Cobalt ( III ) Complexes with Thiosemicarbazones as Potential anti ‐ Mycobacterium tuberculosis Agents

Crystallographic data (excluding structure factors) for the structures in this work were deposited in the Cambridge Crystallographic Data Centre as supplementary publication numbers CCDC 988971 (1·H2O) and 988972 (5·MeOH). Copies of the data can be obtained, free of charge, via www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. E-mail: deposit@ccdc.cam.ac.uk.


Introduction
Tuberculosis (TB) still causes the death of millions of people every year, more than any other disease around the world, as described by the World Health Organization (WHO). 1 The pathogenicity of Mycobacterium tuberculosis (MTB), the human pathogen responsible for TB, is based on the development of methods to survive inside host cells, comprising the capacity to take possession of macrophages. 2Despite a 95% efficacious 6-month treatment, the TB problem is still expanding world-wide. 3t's estimated that one-third of the world's population is infected with dormant forms of MTB, 10% of which will develop the disease among their lives. 4The actual research on TB has been focused on the increased number of multidrug and extensively drug-resistant TB (MDR-and XDR-TB), 5 especially in HIV-positive patients, giving rise to very high mortality. 6The number of promising anti-TB drugs following pre-clinical tests has increased and although they involve diverse possible mechanisms, no one targets dormant bacteria, which means that the latent infections cannot be eliminated by the moment. 7Therefore, Vol. 25, No. 10, 2014   it is mandatory to develop new anti-MTB agents that can solve the current therapy problems and inhibit the growth of pathogenic microorganisms in their latent forms.
3][14] Cobalt is an essential element for life being the main component of Vitamin B12, which is an essential micronutrient that is required for human health and, more importantly, is required in large quantities by cells that are replicating DNA prior to cell division. 15][18][19][20] The interest on the thiosemicarbazones (TSCs) and their metal complexes is due to their versatile chemistry 21 and pharmacological activities, [22][23][24][25][26] which also include anti-TB properties. 11Frequently, the biological activity presented by the free thiosemicarbazone ligands is enhanced upon complexation. 27In an effort to select lead candidates for treatment of TB, in the last few years we have studied and reported complexes derived from thiosemicarbazones which presented a high anti-MTB activity. 11Precisely, V IV and V V , 8 Ni II 14 and Mn II complexes 28 derived from 2-acetylpyridine thiosemicarbazones have shown excellent activity against MTB.In this context, we focused our interest in a continuation of the previous studies with some of the first transition metal row by developing now new Co III compounds, cationic instead of neutral complexes as the Ni II and Mn II compounds studied before. 14,28Hence, believing in the high potential of such compounds as anti-MTB agents and the biochemical features of cobalt, here we describe the preparation of Co III complexes derived from 2-acetylpyridine-thiosemicarbazones, their full characterization as well as the study of their anti-MTB activity and cytotoxicity.

Experimental
Materials 2-Acetylpyridine, thiosemicarbazide, 4-methyl-3-thiosemicarbazide, 4-ethyl-3-thiosemicarbazide, 4-phenyl-3-thiosemicarbazide and analytical reagents grade chemicals and solvents were obtained commercially and used without further purification.4-Cyclohexyl-3thiosemicarbazide was prepared as previously described. 27he ligands Hatc, Hatc-Me, Hatc-Et, Hatc-Ch and Hatc-Ph were prepared by refluxing equimolar ethanolic solutions containing the desired thiosemicarbazide (10 mmol) and 2-acetylpyridine (10 mmol) for 1 h, as reported elsewhere. 29,30struments FTIR spectra were measured as KBr pellets on a Shimadzu IR Prestige-21 spectrophotometer between 400 and 4000 cm −1 .Elemental analyses were determined using a Leco Instrument, model Truspec CHNS-O.The conductivities of the complexes were measured in 1 × 10 -3 mol L -1 MeOH or H 2 O solutions using an Orion Star Series conductometer.UV-visible (UV-Vis) spectra were measured with a Shimadzu UV-1800 spectrophotometer in MeOH solutions.The electrochemical experiments were carried out at room temperature in acetonitrile containing 0.1 mol L -1 tetrabutylammonium perchlorate (PTBA) (Fluka Purum) as supporting electrolyte, using an electrochemical analyzer μAutolab III.Cyclic voltammetry experiments were performed with a glassy carbon (CG) working stationary electrode, a platinum auxiliary electrode and an aqueous Ag/AgCl reference, carried out with a rate sweep of 100 mV s -1 or 200 mV s -1 .The 1 H NMR spectra were acquired using equipment Agilent 400/54 Premium Shielded 9.4 T, working at 399.8 MHz for 1 H.The NMR spectra were internally referenced to TMS.

Crystal structure determination
Brown crystals of [Co(atc)  The structures were solved with SHELXS97 using direct methods, 31 and all non-hydrogen atoms were refined with anisotropic displacement parameters with SHELXL97. 32he hydrogen atoms were calculated at idealized positions using the riding model option of SHELXL97. 32Table 1 presents more detailed information about the structures determination.

Determination of MICs
The anti-MTB activity of the compounds was determined as MIC (minimal inhibitory concentration) values by the REMA (Resazurin Microtiter Assay) method according to Palomino et al. 33

Cytotoxicity assay
In vitro cytotoxicity assays (IC 50 , half maximal inhibitory concentration) were performed first on VERO epithelial cells (ATCC CCL81).Following this approach, the most selective compound (higher SI) was additionally tested on the J774A.1 (ATCC TIB-67) murine macrophage cell line.Both studies were recorded as reported before in a previously work. 28

Selectivity index
The selectivity index (SI) was calculated by dividing IC 50 for VERO cells by the MIC for the pathogen; if SI ≥ 10, the compound is considered suitable for further investigations. 28

Preparations
The Co III complexes were synthesized by adding 0.25 mmol CoCl 2 •6H 2 O to solutions of 0.5 mmol of the desired ligands in EtOH (15 mL).The resulting solutions were stirred for 2 h under reflux.The solutions were kept under slow evaporation at room temperature until brown precipitates were formed.After 3 days the solids were filtered off, washed with hexane and dried under vacuum.

Synthesis of the complexes
Reactions of CoCl 2 •6H 2 O with two equivalents of Hatc-R in EtOH under reflux for 2 h results in microcrystalline precipitates of the cobalt complexes 1-5 in good yields (Scheme 1).Elemental analyses are consistent with the formation of cationic complexes [Co(atc-R) 2 ] + , in accordance with the observed molar conductivity values.All the compounds except [Co(atc-Ph) 2 ]Cl are water soluble.They are very soluble in methanol and dimethyl sulfoxide and sparingly soluble in dichloromethane and chloroform, demonstrating a high hydrophilic character.

Infrared, UV-Vis and 1 H NMR spectroscopies
The IR spectra of the TSC ligands are characterized by strong broad ν(NH) absorptions in the range 3365-3153 cm −1 .One of them, around 3300 cm -1 , is absent Scheme 1. Synthesis of the Co III complexes.
in the spectra of the Co III cationic complexes, according to the monodeprotonation of these ligands.
The ν(C=N) stretching band found around 1580 cm −1 for the free Hatc-R is observed in the 1552-1620 cm -1 range for the complexes.The ν(N-N) band at higher frequencies in the IR spectra of the complexes, between 1035 and 1078 cm -1 , comparing to those observed for the ligands, in the 989-995 cm -1 range, confirms coordination through the azomethine nitrogen atom. 13,34he ν (C=S) bands appear in two regions (1118-1074 cm -1 and 800-846 cm -1 ) for the free thiosemicarbazones, 35 while for the complexes the C=S only one band is observed (752-775 cm -1 ), indicating coordination through the sulfur atom and being consistent with the deprotonation and consequent formation of a C-S single bond in the thiosemicarbazone ligands. 11The IR absorption bands assigned for the free ligands and their cobalt complexes are consistent with the tridentate coordination of the thiosemicarbazone derivatives in a N,N,S-tridentate mode, through the sulfur atom, the azomethine nitrogen and the pyridine nitrogen atoms, forming octahedral complexes.
7][38][39] The spectra of the Co III complexes show the pyridine ring transitions, with the n→π* occurring at higher energies, below 300 nm, confirming the complexes formation. 40Additional bands in the 360-400 nm range are assignable as combinations of d→d transitions with S→Co III and Py→Co III charge transfer transitions. 40herefore, it was not possible to see the Co III 1 T 1g ← 1 A 1g and 1 T 2g ← 1 A 1g allowed transitions usually observed in the visible region. 41e 1 H chemical shift values of the free ligands were previously reported. 8,30,35The Co III complexes show similar 1 H NMR behavior, with the hydrogen signals being found as expected.The NH 2 hydrogens of complex 1 are found at 7.90 ppm as a broad singlet, while the spectra of the complexes 4 and 5 showed the NH signals at 8.86 and 10.37 ppm, respectively.For the complexes 2 and 3, however, two different signals relative to NH hydrogen atoms were observed, at 8.81 and 8.16 ppm for 2 and at 8.86 and 8.17 ppm for 3.This is in accord with the non-equivalence also observed for the methyl and ethyl groups, suggesting that rotation around the C−NHR (R = -CH 3 , −CH 2 CH 3 ) bond is not totally restricted. 42In fact, the methyl groups attached to the NH fragments are also observed at different chemical shifts for 2, one at 2.97 ppm and another one around 2.85 ppm, overlapped with the signal for the methyl group of the CH 3 −C=N moiety.The integration for this broad peak (9 H) is consistent with this overlapping.In the case of the complex 3, however, this observation is difficult to confirm with certainty due to the overlapping of CH 2 peak by residual water, while the methyl group appears as a broad multiplet.For the complexes 1-5, the methyl group (CH 3 −C=N) is observed in the 2.80-2.99ppm region.The aromatic protons are observed between 7.08 and 8.18 ppm for all the complexes.

Crystal structures
ORTEP drawings of complexes 1•H 2 O and 5•MeOH with numbering scheme are represented in Figure 1.Crystal data and structure refinement for both compounds are depicted in Table 1.The cobalt complexes are monocationic, presenting a chloride as counter ion.The thiosemicarbazone ligands are coordinated to the Co III center in N,N,S-tridentate mode and monoanionic form through the pyridine nitrogen atoms N(1A) and N(1B), azomethine atoms N(2A) and N(2B) and sulfur atoms S(1A) and S(1B).
The Co III complexes are clearly characterized by the smaller bond lengths compared to Mn II [Mn(atc-Et) 2 ] compound previously reported. 28This fact is assigned to the change of the oxidation state +II to +III, resulting in a larger attraction of the electrons from donor atoms of the ligand.This fact is easily observed comparing the distances of Co(1)-N(1) and Mn-N(1) 1.952(3) e 2.2806(15) Å, respectively.The distance Co(1)-S(1A) = 2.2035(6) Å is also shorter compared to Mn-S(1A) = 2.5216(5) Å distance.Furthermore, the bond distances C(8A)-S(1A), 1.745(3) and 1.745(2) Å for complexes 1•H 2 O and 5•MeOH, respectively, are consistent with a single bond character.On the other hand, the bond distance N(3A)-C(8A), 1.321(4) Å in 1•H 2 O and 1.314(3) Å in 5•MeOH, shows a double bond character, in accordance with the deprotonation of the TSCs ligands.
The coordination geometry around the metal center is a distorted octahedron with the tridentate thiosemicarbazone ligands perpendicular to each other with N(1A)-Co(1)-N(2B) being close to 90º in both complexes.A quite smaller distortion of the octahedral angles is observed for the Co III complexes when compared with similar Mn II compounds. 28This fact can be observed through the bond angle N(2B)-M(1)-N(2A) that is around 159º in [Mn(atc-Et) 2 ] and 178º in the Co III complexes studied here.The bond lengths are similar to those found for other similar Co III complexes. 43Selected data of interatomic distances and main angles can be found in Table 2.
The crystal structure of 5•MeOH is stabilized by intermolecular hydrogen bonds, as shown in Figure 2. The nitrogen atom N(4A) is H-bonded through H(4a) to the oxygen atom O(1c) from a methanol molecule, while the nitrogen atom N(4b) is H bonded with a chloride ion Cl (1), which also interacts with a solvate molecule.The interactions build a zigzag alignment of the species parallel to the c axis.
The crystal of 1•H 2 O is built up by intermolecular hydrogen bonds in diverse directions (see Supplementary Information), which involve the NH 2 groups, water solvate molecules and chloride ions.

Electrochemical studies
All complexes presented a similar CV behavior, showed exemplarily for 3 in Figure 3.One irreversible process and two well-defined quasi-reversible (i pa /i pc ≈1) waves are detected.The irreversible peak around 1.2 V is assigned as an oxidation process involving the TSC ligand, as previously reported for a similar compound. 44The two cathode processes correspond to the Co III /Co II and Co II /Co I couples, while the two anodic processes correspond to the Co I /Co II and Co II /Co III couples.The complexes presented here have an electrochemical behavior similar to that observed for other Co III complexes already reported. 44hrough the results depicted in Table 3, it is possible to observe the inductive effects of the R group bonded to the N(4) atom of the thiosemicarbazone ligand on the redox potential values.The electron donating group (R = Ch) tends to provide the more negative potential (E 1/2 = −1.10V) while the electron withdrawing group  (R = Ph) shifted the process to a less negative potential (E 1/2 = −0.70V), according to the order: −cyclohexyl < −ethyl < −hydrogen < −methyl < −phenyl relative to Co III /Co II couple.In this context, the process relative to the Co II /Co I couple presents the same trend, demonstrating the same behavior to the first couple.In relation to the second redox pair Co II /Co I the lower half-wave potential is equal to −1.57V (complex 4) and the higher potential is E 1/2 = −1.46V (complex 5).Finally it is evidenced that phenyl stabilizes better the oxidation state +II, while the cyclohexyl group, with electron donating effect, reaches the oxidation state +III easier than the other groups.
We previously reported a similar trend with Mn II complexes relative to oxidation of Mn II /Mn III and Mn III /Mn IV 28 with four of those ligands.However the influence of the groups bonded to N(4) atom observed for Mn II complexes is different for each redox pair.By comparing the values found for manganese complexes, it is possible to conclude that cobalt compounds oxidize much more easily than the manganese ones, since cobalt complexes are stabilized in oxidation state +III.

Biological activity
The biological activity of the compounds was verified by determining the values of MIC against strains of Mycobacterium tuberculosis H 37 Rv ATCC 27294.Synthetic compounds with MIC ≤ 12.5 μg mL -1 are considered of interest to be further evaluated in cytotoxicity tests, which were primarily evaluated using normal epithelial cells (VERO).Complex 5, with SI ≥ 10 (SI = IC 50 /MIC) for VERO cells, was further analyzed against macrophages cells J774A.1 (immunologic system cells).
The biological results (anti-MTB activity and cytotoxicity against VERO cells) are shown in Table 4. Two cobalt complexes present MIC ≤ 12.50 μg mL -1 , 4 and 5, with values of 2.41 μmol L -1 and 9.87 μmol L -1 , respectively.Complex 4 presented a similar activity as the free Hatc-Ch ligand (MIC = 2.82 μmol L -1 ) 27 while complex 5 was more active than the free Hatc-Ph ligand (MIC = 57.75μmol L -1 ), 27 in this case improving the activity by complexation.In the other cases the complexation to the Co III didn't lead to improvement on the activities in relation to the free ligands.The cobalt salt CoCl 2 •6H 2 O was not effectively active (MIC > 105 μmol L -1 ) showing that the activity of the complexes cannot be merely associated to the presence of the metal ion.Complex 5 presented quite low cytotoxicity against VERO cells and therefore was also investigated on macrophages cell line J774A.1 (IC 50 = 988.79μmol L -1 ) resulting in high selectivity (SI = 100).
Ni II and Mn II structurally related compounds studied before 14,28 showed to be more active in vitro than the Co III analogs studied here.This fact can be explained by the increased polarity of the ionic Co III compounds compared with the neutral Ni II and Mn II complexes, which can influence the permeability through the lipid layer of bacterial membrane resulting in a lower cellular inflow of the active species. 45Otherwise, the cationic cobalt complexes are very selective and also more water soluble than the neutral nickel or manganese species, which could enhance their absorption in vivo, compensating the eventual lower cellular permeation.
The high SI found for complex 5 shows its potential for clinical use, with a wide difference between the concentrations regarding the activity on the pathogen and the cytotoxicity on normal epithelial VERO cells, respectively.Furthermore, at the concentration the complex is active on the pathogen it remains innocuous front the

Table 3 .
Cyclic voltammetry for the redox couples Co III /Co II and Co II /Co I for all four complexes, measured in acetonitrile with 0.1 M PTBA as the electrolyte