Bioinorganic Chemistry and Applications

Bioinorganic Chemistry and Applications / 2010 / Article
Special Issue

Special Issue in Honor of Professor Nick Hadjiliadis’ Retirement

View this Special Issue

Research Article | Open Access

Volume 2010 |Article ID 183097 |

Jakob Kljun, Saša Petriček, Dušan Žigon, Rosana Hudej, Damijan Miklavčič, Iztok Turel, "Synthesis and Characterization of Novel Ruthenium(III) Complexes with Histamine", Bioinorganic Chemistry and Applications, vol. 2010, Article ID 183097, 6 pages, 2010.

Synthesis and Characterization of Novel Ruthenium(III) Complexes with Histamine

Academic Editor: Spyros Perlepes
Received30 Dec 2009
Accepted09 Mar 2010
Published02 Jun 2010


Novel ruthenium(III) complexes with histamine (1a) and (1b) have been prepared and characterized by X-ray structure analysis. Their crystal structures are similar and show a protonated amino group on the side chain of the ligand which is not very common for a simple heterocyclic derivative such as histamine. Biological assays to test the cytotoxicity of the compound 1b combined with electroporation were performed to determine its potential for future medical applications in cancer treatment.

1. Introduction

Before the discovery of cisplatin and its successful use as an anticancer agent in the 70s, metal complexes were rarely considered useful for medical applications. Afterwards, new cisplatin analogues (carboplatin, oxaliplatin) were developed and introduced into clinical use [13]. The development of platinum-drug resistance in cancer patients, the general toxicity and severe side effects of platinum drugs however required a different approach in the research of anticancer metal complexes [2].

Complexes of different metals were prepared and tested. Two ruthenium compounds, NAMI-A and KP1019 [46], are currently among the most successful candidates to enter the clinical practice. These two ruthenium(III) complexes have an octahedral geometry and contain four in-plane chlorido ligands as well as one dimethylsulfoxido and one imidazolo (NAMI-A) or two indazolo ligands (KP1019) in trans positions. The aforementioned compounds are the chosen representatives of two larger classes of compounds bearing nitrogen-bound aromatic heterocycles developed by the Alessio and Keppler research groups, respectively [4, 7, 8].

Histamine (4-(2-Aminoethyl)-1H-imidazole, see Scheme 2) is a molecule that performs various functions in the body, the most important being the gastric acid secretion and the triggering of the symptoms of an allergic reaction such as vasodilatation, bronchoconstriction, bronchial muscle contraction, pain, and itching.


Most of the metal complexes in use in current cancer treatment have an intracellular target and the plasma membrane can represent a considerable barrier. Electroporation or electropermeabilization is a process where exposing cells to specific electrical pulses results in temporary formation of hydrophilic pores in the cell membrane. Thus temporally increased cell permeability enables extracellular molecules with otherwise hampered transmembrane transport to enter the cells. Electroporation is used in a variety of biotechnological and medical applications. It has been proven that combining electroporation with chemotherapy potentiates the cytotoxicity of drugs when the drugs' efficacy is limited by its uptake in the cell [913].

The aim of this study was to prepare and characterize new ruthenium NAMI-type compounds, to test their in vitro cytotoxicity and study the influence of electroporation on the cytotoxic activity of the synthesized compounds.

2. Experimental

2.1. Materials and Instruments

Ruthenium(III) chloride hydrate, histamine dihydrochloride, and the solvents (dimethylsulfoxide, concentrated hydrochloric acid, methanol, ethanol, and acetone) were purchased by Sigma-Aldrich and used without further purification.

2.1.1. Infrared Spectroscopy

Infrared spectra (ATR) were recorded on a Perkin-Elmer Spectrum 100 spectrometer. The measurements were made in the range from 4000 to 600 cm-1.

2.1.2. Electrospray Ionization Mass Spectrometry

Mass measurements were run on a hybrid quadrupole time of flight mass spectrometer Q-Tof Premier (Waters Micromass, Manchester, UK), equipped with an orthogonal Z-spray electrospray (ESI) interface.

Water sample solution was introduced directly through syringe pump at a flow rate 5  L/min. Compressed nitrogen (99.999%, Messer Slovenia) was used as both the drying and the nebulizing gas. The nebulizer gas flow rate was set to approximately 20 L/h and the desolvation gas flow rate to 600 L/h. A cone voltage of 30 V and a capillary voltage of 2.9 kV were used in positive ion mode. The desolvation temperature was set to C and the source temperature to C. The mass resolution of approximately 9500 fwhm was used for determination of elemental composition with TOF mass spectrometer. MS and MS/MS spectra were acquired in centroid mode over an m/z range of 50–1000 in scan time 1 s and inter scan time 0.1 s. The detector potential was set to 1850 V. Reproducible and accurate mass measurements at approximate 10000 mass resolution were obtained using an electrospray dual sprayer with leucine enkephalin ( ) as a reference compound, introduced into the mass spectrometer alternating with a sample solution.

The data station operating software was Mass Lynx v. 4.1 (Micromass, Manchester).

Interpretation of peaks in mass spectra and identification of particular fragment ions were confirmed with elemental composition mass measurements of these ions at high resolution.

2.1.3. CHN Elemental Analysis

Elemental analyses were performed on a Perkin-Elmer Elemental analyzer 2400 CHN.

2.1.4. X-Ray Structure Analysis

X-ray diffraction data were collected on a Nonius Kappa CCD diffractometer at 150 K for compound 1a and at room temperature for compound 1b using graphite monochromated Mo-Kα radiation and processed using DENZO [14] program. The structures were solved using SIR92 [15]. A full-matrix least-squares refinement on F magnitudes with anisotropic displacement factors for all nonhydrogen atoms using SHELXL [16] was employed. The drawings were prepared with the Mercury program [17]. Hydrogen atoms were placed in geometrically calculated positions and were refined using a riding model.

The crystallographic data for compounds 1a and 1b have been deposited with the CCDC as supplementary material with the deposition numbers CCDC 759890 and 759818, respectively (see Supplementary Material available online at

2.2. Syntheses

Syntheses of [RuCl4(dmso-S)(histamineH)]·H2O (1a) and [RuCl4(dmso-S)(histamineH)] (1b): the synthesis of compounds 1a and 1b consists of two steps (see Scheme 3). The first step is the preparation of a ruthenium precursor P1 (dmso2H)[trans-RuCl4(dmso-S)2]. This compound was prepared according to the literature [18]. 200 mg of P1 and 70 mg of histamine dihydrochloride were dissolved in 15 mL of methanol and refluxed for 4 hours. After a slow evaporation of the solvent, only a few red crystals of 1a were obtained. The X-ray structure analysis showed the presence of a solvate water in a molecule. Despite considerable effort, we were not able to obtain such crystals again. Crystals of compound 1b were later obtained by adding 15 mL of ethanol to the reaction mixture and the solution was left to stand in an open flask. Overnight snowflake-like crystalline orange solid formed. The crystals were washed with cold acetone and diethylether and dried at C for 30 minutes. Similar crystals although of lower quality were obtained by addition of isopropanol, n-pentanol or ethyl acetate, instead of ethanol, to the reaction mixture.


IR (ATR): 3226 (sh), 3115 (s), 2923 (w), 1597 (w), 1578 (w), 1504 (sh), 1480 (m), 1402 (m), 1231 (w), 1111 (sh), 1082 (s), 1016 (s), 968 (s), 934 (s), 928 (sh), 840 (s), 684 (w), 658 (m) cm-1.

CHN (only 1b): Calc. C 19,41%, H 3,72%, N 9,70%; Found: C 19,78%, H 3,67%, N 9,49%.

ESI-MS (in H2O solution): m/z 361, 325, 299, 264.

2.3. Biological Activity Assays

Murine melanoma cell line B16F1 (European Collection of Cell Cultures, UK) was tested in vitro to determine cytotoxic effect of compound 1b in combination with or without electroporation. B16F1 cell suspension (22 106 cells/mL) was prepared in low conductive electroporation buffer (10 mM (Na2HPO4 /NaH2PO4, pH 7.4) with 1 mM MgCl2 250 mM sucrose). Different concentrations of compound 1b were added to cell suspension to reach final concentrations of 0.01, 0.1, and 1 mM. Immediately after incubation (<0.5 min), a drop of cell suspension was placed between two flat parallel stainless-steel electrodes 2 mm apart and a train of electric pulses (8 pulses, 800 V/cm, 100  s, 1 Hz) was applied with an electroporator Cliniporator (Igea, Carpi, Italy). The same procedure without electric pulses was used for cells exposed to different concentrations of 1b without electroporation. In addition, we tested cytotoxic effect of 1b after prolonged incubation time (60 min, without electroporation). Cell viability was measured 72 h after treatment using the MTS-based Cell Titer 96 One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). Absorption at 490 nm wavelength (A490) was measured with a spectrophotometer Tecan infinite M200 (Tecan, Switzerland). Cell viability (C.V.) of treated cells (tr) was calculated using the formula: , taking the cell viability of the control I as 100%. Statistical analysis was performed using One-Way ANOVA test and SigmaStat statistical software (SPSS, Chicago, USA).

3. Results and Discussion

3.1. Synthesis and Crystal Structure

Several complexes of the NAMI and KP families bearing nitrogen-bound simple heterocycles or smaller biologically active molecules were already synthesized and investigated for biological applications [4, 7, 19, 20]. Most of the NAMI analogues were synthesized by mixing a suspension of P1 in acetone and adding the equivalent amount of the ligand and then recrystallizing the product either from hot acetone or other solvents. In our case this synthetic route was not appropriate due to the low solubility of compound 1b in acetone or other solvents except in water and methanol. The reaction was thus performed in methanol and another less volatile and less polar solvent was added later. Similar crystals although of lower quality were obtained by addition of isopropanol, n-pentanol, or ethyl acetate instead of ethanol to the reaction mixture. The crystals were cut and were suitable for X-ray structure analysis. The experimental data is shown in Table 1.

Compound 1aCompound 1b

Empirical formulaC7H16Cl4N3O2RuSC7H16Cl4N3ORuS
Formula weight449.16 g/mol433.16 g/mol
Temperature150(2) K293(2) K
Wavelength0.71073 Å0.71073 Å
Crystal systemTriclinicMonoclinic
Space groupP 31 2 1C 2/c
Unit cell dimensions
Density (calculated)1.959 g/cm31.845 g/cm3
Absorption coefficient
Crystal size
Theta range for data collection3.26 to 3.22 to
Reflections collected72635747
Independent reflections3830 [R(int)      0.0166]3501 [ int )      0.0290]
Refinement methodFull-matrix least-squares on Full-matrix least-squares on
Goodness-of-fit on F 21.0681.080
Final R indices [I 2 sigma(I)] ,
R indices (all data) ,
Largest diff. peak and hole1.229 and 0.686 e 1.098 and 0.560 e

The asymmetric unit of compound 1b is shown in Figure 1. Selected bond lengths and hydrogen bond short contact distances are presented in Tables 2 and 3, respectively.


2.355 (2)2.359 (2)2.3403 (9)
2.364 (2)2.364 (2)2.3227 (8)
2.356 (2)2.343 (2)2.3588 (9)
2.358 (2)2.356 (2)2.3447 (8)
2.300  (2)2.298 (2)2.2956 (6)
Ru N2.096 (2)2.097 (2)2.081 (2)

AD–A (Å)angle DHA ( )

3.218 (6)151
A 3.331 (6)138
A 3.306 (6)140
B 3.308 (6)149
B 3.349 (6)131
C 2.889 (6)172

The crystal structure of compounds 1a and 1b does not differ significantly from the other NAMI-type compounds. Compounds 1a and 1b have a distorted octahedral geometry with four in-plane chloride anions surrounding the Ru(III) ion in addition to a sulfur atom from an S-bonded dimethylsulfoxide molecule and a nitrogen atom from the histamine molecule in axial positions. The main difference to most of the NAMI-type compounds which are anionic complexes is the protonated amino group on the side chain of the histamine moiety (hence histamineH is used in the formula) that gives compounds 1a and 1b a neutral charge. This structural feature is however more common in NAMI-type compounds with a purine derivative as the nitrogen ligand [21]. The hydrogen atoms on the amino group (H3A, H3B and H3C) form hydrogen bonds with the four chloride atoms and the dmso oxygen. The Cl2 chloride atom forms an additional hydrogen bond with the hydrogen on the imidazole moiety (H2) which results in a slightly longer Ru-Cl2 distance (see Tables 2 and 3 and Figures 1 and 2). Compound 1a exhibits an additional hydrogen bond between the water molecule and one of the chloride anions. It was not possible to locate the positions of two hydrogen atoms bonded to oxygen in a water molecule of the complex 1a by difference Fourier maps.

Another interesting feature of the crystal structure of compound 1a is the formation of channels along z axis where the water molecules are located (see Figure 3).

3.2. Electrospray Ionization Mass Spectrometry

Compound 1b was also characterized by electrospray ionization mass spectrometry. The peaks in the mass spectrum and their respective assignments are presented in Table 4. The data confirm a partial hydrolysis of the compound in water solution. Such behavior is usual for the NAMI-type compounds which undergo a rather quick dissociation of two of the chloride ligands and/or the dmso molecule [23].



3.3. Biological Activity

Previous studies have shown that while NAMI-A is inactive against B16F1 cells in vitro, it shows remarkable activity at relatively low concentrations when combined with electroporation [24]. On the other hand compound 1b shows no activity at any of the tested concentrations (up to 1 mM) either by itself or in combination with electroporation.

As Dyson and Sava suggest [25], the cytotoxicity of a compound as one of the main criteria in the preliminary screenings when looking for a potential drug candidate should be considered with some caution. NAMI-A for example, showed remarkable antimetastatic properties despite very low cytotoxicity. Further investigations of the biological activity of the histaminic analogue and the study of the interactions with different proteins are being planned.


Financial support from the Slovenian Research Agency (ARRS) through projects P1-0175, P2-0249, and J1-0200-0103-008 is gratefully acknowledged. This work was performed within the framework of COST Action D39. Jakob Kljun is grateful to ARRS for funding through a junior researcher grant. Jakob Kljun would also like to thank Marta Kasunič for her aid and advice on crystal structure solving.

Supplementary Materials

CCDC 759890 and 759818 contain the supplementary crystallographic data for 1a and 1b. These data can be obtained free of charge via, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax (+44) 1223-336-033; or e-mail:

  1. Supplementary Material
  2. Supplementary Material


  1. E. Wiltshaw, “Cisplatin in the treatment of cancer: the first metal anti-tumour drug,” Platinum Metals Review, vol. 23, no. 3, pp. 90–98, 1979. View at: Google Scholar
  2. L. Kelland, “The resurgence of platinum-based cancer chemotherapy,” Nature Reviews Cancer, vol. 7, no. 8, pp. 573–584, 2007. View at: Publisher Site | Google Scholar
  3. T. W. Hambley, “Developing new metal-based therapeutics: challenges and opportunities,” Dalton Transactions, no. 43, pp. 4929–4937, 2007. View at: Publisher Site | Google Scholar
  4. E. Alessio, G. Mestroni, A. Bergamo, and G. Sava, “Ruthenium antimetastatic agents,” Current Topics in Medicinal Chemistry, vol. 4, no. 15, pp. 1525–1535, 2004. View at: Publisher Site | Google Scholar
  5. C. G. Hartinger, S. Zorbas-Seifried, M. A. Jakupec, B. Kynast, H. Zorbas, and B. K. Keppler, “From bench to bedside—preclinical and early clinical development of the anticancer agent indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A),” Journal of Inorganic Biochemistry, vol. 100, no. 5-6, pp. 891–904, 2006. View at: Publisher Site | Google Scholar
  6. C. G. Hartinger, M. A. Jakupec, S. Zorbas-Seifried et al., “KP1019, a new redox-active anticancer agent—preclinical development and results of a clinical phase I study in tumor patients,” Chemistry and Biodiversity, vol. 5, no. 10, pp. 2140–2155, 2008. View at: Publisher Site | Google Scholar
  7. E. Reisner, V. B. Arion, A. Eichinger et al., “Tuning of redox properties for the design of ruthenium anticancer drugs—part 2: syntheses, crystal structures, and electrochemistry of potentially antitumor [RuIII/IICl6-n(Azole)n]z (n=3, 4, 6) complexes,” Inorganic Chemistry, vol. 44, no. 19, pp. 6704–6716, 2005. View at: Publisher Site | Google Scholar
  8. I. Bratsos, S. Jedner, T. Gianferrara, and E. Alessio, “Ruthenium anticancer compounds: challenges and expectations,” Chimia, vol. 61, no. 11, pp. 692–697, 2007. View at: Publisher Site | Google Scholar
  9. E. H. Serpersu, K. Kinosita Jr., and T. Y. Tsong, “Reversible and irreversible modification of erythrocyte membrane permeability by electric field,” Biochimica et Biophysica Acta, vol. 812, no. 3, pp. 779–785, 1985. View at: Publisher Site | Google Scholar
  10. J. Teissie, M. Golzio, and M. P. Rols, “Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of ?) knowledge,” Biochimica et Biophysica Acta, vol. 1724, no. 3, pp. 270–280, 2005. View at: Publisher Site | Google Scholar
  11. M. Pavlin and D. Miklavčič, “Theoretical and experimental analysis of conductivity, ion diffusion and molecular transport during cell electroporation–relation between short-lived and long-lived pores,” Bioelectrochemistry, vol. 74, no. 1, pp. 38–46, 2008. View at: Publisher Site | Google Scholar
  12. S. Orlowski, J. Belehradek Jr., C. Paoletti, and L. M. Mir, “Transient electropermeabilization of cells in culture: increase of the cytotoxicity of anticancer drugs,” Biochemical Pharmacology, vol. 37, no. 24, pp. 4727–4733, 1988. View at: Google Scholar
  13. G. Serša, M. Čemažar, and D. Miklavčič, “Antitumor effectiveness of electrochemotherapy with cis-diamminedichloroplatinum(II) in mice,” Cancer Research, vol. 55, no. 15, pp. 3450–3455, 1995. View at: Google Scholar
  14. Z. Otwinowski and W. Minor, “Processing of X-ray diffraction data collected in oscillation mode,” Methods in Enzymology, vol. 276, pp. 307–326, 1997. View at: Publisher Site | Google Scholar
  15. A. Altomare, M. C. Burla, M. Camalli et al., “SIR97: a new tool for crystal structure determination and refinement,” Journal of Applied Crystallography, vol. 32, no. 1, pp. 115–119, 1999. View at: Google Scholar
  16. G. M. Sheldrick, “A short history of SHELX,” Acta Crystallographica Section A, vol. 64, no. 1, pp. 112–122, 2008. View at: Publisher Site | Google Scholar
  17. C. F. Macrae, P. R. Edgington, P. McCabe et al., “Mercury: visualization and analysis of crystal structures,” Journal of Applied Crystallography, vol. 39, no. 3, pp. 453–457, 2006. View at: Publisher Site | Google Scholar
  18. E. Alessio, G. Balducci, M. Calligaris, G. Costa, W. M. Attia, and G. Mestroni, “Synthesis, molecular structure, and chemical behavior of hydrogen trans-bis(dimethyl sulfoxide)tetrachlororuthenate(III) and mer-trichlorotris(dimethyl sulfoxide)ruthenium(III): the first fully characterized chloride-dimethyl sulfoxide-ruthenium(III) complexes,” Inorganic Chemistry, vol. 30, no. 4, pp. 609–618, 1991. View at: Google Scholar
  19. D. Griffith, S. Cecco, E. Zangrando, A. Bergamo, G. Sava, and C. J. Marmion, “Ruthenium(III) dimethyl sulfoxide pyridinehydroxamic acid complexes as potential antimetastatic agents: synthesis, characterisation and in vitro pharmacological evaluation,” Journal of Biological Inorganic Chemistry, vol. 13, no. 4, pp. 511–520, 2008. View at: Publisher Site | Google Scholar
  20. I. Turel, M. Pecanac, A. Golobic et al., “Solution, solid state and biological characterization of ruthenium(III)-DMSO complexes with purine base derivatives,” Journal of Inorganic Biochemistry, vol. 98, no. 2, pp. 393–401, 2004. View at: Publisher Site | Google Scholar
  21. A. García-Raso, J. J. Fiol, A. Tasada et al., “Ruthenium complexes with purine derivatives: syntheses, structural characterization and preliminary studies with plasmidic DNA,” Inorganic Chemistry Communication, vol. 8, no. 9, pp. 800–804, 2005. View at: Publisher Site | Google Scholar
  22. E. Alessio, G. Balducci, A. Lutman, G. Mestroni, M. Calligaris, and W. M. Attia, “Synthesis and characterization of two new classes of ruthenium(III)-sulfoxide complexes with nitrogen donor ligands (L): Na[trans-RuCl4(R2SO)(L)] and mer, cis-RuCl3(R2SO)(R2SO)(L). The crystal structure of Na[trans-RuCl4(DMSO)(NH3)] ·2DMSO, Na[trans-RuCl4(DMSO)(Im)] ·H2O, Me2CO (Im = imidazole) and mer, cis-RuCl3(DMSO)(DMSO)(NH3),” Inorganica Chimica Acta, vol. 203, no. 2, pp. 205–217, 1993. View at: Publisher Site | Google Scholar
  23. G. Mestroni, E. Alessio, G. Sava, S. Pacor, M. Coluccia, and A. Boccarelli, “Water-soluble ruthenium(III)-dimethyl sulfoxide complexes: chemical behaviour and pharmaceutical properties,” Metal-Based Drugs, vol. 1, no. 1, pp. 41–63, 1993. View at: Google Scholar
  24. A. Bicek, I. Turel, M. Kanduser, and D. Miklavcic, “Combined therapy of the antimetastatic compound NAMI-A and electroporation on B16F1 tumour cells in vitro,” Bioelectrochemistry, vol. 71, no. 2, pp. 113–117, 2007. View at: Publisher Site | Google Scholar
  25. P. J. Dyson and G. Sava, “Metal-based antitumour drugs in the post genomic era,” Dalton Transactions, no. 16, pp. 1929–1933, 2006. View at: Publisher Site | Google Scholar

Copyright © 2010 Jakob Kljun et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

More related articles

 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly as possible. We will be providing unlimited waivers of publication charges for accepted research articles as well as case reports and case series related to COVID-19. Review articles are excluded from this waiver policy. Sign up here as a reviewer to help fast-track new submissions.