We prepared four representative square planar gold(III) complexes - [AuCl3(Hpm)], [AuCl2(esal)], [AuCl(dien)]Cl2 and [Au(en)2]Cl3 -and characterized them both in the solid state and in solution. Thereafter, the cytotoxicity of these compounds was evaluated in vitro against the A2780 human ovarian tumor cell line that was used as the reference cell line. Remarkably, ali these gold(III) complexes showed significant cytotoxic effects, [AuCl2(esal)] showing a potency comparable to cisplatin. The present gold(III) complexes were also tested on the corresponding cisplatin-resistant line and revealed they were able to overcome resistance to cisplatin to a large extent. The implications of these findings for the development of new gold(III) complexes to be tested as antitumor agents are discussed

Annalisa Guerri

Bruno Bruni

Giordana Marcon

Luigi Messori

Pierluigi Orioli

English

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ISSN-0011-1643CCA-2572 Author's ReviewStructure and Cytotoxic Properties of Some SelectedGold(III) Complexes*Bruno Bruni, Annalisa Guerri, Giordana Marcon, Luigi Messori,and Pierluigi Orioli**Department of Chemistry, University of Florence, Florence, ItalyReceived November 26, 1998; revised February 23, 1999; accepted May 13, 1999Gold(III) complexes, isostructural and isoelectronic with plati-num(II) complexes, are of potential interest as antitumor drugs.We prepared four representative square planar gold(III) complexes– AuCl3(Hpm), AuCl2(esal), AuCl(dien)Cl2 and Au(en)2Cl3 –and characterized them both in the solid state and in solution.Thereafter, the cytotoxicity of these compounds was evaluated invitro against the A2780 human ovarian tumor cell line that wasused as the reference cell line. Remarkably, all these gold(III) com-plexes showed significant cytotoxic effects, AuCl2(esal) showing apotency comparable to cisplatin. The present gold(III) complexeswere also tested on the corresponding cisplatin-resistant line andrevealed they were able to overcome resistance to cisplatin to alarge extent. The implications of these findings for the develop-ment of new gold(III) complexes to be tested as antitumor agentsare discussed.Key words: gold complexes, X-ray structure, antitumoral compounds,cytotoxicity.INTRODUCTIONThe success of cisplatin in anticancer chemotherapy has raised great in-terest in the study of metal complexes as possible antitumor agents. Themain goals of the intense research activity carried out in the field are theCROATICA CHEMICA ACTA CCACAA 72 (2–3) 221¿229 (1999)* Dedicated to Professor Boris Kamenar on the occasion of his 70th birthday.** Author to whom correspondence should be addressed. (E-mail: orioli@cerm.unifi.it)following: i) search for new metal based anticancer drugs; ii) understandingof their mechanism of action.Within this frame we have synthesized in our laboratory a number ofgold(III) complexes to be tested for antitumor activity in vitro. The choice ofgold(III) derives from the fact that this ion gives rise to complexes that areisoelectronic and isostructural with those of platinum(II), whose antitumoractivity is well established.1 Surprisingly, in spite of the strict chemicalsimilarity, only very few literature data exist on the use of gold(III) com-plexes as anticancer agents. The present knowledge of the antitumor prop-erties of gold(III) complexes was summarized by Sadler.2 From the few dataavailable, and by analogy with the case of platinum(II) complexes, it is hy-pothesized that the biological action of gold(III) compounds is mediated by adirect interaction with DNA.2 For instance, trichloropyridine gold(III) wasreported to react with a number of different conformations of pBR322 DNAand to produce interstrand cross links and single strand breaks;3 Et3PAuBr3is known to bind Hind III/Ncil, a 139 base pair restriction fragment frompBR322.4For a preliminary evaluation of the cytotoxic properties of these com-pounds, we prepared four representative gold(III) complexes – viz. trichloro(2-pyridylmethanol) gold(III), AuCl3(Hpm),5 dichloro(N-ethylsalicylaldiminato)gold(III), AuCl2(esal),5 trichlorodiethylendiamine gold(III), AuCl(dien)Cl26and trichlorobisethylendiamine gold(III), Au(en2)Cl3,7 – characterized their222 B. BRUNI ET AL.c) [AuCl(dien)]Cl2222Cl-AuClN NNH H2+H3Cl-22 22AuNN NN3+H HH Hd) [Au(en)2]Cl3AuClClNCOHCH2CH3b) AuCl2(esal)a) AuCl3(Hpm)AuClClClNOHFigure 1. Schematic drawings of the complexes: a) AuCl3(Hpm), b) AuCl2(esal),c) AuCl(dien)Cl2, and d) Au(en)2Cl3.chemical properties in the solid state and in solution and evaluated theircytotoxic activity in vitro towards the A2780 human ovarian carcinoma cellline.All the mentioned complexes essentially correspond to a square planargeometry but have different sets of donor atoms (Figure 1). The AuCl3(Hpm)contains three chlorides coordinated to the gold(III) center; AuCl2(esal)contains two coordinated chlorides, AuCl(dien)Cl2 a single coordinatedchloride and Au(en2)Cl3 no coordinated chlorides. In all cases, the remain-ing donors are nitrogen atoms – except for AuCl2(esal), which has a nitro-gen and an oxygen – belonging either to amine groups or to heterocycles.Thus, one of the main goals of the present research is to establish whetherany direct correlation exists between the nature of the gold(III) ligands andthe cytotoxic properties of these compounds.EXPERIMENTALSynthesis of the CompoundsThe gold(III) complex AuCl3(Hpm) was synthesized according to the reportedprocedure:5 a solution of 2-pyridylmethanol in water was added dropwise to a stirredsolution of NaAuCl4 and sodium chloride in water at 0 °C. A yellow solid,AuCl3(Hpm), separated immediately; the stirring went on for 45 minutes. The pu-rity of the resulting products was tested through elemental analysis. TheAuCl2(esal) was synthesized according to the reported procedure as well:5 a solu-tion of NaAuCl4 in water was cooled to about –5 °C. To this solution, N-ethylsalicyl-aldimine dissolved in methanol was added dropwise under stirring during 5–10 min.The mixture became cloudy mustard-brown and was allowed to stand in the refrig-erator for a week. The upper aqueous layer was decanted from the dark oil whichhad formed. Chlorobenzene was added to the oil, much of which dissolved to give apurple solution. This solution was evaporated in vacuum to a small volume and thenaddition of diethyl ether gave a green-blue precipitate of the complex AuCl2(esal).The AuCl(dien)Cl2 was prepared according to Ref. 6; the Au(en2)Cl3 was preparedaccording to Ref. 7.X-ray CrystallographyAll data for AuCl3(Hpm) and AuCl2(esal) were collected on an Enraf-NoniusCAD4 diffractometer with graphite-monochromated Cu-K radiation ( = 1.54180 Å)at room temperature. Cell parameters were obtained from 25 well-centered reflec-tions by using least-square refinement. The absorption correction was done with theprogram PSISCAN included in the Enraf-Nonius Structure Determination Package.8The structures were solved using direct methods and successive difference Fouriersynthesis. All non-hydrogen atoms were refined anisotropically and hydrogen atomswere placed in the calculated position and refined isotropically. The calculationswere carried on with the SIR929 and SHELXL9310 programs.CYTOTOXIC GOLD(III) COMPLEXES 223CytotoxicityFor cytotoxicity studies, the human ovarian carcinoma A2780/S cell line wasused. The cisplatin resistant A2780/R cell line was produced by exposure of the sen-sitive parental cell lines to incremental concentrations of cisplatin and was main-tained in culture with 10 and 50 M CDDP, respectively. Cell lines were maintainedin RPMI 1640 medium supplemented with fetal bovine serum and antibiotics at 37°C in a 5% CO2 atmosphere and subcultured twice weekly. Experiments were con-ducted on exponentially growing cells. The SRB assay was conducted in 96-wellplates using RPMI 1640 + 5% FBS. The SRB assay was performed according to theprocedure described by Skehan et al.11RESULTSStructural Description of the Investigated Gold(III) ComplexesThe crystal structures of AuCl(dien)Cl2 and Au(en)2Cl3 were previ-ously solved by other groups and are available in the literature.6,7 The crys-tal structures of AuCl3(Hpm) and AuCl2(esal) have been solved in ourlaboratory and published separately.12,13 ORTEP views of these latter com-plexes are shown in Figure 2. All gold(III) complexes considered in this stu-dy essentially exhibit a square planar geometry with small distortions fromregularity. Au–Cl and Au–N distances fall in the expected range Au–Cl (av)= 2.274 Å; Au–N (av) 2.031 Å. In AuCl3(Hpm) the coordination planemakes an angle of 73.0° with the pyridine ring, a position that minimizesthe repulsion energy. The coordination plane in AuCl2(esal) is not coplanar224 B. BRUNI ET AL.Figure 2. ORTEP diagrams of the complexes: a) AuCl2(esal) and b) AuCl3(Hpm).The ellipsoids show 50% of probability.with the aromatic moiety of the salicylaldiminate ligand either, the anglebeing 152°. Interestingly enough, in AuCl(dien)Cl2 gold(III) coordination iscompleted by two chlorides at 3.12 and 3.18 Å.Solution PropertiesThe Au(en)2Cl3 and AuCl(dien)Cl2 are fairly soluble in water; in con-trast, AuCl3(Hpm) and AuCl2(esal) are poorly soluble in water but dis-solve easily in DMSO. The stability of the four complexes in a physiologicalbuffer was investigated spectrophotometrically. In fact, all complexes ex-hibit characteristic ligand to metal charge transfer bands in the visible re-gion, which allow direct monitoring of the gold(III) chromophore. It is ob-CYTOTOXIC GOLD(III) COMPLEXES 225Figure 3. UV spectra of Au(en)2Cl3 and AuCl(dien)Cl2 in physiological buffer a) atmixing, and b) after one hour.served that Au(en)2Cl3 and AuCl(dien)Cl2 are stable for several hourswhen dissolved within a physiological buffer (Figure 3); in contrast,AuCl3(Hpm) and AuCl2(esal), which are quite stable in DMSO, undergoquick transformation and degradation when dissolved in the same buffer.14Cytotoxic PropertiesThe cytotoxic properties of the four complexes were analyzed with re-spect to the A2780 human ovarian carcinoma cell line, either sensitive or re-sistant to cisplatin. The results are shown in Figure 4 and Table I. All com-plexes exhibit significant cytotoxic properties with IC50 values falling in the5–15 micromolar range. The order of potency shown by the four complexes isthe following: AuCl2(esal) > Au(en)2Cl3 > AuCl(dien)Cl2 > AuCl3(Hpm).226 B. BRUNI ET AL.0,01 0,1 1 10 100020406080100Au-HpmAu-dienAu-enAuesal[drug] x 10Cellulargrowth(%ofcontrol)vs.6Figure 4. Cytotoxicity curves of the various gold(III) complexes.TABLE IIC50 values for the listed complexes: average data of 3 experimentsAu(Hpm) Au(esal) Au(dien) Au(en)2 CDDPA2780/S 10.1  1.0 2.1  0.7 8.2  1.3 8.4  1.1 1.6  1.3A2780/R 21.0a 3.8  1.4 18.7  3.0 17.0  6.0 16.1  8.7Ratio R/S 2.08 1.81 2.28 2.02 10.06a 2 experiments only.Remarkably, all complexes exhibit favorable cytotoxic properties toward thecisplatin resistant cell line. The decrease in sensitivity to gold(III) com-plexes when passing from the A2780S to the A2780R line is on average lessthan a factor two whereas sensitivity to cisplatin decreases by more thanone order of magnitude.DISCUSSION AND CONCLUSIONSGold(III) complexes, isostructural and isoelectronic with Pt(II) com-pounds, are potentially attractive as antitumor agents. Given the strict si-milarity to cisplatin, gold(III) complexes are of interest for two main rea-sons: on the one side, they may constitute a new class of anticancercompounds with a novel profile of antitumor activity; on the other side, theyrepresent a further attempt to elucidate the mechanism of action of antitu-mor d8 square planar metal complexes, which still remains largely unknown.In this context, it is of fundamental importance to determine to what extentgold(III) compounds are cytotoxic and to define their structure-function re-lationships. Previous investigations had already revealed favorable cyto-toxic properties for some gold(III) complexes; however, the data present inthe literature are still very scarce, probably as a consequence of the high re-activity of gold(III) complexes.In any case, the evaluation of the cytotoxic properties of a series ofgold(III) compounds synthesized in our laboratory has provided satisfactoryand even surprising results. Indeed, analysis of the activity profiles sug-gests that in some cases the intrinsic cytotoxicity of the present compoundsis comparable to or only slightly lower than that of cisplatin (IC50 values inthe 1–10 M range). In some cases, these compounds exhibit a cytotoxic ac-tion that even exceeds that of cisplatin itself. Since all the investigated com-plexes exhibit IC50 values roughly falling within a single order of magni-tude, it may be ruled out that there is a direct correlation between thenumber of gold-coordinated chloride groups and the cytotoxic effects. Infact, either AuCl(dien)Cl2 or Au(en)2Cl3 bearing only a single or no goldcoordinated chloride exhibit cytotoxic properties comparable to those ofAuCl3(Hpm) and AuCl2(esal) with two or three gold(III) coordinated chlo-rides. Such a finding allows us to state that the presence of one or moregold(III) coordinated halides is not an essential requirement for cytotoxcityin gold(III) complexes. Apparently, more complex effects are responsible forthe observed biological acitivity. In addition, it has been recently reported14that the gold(III) complex Au(cyclam)(ClO4)2Cl containing four nitrogendonor atoms is virtually devoid of cytotoxicity toward the same tumor cellline. Even the favorable cytotoxic properties of AuCl2(esal) are a surpriseif one considers the high chemical reactivity of this compound. Indeed, un-CYTOTOXIC GOLD(III) COMPLEXES 227der physiological conditions, AuCl2(esal) readily hydrolyzes and partiallytransforms into a reduced gold(I) species. The fact that the measured cyto-toxicity is nevertheless relevant may imply that this complex either re-mains in part as a gold(III) species and is quickly taken up by cells as suchor that one of its metabolites is highly cytotoxic.Acknowledgment. – Cassa di Risparmio di Firenze is gratefully acknowledged fora generous grant in support of this research.REFERENCES1. R. J. Puddephatt, The Chemistry of Gold, Elsevier, Amsterdam, 1978.2. P. J. Sadler and O. M. Ni Dhubhghaill, in: B. K. Keppler (Ed.), Metal Complexes inCancer Chemotherapy, VCH, New York, 1993, pp. 221–228.3. P. J. Sadler, M. Nasr, and V. L. Narayanan, in: E. B. Douple and I. H. Krakhoff(Eds.), Platinum Coordination Complexes in Cancer Chemotherapy, Nijhoff Pub-lishers, Boston, 1984, pp. 290–304.4. B. Ward and J. C. Dabrowiak, J. Am. Chem. Soc. 109 (1987) 3810–3811.5. A. Dar, K. Moss, S. M. Cottrill, R. V. Parish, C. A. McAuliffe, R. G. Pritchard, B.Beagley, and J. Sandbank, J. Chem. Soc., Dalton Trans. (1992) 1907–1913.6. B. P. Block and J. C. Bailar, J. Am. Chem. Soc. 73 (1951) 4722–4725.7. G. Nardin, L. Randaccio, G. Annibale, G. Natile, and B. Pitteri, J. Chem. Soc.,Dalton Trans. (1979) 220–222.8. B. A. Frenz, Enraf-Nonius Structure Determination Package; Enraf-Nonius Delft,Holland, 1985.9. M. C. Burla, M. Camalli, G. Cascarano, C. Giacovazzo, G. Polidori, R. Spagna, andD. Viterbo, J. Appl. Crystallogr. 22 (1989) 389–394.10. G. M. Sheldrick, SHELXL 93. A Program for Structure Refinement, University ofGöttingen, Germany, 1993.11. P. Skehan, R. Stroreng, D. Scudiero, A. Monks, J. Mcmahon, D. Vistica, J. T. War-ren, H. Bokesch, S. Kenney, and M. R. Boyd, J. Natl. Cancer Inst. 82 (1996) 1107–1112.12. B. Bruni, M. Ferraroni, P. Orioli, and G. P. Speroni, Acta Crystallogr., Sect. C 52(1996) 1423–1424.13. P. Calamai, A. Guerri, L. Messori, P. Orioli, and G. P. Speroni, Inorg. Chim. Acta285/2 (1999) 309–312.14. S. Carotti, A. Guerri, T. Mazzei, L. Messori, E. Mini, and P. Orioli, Inorg. Chim.Acta 281 (1998) 90–94.228 B. BRUNI ET AL.SA@ETAKStruktura i citotoksi~na svojstva nekih izabranihzlatovih(III) kompleksaBruno Bruni, Annalisa Guerri, Giordana Marcon, Luigi Messori i Pierluigi OrioliZlatovi(III) kompleksi koji su izostrukturni i izoelektronski s platinskim(II)kompleksima potencijalni su antitumorni lijekovi. Prire|ena su ~etiri tipi~nakompleksa trovalentnog zlata kvadratne koordinacije – AuCl3(Hpm), AuCl2(esal),AuCl(dien)Cl2 and Au(en)2Cl3 – i potanko prou~ena, kako u kristalnom stanju ta-ko i u otopini. Nadalje, citotoksi~nost tih spojeva procijenjena je in vitro na ljudskimtumornim stanicama jajnika A2780 koje su bile kori{tene kao referentna skupinastanica. Znakovito je da su svi ti zlatovi(III) kompleksi pokazivali znatne citotok-si~ne u~inke od kojih je AuCl2(esal) pokazivao djelovanje usporedljivo onom cispla-tina. Kada su zlatovi(III) kompleksi bili ispitani na odgovaraju}oj skupini stanicakoje su bile rezistentne na cisplatin, rezultati su znatno prema{ivali otpornost pre-ma cisplatinu. Raspravlja se o zna~enju rezultata ovih istra`ivanja na razvoj novihkompleksa trovalentnog zlata koji bi se mogli uporabiti kao antitumorni agensi.CYTOTOXIC GOLD(III) COMPLEXES 229

Structure and Cytotoxic Properties of Some Selected Gold(III) Complexes

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