Michał ŁOMZIK, Małgorzata BRINDELL ? Inorganic Chemistry Department, Faculty of Chemistry, Jagiellonian University, Cracow, Poland
Previous studies have shown that some thiosemicarbazones have antitumor activity as inhibitors of ribonucleotide reductase and their reactivity can be enhanced by coordination to Ru ion. The following paper describes the synthesis of N,N-p-dimethylaminbenzaldehyde thiosemicarbazone and its Ru(II) complex. In addition, the interaction of the obtained compounds with albumin was carried out using fluorescence spectroscopy. It has been shown that coordination of ligand to ruthenium decrease its reactivity towards albumin and therefore the better bioavailability is expected.
Please cite as: CHEMIK 2013, 67, 2, 91-98
Cancer is defined as a group of diseases characterized by uncontrolled cell division. As a result, the growth of tumour tissue is observed, and very often it leads to an impairment of an organ on which the tumour is growing. One of the main problem in anticancer therapy is the variety of cancer types. Therefore, it is difficult to design a universal drug. The discovery in the 60s of the last century the cisplatin significantly facilitated the battle against cancer. But nowadays we need much more effective and better acting drugs than cisplatin. One way to obtain new anticancer compounds was based on a modification of substituent in the cisplatin. This provided a number of currently used chemotherapeutic agents . However, the search for other biologically active compounds that can be used in the treatment of cancer is still in progress. For several years, scientists are interested in other metals for use them in anticancer therapy. Particularly ruthenium complexes are promising. In 2004 two ruthenium(III) complexes were induced to phase I clinical trial and two years later (in 2006) were accepted to phase II clinical trial . Those two complexes are namely NAMI-A and KP1019 as presented in Figure1.
It is suggested that complexes of ruthenium(III) have a little biological activity, however at physiological conditions, where concentration of reducing agents such as ascorbic acid, cysteine or glutathione is significant, they can activate these complexes via their reduction. Research in our group confirmed that NAMI-A at physiological conditions can be reduced from ruthenium(III) to ruthenium(II) .
Previous studies indicate that one of the approaches to design new, more effective drugs is the combining the metal complex with biologically active compound as its ligand. One of the expected advantages would be the enhancement of their biological activity. For several years, as ligands for the ruthenium ion the biologically active thiosemicarbazones are under investigation. Thiosemicarbazones are well known in chemistry for a long time. They are formed in condensation reaction between aldehyde or keton and thiosemicarbazide. Due to this reaction, in the old times, thiosemicarbazide and semicarbazide were used in analytical chemistry for the detection of compounds with a carbonyl group ? aldehydes and ketones. Research in the field of biological activity of thiosemicarbazones has shown that many of them have antibacterial, antifungal and antiviral activity. Some thiosemicarbazones possess anticancer activity. It is proposed that their activity arise mostly from the inhibition of the enzyme ribonucleotide reductase, which is responsible for the synthesis of deoxyribonucleotides needed for DNA synthesis . Recent literature reports have shown that binding thiosemicarbazone to ruthenium(II) and forming complex leads to higher cytotoxicity than found for the free ligands. In vitro studies on human cells Molt 4/C8, CEM T-lymphocytes and L1210 have shown that for selected systems comprising ruthenium and thiosemicarbazone the cytotoxicity is increased almost 100 times relative to free ligand .
In this article we describe the modified method for synthesis of N,N-p-dimethylaminbenzaldehyde thiosemicarbazone (NmbaTSC). Recent studies have shown that this thiosemicarbazone possess some antimalarial effect . Moreover molecular modelling methods, 3D-QSAR and experimental studies have proved that it also has the ability to inhibit tyrosinase . This enzyme is necessary in synthesis of many compounds responsible for such diseases as e.g. Parkinson?s diseases . Related to this biological activity the synthesis of the complex with ruthenium(II) viz. [Ru(bpy)2(NmbaTSC)](ClO4)2 was undertaken. In this article we focus on an examination of the interaction of albumin with both the free ligand, and after coordinated to ruthenium(II) ion. Albumin is the major plasma protein, and significantly affect the pharmacokinetics and bio-distribution of drugs in the body, therefore it is important to know how strongly a potential drug can bind.
All reagents used for synthesis were purchased from Sigma-Aldrich. Organic solvents were purchased from POCH and were used without prior distillation.
N,N-p-dimethylaminbenzaldehyde thiosemicarbazone (NmbaTSC) (Fig. 2a)
The synthesis was based on the literature procedure  with some changes as outlined below. Into hot solution of N,Np- dimethylaminbenzaldehyde (4.81 g, 0.032 mol, 1 eq) in 15 ml ethanol, solution of thiosemicarbazide (3.00 g, 0.033 mol, 1.02 eq) in 20 ml ethanol was added. Mixture was refluxed under argon conditions for 3 h. After cooling down to ca. 0°C the yellow crystal needles that formed were collected by filtration and washed with cold ethanol. Crude product was crystallized from mixture ethanolwater, washed with diethyl ether and dried. Elemental analysis. Exp.: C 53.60; H 6.23; N 24.54; S 15.43; Calc.: C 54.03; H 6.35; N 25.20; S 14.42. 1H, NMR (300 MHz, CD3CN) ä 9.42 (s, 1H, HC=N), 7.83 (s, 1H, NH), 7.57 (d, J = 9.0 Hz, 2H, Har), 7.32 (s, 1H, NH2), 6.74 (d, 2H, Har), 6.62 (s, 1H, NH2), 2.99 (s, 6H, CH3). IR (cm-1): 3374 (NH2), 3331 (NH), 1600 (C=N), 1369, (C=S) Complex [Ru(bpy)2(NmbaTSC)](ClO4)2 (Fig. 2b) Into solution of silver nitrate (70 mg, 0.4 mmol, 2 eq) in 30 ml ethanol, cis-[Ru(bpy)2Cl2]×2H2O (100 mg, 0.2 mmol, 1 eq) was added. Mixture was refluxed for 30 min and white precipitate of silver chloride was removed by filtration. To the filtrated solution 0.5 ml of triethylamine and N,N-p-dimethylaminbenzaldehyde thiosemicarbazone (46.62 mg, 0.2 mmol, 1 eq) were added and refluxed under argon conditions for 3 h. Solution was concentrated under reduced pressure to volume of ca. 10 ml and cooled down in ice-bath. The saturated solution of sodium perchlorate ca.1ml was added into this solution and allowed to crystallize. Crude product was crystallized from acetonitrile. Elemental analysis. Exp.: C 44.02; H 3.22; N 14.34; S 4.11; Calc.: C 43.59; H 2.68; N 13.56; S 3.86. IR (cm-1): 1588 (C=N coordinated to Ru), 1362, (C=S coordinated to Ru) . TLC: mixture ethanol: 10% water solution of sodium chloride 7:3 Rf = 0.625. UV-Vis: (CH3CN) ?max (nm/M-1 cm-1) 242/37000, 296/58000, 370/32000, 524/8000.
Emission spectra were measured on Perkin Elmer LS55, Fluorescence spectrometer in the range between 310 and 560 nm upon excitation at 295 nm. Samples were thermostated using a circulating flow PolyScience 9106 thermostat. Absorption spectra in UV-Vis range were measured on Perkin Elmer Lambda 35 UV-Vis spectrometer equipped with PTP 6+6 thermostat. Measurements were performed in the range of wavelength from 250-800 nm. All studies were carried out at 37°C, in 0.1 M TRIS/HCl buffer pH 7.4 in the presence of 0.1 M NaCl. Both NmbaTSC ligand and its complex of ruthenium are poorly soluble in water therefore, the stock solutions of ca. 2 mM were prepared by dissolving in acetonitrile or DMSO. These solutions were further diluted in buffer to obtained appropriate final concentration. Bovine serum albumin (Sigma-Aldrich) was dissolved in buffer and its concentration was determined spectrophotometrically .
Results and discussion
Albumin is one of the major proteins occurring in the blood and it has a significant impact on the transport and bioavailability of drugs administered intravenously. Thus, it is essential to study reactivity of potential drugs towards this protein. In this study the bovine serum albumin (BSA) was used due to its low cost, high availability and structural similarities with human serum albumin. Interaction of free ligand NmbaTSC, and its complex viz. [Ru(bpy)2(NmbaTSC)] (ClO4)2 with albumin was followed using fluorescence properties of protein. Albumin contains one tryptophan residues in its structure and exhibits a significant fluorescence when excited at 295 nm. As is shown in Figures 3 and 4 both, ligand and its complex with ruthenium strongly quench fluorescence of albumin.
Titration of albumin with studied compounds leads to change a clear fluorescence maximum at 355 nm (Fig. 3 spectrum 1) into very broad band (an increasing contribution of bands at shorter over those at longer wavelength is observed), which may indicate a selective quenching of those tryptophan residues that are more exposed to the polar environment and therefore more readily available. From the literature it is known that the fluorescent properties of tryptophan residues are extremely sensitive to changes in polarity of the microenvironment, and even for proteins having only one tryptophan residue, as in the case of albumin, a complex fluorescence spectrum is observed which is a sum of different conformers present in solution. This heterogeneity is particularly evident when measuring the average fluorescence lifetime . It should also be noted that studied thiosemicarbazone also exhibits fluorescence with a maximum emission at 450 nm when excited at 295 nm. This band, however, is quite well separated from the band assigned to protein, which allows the use of these results for further analysis. The selective excitation of ligand in the presence of protein is not possible and the obtained results are sum of emission spectra of both components which does not allow its use for further analysis (results not shown). The presence of protein in solution does not alter emission maximum for ligand and the intensity is almost unchanged (Fig. 5).
To define fluorescence quenching mechanism for studied compounds the obtained results were analysed using Stern-Volmer?s equation:
where: F0 and F- fluorescence intensity of albumin in the absence and the presence of quencher, respectively, [Q] ? concentration of quencher in mol/dm3, KSV ? Stern-Volmer?s constant. As is shown in Figure 6 the plot of F0/F from quencher concentration is linear in the whole range of studied concentrations. This finding proves that only one type of quenching mechanism is observed.
N,N-p-dimethylaminbenzaldehyde thiosemicarbazone has a much higher capacity for quenching the fluorescence of albumin than its complex with ruthenium(II) ion. The calculated Stern- Volmer?s constants have values of (2.26 ? 0.05) × 105 M-1 for ligand and (1.41 ? 0.03) × 105 M-1 for ruthenium complex. Such large constants indicate that these compounds quench the fluorescence of albumin by static (i.e. formation of non-fluorescent proteincompound complex) and not dynamic (due to collisions) mechanism . The obtained Stern-Volmer?s constants define the constant for quencher-protein complex formation and in this case it concerns the formation of adducts between albumin and thiosemicarbazone or its complex with ruthenium. It must be noted that, on this basis cannot be determined neither stoichiometry nor real binding constants. They can be rather called as association constants which characterized affinity of compound to protein not including its character or stoichiometry and is often used for studies of interactions with macromolecules. It is likely, that van der Waals interactions as well as hydrogen bonds have a key role in the interaction with protein for both compounds, however for ruthenium complex which is positively charged also the electrostatic interactions have to be taken into account. The formation of coordination bonds between ruthenium and protein is rather not feasible since ruthenium complex has no labile coordination sites. All ligands are bidentate and, as depicted by spectrophotometric measurements, within 24 h of monitoring the UV-Vis spectrum remains unchanged (results not shown), which indicates its high stability. In the literature reports a similar value of the Stern-Volmer?s constant (1.4 × 105 M-1) was found for the interaction of albumin with an organometallic complex of ruthenium, having as a ligand thiosemicarbazone containing in its structure anthracene . Stronger interaction of ligand compare to its complex with ruthenium can arise from its higher hydrophobicity and better docking into protein due to its size.
The application of albumin fluorescence quenching phenomenon by various potential drugs for testing their affinity towards albumin is relatively fast and accurate method. High values of Stern-Volmer?s constants indicate very strong protein interaction either with free ligand or with its complex with ruthenium(II). Due to high concentration of albumin in the blood it is of great importance to assess quantitatively such interactions. The compounds which are strongly bound to albumin are expected to have worse bioavailability than those weaker bounded after intravenous administration. So significant reduction in affinity of ligand towards albumin through its coordination to the ruthenium ion is beneficial. Ruthenium(II) complex with N,N-p-dimethyloaminbenzaldehyde thiosemicarbazone has lower affinity to albumin then free ligand. As a result after administration of this complex it can bind to albumin weaker than free ligand, and this can lead to increased accumulation in the cells. Further studies planned for the presented ligand and ruthenium complex include examination of the cytotoxicity and in case of positive results also determination of cell targets.
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Translation into English by the Author
Michał ŁOMZIK is a student with an individual program of study at the Department of Chemistry, Jagiellonian University (now 2nd year of M. Sc.). He takes the part in a double diploma program in collaboration with the University of Orleans. Works scientifically from his first year of study, and is active in the Scientific Association of Chemistry Students at JU. Scientific interests: ruthenium complexes as potential anticancer drugs, synthesis of new ligand for ruthenium complexes. Co-author of two posters presentations at national conferences.
Małgorzata BRINDELL ? M.Sc., (Ph.D.) , graduateed from the Faculty of Chemistry, Jagiellonian University (1999). The doctoral degree she received at JU was carried out in collaboration with University of Lund (Sweden) and University of Ferrara (Italy) (2004). She is awarded 2 times by Ministry. Scientific interests: bioinorganic chemistry, anticancer drugs, interactions macromolecules-ligands. She is co-author of 1 book (published by Wiley), 14 scientific papers in international publishing and 41 orals or poster presentation at national and international conferences.
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