Potential new inorganic antitumour agents from combining the anticancer traditional Chinese medicine (TCM) liriodenine with metal ions, and DNA binding studies

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Journal of Inorganic Biochemistry 105 (2011) 171–180

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Journal of Inorganic Biochemistry

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Potential new inorganic antitumour agents from combining the anticancertraditional Chinese medicine (TCM) matrine with Ga(III), Au(III),Sn(IV) ions, and DNA binding studies

Zhen-Feng Chen a,⁎, Li Mao a, Li-Min Liu b, Yan-Cheng Liu a, Yan Peng a, Xue Hong a, Hong-Hong Wang a,Hua-Gang Liu b, Hong Liang a,⁎a Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China),School of Chemistry & Chemical Engineering of Guangxi Normal University, Guilin 541004, PR Chinab School of Pharmacy, Guangxi Medical University, Nanning 530021, PR China

⁎ Corresponding author. Fax: +86 773 2120958.E-mail address: [email protected] (Z.-F. Chen).

0162-0134/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.jinorgbio.2010.10.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 June 2010Received in revised form 4 October 2010Accepted 6 October 2010Available online xxxx

Keywords:Metal complexIonic compoundCrystal structureMatrineDNA bindingAntitumour agent

Three new compounds of Ga(III), Au(III), Sn(IV) withmatrine (MT), [H-MT][GaCl4] (1), [H-MT][AuCl4] (2) and[Sn(H-MT)Cl5] (3), have been synthesized and characterized by elemental analysis, IR, ESI-MS and singlecrystal X-ray diffraction methods. The crystal structural analyses indicate that 1 and 2 are ionic compounds,whereas 3 is a tin(IV) complex formed by the monodentate MT via its carbonyl oxygen atom of MTcoordinating to Sn(IV). Their in vitro cytotoxicity towards eight selected tumour cell lines has been evaluatedby MTT (3-[4,5-Dimentylthiazole-2-yl]-2,5-diphenpyltetra-zolium bromide) method, and compounds 1 and2 exhibit enhanced activity, such as 1 to SW480, 2 to HeLa, HepG2 and MCF-7, which exceeds matrine andcisplatin, and display synergistic contribution of their components. The cell cycle analyses show thatcompounds 1, 3 and MT exhibit cell cycle arrest at the G2/M phase. Interactions of these compounds with calfthymus DNA (ct-DNA) have been investigated by spectroscopic analyses. The planar extension of theintercalative metal–matrine compounds increases the interaction of the metal–matrine with DNA, indicatingthat the cationic metal ions and configuration of the intercalated metal–matrine will affect the extent ofinteraction. Compound 2, [H-MT][AuCl4], exhibits more intensive binding ability to DNA, which may correlatewith intercalation and other action mode. The circular dichroism spectra of the ct-DNA bound with metal–MTcompounds also suggest that ct-DNA interacted with 1, 2, 3 does not influence its secondary structure.Furthermore, both compounds 1 and 2 exhibit effective inhibition ability to topoisomerase (TOPO I) atconcentration of 50 μM, while matrine and compound 3 do not.

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1. Introduction

Although the platinum-based anticancer drug such as cisplatin,carboplatin and oxaliplatin have been widely applied for many yearsto treat cancer, the treatment is limited by severe side effectsincluding nephrotoxicity, emetogenesis and neurotoxicity [1,2] aswell as acquired and/or intrinsic resistance against the drugs [1,3].Efforts towards the development of other active transition metalanticancer complexes with better efficiency and new action mecha-nism overcoming the aforementioned limitations have attractedmany bioinorganic chemists' interests and became one of the focusedresearch fields of bioinorganic chemistry [1,2]. The high antitumouractivity of GaCl3 and Ga(NO3)3 which has been tested in cancerpatients [4], but their unfavourable pharmacokinetic properties have

prevented them widespread use in systemic chemotherapy of cancerpatients [5]. Therefore development of tumour-inhibiting galliumcomplexes has been pursued as a strategy to circumvent thelimitations faced with simple gallium salts [6]. The positive synergisticeffect of gallium nitrate and vinblastine has been found on metastasisof bladder epithelial tumour and resistance of ovarian cancer tocisplatin [7]. Gold compounds are recognized as promising anticanceragents [8], because they are the isoelectronic configuration (d8) andstructural characteristics (square planar), similar to Pt(II) [9]. The gold(I) complexes auranofin and aurothioglucose as well as the gold(III)chloride significantly inhibit the thioredoxin reductase which isinvolved in cancer cell growth [10]. Besides gallium and goldcompounds, recent studies have shown very promising in vitroantitumour properties of organotin compounds against a wide panelof tumour cell lines of human origin [11,12].

Recently, our group has embarked on the metal-based anticanceragents by combining the anticancer traditional Chinese medicines(TCMs) with metal ions [13,14]. As ongoing research, a quinolizidine

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Table 1Crystallographic data and refinements of compounds 1, 2, 3.

1 2 3

Formula C15H25Cl4GaN2O C15H25AuCl4N2O C16H29Cl5N2O2SnMr 460.89 588.14 577.35Crystal size/mm 0.28×0.21×0.14 0.26×0.20×0.14 0.27×0.19×0.11Crystal system Orthorhombic Orthorhombic OrthorhombicSpace group P2(1)2(1)2(1) P2(1)2(1)2(1) P2(1)2(1)2(1)a/Å 8.480(4) 8.0565(13) 9.6971(12)b/Å 12.940(7) 10.4684(17) 12.6070(15)c/Å 18.151(9) 23.232(4) 18.333(2)α=β= γ/° 90 90 90V/Å3 1991.6(17) 1959.3(5) 2241.2(5)T/K 296(2) 296(2) 296(2)Z 4 4 4Dc/g cm−3 1.537 1.994 1.7112θ/° 3.86 to 50.10 3.50 to 50.08 3.92 to 50.10F(000) 944 1136 1160μ(Mo Kα)/mm−1 1.922 8.058 1.749Total no. reflns 9691 9793 11080No. indep. reflns 3527 3462 3962Rint 0.0462 0.0403 0.0270R1 [IN2σ(I)] 0.0396 0.0277 0.0221wR2(all data) 0.0889 0.0556 0.0459Gof(F2) 0.993 0.982 0.992

172 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 171–180

alkaloid matrine (MT), the main component of root of Chinese herbsSophora plants including Sophora flavescens and Sophora tonkinensis,was selected as an active ligand. Matrine has been extensively used inChina for the treatment of viral hepatitis and cardiac diseases [15].Matrine also exhibits inhibition activity towards many tumour cells(such as HeLa cell and gastric cancer MKN45 cell) [16,17]. Althoughthe crystal structure and antitumour activity of ionic compound[H-MT][FeCl4] [18], and the crystal structure of matrine [19], [H-MT][ZnCl4] [20] and different stereoisomer of matrine: isosophoridine[21], cis-matrine [22], tetrahydoneosophoramine [23] have beenreported, up to now, matrine–metal compounds have not far beenexplored, especially their antitumour activity and interaction withbiomolecules. Herein, we report the synthesis and characterization ofGa(III), Au(III) and Sn(IV) compounds with matrine, along with theirin vitro antitumour activity, interaction with DNA and their inhibitionability to Topoisomerase I (TOPO I).

2. Experimental

2.1. Materials

Matrine, HAuCl4⋅4H2O, Ga(NO3)3⋅xH2O, SnCl4⋅5H2O, as well asthe organic solvents of analytical grade in the experiments werecommercially available and used as received without furtherpurification unless noted specifically.

In DNA binding studies, all the synthetic metal–MT compounds aswell as ligand were dissolved in DMSO for preparation of stocksolution at 2.0 mM. Calf thymus DNA (ct-DNA) was purchased fromSigma and pUC19 plasmid DNA as stock solution of 250 ng/μL waspurchased from Takara Biotech (Dalian of China).

2.2. Instrumentation and methods

Elemental analyses (C, H and N) were recorded on a PerkinElmerSeries II CHNS/O 2400 elemental analyzer. Infrared spectra wererecorded on a PerkinElmer FT-IR Spectrometer. UV–Vis absorptiontitration was performed on a Cary 100 Conc. UV–visible spectropho-tometer. ESI-MS spectra were recorded on a Bruker HCT ElectrosprayIonization Mass Spectrometer. Fluorescence emission titration wasperformed on a Shimadzu RF-5301/PC spectrofluorometer. ThepH value of buffer solution was measured on Sartorius ProfessionalpH-Meter PP-20. The circular dichroism (CD) spectra were performedon JASCO J-810-150L Spectropolarimeter.

2.3. Synthesis

[H-MT][GaCl4] (1): A mixture of Ga(NO3)3·xH2O (0.128 g,0.5 mmol), matrine (0.124 g, 0.5 mmol) dissolved in ethanol (20 mL)was refluxed with stirring; a light-yellow precipitate appeared after afewmin. Then HCl solution (1 M)was dropped until themixed solutionbecame clear, and continuously refluxed in water-bath at 80 °C for 5 h.The resultant solution was filtered and concentrated by evaporationaffording colorless prismatic crystals suitable for X-ray diffractionanalysis. Yield: 0.14 g, 61%. Anal. Found(%): C, 39.15; H, 5.56; N, 6.12.Calculated(%) for C15H25Cl4GaN2O: C, 39.09; H, 5.47; N, 6.08. IR(v, cm−1,KBr, m=moderate, s=strong, w=weak): 3438m, 3013m(–CH2–),2930s(–CH2–), 2870m(–CH2–), 1576s(C O), 1467m, 1406m, 1337(s),1252w, 1176w, 1102m, 1084m, 803m. ESI-MS: 210.5 (−MS, calculated210.8 for [GaCl4]−), 248.9 (+MS, calculated 249.2 for [H-MT]+).

[H-MT][AuCl4] (2): This compound was synthesized using thesimilar procedure as to 1, with HAuCl4·4H2O (0.5 mmol, 0.206 g)being used instead of Ga(NO3)3·xH2O. The difference is the reactionsolution of 2 was stirred at room temperature for 15 h. The resultantsolution was filtered and concentrated by evaporation affordingyellow prismatic crystals suitable for X-ray diffraction analysis. Yield:0.203 g, 69%. Anal. Found(%): C, 30.75; H, 4.23; N, 4.72. Calculated(%)

for C15H25AuCl4N2O: C, 30.63; H, 4.28; N, 4.76. IR(v, cm−1, KBr,m=moderate, s=strong, w=weak) 3440m, 3055w(–CH2–), 2927m(–CH2–), 2868w(–CH2–), 1603s(C O), 1468(s), 1335(m), 1204(m),1108(m), 1060(m), 618w. ESI-MS: 338.7 (−MS, calculated 338.8 for[AuCl4]−), 248.9 (+MS, calculated 249.2 for [H-MT]+).

[Sn(H-MT)Cl5] (3): A mixture of SnCl4.5H2O (0.2 mmol, 0.069 g),matrine (0.2 mmol, 0.049 g) and 0.5 mL of methonal were placed in athick Pyrex tube (ca 20 cm long). The mixture was frozen by liquid N2

and evacuated under vacuum and sealed. Then it was heated at 110 °Cfor five days. Light-yellow block crystals suitable for X-ray diffractionanalysis were obtained. Yield: 0.064 g, 55%. Anal. Found(%): C, 33.35;H, 5.12; N, 4.82. Calculated(%) for C16H29Cl5N2O2Sn: C, 33.28; H, 5.06;N, 4.85. IR (v, cm−1, KBr, m=moderate, s=strong, w=weak):3510s, 3055w(–CH2–), 2950m(–CH2–), 2873w(–CH2–), 1576s (C O),1475s, 1456s, 1424m, 1203m, 1060m, 1009s, 804w. ESI-MS: 296.6(−MS, calculated 296.7 for [SnCl5]−), 248.9 (+MS, calculated 249.2for [H-MT]+).

2.4. X-Ray crystallography

Crystal data were collected on a Bruker Smart Apex II CCDdiffractometer equipped with graphite monochromated Mo-Kαradiation (λ=0.71073 Å) at room temperature. The structures weresolved with direct methods and refined using SHELX-97 programs[24]. The non-hydrogen atoms were located in successive differenceFourier synthesis. The final refinement was performed by full-matrixleast-squares methods with anisotropic thermal parameters for no-hydrogen atoms on F2. The hydrogen atoms were added theoreticallyand riding on the concerned atoms. The crystallographic data andrefinement details of the structure analyses are summarized inTable 1.

2.5. Cytotoxicity assay

Cell lines: CNE1, HeLa, NCI-H460, MCF-7, SGC7901, Ketr-3, SW480,HepG-2 were obtained from the Shanghai Cell Bank in the ChineseAcademy Sciences. Tumour cell lines were grown in RPMI-1640medium supplemented with 10% (v/v) fetal bovine serum, 2 mMglutamine, 100 U/mL penicillin, and 100 U/mL streptomycinin at37 °C, in a highly humidified atmosphere of 95% air/5% CO2. Thecytotoxicity ofMT and title compounds against CNE1, HeLa, NCI-H460,MCF-7, SGC7901, Ketr-3, SW480, HepG-2 cell lines were examined by

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Scheme 1. Two binding modes of matrine in metal–matrine compounds.

173Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 171–180

themicroculture tetrozolium (MTT) assay [25]. The experiments werecarried out using reported procedure [14]. The growth inhibitory rateof treated cells was calculated using the data from three replicate testsby (ODcontrol−ODtest)/ODcontrol×100%. These compounds incubatedwith cell lines, respectively, for 24 h, 48 h and 72 hwith concentrationgradient of 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL.

2.6. Cell cycle analysis

HepG2 cell lines were maintained in Dulbecco's modified Eagle'smedium with 10% fetal calf serum in 5% CO2 at 37 °C. Cells wereharvested by trypsinization and rinsed with PBS. After centrifugation,the pellet (105–106 cells) was suspended in 1 mL of PBS and kept onice for 5 min. The cell suspension was then fixed by dropwise additionof 9 mL of precooled (4 °C) 100% ethanol under violent shaking. Fixedsamples were kept at 4 °C until use. For staining, cells werecentrifuged, resuspended in PBS, digested with 150 mL RNAse A(250 μg/mL), and treated with 150 mL P1 (100 μg/mL), then incubat-ed for 30 min. at 4 °C. PI-positive cells were counted with a FACScanfluorescence-activated cell sorter (FACS). The population of cells ineach cell cycle phase was determined using Cell Modi FIT software(Becton Dickinson).

2.7. Spectroscopic studies on DNA interaction

All the experiments involving the interaction of the compoundswith DNA were examined in the mixed solution of Tris–HCl buffer(50 mM NaCl, 5 mM Tris, pH 7.35) containing 0.2% DMSO. Theconcentration of calf thymus DNA (ct-DNA) per nucleotide wasdetermined by the UV absorption at 260 nm using the molar

Table 2Selected bond lengths (Å) and bond angles (°) for compounds 1, 2 and 3.

Compound 1

Ga(1)−Cl(1) 2.1694(15) Ga(1)−Cl(2) 2.1656(16)Cl(1)−Ga(1)−Cl(2) 108.80(7) Cl(1)−Ga(1)−Cl(3) 108.56(6)Cl(1)−Ga(1)−Cl(4) 109.42(7) Cl(2)−Ga(1)−Cl(4) 110.02(7)

Compound 2

Au(1)−Cl(1) 2.2760(17) Au(1)−Cl(2) 2.2719(18)Cl(2)−Au(1)−Cl(1) 89.29(7) Cl(2)−Au(1)−Cl(3) 89.73(7)Cl(4)−Au(1)−Cl(1) 90.40(7) Cl(4)−Au(1)−Cl(3) 90.65(7)

Compound 3

Sn(1)−O(1) 2.1342(19) Sn(1)−Cl(1) 2.4237(7)Sn(1)−Cl(4) 2.3993(8) Sn(1)−Cl(5) 2.4140(10)O(1)−Sn(1)−Cl(1) 86.83(5) O(1)−Sn(1)−Cl(2) 89.35(6)O(1)−Sn(1)−Cl(5) 87.23(6) Cl(2)−Sn(1)−Cl(1) 90.31(3)Cl(3)−Sn(1)−Cl(2) 90.31(4) Cl(3)−Sn(1)−Cl(5) 90.80(4)Cl(4)−Sn(1)−Cl(3) 95.33(3) Cl(4)−Sn(1)−Cl(5) 91.70(4)

absorption coefficient of 6600 M−1 cm−1 [26]. The UV absorbanceat 260 and 280 nm of the ct-DNA solution in Tris–HCl buffer gives aratio of 1.85, indicating that the DNA was sufficiently free of protein[27]. A solution system of 10−4 M DNA and 10−5 M EB with [DNA]/[EB] ratio of 10:1 was prepared for EB–DNA competitive bindingstudies on spectrofluorometers.

2.8. Agarose gel electrophoretic assay [28]

In plasmid DNA unwinding experiments, all compounds weredissolved in DMSO at concentration of 2×10−3 M as stock solution,and were diluted to 10 and 100 μM by 1× TBE buffer. Compounds ofvarious concentrations were mixed with 0.5 μg DNA and made up to atotal 25 μL by TBE buffer, so that the same experiment can be repeatedtwice. All samples were incubated at 25 °C in dark for 4 h. Then 12 μLof each sample mixed with 2 μL DNA loading buffer was electro-phoresed at 5 V/cm through 0.8% agarose gel immersed in 1× TBEbuffer solution for 60 min. Finally, the gel was stained with EB(0.5 mg/L) in dark for 30 min, followed by visualized on a BIO-RADimaging system with a UV–Vis transilluminator.

Notably, in TOPO I inhibition experiments, a total 12 μL reactionsolution of each sample contains 0.25 μg pUC19 plasmid DNA, 1 UTOPO I, 1 μL TOPO I buffer, 1 μL BSA and compounds with variousconcentrations, with 10-hydroxy camptothecin (HCPT) as control.The samples were incubated for 30 min at 37 °C and then for 5 min at55 °C. Since DMSO stock solution of the compound is 2×10−3 M, thefinal percentage of DMSO in reaction solution is no more than 5%.

3. Results and discussion

3.1. Synthesis

In the presence of 1 M HCl, matrine reacted with Ga(NO3)3 andAuCl3 to generate ionic compounds with matrine taking place inprotonation, in which the carbonyl O did not coordinate to metalcenter directly. Whereas under neutral or weak acidic condition,similar to [Zn(Cl4)(H-MT)] [20], matrine reacted with SnCl4 to formSn–MT complex with zwitterionic matrine directly bonding to Sn(IV)via its carbonyl O.

Important IR spectral bands of compounds 1, 2, 3 show that thestrong carbonyl stretching band is red-shifted from 1633 cm−1 formatrine to 1576, 1603 and 1576 cm−1 for compounds 1, 2, 3,respectively, due to the lengthening of the carbonyl bond. Othervibrations are mainly due to –CH2– stretching and deformationvibrations of trans quinolizidine group [20]. The broad bands centeredat 3510–3438 cm−1 are due tomoisture. The IR spectra of compounds

Ga(1)−Cl(3) 2.1430(16) Ga(1)−Cl(4) 2.1471(17)Cl(2)−Ga(1)−Cl(3) 109.67(6) Cl(3)−Ga(1)−Cl(4) 110.34(8)

Au(1)−Cl(3) 2.2759(17) Au(1)−Cl(4) 2.2746(18)Cl(2)−Au(1)−Cl(4) 177.10(7) Cl(3)−Au(1)−Cl(1) 178.19(7)

Sn(1)−Cl(2) 2.4061(9) Sn(1)−Cl(3) 2.4057(9)C(1)−O(1) 1.288(3)O(1)−Sn(1)−Cl(3) 87.86(6) O(1)−Sn(1)−Cl(4) 176.65(6)Cl(2)−Sn(1)−Cl(5) 176.37(3) Cl(3)−Sn(1)−Cl(1) 174.65(3)Cl(4)−Sn(1)−Cl(1) 89.96(3) Cl(4)−Sn(1)−Cl(2) 91.64(3)Cl(5)−Sn(1)−Cl(1) 88.27(3) C(1)−O(1)−Sn(1) 128.48(17)

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Fig. 1. ORTEP drawing (left) and space filling (right, [GaCl4]− has been omitted) of [H-MT][GaCl4] (1). Ellipsoids are plotted at the 50% level.

174 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 171–180

1, 2, 3 present asymmetric (–CH2–) stretching vibrations at 3055–3013 cm−1 and 2950–2927 cm−1. The symmetric (–CH2–) stretchingvibrations are at 2873–2868 cm−1. The –CH2– scissor band appears at1475–1467 cm−1 which is comparable to that at 1463 cm−1 formatrine. The bands around 1456–1337 cm−1 can be assigned towagging or twisting of CH2–N, CH2–CO and CH2, respectively [20].

3.2. Crystal structure characterization

Owing to matrine only having one carbonyl O as donor atom, andits N easily taking its place in protonation, therefore, as shown inScheme 1, matrine reacts with metal salts generally to give rise to theionic compounds and monodentate metal–matrine complex. Theselected bond lengths (Å) and bond angles (°) for compounds 1, 2, 3are given in Table 2.

3.2.1. [H-MT][GaCl4] (1)As shown in Fig. 1, similar to [4-NH2PyH][GaCl4] [29], in 1, matrine

(MT) is protonated at N(2) to give a cationmatrinium, does not contactGa(III) directly, and tetrachloride gallium(III) acts as an anion to balancethe charge. This coordination mode of MT towards Ga(III) is known inmatrinium tetrachloroferrate(III) [18]. The expected intra-molecularbond lengths and angles in [H-MT][GaCl4] show an almost tetrahedralgeometrywithGa–Cl bond lengths in the rangeof 2.1430−2.1694 Åandangular [Cl(2)−Ga(1)−Cl(4) 110.02(7)°, Cl(3)−Ga(1)−Cl(4) 110.34(8)°], which agree well with the geometry parameters of K[GaCl4] [30].Likematrinium tetrachloroferrate(III) [18], the rings A, B, C, andD of thematrinium cation exist in the chair, chair, chair, and half-chairconformation, respectively. The A/B-, B/C-, and C/D-ring fusions aretrans, cis, cis, and trans, respectively. The chiral C5(R), C6(R), C7(S) andC8(S) atoms have the same absolute configurations as those reportedpreviously [19]. The bond distances and angles in the matrinium cationare consistentwith the previous reported values [18]. As shown in Fig. 1,viewing from the space filling drawing of the matrinium cation, itpossesses an approximate planarity.

Fig. 2. ORTEP drawing (left) and space filling (right, [AuCl4]− has been o

3.2.2. [H-MT][AuCl4] (2)As shown in Fig. 2, similar to compound 1, compound 2 comprises

one matrinium cation and one [AuCl4]− anion. The [AuCl4]− anion isthe square planar structure, which is commonly observed for gold (III)[31]. The Au−Cl bond lengths in the anion (2.2719−2.2760 Å) areanalogous those previously reported for other chlorogold(III) com-plexes [32]. Resembling matrinium tetrachloroferrate(III) [18] andcompound 1, the rings A, B, C, and D of the matrinium cation exist inthe chair, chair, chair, and half-chair conformation, respectively. TheA/B-, B/C-, and C/D-ring fusions are trans, cis, cis, and trans,respectively. The chiral C4(R), C5(R), C11(S) and C15(S) atoms havethe same absolute configurations as those reported previously [19].The bond distances and angles in the matrinium cation are consistentwith the previous reported values [18]. As shown in Fig. 2, viewingfrom the space filling drawing of thematrinium cation, it suggests thatthe matrinium displays an approximate planarity.

3.2.3. [SnCl5(H-MT)]⋅CH3OH (3)As shown in Fig. 3, compound 3 consists of [SnCl5(H-MT)] and co-

crystallized methanol solvent molecule. Unlike ionic compounds 1and 2, but similar to matrin-1-ium-trichlorozincate [20], compound 3is a zwitterionic complex, in which a positive charge locates on N(2)and a negative charge lies on the SnCl5 group. In 3, the matrinemolecule is protonated at N(2) and acts as a ligand coordinating to Snatom directly (with Sn−O coordination bond of 2.1342(19) Å); andthe Sn atom is further coordinated by five Cl atoms and results in adistorted octahedral geometry, in which cis Cl−Sn−Cl bond anglesappear in the range 88.27(3) to 95.33(3)°, with a trans O−Sn−Cllinkage 176.65(6)°. They fall within the expected range and arecomparable to octahedral tin(IV) species, e.g. [SnCl5(thf)]− [33]. Likematrin-1-ium-trichlorozincate [20], the carbonyl bond length (1.288(3) Å) in matrinium of 3 is significantly longer than that in matriniumof 1 and 2, apparently due to the formation of coordination bond,which is consistent with the fact that the strong carbonyl stretchingband is red-shifted from 1633 cm−1 for matrine to 1576 cm−1 [20].The D ring of matrinium is a distorted sofa conformation, however the

mitted) of [H-MT][AuCl4] (2). Ellipsoids are plotted at the 50% level.

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Fig. 3. ORTEP drawing (left) and Space filling (right) of [SnCl5(H-MT)]⋅CH3OH (3). Ellipsoids are plotted at the 50% level. For clarity, the solvent molecule was omitted.

175Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 171–180

other rings adopt chair formation. The A/B-, B/C-, and C/D-ring fusionsare trans, cis, cis, and trans, respectively. Because of the conjugationbetween the C O group and the lone electron pair of nitrogen atomwithin the cyclic amide frame, it leads to an approximate planarity ofthe fragment comprising atoms C1, C5, C16, N1, and O1, which altersthe chirality of chiral carbon atoms as C5(R), C6(S), C14(S) and C15(S)atoms have the same absolute configurations as those reportedpreviously [20]. As shown in Fig. 3, viewing from the space fillingdrawing of 3, it is found that 3 has an approximate planarity.

Although the matrine in above mentioned three matrine–metalcompounds displays in two modes: uncoordinated matrinium andcoordinated matrinium, the matrine molecules still retain theiroriginal conformation of junction of sofa and chair with anapproximate but not exact planarity [20], which can be observedfrom the space filling drawing. This structural feature facilitates itsintercalation to the base pairs of DNA. Whereas, from the ESI-MSanalysis results, it was found that the main species in solution ofcompounds 1, 2were almost same as the solid state structure, namelycontaining [H-MT]+ cations, and [GaCl4]−, [AuCl4]− anions; while inthe water solution of compound 3, the disassociated species differentfrom solid state structure were observed, mainly including [H-MT]+

cations, and [SnCl5]− anions.

3.3. In vitro antitumour activity assay and cell cycle analysis

The cell growth inhibition rates of compounds 1, 2, 3 against eighttumour cell lines were evaluated by MTT methods, as shown inFigs. 4–6 and Table 3, respectively. After incubation of tumour cellsand each compound at concentration of 50 μg mL−1 for 72 h as

Fig. 4. Cytotoxic activity of [H-MT][GaCl4] (1), MT and cisplatin against eight humantumour cell lines.

identical experimental conditions (in some case for 48 h), thesecompounds exhibit different antitumour activity.

As shown in Fig. 4 and Table 3, the inhibition rates of compound 1towards NCI-H460 and HepG2 are 56% and 71.4%, respectively, andboth higher than 50%. Especially, in contrast to matrine (34.8%) andcisplatin (41.8%), compound 1 almost completely inhibits the growthof SW480, with an inhibition rate of 99.4%, which exhibits signif-icantly enhanced effect after matrine combination with GaCl3. Incomparison with matrine and cisplatin, against the other five tumourcell lines, the inhibition rates of compound 1 are moderate and nohigher than 50%. In some case, the inhibition rates of compound 1 arehigher than those of matrine and cisplatin; but in the other cases, theyare not.

As shown in Fig. 5 and Table 3, in contrast to compound 1, matrineand cisplatin, compound 2 exhibits higher inhibition activity againstsix tumour cell lines than that of 1, matrine and cisplatin, except forNCI-H460 and SW480. Compound 2 can effectively inhibit the growthof HeLa, Ketr-3, HepG2 and MCF-7 cell lines with inhibition rates over80%.

As shown in Fig. 6 and Table 3, comparing with compounds 1, 2,matrine and cisplatin, compound 3 exhibits lower inhibition ratesagainst most tested tumour cell lines, except for Ketr-3 with 64.2%.The inhibition rates of 3 to the other seven tumour cell lines are closeto those of matrine or cisplatin or lower than those of both matrineand cisplatin.

Furthermore, in order to investigate the influence of themetal saltson the antitumour activity of the compounds 1, 2, 3, the activity of thetumour cell growth inhibition rates of the corresponding metal saltsagainst six tested tumour cell lines at concentration of 100 μM was

Fig. 5. Cytotoxic activity of [H-MT][AuCl4] (2), MT and cisplatin against eight humantumour cell lines.

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Fig. 6. Cytotoxic activity of [SnCl5(H-MT)]⋅CH3OH (3), MT and cisplatin against eighthuman tumour cell lines.

176 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 171–180

assayed, as listed in Table 4. From Table 4, it was found that none ofthese metal salts exhibited inhibition rates to the six tumour cell linestested were over 30% at the concentration of 100 μM (for comparison,the molar concentrations of 1, 2, 3 are 108, 85, 92 μM, respectively).These experimental results suggest that the corresponding metal saltsalone do not play the key role to antitumour function under theapproximately same concentration magnitude, even though thesemetal salts generally are accepted as potent antitumour agents.Whereas the combination of themwith matrine gives rise to the ionicmetal–matrinium compounds, in some cases they exhibit significantenhanced antitumour activity, such as 1 to SW480 and 2 to HeLa,HepG2 and MCF-7, which exceeds matrine and cisplatin, and displaysynergistic contribution of their components. Such synergistic effecthas been observed in the combination of gallium nitrate withvinblastine to treat of metastasis of bladder epithelial tumour andresistance of ovarian cancer to cisplatin [7].

In order to investigate the reason of the higher cytotoxicity of thetitle compounds, cell cycle-arrest studies were undergone. The cellcycle analysis based on DNA content detected by FACS was performedwith HepG2 cells after treated with compounds 1, 3 and MT (50 μg/mL) for 24 h. As shown in Fig. 7, there are apparent transitions from anS-phase accumulation to a G2/M arrest, resulting in concomitantincreases in the G2-phase population (from 18.85 to 30.82% for 1,31.72% for 3 and 35.55% for MT) and leading to cell cycle arrest at theG2/M phase. This finding was supported by following DNA bindingstudies.

3.4. Topoisomerase I inhibition studies

Topoisomerases are ubiquitous molecules that relieve the torsion-al stress in the DNA helix generated as a result of replication,transcription and other nuclear processes. They are also specifictargets for a number of anticancer agents, including the camptothe-

Table 3Inhibition rates of matrine and compounds 1, 2, 3 towards eight selected tumour cell lines

Compounds Inhibition rate (%)

HeLa SGC7901 NCI-H460 SW

Matrine 42.0±6.5 18.7±2.7 63.1±6.6a 34.[H-MT][GaCl4] (1) 42.5±6.3 24.9±5.1 56.0±5.1 99.[H-MT][AuCl4] (2) 80.5±2.6 34.1±9.7a 43.8±6.7 5.[Sn(H-MT)Cl5] (3) 40.2±1.8 23.2±7.9 43.4±1.5 40.Cisplatin 29.6±4.4 11.3±2.2 48.6±8.7 41.

Note: “a” represents that the incubation time of tumour cell line and the compound is 48 h

cins, indolocarbazoles, and indenoisoquinolines [34]. These com-pounds bind to a transient topoisomerase I (TOPO I)–DNA covalentcomplex and inhibit the resealing of a single-strand nick that theenzyme creates to relieve superhelical tension in duplex DNA [35,36].

The inhibition abilities of MT and compounds 1, 2, 3 to TOPO I wereevaluated by agarose gel electrophoresis. As shown in Fig. 8, MT alonecannot inhibit TOPO I to cleave and unwind plasmid DNA atconcentration range of 10 and 100 μM. While compounds 1 and 2both exhibit significant TOPO I inhibition ability at 50 μM. It indicatesthat either the cationic form of protonatedMT or [GaCl4]− and [AuCl4]−

as counterionshas potent TOPO I inhibition ability, and synergistic effectbetween these two parts also cannot be excluded. Moreover, no TOPO Iinhibition ability was observed for compound 3, which might correlateto its different structure.

3.5. DNA binding properties

Although there is some evidence to suggest that other biologicaltargets, including RNA or proteins, may be important in the cisplatinmechanism, it is generally accepted that DNA is the primary target[37]. Similarly, interactions between small molecules and DNA rankamong the primary action mechanisms of antitumour activity. DNAreplication in tumour cells will be blocked by the intercalation of smallmolecules into the base pairs of DNA. There is research indicating thatDNA intercalation plays an important role in naturally derivedcompounds to exhibit their anti-proliferative activities and apoptosiseffects on tumour cells [35,38]. Besides the intercalation, the DNAbinding modes of small molecules include groove binding, covalentbinding, electrostatic interaction as well as hydrogen-binding [39].Generally, the active compounds with intercalation ability arerequired to possess an approximate planar structure, with amedium-sized planar area and some hydrophobic character [39]. Inorder to investigate the binding properties of matrine and its metalcompounds 1, 2, 3 to DNA, the classic spectroscopic studies includingUV–Vis, fluorescence and CD, as well as agarose gel electrophoresis,were carried out.

3.5.1. UV–visible (UV–Vis) absorption titration analysisUV–Vis spectra of MT and compound 3 are shown in Fig. S1 (ESI†).

MT exhibits its characteristic absorption in water solution as a broadband in the range of 200–220 nm, which can be ascribed to the sumabsorptions of n(N)→π⁎(C O) and n(O)→π⁎(C O) electron transition[40]. With increasing concentration of ct-DNA added, significanthypochromicity and red-shift could be observed. At the ratio of [DNA]/[MT] up to 20 times, 56% hypochromicity with a 7 nm red-shift (from210 to 217 nm) was achieved. Considering the approximate planarstructure of MT, it suggests that the main part of MT, especially thecarbonyl group (C O), should have intercalated the neighbouring basepairs of ct-DNA, and the π orbital coupling between π⁎ orbital of C Oand π orbitals of DNA bases could exist, which may be interpreted viamixed QM/MM molecular dynamics simulations [41].

The UV–Vis spectral titration analysis between ct-DNA andcompounds 1, 2 are shown in Fig. 9, and similar broad absorptionbands as MT in the range of 200–220 nm are observed. The absorption

at 50μg⋅ml−1 for 72 h.

480 Ketr-3 CNE-1 HepG2 MCF-7

8±7.6 23.7±2.3 59.7±8.6 69.5±4.1 52.1±6.14±10.9 34.2±2.2 30.2±3.7 71.4±2.4 45.8±5.07±2.2 86.9±5.3a 71.0±3.7 86.4±4.6 85.9±9.12±5.0 64.2±4.4 38.4±5.6 23.9±5.5 47.9±5.18±0.9 45.8±5.5 45.5±6.6 25.9±3.9 35.4±5.3

.

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Table 4Inhibition rates of threemetallic materials to six selected tumour cell lines at 100 μM for72 h.

Metallicmaterials

Inhibition rate (%)

HeLa SGC7901 NCI-H460 CNE-1 HepG2 MCF-7

Ga(NO3)3 25.13 6.99 ND ND ND 29.56NaAuCl4 21.02 −0.35 5.20 −3.62 12.08 24.40SnCl4(DMSO)2 ND ND 22.89 −11.77 18.47 26.60

ND: not determined.

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bands of 1 and 2 also show significant hypochromicities and red shiftswith the increasing ratio of [DNA]/[complex]. The peak absorptionintensities of compounds 1 and 2 decreased about 46.6% with a red-shift of 11 nm (from 217 to 228 nm) and 50.6% with a red-shift of10 nm (from 212 to 222 nm), respectively, when the ratio of [DNA]/[compound] increased from 0 to 20. Comparing with that of MT, thechanges on absorption spectra of MT and compounds 1, 2 are at samemagnitude, which suggests the binding of 1 and 2 to ct-DNA is mainlyattributed to the H-MT intercalation into neighbouring base pairs ofct-DNA [42], due to the approximate planarity of the matrinium

Fig. 7. Induction of cell cycle arrest in HepG2 cells after treatment with 1, 3 andMT. Untreatepropidium iode staining and flow cytometric analysis. G0/G1, G2/M, and S indicate the cell

(Figs. 1 and 2). While the protonation of nitrogen of matrine in 1 and2, which results in the cationic form of H-MT, also suggests thatexternal electrostatic interaction between compounds 1, 2 andpolyanionic backbone of ct-DNA should be considered.

However, compound 3 does not show any absorption band in theUV absorption region above 190 nm, which may be due to thecoordination of carbonyl O atom of MT to tin(IV), which weakens thedouble bond of C O and leads to the hypochromatic shift of itsabsorption, or due to its different disassociated [SnCl5]− anionexistence in solution. Therefore, the interaction between ct-DNAand compound 3 by UV–Vis spectral analysis was not discussed here.

3.5.2. EB-competitive binding studiesMatrine and its metal compounds exhibit no significant fluores-

cence emission because they do not contain aromatic moieties.Therefore the binary interaction system of matrine/compound and ct-DNA is not suitable for DNA binding studies. Thus EB–DNA–compoundternary systemwas selected to probe the DNA binding modes of thesemetal–MT compounds. Ethidium bromide (EB) is a classic intercalatorthat gives significant fluorescence emission intensity when inter-calates into base pairs of DNA. When it is replaced or excluded frominternal hydrophobic circumstance of DNA double helix by other

d cells and cells treated with 1, 3 andMT (50 μg/mL) were collected at 24 h, subjected tophase. Data shown is a representative experiment repeated twice with similar results.

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Fig. 8. TOPO I inhibition pUC19 DNA in the presence of MT and its compounds 1, 2, 3 at various concentrations. Lanes 1 and 2 refer to DNA+TOPO I with MT of 100 μM and 10 μM,respectively; Lanes 3–6, 7–10, and 11–14 represent DNA+TOPO I with compounds 1, 2 and 3 of 100, 50, 10 and 5 μM; Lane 15, DNA alone; Lane 16, DNA+TOPO I; Lane 17,DNA+TOPO I+HCPT as control; SC = supercoiled DNA; RX = relaxed DNA).

178 Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 171–180

small molecules, its fluorescence emission is effectively quenched bythe external polar solvent molecules (such as H2O) [27].

As shown in Fig. 10 and Fig. S2(ESI†), the EB–DNA system showscharacteristic strong emission at about 585 nm when excited at345 nm, indicating that the intercalated EB molecules have beensufficiently protected by the neighbouring DNA base pairs from beingquenched by H2O. With increasing concentration of MT or its metalcompounds added by titration, the emission intensity of EB decreasedintensively. Under the same condition of [DNA]/[EB]/[compound]from 10/1/0.2 up to 10/1/10, calculated decreases of EB emissionintensity at 585 nmbyMT and compounds 1, 2, 3were of 29.1%, 87.8%,66.7% and 54.3%, respectively. These results indicate that all the three

Fig. 9. UV absorption spectra of complex 1 and 2 with increasing concentration ofct-DNA (concentration of complex is 2.0×10−5 M, [ct-DNA]/[compound] ranged from0 to 20).

compounds exhibit higher competitive binding ability with EB in ct-DNA system than MT alone does, and hence more intensiveintercalation binding of metal–MT compounds than free MT issuggested, because compounds 1, 2, 3 retain approximate planarity(Figs. 1–3) [43]. In addition, it may be due to the cationic type of H-MTformed in these compounds, which affords external electrostaticinteraction as additive stabilizing energy with ct-DNA polyanionicbackbone.

3.5.3. Circular dichroism absorption spectral analysisTo gain further information of DNA conformation altered by the

title compounds, we recorded CD spectra of DNA modified bycompounds 1, 2 and 3. In the CD spectrum of ct-DNA, a positiveband at 276 nm due to base stacking and a negative band at 246 nmdue to the right-handed helicity can be observed, which indicates Bform of DNA in solution system. Generally accepted, the CDabsorption spectrum of DNA is very sensitive to its conformationalchanges. So only when small molecules bind to ct-DNA byintercalation or covalent binding, which may result in the DNAconformational modifications or inducing significant CD spectralperturbation of ct-DNA [44].

As shown in Fig. 11 and Figs. S3–S5 (see ESI†), increasingconcentration of MT, no obvious spectral perturbation is observedon both of the positive and negative absorption. Spectral analyses onUV–Vis and EB binding indicate that MT mainly bind to ct-DNA byintercalation and most possibly via carbonyl group, which does notsignificantly influence the base stacking and conformation of ct-DNA,and the unchanged CD spectrum of ct-DNA bound byMT hence can beunderstood [44]. Similar to that of MT, binding of compounds 1 and 3also does not induce significant CD spectral perturbation of ct-DNA.However, hypochromicity and red-shift on positive absorption bandof ct-DNA can be observed when bound with 2 with increasingconcentration, which suggests some other binding modes should beexisting between 2 and ct-DNA, and [AuCl4]− may exert its specialaction mechanism to ct-DNA. But it needs detailed investigation infuture publication.

3.5.4. Agarose gel electrophoresis assayAgarose gel electrophoresis assay is a useful method to investigate

various binding modes of small molecules to supercoiled DNA.Natural-derived plasmid DNA mainly has a closed-circle supercoiledform (form I), as well as nicked form (form II) and linear form (formIII) as small fractions. Intercalation of small molecules to plasmid DNAcan loosen or cleave the supercoiled form DNA, which decreases itsmobility rate and can be separately visualized by agarose gelelectrophoresis method. Whereas simple electrostatic interaction ofsmall molecules to DNA does not significantly influence the super-coiled form of plasmid DNA, thus the mobility of supercoiled DNAdoes not be changed.

As shown in Fig. 12, binding with MT does not change the mobilityrate of closed-circle supercoiled DNA. It indicates that MT cannot

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Fig. 10. Fluorescence emission spectra of EB-ct-DNA system (EB, 2×10−5 M; ct-DNA,2×10−4 M) in the absence (dashed line) and presence (solid line) of compounds([DNA]/[EB]/[1/2/3]=10/1/0.2–10/1/10)

Fig. 11. Circular dichroism spectra of ct-DNA in the absence (dashed line) and presence(solid line) of compound1, respectively ([ct-DNA]=5×10−5 M. r=[compound]:[ct-DNA](r=0, 1, 4, 6, 8, 10)).

Fig. 12. Gel electrophoretic assay for MT and its compounds 1, 2, 3with pUC19 plasmidDNA. (Lane 1, DNA alone; Lanes 2–3, 4–5, 6–7, 8–9 refers to pUC19 DNA boundwithMT,compounds 1, 2 and 3 of 10 μM and 100 μM, respectively.)

179Z.-F. Chen et al. / Journal of Inorganic Biochemistry 105 (2011) 171–180

intensively destroy the conformation of supercoiled form DNA. WhenDNA bound by compounds 1 and 3, slight decrease on mobility rate ofsupercoiled DNA can be observed, which can be ascribed to anintercalation binding mode of H-MT [28]. Notably, 2 at a highconcentration of 100 μM can obviously decrease the mobility rate ofsupercoiled form DNA, as shown in lane 7, which suggests a moderateunwinding ability of 2 to supercoiled DNA, as well as an intercalationbindingmode of H-MT should exist. The exceptional behavior of 2 thatis in agreement with the exhibition of 2 in CD spectral analysis, maycorrelate with the square planar [AuCl4]− anion.

In summary, the UV–Vis spectral titration analyses reveal that thebinding of 1 and 2 to ct-DNA is mainly attributed to H-MTintercalation into neighbouring base pairs of ct-DNA; furthermore,the EB-competitive binding studies indicate that compounds 1, 2, 3exhibit higher competitive binding ability with EB in ct-DNA systemthan MT alone does; and compound 2 induces significant CD spectralperturbation of ct-DNA, but compounds 1 and 3 do not, which isfurther confirmed by the fact that DNA bound by compounds 1 and 3slightly decreased the mobility rate of supercoiled DNA, whereas 2 athigh concentration could obviously decrease the mobility rate ofsupercoiled formDNA. Thus it can be seen that compound 2 binding toDNA is the most powerful. These findings agree well with the fact thatthe cytotoxicities of compounds 1, 2, 3 towards most tumour cell linestested are more sensitive than those of MT alone, and compound 2 isthe most one, which are mainly caused by the approximate planarmatrinium in compounds 1, 2, 3 bindingmore powerfully to DNA thanMT, and themetal chloride anions also playing key roles in their actionmechanisms.

4. Conclusion remarks

Three new matrinium–metal compounds of Ga(III), Au(III) and Sn(IV) have been synthesized and characterized. Their crystal structuresare determined by single crystal X-ray diffraction analysis. Com-pounds 1 and 2 are ionic compound, whereas 3 is a coordinationcompound formed by monodentate matrinium via its carbonyl O.But the ESI-MS results show that they may exist with ionic species:[H-MT]+, [GaCl4]−, [AuCl4]− and [SnCl5]− in water solution. The invitro cytotoxicities of 1, 2 and 3 against eight selected human tumourcell lines are different. The combination of [GaCl4]− and [AuCl4]− withmatrinium gives rise to the ionic metal–matrinium compounds: 1 and

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2, in some case, they exhibit significant enhanced antitumour activity,such as 1 to SW480, 2 to HeLa, HepG2 and MCF-7, which exceedsmatrine and cisplatin, and displays synergistic contribution of theircomponents. However, in the case of 3, such synergistic effect couldnot be observed due to the electronic structure alteration of lactamgroup of matrinium, thus 3 exhibits lower antitumour activity thanthat of compounds 1 and 2. Although the spectroscopic and agarosegel electrophoresis assay show that these compounds bind to DNAinducing only small structural changes in the duplex, it could lead to adifferent cellular response [41]. The cell cycle analyses show thatcompounds 1, 3 and MT exhibit cell cycle arrest at the G2/M phase.Their interactions with ct-DNA indicate that these metal–matrinecompounds may act mainly via intercalation mode of H-MT. Inaddition, 1 and 2 exhibit potent TOPO I inhibition ability, implyingtopoisomerase I may be another molecular target. However, the exactmolecular mechanism requires further detailed investigation.

Abbreviations

BSA bovine serum albuminSGC7901 human gastric adenocarcinoma cellsCD circular dichroismct-DNA calf thymus DNAEB ethidium bromideESI-MS electrospray ionization mass spectrumHCPT 10-hydroxy camptothecinHeLa human cervical cancer cellsHepG2 human hepatoma cellsKetr-3 renal carcinoma cellsMCF-7 human breast adenocarcinoma cellsMKN45 gastric cancer cellsMT matrineMTT 3-[4,5-Dimentylthiazole-2-yl]-2,5-diphenpyltetra-zolium

bromideNCI-H460 large cell lung cancer cellsPBS phosphate-buffered salineQM/MM quantum mechanics/molecular mechanicsRPMI Roswell Part Memorial InstituteSGC7901 human gastric adenocarcinoma cellsSW480 colorectal adenocarcinoma cellsTBE tris, boric acid, edtaTOPO I topsoisomerase type ITris tris(hydroxymethyl)aminomethane

Acknowledgements

This work was supported by National Basic Research Program ofChina (2009CB526503, 2010CB534911, and 2010CB933902), theNational Natural Science Foundation of China (No. 20861002), andNatural Science Foundation of Guangxi Province (Nos. 0991012Z,0991003, and 2010GXNSFF013001). We also thank Prof. Dr. Huai-Ming Hu for the crystal structures determination.

Appendix A. Supplementary Data

Crystallographic data for the structural analysis have beendeposited with the Cambridge Crystallographic Data Centre, CCDCNo. 670188–670190 for compounds 1, 2, 3. The data can be obtainedfree of charge via http://www.ccdc.cam.ac.uk, or from the Cambridge

Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK;fax: (+44) 1223-336-033; or e-mail:[email protected].

Supplementary data to this article can be found online at doi:10.1016/j.jinorgbio.2010.10.007.

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