Lanthanide-Connecting and Lone-Electron-Pair Active Trigonal-Pyramidal-AsO3 Inducing Nanosized Poly(polyoxotungstate) Aggregates and Their Anticancer Activities

By virtue of the stereochemical effect of the lone-electron pair located on the trigonal-pyramidal-AsO3 groups and the one-pot self-assembly strategy in the conventional aqueous solution, a series of novel lanthanide-bridging and lone-electron-pair active trigonal-pyramidal-AsO3 inducing nanosized poly(polyoxotungstate) aggregates [H2N(CH3)2]6 Na24H16{[Ln10W16(H2O)30O50](B-α-AsW9O33)8}·97H2O [Ln = EuIII (1), SmIII (2), GdIII (3), TbIII (4), DyIII (5), HoIII (6), ErIII (7), TmIII (8)] were prepared and further characterized by elemental analyses, IR spectra, UV spectra, thermogravimetric (TG) analyses and single-crystal X-ray diffraction. The most remarkable structural feature is that the polyanionic skeleton of {[Ln10W16(H2O)30O50](B-α-AsW9O33)8}46− is constructed from eight trivacant Keggin [B-α-AsW9O33]9− fragments through ten Ln centers and sixteen bridging W atoms in the participation of fifty extraneous oxygen atoms. Notably, 4 and 8 can be stable in the aqueous solution not only for eight days but also in the range of pH = 3.9–7.5. Moreover, the cytotoxicity tests of 4 and 8 toward human cervical cancer (HeLa) cells, human breast cancer (MCF–7) cells and mouse fibroblast (L929) cells were performed by the 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the cell apoptosis processes were characterized by calcein AM/PI staining experiments, annexin V-FITC/PI staining experiments and morphological changes.


Results and Discussion
Structural description. The good phase purity of 1-8 is verified by the consistency of powder X-ray diffraction patterns (PXRD) of the as-prepared samples of 1-8 with the simulated XRD patterns derived from single-crystal structural analyses ( Figure S1). X-ray diffraction structural analysis indicates that 1-8 are isomorphous and crystallize in the triclinic space group P− 1. Thus, the structure of 1 is herein discussed as an example below. The centrosymmetric octameric polyoxoanionic framework {[Eu 10 W 16 (H 2 O) 30  Obviously, the formation of the stable trivacant [B-α -AsW 9 O 33 ] 9− moiety with six exposed surface oxygen atoms in the trivacant position is benefited from the inducing effect of the lone-electron-pair active trigonal-pyramidal-AsO 3 group 14,22 . The intriguing rectangular [Eu 10 W 16 (H 2 O) 30 O 50 ] 26+ cluster core (Fig. 1c,d) can be viewed as a combination of four {W 3 Eu 2 } (namely W1W3W7Eu3Eu4, W4W5W12Eu1Eu2, W1AW3AW7AEu3AEu4A, W4AW5W12AEu1AEu2A) and two {W 2 Eu 1 } (namely W2W11Eu5, W2AW11 AEu5A) segments (Fig. 1e,f) (Fig. 2h) 36 . The remarkable differences between 1c (Fig. 2i) and the above-mentioned four pentanuclear sandwich-type POMs lie in two aspects: a) 1c own a heterometallic pentanuclear central core (Fig. 2j) whereas others have the isometallic pentanuclear cores, b) 1c was prepared from the one-pot reaction of simple materials of Na 2 WO 4 ·2H 2 O and NaAsO 2 while others were made by the prefabricated precursors such as K 10 15 Figure S7a), two types of heterometallic sandwich-type segments in 1b are almost mirror-symmetric to each other ( Figure S7b). Interestingly, after the removal of Eu III ions, the two remaining [As 2 W 21 O 76 ] 20− units in 1b still keep this mirror-symmetry ( Figure S7c). However, the centrosymmetry of the whole polyanionic framework leads to the racemization of 1a, and thus 1 can't show the chirality and the circular dichroism optical activity. Above all, the skeleton of the giant tungsten cluster of 1a with the omission of ten Eu III centers not only demonstrates the existence of eighty-eight W centers, but also highlights the structure-stabilizing effect of Eu III ions in the formation of the giant tungsten cluster (Figures S8b,c)  Aqueous solution stability and anticancer activities. In order to study the aqueous solution stability of 1-8, the UV spectra of 4 and 8 in the aqueous solution as representatives have been investigated in the range of 190-400 nm at room temperature. Both UV spectra exhibit a strong absorption band peak at ca. 194 nm (4) and ca. 195 (8) that can be ascribed to the O t → W pπ -dπ charge-transfer transitions and a weaker absorption band at ca. 248 nm (4) and ca. 247 nm (8) that can be attributed to the O b(c) → W pπ -dπ charge-transfer transitions ( Figure S11) 40 . It is noteworthy that the UV spectra of 4 and 8 almost remain unchanged at room temperature for eight days (Figs 4a and S12a), which preliminarily imply that 4 and 8 are stable in aqueous solution within eight days and provide a necessity for performing their biological evaluation. To further probe the dependence of 4 and 8 on the pH variation in aqueous solution, the UV spectra of 4 and 8 in acidic and alkaline regions have been measured. The pH values are adjusted by using diluted H 2 SO 4 and NaOH. It should be noted that the initial pH values of 4 and 8 dissolved in aqueous solution are about 5.94 and 5.88, respectively. Experimental results indicate that the UV spectrum of 4 has no conspicuous change in the pH scope of 3.90-7.50. However, the intensity of the O b(c) → W absorption band decreases and a new broad centered at 260 nm comes to appear when the pH is gradually lower than 3.90 (Fig. 4b) whereas the O b(c) → W absorption band gradually becomes weaker until disappearing and the O t → W absorption band become more and more stronger upon the pH being higher than 7.50 (Fig. 4c). Therefore, a conclusion could be drawn that the pH stable range of 4 in aqueous solution is about 3.9-7.5. Similarly, 8 is stable in the pH scope of ca. 3.9-7.4 ( Figures S12-c). This fact suggests that 4 and 8 can be stable in human blood environment (pH = 7.3-7.5), which provides a clear guidance that 4 and 8 can be utilized  (Table S2). In comparison with IC 50 values of 4 and 8 against normal L929 cells (59.68 μ g/mL and 58.04 μ g/mL, respectively) ( Fig. 5e,f, Table S2), it can be concluded that 4 and 8 exhibit the higher cytotoxicity against HeLa and MCF-7 cells than against normal L929 cells, indicating that 4 and 8 behave as considerable anticancer activities in killing HeLa and MCF-7 cells. It is well known that arsenic compounds have been extensively exploited as anti-proliferative drugs and can induce complete remission of the cancer patients with relapsed 41,42 Fig. 6) that the control cells emit green fluorescence, which signifies that they are alive. However, most HeLa and MCF-7 cells incubated by 4 and 8 with the concentration of 1 mg/mL after 6 h exhibit the fluorescence color change from green to red, which indicate that they have been dead 44 . Numerous studies have shown that apoptosis is a typical form for chemotherapy drug-induced cell death. For example, cisplatin and its generation analogues can induce DNA damage and then arrest the cancer cells at the G2/M phase of the whole cell cycle 45,46 . Arsenic trioxide can trigger apoptosis and autophagy of leukemia cell lines 47,48 . For the purpose of verifying that apoptosis induces the cell death for HeLa and MCF-7 cells, the widely used fluorescent staining of Annexin V-FITC together with PI were used to 4 and 8. Generally, after staining a cell population with Annexin V-FITC and PI, apoptotic cells show green fluorescence, dead cells or necrosis cells emit red fluorescence, and live cells exhibit little or no fluorescence. Thereby, Annexin V-FITC/PI staining method can distinguish apoptosis cells and necrosis cells. As shown in the bottom of Fig. 6,    The thermostability of 3-6 has been also probed by multiply techniques including TG analyses, variable temperature powder X-ray diffraction (VTPXRD) patterns and variable temperature IR (VTIR) spectra. First of all, the TG analyses of 1-8 have been examined on the pure crystalline samples under the flowing nitrogen atmosphere in the temperature range of 25-900 °C with the heating rate of 10 °C min −1 ( Figure S16). Obviously, the TG curves of 1-8 can be divided into three steps. The first weight loss of 6.35% (calcd. 6.61%) for 1, 6.66% (calcd. 6.61%) for 2, 6.52% (calcd. 6.59%) for 3, 6.47% (calcd. 6.59%) for 4, 6.55% (calcd. 6.58%) for 5, 6.29% (calcd. 6.57%) for 6, 6.28% (calcd. 6.57%) for 7 and 6.46% (calcd. 6 Figure S17, all diffraction peaks almost retain unchangeable before 100 °C, being indicative of the good crystallinity of 3-6, which further illustrate that the structures of 3-6 are almost no change except for the loss of some lattice water molecules. Upon heating to ca. 430 °C, most of characteristic diffraction peaks gradually disappear, which principally originates from the fact that the crystalline samples of 3-6 have been efflorescent and led to the very bad crystallinity of 3-6 when all the lattice water molecules and some coordinate water molecules are removed away from of 3-6. This fact can be also confirmed by the results of TG analyses and VTIR spectra. After 600 °C, the occurrence of some new diffraction peaks in the PXRD patterns at 600 and 700 °C reveals that new decomposition phases come to emerge, which demonstrates that the dehydration of protons and the sublimation of part As 2 O 3 result in the decomposition of polyoxoanionic skeletons of 3-6. This observation is also consolidated by the apparent distinction of IR spectra at 600 and 700 °C from those at 25 (2), Gd III (3), Tb III (4), Dy III (5), Ho III (6), Er III (7), Tm III (8)] have been successfully isolated based on the stereochemical effect of the lone-electron pairs located on trigonal pyramidal AsO 3 groups located on polyoxtungstate fragments and the connection role of Ln cations. Intriguingly, the multi-Ln incorporated octameric framework {[Ln 10 W 16 (H 2 O) 30 O 50 ](B-α -AsW 9 O 33 ) 8 } 46− consists of eight trivacant Keggin [B-α -AsW 9 O 33 ] 9− fragments linked by ten Ln ions and sixteen bridging W atoms in the presence of fifty extraneous oxygen atoms. Moreover, the aqueous solution stability and thermostability of some representatives have been investigated. Furthermore, the cytotoxicity tests of 4 and 8 toward HeLa, MCF-7 and L929 cells have been examined by the MTT assay and the cell apoptosis processes have been characterized by calcein AM/PI staining experiments, annexin V-FITC/PI staining experiments and morphological changes. This finding opens the door to the research on medical activities of multi-Ln incorporated POMs and expands the research domain of POM chemistry. Our following work will be concentrated on expanding the designed syntheses and pharmaceutical activity evaluation of much more high nuclear LENPs (X = As III , Sb III , Bi III , Se IV , Te IV ). Emphasis will put on investigating cancer cell apoptosis process and apoptosis mechanism.

Materials.
All the reagents were purchased commercially and used without further purification. (1.400 g, 4.240 mmol) and dimethylamine hydrochloride (0.502 g, 6.156 mmol) were dissolved in water (20 mL) under stirring and NaAsO 2 (0.5 mL, 1 moL·L −1 ) was added. After the pH of the resulting solution was adjusted to 4.0 by using hydrochloric acid (6.0 moL·L −1 ), Eu(NO 3 ) 3 ·6H 2 O (0.198 g, 0.444 mmol) was then added and the pH was again adjusted to 4.0. After stirring for 30 min, the solution was filtered and left at room temperature. Slow   (Figure S16).   Figure S16).     Figure S16).  (Figure S16).

Preparation of [H 2 N(CH
Single-crystal X-ray diffraction. Good-quality single crystals for 1-8 were carefully chosen from their mother liquids under the optical microscope and sealed in a capillary. Their diffraction data were collected on a Bruker Apex II diffractometer with the graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 296(2) K. Intensity data were corrected by Lorentz and polarization effect and empirical absorption on the base of the multi-scan technique. Their structures were solved by direct methods. The heavy atoms were located using the SHELXTL-97 program package 52,53 , and the remaining atoms were found from successive full-matrix least-squares refinements on F 2 and Fourier syntheses. Those H atoms attached to C and N atoms were added in idealized geometrical positions. No H atoms linking to H 2 O molecules were found from the difference Fourier map. The non-H atoms were refined anisotropically except for some O, C, N atoms and H 2 O molecules. Solvent accessible voids are observed in the check cif reports of 1-8, indicating that some highly disordered water molecules that can't be found from the weak residual electron peaks may exist in their structures. We tried to locate and refine them, but we failed. Finally, according to the results of elemental analyses and TG measurements, seventy-six water molecules were directly added to each molecular formula. This phenomenon is very common in POM chemistry 54 . Crystallographic data and structural refinement parameters for 1-8 are listed in Table S3. CCDC-1421345 (1), 1421346 (2), 1421347 (3), 1421348 (4), 1421349 (5), 1421350 (6), 1421351 (7) and 1421352 (8) contain the supplementary crystallographic data for this paper. These data can be also obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Elemental analyses.
Elemental analyses (C, H, N) were performed using a Perkin-Elmer 240C elemental analyzer. Inductively coupled plasma atomic emission spectrometry (ICP-AES) was performed on a Perkin-Elmer Optima 2000 ICP-AES spectrometer. IR spectra. IR spectra were recorded from a powdered sample pelletized with KBr on a Nicolet 170 SXFT-IR spectrometer in the range of 400-4000 m −1 .
TG analyses. TG analyses were measured under a N 2 atmosphere on a Mettler-Toledo TGA/SDTA 851 e instrument with a heating rate of 10 °C·min −1 .
Cell culture. HeLa, MCF-7 and L929 cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). The cells were cultured at 37 °C under 5% CO 2 atmosphere with the culture medium replaced once every day.
MTT experiments. Cells harvested in a logarithmic growth phase were seeded in 96-well plates at a density of 105 cells per well and incubated in DMEM for 24 h. The medium was then replaced by 4 and 8 at various concentrations. The incubation was continued for 48 h. Then, 20 μ L of MTT solution in phosphate buffered saline (PBS) with the concentration of 5 mg/mL was added and the plates were incubated for another 4 h at 37 °C, followed by removal of the culture medium containing MTT and addition of 150 μ L of DMSO to each well to dissolve the formazan crystals formed. Finally, the plates were shaken for 5 min, and the absorbance of formazan product was measured at 490 nm by a microplate reader.
Optical microscope observation. The cells were observed with an optical microscope (Nikon Eclipse Ti, Optical Apparatus Co., Ardmore, PA, USA).