Trithiocyanurate Complexes of Iron, Manganese and Nickel and Their Anticholinesterase Activity

The complexes of Fe(II), Mn(II) and Ni(II) with a combination of a Schiff base, nitrogen-donor ligand or macrocyclic ligand and trithiocyanuric acid (ttcH3) were prepared and characterized by elemental analysis and spectroscopies. Crystal and molecular structures of the iron complex of composition [Fe(L1)](ttcH2)(ClO4)·EtOH·H2O (1), where L1 is Schiff base derived from tris(2-aminoethyl)amine and 2-pyridinecarboxaldehyde, were solved. It was found that the Schiff base is coordinated to the central iron atom by six nitrogens forming deformed octahedral arrangement, whereas trithiocyanurate(1-) anion, perchlorate and solvent molecules are not coordinated. The X-ray structure of the Schiff base sodium salt is also presented and compared with the iron complex. The anticholinesterase activity of the complexes was also studied.


Introduction
The sodium salt of trithiocyanuric acid (ttcH 3 = trithiocyanuric acid, also named as 2,4,6-trimercapto-1,3,5-triazine (TMT)) readily forms precipitates with heavy metal ions and that is why it is used for removal of heavy metal ions from industrial wastewater. The effectiveness of heavy metal removal was widely studied by Atwood et al. [1][2][3] and other groups [4]. Removal of residual palladium and its compounds from reaction mixtures in preparation of drugs, in which palladium is used as a catalyst, is also very important [5,6].
Biological activity of trithiocyanuric compound was also evaluated as it can serve as a ligand of Toxoplasma gondii orotate phosphoribosyltransferase [7][8][9]. This enzyme is necessary for replication of the parasitic protozoan Toxoplasma gondii, which causes the disease toxoplasmosis. It was proved that trithiocyanuric acid is a better ligand for the enzyme than 5-fluorouracil and emimycin, which are used for clinical treatment of toxoplasmosis. Kar et al. prepared a series of trinuclear Ru(II) complexes of a composition [{Ru(L) 2 } 3 (ttc)](ClO 4 ) 3 , where L = 2,2'-bipyridine, 1,10-phenanthroline and arylazopyridine, which contain trithiocyanurate(3-) bridge bounding Ru(II) centers by chelating S,N donor sets of the anion [10,11]. In addition to the structural, electrochemical and spectral study, interaction of the complexes with the circular and linear forms of p-Bluescript DNA was reported. The Ru(II) complexes reduced the fluorescence intensity of both circular and linear DNA. Zn(II), Fe(II) and Mn(II) complexes with a combination of nitrogen-donor ligands and ttcH 3 were prepared and their antitumor and antimicrobial activities were assayed [12]. The IC 50 values of the Fe(II) and Mn(II) compounds turned out to be lower than those of cisplatin and oxaliplatin.
Potentially six donor atoms can be used for coordination to metal centres. It is always difficult to avoid the formation of precipitates of unknown and probably polymeric structure with metal ions in the presence of deprotonated trithiocyanuric acid. Mostly blocking ligands on metal centres must be coordinated. Despite of that, bonding properties of trithiocyanuric acid complexes were proved by single crystal X-ray analysis. In some compounds, only deprotonated trithiocyanuric acid is present as anion not bonded to central atoms [13]. Mononuclear nickel and zinc complexes with nitrogen donor ligands and trithiocyanurate(2-) bonded by S and N have been structurally characterized [14][15][16][17][18] [20,21]. The hexanuclear [{AgPPh 3 } 6 (-ttc) 2 ] complex with two parallel triazine rings held by six Cu-S bridges was characterized [22], as well as the Au(I) cluster [23] and Cu(I) polymer [24]. Trinuclear cyclopentadienyl complexes of rhodium and iridium were also reported [25,26]. Magnetic and structural studies on trinuclear copper complex with 1,3-bis(2-(4methylpyridyl)imino)isoindoline as blocking ligand and ttc were reported [27]. Pmdien (N,N,N',N'',N''pentamethyldiethylenetriamine) was proven to be a very good terdentate ligand for complexes with ttc.

Synthesis and Spectral Study
[Fe(L 1 )](ttcH 2 )(ClO 4 )·EtOH·H 2 O (1) was prepared by the reaction of iron perchlorate, Schiff base (formed in situ), and ttcNa 3 in an ethanol-water mixture. Although we expected the formation of a binuclear or polynuclear complex with a trithiocyanurate bridge, only a mononuclear Fe(II) complex was formed. Its composition was proposed on the base of elemental analysis and unambiguously confirmed by single crystal X-ray analysis. The deformed octahedral coordination of the central Fe(II) was also confirmed by Mössbauer spectroscopy (see Figure 2). The room temperature Mössbauer spectrum of 1 is composed of two doublets with the isomer shift values (0.28 and 0.14 mm s −1 ) typical of octahedral low-spin iron(II) complexes [33,34]. The doublet I with relative spectrum area A = 91.4% has a higher value of the quadrupole splitting parameter (q.s. = 0.28 mm s −1 ) than the doublet II (A = 8.6%, q.s. = 0.20 mm s −1 ). The two different values of quadrupole splitting show that there are two octahedrally coordinated iron centers with lower and higher distortion from the ideal octahedral arrangement, found in the polycrystalline material, but one arrangement is dominant. Similar spectra with two doublets were also found as a result of the octahedral arrangement distortion of the central atoms in Fe(II) complexes [12,13]. Our attempts to prepare Schiff base L 1 in solid form were unsuccessful, but finally its sodium complex Na(L 1 )ClO 4 (4) was obtained from the reaction mixture as light-yellow crystals, suitable for X-ray study. Complex 4 was also obtained by the reaction of L 1 with sodium perchlorate. Its structure is discussed hereinafter. The complex [Mn 3 (phen) 6 (ttc)](ClO 4 ) 3 (2) was prepared according to Cermakova [35]. The complex was characterized by FTIR and Raman spectroscopies, MALDI-TOF mass spectrometry, magnetic and conductivity measurements. On the basis of different techniques, the trinuclear structure of complex was proposed, where three central manganese atoms are connected by trithiocyanurate(3-) bridge.
Complex Ni 2 (L 2 )(ttcH)(ClO 4 ) 2 ·6H 2 O·EtOH (3) was prepared from Ni(bapen)(ClO 4 ) 2 (bapen = N,N'bis(3-aminopropyl)ethylenediamine) and in situ formation of macrocyclic ligand L 2 by the condensation reaction of the terminal amino groups of bapen and ethylenediamine with formaldehyde. A similar preparation of macrocyclic complexes was for example published by Comba et al. [36]. Addition of ttcNa 3 led to a formation of violet crystalline product. As our attempts to prepare crystals for X-ray analysis were unsuccessful we used mass spectroscopy to confirm the composition of 3.
The ESI − mass spectra displays intense peaks at m/z = 947 and m/z = 848, corresponding to the binuclear molecular ion with ClO 4 − adducts of composition [Ni 2 (L 2 )(ttcH)(ClO 4 ) 2 H − ] − and [Ni 2 (L 2 )(ttcH)(ClO 4 )H − ] − , respectively. The formation of perchlorate ion adducts is well known for such kinds of complex ions [31,37]. The peaks observed at lower m/z = 514, 455, 233 and 99, correspond to different fragments of the complex and its organic parts. The value of effective magnetic moment calculated per nickel(II) (μ eff = 3.31 BM) for 3 is higher than that expected for the spin only value of octahedral or pentacoordinated nickel central atoms (μ so = 2.83 BM). The higher value of the magnetic moment can be explained by a spin-orbital contribution to the spin only value. We can assume that the central nickel atoms are coordinated by four N atoms of macrocyclic ligand and by N or N, S set of donor atoms of ttcH − anion.

Anticholinesterase Activity
The anticholinesterase activity of the complexes 1-3 and Fe(II), Mn(II) and Ni(II) salts were studied. The results of the study are presented in Table 5 and in Figure 7. As it is clearly seen, the newly prepared complexes of Fe(II) and Mn(II) were more than one hundred times and Ni(II) complex one thousand times stronger inhibitors if compared with corresponding standards (FeSO 4 , MnSO 4 , NiSO 4 ).
All the complexes show low solubility in water and are well soluble in DMF and DMSO. From the composition of [Fe(L 1 )](ttcH 2 )(ClO 4 )·EtOH·H 2 O (1) proved by X-ray it is obvious that in solution, complex cation and ttcH 2 and ClO 4 anions are formed. The complex cation is very stable due to the chelating Shiff base on the iron(II) center as was demonstrated, for example, in a study of oxygen bridged [(salen)FeOFe(salen)] (H 2 salen = N,N'-bis(salicylidene)ethylene diamine) complex [38]. Also in complexes [Fe(bpy) 3 ](ttcH)·2bpy·7H 2 O and [Fe(phen) 3 ](ttcH 2 )(ClO 4 )·2CH 3 OH·2H 2 O, where bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, the strong N-N ligands prevent the coordination of ttc anion to the metal center [13]. In the case of 1, we can assume that biological activity is caused by a combined effect of the individual components presented within the corresponding mixture in the medium used.  We can assume the stability of [Mn 3 (phen) 6 (ttc)](ClO 4 ) 3 (2) from the MALDI-TOF mass spectra, where the molecular peak was found [35]. Fragments of the complex were present in the spectra but we can expect that once the ttc bridge is formed it is bonded to the metal centre. We proved this by our attempt to prepare single crystals of 2. We dissolved the complex in DMSO and added diethyl ether to induce crystallization. After two weeks, we have only obtained from the solution single crystals of the dinuclear complex [Mn 2 (phen) 4 (ttc)](ClO 4 ) (its structure will be published elsewhere), so it can be suggested that the complex undergoes dissociation but it can be considered as stable enough for biological activity testing. The complex Ni 2 (L 2 )(ttcH)(ClO 4 ) 2 ·6H 2 O·EtOH (3) was studied by ESI − mass spectroscopy and a molecular peak was found. Macrocyclic ligands form very stable complexes as it can be demonstrated on multinuclear zinc cyclen (1,4,7,10-tetraazacyclododecane) complexes with ttc bridges, which are stable in water at neutral pH [39]. In this case of 3, the biological activity is caused either by a combined effect of macrocyclic complex and ttc anion or by complex with coordinated ttc ligand.
Due to the data obtained, further investigation of the anticholinesterase activity of the prepared complexes should be done. Because of the potency of tested compounds to inhibit cholinesterases, it could be considered to design structurally related complexes as potential drugs for Alzheimer´s disease or as prophylactics in case of nerve agent or pesticide poisoning.

Materials and Methods
Safety note: Caution! Perchlorate salts of metal complexes with organic ligands are potentially explosive and should be handled with great care. The chemicals and solvents were supplied by Aldrich (St. Louis, MO, USA) and used without further purification. The C, H, N, and S analyses were carried out on an EA 1108 instrument (Fisons Instruments, Rodano, Italy). The magnetochemical data were obtained by Faraday method at 293 K using a M-25D electrobalance (Sartorius, Elk Grove, IL, USA). Hg[Co(SCN) 4 ] was used as a calibrant. The correction for diamagnetism was calculated using Pascal's constants. The transmission Mössbauer spectrum was recorded using a Mössbauer spectrometer in constant acceleration mode with a 57 Co(Rh) source. Isomer shift parameters are related to metallic iron (the calibration temperature of 300 K).
The ESImass spectra were recorded on a ZMD 2000 mass spectrometer (Waters, Milford, MA, USA). The mass-monitoring interval was m/z 10-1500. The spectra were collected using 3.0 s cyclical scans and applying the sample cone voltages 20, 30 or 40 V, at the source block temperature 80 °C, desolvation temperature 150 °C and desolvation gas flow rate 200 l/h. The mass spectrometer was directly coupled to a MassLynx data system. All m/z interpretations were based on 35 Cl and 58 Ni, respectively.
The crystallographic data for the structures 1 and 4 has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. 960842 and 960843. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).

X-ray Crystallography
X-ray data of 1 and Na(L 1 )ClO 4 (4) were collected on a SMART CCD diffractometer (Siemens, Madison, WI, USA) with Mo-Kα radiation (λ = 0.71073 Å, graphite monochromator). The crystal was cooled to 173(2) K by a flow of nitrogen gas using the LT-2A device. A full sphere of reciprocal space was scanned by 0.3 steps in ω with a crystal-to-detector distance of 3.97 cm. Preliminary orientation matrices were obtained from the first frames using SMART [40]. The collected frames were integrated using the preliminary orientation matrix which was updated every 100 frames. Final cell parameters were obtained by refinement of the positions of reflections with I > 10σ (I) after integration of all the frames using SAINT software [40]. The data were empirically corrected for absorption and other effects using the SADABS program [41]. The structures were solved by direct methods and refined by full-matrix least squares on all |F 2 | data using SHELXTL software [42].

Conclusions
Metal based complexes play important roles in numerous applications, including drugs. Their effects on enzyme pathways can be reversible and/or irreversible, which is of great interest for physicians, because such compounds can help alter disease-connected pathways. In this study, we prepared and characterized complexes of Fe(II), Mn(II) and Ni(II) with a combination of Schiff base, nitrogen-donor ligand or macrocyclic ligand and trithiocyanuric acid (ttcH 3 ). Besides their structural characterization, their effect on anticholinesterase activity was also examined.

Author Contributions
Pavel Kopel synthesized complexes, participated in design and coordination of the study and drafted manuscript. Karel Dolezal characterized complexes using mass spectroscopy. Vratislav Langer characterized complexes using X-ray crystallography. Daniel Jun participated in testing of biological activity of the complexes. Kamila Kuca participated in preparation of the manuscript and in the design of biochemical experiment. Rene Kizek participated in design and coordination of the study. Vojtech Adam participated in design of study and in drafting the manuscript.