- Email: [email protected]
Crystal structure and solution chemistry of the cytotoxic complex 1,2-dichloro(o-phenanthroline)gold(III) chloride
www.elsevier.nl/locate/ica Inorganica Chimica Acta 311 (2000) 1 – 5
Crystal structure and solution chemistry of the cytotoxic complex 1,2-dichloro(o-phenanthroline)gold(III) chloride F. Abbate a, P. Orioli a,*, B. Bruni a, G. Marcon b, L. Messori a a
Department of Chemistry, Uni6ersity of Florence, 6ia Gino Capponi 7, 50121 Florence, Italy b CIRCMSB, Unit of Florence, Florence, Italy Received 29 May 2000; accepted 24 July 2000
Abstract The crystal structure of the cytotoxic complex 1,2-dichloro(o-phenanthroline)gold(III) chloride ([AuphenCl2]Cl) has been solved through single crystal X-ray diffraction methods. The complex is square planar and exhibits a quite regular geometry. Crystals of the compound belong to the space group P21/n with a= 12.632(5), b=16.916(3), c= 12.902(6) A, , i= 91.31(3)° and Z =8. The coordination of the two gold(III) ions in the asymmetric unit is completed by two chloride ions at 2.972(3) and 3.043(3) A, , respectively, forming a distorted square pyramid. The behavior in solution of [AuphenCl2]Cl was further analyzed through 1H NMR spectroscopy. Results point out that the [Au(III)phen]3 + molecular fragment is stable in solution for several hours, even under physiological conditions, whereas the two chloride ligands are released within approximately 30 min after dissolution in the buffer, at 25°C. The gold(III) chromophore is easily and quickly reduced by addition of stoichiometric amounts of sodium ascorbate; metallic gold is formed and free phenanthroline liberates. The implications of these findings for the biological properties of the [Au(III)phen]3 + species are discussed. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Gold complexes; Phenanthroline; X-ray crystal structures
1. Introduction For the last 5 years new interest has focused on gold(III) complexes as possible cytotoxic and antitumor agents [1–3]. Indeed, a few gold(III) complexes were shown to be sufficiently stable in buffered solutions, owing to stabilization effects played by the ligands, and to exhibit interesting cytotoxic properties when tested in vitro on selected tumor cell lines [1]. For instance Buckley et al. reported that the complex Audamp shows promising antitumor properties both in vitro and in vivo [4]. Henderson et al. prepared and characterized a number of organogold(III) complexes that showed interesting antibacterial, antitumor and antifungal properties [5]. Three representative gold(III) polyamine complexes were recently investigated in our laboratory, and two of them turned out to exhibit relevant cytotoxic properties [6]. Moreover, we have shown that the
complex [AuphenCl2]Cl is highly cytotoxic toward the A2780 tumor cell line either sensitive or resistant to cisplatin; some aspects of the solution chemistry of [AuphenCl2]Cl were also described [7]. To further elucidate the relationships existing between the biological activity and the chemical properties of [AuphenCl2]Cl, we report here the crystal structure of this complex and describe in more detail its solution behavior. In particular, our interest focuses on the identification of the dominant species existing in solution, under physiological conditions. The chemical and biological properties of [AuphenCl2]Cl are critically compared to those of similar metallo-phenanthroline complexes.
2. Experimental
2.1. Synthesis of [AuphenCl2]Cl * Corresponding author. Tel.: + 39-055-275 7554; fax: + 39-055275 7555. E-mail address: [email protected] (P. Orioli).
[AuphenCl2]Cl was prepared by addition of phenantroline·HCl to a HAuCl4 solution at a 1:1 stoi-
0020-1693/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 0 - 1 6 9 3 ( 0 0 ) 0 0 2 9 9 - 1
F. Abbate et al. / Inorganica Chimica Acta 311 (2000) 1–5
2
chiometry according to the procedure reported in Ref. [8] (using ethanol as solvent). The obtained product was checked by elemental analysis; the purity of the compound was higher than 95%. Further evidence for the correct identification of the obtained compound is provided by the electronic and mass spectra.
2.2. X-ray structure determination Crystals of [AuphenCl2]Cl were obtained from the mother liquor of the reaction. X-ray diffraction data were collected on a single crystal X-ray Enraf-Nonius CAD4 diffractometer using the Ka radiation of Mo (u= 0.71073 A, ) at room temperature (r.t.). Cell parameters were obtained from 25 well-centered reflections by using least-square refinement in the range 85 q 515; relevant crystal data are reported in Table 1. The absorption correction was done by using the PSISCAN procedure. The structure was solved by using direct methods and successive difference Fourier synthesis; an alternative approach was also used starting with Patterson method. All non-hydrogen atoms were refined anisotropically and the hydrogen atoms were placed in calculated positions. Calculations were carried on with the SIR-97 [9] and SHELX-97 [10] programs.
2.3. Solution studies The absorption spectra in the UV – Vis region were recorded on a Perkin – Elmer Bio 20 spectrophotometer operating at r.t. The gold(III) complex [AuphenCl2]Cl is soluble in water (10 − 2 M solutions are easily obtained); electronic spectra were recorded on freshly prepared buffered solutions at r.t. The hydrolysis experiments were carried out by adding small amounts of Table 1 Crystallographic data for [Au(phen)Cl2]Cl Formula M Space group Unit cell parameters a (A, ) b (A, ) c (A, ) i (°) V (A, 3) Z Dcalc (g cm−3) F(000) v (Mo Ka) (mm−1) u (A, ) qmin–qmax Number of atoms Independent reflections R1 R1
C12H8N2Cl3Au1 483.5 P21/n 12.632(5) 16.916(3) 12.902(6) 91.31(3) 2756.21(1.75) 8 2.33 1872 11.25 0.71069 2–25 54 4823 (unique reflections) 0.0415 for 3861 Fo\4|(Fo) 0.0571 for all 4823 data
freshly prepared, concentrated water solutions of [AuphenCl2]Cl to the reference buffer (50 mM phosphate, 4 mM NaCl, pH 7.4 at 25°C) and monitoring the electronic spectra of the resulting mixtures over 72 h. The stability of the chromophore in water was also investigated. Potentiometric measurements of chloride release were performed by using a chloride selective electrode interfaced to a Hanna potentiometer. The apparatus was calibrated with sodium chloride solutions of known concentration.
2.4. 1H NMR studies Solution 1H NMR spectra of [AuphenCl2]Cl were recorded on a Bruker Avance spectrometer operating at 600 MHz. Solutions (1.0 mM) were prepared in D2O and in 50 mM PO43 − , 100 mM NaCl, pH 7.4 buffer. The spectra were recorded immediately after dissolution and over a period of 1–3 h.
3. Results and discussion
3.1. Crystal structure of [AuphenCl2]Cl The crystal structure of [Au(phen)Cl2]Cl shows the presence of two molecules of the complex in the asymmetric unit (Fig. 1). Analogous bond lengths and angles in the two molecules show only differences within few standard deviations. In both molecules the gold(III) ions show a slightly distorted square planar coordination formed by two chloride ions and the two nitrogen atoms of the bidentate 1,10-N,N%-phenanthroline ligand. Bond lengths and angles around the gold(III) ions and in the phenanthroline ligands are in agreement with values from the literature (Tables 2 and 3) [11]. The least squares planes through the coordination planes and the phenanthroline ligands show deviations from planarity within a few standard deviations. The angles between the coordination and the phenanthroline planes are 4.4° and 1.1°, respectively, for the two complex molecules. The two molecules are not totally parallel, indeed the angle between the mean plane of the two molecules is 4.5°. In both molecules the gold(III) ions are additionally coordinated by chloride ions at 2.972(3) and 3.043(3) A, , respectively, for the two molecules, forming the apices of distorted square pyramids. The sixth positions are filled by a chloride ion at 3.54 A, for one gold(III) ion and by an aromatic ring of a phenanthroline ligand for the other gold(III) ion. The chloride ions at the apices of the square pyramids are hydrogen bonded to the water molecules (Cl···O= 3.12 and 3.15 A, ). The square pyramidal or tetragonal bipyramidal stereochemistries attained with secondary bonds are quite common for gold(III) complexes as it can be seen
F. Abbate et al. / Inorganica Chimica Acta 311 (2000) 1–5
3
a distance of 2.58 A, from the gold(III) ion. The difference between the stereochemistries of the two complexes is undoubtedly attributable to the steric hindrance of the bulky methyl groups which do not allow the coplanarity of the dmphen ligand with the chloride ions.
3.2. Solution chemistry of [AuphenCl2]Cl [AuphenCl2]Cl is pretty soluble in water and within a standard physiological buffer; freshly prepared solutions of [AuphenCl2]Cl are bright yellow. As previously reported, the electronic spectrum of [AuphenCl2]Cl in water and in the buffer is characterized by a number of intense bands in the visible falling between 300 and 450 nm [7], assigned as N-to-gold(III) charge transfer Table 2 Selected bond lengths (A, ) of the two molecules in the asymmetric unit for [Au(phen)Cl2]Cl Au1–Cl1 Au1–Cl2 Au1–Cl3 Au1–N1 Au1–N2 N1–C1 N1–C2 N2–C11 N2–C12 C1–C12 C1–C5 C2–C3 C3–C4 C4–C5 C5–C6 C6–C7 C7–C8 C8–C9 C8–C12 C9–C10 C10–C11
Fig. 1. Crystal structure of [AuphenCl2]Cl.
by comparison with the analogous structure of [Auphen(CN)2]Br [11]. In the paper, which describes this structure, the authors make an interesting survey of 102 square planar gold(III) complexes and find that nearly 15% of them display secondary coordination. It is also interesting to compare the present structure with that of trichloro-(2,9-dimethyl-1,10-phenanthroline)gold(III) [12]. In this structure the gold(III) ion shows a distorted square pyramidal coordination, but the three chloride ions lie in the coordination plane with one nitrogen atom of the 2,9-dimethyl-1,10phenanthroline (dmphen) ligand, whereas the second nitrogen atom of the ligand is in the apical position at
2.972(3) 2.266(3) 2.263(3) 2.033(8) 2.056(8) 1.38(1) 1.34(1) 1.30(1) 1.37(1) 1.42(1) 1.41(1) 1.40(1) 1.35(2) 1.41(1) 1.41(1) 1.37(2) 1.45(1) 1.39(2) 1.40(1) 1.36(2) 1.41(1)
Au1%–Cl1% Au1%–Cl2% Au1%–Cl3% Au1%–N1% Au1%–N2% N1%–C1% N1%–C2% N2%–C11% N2%–C12% C1%–C12% C1%–C5% C2%–C3% C3%–C4% C4%–C5% C5%–C6% C6%–C7% C7%–C8% C8%–C9% C8%–C12% C9%–C10% C10%–C11%
3.043(3) 2.256(3) 2.268(3) 2.044(8) 2.045(8) 1.37(1) 1.32(1) 1.34(1) 1.38(1) 1.40(1) 1.40(1) 1.39(2) 1.37(2) 1.40(2) 1.46(2) 1.34(1) 1.43(1) 1.40(1) 1.39(1) 1.38(2) 1.38(2)
Table 3 Selected angles (°) of the two molecules in the asymmetric unit for [Au(phen)Cl2]Cl Cl1–Au1–Cl2 Cl1–Au1–Cl3 Cl1–Au1–N2 Cl1–Au1–N1 Cl2–Au1–Cl3 Cl2–Au1–N1 Cl2–Au1–N2 Cl3–Au1–N1 Cl3–Au1–N2 Au1–N1–C1 Au1–N1–C2 Au1–N2–C12 Au1–N2–C11 N1–Au1–N2
93.1(1) 97.0(1) 90.7(2) 86.7(2) 89.5(1) 94.2(2) 174.5(2) 174.6(2) 94.0(2) 112.1(6) 129.0(7) 111.1(6) 127.6(7) 82.0(3)
Cl1%–Au1%–Cl2% Cl1%–Au1%–Cl3% Cl1%–Au1%–N2% Cl1%–Au1%–N1% Cl2%–Au1%–Cl3% Cl2%–Au1%–N1% Cl2%–Au1%–N2% Cl3%–Au1%–N1% Cl3%–Au1%–N2% Au1%–N1%–C1% Au1%–N1%–C2% Au1%–N2%–C12% Au1%–N2%–C11% N1%–Au1%–N2%
92.9(1) 91.1(0) 89.7(2) 91.6(2) 90.1(1) 93.6(2) 175.2(2) 175.2(2) 93.9(2) 110.6(6) 129.4(7) 111.2(6) 128.4(7) 82.3(3)
4
F. Abbate et al. / Inorganica Chimica Acta 311 (2000) 1–5
Fig. 2. Potentiometric profiles of a freshly prepared [AuphenCl2]Cl solution. The graph shows the values of the electrode potentials of a chloride selective electrode against time. Experimental conditions: 1 mM [AuphenCl2]Cl was dissolved in a buffer 50 mM phosphate, pH 7.4 at 25°C and the electrode potential was monitored continuously for 3 h.
Fig. 3. 600 MHz 1H NMR spectra of [AuphenCl2]Cl in the buffer (50 mM phosphate, 100 mM NaCl, pH 7.4) at mixing (a) and after 2 h (b); the individual species are labeled as follows: [AuphenCl2]+ ( ), [Auphen(OH)Cl]+ (), [Auphen(OH)2]+ ( ).
bands. Notably the visible spectra of [AuphenCl2]Cl, in water and in the buffer, do not change appreciably with time when monitored over a time period of 24 h, implying that the [Au(phen)]3 + chromophore is stable. These results point out that the same chromophore
containing gold in the oxidation state + 3 is present either in water or in the buffer. Potentiometric experiments were carried out, with the aid of a chloride-selective electrode, to establish whether the dominant species in solution still contains gold(III)-coordinated chlorides. Potentiometric profiles are shown in Fig. 2. Notably, a progressive large decrease of the electrode potential is observed within approximately 80 min after dissolution, indicating an increase of chloride concentration. The total change of potential equals 40 mV: such a value roughly corresponds to an increase in chloride concentration from 1 to 2.8 mM. From analysis of this data it is inferred that the [AuphenCl2]+ species slowly releases both gold-coordinated Cl groups. The hydrolysis process is clearly biphasic, as it was observed also for [Au(Oac)2(damp) [13], with the fast phase lasting approximately within 15 min and the slow phase reaching completion within 60 min. It is straightforward to assign the fast phase to the release of the first Cl group and the slow phase to the release of the second Cl group. The solution behavior of [AuphenCl2]Cl was further analyzed by 1H NMR spectroscopy. Spectra are shown in Fig. 3. Upon dissolving [AuphenCl2]Cl in the reference physiological buffer (50 mM phosphate, 100 mM NaCl, pH 7.4) several signals, of variable intensity, are observed in the aromatic region, that are suggestive of the simultaneous presence of at least three species under slow exchange conditions (Fig. 3(a)). A significant spectral simplification then takes places within 2 h after dissolution (at r.t.) indicating that one species becomes dominant (Fig. 3(b)). Careful analysis of the 1H NMR spectra allows assignment of the 1H NMR signals to three well distinct species most likely corresponding to the dichloro species [AuphenCl2]+, the monochloro species [Auphen(OH)Cl]+ and the dihydroxo species [Auphen(OH)2]+. Signals are attributed to each species by analysis of the time dependent changes in intensity and are labeled accordingly. Notably 1H NMR signals attributed to the [AuphenCl2]+ and [Auphen(OH)Cl]+ species decrease progressively in intensity with time whereas signals assigned to the [Auphen(OH)2]+ species become dominant after approximately 30 min. Later on, the spectrum remains stable for hours. The final 1H NMR spectrum corresponding to the end-point of the kinetic process shows that, under the experimental conditions employed, an equilibrium is reached where the dihydroxo species is largely predominant but signals of the monochloro and dichloro species are still observed. The effects of the addition of the reducing agent sodium ascorbate on the spectral properties of [AuphenCl2]Cl were also analyzed. The electronic spectra show immediate disappearance of the characteristic LMCT transitions suggesting that sodium ascorbate
F. Abbate et al. / Inorganica Chimica Acta 311 (2000) 1–5
causes immediate and complete reduction of the gold(III) center; accordingly the 1H NMR spectra show that the 1 H NMR signals of [AuphenCl2]Cl suddenly disappear. It is inferred that, upon reduction, the complex breaks down and free phenanthroline is released. In nice agreement with this hypothesis, the 1H NMR signals of free phenanthroline are newly detected after reduction; simultaneously formation of characteristic Cassius porpora (colloidal gold) is observed.
5
Acknowledgements MURST is acknowledged for financial support in the frame of the project ‘Pharmacological and diagnostic aspects of metal complexes’. The Cassa di Risparmio di Firenze is gratefully acknowledged for a generous grant. We thank Professor Marco Mascini, Dr. Ilaria Palchetti and Dr. Costanza Landi for helping with chloride selective potentiometric measurements.
References
4. Conclusions [AuphenCl2]Cl exhibits relevant cytotoxic properties toward cultured human tumor cells. Single crystal Xray diffraction studies demonstrate that [AuphenCl2]Cl is a classical square planar gold(III) complex. Solution studies show that the molecular fragment [Au(phen)]3 + is relatively stable under physiological conditions whereas the chloride groups are slowly released after dissolution in the buffer. The predominant species existing in solution under physiological conditions is likely to be [Auphen(OH)2]+; this species may interact with biomolecules through two mechanisms: (i) formation of monofunctional or bifunctional coordination bonds at the two (labile) cis positions on the gold(III) center; (ii) intercalation of the phenanthroline moiety. Nevertheless, it must be remembered that the complex investigated is promptly reduced by a relatively mild reducing agent such as ascorbate, implying that the biological properties of [AuphenCl2]Cl might derive at least partially from free phenanthroline. Indeed, previous studies showed that the free ligand phenanthroline is endowed with significant cytotoxic properties [7]. Further studies are now needed to identify unambiguously the species that are actually responsible for the cytotoxic effects.
.
[1] B. Bruni, A. Guerri, G. Marcon, L. Messori, P. Orioli, Croat. Chem. Acta 72 (1999) 221. [2] P. Calamai, S. Carotti, A. Guerri, L. Messori, E. Mini, P. Orioli, G.P. Speroni, J. Inorg. Biochem. 66 (1997) 103. [3] P. Calamai, A. Guerri, L. Messori, P. Orioli, G.P. Speroni, Inorg. Chim. Acta 285 (1999) 309. [4] (a) R.G. Buckley, A.M. Elsome, S.P. Fricker, G.R. Henderson, B.R.C. Theobald, R.V. Parish, B.P. Howe, L.R. Kelland, J. Med. Chem. 39 (1996) 5208. (b) R.V. Parish, B.P. Howe, J.P. Wright, J. Mack, R.G. Pritchard, R.G. Buckley, A.M. Elsome, S.P. Fricker, Inorg. Chem. 35 (1996) 1659. [5] (a) M.B. Dinger, W. Henderson, J. Organomet. Chem. 557 (1998) 231. (b) M.B. Dinger, W. Henderson, J. Organomet. Chem. 560 (1998) 233. [6] S. Carotti, A. Guerri, T. Mazzei, L. Messori, E. Mini, P. Orioli, Inorg. Chim. Acta 281 (1998) 90. [7] L. Messori, F. Abbate, G. Marcon, P. Orioli, M. Fontani, E. Mini, T. Mazzei, S. Carotti, T. O’Connell, P. Zanello, J. Med. Chem. 43 (2000) 3541. [8] B.P. Block, J.C. Bailar, J. Am. Chem. Soc. 73 (1951) 4722. [9] M.C. Burla, M. Camalli, G. Cascarano, C. Giacovazzo, G. Polidori, R. Spagna, D. Viterbo, J. Appl. Crystallogr. 22 (1989) 389. [10] G.M. Sheldrick, A Program for Structure Refinement, University of Go¨ttingen, Germany, 1993. [11] G. Marangoni, B. Pitteri, V. Bartolasi, V. Ferretti, G. Gilli, J. Chem. Soc., Dalton Trans. (1987) 2235. [12] W.T. Robinson, E. Sinn, J. Chem. Soc., Dalton Trans. (1975) 726. [13] R.V. Parish, J. Meck, L. Hargreaves, J.P. Wright, R.G. Buckley, A.M. Elsome, S.P. Fricker, B.R.C. Theobald, J. Chem. Soc., Dalton Trans. (1996) 69.
Crystal structure and solution chemistry of the cytotoxic complex 1,2-dichloro(o-phenanthroline)gold(III) chloride F. Abbate a, P. Orioli a,*, B. Bruni a, G. Marcon b, L. Messori a a
Department of Chemistry, Uni6ersity of Florence, 6ia Gino Capponi 7, 50121 Florence, Italy b CIRCMSB, Unit of Florence, Florence, Italy Received 29 May 2000; accepted 24 July 2000
Abstract The crystal structure of the cytotoxic complex 1,2-dichloro(o-phenanthroline)gold(III) chloride ([AuphenCl2]Cl) has been solved through single crystal X-ray diffraction methods. The complex is square planar and exhibits a quite regular geometry. Crystals of the compound belong to the space group P21/n with a= 12.632(5), b=16.916(3), c= 12.902(6) A, , i= 91.31(3)° and Z =8. The coordination of the two gold(III) ions in the asymmetric unit is completed by two chloride ions at 2.972(3) and 3.043(3) A, , respectively, forming a distorted square pyramid. The behavior in solution of [AuphenCl2]Cl was further analyzed through 1H NMR spectroscopy. Results point out that the [Au(III)phen]3 + molecular fragment is stable in solution for several hours, even under physiological conditions, whereas the two chloride ligands are released within approximately 30 min after dissolution in the buffer, at 25°C. The gold(III) chromophore is easily and quickly reduced by addition of stoichiometric amounts of sodium ascorbate; metallic gold is formed and free phenanthroline liberates. The implications of these findings for the biological properties of the [Au(III)phen]3 + species are discussed. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Gold complexes; Phenanthroline; X-ray crystal structures
1. Introduction For the last 5 years new interest has focused on gold(III) complexes as possible cytotoxic and antitumor agents [1–3]. Indeed, a few gold(III) complexes were shown to be sufficiently stable in buffered solutions, owing to stabilization effects played by the ligands, and to exhibit interesting cytotoxic properties when tested in vitro on selected tumor cell lines [1]. For instance Buckley et al. reported that the complex Audamp shows promising antitumor properties both in vitro and in vivo [4]. Henderson et al. prepared and characterized a number of organogold(III) complexes that showed interesting antibacterial, antitumor and antifungal properties [5]. Three representative gold(III) polyamine complexes were recently investigated in our laboratory, and two of them turned out to exhibit relevant cytotoxic properties [6]. Moreover, we have shown that the
complex [AuphenCl2]Cl is highly cytotoxic toward the A2780 tumor cell line either sensitive or resistant to cisplatin; some aspects of the solution chemistry of [AuphenCl2]Cl were also described [7]. To further elucidate the relationships existing between the biological activity and the chemical properties of [AuphenCl2]Cl, we report here the crystal structure of this complex and describe in more detail its solution behavior. In particular, our interest focuses on the identification of the dominant species existing in solution, under physiological conditions. The chemical and biological properties of [AuphenCl2]Cl are critically compared to those of similar metallo-phenanthroline complexes.
2. Experimental
2.1. Synthesis of [AuphenCl2]Cl * Corresponding author. Tel.: + 39-055-275 7554; fax: + 39-055275 7555. E-mail address: [email protected] (P. Orioli).
[AuphenCl2]Cl was prepared by addition of phenantroline·HCl to a HAuCl4 solution at a 1:1 stoi-
0020-1693/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 0 - 1 6 9 3 ( 0 0 ) 0 0 2 9 9 - 1
F. Abbate et al. / Inorganica Chimica Acta 311 (2000) 1–5
2
chiometry according to the procedure reported in Ref. [8] (using ethanol as solvent). The obtained product was checked by elemental analysis; the purity of the compound was higher than 95%. Further evidence for the correct identification of the obtained compound is provided by the electronic and mass spectra.
2.2. X-ray structure determination Crystals of [AuphenCl2]Cl were obtained from the mother liquor of the reaction. X-ray diffraction data were collected on a single crystal X-ray Enraf-Nonius CAD4 diffractometer using the Ka radiation of Mo (u= 0.71073 A, ) at room temperature (r.t.). Cell parameters were obtained from 25 well-centered reflections by using least-square refinement in the range 85 q 515; relevant crystal data are reported in Table 1. The absorption correction was done by using the PSISCAN procedure. The structure was solved by using direct methods and successive difference Fourier synthesis; an alternative approach was also used starting with Patterson method. All non-hydrogen atoms were refined anisotropically and the hydrogen atoms were placed in calculated positions. Calculations were carried on with the SIR-97 [9] and SHELX-97 [10] programs.
2.3. Solution studies The absorption spectra in the UV – Vis region were recorded on a Perkin – Elmer Bio 20 spectrophotometer operating at r.t. The gold(III) complex [AuphenCl2]Cl is soluble in water (10 − 2 M solutions are easily obtained); electronic spectra were recorded on freshly prepared buffered solutions at r.t. The hydrolysis experiments were carried out by adding small amounts of Table 1 Crystallographic data for [Au(phen)Cl2]Cl Formula M Space group Unit cell parameters a (A, ) b (A, ) c (A, ) i (°) V (A, 3) Z Dcalc (g cm−3) F(000) v (Mo Ka) (mm−1) u (A, ) qmin–qmax Number of atoms Independent reflections R1 R1
C12H8N2Cl3Au1 483.5 P21/n 12.632(5) 16.916(3) 12.902(6) 91.31(3) 2756.21(1.75) 8 2.33 1872 11.25 0.71069 2–25 54 4823 (unique reflections) 0.0415 for 3861 Fo\4|(Fo) 0.0571 for all 4823 data
freshly prepared, concentrated water solutions of [AuphenCl2]Cl to the reference buffer (50 mM phosphate, 4 mM NaCl, pH 7.4 at 25°C) and monitoring the electronic spectra of the resulting mixtures over 72 h. The stability of the chromophore in water was also investigated. Potentiometric measurements of chloride release were performed by using a chloride selective electrode interfaced to a Hanna potentiometer. The apparatus was calibrated with sodium chloride solutions of known concentration.
2.4. 1H NMR studies Solution 1H NMR spectra of [AuphenCl2]Cl were recorded on a Bruker Avance spectrometer operating at 600 MHz. Solutions (1.0 mM) were prepared in D2O and in 50 mM PO43 − , 100 mM NaCl, pH 7.4 buffer. The spectra were recorded immediately after dissolution and over a period of 1–3 h.
3. Results and discussion
3.1. Crystal structure of [AuphenCl2]Cl The crystal structure of [Au(phen)Cl2]Cl shows the presence of two molecules of the complex in the asymmetric unit (Fig. 1). Analogous bond lengths and angles in the two molecules show only differences within few standard deviations. In both molecules the gold(III) ions show a slightly distorted square planar coordination formed by two chloride ions and the two nitrogen atoms of the bidentate 1,10-N,N%-phenanthroline ligand. Bond lengths and angles around the gold(III) ions and in the phenanthroline ligands are in agreement with values from the literature (Tables 2 and 3) [11]. The least squares planes through the coordination planes and the phenanthroline ligands show deviations from planarity within a few standard deviations. The angles between the coordination and the phenanthroline planes are 4.4° and 1.1°, respectively, for the two complex molecules. The two molecules are not totally parallel, indeed the angle between the mean plane of the two molecules is 4.5°. In both molecules the gold(III) ions are additionally coordinated by chloride ions at 2.972(3) and 3.043(3) A, , respectively, for the two molecules, forming the apices of distorted square pyramids. The sixth positions are filled by a chloride ion at 3.54 A, for one gold(III) ion and by an aromatic ring of a phenanthroline ligand for the other gold(III) ion. The chloride ions at the apices of the square pyramids are hydrogen bonded to the water molecules (Cl···O= 3.12 and 3.15 A, ). The square pyramidal or tetragonal bipyramidal stereochemistries attained with secondary bonds are quite common for gold(III) complexes as it can be seen
F. Abbate et al. / Inorganica Chimica Acta 311 (2000) 1–5
3
a distance of 2.58 A, from the gold(III) ion. The difference between the stereochemistries of the two complexes is undoubtedly attributable to the steric hindrance of the bulky methyl groups which do not allow the coplanarity of the dmphen ligand with the chloride ions.
3.2. Solution chemistry of [AuphenCl2]Cl [AuphenCl2]Cl is pretty soluble in water and within a standard physiological buffer; freshly prepared solutions of [AuphenCl2]Cl are bright yellow. As previously reported, the electronic spectrum of [AuphenCl2]Cl in water and in the buffer is characterized by a number of intense bands in the visible falling between 300 and 450 nm [7], assigned as N-to-gold(III) charge transfer Table 2 Selected bond lengths (A, ) of the two molecules in the asymmetric unit for [Au(phen)Cl2]Cl Au1–Cl1 Au1–Cl2 Au1–Cl3 Au1–N1 Au1–N2 N1–C1 N1–C2 N2–C11 N2–C12 C1–C12 C1–C5 C2–C3 C3–C4 C4–C5 C5–C6 C6–C7 C7–C8 C8–C9 C8–C12 C9–C10 C10–C11
Fig. 1. Crystal structure of [AuphenCl2]Cl.
by comparison with the analogous structure of [Auphen(CN)2]Br [11]. In the paper, which describes this structure, the authors make an interesting survey of 102 square planar gold(III) complexes and find that nearly 15% of them display secondary coordination. It is also interesting to compare the present structure with that of trichloro-(2,9-dimethyl-1,10-phenanthroline)gold(III) [12]. In this structure the gold(III) ion shows a distorted square pyramidal coordination, but the three chloride ions lie in the coordination plane with one nitrogen atom of the 2,9-dimethyl-1,10phenanthroline (dmphen) ligand, whereas the second nitrogen atom of the ligand is in the apical position at
2.972(3) 2.266(3) 2.263(3) 2.033(8) 2.056(8) 1.38(1) 1.34(1) 1.30(1) 1.37(1) 1.42(1) 1.41(1) 1.40(1) 1.35(2) 1.41(1) 1.41(1) 1.37(2) 1.45(1) 1.39(2) 1.40(1) 1.36(2) 1.41(1)
Au1%–Cl1% Au1%–Cl2% Au1%–Cl3% Au1%–N1% Au1%–N2% N1%–C1% N1%–C2% N2%–C11% N2%–C12% C1%–C12% C1%–C5% C2%–C3% C3%–C4% C4%–C5% C5%–C6% C6%–C7% C7%–C8% C8%–C9% C8%–C12% C9%–C10% C10%–C11%
3.043(3) 2.256(3) 2.268(3) 2.044(8) 2.045(8) 1.37(1) 1.32(1) 1.34(1) 1.38(1) 1.40(1) 1.40(1) 1.39(2) 1.37(2) 1.40(2) 1.46(2) 1.34(1) 1.43(1) 1.40(1) 1.39(1) 1.38(2) 1.38(2)
Table 3 Selected angles (°) of the two molecules in the asymmetric unit for [Au(phen)Cl2]Cl Cl1–Au1–Cl2 Cl1–Au1–Cl3 Cl1–Au1–N2 Cl1–Au1–N1 Cl2–Au1–Cl3 Cl2–Au1–N1 Cl2–Au1–N2 Cl3–Au1–N1 Cl3–Au1–N2 Au1–N1–C1 Au1–N1–C2 Au1–N2–C12 Au1–N2–C11 N1–Au1–N2
93.1(1) 97.0(1) 90.7(2) 86.7(2) 89.5(1) 94.2(2) 174.5(2) 174.6(2) 94.0(2) 112.1(6) 129.0(7) 111.1(6) 127.6(7) 82.0(3)
Cl1%–Au1%–Cl2% Cl1%–Au1%–Cl3% Cl1%–Au1%–N2% Cl1%–Au1%–N1% Cl2%–Au1%–Cl3% Cl2%–Au1%–N1% Cl2%–Au1%–N2% Cl3%–Au1%–N1% Cl3%–Au1%–N2% Au1%–N1%–C1% Au1%–N1%–C2% Au1%–N2%–C12% Au1%–N2%–C11% N1%–Au1%–N2%
92.9(1) 91.1(0) 89.7(2) 91.6(2) 90.1(1) 93.6(2) 175.2(2) 175.2(2) 93.9(2) 110.6(6) 129.4(7) 111.2(6) 128.4(7) 82.3(3)
4
F. Abbate et al. / Inorganica Chimica Acta 311 (2000) 1–5
Fig. 2. Potentiometric profiles of a freshly prepared [AuphenCl2]Cl solution. The graph shows the values of the electrode potentials of a chloride selective electrode against time. Experimental conditions: 1 mM [AuphenCl2]Cl was dissolved in a buffer 50 mM phosphate, pH 7.4 at 25°C and the electrode potential was monitored continuously for 3 h.
Fig. 3. 600 MHz 1H NMR spectra of [AuphenCl2]Cl in the buffer (50 mM phosphate, 100 mM NaCl, pH 7.4) at mixing (a) and after 2 h (b); the individual species are labeled as follows: [AuphenCl2]+ ( ), [Auphen(OH)Cl]+ (), [Auphen(OH)2]+ ( ).
bands. Notably the visible spectra of [AuphenCl2]Cl, in water and in the buffer, do not change appreciably with time when monitored over a time period of 24 h, implying that the [Au(phen)]3 + chromophore is stable. These results point out that the same chromophore
containing gold in the oxidation state + 3 is present either in water or in the buffer. Potentiometric experiments were carried out, with the aid of a chloride-selective electrode, to establish whether the dominant species in solution still contains gold(III)-coordinated chlorides. Potentiometric profiles are shown in Fig. 2. Notably, a progressive large decrease of the electrode potential is observed within approximately 80 min after dissolution, indicating an increase of chloride concentration. The total change of potential equals 40 mV: such a value roughly corresponds to an increase in chloride concentration from 1 to 2.8 mM. From analysis of this data it is inferred that the [AuphenCl2]+ species slowly releases both gold-coordinated Cl groups. The hydrolysis process is clearly biphasic, as it was observed also for [Au(Oac)2(damp) [13], with the fast phase lasting approximately within 15 min and the slow phase reaching completion within 60 min. It is straightforward to assign the fast phase to the release of the first Cl group and the slow phase to the release of the second Cl group. The solution behavior of [AuphenCl2]Cl was further analyzed by 1H NMR spectroscopy. Spectra are shown in Fig. 3. Upon dissolving [AuphenCl2]Cl in the reference physiological buffer (50 mM phosphate, 100 mM NaCl, pH 7.4) several signals, of variable intensity, are observed in the aromatic region, that are suggestive of the simultaneous presence of at least three species under slow exchange conditions (Fig. 3(a)). A significant spectral simplification then takes places within 2 h after dissolution (at r.t.) indicating that one species becomes dominant (Fig. 3(b)). Careful analysis of the 1H NMR spectra allows assignment of the 1H NMR signals to three well distinct species most likely corresponding to the dichloro species [AuphenCl2]+, the monochloro species [Auphen(OH)Cl]+ and the dihydroxo species [Auphen(OH)2]+. Signals are attributed to each species by analysis of the time dependent changes in intensity and are labeled accordingly. Notably 1H NMR signals attributed to the [AuphenCl2]+ and [Auphen(OH)Cl]+ species decrease progressively in intensity with time whereas signals assigned to the [Auphen(OH)2]+ species become dominant after approximately 30 min. Later on, the spectrum remains stable for hours. The final 1H NMR spectrum corresponding to the end-point of the kinetic process shows that, under the experimental conditions employed, an equilibrium is reached where the dihydroxo species is largely predominant but signals of the monochloro and dichloro species are still observed. The effects of the addition of the reducing agent sodium ascorbate on the spectral properties of [AuphenCl2]Cl were also analyzed. The electronic spectra show immediate disappearance of the characteristic LMCT transitions suggesting that sodium ascorbate
F. Abbate et al. / Inorganica Chimica Acta 311 (2000) 1–5
causes immediate and complete reduction of the gold(III) center; accordingly the 1H NMR spectra show that the 1 H NMR signals of [AuphenCl2]Cl suddenly disappear. It is inferred that, upon reduction, the complex breaks down and free phenanthroline is released. In nice agreement with this hypothesis, the 1H NMR signals of free phenanthroline are newly detected after reduction; simultaneously formation of characteristic Cassius porpora (colloidal gold) is observed.
5
Acknowledgements MURST is acknowledged for financial support in the frame of the project ‘Pharmacological and diagnostic aspects of metal complexes’. The Cassa di Risparmio di Firenze is gratefully acknowledged for a generous grant. We thank Professor Marco Mascini, Dr. Ilaria Palchetti and Dr. Costanza Landi for helping with chloride selective potentiometric measurements.
References
4. Conclusions [AuphenCl2]Cl exhibits relevant cytotoxic properties toward cultured human tumor cells. Single crystal Xray diffraction studies demonstrate that [AuphenCl2]Cl is a classical square planar gold(III) complex. Solution studies show that the molecular fragment [Au(phen)]3 + is relatively stable under physiological conditions whereas the chloride groups are slowly released after dissolution in the buffer. The predominant species existing in solution under physiological conditions is likely to be [Auphen(OH)2]+; this species may interact with biomolecules through two mechanisms: (i) formation of monofunctional or bifunctional coordination bonds at the two (labile) cis positions on the gold(III) center; (ii) intercalation of the phenanthroline moiety. Nevertheless, it must be remembered that the complex investigated is promptly reduced by a relatively mild reducing agent such as ascorbate, implying that the biological properties of [AuphenCl2]Cl might derive at least partially from free phenanthroline. Indeed, previous studies showed that the free ligand phenanthroline is endowed with significant cytotoxic properties [7]. Further studies are now needed to identify unambiguously the species that are actually responsible for the cytotoxic effects.
.
[1] B. Bruni, A. Guerri, G. Marcon, L. Messori, P. Orioli, Croat. Chem. Acta 72 (1999) 221. [2] P. Calamai, S. Carotti, A. Guerri, L. Messori, E. Mini, P. Orioli, G.P. Speroni, J. Inorg. Biochem. 66 (1997) 103. [3] P. Calamai, A. Guerri, L. Messori, P. Orioli, G.P. Speroni, Inorg. Chim. Acta 285 (1999) 309. [4] (a) R.G. Buckley, A.M. Elsome, S.P. Fricker, G.R. Henderson, B.R.C. Theobald, R.V. Parish, B.P. Howe, L.R. Kelland, J. Med. Chem. 39 (1996) 5208. (b) R.V. Parish, B.P. Howe, J.P. Wright, J. Mack, R.G. Pritchard, R.G. Buckley, A.M. Elsome, S.P. Fricker, Inorg. Chem. 35 (1996) 1659. [5] (a) M.B. Dinger, W. Henderson, J. Organomet. Chem. 557 (1998) 231. (b) M.B. Dinger, W. Henderson, J. Organomet. Chem. 560 (1998) 233. [6] S. Carotti, A. Guerri, T. Mazzei, L. Messori, E. Mini, P. Orioli, Inorg. Chim. Acta 281 (1998) 90. [7] L. Messori, F. Abbate, G. Marcon, P. Orioli, M. Fontani, E. Mini, T. Mazzei, S. Carotti, T. O’Connell, P. Zanello, J. Med. Chem. 43 (2000) 3541. [8] B.P. Block, J.C. Bailar, J. Am. Chem. Soc. 73 (1951) 4722. [9] M.C. Burla, M. Camalli, G. Cascarano, C. Giacovazzo, G. Polidori, R. Spagna, D. Viterbo, J. Appl. Crystallogr. 22 (1989) 389. [10] G.M. Sheldrick, A Program for Structure Refinement, University of Go¨ttingen, Germany, 1993. [11] G. Marangoni, B. Pitteri, V. Bartolasi, V. Ferretti, G. Gilli, J. Chem. Soc., Dalton Trans. (1987) 2235. [12] W.T. Robinson, E. Sinn, J. Chem. Soc., Dalton Trans. (1975) 726. [13] R.V. Parish, J. Meck, L. Hargreaves, J.P. Wright, R.G. Buckley, A.M. Elsome, S.P. Fricker, B.R.C. Theobald, J. Chem. Soc., Dalton Trans. (1996) 69.