Ternary Phenolate-Based Thiosemicarbazone Complexes of Copper(II): Magnetostructural Properties, Spectroscopic Features and Marked Selective Antiproliferative Activity against Cancer Cells

The new diprotic ligand 3,5-di-tert-butylsalicylaldehyde 4-ethyl-3-thiosemicarbazone, abbreviated H2(3,5-t-Bu2)-sal4eT, exists as the thio-keto tautomer and adopts the E-configuration with respect to the imine double bond, as evidenced by single-crystal X-ray analysis and corroborated by spectroscopic characterisation. Upon treatment with Cu(OAc)2·H2O in the presence of either 2,9-dimethyl-1,10-phenanthroline (2,9-Me2-phen) or 1,10-phenanthroline (phen) as a co-ligand in MeOH, this thiosemicarbazone undergoes conformational transformation (relative donor-atom orientations: syn,anti → syn,syn) concomitantly with tautomerisation and double deprotonation to afford the ternary copper(II) complexes [Cu{(3,5-t-Bu2)-sal4eT}(2,9-Me2-phen)] (1) and [Cu2{3,5-t-Bu2)-sal4eT}2(phen)] (2). Crystallographic elucidation has revealed that complex 1 is a centrosymmetric dimer of mononuclear copper(II) complex molecules brought about by intermolecular H-bonding. The coordination geometry at the copper(II) centre is best described as distorted square pyramidal in accordance with the trigonality index (τ = 0.14). The co-ligand adopts an axial–equatorial coordination mode; hence, there is a disparity between its two Cu–N coordinate bonds arising from weakening of the apical one as a consequence of the tetragonal distortion. The axial X-band ESR spectrum of complex 1 is consistent with retention of this structure in solution. Complex 2 is a centrosymmetric dimer of dinuclear copper(II) complex molecules exhibiting intermolecular H-bonding and π-π-stacking interactions. The two copper(II) centres, which are 4.8067(18) Å apart and bridged by the thio-enolate nitrogen of the quadridentate thiosemicarbazonate ligand, display two different coordination geometries, one distorted square planar (τ4 = 0.082) and the other distorted square pyramidal (τ5 = 0.33). Such dinuclear copper(II) thiosemicarbazone complexes, which are crystallographically characterised, are extremely rare. In vitro, complexes 1 and 2 outperform cisplatin as antiproliferative agents in terms of potency and selectivity towards HeLa and MCF-7 cancer cell lines.


Synthesis and Chemical Identification of the Thiosemicarbazone Ligand
The thiosemicarbazone H 2 (3,5-t-Bu 2 )-sal4eT was synthesised from equimolar amounts of 3,5-di-tert-butylsalicylaldehyde and 4-ethyl-3-thiosemicarbazide through the usual single-step Schiff-base condensation reaction in refluxing absolute ethanol.The resultant light yellow solution afforded long lustrous colourless needles upon slow evaporation of the solvent under ambient conditions over a span of several days.Unlike the synthesis of pyridyl-based thiosemicarbazones [8,[13][14][15], this phenolic thiosemicarbazone was produced straightforwardly in high yield without requiring acid-catalysis (Scheme 1).The chemical identity of this ligand was ascertained through microanalysis along with electrospray ionisation (ESI) mass spectrometry.The ESI mass spectrum exhibits a parent peak at m/z = 334.3[M − H + ] − in the negative-ion mode (Figure S1) and at m/z = 336.3[M + H + ] + in the positive-ion mode, consistent with the molecular mass (M = 335.51amu).

Synthesis and Chemical Identification of the Thiosemicarbazone Ligand
The thiosemicarbazone H2(3,5-t-Bu2)-sal4eT was synthesised from equim amounts of 3,5-di-tert-butylsalicylaldehyde and 4-ethyl-3-thiosemicarbazide throug usual single-step Schiff-base condensation reaction in refluxing absolute ethanol.Th sultant light yellow solution afforded long lustrous colourless needles upon slow ev ration of the solvent under ambient conditions over a span of several days.Unlik synthesis of pyridyl-based thiosemicarbazones [8,[13][14][15], this phenolic thiosemicarba was produced straightforwardly in high yield without requiring acid-catalysis (Sch 1).The chemical identity of this ligand was ascertained through microanalysis along electrospray ionisation (ESI) mass spectrometry.The ESI mass spectrum exhibits a p peak at m/z = 334.3[M − H + ] − in the negative-ion mode (Figure S1) and at m/z = 336.3H + ] + in the positive-ion mode, consistent with the molecular mass (M = 335.51amu).

FT-IR and NMR Spectroscopic Characterisation of the Thiosemicarbazone Ligand
That H 2 (3,5-t-Bu 2 )-sal4eT exists as the thio-keto (thione) tautomer in the solid state is demonstrated by the prominent IR absorption band at 3157 cm −1 , indicative of the thio-amide ν(N-H) (Figures 1 and S2a).The associated thio-carbonyl bond is characterised by the vibrational band with a stretching frequency of 1032 cm −1 .Indeed, the absence of a vibrational band at around 2600 cm −1 due to ν(S-H) [10] excludes the possibility of the occurrence of the thio-enol tautomer.At 3320 cm −1 in the IR spectrum, an absorption band occurs that is ascribable to the N-H stretching of the terminal secondary amino group of the thiosemicarbazone.A characteristic feature of Schiff bases is the imine bond whose presence in H 2 (3,5-t-Bu 2 )-sal4eT is evidenced by the absorption at 1609 cm −1 typifying ν(C=N).The tert-buyl C-H stretches are conspicuous given their characteristic pattern of absorption bands in the range of 2867-2960 cm −1 [59].Contributing to the intensity of these absorptions are the C-H vibrations of the N-ethyl substituent group.The sharp absorption band with a stretching frequency of 3013 cm −1 is attributable to the aromatic ν(C-H).Finally, the broad band at around 3500 cm −1 is typical of phenolic O-H vibrations.
the vibrational band with a stretching frequency of 1032 cm −1 .Indeed, the absence of a vibrational band at around 2600 cm −1 due to ν(S-H) [10] excludes the possibility of the occurrence of the thio-enol tautomer.At 3320 cm −1 in the IR spectrum, an absorption band occurs that is ascribable to the N-H stretching of the terminal secondary amino group of the thiosemicarbazone.A characteristic feature of Schiff bases is the imine bond whose presence in H2(3,5-t-Bu2)-sal4eT is evidenced by the absorption at 1609 cm −1 typifying ν(C=N).The tert-buyl C-H stretches are conspicuous given their characteristic pattern of absorption bands in the range of 2867-2960 cm −1 [59].Contributing to the intensity of these absorptions are the C-H vibrations of the N-ethyl substituent group.The sharp absorption band with a stretching frequency of 3013 cm −1 is attributable to the aromatic ν(C-H).Finally, the broad band at around 3500 cm −1 is typical of phenolic O-H vibrations.The 1 H-NMR spectrum of H2(3,5-t-Bu2)-sal4eT was recorded in DMSO-d6 (Figure S3a) at a radiofrequency of 700 MHz with TMS as an internal reference standard (δ = 0).The broad peak at δ 9.98 assignable to the hydrazinic proton reveals that the thione tautomer of this thiosemicarbazone remains intact in solution.The phenolic proton, which is represented by the singlet at δ 11.27, is the most deshielded on account of its intramolecular interaction with the imine nitrogen atom.The aldimine proton is associated with the sharp singlet at δ 8.28.A broad resonance shaped like an unresolved triplet occurs at δ 8.48 and is attributable to the proton of the amino group between the thio-carbonyl and ethyl groups.The aromatic protons in positions 4 and 6 resonate as doublets at δ 7.13 and 7.29, respectively, with identical coupling constants (J = 2.38 Hz).The N-ethyl group is characterised by partially overlapping quartet signals at δ 3.59 (J = 6.55 Hz) and a triplet resonance at δ 1.16 (J = 7.14 Hz) corresponding to the methylene protons (in non-equivalent environments) and the methyl protons, respectively.Finally, the protons of the 3-tert-butyl and 5-tert-butyl substituent groups have singlet signals with the chemical shifts at δ 1.41 and 1.27, respectively.
Interestingly, when the 1 H-NMR spectrum of H2(3,5-t-Bu2)-sal4eT is measured in deuterated methanol (CD3OD), the resonances of the -OH and the two -NH protons disappear (Figure S3b), indicative of rapid exchange of each of these for deuterium on the NMR timescale.The chemical shift at δ 8.14 of the imine -CH=N singlet is the most downfield in this spectrum.The aromatic protons in positions 4 and 6 appear as doublets (δ 7.14, J = 2.38 Hz and δ 7.38, J = 2.44 Hz, respectively).The ethyl -N(H)-CH2CH3 protons  The 1 H-NMR spectrum of H 2 (3,5-t-Bu 2 )-sal4eT was recorded in DMSO-d 6 (Figure S3a) at a radiofrequency of 700 MHz with TMS as an internal reference standard (δ = 0).The broad peak at δ 9.98 assignable to the hydrazinic proton reveals that the thione tautomer of this thiosemicarbazone remains intact in solution.The phenolic proton, which is represented by the singlet at δ 11.27, is the most deshielded on account of its intramolecular interaction with the imine nitrogen atom.The aldimine proton is associated with the sharp singlet at δ 8.28.A broad resonance shaped like an unresolved triplet occurs at δ 8.48 and is attributable to the proton of the amino group between the thio-carbonyl and ethyl groups.The aromatic protons in positions 4 and 6 resonate as doublets at δ 7.13 and 7.29, respectively, with identical coupling constants (J = 2.38 Hz).The N-ethyl group is characterised by partially overlapping quartet signals at δ 3.59 (J = 6.55 Hz) and a triplet resonance at δ 1.16 (J = 7.14 Hz) corresponding to the methylene protons (in non-equivalent environments) and the methyl protons, respectively.Finally, the protons of the 3-tert-butyl and 5-tert-butyl substituent groups have singlet signals with the chemical shifts at δ 1.41 and 1.27, respectively.
Interestingly, when the 1 H-NMR spectrum of H 2 (3,5-t-Bu 2 )-sal4eT is measured in deuterated methanol (CD 3 OD), the resonances of the -OH and the two -NH protons disappear (Figure S3b), indicative of rapid exchange of each of these for deuterium on the NMR timescale.The chemical shift at δ 8.14 of the imine -CH=N singlet is the most downfield in this spectrum.The aromatic protons in positions 4 and 6 appear as doublets (δ 7.14, J = 2.38 Hz and δ 7.38, J = 2.44 Hz, respectively).The ethyl -N(H)-CH 2 CH 3 protons are observed as quartet (δ 3.70, J = 7.18 Hz) and triplet (δ 1.24, J = 7.19 Hz) signals, respectively, whereas the 3-tert-butyl and 5-tert-butyl protons occur as singlet peaks at δ 1.44 and 1.30, respectively.
The 13 C-NMR spectrum of H 2 (3,5-t-Bu 2 )-sal4eT, recorded in DMSO-d 6 at 176 MHz (Figure S4), exhibits a resonance for the thio-carbonyl carbon at δ 176.29, confirming the existence of this thiosemicarbazone as the thione tautomer in this solution.At δ 147.54, a signal occurs representing the imine carbon atom.All the aromatic carbon atoms are accounted for in the range of δ 117.75-153.11,with the phenolic carbon being the most deshielded.The signals of the N-ethyl methylene and methyl carbon atoms appear at δ 38.73 and 14.59, respectively.Finally, the tert-butyl carbon atoms have resonances in the range of δ 29.44-34.71.

Single-Crystal X-ray Structural Determination of the Thiosemicarbazone Ligand
Definitive evidence for the solid-state 3D structure of the ligand was obtained through single-crystal X-ray analysis.A colourless needle amenable to X-ray diffraction was grown from a solution of H 2 (3,5-t-Bu 2 )-sal4eT in EtOH at room temperature.X-ray data collection was performed at 100 K. Crystal data, details of data collection and parameters for structural solution and refinement are compiled in Table 1.Evidently, this thiosemicarbazone ligand crystallised in the monoclinic space group P2 1 /c with four molecules in the unit cell.The X-ray crystal structure is depicted in Figure 2 while selected bond distances and angles are presented in Table 2.The distance of the Schiff-base bond C=N [C(15)-N(1) = 1.2904( 19) Å] lies within the range observed for normal imine bonds [1.26-1.30Å] [ [13][14][15]19,20,[60][61][62][63][64][65][66][67][68][69] in non-coordinated ligands.Upon reduction in the Schiff base, the imine double bond becomes a single bond (C-N) with a distance of ~1.47 Å [65].The C(16)-S(1) distance of 1.7029( 14) Å verifies the occurrence of the thione tautomer.Literature values for the distance of the thio-carbonyl bond in free thiosemicarbazone ligands range typically from 1.65 to 1.70 Å [13][14][15]19,20,[60][61][62][63][64][65][66][67][68][69] (even longer if involved in bifurcated H-bonding) [67].Both imine nitrogen and thio-carbonyl carbon are sp 2 -hybridised and the angles around them reflect the angular (with a lone pair) and trigonal planar geometries about these two atoms.The hydrazinic N-N bond [N(1)-N(2) = 1.3854( 16) Å] is somewhat longer than most of those reported for other non-coordinated thiosemicarbazones and is consistent with single-bond character.16) in this structure) single b Thus the potential donor atoms can be positioned anti or syn relative to each other.E ples of crystallographically observed orientations of phenolic thiosemicarbazones E(syn,anti) [60,62,63], E(syn,syn) [61,64] and E(ant,anti) [63], are shown in Figure S5 structure of H2(3,5-t-Bu2)-sal4eT is consistent with the E-configuration; the phenolic group and the imine nitrogen are positioned syn to each other while the thione sul points to the opposite side in an anti-orientation relative to the imine nitrogen.16) in this structure) single bond.Thus the potential donor atoms can be positioned anti or syn relative to each other.Examples of crystallographically observed orientations of phenolic thiosemicarbazones, viz.E(syn,anti) [60,62,63], E(syn,syn) [61,64] and E(ant,anti) [63], are shown in Figure S5.The structure of H 2 (3,5-t-Bu 2 )-sal4eT is consistent with the E-configuration; the phenolic -OH group and the imine nitrogen are positioned syn to each other while the thione sulphur points to the opposite side in an anti-orientation relative to the imine nitrogen.2), respectively.The chemical formulations of these two ternary complexes were established through elemental analyses.The mass spectra of the complexes were measured in MeOH.The positive-ion ESI mass spectrum of complex 1 presented in Figure 3a shows a molecular peak at m/z = 605.4 in agreement with the molecular mass of this complex (605.25 amu).As regards the dinuclear complex (2), the parent ion was not detected; however, the ESI spectrum revealed important structural information from the fragmentation pattern.In the negative mode, the spectrum shows a minor peak at m/z = 792.5 consistent with the loss of the phen co-ligand.At m/z = 730.5, a major peak occurs that is ascribable to the fragment [Cu{H(3,5-Bu 2 )-sal4eT}{(3,5-Bu 2 )-sal4eT}] − (Figure 3b).On the other hand, the positive-ion ESI spectrum exhibits a peak at m/z = 732.6 attributable to the fragment [Cu{

FT-IR Spectroscopy and Magnetic Susceptibility Measurements
A comparison of the IR spectra of the thiosemicarbazone ligand and its copper(II) complexes (1 and 2) in Figures 1 and S2 clearly shows the absence of the vibrational band of the hydrazinic N-H bond from the spectra of the complexes.The disappearance of the hydrazinic proton coupled with the shift in the stretching frequency of the absorption band of the carbon-sulphur bond from 1032 cm −1 for the ligand to 842 and 858 cm −1 for 1 and 2, respectively, is indicative of tautomerisation and deprotonation of the thiosemicarbazone upon coordination to the copper(II) ion, as is indeed necessary for charge-neutrality of the resultant complexes.The presence of the N-H group attached to the terminal ethyl group is proven by the occurrence of sharp absorption bands at 3398 and 3342 cm −1 in the spectra of 1 and 2, respectively.The wavenumbers of the imine bond for 1 and 2

FT-IR Spectroscopy and Magnetic Susceptibility Measurements
A comparison of the IR spectra of the thiosemicarbazone ligand and its copper(II) complexes (1 and 2) in Figures 1 and S2a clearly shows the absence of the vibrational band of the hydrazinic N-H bond from the spectra of the complexes.The disappearance of the hydrazinic proton coupled with the shift in the stretching frequency of the absorption band of the carbon-sulphur bond from 1032 cm −1 for the ligand to 842 and 858 cm −1 for 1 and 2, respectively, is indicative of tautomerisation and deprotonation of the thiosemicarbazone upon coordination to the copper(II) ion, as is indeed necessary for charge-neutrality of the resultant complexes.The presence of the N-H group attached to the terminal ethyl group is proven by the occurrence of sharp absorption bands at 3398 and 3342 cm −1 in the spectra of 1 and 2, respectively.The wavenumbers of the imine bond for 1 and 2 complexes are somewhat lower than that of the free ligand [ν(C=N): 1598 and 1599 cm −1 vs. 1609 cm −1 ], consistent with coordination of the imine donor atom.Interestingly, the ν(N-N) absorptions for the ligand and complexes 1 and 2 virtually coincide (1172, 1170 and 1169 cm −1 , respectively), implying minimal delocalisation of π-electrons, if any, along the ligand backbone in the complexes.Finally, the other ligand IR absorption patterns, especially those of the tert-butyl C-H bonds (2850-2960 cm −1 ), are retained.

Single-Crystal X-ray Analyses of the Ternary Copper(II) Complexes
For the complexes [Cu{(3,5-t-Bu 2 )-sal4eT}(2,9-Me 2 -phen)] ( 1) and [Cu 2 {(3,5-t-Bu 2 )-sal4eT} 2 (phen)] (2), X-ray diffraction data were collected on a single crystal at 100 K employing Cu-Kα radiation (λ = 1.54178Å).Crystal data together with details of data collection and structural refinement are presented in Table 1.Selected bond distances and angles are given in Table 3. Whereas complex 1 crystallised in the monoclinic space group P2 1 /n with Z = 4, complex 2 did so in the triclinic space group P1 with two complex molecules in the unit cell.Both complexes 1 and 2 are free of solvent molecules of crystallisation.The crystal structure of [Cu{(3,5-t-Bu 2 )-sal4eT}(2,9-Me 2 -phen)], depicted in Figure 4, reveals that this complex exists as a centrosymmetric dimer of mononuclear molecular ternary complexes of copper(II).The dimerisation occurs via two intermolecular hydrogenbonding interactions involving the -N 4 -H group of one complex molecule and the thioenolate sulphur of the other complex molecule (Figure 4b) The charge-neutrality of this ternary complex implies that the thiosemicarbazone been doubly deprotonated upon complexation.Indeed, transformation of the ligand from the thio-keto tautomer to the thio-enolate anion is demonstrated through the changes to the lengths of the pertinent bonds of the thio-amide.The thio-amide N-C bond [N(2)-C( 16 Carbon-nitrogen bonds with double-bond character have been reported to have distances in the range 1.27-1.32Å [66,[71][72][73] when the N donor atom is coordinated to a central metal ion.On the other hand, typical lengths of carbon-sulphur bonds with single-bond character in thio-enolate complexes are in the range 1.72-1.77Å [66,[71][72][73].The distances of the imine C=N [C(7)-N(1) = 1.296(4)Å] and the hydrazinic N-N [N(1)-N(2) = 1.400(4)Å] in 1 are normal with regard to their respective bond orders.
The magnitude of the disparity in the Cu-N distances of the asymmetrically coordinated N,N-donor co-ligand is in the range of ~0.22-0.32Å.Moreover, the complex cation of tris(1,10-phenanthroline)copper(II) perchlorate [76] exhibits Jahn-Teller distortion (Cu-N bond averages: Cu-N ax ~2.33 Å vs. Cu-N eq ~2.04 Å) whereby the axial Cu-N phen bonds are elongated to the same extent as those in the above-mentioned square pyramidal copper(II) ternary complexes.In contrast, it has been crystallographically proven that the two Cu-N bonds of N,N-donor co-ligands in square pyramidal and octahedral complexes where they lie on the equatorial plane are virtually equivalent as neither is subject to the Jahn-Teller effect [54-58].In addition, in the complex [Cu{N(CN) 2 }(phen) 2 ] + [77]   The five-coordinate geometry at the copper(II) centre arises from the tridentate coordination of the thiosemicarbazonate ligand with the donor atoms, namely phenolate oxygen, imine nitrogen and thio-enolate sulphur, arranged meridionally, and the bidentate coordination of the 2,9-Me2-phen co-ligand oriented nearly perpendicularly relative to the primary ligand.The axial-equatorial coordination mode of the pyridyl nitrogen atoms of As can be seen from Figure 5, [Cu 2 {(3,5-t-Bu 2 )-sal4eT} 2 (phen)] (2) exists in the crystal lattice as a centrosymmetric dimer of dinuclear molecular ternary complexes of copper(II) stabilised mainly by two types of intermolecular forces.The linkage of two dinuclear complex molecules occurs through two H-bonds between the N 4     .The two associated thiosemicarbazonate ligands exhibit different denticities: one coordinates in a tridentate fashion to the metal centre (Cu(2) in Figure 5) with coordination number 4, whereas the other adopts the relatively unusual quadridentate coordination mode to bridge the two metal centres with the thio-enolate nitrogen atom, N(2), and coordinate meridionally to the other metal centre (Cu(1) in Figure 5).For the tridentate ligand, coordinated to Cu(2), the distance of the newly formed thio-enolate N=C bond is virtually indistinguishable from that of the imine C=N bond [cf.N( 7 9) Å] is normal for complexed thiosemicarbazonate ligands [66,[71][72][73].It is noteworthy that the lengths of the hydrazinic N-N bonds in this dinuclear complex [N(1)-N(2) = 1.381(11)Å and N(6)-N(7) = 1.391( 11) Å] are very similar to that observed in the free ligand as a thio-keto (thione) tautomer (1.3854( 16) Å), suggesting that there is no delocalisation of electrons involving this chemical bond in the thiosemicarbazonate backbone.
Their selectivity over non-cancerous cells such as the human breast epithelial cell line (MCF-10A) has yet to be determined.It is noteworthy that in vitro complexes 1 and 2 are more efficacious as antiproliferative agents than cisplatin.Moreover, cisplatin lacks the cancer-specificity that these complexes possess.They also exhibit higher potent anticancer activity than the standards.Although the study of the mode of action of complexes 1 and 2 as anticancer agents is beyond the scope of this work, it has been amply demonstrated pre-viously for a diverse range of copper(II) complexes, including those of thiosemicarbazones, that the potentials of the Cu II /Cu I redox couple [8,15,18,20,30] lie within the biologically accessible redox potential window leading to the generation of reactive oxygen species (ROS) that cause apoptotic cell death.copper(II) reacts readily with 2,9-Me2-phen to form the red copper(I) complex [Cu(2,9-Me2-phen)2] + .We are not aware of any previous studies that compared anticancer activities of ternary copper(II) complexes with binary copper(II)/bipy or copper(II)/phen complexes.However, it has recently been shown experimentally that free phen, bipy and Cu II exhibit much lower cytotoxicity towards MCF-7 cells than the relevant ternary copper(II) complexes with trien as a primary ligand [53].Finally, carefully designed series of mononuclear and dinuclear ternary phenolate-based thiosemicarbazone complexes similar to 1 and 2, respectively, are required for a systematic study of antiproliferative activity.Previous studies of such phenolate-containing thiosemicarbazone complexes of copper(II) have focused on DNA cleavage, antimicrobial activity and modelling catalytic activity of metallo-enzymes [33][34][35][36][37].The differences between the anticancer activities of complexes 1 and 2 are fascinating but complicated.Presumably, they arise from the structural differences (mononuclear vs. dinuclear) and the nature of the bidentate N,N-donor co-ligand (phen vs. 2,9-Me 2 -phen).The cause of cell death is associated with DNA cleavage, ROS generation and the ability of a drug to enter the cancer cell and cause damage.Unfortunately, comparison of this work with previous studies is limited given that [Cu 2 (sal4eT) 2 (bipy)] [4], the only closely related dinuclear complex to [Cu 2 {(3,5-t-Bu 2 )-sal4eT} 2 (phen)] (2), was not investigated for anticancer activity.Likewise, the reported crystallographically characterised ternary phenolate-based thiosemicarbazone copper(II) complexes that we are aware of were not tested for cytotoxicity against cancer cells [31][32][33][34][35][36][37][38].Recently, a paper reported the cytotoxicity of a series of 2-formylpyridyl-based thiosemicarbazone ternary copper(II) complexes with bipy, phen and their derivatives in the HeLa cell line only and observed the effect of the nature of the co-ligands [39].It is now well-established, as we also observed in this study, that the antiproliferative activity of thiosemicarbazones is enhanced on complexation with bioactive metal ions.As far as we are aware, binary copper(II) complexes of the type [Cu(R 1 ,R 2bipy/R 1 ,R 2 -phen) n ] 2+ have not been investigated for anticancer activity.In fact, copper(II) reacts readily with 2,9-Me 2 -phen to form the red copper(I) complex [Cu(2,9-Me 2 -phen) 2 ] + .We are not aware of any previous studies that compared anticancer activities of ternary copper(II) complexes with binary copper(II)/bipy or copper(II)/phen complexes.However, it has recently been shown experimentally that free phen, bipy and Cu II exhibit much lower cytotoxicity towards MCF-7 cells than the relevant ternary copper(II) complexes with trien as a primary ligand [53].Finally, carefully designed series of mononuclear and dinuclear ternary phenolate-based thiosemicarbazone complexes similar to 1 and 2, respectively, are required for a systematic study of antiproliferative activity.Previous studies of such phenolate-containing thiosemicarbazone complexes of copper(II) have focused on DNA cleavage, antimicrobial activity and modelling catalytic activity of metallo-enzymes [33][34][35][36][37].

Materials and Physical Techniques
All pertinent chemicals, reagents and solvents (HPLC/AR-grade) were purchased from Sigma-Aldrich (Burlington, MA, USA) and used as received.Microanalyses (CHN) were performed on a EuroVector elemental analyser (EuroVector, Pavia, Italy).ESI mass spectra were measured with an Agilent 6460 Triple Quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) using MeOH as the matrix.Electrical conductivities of the complexes were determined with a JENWAY 4520 conductivity meter (Cole-Parmer, Vernon Hills, IL, USA) at room temperature using freshly prepared solutions (1 mM) in MeOH, EtO, DMF and DMSO.FT-IR spectra were recorded on a Perkin-Elmer spectrophotometer (4000-400 cm −1 ) (Perkin-Elmer, Waltham, MA, USA) with the samples compressed as KBr discs using a Specac press (Specac Ltd., Orpington, UK). 1 H-and 13 C-NMR spectroscopic measurements were carried out at room temperature in DMSO-d 6 on a Bruker ASCEN 700 spectrometer (Bruker, Billerica, MA, USA) operating at a radiofrequency of 700 MHz; the chemical shifts are referenced to TMS as an internal standard (δ = 0).X-band ESR spectra of the copper(II) complexes were recorded on a Bruker ELEXSYS E580X FT CW spectrometer (ν ~9.4 GHz).Electronic absorption spectra were measured with a Shimadzu 2450 UV-visible spectrophotometer (190-1000 nm) (Shimadzu, Tokyo, Japan) using freshly prepared solutions.Magnetic susceptibility measurements were carried out at room temperature using a Sherwood Scientific magnetic susceptibility balance (Sherwood Scientific, Cambridge, UK).The magnetic data were corrected for diamagnetism using Pascal's constants in the usual way (x para = x meas − x dia ).Single-crystal X-ray structural determinations were carried out on a Bruker APEX-II CCD area-detector diffractometer or a Bruker D8 Venture CMOS Photon 100 diffractometer.The crystals were mounted in Fomblin oil and cooled in a stream of cold N 2 .Data were corrected for absorption using empirical methods (SADABS) [84] based upon symmetry equivalent reflections combined with measurements at different azimuthal angles [85].The crystal structures were solved and refined against F 2 values using ShelXT [86] for solution and ShelXL [87] for refinement (using least squares minimisation), accessed via the Olex2 programme [88].
Three controls were prepared within each 96-well culture plate: DMSO solvent as a negative control and the standards paclitaxel and docetaxel as positive controls.Approximately 10,000 viable cells were seeded per well in 96-well culture and incubated for 24 h at 37 • C and 5% CO 2 .Solutions of the compounds with different concentrations (Figure 8) were prepared in DMSO and added to each well.The cells were then incubated with the compounds for another 48 h, after which the cytotoxicity was evaluated.Initially, the cells were washed with phosphate-buffered saline (PBS) and then 20 µL of the MTT reagent (5 mg/mL) in PBS was added to each well.After 4 h of incubation at 37 • C, the culture medium was discarded, and then the purple formazan crystals were dissolved in DMSO (100 µL) in each well.The absorbance of each well was measured using a plate reader (Anthous 2020; Austria) at λ = 550 nm against a standard reference solution at 690 nm.Assays were carried out in triplicate in three independent experiments.The concentration required for 50% inhibitory activity (IC 50 ) was determined from a plot of the percentage cytotoxicity versus the concentration on a logarithmic graph.
with a distorted trigonal bipyramidal geometry at the metal centre, the phen Cu-N distances are virtually indistinguishable from each other as the Jahn-Teller effect does not apply.The copper(II) ion in [Cu{(3,5-t-Bu 2 )-sal4eT}(2,9-Me 2 -phen)] (1) is displaced out of the mean basal plane [N(1), S(1), O(1), N(4)] towards the apical phen N(5) donor atom by 0.1998(12) Å.Finally, the magnetostructural behaviour of this complex is consistent with half occupancy of the d x 2 -y 2 orbital in the ground state.
One of the prominent structural features of interest is the