{In vitro antimicrobial activity and cytotoxicity of nickel(II) complexes with different diamine ligands} J. Serb. Chem. Soc. 82 (4) 389–398 (2017) UDC 546.742+547.415.1:615.28–188:576+615.9 JSCS–4974 Original scientific paper 389 In vitro antimicrobial activity and cytotoxicity of nickel(II) complexes with different diamine ligands NENAD S. DRAŠKOVIĆ1#, BILJANA Đ. GLIŠIĆ2*#, SANDRA VOJNOVIC3, JASMINA NIKODINOVIC-RUNIC3** and MILOŠ I. DJURAN2# 1University of Priština, Faculty of Agriculture, Kopaonička bb, 38228 Lešak, Serbia, 2Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovića 12, 34000 Kragujevac, Serbia and 3Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia (Received 13 January, revised 30 January, accepted 13 February 2017) Abstract: Three diamines, 1,3-propanediamine (1,3-pd), 2,2-dimethyl-1,3-pro- panediamine (2,2-diMe-1,3-pd) and (±)-1,3-pentanediamine (1,3-pnd), were used for the synthesis of nickel(II) complexes 1–3, respectively, of the general formula [Ni(L)2(H2O)2]Cl2. The stoichiometries of the complexes were con- firmed by elemental microanalysis, and their structures were elucidated by spectroscopic (UV–Vis and IR) and molar conductivity measurements. The complexes 1–3, along with NiCl2·6H2O and the diamine ligands, were eva- luated against a panel of microbial strains that are associated with skin, wound, urinary tract and nosocomial infections. The obtained results revealed no sig- nificant activity of 1–3 against the investigated bacterial strains. On the other hand, they showed good antifungal activity against pathogenic Candida strains, with minimum inhibitory concentration (MIC) values in the range from 15.6 to 62.5 µg mL-1. The best anti-Candida activity was observed for complex 2 against C. parapsilosis, while the least susceptible to the effect of the com- plexes was C. krusei. The antiproliferative effect on normal human lung fibro- blast cell line MRC-5 was also evaluated in order to determine the therapeutic potential of nickel(II) complexes 1–3. These complexes showed lower negative effects on the viability of the MRC-5 cell line than the clinically used nystatin and comparable selectivity indexes to that of this antifungal drug. Keywords: nickel(II) complexes; diamines; antimicrobial activity; Candida; cytotoxicity. INTRODUCTION The field of medicinal application of metal-based compounds has attracted widespread attention of researchers over the decades.1 This broad interest is due *,** Corresponding authors. E-mail: (*)bglisic@kg.ac.rs; (**)jasmina.nikodinovic@gmail.com # Serbian Chemical Society member. https://doi.org/10.2298/JSC170113026D 390 DRAŠKOVIĆ et al. to the constant demand of medicinal chemistry for innovation and input of novel metal-based compounds to cope with very important challenges, such as broader spectrum of activity, selectivity, reduced toxicity and emerging resistance. The development of a new therapeutic agent is a multi-stage process involving syn- thesis, characterization, evaluation of biological activity, and pre-clinical and cli- nical testing. Considering the fact that this process becomes very expensive in the later stage, it is important to use the best possible models in the identification of those compounds that have the desired biological activity. Furthermore, it was found previously that microorganisms and infections play major roles in carcino- genesis, as well as in antitumor response.2 Of the 12.7 million new cancer cases that occurred in 2008, around 2 million could be attributed to infections. Bacter- emia is a major cause of life-threatening complications in patients with cancer, who are at extremely high risk for infections caused by antibiotic-resistant Gram- -negative bacteria. Invasive candidiasis is the fourth most common bloodstream infection (surpassing many bacterial pathogens) with mortality rates remaining disturbingly high at 40 %.3 More than 17 different Candida species are known to be etiological agents of human infection, however, more than 90 % of invasive infections are caused by C. albicans, C. glabrata, C. parapsilosis and C. krusei.4 Considering this and in the view of the global problem of multi-drug resistant microbial strains, the search for new antibacterial and antifungal therapeutics is of paramount importance. Nickel was considered for many years as an element without important bio- logical significance, until its existence in the active center of the enzyme urease was established in 1975.5,6 Since then, Ni2+ was confirmed to be competitive antagonists with both Mg2+ and Ca2+ and the presence of nickel was established in the active sites of different metallo-enzymes, and hence, interest for the eva- luation of its biological properties has rapidly expanded.7,8 It was found that chronic exposure to nickel could be connected with increased risk of lung cancer, cardiovascular disease, neurological deficits, developmental deficits in childhood and high blood pressure.9 Nickel is also considered as a potential allergen, which may cause contact dermatitis.10 Nevertheless, a broad spectrum of beneficial bio- logical activities of various nickel(II) complexes has hitherto been reported. Nickel(II) complexes were reported to act as anticonvulsant,11 antiepileptic,12 antibacterial,13 antifungal,14 antileishmanial,15 antioxidant16,17 and antiprolifer- ative agents.18 Considering this, in the present study, three diamines, 1,3-pro- panediamine (1,3-pd), 2,2-dimethyl-1,3-propanediamine (2,2-diMe-1,3-pd) and (±)-1,3-pentanediamine (1,3-pnd) were used for the synthesis of nickel(II) complexes of the general formula [Ni(L)2(H2O)2]Cl2 (Scheme 1). Although the synthesis of these complexes was reported previously,19–22 their antimicrobial effects have not been investigated. In order to determine the therapeutic potential BIOLOGICAL EVALUATION OF BIS(DIAMINE)NICKEL(II) COMPLEXES 391 of these complexes, their antiproliferative effect on the normal human lung fib- roblast cell line MRC-5 was also evaluated. Scheme 1. Structural representation of [Ni(L)2(H2O)2]Cl2 complexes 1–3 (L = 1,3-pd (1), 2,2-diMe-1,3-pd (2) and 1,3-pnd (3)). EXPERIMENTAL Reagents Distilled water was demineralized and purified to a resistance of greater than 10 MΩ cm. Nickel(II) chloride hexahydrate, 1,3-propanediamine (1,3-pd), 2,2-dimethyl-1,3-propanedi- amine (2,2-diMe-1,3-pd) and (±)-1,3-pentanediamine (1,3-pnd) were purchased from Sigma– –Aldrich. All the employed chemicals were of analytical reagent grade. Synthesis of the nickel(II) complexes 1–3 The nickel(II) complexes with the above-mentioned diamine ligands were synthesized by modification of a previously described method.19 The corresponding diamine (0.02 mol; 1.7 mL of 97 % 1,3-pd, ρ = 0.887 g mL-1; 2.4 mL of 99 % 2,2-diMe-1,3-pd, ρ = 0.851 g mL-1 and 2.4 mL of 98 % 1,3-pnd, ρ = 0.855 g mL-1) was added slowly under stirring to a solution containing 0.01 mol of NiCl2·6H2O (2.38 g) in 10.0 mL of water. The formed nickel(II) hyd- roxide was removed by filtration and the filtrate was stirred at 40 °C for 15 min, and then left standing at ambient temperature to evaporate slowly to a volume of 3.0 mL. The concentrated solution was then stored in refrigerator and purple crystals of the nickel(II) complexes had formed after two days. These crystals were filtered off and dried at ambient temperature. The yield was 83 % for [Ni(1,3-pd)2(H2O)2]Cl2 (1; 2.61 g), 77 % for [Ni(2,2-diMe-1,3- -pd)2(H2O)2]Cl2 (2; 2.85 g) and 79 % for [Ni(1,3-pnd)2(H2O)2]Cl2 (3; 2.92 g). Measurements Elemental microanalyses of the nickel(II) complexes for carbon, hydrogen and nitrogen were performed by the Microanalytical Laboratory, Faculty of Chemistry, University of Bel- grade. The IR spectra were recorded as KBr pellets on a Perkin Elmer Spectrum One spectro- meter over the wavenumber range 4000–450 cm-1. The UV–Vis spectra were recorded over the wavelength range of 1100–190 nm on a Shimadzu UV-1800 spectrophotometer after dis- solving the corresponding nickel(II) complex in water. The concentration of the nickel(II) 392 DRAŠKOVIĆ et al. complexes was 5×10-2 M. The molar conductivities were measured at room temperature on a Crison multimeter MM 41 digital conductivity-meter. The concentration of the aqueous sol- utions of nickel(II) complexes used for conductivity measurements was 1×10-3 M. Analytical and spectral data of the synthesized compounds are given in the Supple- mentary material to this paper. Determination of the biological activity Nickel(II) complexes 1–3, NiCl2·6H2O and the diamine ligands were dissolved in distil- led water to give stock solutions of 50 mg mL-1, which were used immediately for biological assessment of their activities. The MIC concentrations (concentration value corresponding to the lowest concentration that inhibited the growth after 24 h at 37 °C) were determined according to the standard broth microdilution assays, recommended by the National Commit- tee for Clinical Laboratory Standards (M07-A8) for bacteria and Standards of the European Committee on Antimicrobial Susceptibility Testing (EDef 7.1.). The highest concentration used was 500 µg mL-1. The test organisms included Pseudomonas aeruginosa PAO1 (NCTC 10332), Staphylococcus aureus (NCTC 6571), Candida albicans (ATCC 10231), C. glabrata (ATCC 2001), C. parapsilosis (ATCC 22019) and C. krusei (ATCC 14243). The inoculums were 105 colony-forming units, CFU, per mL for the bacteria and 104 CFU mL-1 for the Candida strains. Cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.23 The assay was performed using human lung fibroblasts (MRC-5) after 48 h of cell incubation in the media containing the compounds at concentrations ranging from 0.1–500 µg mL-1. The MRC-5 cell line was maintained in RPMI-1640 medium supple- mented with 100 μg mL-1 streptomycin, 100 U mL-1 penicillin and 10 vol.% fetal bovine serum (FBS), all from Sigma, as a monolayer (1×104 cells per well) and grown in a humid- ified atmosphere of 95 % air and 5 % CO2 at 37 °C. The extent of MTT reduction was measured spectrophotometrically at 540 nm using a Tecan Infinite 200 Pro multiplate reader (Tecan Group, Männedorf, Switzerland), and the cell survival was expressed as percentage of the control (untreated cells). RESULTS AND DISCUSSION Synthesis and structural features of the nickel(II) complexes 1–3 Three nickel(II) complexes of the general formula [Ni(L)2(H2O)2]Cl2, where L stands for bidentately coordinated 1,3-pd (1), 2,2-diMe-1,3-pd (2) or 1,3-pnd (3), were prepared in high yields (≈80 %) by reacting NiCl2·6H2O with the cor- responding diamine in 1:2 mole ratio in water using a modified procedure rep- orted previously.19 The crystal structures of the [Ni(L)2(H2O)2]2+ complexes were previously determined by single-crystal X-ray diffraction analysis.19–22 In this study, spectroscopic (UV–Vis and IR) and conductivity measurements were used for structural characterization of the [Ni(L)2(H2O)2]Cl2 complexes. The UV–Vis spectra of the investigated complexes 1–3 are presented in Fig. 1, while the wavelengths of the maximum absorption (λmax / nm) and molar ext- inction coefficients (ε / M–1 cm–1), determined immediately after dissolution of the complexes, are listed in Table I. The shape of the UV–Vis spectra for the investigated complexes was similar to that of the octahedral [Ni(en)2(H2O)2]CO3 BIOLOGICAL EVALUATION OF BIS(DIAMINE)NICKEL(II) COMPLEXES 393 complex (en is bidentately coordinated ethylenediamine) with the same N4O2 coordination environment.24 In accordance with the previously established results for the [Ni(en)2(H2O)2]CO3 complex,24 the interpretation of UV–Vis spectra of the presently investigated complexes 1–3 was realized using an octa- hedral model (Oh): 3A2g → 3T2g(F) (band I), 3A2g → 3T1g(F) (band III) and 3A2g → 3T1g(P) (band IV). In addition, in each spectrum, there was a shoulder at approximately 735 nm (band II, Table I), which occurs on the higher-energy side of the spin allowed band I. As was previously found, this shoulder arises from a spin forbidden triplet-to-singlet transition, 3A2g → 1Eg(D).24,25 As could be seen from Fig. 1, the absorption maxima of the bands I and III for the investigated nickel(II) complexes were slightly shifted to higher energy in the following order 1 > 3 > 2. Moreover, the molar absorptivity of the absorption maxima for these bands increased in the same order. These differences in the spectra could be attributed to the presence of the substituent in the six-membered 1,3-propane- diamine ring of the corresponding nickel(II) complex, i.e., two methyls for 2 and an ethyl for 3. It could be assumed that these substituents affect some changes in the strain of the six-membered 1,3-propandiamine ring. Moreover, all absorption maxima of the investigated complexes 1–3 were shifted to lower energies with res- pect to those for the [Ni(en)2(H2O)2]2+ complex.24 This shifting results from the presence of a six-membered 1,3-propanediamine ring in 1–3, which is less strained than the five-membered ethylenediamine ring in the [Ni(en)2(H2O)2]2+ complex. Fig. 1. Electronic absorption spectra of the investigated nickel(II) complexes 1–3 measured in water (c = 5×10-2 M). The IR spectroscopic data for the nickel(II) complexes are listed in the Sup- plementary material to this paper and are consistent with the structural formula presented in Scheme 1. The IR spectra of these complexes recorded in the range 394 DRAŠKOVIĆ et al. of 4000–450 cm–1 showed the expected peaks attributable to the coordinated diamine and water ligands. Thus, a broad absorption in the 3400–3300 cm–1 region attributed to the stretching vibration of OH confirmed the presence of a coordinated water molecule.26 Moreover, the complexes exhibited two very strong and sharp bands at approximately 3300 and 3200 cm–1, which were assigned to the asymmetric and symmetric stretching vibration of the coordinated amino group, respectively.22 TABLE I. Electronic absorption data for the nickel(II) complexes 1–3. For comparison the corresponding data for the previously reported [Ni(en)2(H2O)2]CO3 complex is given24 Complex Absorption Assignments λ / nm ε / M-1 cm-1 [Ni(en)2(H2O)2]CO3 I 905 – 3A2g → 3T2g(F) II 690 – → 1Eg(D) III 555 – → 3T1g(F) IV 349 – → 3T1g(P) [Ni(1,3-pd)2(H2O)2]Cl2 (1) I 946 5.1 II 738 2.0 III 581 9.7 IV 356 20.7 [Ni(2,2-diMe-1,3-pd)2(H2O)2]Cl2 (2) I 931 7.5 II 734 4.3 III 575 13.9 IV 358 22.7 [Ni(1,3-pnd)2(H2O)2]Cl2 (3) I 941 5.8 II 734 2.6 III 580 11.2 IV 361 16.8 Molar conductivity values for the nickel(II) complexes 1–3, being approx- imately 250 Ω–1 cm2 mol–1 (see Supplementary material), are sufficiently high to assess the non-coordinated nature of the two chloride anions, i.e., these values in water solvent are in agreement with 1:2 electrolytic nature of the synthesized complexes.27 Biological activity of the nickel(II) complexes 1–3 In vitro antimicrobial activity assays of nickel(II) complexes 1–3, NiCl2·6H2O and the corresponding diamine ligands revealed no significant activity against two bacterial strains (P. aeruginosa PAO1 and S. aureus) even at 500 µg mL–1 (data not shown), while MIC values against the pathogenic Candida strains were between 15.6–62.5 µg mL–1 for complexes 1–3 and 250 µg mL–1 for the inorg- anic salt (Table II). Therefore, a certain level of selectivity of 1–3 towards fungal strains could be concluded. The best anti-Candida activity was that of complex 2 against C. parapsilosis, while C. krusei was the least susceptible to the effects of BIOLOGICAL EVALUATION OF BIS(DIAMINE)NICKEL(II) COMPLEXES 395 the complexes. In contrast, nickel(II) thiohydrazide and thiodiamine complexes exhibited significant activity towards P. aeruginosa and Escherichia coli, and comparable activity against a selection of fungal Aspergillus strains.28 Antifungal activity of NiCl2·6H2O and nickel(II) complexes derived from amino sugars against C. albicans was reported by Yano et al. with MIC values in the 200–250 µM range, which is 2.5–5-fold higher in comparison to the MIC values of 1–3.29 Furthermore, the complexes from the present study showed better anti-Candida activities in comparison to nickel(II) complexes with pyrazoline-based ligand, which had MIC values ranging from 100 – 1000 µg mL–1.30 It was shown that certain selectivity against Candida strains by nickel(II) compounds was due to the competitive inhibition of fungal chitinase (chitin-degradation enzyme).29 TABLE II. Minimal inhibitory concentrations (MIC / µg mL-1) against Candida strains and IC50 values against MRC-5 cells (concentration that inhibits 50 % of cell growth after treat- ment with the tested compounds, µg mL-1); the results are from three independent experi- ments, each performed in triplicate. Standard deviations were within 1–3 % Compound C. albicans ATCC 10231 C. glabrata ATCC 2001 C. parapsilosis ATCC 22019 C. krusei ATCC 14243 MRC-5 1 31.2 31.2 31.2 62.5 500 2 31.2 31.2 15.6 62.5 80 3 31.2 31.2 31.2 62.5 500 NiCl2·6H2O 250 250 250 250 100 1,3-pd >500 >500 >500 >500 >500 2,2-diMe-1,3-pd >500 >500 >500 >500 100 1,3-pnd >500 >500 >500 >500 50 Nystatin 4 2 2 8 40 In parallel, to determine the applicability of complexes 1–3 as potential anti- fungals, their in vitro cytotoxicity against healthy human lung fibroblasts was examined (Table II, Fig. 2). While the ligands exerted no activity against Can- dida strains at 500 µg mL–1, 2,2-diMe-1,3-pd and 1,3-pnd had IC50 values of 100 and 50 µg mL–1, respectively. 1,3-Propanediamine was not cytotoxic even at 500 µg mL–1 (Table II). Accordingly, 2 was the most cytotoxic of the nickel(II) complexes, while 1 and 3 had IC50 values of 500 µg mL–1. The inorganic salt NiCl2·6H2O had a toxic effect on the cells in a dose-dependent manner (IC50 value of 100 µg mL–1), while the cytotoxicity of 1–3 did not follow this trend (Fig. 2). This may be due to differing dissociation dynamics of the complexes and different toxicity of the ligands. Thus, the selectivity index for the complexes was between 1.3 and 16, while the antiproliferative effect of the inorganic salt was higher than its antifungal effect (Table II). Although the MIC values of 1–3 were 8–16-fold higher in comparison to that of the clinically used nystatin, the selectivity indexes were comparable. This finding is encouraging for further dev- elopment of nickel(II)-based complexes for antifungal treatment. 396 DRAŠKOVIĆ et al. Fig. 2. In vitro cytotoxic effect on healthy human fibroblasts (MRC-5) of various concentrations of nickel(II) compounds upon 48 h treatment. CONCLUSIONS This work presents a modified procedure for the preparation of [Ni(L)2(H2O)2]Cl2 complexes 1–3 in high yields. The octahedral geometry of these complexes was confirmed by spectroscopic and conductivity measure- ments. In vitro antimicrobial activity assays of these complexes showed their good selectivity towards the investigated Candida strains. The best anti-Candida activity was observed for complex 2 against C. parapsilosis, while the least sus- ceptible to the effect of complexes was C. krusei. Moreover, an in vitro cytotox- icity study showed that complex 2 was the most cytotoxic against healthy human lung fibroblasts. This arises from the presence of two methyl substituents in the six-membered 1,3-propanediamine ring of 2, indicating that better antimicrobial and cytotoxic activities of bis(diamine)nickel(II) complexes could be achieved by structural modification of the chelated diamine ligand. The obtained results are encouraging for further development of nickel(II) complexes with diamine lig- ands as antifungal agents. A study in this sense is in progress. SUPPLEMENTARY MATERIAL Analytical and spectral data of the synthesized compounds are available electronically at the pages of the journal website: http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgement. This work was funded in part by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project Nos. 172036 and 173048). BIOLOGICAL EVALUATION OF BIS(DIAMINE)NICKEL(II) COMPLEXES 397 И З В О Д IN VITRO АНТИМИКРОБНА АКТИВНОСТ И ЦИТОТОКСИЧНОСТ КОМПЛЕКСА НИКЛА(II) СА РАЗЛИЧИТИМ ДИАМИНСКИМ ЛИГАНДИМА НЕНАД С. ДРАШКОВИЋ1, БИЉАНА Ђ. ГЛИШИЋ2, САНДРА ВОЈНОВИЋ3, ЈАСМИНА НИКОДИНОВИЋ-РУНИЋ3 И МИЛОШ И. ЂУРАН2 1 Пољопривредни факултет, Универзитет у Приштини, Копаоничка бб, 38228 Лешак, 2 Институт за хемију, Природно–математички факултет, Универзитет у Крагујевцу, Р. Домановића 12, 34000 Крагујевац и 3 Институт за молекуларну генетику и генетичко инжењерство, Универзитет у Београду, Војводе Степе 444а, 11000 Београд Три диамина, 1,3-пропандиамин (1,3-pd), 2,2-диметил-1,3-пропандиамин (2,2- -diMe-1,3-pd) и (±)-1,3-пентандиамин (1,3-pnd), коришћена су за синтезу никaл(II) ком- плекса 1–3 опште формуле [Ni(L)2(H2O)2]Cl2. Комплекси су окарактерисани применом елементалне микроанализе, UV–Vis и IR спектроскопије и мерењем моларне провод- љивости. Никал(II) комплекси 1–3, NiCl2·6H2O и одговарајући диамини су испитивани као потенцијални антимикробни агенси према различитим сојевима бактерија и гљива, који могу узроковати инфекције коже и рана, као и уринарне и интрахоспиталне инфек- ције. Добијени резултати су показали да комплекси 1–3 немају значајну активност према испитиваним сојевима бактерија. Насупрот томе, ови комплекси показују добру активност према испитиваним патогеним сојевима гљива, при чему су вредности мини- малне инхибиторне концентрације (MIC) у опсегу од 15,6 до 62,5 μg mL-1. Највећу анти- фунгалну активност према C. parapsilosis показује комплекс 2, док је активност ком- плекса најмања према C. krusei. У циљу одређивања терапеутског потенцијала ових ком- плекса, испитивана је њихова антипролиферативна активност према нормалној ћелиј- ској линији фибробласта плућа. Добијени резултати су показали да су комплекси никла(II) мање токсични на MRC-5 ћелијској линији у односу на нистатин и да имају индексе селективности сличне овом антифунгалном агенсу. (Примљено 13. јануара, ревидирано 30. јануара, прихваћено 13. фебруара 2017) REFERENCES 1. E. Alessio, Bioinorganic Medicinal Chemistry, Wiley–VCH, Weinheim, Germany, 2011 2. N. D. Savić, D. R. Milivojevic, B. Đ. Glišić, T. Ilic-Tomic, J. Veselinovic, A. Pavic, B. Vasiljevic, J. Nikodinovic-Runic, M. I. Djuran, RSC Adv. 6 (2016) 13193 3. M. A. Pfaller, S. A. Messer, G. J. Moet, R. N. Jones, M. Castanheira, Int. J. Antimicrob. Agents 38 (2011) 65 4. J. C. Sardi, L. Scorzoni, T. Bernardi, A. M. Fusco-Almeida, M. J. Mendes Giannini, J. Med. Microbiol. 62 (2013) 10 5. N. E. Dixon, C. Gazzola, R. L. Blakeley, B. Zerner, J. Am. Chem. Soc. 97 (1975) 4131 6. C. Tserkezidou, A. G. Hatzidimitriou, G. Psomas, Polyhedron 117 (2016) 184 7. R. R. 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