradication of viable cells at concentrations exceeding 50 μM. In both cell lines the Ru(III) complex 2 with 2-methylbenzothiazole ligands proved to significantly outclass both other coordination compounds, actually its relative cytotoxicity was comparable to that of the reference ▶ Table 1). Complexes 1 and 3 displayed cytotoxic drug cisplatin (● similar activity. In general the tested complexes were more active against the lymphoid KE-37 cells as compared to the myeloid K-562 cells. Throughout the MTT-bioassay the ligands displayed only marginal inhibitory effects against the human ▶ Fig. 3. leukemic cell lines. The results are shown in ● The MTT-data were corroborated by another concentrationresponse experiment using a commercially available “Cell death detection ELISA” kit. Following a 24 h treatment period, the novel complexes evoked concentration-dependent oligonucleosomal DNA fragmentation in K-562 cells which indicates that their cytotoxicity is at least partially mediated by induction of cell death through apoptosis. The apoptogenic effects of the tested agents decreased in the following order: cisplatin > com▶ Fig. 4. plex 2 > complexes 1, 3, as they are shown in ●
Conclusion
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3 new Ru(III) complexes with benzothiazole and some its derivatives were synthesized. The stoichiometry and physicochemical studies reveal the formation of monometallic complexes of Ru (III). The ligands coordinate to the metal ion through the N-atom and act as monodentate once. Octahedral stereochemistry around the metal ion is proposed.
Nikolova A et al. Ru(III) Complexes with Benzothiazoles … Drug Res 2015; 65: 317–322
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of each complex, which can be assigned to ν(Ru-N) in 237– 260 cm − 1 range when it was from the heterocyclic ring [29]. 1 H-NMR spectra for complexes 1, 2 and 3 are displayed between 10 and 2 ppm. In general, all complexes showed very similar spectra. The signals of the protons in the spectra of the complexes downfield shifted compared to the corresponding signals of the protons in uncoordinated ligands, as expected. In particular, the signal of the proton from the thiazole ring is shifted from 9.42 ppm in the spectra of free ligand to 9.92 ppm in the complex 1. In all complexes the signal for the most downfield shifted proton H-4, which is the closest proton to the metal ion, and expected to be most affected by its paramagnetic influence. Notably, the signals of H-4 were shifted from 8.11 ppm in the spectra of the free ligand to 8.46 ppm in the spectra of complex 1, from 7.73 ppm to 8.06 ppm in complex 2 and from 7.61 ppm to 7.93 ppm in the spectra of complex 3. The differences between the chemical shifts of the protons H-4 of the ligands and those of the corresponding complexes are noticeable at 0.35, 0.33 and 0.32 ppm. The other protons are less influenced by the complex formation as the signals of H-7 protons in the spectra of all complexes downfield shifted with 0.2 ppm and the signals of H-5 and H-6 protons are downfield shifted only with 0.2 ppm. The biggest displacement for the signal of H-4 was due to the coordination of the ligand through the nitrogen atom of the heterocycle. In 13C-NMR spectra of the complexes downfield shift with 6 ppm of the signals for carbon atoms C-2 and C-9 was observed. C-2 showed absorptions at 155.8 ppm for the free benzothiazole and 161.8 ppm for complex 1, 166.6 ppm for free 2-methylbenzothiazole and 172.8 ppm for complex 2, 189.8 ppm for free 2-mercaptobenzothiazole and 196.1 ppm for complex 3. C-9 exhibited absorptions at 153.1 ppm for the free benzothiazole and 159.1 ppm for complex 1, 153.4 ppm for free 2-methylbenzothiazole and 159.2 ppm for complex 2, 141.2 ppm for free 2-mercaptobenzothiazole and 147.3 ppm for complex 3. The signal for the carbon atom C-8, which is connected to the sulphur is shifted to lower field also, but only with 4 ppm. In particular the signals in the spectra of the free ligands are 133.6 ppm, 135.6 ppm and 129.4 ppm. In the spectra of the complexes 1, 2 and 3 the signals appear respectively at 137.6, 139.5 and 133.5 ppm. The signals for the other carbon atoms are influenced insignificantly. The large displacement of the signals for C-2 and C-9 that are directly related to the nitrogen atom was due to monodentate linking of the ligand through the nitrogen atom [30–37]. Absorption spectra of all complexes HL[Ru(L)2Cl4] in DMSO solutions (10 − 4 mol.dm − 3) were recorded. For comparison, the electronic spectra of the uncoordinated ligands (L1, L2 and L3 were obtained at the same experimental conditions. The electronic spectra of the free ligands exhibit absorption bands around 220 nm and 280 nm, which are corresponding to π–π* transitions of the benzenoid system of the benzothiazole moiety. The absorption band around 250 nm is due to the π–π* transitions of the thiazole moiety. These bands were found in the spectra of all complexes with a small shift. This may be attributed to the donation of lone pairs of electrons of the nitrogen atom of the ligand to the metal ion. Octahedral low spin d5 complexes have the 2T2 g ground state corresponding to the t52 g electronic state. In fact, the electronic spectra of all complexes show new intense broad absorption bands in the visible region (λmax = 406 nm for complex 1, λmax = 410 nm for complex 2 and λmax = 425 nm for complex 3, in DMSO) attributed to ligand – metal charge transfer which was influenced by the formation of a chemical bond between the metal ion and the corresponding ligand [38–40].
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Fig. 4 Apoptotic oligonucleosomal DNA fragmentation in K-562 cells after 24 h exposure to the tested ruthenium(III) complexes 1–3, and the reference cytotoxic drug cisplatin (“Cell death detection ELISA” kit).
Our data indicate that the presented series of Ru(III) complexes with heterocyclic ligands exert strong inhibitory effects against human leukemic cell lines. The established cytotoxicity at low micromolar concentrations gives us a reason to conclude that the obtained compounds necessitate further, more detailed pharmacological evaluation.
Acknowledgment
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We are grateful to the Faculty of Pharmacy, Medical University of Sofia, Bulgaria for the financial support of this research.
Conflict of Interest
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The authors declare that there are no financial/commercial conflicts of interest.
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Fig. 3 Concentration-response curves of the tested ruthenium(III) complexes 1–3, the corresponding ligands and the reference cytotoxic drug cisplatin after 72 h treatment (MTT-dye reduction assay).
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32 Marchesi E, Marchi A, Marvelli L et al. Rhenium(III) and technetium(III) complexes with 2-mercapto-1,3-azole ligands and X-ray crystal structures. Inorg Chim Acta 2005; 358: 352–362 33 Yousif E, Farina Y, Kasar K et al. Complexes of 2-thioacetic acid benzothiazole with some metal ions. Am J Applied Sci 2009; 6: 582–585 34 Marjani K, Mousavi M, Hughes DL. Synthesis and crystal structure determination of copper(II) and iron(III) complexes of 2-(2-pyridyl) benzothiazole. Trans Met Chem 2009; 34: 85–89 35 Cesar ML, Cox O, Muir MM et al. Cytotoxic anionic tribromo platinum(II) complexes containing benzothiazole and benzoxazole donors: synthesis, characterization, and structure-activity correlation. Inorg Chim Acta 1998; 271: 137–144 36 Racane L, Mihalic Z, Ceric H et al. Synthesis, structure and tautomerism of two benzothiazolyl azo derivatives of 2-naphthol: A crystallographic, NMR and computational study. Dye Pigment 2013; 96: 672–678 37 Machura B, Wolff M, Benoist E et al. Tricarbonyl rhenium(I) complex of benzothiazole – Synthesis, spectroscopic characterization, X-ray crystal structure and DFT calculations. J Organomet Chem 2013; 724: 82–87 38 Etaiw SE, Farag RS, El-Atrash AM et al. Spectrophotometric studies of some thiazole and benzothiazole derivatives. Spectrochim Acta A 1992; 48: 025–1026 39 Ibrahim AMA, Etaiw SEH. Paramagnetic charge transfer complexes: new molecular composites via intercalation of thiazole and benzothiazole derivatives within the cavities of the 3D host polymers (Me3E)3Fe(CN)6]∞; E – Sn or Pb. Polyhedron 1997; 16: 1585–1594 40 Neelakantan MA, Marriappan SS, Dharmaraja J et al. Spectral, XRD, SEM and biological activities of transition metal complexes of polydentate ligands containing thiazole moiety. Spectrochim Acta A 2008; 71: 628–635 41 Geary WJ. The use of conductivity measurements in organic solvents for the characterisation of coordination compounds. Coord Chem Rev 1971; 7: 81–122
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322 Original Article
Novel Ru(III) Complexes with Some Benzothiazole Derivatives: Synthesis, Physicochemical and Pharmacological Investigations
Affiliations
Key words
▶ cytotoxicity ● ▶ ruthenium(III) complexes ● ▶ benzothiazole ● ▶ synthesis ●
A. Nikolova1, G. Momekov2, A. Bakalova1, K. Nikolova1, D. Ivanov1 1 2
Department of Chemistry, Faculty of Pharmacy, Medical University – Sofia, Sofia, Bulgaria Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University – Sofia, Sofia, Bulgaria
Abstract
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In this work we present 3 new complexes of Ruthenium (III) with a general formula HL[Ru(L)2Cl4], where L = benzothiazole, 2-methylbenzothiazole and 2-mercaptobenzothiazole. The syntheses were carried out in polar medium under argon. The compounds obtained were characterised by IR-, 1H-NMR- 13C-NMR-, UVVIS-spectroscopy and conductivity measurements. The ligands behaved as monodentate, bounding Ru(III) through the nitrogen atoms from the heterocycle. The cytotoxicity of the new complexes was tested against 2 human leukemic
Introduction
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received 07.02.2014 accepted 29.05.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382052 Published online: July 3, 2014 Drug Res 2015; 65: 317–322 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence A. Nikolova Department of Chemistry Faculty of Pharmacy Medical University – Sofia 2 Dunav Street 1000 Sofia Bulgaria Tel.: + 359/2/9236 595 Fax: + 359/2/9879 874 [email protected]
Since the discovery of Rosenberg et al. as that Cisplatin has ability to inhibit tumors [1] many different types of coordination compounds have been investigated like potential anticancer agents. Despite that, cisplatin and other platinum drugs have high antitumor as activity their clinical application is limited from several side effects [2]. In recent years, Ru(III) complexes have emerged as a new class of effective anticancer agents against tumors that proved to be resistant to all other chemotherapeutic drugs currently in clinical use. Furthermore, ruthenium complexes generally have less toxic side effects than platinum – based drugs [3]. Some ruthenium (III) complexes [ImH][trans-RuCl4(Im)2] (ICR), [IndH] [trans-RuCl4(Ind)2] (KP1019), and [ImH][transRuCl4(DMSO)Im] (NAMI-A) have been tested as drug candidates in the clinic [4–6]. In the search for novel ruthenium-based complexes with better pharmacological properties, the thiazole analogues of antitumor ICR and NAMI-A were synthesized [7]. Replacement of imidazole with less basic thiazole ligands results in kinetic properties of potential antitumor Ru(III) complexes [8, 9]. Although the molecular
cell lines (K-562 and KE-37), using the MTT-dye reduction assay. The Ru(III) coordination compound with 2-methylbenzothiazole displayed superior activity compared to the other novel complexes. Its IC50 values were comparable to that of the reference cytotoxic drug cisplatin. In general, the ligands displayed only marginal inhibitory effects on the human leukemic cell lines. Moreover, the ability of the complexes to trigger apoptosis was evaluated using a commercially available DNA-fragmentation ELISA kit and the obtained data indicated that their proapoptotic effects well correlate to the MTT-bioassay data.
mechanism of action of the ruthenium complexes is not completely investigated it is presumed that it is similar to that of cisplatin. The complexes of Ru(III) are often fairly inert to ligand substitution due to strong ligand-field stabilization. We expected that the complexes with a less basic ligands such as derivatives of the benzothiazole would have less value of the stability constants, higher rate of hydrolysis, and higher rate of binding to human serum albumin (HAS) and transferrin. We assume that the modified kinetic properties will have as result increased cytotoxicity of ruthenium complexes [3]. On the other hand, compounds containing the benzothiazole (bt) moiety constantly attract the interest of chemists and pharmacists due to their versatile biological activity and industrial applications. Most of them exhibit anticancer [10], antitumor [11], antimicrobial [12], antifungal [13], anti-inflammatory [14] and anti-allergic [15] activities. Furthermore, possessing 2 endocyclic heteroatoms with pronounced donor abilities, e. g., S and N, they are of significant interest in coordination chemistry, binding to the metal centres in a monodentate way or forming supramolecular arrangements [16] and [17].
Nikolova A et al. Ru(III) Complexes with Benzothiazoles … Drug Res 2015; 65: 317–322
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Authors
318 Original Article
Materials and Methods
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All chemicals were purchased from Sigma-Aldrich, reagent grade and were used without further purification. The synthesis of initial solutions of Ru(III) chloride and the metal complexes were carried out under argon atmosphere. The melting points were determined on an electro thermal apparatus (Büchi 535 in an open capillary tube and are not corrected. The chemical analyses were performed on a Euro EA 3000 – Single, Euro Vector SpA (for C, H, N, S and Cl). The content of Ru(III) was determined spectrophotometrically [18]. The IR spectra in the region 4 500–400 cm − 1 (KI palettes) were recorded on a Thermo scientific Nicolet in the region 400–150 cm − 1 (CsI palettes) on a Bruker IFS 113v. Only significant bands are cited in the text. The 1 H NMR and 13C NMR spectra were registered on a Bruker 250 (250 MHz) spectrometer in DMSO-d6. The chemical shifts were expressed as δ values in ppm against TMS as an internal standard. The UV-VIS spectra of 10 − 4 mol/l solutions in DMSO were obtained on a Hewlett Packard-8452 spectrophotometer. The molar conductivity of 10 − 3 mol/l solutions in DMSO was measured by a Metrohm conductomether 660 (cell constant – 0.82 − 1). The cells lines used in this study, namely KE-37 and K-562 were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig, Germany). The cells were grown as suspension type cultures in controlled environment – cell culture flasks at 37 °C in an incubator ̔BB 16-Function Line̛ Heraeus (Kendro, Hanau, Germany) with humidified atmosphere and 5 % CO2. The growth medium was 90 % RPMI1640 supplemented with 10 % FBS and L-glutamine. Cells were maintained in log phase by supplementation with fresh medium, 2 or 3 times a week. The cytotoxicity of the compounds was assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye reduction assay as described by Mossman [19] with some modifications [20].
General procedure for synthesis of the complexes (1–3) The starting solution of RuCl3 was prepared according to a method given in literature [21]. The 3 new complexes of Ru (III) with benzothiazole (L1), 2-methylbenzothiazole (L2) and 2-mercaptobenzothiazole (L3) are synthesized by a modified procedure in regard to the method reported by Keppler [21]: 95 % ethanol (20 ml) was added to the corresponding ligand (L1, L2 or L3, 15 284 mmol) and the solution was stirred for 10 min at room temperature. To this solution of the ligand, starting solution of RuCl3 (8 ml, 2 549 mmol) was added. The molar ratio of Ru (III): L is 1:6. The reaction mixture was stirred at room temperature and evaporated to half of the volume. The initial brown coloured solution slowly changed. The solid products were formed after a week. The residues were filtered off, washed with several small amounts of distilled water and ethanol. The crystals are dried in a desiccator over P2O5. The new complexes exhibited low solubility in water, ethanol, methanol (M < 1.10–3) and were readily soluble in DMSO.
Benzothiazoliumbis(bisbenzothiazole)tetrachlororutenate(III) (1): Light brown powder: 78 % yield, m. p. 169–171 °C. IR (CsI, ν,cm − 1): 311 (Ru-N), IR (nujol, ν,cm − 1): 924 and 1 273 (CNS),
1 480 (C = N), 3 438 (NH). 1H-NMR (DMSO-d6, Me4Si, δ, ppm): 9.92 (s,1H, N = CH), 8.48 (d, 1H, J = 8.1Hz, H-7), 8.38 (d, 1H, J = 8.8Hz, H-4), 7.58 (t, 1H, J = .7Hz, H-5), 7.46 (t, 1H, J = 8.7Hz, H-6), 6.63 (bs,1H, HN). 13C-NMR (DMSO-d6, Me4Si, δ, ppm): 161.8 (C-2), 159.1 (C-9), 137.6 (C-8), 128.0 (C-5), 127.2 (C-6), 125.3 (C-4), 125.3 (C-7). UV-Vis in DMSO λmax/nm (ε/dm3 M − 1 cm − 1): 218 (11629), 254 (6931), 280 (2231), 406 (very broad band 701). Molar conductance, ΛM (DMSO): 42.35 Ω − 1cm2mol − 1. Anal. calcd. for C21H16N3S3Cl4Ru (649.44 g/mol): C, 38.80; H, 2.46; N, 6.47; S, 14.80; Cl, 21.83; Ru, 15.56. Found: C, 38.43; H, 2.31; N, 6.02; S, 14.73; Cl, 22.12; Ru, 14.79 %.
2-methylbenzothiazoliumbis(bis-2-methylbenzothiazole) tetrachlororutenate(III) (2): Dark brown powder: 66 % yield, m. p. 216–218 °C. IR (CsI, ν,cm − 1) 293 (Ru-N). IR (nujol, ν,cm − 1): 928 and 1 277 (CNS), 1 478 (C = N), 3 445 (NH). 1H-NMR (DMSOd6, Me4Si, δ, ppm): 8.12, (d, 1H, J = 8.3Hz, H-7), 8.06 (d, 1H, J = 8.7Hz, H-4), 7.51(t, 1H, J = 8.8Hz, H-5), 7.42 (t, 1H, J = 8.8Hz, H-6), 6.63 (bs, 1H, HN), 3.01 (s, 3H, CH3). 13C-NMR (DMSO-d6, Me4Si, δ, ppm): 172.8 (C-2), 159.2 (C-9), 139.5 (C-8), 127.8 (C-5), 126.6 (C-6), 123.3 (C-4), 123.2 (C-7), 27.1 (C-10). UV-Vis in DMSO λmax/nm (ε/M − 1 cm − 1): 219 (11500), 262 (6800), 282 (2340), 410 (very broad band 680). Molar conductance, ΛM (DMSO): 40.05 Ω − 1 cm2 mol − 1; Anal. calcd. for C24H22N3S3Cl4Ru (691.44 g/mol): C, 41.65; H, 3.18; N, 6.07; S, 13.91; Cl, 20.51; Ru, 14.62. Found: C, 41.95; H, 3.25; N, 6.2; S, 13.56; Cl, 20.73; Ru, 14.95 %. 2-mercaptobenzothiazoliumbis(bis-2-mercaptobenzothiazole) tetrachlororutenate(III) (3): Dark green powder: 72 % yield, m. p. 245–247 °C. IR (CsI, ν,cm − 1): 282 (Ru-N). IR (nujol, ν,cm − 1): 960 and 1 280 (CNS), 1 485 (C = N), 3 438 (NH). 1H-NMR (DMSO-d6, Me4Si, δ, ppm): 8.08 (d, 1H, J = 8.2Hz, H-7), 7.93 (d, 1H, J = 8.7Hz, H-4), 7.58 (t, 1H, J = 8.8Hz, H-5), 7.26 (t, 1H, J = 8.7Hz, H-6), 13.76 (bs,1H, HN), 3.72(bs,1H, SH). 13C-NMR (DMSO-d6, Me4Si, δ, ppm): 196.1 (C-2), 147.3 (C-9), 133.5 (C-8), 129.2 (C-5), 126.0 (C-6) 123.2 (C-7), 117.1 (C-4). UV-Vis in DMSO λmax/nm (ε/M − 1 cm − 1): 208 (5150), 240 (6320), 286 (9391), 418 (very broad band 902). Molar conductance, ΛM (DMSO): 38.06 Ω − 1 cm2 mol − 1. Anal. calcd. for C21H16N3S6Cl4Ru (745.62 g/mol ): C, 33.80; H, 2.15; N, 5.63; S, 25.80; Cl, 19.02; Ru,13.56. Found: C, 34.20; H, 2.23; N, 5.71; S, 26.02; Cl, 18.48; Ru, 13.12 %.
Cell studies In brief, exponentially growing cells were seeded in 96-well microplates (100 μl/well at a density of 3.5 × 105 cell/ml for the adherent and 1 × 105 cells/ml for the suspension cell lines) and allowed to grow for 24 h prior the exposure to the studied compounds. Stock solutions of the ruthenium complexes, the ligands and the reference anticancer drug (cisplatin) were freshly prepared in DMSO and then diluted with RPMI-1640 growth medium. At the final dilutions the solvent concentration never exceeded 0.5 %. Cells were exposed to the tested agents for 72 h, whereby for each concentration a set of 8 separate wells were used, and each MTT-bioassay was run in triplicate. After incubation with the tested compounds MTT solution (10 mg/ml in PBS) aliquots were added to each well. The plates were further incubated for 4 h at 37 °C and the formazan crystals formed were dissolved by adding 110 μl of 5 % HCOOH in 2-propanol. Absorption of the samples was measured using DTX-880 multimode microplate reader. The survival fractions were calculated as percentage of the untreated control, set as 100 % viable. The experimental data were processed
Nikolova A et al. Ru(III) Complexes with Benzothiazoles … Drug Res 2015; 65: 317–322
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The aim of this study was to synthesize and characterize new ruthenium compounds and to test their in vitro cytotoxicity.
using GraphPad Prizm software and were fitted to sigmoidal concentration/response curves, using non-linear regression. The DNA fragmentation as a quantitative merit of the ability of tested compounds to induce apoptosis was detected using a commercially available “Cell Death Detection” ELISA kit (Roche Applied Science), according to the manufacturer’s instructions. Each test was run in triplicate.
Results and Discussion
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Chemistry The new 3 complexes with general formula HL[Ru(L)2Cl4] where L1 = benzothiazole, L2 = 2-methylbenzothiazole and L3 = 2-mer▶ Fig. 1. captobenzothiazole were synthesized, as shown in ● These reactions were performed under argon atmosphere. As a distinct from the described procedures till now, where the ratio of Ru (III): ligand is from 1:10 to 1:19 [7, 21, 29], the new complexes are synthesized in ratio 1:6. Furthermore, instead of a suspension of the ligand in conc. HCl (6 N, 8 N) our ligands were dissolved in ethanol. We think that lower concentration of hydrochloric acid facilitates the substitution of the chloride ions with benzothiazole moiety and because of that the excess of ligand is smaller. The evaporation half of the volume of the solution is in order to increase the yield. Unlike the red brown initial solution of Ru(III) chloride the complex (1) is light brown, (2) is dark brown and (3) is dark green. The schematic structures of ▶ Fig. 2. The comthe complexes obtained are presented in ●
pounds are soluble in DMSO, partially soluble in alcoholic solution and slightly soluble in water. The data from the elemental analysis for the new complexes and the calculated data are in a good agreement. The coordination with Ru(III) ion led to the appearance of new IR-bands in the spectra of the complexes in comparison to the spectra of the free ligands. A new weak broad band at 3 440 cm − 1 due to ν(NH) appeared which was an evidence that the nitrogen atom in the complexes is protonated. In each of the spectra of the free ligands in the range 1 450–1 590 cm − 1, 2 bands that are characteristic for stretching vibrations ν(C = C) and ν(C = N) were observed. In the spectra of the complexes only one peak was observed, which is with lower intensity in comparison to the free ligands. Moreover, in the spectra of each of the free ligands, 2 peaks were observed around 1 010 m and 1 330 s cm − 1, characteristic to stretching vibrations of ν(CNS). In the spectra of the complexes they have shifted with more than 50 cm − 1 to lower frequencies. The formation of the bond between the ruthenium ion and the ligands is confirmed by the appearance of new band in the far IR-spectra of each complex, which can be assigned to ν(Ru-N) in the range 237–260 cm − 1 when it was from the heterocyclic ring. Also in the spectra of the complexes were seen many changes in the range 400–600 cm − 1. Bands in this range are characteristic of ν(C-S) and δ(C-N). These spectral changes probably were due to the formation of a bond between the ruthenium ion and the ligands [22–28]. Certainly, this fact is confirmed by the appearance of a new band in the far IR-spectra
Fig. 1 Synthetic scheme of Ruthenium complexes.
Fig. 2 Schematic structures of the complexes obtained: [1-] benzothiazoliumbis(bisbenzothiazole) tetrachlororutenate(III); [2-] 2-methylbenzothiazoliumbis(bis-2-methylbenzothiazole)tetrachlororutenate(III); [3-] 2-mercaptobenzothiazoliumbis(bis-2-mercaptobenzothiazole)tetrachlororutenate(III).
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320 Original Article
Table 1 Cytotoxic effects of the tested complexes, the corresponding ligands and the reference cytotoxic drug cisplatin after 72 h treatment (MTT-dye reduction assay). Compounds Complex 1 Complex 2 Complex 3 Ligand 1 Ligand 2 Ligand 3 Cisplatin
IC50 (μM) KE-37
K-562
22.34 ± 0.98* 7.74 ± 2.50* 20.97 ± 3.71* > 200 > 200 > 200 4.43 ± 1.41
32.80 ± 5.09* 16.21 ± 2.33* 29.40 ± 4.29* > 200 > 200 > 200 8.61 ± 2.41
Values were the means of 8 replicates ± SD, run in triplicate. The asterisk (*) denotes statistical significance vs. cisplatin (at p ≤ 0.05 set as significance level; paired t-test)
The molar conductivities of freshly prepared 10 − 3 M solutions in DMSO were measured at room temperature. The values of 42.35 ΛM Ω − 1 mol − 1 cm2 for 1, 40.05 for 2 and 37.06 for 3 indicated the ionic nature of these complexes [41]. Based on the results of the physicochemical studies the following structures for Ru (III) ▶ Fig. 2). complexes have been proposed (●
Cell studies The cytotoxicity of the newly described ruthenium(III) coordination compounds and the corresponding ligands were assessed against 2 human leukemic cell lines, namely KE-37 (T-cell leukemia) and K-562 (chronic myeloid leukemia), using the MTT-dye reduction assay. Following a 72 h exposure period the tested complex compounds displayed prominent cytotoxic effects upon the used cell lines, in a concentration-dependent manner, causing 50 % inhibition of the cellular viability at low micromolar concentrations and almost total eradication of viable cells at concentrations exceeding 50 μM. In both cell lines the Ru(III) complex 2 with 2-methylbenzothiazole ligands proved to significantly outclass both other coordination compounds, actually its relative cytotoxicity was comparable to that of the reference ▶ Table 1). Complexes 1 and 3 displayed cytotoxic drug cisplatin (● similar activity. In general the tested complexes were more active against the lymphoid KE-37 cells as compared to the myeloid K-562 cells. Throughout the MTT-bioassay the ligands displayed only marginal inhibitory effects against the human ▶ Fig. 3. leukemic cell lines. The results are shown in ● The MTT-data were corroborated by another concentrationresponse experiment using a commercially available “Cell death detection ELISA” kit. Following a 24 h treatment period, the novel complexes evoked concentration-dependent oligonucleosomal DNA fragmentation in K-562 cells which indicates that their cytotoxicity is at least partially mediated by induction of cell death through apoptosis. The apoptogenic effects of the tested agents decreased in the following order: cisplatin > com▶ Fig. 4. plex 2 > complexes 1, 3, as they are shown in ●
Conclusion
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3 new Ru(III) complexes with benzothiazole and some its derivatives were synthesized. The stoichiometry and physicochemical studies reveal the formation of monometallic complexes of Ru (III). The ligands coordinate to the metal ion through the N-atom and act as monodentate once. Octahedral stereochemistry around the metal ion is proposed.
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of each complex, which can be assigned to ν(Ru-N) in 237– 260 cm − 1 range when it was from the heterocyclic ring [29]. 1 H-NMR spectra for complexes 1, 2 and 3 are displayed between 10 and 2 ppm. In general, all complexes showed very similar spectra. The signals of the protons in the spectra of the complexes downfield shifted compared to the corresponding signals of the protons in uncoordinated ligands, as expected. In particular, the signal of the proton from the thiazole ring is shifted from 9.42 ppm in the spectra of free ligand to 9.92 ppm in the complex 1. In all complexes the signal for the most downfield shifted proton H-4, which is the closest proton to the metal ion, and expected to be most affected by its paramagnetic influence. Notably, the signals of H-4 were shifted from 8.11 ppm in the spectra of the free ligand to 8.46 ppm in the spectra of complex 1, from 7.73 ppm to 8.06 ppm in complex 2 and from 7.61 ppm to 7.93 ppm in the spectra of complex 3. The differences between the chemical shifts of the protons H-4 of the ligands and those of the corresponding complexes are noticeable at 0.35, 0.33 and 0.32 ppm. The other protons are less influenced by the complex formation as the signals of H-7 protons in the spectra of all complexes downfield shifted with 0.2 ppm and the signals of H-5 and H-6 protons are downfield shifted only with 0.2 ppm. The biggest displacement for the signal of H-4 was due to the coordination of the ligand through the nitrogen atom of the heterocycle. In 13C-NMR spectra of the complexes downfield shift with 6 ppm of the signals for carbon atoms C-2 and C-9 was observed. C-2 showed absorptions at 155.8 ppm for the free benzothiazole and 161.8 ppm for complex 1, 166.6 ppm for free 2-methylbenzothiazole and 172.8 ppm for complex 2, 189.8 ppm for free 2-mercaptobenzothiazole and 196.1 ppm for complex 3. C-9 exhibited absorptions at 153.1 ppm for the free benzothiazole and 159.1 ppm for complex 1, 153.4 ppm for free 2-methylbenzothiazole and 159.2 ppm for complex 2, 141.2 ppm for free 2-mercaptobenzothiazole and 147.3 ppm for complex 3. The signal for the carbon atom C-8, which is connected to the sulphur is shifted to lower field also, but only with 4 ppm. In particular the signals in the spectra of the free ligands are 133.6 ppm, 135.6 ppm and 129.4 ppm. In the spectra of the complexes 1, 2 and 3 the signals appear respectively at 137.6, 139.5 and 133.5 ppm. The signals for the other carbon atoms are influenced insignificantly. The large displacement of the signals for C-2 and C-9 that are directly related to the nitrogen atom was due to monodentate linking of the ligand through the nitrogen atom [30–37]. Absorption spectra of all complexes HL[Ru(L)2Cl4] in DMSO solutions (10 − 4 mol.dm − 3) were recorded. For comparison, the electronic spectra of the uncoordinated ligands (L1, L2 and L3 were obtained at the same experimental conditions. The electronic spectra of the free ligands exhibit absorption bands around 220 nm and 280 nm, which are corresponding to π–π* transitions of the benzenoid system of the benzothiazole moiety. The absorption band around 250 nm is due to the π–π* transitions of the thiazole moiety. These bands were found in the spectra of all complexes with a small shift. This may be attributed to the donation of lone pairs of electrons of the nitrogen atom of the ligand to the metal ion. Octahedral low spin d5 complexes have the 2T2 g ground state corresponding to the t52 g electronic state. In fact, the electronic spectra of all complexes show new intense broad absorption bands in the visible region (λmax = 406 nm for complex 1, λmax = 410 nm for complex 2 and λmax = 425 nm for complex 3, in DMSO) attributed to ligand – metal charge transfer which was influenced by the formation of a chemical bond between the metal ion and the corresponding ligand [38–40].
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Fig. 4 Apoptotic oligonucleosomal DNA fragmentation in K-562 cells after 24 h exposure to the tested ruthenium(III) complexes 1–3, and the reference cytotoxic drug cisplatin (“Cell death detection ELISA” kit).
Our data indicate that the presented series of Ru(III) complexes with heterocyclic ligands exert strong inhibitory effects against human leukemic cell lines. The established cytotoxicity at low micromolar concentrations gives us a reason to conclude that the obtained compounds necessitate further, more detailed pharmacological evaluation.
Acknowledgment
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We are grateful to the Faculty of Pharmacy, Medical University of Sofia, Bulgaria for the financial support of this research.
Conflict of Interest
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The authors declare that there are no financial/commercial conflicts of interest.
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Fig. 3 Concentration-response curves of the tested ruthenium(III) complexes 1–3, the corresponding ligands and the reference cytotoxic drug cisplatin after 72 h treatment (MTT-dye reduction assay).
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322 Original Article
