Structural and antitumor activity study of γ-octamolybdates containing aminoacids and peptides

Inorganica Chimica Acta 359 (2006) 1673–1680 www.elsevier.com/locate/ica

Note

Structural and antitumor activity study of c-octamolybdates containing aminoacids and peptides Marina Cindric´ a b

a,*

, Tanja Kajfezˇ Novak a, Sandra Kraljevic´ b, Marijeta Kralj b, Boris Kamenar a

Laboratory of General and Inorganic Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia Laboratory of Functional Genomics, Division of Molecular Medicine, Rud-er Bosˇkovic´ Institute, Bijenicˇka 54, HR-10000 Zagreb, Croatia Received 18 April 2005; accepted 8 October 2005 Available online 15 February 2006

Abstract c-Type octamolybdates of the formulae, Na4[Mo8O26(alaO)2] Æ 18H2O (I), Na4[Mo8O26(glyglyO)2] Æ 15H2O (II) and Na4[Mo8O26(glyglyO)2] Æ 12H2O (III) have been prepared from sodium molybdate in aqueous solution by adding DL-alanine or glycylglycine. Their crystal structures have been determined by X-ray structure analysis. DL-alanine and glycylglycine coordinate molybdenum atom in c-octamolybdate [Mo8O26]4 anions via monodentate carboxylate-oxygen atom. The prepared octamolybdates were screened for the possible antiproliferative activity on a panel of five tumor cell lines and on a normal cell line. All tested compounds showed a differential cell-growth inhibition in a dose-dependent manner selectively on hepatocellular carcinoma cell line (HepG2) and breast cancer cell line (MCF-7).  2005 Elsevier B.V. All rights reserved. Keywords: Octamolybdates; Peptides; Crystal structures; Antitumor activity

1. Introduction Heteropoly- and isopolyoxomolybdates represent a class of polyanionic compounds with a variety of important biological activities such as the inhibition of specific enzymes or antiviral and antitumor effects [1,2]. The biomedical investigations of polyoxomolybdates containing aminoacids or peptides have been focused upon finding polyoxomolybdates with both good activity against cancer and improved clinical safety profiles [3–6]. The heptamolybdate, [NH3Pri]6[Mo7O24] Æ 3H2O, has been found to represent a potent antitumor activity in vivo [7] and such activity has been explained by repeated redox cycles of [Mo7O24]6 in tumor cells which inhibit the ATP generation [8]. *

Corresponding author. Tel.: +385 1 461 1191. E-mail address: [email protected] (M. Cindric´).

0020-1693/$ - see front matter  2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2005.10.061

This paper deals with syntheses, crystal structures and antitumor screening of c-type octamolybdates coordinated by DL-alanine, Na4[Mo8O26(alaO)2] Æ 18H2O (I) and glicylglycine, Na4[Mo8O26(glyglyO)2] Æ 15H2O (II) and Na4[Mo8O26(glyglyO)2] Æ 12H2O (III). Their crystal structures consist of c-octamolybdate [Mo8O26]4anions, and DL-alanine or glycylglycine coordinated to molybdenum via monodentate carboxylate-oxygen atoms. Similar c-type octamolybdates, such as [Hmorph]4[Mo8O24(OH)2(metO)2] Æ 4H2O (IV), [Hmorph]4[Mo8O24(OH)2(metO)2] Æ 4CH3OH (V) and [Hmorph]4[Mo8O24(OH)2(alaO)2] Æ 4CH3OH (VI), already known from the literature [9,10], were also part of our tests against carcinoma cells. In this way, structural chemistry of polyoxomolybdates and their complexes with peptides or amino acids might contribute to a better understanding of the reasons for antitumor/viral activities of this class of compounds.

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M. Cindric´ et al. / Inorganica Chimica Acta 359 (2006) 1673–1680 Table 2 ˚ ) in the coordination sphere around molybdenum atoms Bond lengths (A in c-octamolybdates I, II and III

2. Experimental 2.1. Materials and measurements All chemicals were of reagent grade and used as purchased from commercial sources. The IR spectra were recorded in KBr with FTIR 1600 Fourier-transform spectrophotometer in the 4500– 450 cm1 region. Molybdenum was analytically determined according to the procedure described in the literature [11]. 2.2. Synthesis 2.2.1. Na4[Mo8O26(alaO)2] Æ 18H2O (I) DL-Alanine (0.18 g, 2 mmol) was added to an aqueous solution of Na2MoO4 (0.5 g, 2 mmol) and the solution was acidified by addition of HNO3 to pH 3.4. After standing for 10 days at room temperature colorless crystals were obtained. Yield: 0.35 g, 76.1%. Anal. Calc. for C6H50Mo8N2Na4O48: C, 4.05; H, 2.81; N, 1.57; Mo, 43.17. Found: C, 3.46; H, 3.22; N, 1.32; Mo, 42.70%. IR (KBr pellet, cm1): 1660s, 1431m, 976s, 912s, 696s, 644m, 532m. 2.2.2. Na4[Mo8O26(glyglyO)2] Æ 15H2O (II) and Na4[Mo8O26(glyglyO)2] Æ 12H2O (III) Glycylglycine (0.25 g, 2 mmol) was added to an aqueous solution of Na2MoO4 (0.5 g, 2 mmol) and the solution was acidified to pH 3.4 (by addition of HClO4 for complex II or of HNO3 for complex III). After standing for 12 (or 10) days at room temperature colorless crystals of II (or III) were isolated. Yield for II: 0.40 g, 85.1%. Anal. Calc. for C8H46Mo8N4Na4O47: C, 5.32; H, 2.57; N, 3.10; Mo, 42.54. Found:

I

II

III

Mo(1)–O(1) Mo(1)–O(11) Mo(1)–O(12) Mo(1)–O(3) Mo(1)–O(4) Mo(1)–O(8)

2.107(2) 1.724(2) 1.697(2) 1.901(2) 2.258(2) 2.095(1)

2.065(2) 1.714(2) 1.705(2) 1.917(2) 2.277(2) 2.123(2)

2.096(2) 1.706(2) 1.709(2) 1.911(2) 2.275(2) 2.113(2)

Mo(2)–O(21) Mo(2)–O(22) Mo(2)–O(6) Mo(2)–O(3) Mo(2)–O(5) Mo(2)–O(4)

1.718(2) 1.708(2) 1.943(2) 1.948(1) 2.211(1) 2.317(2)

1.702(2) 1.720(2) 1.958(2) 1.947(2) 2.193(2) 2.299(2)

1.709(2) 1.714(2) 1.954(2) 1.952(2) 2.250(2) 2.295(2)

Mo(3)–O(31) Mo(3)–O(7) Mo(3)–O(4) Mo(3)–O(5) Mo(3)–O(8) Mo(3)–O(5)i

1.706(1) 1.752(1) 1.887(1) 1.945(1) 2.158(2) 2.445(2)

1.704(2) 1.750(2) 1.876(2) 1.967(2) 2.136(2) 2.452(2)

1.708(2) 1.751(2) 1.893(2) 1.955(2) 2.136(2) 2.454(2)

Mo(4)–O(41) Mo(4)–O(42) Mo(4)–O(6) Mo(4)–O(8) Mo(4)–O(5) Mo(4)–O(7)

1.721(2) 1.724(2) 1.911(1) 1.922(1) 2.235(1) 2.318(2)

1.708(2) 1.730(2) 1.922(2) 1.907(2) 2.215(2) 2.380(2)

1.705(2) 1.706(2) 1.945(2) 1.911(2) 2.237(2) 2.408(2)

(i) x, y + 1, z.

C, 5.81; H, 2.13; N, 3.14; Mo, 42.96%. IR (KBr pellet, cm1): 1636s, 1382m, 1576s, 933s, 892s, 690s, 551m. Yield for III: 0.17 g, 37.7%. Anal. Calc. for C8H40Mo8N4Na4O44: C, 5.46; H, 2.29; N, 3.19; Mo, 43.71.

Table 1 Crystallographic data for c-octamolybdates I, II and III

Chemical formula Formula weight Crystal system Crystal size (mm) Colour Space group ˚) a (A ˚) b (A ˚) c (A a () b () c () ˚ 3) V (A Z qCalc (g cm3) l (mm1) Data measured Unique data Observed data [I > 2r(I)] Number of variables ˚ 3) Maximum/minimum Dq (e3 A R (Fo) wR ðF 2o Þ

I

II

III

C6H50Mo8N2Na4O48 1777.96 triclinic 0.27 · 0.25 · 0.22 colorless P 1 8.8650(18) 11.000(2) 14.115(3) 77.39(3) 74.71(3) 71.12(3) 1242.9(5) 1 2.375 2.105 14 117 7535 7205 321 2.088/0.807 0.0202 0.0537

C8H46Mo8N4Na4O47 1803.92 triclinic 0.22 · 0.24 · 0.27 colorless P 1 10.946(5) 11.497(4) 11.869(5) 64.70(3) 64.30(3) 82.78(3) 1213.6(9) 1 2.468 2.158 7237 6976 6435 335 0.769/0.699 0.0270 0.0724

C8H40Mo8N4Na4O44 1755.92 triclinic 0.24 · 0.23 · 0.20 colorless P 1 9.4207(8) 11.3205(8) 11.8201(11) 63.896(6) 74.109(7) 82.371(7) 1088.6(2) 1 2.678 2.397 7807 6282 5547 320 0.609/0.701 0.0233 0.0612

M. Cindric´ et al. / Inorganica Chimica Acta 359 (2006) 1673–1680

Found: C, 5.64; H, 2.22; N, 3.09; Mo, 43.45%. IR (KBr pellet, cm1): 1632s, 1381m, 1577s, 930s, 900m, 692s, 550m. 2.3. Crystal structure determination Intensity data for structure I were collected at low temperature (200 K) on a Nonius Kappa CCD diffractometer with graphite-monochromated Mo Ka radiation (k = ˚ ) and reduced using DENZO programme [12]. 0.7107 A Diffraction data for II and III were collected at room temperature on a Philips PW1100 diffractometer updated by STOE with graphite-monochromated Mo Ka radiation ˚ ). The data were corrected for Lorentz and (k = 0.7107 A polarization effects by the X-RED programme [13]. The structures of all complexes were solved by Patterson and Fourier methods using SHELXS97 [14] programme. The refinements were performed by full-matrix least-squares method assuming anisotropic temperature factors for all non-H atoms using SHELXL97 [15].

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Hydrogen atoms bonded to carbon atoms in all structures were generated on geometrical grounds. Hydrogen atoms of amino groups were found in difference Fourier maps and their positions refined, while water hydrogen atoms were also located in difference Fourier maps but kept fixed during the refinement. Crystal parameters, data collection details and the results of refinements are summarized in Table 1. Selected bond lengths and bond angles are listed in Table 2. The molecular structure drawings were prepared using ORTEP [16] and PLATON [17]. 2.4. Antitumor activity 2.4.1. Cell culturing The cell lines HeLa (cervical carcinoma), SW 620 (colon carcinoma), HepG2 (hepatocellular carcinoma), Hep-2 (laryngeal carcinoma), MCF-7 (breast carcinoma) and

0 b c O41

Mo4 O42

O21

O5

O6 O22 O3

Mo3

a

Mo2

O7 O4 Mo1 O11

O8 O31

O12 O1

O2

C1 O9

C2

C3 N2 N1 C4 Fig. 1. ORTEP presentation of the octamolybdate anion with glycylglycine coordinated via carboxylate oxygen atom in structure II with 50% probability displacement ellipsoids. Atomic labelling according to Table 2. The c-octamolybdate anions in I and III have the same structural and coordination feature but differing in the type of the ligands (DL-alanine, glycylglycine).

Fig. 2. Packing of c-octamolybdate anions coordinated by DL-alanine molecule in structure I. Solvated water molecules build layers along c axis (circled). Sodium cations are ommited for clarity.

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WI 38 (normal diploid human fibroblasts), were cultured as monolayers and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 lg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37 C.

linear regression analysis by fitting the test concentrations that give PG values above and below the reference value (i.e., 50%). Each test point was performed in quadruplicate in three individual experiments. 2.4.3. Cell cycle analysis 0.5 · 106 cells were seeded per 100 mm plate. After 24 h the solutions of the compounds were added at concentrations 105 and 5 · 106 M. After 72 h the attached cells were trypsinized, combined with floating cells, washed with phosphate buffer saline (PBS), fixed with 70% ethanol and stored at 20 C. Immediately before the analysis, the cells were washed with PBS and stained with 1 lg/ml of propidium iodide (PI) with the addition of 0.2 lg/ll of RNAse A (Sigma). The stained cells were then analyzed with Becton Dickinson FACScalibur flow cytometer (20 000 counts were measured). The percentage of the cells in each cell cycle phase was determined using the WinMDI software based on the DNA histograms. Statistical analysis was performed in Microsoft Excel by using the ANOVA single factor test.

2.4.2. Proliferation assays The panel cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0, at 1.5 · 104– 3 · 104 cells/ml, depending on the doubling times of a specific cell line. Test agents (aqueous stock solutions) were then added in five 10-fold dilutions (108–104 M) and incubated for a further 72 h. Working dilutions were freshly prepared on the day of testing. After 72 h of incubation, the cell growth rate was evaluated by performing the MTT assay, which detects dehydrogenase activity in viable cells. The MTT cell proliferation assay is a colorimetric assay system, which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells [18]. The absorbency (OD, optical density) was measured on a microplate reader at 570 nm. The absorbency is directly proportional to the cell viability. The IC50 value for each compound was calculated from dose–response curves using

2.4.4. Annexin-V test Detection and quantification of apoptotic cells at single cell level, were performed using Annexin-V-FLUOS stain-

b

N1

C1

C3 C4

c

O1

C2

O9 O2

0 N2 a

Fig. 3. Packing diagram of isolated glycylglycine ligand molecules in crystal structure II. The ligand molecules are forming 18-membered dimers through intermolecular hydrogen bonds thus making infinite chanells along c axis. The octamolybdate parts of compounds and sodium ions are omitted for clarity.

M. Cindric´ et al. / Inorganica Chimica Acta 359 (2006) 1673–1680

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c

a O2 O1

0

C1

C2 N1

C3

O9 N2

C4

b

Fig. 4. Packing diagram of isolated glycylglycine ligand molecules in crystal structure III. The ligand molecules are planar molecules packing in antiparallel sheets along b axis. The octamolybdate of compounds and sodium ions are omitted for clarity.

ing kit (Roche), according to the manufacturer’s recommendations. After 72 h, both floating and attached cells were collected. The cells were then washed with PBS, pelleted and resuspended in staining-solution (Annexin-Vfluorescein labeling reagent and propidium iodide (PI) in Hepes buffer). The cells were then analyzed under a fluorescence microscope. Annexin-V (green fluorescent) cells were determined to be apoptotic and Annexin-V and PI cells were determined to be necrotic. Percentage of apoptotic cells was expressed as the number of fluorescent cells in relation to the total cell number (fluorescent and non-fluorescent cells), which was expressed as 100%. 3. Results and discussion 3.1. Synthesis and characterization of octamolybdates Three new c-type octamolybdates were prepared by the reaction of Na2MoO4 and DL-alanine (I) or glycylglycine (II and III) in aqueous solution at pH = 3.4. The formulae of Na4[Mo8O26(alaO)2] Æ 18H2O (I), Na4[Mo8O26(glyglyO)2] Æ 15H2O (II) and Na4[Mo8O26(glyglyO)2] Æ 12H2O (III) were proved by chemical analysis, the IR spectra and X-ray structure analysis. The strong absorption

maxima at 976, 912, 696 (for I), at 933, 892, 690 (for II) and at 930, 900, 692 (for III) confirm the presence of Mo–Oterminal and Mo–Obridging groups [9], while the absorption maxima at 1660 and 1431 cm1 (for I), at 1636 and 1381 cm1 (for II) or at 1632 and 1381 cm1 (for III) are indicative of COOas and COOsym stretching frequencies [9]. All our attempts to prepare and isolate the complexes coordinated with DL-alanine and glycylglycine at other pH and temperature values were unsuccessful. We propose according to the literature that the main reason is stability of the molybdenum(VI) species present in solution [2]. All three crystal structures are composed of c-octamolybdate complex anions (Fig. 1), sodium cations and water molecules. Since the anion is centrosymmetric, each asymmetric unit contains half a molecule. c-Octamolybdate anions consist of eight condensed edge-sharing MoO6 octahedra. They belong to the proposed c[Mo8O26]4 type but with two additional terminal positions, thus satisfying the octahedral co-ordination of all eight molybdenum atoms There are 16 terminal positions, and 14 of them are occupied by oxo-oxygen and by two carboxylato-oxygen atoms like in Na2[Mo8O26-

M. Cindric´ et al. / Inorganica Chimica Acta 359 (2006) 1673–1680 Table 3 Growth inhibition of c-octamolybdates tested on the six-panel cell-line expressed as IC50 values Structure modification

Cell line HeLa

HepG2

Hep-2

SW 620

MCF-7

WI 38

>100 69 ± 0.4 >100 >100 >100 >100

31 ± 38 23 ± 29 56 ± 0.7 35 ± 28 47 ± 75 27 ± 30

>100 P100 >100 >100 >100 >100

>100 >100 >100 >100 >100 >100

69 ± 18 52 ± 30 >100 54 ± 37 70 ± 19 32 ± 6

>100 >100 >100 >100 >100 >100

a

IC50 (lM) I II III IV V VI a

IC50, the concentration that causes 50% growth inhibition.

of normal fibroblasts WI 38 was observed for any of the tested compounds (Fig. 5A–C). 3.3. Cell cycle analysis Flow cytometric analysis was performed for compounds I and II to identify whether the cell growth inhiA

(I) 150 Hep-2

100

HeLa PG (%)

(L-lysH2)2] Æ 8H2O or [Hmorph]4[Mo8O24(OH)2(metO)2] Æ 4H2O [5,9]. There are 12 bridging oxygen atoms, six of them [O(3), O(6), O(7) and their centrosymmetric pairs] bridge two Mo atoms, four [O(4), O(8) and their centrosymmetric pairs] bridge three Mo atoms, while two [O(5) and centrosymmetric O 0 (5)] bridge four Mo atoms. As it can be seen from Table 2, the bond lengths in all three octamolybdate anions are mutually in very good agreement. Also, these values agree very well with those found and described in similar structures [3–6]. The fact that all three compounds were prepared at pH 3.4, indicates that nitrogen atom of a-amino group is protonated ½pK a ða-NH3 þ Þ  9:0. This was also confirmed by locating these hydrogen atoms in electron density maps. The amino acid (DL-alanine) and peptide (glycylglycine) molecules in all three crystal structures are coordinated via monodentate carboxylate-oxygen to molybdenum (Mo1) atom. Main structural difference in all three structures is observed in their packing diagrams. In crystal structure I, solvated water molecules are packed along the c axis (Fig. 2) between c-octamolybdate anions thus making these crystals relatively unstable. The glycylglycine-ligands in structures II and III have different conformations which results in different packing mode of both structures. In structure II torsion angle C(3)–N(1)–C(2)–C(1) is 76.09 and in structure III is 174.23, respectively. These values indicate that the ligand molecule in structure II shows more torsion flexibility while in structure III the ligand molecules are almost planar. In structure II the dipeptide molecules form dimers through the intermolecular N2–H22  O2i, [(i) ˚ . As 1  x, y + 1, z] hydrogen bond of 3.065(5) A seen in Fig. 3, such dimers build infinite channels along the c axis. In structure III dipeptide molecules are planar and therefore packed in antiparallel sheets along the b axis (Fig. 4) and with no similar infinite channels as observed in structure II. Yamase and coworkers [4] gave only the structural parameters for the compound which has the same chemical formula as III. The difference is only in c-angle but they did not describe the coordination around molybdenum atoms, bond lengths and bond angles in the structure.

50

SW 620 WI 38

0

MCF-7

-50

HepG2

-100 -9

-8

-7

-6

-5

-4

-3

log 10 concentration (M)

B

(II) 150 Hep-2

100 PG (%)

1678

HeLa SW 620

50

WI 38

0

MCF-7 HepG2

-50 -100 -9

-8

-7

-6

-5

-4

-3

log 10 concentration (M)

C

3.2. Antiproliferative effect

(III) 150 Hep-2

PG (%)

100

All tested compounds showed a certain antiproliferative effect on the presented panel cell lines (Table 3. and Fig. 5A–C). However, all tested compounds markedly inhibited the growth of HepG2 cells (IC50 values ranging from 23 ± 29 to 56 ± 0.7 lM), and MCF-7 cells (IC50 values ranging from 23 ± 29 to 56 ± 0.7 lM) in a dose-dependant manner. Additionally, minor growth inhibitory effect on HeLa was observed for compounds II and IV (Table 3 and Fig. 5B), while compound II slightly inhibited the growth of Hep-2 (Fig. 5B). No substantial growth inhibition

HeLa 50

SW 620 WI 38

0

MCF-7

-50

HepG2

-100 -9

-8

-7

-6

-5

-4

-3

log 10 concentration

Fig. 5. The dose-response profiles for c-octamolybdates tested in vitro on presented 6-cell line panel at concentrations 104 to 108 M. The results are shown as percentages of growth (PG) for each cell line.

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Table 4 Flow cytometric analysis of HepG2 after addition of I and II Cell cycle phase (%)

Sub G1 G0/G1 S G2/M

II

I 6

Control (%)

5 · 10

3.6 68.9 4.1 20.3

3.3 66.7 4.29 21.3

M (%)

5

10

M (%)

3.9 66 4.2 21

Control (%)

5 · 106 M (%)

105 M (%)

7.5 63.6 3.2 19.9

6.5 66.6 2.8 20.1

7.2 69.8* 3.4 17.2*

The results are shown as percentages of cell population in each cell cycle phase. * Statistically significant at p < 0.05.

bition was caused by specific perturbation of the cell cycle-related events. HepG2 cells were chosen because they were the most sensitive to the antiproliferative effects of the tested compounds, and WI 38 cells, because they represent normal cells. DNA contents of HepG2 and WI 38 were measured 72 h after the addition of the tested compounds at concentrations 5 · 106 and 105 M. DNA histograms of HepG2 for I at the concentration 105 M showed a slight accumulation of cells in the G0/G1 phase of the cell cycle (69.8% versus 63.6%) and a subsequent decrease of G2/M cell population (17.2% versus 19.9%), (Table 4). Interestingly, no changes in the cell cycle populations of HepG2 were observed after treatment with II at both tested concentrations (Table 4). Also, no significant changes were observed in the cell cycle of WI 38 at both tested concentrations of I and II (data not shown). Both I and II did not induce apoptosis (changes in the subG1 population) at both tested concentrations, in HepG2 and WI38 cells, which was additionally confirmed by Annexin-V test (data not shown). HepG2 cells are tumor-derived cells of hepatic origin. Those cells exhibit most of the characteristics of the differentiated hepatocyte and they express a wide range of enzymes important in detoxification of xenobiotics such as cytochrome P450 monooxigenases (CYP), glutathionS-tranferase, catalase, peroxidase, cytochrome c reductase, cytochrome P450 reductase, etc. Due to this, they are often used for toxicity studies [19,20]. Further on, the human breast cancer cell line MCF-7 has a proficient stress-induced cell cycle checkpoint activation, well-characterized genotoxic stress response, and proficient DNA repair. MCF-7 is representative of a tumor cell type that does not readily undergo p53-dependent apoptosis [19,21]. In comparison with other tested tumor cell lines, HepG2 and MCF-7 are more susceptible to toxic agents. Hence, in accordance with the flow cytometric analysis and Annexin test results, we deduce that the accentuated antiproliferative effect of the tested compounds on HepG2 and MCF-7 was not a cell cycle-related event and that it was not caused by induction of apoptosis. The minor increase of G0/G1 cell population after treatment with compound I could not be described as an important mechanism of cell growth inhibition. Therefore, we assume that the observed antiproliferative effect on these cell lines might be the result of the induced repeated

redox cycles in the cell lines induced by polyoxometalles previously described in [1,2]. 4. Conclusions In all c-octamolybdate complex anions [Mo8O24(OH)2(metO)2]4 (IV) [9], [Mo8O24(OH)2(alaO)2]4 (VI) [10], [Mo8O26(glyglyO)2]4 (II or III) and [Mo8O26(alaO)2]4 (I) amino acids and peptide molecules are coordinated via monodentate carboxylate-oxygen to molybdenum. Main structural difference in all described structures (I, II and III) is observed in their packing diagrams. The methioninato (IV) [9], and alaninato (VI) [10] or (I) octamolybdates are unstable compounds, they easily loose solvent molecules and we could propose the similarity in their crystal packing. Based on presented in vitro screening results we may conclude that all tested compounds showed a differential cell-growth inhibition in a dose-dependent manner selectively on HepG2 and MCF-7 cell lines. However, this effect was not a cell cycle-related event and it was not caused by the induction of apoptosis. These results are in accordance with the results described in the literature. In addition, since a key point in all biological studies of polyoxometallates is the issue of whether the complexes stay intact during the treatment, we always used freshly prepared stock solutions, as well as working dilutions. However, as the biological experiments lasted for 72 h, it is possible that the octamolybdate framework is partially or fully dissociated into monomeric molybdenum(VI) species. Therefore, it is likely that some other inorganic and/or organic dissociation products are responsible for the observed antiproliferative activity. Further stability studies are currently being performed. 5. Supplementary materials CCDC 256958, 256959 and 256960 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336 033; or e-mail: deposit@ccdc. cam.ac.uk).

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Acknowledgements This research was supported by the Ministry of Science and Technology of the Republic of Croatia, Grant Nos. 0119631 and 00981104. We wish to thank Dr. Gerald Giester from the Institute for Mineralogy and Crystallography, University of Vienna, Austria, for the data collection of compound I. References [1] J.T. Rhule, C.L. Hill, D.A. Judd, Chem. Rev. 98 (1998) 327. [2] M.T. Pope, A. Mueller (Eds.), Polyoxometallates: From Platonic Solids to Anti-Retroviral Activity, Kluwer, Dordrecht, The Netherlands, 1994. [3] J. Lu, Y. Xu, Chem. Mater. 10 (1998) 4141. [4] T. Yamase, M. Inoue, H. Naruke, K. Fukaya, Chem. Lett. (1999) 563. [5] M. Inoue, T. Yamase, Bull. Chem. Soc. Jpn. 68 (1995) 3055. [6] G. Liu, S. Zhang, Y. Tang, Z. Anorg. Allg. Chem. 627 (2001) 1077. [7] T. Yamase, Mol. Eng. 3 (1993) 241. [8] J.T. Rhule, C.L. Hill, D.A. Judd, R.F. Shinazi, Chem. Rev. 98 (1) (1998) 327.

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