Copper(I) halide complexes of 5-carbethoxy-2-thiouracil: Synthesis, structure and in vitro cytotoxicity

Accepted Manuscript

Copper(I) halide complexes of 5-carbethoxy-2-thiouracil: Synthesis, structure and in

vitro cytotoxicity

I. Papazoglou, P.J. Cox, A.G. Hatzidimitriou, C. Kokotidou, T. Choli-Papadopoulou, P.

Aslanidis

PII:

S0223-5234(14)00265-7

10.1016/j.ejmech.2014.03.052

EJMECH 6835

DOI:

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 20 September 2013

Revised Date: 13 March 2014

Accepted Date: 16 March 2014

Please cite this article as: I. Papazoglou, P.J. Cox, A.G. Hatzidimitriou, C. Kokotidou, T. Choli-

Papadopoulou, P. Aslanidis, Copper(I) halide complexes of 5-carbethoxy-2-thiouracil: Synthesis,

structure and in vitro cytotoxicity, European Journal of Medicinal Chemistry (2014), doi: 10.1016/

j.ejmech.2014.03.052.

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ACCEPTED MANUSCRIPT

Graphical Abstract

5-carbethoxy-2-thiouracil as ligand in new dinuclear and mononuclear copper(I) halide

complexes of composition [CuX(eitotH₂)₂]₂and [CuX(eitotH₂)(PPh₃)₂] respectively, with

remarkably high in vitro cytotoxic activity against human pulmonary carcinoma cells,

human fetal lung fibroblast and human epithelial carcinoma cells.

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Highlights

•

New copper(I) complexes containing 5-carbethoxy-2-thiouracil and

triphenylphosphine.

•

Remarkable in vitro cytotoxicity against two carcinoma cell lines.

The mononuclear complexes possessing triphenylphosphine are highly

cytotoxic.

•

The phosphine-free dicopper(I) complexes are only moderately cytotoxic.

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Copper(I) halide complexes of 5-carbethoxy-2-thiouracil:

Synthesis, structure and in vitro cytotoxicity

I. Papazoglou^[a], P.J. Cox^[b], A.G. Hatzidimitriou^[a], C. Kokotidou ,^[c]T. Choli-

[c]*

Papadopoulou

and P. Aslanidis^[a]*

^*[a]Aristotle University of Thessaloniki, Faculty of Chemistry, Inorganic Chemistry Laboratory

P.O. Box 135, GR- 541 24 Thessaloniki, Greece; E-mail: aslanidi@chem.auth.gr

^[b]School of Pharmacy and Life Sciences, Riverside East, Robert Gordon University, Garthdee

Road, Aberdeen AB10 7GJ, Scotland.

^*[c]Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, GR- 541 24

Thessaloniki, Greece; E-mail: tcholi@chem.auth.gr

____________________________________________________________________

Abstract

5-carbethoxy-2-thiouracil (eitotH₂) reacts with copper(I) halides CuX (X = Cl, Br, I) to give

dinuclear complexes of the formula [CuX(eitotH₂)₂]₂while mononuclear mixed-ligand complexes

of the formula [CuX(PPh₃)₂(eitotH₂)] result when the reactions are performed in the presence of

two equivalents of triphenylphosphine (PPh₃). The molecular structures of representative

compounds from each of the above types of complexes, namely [CuI(eitotH₂)₂]₂,

[CuCl(PPh₃)₂(eitotH₂)] and [CuBr(PPh₃)₂(eitotH₂)] have been established by single-crystal X-ray

diffraction. The new copper(I) complexes were evaluated for in vitro antitumor properties against

two tumor cell lines, A549 (human pulmonary carcinoma cell line) and HeLa (human epithelial

carcinoma cell line) and one normal immortalized cell line MRC5 (human fetal lung fibroblast).

The mixed-ligand complexes possessing triphenylphosphine were found to be highly cytotoxic in

contrast to the phosphine-free ones which inhibited cell proliferation only in relatively high

concentrations.

Keywords: Copper(I); 5-carbethoxy-2-thiouracil; triphenylphosphine; cytotoxicity.

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1. Introduction

The biochemical key role of copper both as an essential trace metal bound to several

metalloenzymes and as a constituent of exogenously administered compounds in humans – mainly

in form of complexes that can interact with biomolecules – is well known. Numerous copper

enzymes and proteins, many of them containing a bimetallic copper center in their active site, play

crucial roles in biological systems [1]. Thus, bimetallic copper complexes are of particular interest,

since they can serve as models for a number of important biological systems. For example, the role

of dicopper sites in electron transfer processes in the biological binding, activation and reduction of

dioxygen is a subject of intense research during the last two decades [2–5]. Current interest in

copper complexes includes their potential use as antimicrobial [6–8], anti-inflammatory [9, 10] and

antitumor [11–14] agents, however, most of the compounds tested are copper(II) complexes.

Similar studies on copper(I) derivatives are less widespread, and the complexes normally contain

planar aromatic chelating ligands [15–17] or ligands capable of stabilizing the low oxidation state

of the metal ion in aqueous media [18].

Orotic acid (6-carboxyuracil) is a biologically very important molecule, being the only

effective precursor of the pyrimidine bases for nucleic acid formation [19, 20]. For this reason,

orotic acid plus its derivatives, and metal complexes, have been the subject of intensive studies

[21–24]. On the other hand, relatively less attention has been paid to the coordination chemistry of

isoorotic acid (5-carboxyuracil), despite its anticancer, antibacterial and antihypertensive properties

[25, 26]. This is even more remarkable when the notable and promising results of the few related

studies are considered, for example, antibacterial activity was found for a copper(II) complex of

isoorotic acid [27], while a platinum(II) complex of ethyl-isoorotate showed antitumor,

antibacterial and antiviral properties [28].

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Some reports have been concerned with the biological evaluation of not only thiolated orotic

acid derivatives [29–31] but also coordination compounds with 2-thioorotic acid as ligand [32–34].

On the other hand, only a small number of reports are related to thioisoorotic acid and its

derivatives. These are quite limited to some structural and biological studies on first row transition

metal complexes containing 2-thioisoorotato ligands that appeared about two decades ago [35–38].

With these considerations in mind, we decided to include 5-carbethoxy-2-thiouracil (eitotH₂)

[shown in Scheme 1], in our investigations. In preferring the ester instead of the free 5-carboxy-2-

thiouracil we intended to ensure the well-known thione-S coordination to the soft copper(I) ion

avoiding any possible side effects, for example, oxidation of the metal. Despite its biological

importance [39], 5-carbethoxy-2-thiouracil has received little attention so far, as there are

practically no references about its coordinating behavior.

In this work we report the synthesis and characterization of copper(I) halide complexes

[CuX(eitotH₂)₂]₂and [CuX(eitotH₂)(PPh₃)₂] and the structural characterization of representative

compounds of each type. Further we investigated the cytotoxic activity of the new compounds

against two tumor cell lines, A549 (human pulmonary carcinoma cell line) and HeLa (human

epithelial carcinoma cell line) and one normal immortalized cell line MRC5 (human fetal lung

fibroblast).

(Scheme 1)

2. Experimental section

2.1. Materials for the Synthesis and Instrumentation

Commercially available copper (I) halides, triphenylphosphine and 5-carbethoxy-2-thiouracil

were purchased as reagent grade and were used as supplied, whereas the solvents were purified

according to standard procedures. Infra-red spectra in the region of 4000–200 cm^-1were obtained in

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KBr discs with a Nicolet FT-IR 6700 spectrophotometer, while a Shimadzu 160A

spectrophotometer was used to obtain the electronic absorption spectra. Melting points were

determined in open tubes with a STUART scientific instrument and are uncorrected.

2.2. Synthesis

Synthesis of complexes 1–3: To a solution of (0.5 mmol) of the copper(I) halide (49 mg for CuCl,

71.7 mg for CuBr, 95.2 mg for CuI) in 30 cm³of dry acetonitrile, a solution of 200,2 mg (1 mmol)

of 5-carbethoxy-2-thiouracil in 20 cm³of methanol was added and the mixture was stirred for 1 h at

ambient temperature. The resulting bright yellow solution was filtered off and left to stand at

ambient, whereupon yellow crystals deposited which were filtered off and dried in vacuo.

o

[CuCl(eitotH₂)₂]₂(1): Yellow powder (112 mg, 45 %), m.p. 270 C; Anal. Calc. For

C₂₈H₃₂Cu₂Cl₂N₈O₁₂S₄: C, 33.67; H, 3.23; N, 11.22. Found: C, 33.47; H, 3.12; N, 11.08. IR (cm^-1):

3119m, 3064m, 2934m, 1715vs, 1747vs, 1617s, 1562vs, 1464s, 1372m, 1293s, 1140vs, 1061m,

1010m, 802s, 746m, 584s; UV-Vis (λ_max, log ε): 264 (4.23), 311 (4.37).

o

[CuBr(eitotH₂)₂]₂(2): Yellow crystals (207 mg, 70 %), m.p. 271 C; Anal. Calc. For

C₂₈H₃₂Cu₂Br₂N₈O₁₂S₄: C, 30.92; H, 2.97; N, 10.30. Found: C, 31.17; H, 3.03; N, 10.18. IR (cm^-1):

3142m, 3050m, 2999m, 1733vs, 1622vs, 1552vs, 1529vs, 1460s, 1395s, 1302vs, 1210vs, 1145vs,

1010m, 886m, 792m, 602s, 510m; UV-Vis (λ_max, log ε): 263 (4.41), 310 (4.67).

o

[CuI(eitotH₂)₂]₂(3): Yellow crystals (221 mg, 75 %), m.p. 274 C; Anal. Calc. For

C₂₈H₃₂Cu₂I₂N₈O₁₂S₄: C, 28.46; H, 2.73; N, 9.48. Found: C, 28.55; H, 2.78; N, 9.41. IR (cm^-1):

3129m, 3050m, 2911m, 1733vs, 1617vs, 1556vs, 1524vs, 1460vs, 139v0s, 1302vs, 1210vs, 1176s,

1149vs, 1010m, 886m, 866s, 792s, 602vs, 593vs, 514vs; UV-Vis (λ_max, log ε): 264 (3.94), 311

(4.11).

Synthesis of complexes 4–6: To a solution of (0.25 mmol) of the copper(I) halide (24.5 mg for

CuCl, 35.8 mg for CuBr, 47.6 mg for CuI) in 30 cm³of dry acetonitrile, triphenylphosphine (131

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mg, 0.5 mmol) was added and the solution was stirred until a white precipitate was formed. A

solution of 50 mg (0.25 mmol) of 5-carbethoxy-2-thiouracil in 20 cm³of methanol was added and

the mixture was stirred for 1 h at room temperature. The resulting bright yellow clear solution was

filtered off and kept at ambient, whereupon pale yellow crystals of the respective product

deposited, which were filtered off and dried in vacuo.

o

[CuCl(PPh₃)₂(eitotH₂)]^.0.5 CH₃C(O)CH₃(4): Yellow crystals (134 mg, 65 %), m.p. 147 C; Anal.

Calc. For C_43.5H₄₀CuClN₂P₂O_3.5S: C, 62.67; H, 4.85; N, 3.28. Found: C, 62.47; H, 4.82; N, 3.32. IR

(cm^-1): 3050m, 2984m, 1752vs, 1723vs, 1695vs, 1616s, 1565, 1540 vs, 1480s, 1461vs, 1435vs,

1410s, 1369s, 1296vs, 1226vs, 1147vs, 1093vs, 1061m, 1030m, 796s, 742vs, 694vs, 590s, 517vs,

501vs; UV-Vis (λ_max, log ε): 262 (4.48), 310 (4.39).

[CuBr(PPh₃)₂(eitotH₂)]^.CH₃CN (5): Yellow crystals (173 mg, 80 %), m.p. 154 ^oC; Anal. Calc. For

C₄₅H₄₁CuBrN₃P₂O₃S: C, 59.44; H, 4.54; N, 4.62. Found: C, 59.17; H, 4.61; N, 4.64. IR (cm^-1):

3148m, 3049m, 2936m, 1752vs, 1730vs, 1619s, 1566, 1556vs, 1482vs, 1458s, 1435vs, 1407s,

1367s, 1293vs, 1223vs, 1150vs, 1097vs, 1061m, 1026m, 846m, 745vs, 694vs, 587s, 517vs, 498vs;

UV-Vis (λ_max, log ε): 263 (4.56), 308 (4.49).

o

[CuI(PPh₃)₂(eitotH₂)] (6): Yellow powder (176 mg, 77 %), m.p. 191 C; Anal. Calc. For

C₄₃H₃₈CuIN₂P₂O₃S: C, 56.43; H, 4.18; N, 3.06. Found: C, 56.15; H, 4.10; N, 3.01. IR (cm^-1):

3050m, 2932m, 1755vs, 1736vs, 1616s, 1556vs, 1477vs, 1457s, 1435vs, 1401s, 139v0s, 1293vs,

1226vs, 1144vs, 1093s, 1024m, 886m, 846m, 799s, 742vs, 694vs, 587s, 517vs, 501vs, 489s; UV-

Vis (λ_max, log ε): 264sh (5.11), 309 (4.88).

2.3. Crystal Structure Determination

Single crystals of 3 suitable for crystal structure analysis were obtained by slow evaporation

of the CH₃CN/CH₃OH mother liquid at room temperature. X-ray diffraction data were collected on

a Rigaku Saturn 724+ diffractometer by the NCS [40] using CrystalClear-SM Expert [41] for cell

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refinement, data collection and data reduction. The structure was solved with SHELXS97 [42] and

refined with SHELXL97. The program used for molecular graphics was PLATON [43]. Tables for

publication were prepared with SHELXS97 and WINGX [44]. Single crystals of compound 5

suitable for crystal structure analysis were obtained by slow evaporation of the CH₃CN/CH₃OH

mother liquid at room temperature, whereas compound 4 was recrystallized from acetone. For the

structure determination the single crystals of 4 and 5 were mounted on a Bruker Kappa APEX II

diffractometer equipped with a triumph monochromator. Both structures were solved using

SUPERFLIP package [45] and refined by full-matrix least-squares method on F²using the

CRYSTALS package version 14.40 [46]. In structure of 4 five of the six atoms of one of the

phosphane phenyl rings were found disordered over two positions with equal occupation factors.

The whole acetone solvent molecule was also found disordered over two positions with equal

occupation factors. The disordered atoms have been refined isotropically. All the remaining non-

hydrogen atoms of the main residue have been found and refined anisotropically. All hydrogen

atoms of the main residue were found at their expected positions and refined using soft constraints.

All hydrogen atoms connected to the disordered atoms were positioned geometrically. By the end

of the refinement all hydrogen atoms were positioned using riding constraints to the parent atoms.

The crystals data and some details of the data collection and structure refinement for both

compounds are given in Table 1.

2.4. Materials and methods for biological tests

Materials: Culture medium DMEM (Dulbecco’s Modified Eagle Medium with L-glutamin), Fetal

Bovine Serum, Antibiotic-Antimycotic and PBS (pH=7, 2) were from

E.U Gibco BRL.

Trypsin+EDTA 1x was purchased from Invitrogen. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-

Diphenyltetrazolium Bromide) powder 1g, was purchased from Invitrogen. All other chemicals

used were of high purity and available from commercial sources.

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Cell cultures: Human cell lines from three different sources, namely A549 (human pulmonary

carcinoma cell line), immortalized MRC5 (human fetal lung fibroblast) and HeLa (human

epithelial carcinoma cell line) were cultured in DMEM (Dulbecco’s modified Eagle’s medium)

supplemented with 10% fetal bovine serum and antibiotics penicillin and streptomycin. Cell line

was maintained in continuous culture at 37^oC, in 5% CO₂, in a fully humidified incubator, using

standard aseptic techniques and cell growth was monitored by determining the cell number/ml with

the use of a Coulter counter model ZBI.

MTT cytotoxicity assay: The growth inhibitory effect towards tumor cell lines was evaluated by

means of MTT assay. Cell proliferation was assessed by monitoring the conversion of 3-(4,5-

dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan. The reduction of

MTT is catalyzed by mitochondrial dehydrogenase enzyme and is therefore a measure for cell

viability [47]. 1x10⁴cells /well (A549) and 5 x 10³cells /well (HeLa and MRC5) were seeded in

96-well plates and after 24 hrs were treated with varying concentrations (5, 10, 15, 20, 30, 40, 60

and 120 ꢀM) of copper complexes and their uncoordinated ligands(5-carbethoxy-2-thiouracil and

triphenylphosphine) which were dissolved prior in DMSO.

Cells which were not exposed to copper complexes served as the control. The complexes

incubation with the cells for 48 hrs was followed by medium removal from each well and its

replacement with 100 ꢀl fresh medium and 10ꢀl MTT 12 mM (5 mg/ml) per well. After another

short incubation for 4 h at 37 °C, until the development of the purple-colored formazan product,

100 ꢀl of SDS10%-HCl 0,01 M was added to the cells and left under the same growth conditions

for 15–18 hrs.

The Organon Teknika@ Enterprise Ireland plate was used to measure the absorbance of the

plates at 570 nm. Values shown are the mean values of at least three measurements. IC50 values

represent the copper complexes concentrations that reduced the mean absorbance at 570 nm to 50%

of those in the untreated control wells.

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3. Results and Discussion

3.1. Synthesis

As expected, the interaction of heterocyclic thiones, which are versatile S- and/or N-donor

ligands, with a soft Lewis acid like monovalent copper is highly flexible, resulting in the formation

of a rich variety of coordination compounds ranging from mono- and dinuclear complexes to

polynuclear networks. In the specific case of copper(I) halides, the result of the reactions depends

on the stoichiometry and the conditions chosen, usually leading to four-coordination which is most

favoured for copper(I). Although steric demands of the ligated molecules are expected to influence

both the local and the overall structure of a coordination compound, during our previous work we

have never noticed a decrease of the coordination number in copper(I) complexes by introducing

bulky ligands. Quite to the contrary, we recently observed the formation of three-coordinated

mononuclear species [CuX(th)₂] for the sterically undemanding 2-thiohydantoin [48], which

implies that electronic interactions should also be taken into consideration.

Further, dimerization within complexes of monovalent copper halides with heterocyclic

thioamides is quite common as it allows the copper ion to obtain a pseudotetrahedral environment.

This was also the case for the reactions within the present study, which in addition turned out to be

independent from the ratio of reactants chosen (CuX: eitotH₂= 1:2 or 1:3) the composition of

compounds 1–3 being always the same, namely [CuX(eitotH₂)₂]₂.

Aiming to obtain phosphine/thione mixed-ligand complexes, we treated a 1:2 mixture of CuX

and PPh₃in dry acetonitrile with a methanolic solution of one equivalent of 5-carbethoxy-2-

thiouracil. Stirring this mixture at 50^oC produced a clear yellow solution, from which, on slow

evaporation, the corresponding desired microcrystalline compound was deposited. Interestingly,

while compounds 4–6 were easily obtained and in high yield by this simple one-top two-step

synthetic procedure, attempts to prepare complexes using compounds 1–3 as precursors remained

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unsuccessful. All prepared compounds are air stable diamagnetic solids, moderately soluble in

acetonitrile, chloroform or acetone.

3.2. Spectroscopy

The electronic absorption spectra of complexes 1–6, recorded in acetonitrile at room

temperature, show two intense broad bands with maxima at ~264 and ~310 nm. With reference to

the absorption spectrum of the uncoordinated 5-carbethoxy-2-thiouracil, the high energy band can

be attributed to intraligand π→π* transitions on the thione ligand, whereas the lower energy band

can be considered as a thione originating CT transition at the C=S bond [49, 50], which may

possess partial CT character, as it lies in the region where the free ligand absorbs, expressing a

small red shift as a consequence of the coordination to copper. In the spectra of compounds 4–6 the

absorption due to the intraligand transition within the triphenylphosphine completely overlaps with

the high energy band assigned to the intraligand π→π* transitions on the 5-carbethoxy-2-thiouracil

ligand, resulting in a broad band of increased intensity relatively to that observed in the spectra of

compounds 1–3. The absence of any d-d transitions confirms the diamagnetic nature of the

complexes.

-1

The infrared spectra of compounds 1–6, recorded in the range 4000-250 cm contain all the

characteristic bands of the 5-carbethoxy-2-thiouracil ligand with shifts indicating an exclusive S-

coordination. In particular, the intense bands at 1755 cm^-1and 1733 cm^-1in the spectrum of free

eitotH₂, attributed to ν(C=O) stretching vibrations, appear slightly shifted to higher energies in the

spectra of all complexes. On the other hand, the very strong band at 1565 cm^-1known as

“thioamide I” band, having contributions from ν(C–N) and δ(C–Η), remains almost unshifted, but

the band at 1163 cm^-1assigned to the “thioamide III”, which involves major contributions from

ν(C=S), appears clearly shifted (by ca. 20 cm^-1) towards lower energies upon coordination. Finally,

shifts of the ν(N–H) vibration band towards lower wavenumbers, in some cases accompanied by a

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broadening, indicates involvement of this group in Ν–Η^….Χ hydrogen bridges, a fact that is also

clearly confirmed by the crystal structure investigations.

3.3. X-ray structural investigations

The structures of [CuI(eitotH₂)₂]₂(3), [CuCl(PPh₃)₂(eitotH₂)] (4) and [CuBr(PPh₃)₂(eitotH₂)]

(5) (details of crystal and structure refinement are given in Table 1) were established by single

crystal X-ray diffraction.

Compound 3 crystallizes in the triclinic system Pī, with one discrete molecule in the unit cell.

Main bond lengths and angles are given in Table 2 and a perspective drawing showing the atom

numbering is shown in Figure 1. The basic structural unit is a dimer in which the two copper atoms,

separated by 3.555(1) Å, are doubly bridged by two S atoms of two thione ligands to form a strictly

planar four-membered Cu₂S₂core. The highly distorted tetrahedral coordination around each

copper is completed by the S atom of a terminally bonded thione ligand and one iodine atom.

(Figure 1)

(Table 2)

Each of the two trans positioned iodine atoms stabilize the complex with two strong

…

_

intramolecular hydrogen bonds [N(1) I(1) = 3.809(2) Å, H(1) I(1) = 2.794 Å, N(1) H(1) I(1) =

o

…

o

_

169.6 and [N(4) I(1) = 3.636(2) Å, H(4) I(1) = 2.76 Å, N(4) H(4) I(1) = 174.3 ]. Further strong

intramolecular hydrogen bonds exist between pairs of bridging and terminal thione units (see Table

2), which are essentially parallel to each other.

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Within the Cu₂S₂core, there is an extremely large asymmetry in the two bridging Cu–S bonds

distances [2.2598(9) and 2.9447(11) Å]. By comparison, the corresponding distances are 2.2727(9)

and 2.5634(10) Å in bis[ꢀ-S(pyrimidine-2-thione)(pyrimidine-2-thione)copper(I)bromide] [51] and

2.3348(9) and 2.4879(7) Å in bis[ꢀ-S(pyridine-2-thione)(tmtp)copper(I)chloride] [52]. The two

_

terminally bonded thione ligands are trans disposed with a Cu S bond distance of 2.2475(9) Å,

_ _

_

which is close to the “short” one in the Cu S Cu bridge. The Cu I bond distance of 2.5089(7) Å is

somewhat shorter than the values observed in dimeric copper(I) iodide compounds with double

bridging sulphur atoms or in monomeric copper(I) iodide complexes bearing terminally bonded

thione ligands [53, 54]. Finally, the S–Cu–S and Cu–S–Cu angles of 103.89(3) and 85.08(3)^o

respectively deviate from the ideal values (109.5 and 70.5^o) predicted for symmetric dimers,

reflecting the distortions induced by the intramolecular hydrogen bridges, but are close to the ideal

geometry for Y₂MX₂MY₂dimers according to theoretically proposed criteria [55].

Pale yellow crystals of [CuCl(PPh₃)₂(eitotH₂)] (4) and [CuBr(PPh₃)₂(eitotH₂)] (5) have been

obtained by slow evaporation at room temperature of their saturated acetone and acetonitrile

solutions respectively. Both compounds crystallize in the monoclinic system C2/c, with 4 discrete

molecules in the unit cell. Main bond lengths and angles are given in Tables 3 and 4 and drawings

showing the atom numbering are shown in Figures 2 and 3 respectively.

(Figure 2 + Figure 3)

(Table 3 + Table 4)

The geometrical characteristics of both the four-coordinate monomers generally refer to those

of other copper(I) halide complexes tetrahedrally coordinated by thione and phosphine ligands.

Three of the interbond angles around the central copper atom deviate moderately from the idealized

tetrahedral geometry while the P1–Cu1–P2 angle is opened up to a value of 122.71(3)^oand

123.47(3)^oin 4 and 5 respectively, reflecting the steric imposition of the bulky phosphine ligands.

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The Cu–S distance of 2.4083(8) Å and 2.3931(8) Å are quite large, but the Cu–Cl and Cu–Br bond

lengths as well as the two individual Cu–P distances in both complexes are comparable to the

corresponding values previously found in related four coordinate complexes with terminal chlorine,

bromine and thione-S donors [41, 56, 57].

3.4. Cytotoxicity assay

The antitumor activity of the compounds has been estimated by assessing their ability to

inhibit the growth of the tumor cells in the culture medium DMEM (Dulbecco’s Modified Eagle

Medium with L-glutamin) supplemented with 10% fetal bovine serum and the antibiotics penicillin

and streptomycin. The copper compounds 1–6 and the corresponding free ligands eitotH₂and PPh₃

were subjected to MTT assay and their cytotoxic properties were investigated against a panel of

two tumor cell lines, A549 (human pulmonary carcinoma cell line) and HeLa (human epithelial

carcinoma cell line) and furthermore in one immortalized normal cell line MRC5 (human fetal lung

fibroblast). IC50 values, calculated from the dose-survival curves obtained after 48 h complex

solution treatment from MTT are shown in Table 5.

(Table 5)

As it can be clearly seen from Table 5 and through Figures 4–9, free 5-carbethoxy-2-

thiouracil (eitotH₂) and triphenylphosphine (PPh₃) proved to be quite ineffective in all cell lines

used. In particular PPh₃gave detectable IC50 values only in higher concentrations. On the contrary,

all the tested complexes showed a growth inhibitory potency in the micromolar range towards the

different cell lines. Especially the three mixed-ligand complexes [CuI(PPh₃)₂(eitotH₂)],

[CuCl(PPh₃)₂(eitotH₂)] and [CuBr(PPh₃)₂(eitotH₂)] (Figures 5, 7 and 9) were appreciably more

effective against all cell lines and noteworthy against HeLa cell line exhibiting IC50 values of 2.55

ꢀΜ, 3.63 ꢀΜ and 3.36 ꢀΜ, respectively (Table 5). Interestingly, the copper halide complexes of 5-

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carbethoxy-2-thiouracil (compounds 1–3) are appreciably less effective towards HeLa, A549

cancer cell lines as well as against MRC5, which is a non cancer cell line, than their

triphenylphosphine containing counterparts (compounds 4–6) which are highly toxic even at low

concentrations.

Among the three homoleptic copper (I) halide complexes 1–3 the cytotoxic action follows the

order of [CuI(eitotH₂)₂]₂> [CuBr(eitotH₂)₂]₂> [CuCl(eitotH₂)₂]₂with half minimum inhibitory

concentrations (IC₅₀) ranging from 45.5–86.6 ꢀΜ for HeLa, to 54.25–89.56 for MRC5 and to 41.3–

110 for A549.

Considering the three heteroleptic complexes 4–6, the values of the half minimum inhibitory

concentrations (IC₅₀) are significantly lower ranging from 2.55–3.6 ꢀΜ for HeLa, 4.77–6.84 ꢀΜ

for MRC5 and 4.65–5.0 for A549. The in vitro cytotoxicity of all three complexes against A549

exceeds that recently reported for cisplatin by a factor of about 2 [58]. In contrast with the case of

compounds 1–3, there is no clear trend in the cell killing effect observed on going from chloro to

bromo to iodo derivative.

(Figures 4 – 9)

Currently, extensive search for anticancer agents as alternative to platinum drugs is in full

swing. In the quest for new metal-based drugs, a variety of copper-based drugs have been examined

and proved to be very promising due to their selective cytotoxicity against tumor cells [59]. The use

of phosphine containing complexes as potential anticancer agents is well documented [60–62]. In an

early study the cytotoxicity/anti-tumor activity of a large number of auronofin analogues was

evaluated [63]. It was observed that in most cases the presence of a phosphine in these gold

thiolates caused an enhanced cytotoxicity compared with the other phosphine free species.

Meanwhile, research on potential cytotoxic compounds has been focused, among others, on the use

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of copper(I) compounds bearing tertiary phosphine ligands [64–66]. It is obvious that the

particularly high cytotoxicity of the mixed-ligand complexes 4–6, compared to that of compounds

1–3, is due to the presence of the triphenylphosphine ligand. This finding is in good agreement with

earlier observations on gold(I) compounds [5] but also recently on phosphine containing mixed-

ligand complexes of copper(I) [57]. For these later copper(I) phosphine complexes the cytotoxic

activity was correlated with their ability to act as endoplasmic reticulum stress inducers, and a

nonapoptotic mechanism of programmed cell death (paraptosis) was proposed to be likely [67].

It is known that, like other established proteasome inhibitors, copper binding compounds are

only effective in inducing ubiquinated protein accumulation and apoptosis in tumor, but not in non-

trasformed cells. The anti-angiogenesis effects, as well as the potential use of proteasome inhibitors

in cancer therapies, have been extensively reviewed [68, 69]. However, the ability of some copper

binding compounds to inhibit the proteasome and induce apoptosis by themselves in copper-

enriched cancer cells is promising for their development as anticancer compounds in a nontoxic

chemotherapeutic strategy [70]. The mechanism of action of the copper complexes presented in this

work has not been elucidated at the molecular level. Investigations involving DNA breakage or

copper substitution of copper harbouring enzymes, ROS production enhancement or alteration in

copper metabolism in general need to be performed. However, our results along with recent studies

concerning copper(I) derivatives are encouraging as we can consider these new derivatives as lead

compounds to develop new potential drugs against cancer.

Conclusions

5-carbethoxy-2-thiouracil (eitotH₂) coordinates to copper(I) halides exclusively via the “soft”

thione-S atom forming dinuclear complexes of composition [CuX(eitotH₂)₂]₂with the two metal

ions in a strongly distorted tetrahedral environment, doubly bridged by thione sulfur atoms. These

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dimers are used as precursors for the preparation of mononuclear four-coordinate phosphine/thione

mixed-ligand complexes of the type [CuX(eitotH₂)(PPh₃)₂].

Evaluation of the cytotoxicity results shows that the presence of triphenylphosphine in

complexes 4–6 results in a significant increase of cytotoxic activity when compared to the activity

of the phosphine-free compounds 1–3. Moreover, the cytotoxicity of all the above complexes is

higher in all the cell lines that were tested, in comparison to that of each of the free ligands. With

reference to literature information available so far, the remarkable antitumor activity of

[CuCl(PPh₃)₂(eitotH₂)], [CuBr(PPh₃)₂(eitotH₂)] and [CuI(PPh₃)₂(eitotH₂)], which is due to the

presence of PPh₃, could be considered indicative of their ability to induce apoptosis.

Acknowledgments

We thank the EPSRC UK National Crystallographic Service at the University of Southampton for

the collection of the crystallographic data.

Appendix. Crystallographic data

Crystallographic data (without structure factors) for the structures reported in this paper have been

deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication

no. CCDC-945117 (3), CCDC-944792 (4) and CCDC-944791 (5). Copies of the data can be

obtained free of charge from the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: +44-

1223-336408; Fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk; Web site:

http://www.ccdc.ca.ac.uk).

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Scheme/Figure Captions

Scheme 1. The heterocyclic thione 5-carbethoxy-2-thiouracil (eitotH₂) used as ligand.

Figure 1. A view of compound 3 with atom labels. Displacement ellipsoids are shown in the 50%

probability level.

Figure 2. A view of compound 4 with atom labels. Displacement ellipsoids are shown in the 50%

probability level

.

Figure 3. A view of compound 5 with atom labels. Displacement ellipsoids are shown in the 50%

probability level.

Figure 4. Dose-effect survival plots for the three homoleptic complexes [1–3] against A549 cell

line 48 h after the administration of the agents. Cytotoxicity was estimated via MTT assay (each