N,N´-substituted thioureas and their metal complexes: syntheses, structures and electronic properties

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Introduction
The acylthiourea group is a versatile and readily synthesised functional group which has been utilised in synthesis and incorporated into various molecules, including co-ordination complexes.Such systems have attracted significant attention due to these complexes having potential applications in medicinal chemistry as pesticides, 1 bacteriocides, 2,3 antiviral, 4 antihelmintic, 5 antifungal 6 and antimalarial 7 compounds.Additionally, specific studies have shown a variety of acyl thiourea compounds to be extremely important in a biological context as they are, for example: (i) selective inhibitors of platelet-derived growth factor (PDGF) receptor; 8 (ii) potent inhibitors of the Hedgehog signaling pathway; 9 (iii) a c-Met (RTK or HGFR) inhibitor, for deactivating mechanisms by which various tumours and cancer stem cells promote angiogenesis and metastasis; 10 and (iv) an HDAC8 activator in non-catalytic HDAC8 mutants for research models of cohesinopathies. 11n addition to these biological applications, the structural rigidity of the acylthiourea group and its potential as a multiple hydrogen bond donor has prompted investigations into its behaviour as an anion binder, 12 including via a multi-array of hydrogen bond donors, 13 and as a ligand for the construction of polynuclear complexes. 14n its role in co-ordination chemistry the acylthiourea group is potentially a bidentate ligand, co-ordinating to metal ions via both the hard oxygen and soft sulfur donor atoms. 15,168] When an additional donor atom (e.g.pyridine) is placed on the thiourea framework, the ligand can then bind in a bidentate fashion in both cases, as shown in Figure 1, with the structures observed being dependent on the nature of the metal ion.
With widespread and important biological activity, it is clearly advantageous to be able to modify these acylthiourea derived ligands and understand their co-ordination chemistry to a variety of metal centres.Clearly a better understanding of the nature of these complexes and properties is of potential value.Herein, we present an extensive structural and spectroscopic investigation into a series of metal complexes that incorporate This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins the acylthiourea moiety.Importantly, we report interesting reactivities that can result in ligand cleavage and rearrangements which will have significant implications in the design and biological application of such systems in the future.

Ligand Synthesis
Two series of substituted thiourea derived ligands were prepared.The synthesis of L 1a -L 3a and L 1b -L 3b required the preparation of the benzoyl isothiocyanate and pivaloyl isothiocyanate precursors through the reaction of the corresponding acid chloride with potassium thiocyanate.The target ligands L 1a -L 3a and L 1b -L 3b were obtained by reaction of either 2-amino-5-methyl pyridine or 2,6-diaminopyridine with benzoyl isothiocyanate or pivaloyl isothiocyanate in acetonitrile (Scheme 1).The purification and recrystallisation approaches for these various ligands are described in the Experimental Section.All samples were gently heated under vacuum prior to elemental analyses.

Co-ordination chemistry
The chelating properties of the new ligands were investigated using a variety of Cu(II), Ni(II), and Zn(II) sources.Schemes 2-7 provide the reaction conditions for the proposed complex formulations.Table 1 also summarises the isolated complexes and their respective ligand formulations obtained in this study.For example, reaction of the different ligands with Cu(ClO4)2.6H2O in a 2:1 stoichiometry was carried out in DMF/water at room temperature, yielding Cu(I) complexes of L 1a and L 1b (1 and 15), L 2a and L 2b (6 and 18), L 3a and L 3b (12 and  22).In all cases, analytical, spectral and crystallographic data Please do not adjust margins Please do not adjust margins (discussed later) indicated the reduction of the source Cu(II) to form [Cu(L)2](ClO4) species.These Cu(I) complexes were isolated as red, orange or yellow solids, and were stable at room temperature.In the cases of L 3a and L 3b , when the reaction was repeated using a metal:ligand ratio of 1:1, Cu(I) complexes 11 and 21 were successfully isolated in the form of [Cu(L)](ClO4).Interestingly, using CuCl2 instead of Cu(ClO4)2.6H2Owith L 2b resulted in a Cu(II) complex, 19, where the ligand had undergone a transformation to give L 4 (Scheme 6).The characterization of 19 revealed that the pyridylthiourea group of the ligand had undergone a Hugershoff reaction 19,20 .Previously, the oxidation of pyridylthioureas by bromine has been reported to yield the related heterocyclic 1,2,4thiadiazolo[2,3-a]pyridine 21,23 and here it is assumed that CuCl2 acts as an oxidant that promotes the rearrangement of the ligand.We postulate that formation of L 4 , which results in the loss of the thiourea donor, is subsequently less suitable for supporting a Cu(I) centre.The analysis of 19, supported by X-ray crystallographic studies (see below), suggests the Cu(II) ion is four co-ordinate with two trans arranged monodentate L 4 ligands and two co-ordinated chloride ions.While not in the scope of this current work, clearly future studies will address the limitations of this reaction with related ligands and varying conditions.
Reaction of the ligands with Ni(ClO4)2.6H2O in a 2:1 ratio were initially undertaken at 75 C in CHCl3/methanol.Characterisation of these Ni(II) complexes indicated cleavage (these coordinated ligands are thus referred to as L 1c , L 2c , L 3c ) of one or two of the benzoyl or pivaloyl groups (see Scheme 1).
Repeating the reaction at 50 C prevented cleavage of these groups and gave the anticipated complexes.The Ni(II) complexes were collected as brown or green coloured solids and were stable at room temperature.
Mononuclear Zn(II) complexes were formed as colourless powders by the reaction of the ligands with Zn(ClO4)2.6H2O(2:1 molar ratio) in a mixture of CHCl3/methanol at room temperature and 75 C.As observed with the Ni(II) coordination chemistry, the Zn(II) complexes again formed with cleavage (giving complexes of L 1c , L 2c , L 3c ) of one or both acyl moieties from the co-ordinated ligands.all were tetrahedral and the metal ion was coordinated by the pyridyl group and the sulfur donor.Typical for this class of ligand, intermolecular hydrogen bond interactions were also observed.More specifically, and typical of acyl thioureas, hydrogen bonding between the C=O and an N-H group results in the formation of a six-membered ring (N2−H2 ... O1 and N22−H22 ... O21).Furthermore, there are additional weaker intermolecular hydrogen bonds between the ligand and the perchlorate counter anion.

X-ray crystal structure of [Zn(L 1c
)2](ClO4)2 (4) Monoclinic colourless crystals of 4 were obtained by vapour diffusion of diethyl ether into an acetonitrile solution of 4 (Figure 3).Selected bond distances and bond angles are given in Table 3.The complex crystallises in the monoclinic space group P21.The Zn(II) ion displays a tetrahedral geometry, with bond angles about the metal centre ranging from 99.33(14)-115.51(18).The variation from tetrahedral geometry is less pronounced that that seen in 1 and 15.The metal ion in 4 is coordinated by two bidentate ligands via pyridine and sulfur donor atoms.The structure clearly shows that the ligand has hydrolysed in the reaction to give L 1c and benzoic acid.There are extensive intermolecular hydrogen bond interactions between the complex and the perchlorate counter-ions.One interaction is between a thiourea, acting as a bidentate hydrogen bond donor, and a perchlorate (N1−H1b ... O22, N2−H2 ... O23) and the other interaction is a perchlorate ion hydrogen bonding with two separate thioureas, each acting as a monodentate donor (N1−H1a ... O31 i and N11-H11b i… O32) were observed between the thiourea N-H and the perchlorate oxygen atom.(Symmetry transformation to generate O31 i : x+1,y,z)

X-ray crystal structure of [Cu(L 2a )2]ClO4 (6)
Please do not adjust margins Please do not adjust margins Monoclinic dark orange crystals of 6 were obtained by vapour diffusion of diethyl ether into an acetonitrile/methanol (2:1) solution of the complex.The molecular structure of the complex [Cu(L 2a )2]ClO4 (6) was established by X-ray crystallography and is illustrated in Figure 4, along with key bond lengths and angles in Table 4.The Cu(I) centre is chelated by two L 2a ligands resulting in a distorted structure with three short Cu-S or Cu-N bonds and one longer Cu-O bond (2.3703(16) Å).Of note, L 2a and L 1a are similar except for the substitution of the pyridine ring, with L 1a having a methyl group in the 5-position and L 2a an amino group in the 6-position.These groups play no direct role in the co-ordination of the metal centre, and the amino group does not appear to be involved in any strong hydrogen bonding interactions, suggesting the differences between the two structures, 1 and 6, appears to be due to the differing steric demands of the two ligands.More specifically, the greater steric demand of L 2a results in the ligand having two different coordination modes in 6.One ligand co-ordinates to the metal in an identical manner to that observed in 1, while the second ligand also co-ordinates in a bidentate manner, but through the sulfur and oxygen donors.In this co-ordination mode, the pyridine nitrogen is now forming an intramolecular hydrogen bond with the thiourea NH groups (N21-H21 … N23).
Presumably, the soft Cu(I) centre forms the Cu(I)-O interaction in the absence of other possible softer donors.The Cu-O bond is significantly longer than the other Cu-donor interactions and this 3+1 interaction might be considered as a trigonal planar structure or as a highly distorted tetrahedral geometry.

X-ray structures of [Ni(L 2c )2](ClO4)2•H2O•EtOH (7)
Monoclinic red coloured crystals of 7 were obtained by vapour diffusion of diethyl ether into an ethyl acetate solution of 7. The selected bond distances and bond angles are given in Table 5 and the structure of 7 is shown in Figure 5.The complex contains two bidentate ligands, each demonstrating loss of the benzoyl group.The ligands again co-ordinate through the pyridine ring and sulfur atom of the thiourea.The Ni(II) complex has a slightly distorted square planar geometry: the torsion angle between the four donors range from 13.4-14.5(5)    (Symmetry transformations used to generate equivalent atoms: Cl1ii: x,-y+1/2,z+1/2; Cl1iii: -x+2,y-1/2,-z+1/2   8.The metal centre (Figure 8) is co-ordinated by a single ligand that acts as a tridentate ligand through a nitrogen pyridine ring and the sulfur atoms of the two thioureas.The Cu(I) centre is approximately trigonal planar, although the bond angles around Cu(I) (107.40 (18), 107.69 (18) and 144.87(7)°) highlight the distortion caused by the strain in the sixmembered chelate rings.This geometry is not uncommon for Cu(I), and an analysis of monodentate homoleptic donor sets with Cu(I) has revealed a significant propensity to form trigonal planar complexes, 25 and examples exist for Cu(I) trigonal planar complexes with a NS2 donor set. 26,27

Spectroscopic Characterization of the Complexes 1 H and 13 C{ 1 H} NMR Spectroscopy
The 1 H and  34-180.65 ppm, respectively.In the complexes, these resonances shift significantly indicating coordination of the ligands to the metal centre via the S donor of the thiourea group. 13C{ 1 H} NMR spectra for complexes 4, 7, and 9 showed the loss of the C=O signals consistent with the cleavage of the benzoyl/pivaloyl groups from those ligands.

Infrared spectra
IR spectra were recorded in the solid state.The spectra of the free ligands (L 1a -L 3b ) showed characteristic bands at 3298-3485 (for N-H), 1672-1692 (C=O), 1468-1512 (C=N) and 1325-1366 cm -1 (C=S).The IR spectra of thiourea complexes (1-24) showed these absorption bands at 3267-3485 (N-H), 1636-1692 (C=O), 1439-1504 (C=N) and 1252-1287 cm -1 (C=S).In particular, a weakening of the C=S bond upon co-ordination is consistent with these observations.The IR spectra of complexes of L 2a (6 and 8) show considerable lowering of the C=O stretching frequency (~1660 cm -1 cf ~1670 cm -1 ), suggesting co-ordination with the metal centre through the carbonyl group, which is consistent with solid state the X-ray structure for 6.Complex 20 has a C=O stretching frequency of 1664 cm -1 suggesting that this ligand binds in a similar manner (bidentate through O and S).For complexes 4, 7, and 9, the most striking change is the loss of the C=O indicating cleavage of the benzoyl and pivaloyl groups from the complexes.The complexes 1-3, 5, 10-17, 18 and 21-23 show little change in C=O stretching frequency when compared to the corresponding free ligand, implying the carbonyl group is non-co-ordinating for these complexes.The presence of two characteristic bands at 1053-1096 and 621-627 cm -1 indicated that the Td symmetry of ClO4 -is maintained in all cationic complexes and thus implies non-co-ordinating ClO4 -. 28,29

Electronic spectra
The UV-vis.spectra of the ligands L 1a -L 3b and their complexes were recorded as DMF solutions and their full data are presented in the Experimental Section.Spectra of the free ligands L 1a -L 3b showed strong absorption bands at λmax 214-293 nm and 299-367 nm which are attributed to the presence of allowed ligand-centred π → π* transitions.The corresponding spectra for the d 10 Cu(I) and Zn(II) complexes are thus dominated by the ligand-centred bands in the UV region, with minor shifts noted upon coordination.In contrast, the spectra for the square planar Ni(II) complexes 2, 3, 7, 8, 17 and 20 also display weaker additional bands between 390-421 nm (ca.400 M -1 cm -1 ) and around 584-612 nm (< 100 M -1 cm -1 ).The latter of these bands is likely to be the d(xy) → d(x 2 -y 2 ) transitions which is in good agreement with previously reported Ni(II) complexes. 30,31The spectra of the six co-ordinate, pseudo octahedral Ni(II) complexes 13 and 23 also showed two very weak d-d bands in the visible and near-IR region, assigned to the 3 A2g→ 3 T1g(F) and 3 A2g→ 3 T2g transitions, respectively.From these ligand field spectra it was possible to deduce (assuming Oh geometry) Dq, B and β parameters, of 1068 cm -1 , 855 cm -1 and 0.82 for 13, and 1050 cm -1 , 875 cm -1 and 0.84 for 23.

Magnetic susceptibility measurements
The observed magnetic moments of Ni(II) complexes 2, 3, 7, 8, 17 and 20 were measured using the Evan's method and suggest a diamagnetic square planar structure, while the magnetic moments for complexes 13 and 23 are 2.94 and 2.98 BM and, though lower than might be expected, are more consistent with a pseudo octahedral geometry at Ni(II).The magnetic moment of complex 19 is 1.80 BM, consistent with a Cu(II) oxidation state.typically showing a quasi-reversible process between -0.20 to -0.45 V (vs Fc/Fc + ) with large peak-to-peak separations being observed (200-350 mV).This potential is typical for a Cu(I)/Cu(II) couple and the peak to peak separations, being much greater than those observed with the ferrocene internal standard, suggest a quasi-reversible nature.This is possibly due to a structural/geometric rearrangement induced between the Cu(I) and Cu(II) oxidation states and their resultant geometric preferences.Furthermore, it was noted that the application of high negative potentials for prolonged periods caused a sharp anodic peak between -0.5 to -0.7 V suggestive of a species being deposited upon the electrode.All Cu(I) complexes except 16 showed an irreversible oxidative process in the range +0.520 to +1.08 V. Interestingly, 19 was unique among these copper complexes as the voltammogram displayed a single reversible process at +0.250 V vs Fc/Fc + ; (ΔE = 72 mV).To the best of our knowledge, the electrochemical properties of the heterocycle within 19 has not been reported and therefore we tentatively assign the signal at +0.250 V to this moiety.The cyclic voltammograms of the Ni(II) complexes 2, 3, 7, 8, 13, 17, 20 and 23 revealed two irreversible reductions at -1.11 to -1.34 V and -1.63 to -1.99 V vs Fc/Fc + .Similar behaviour has been reported by Saad et al. for the Ni(II) complex of bis(6benzoylthiourea-2-pyridylmethyl)(2-pyridyl methyl)amine which showed two similar irreversible reductions at -1.37 V and -1.78 V in acetonitrile. 13][3][4][5][6][7] However, once coordinated to the metal centre, this group may become less accessible.Therefore we performed preliminary studies to ascertain the biological activity of some selected complexes.Affecting some 150-300 million people and causing the death of approximately 425,000 in 2015, malaria is one of the most important diseases of the developing world. 34With concerns regarding the recent treatment failure of, and now resistance to, artemisinin combination therapies, there is an urgent demand to identify novel chemotypes to target this devastating disease.Thus, a selection of complexes was tested here, using the Malaria Sybr Green I Fluorescence assay 33 including the Ni(II), Cu(I) and Zn(II) salts of L 1a , L 2a and L 3b .The tabaluated results (Table 3) show the 50% Effective Concentration (EC50) and the 95% Confidence Intervals (CI) for each selected compound.The EC50 value is therefore the concentration of compound required to inhibit growth of the intraerythrocytic asexual stage of Plasmodium falciparum by 50%.The biological testing results should be placed in context with previous discussion on the hydrolysis of the thiourea ligand in the zinc complexes which may not, therefore, allow a direct comparison between the different coordinated metals.

Electrochemical measurements
From the preliminary data it is clear that the EC50 values of all compounds were > 1 μM, with complex 22 demonstrating the most significant activity (EC50 = 1.2 μM).Overall, the results suggest that the more labile d 10 complexes of Zn(II) and Cu(I) yield more active complexes, whereas the Ni(II) variants of all ligands appear to be less potent among the series of compounds.For example, comparison of 1 vs 3 and 6 vs 8 shows that for a given ligand the Cu(I) species is much more active.Interestingly, comparison of 21 and 22 suggest that doubling the molar equivalents of the ligand (from 1:1 to 2:1) within the complex increases potency again alluding to the biological activity of the ligand.However, while we wished to compare these activities of the complexes against the free ligand, the low solubility of the ligand once dilituted in aqueous media meant such a comparison would not be valid.Overall, these preliminary studies suggest that in the d 10 complexes the C=S group may be more biologically accessible via partial or complete dissociation of the complex suggesting an avenue for further study.

Conclusions
In this study, six new N,N'-disubstituted thiourea derivatives (L 1a -L 3b ) and the products resulting from their reactions with Cu(I), Cu(II), Ni(II) and Zn(II) yielded complexes (1-24).The synthesis and reactivity of the ligands has been explored revealing a range of metal-ligand interactions as well as varied reactivity, including controllable cleavage of ligand substituents, and heterocycle formation which can be promoted and controlled by the choice of metal ion source.Xray crystallographic studies confirm the versatility of these ligands to form stable complexes with a range of transition metal ions.Of particular interest is the flexible binding modes of the ligand and how these are supported by commensurate Hbonding within the structure.Ease of cleavage of the acyl groups from the ligand is dependant on the metal ion coordinated: no cleavage was observed with Cu(II/I), where as Ni(II) facilitates cleavage at raised temperature and Zn(II) causes cleavage at room temperature.It is conceivable that ligands with such behaviour may, in future, be incorporated into metal ion sensing molecules or for thermally sensitive drug delivery devices.

Please do not adjust margins
Please do not adjust margins Experimental All reagents and solvents were of reagent grade quality and obtained from commercial suppliers, and used without further purification.Elemental analyses for carbon, hydrogen and nitrogen were performed on London Metropolitan University.Magnetic susceptibilities were determined at room temperature (20 C) using the Evans Method. 34Electronic spectra were recorded using a Shimadzu UV-1800 UV spectrophotometer with complexes were dissolved in CH3CN unless otherwise stated.IR spectra were carried out with a Shimadzu IR AFFINITY-1S.Electrospray mass spectroscopy (ESI-MS) was measured on a Waters LCT Premier XE (oa-TOF) mass spectrometer.The 1 H and 13 C NMR spectra were obtained on a Bruker AC 250 instrument using CDCl3, DMSO-d 6 and acetonitrile-d 3 as solvents.Cyclic voltammetric measurements were performed using a PARSTAT potentiometer with a single compartment three-electrode cell using a platinum disk (2 mm diameter) working electrode and a platinum wire auxiliary electrode with a a non aqueous Ag/AgNO3 reference electrode.The ferrocene/ferrocenium couple was used as an internal reference at the end of each set of measurements.All experiments were carried out under an atmosphere of dry nitrogen.The concentration of electroactive species was approximately 1.5  10 -3 M with tetrabutylammonium hexafluorophosphate (0.1 M) as the supporting electrolyte.

Synthesis of N-((5-methylpyridin-2-yl)carbamothioyl)benzamide (L 1a )
The ligand was synthesised by a modification to a previously described method. 40To a suspension of potassium thiocyanate (3.89 g, 40 mmol) in acetonitrile (40 cm 3 ) was added dropwise a solution of benzoyl chloride (5.62 g, 40 mmol) in acetonitrile (10 cm 3 ).The reaction mixture was heated to reflux for 3 hrs.After this time, the mixture was a yellow solution with a white precipitate.The mixture was filtered to remove the white KCl precipitate.The yellow solution was added to a solution of 2amino-5-methyl pyridine (4.32 g, 40 mmol) in acetonitrile (15 cm 3 ) and the reaction mixture was heated to reflux for a further 15 hrs.The solution was left to cool and the resultant white precipitate was collected by filtration.This product was washed with acetonitrile (30 cm 3 ) and purified by recrystallization from chloroform:ethanol (1:1) to obtain white crystals (yield: 3.7 g, 85%).Melting point = 158-159 ⁰C.

Synthesis of N-((5-methylpyridin-2-yl)carbamothioyl)pival amide (L 1b )
To a suspension of potassium thiocyanate (0.81 g, 8.3 mmol) in acetonitrile (15 cm 3 ) was added dropwise the solution of trimethyl acetyl chloride (1 g, 8.3 mmol) in acetonitrile (10 cm 3 ).The reaction mixture was heated to reflux for 3 hrs.The mixture was filtered to remove the white KCl precipitate.The yellow solution was then added to a solution of 2-amino-5-methyl pyridine (0.9 g, 8.3 mmol) in acetonitrile (5 cm 3 ) and the reaction mixture was heated to reflux for 21 hrs.The solution was concentrated to half of its original volume and the white precipitate of product was collected by filtration.The product was washed with cold acetonitrile (5 cm 3 ) and purified by recrystallization from ethanol (yield: 0.78 g, 87%).Melting point = 86-88 ⁰C.

Synthesis of the complexes 1 -24
CAUTION: Perchlorate compounds of metal ions are potentially explosive especially in presence of organic ligands.Only a small amount of material should be prepared and handled with care.All yields are calculated with respect to the moles of metal ions isolated from the reaction mixture.

Synthesis of [Cu(L 1a )2]ClO4 (1)
A solution of Cu(ClO4)2.6H2O(0.185 g, 0.5 mmol) in H2O (3 cm 3 ) was added to a solution of L 1a (0.272 g, 1.0 mmol) in DMF (4 cm 3 ).The mixture was stirred at room temperature for 3 hrs.The colourless solution turned orange and formed a precipitate.The resultant orange precipitate was filtered, washed with CHCl3 (20 cm 3 ) to remove unreacted ligand and dried under vacuum.Red crystals of 1 were grown at room temperature by the diffusion of diethyl ether vapour into an acetonitrile solution (yield: 0.25 g, 70 %). 1

Figure 7 .
Figure 7.The asymmetric unit of 19.Displacement ellipsoids are shown at 50% probability.X-ray structure of [Cu(L 3b )](ClO4) (21) Monoclinic yellow crystals of 21 were obtained by vapour diffusion of diethyl ether into an ethanol:dichloromethane (3:1) solution of 21.Selected bond lengths and angles are given in Table8.The metal centre (Figure8) is co-ordinated by a single ligand that acts as a tridentate ligand through a nitrogen pyridine ring and the sulfur atoms of the two thioureas.The Cu(I) centre is approximately trigonal planar, although the bond angles around Cu(I) (107.40(18),107.69(18) and 144.87(7)°) highlight the distortion caused by the strain in the sixmembered chelate rings.This geometry is not uncommon for Cu(I), and an analysis of monodentate homoleptic donor sets with Cu(I) has revealed a significant propensity to form trigonal planar complexes,25 and examples exist for Cu(I) trigonal planar complexes with a NS2 donor set.26,27

Table 1 .
Summary of the metal complexes isolated in this work.

Table 2 .
Selected bond lengths and angles for structures 1 and 15.

Table 3 .
Selected bond lengths and angles for structure 4.

Table 4 .
Selected bond lengths and angles for structure 6.

Table 5 .
Selected bond lengths and angles for structure 7.

Table 6 .
Selected bond lengths and angles for structure 16.

Table 7 .
Selected bond lengths and angles for structure 19.

Table 8 .
Selected bond lengths and angles for structure 21.
1H NMR spectra for these complexes also show the disappearance of the proton signals of the phenyl or tert-butyl groups in complexes 2, 4, 5, 7, 9, 10 and 14.