Anticancer Activity of Polyoxometalate-Bisphosphonate Complexes: Synthesis, Characterization, in Vitro and in Vivo Results

Article abstract of DOI:10.1021/acs.inorgchem.7b01114

We synthesized a series of polyoxometalate-bisphosphonate complexes containing Mo^VIO₆ octahedra, zoledronate, or an N-alkyl (n-C₆ or n-C₈) zoledronate analogue, and in two cases, Mn as a heterometal. Mo₆L₂ (L = Zol, ZolC₆, ZolC₈) and Mo₄L₂Mn (L = Zol, ZolC₈) were characterized by using single-crystal X-ray crystallography and/or IR spectroscopy, elemental and energy dispersive X-ray analysis and ³¹P NMR. We found promising activity against human nonsmall cell lung cancer (NCI-H460) cells with IC₅₀ values for growth inhibition of ～5 μM per bisphosphonate ligand. The effects of bisphosphonate complexation on IC₅₀ decreased with increasing bisphosphonate chain length: C₀ ≈ 6.1×, C₆ ≈ 3.4×, and C₈ ≈ 1.1×. We then determined the activity of one of the most potent compounds in the series, Mo₄Zol₂Mn(III), against SK-ES-1 sarcoma cells in a mouse xenograft system finding a ～5× decrease in tumor volume than found with the parent compound zoledronate at the same compound dosing (5 μg/mouse). Overall, the results are of interest since we show for the first time that heteropolyoxomolybdate-bisphosphonate hybrids kill tumor cells in vitro and significantly decrease tumor growth, in vivo, opening up new possibilities for targeting both Ras as well as epidermal growth factor receptor driven cancers.

Full text of DOI:10.1021/acs.inorgchem.7b01114

Article
pubs.acs.org/IC
Anticancer Activity of Polyoxometalate-Bisphosphonate Complexes:
Synthesis, Characterization, In Vitro and In Vivo Results
Amandine Boulmier,† Xinxin Feng,∥ Olivier Oms,† Pierre Mialane,† Eric Riviere,⊥ Christopher J. Shin,∥
̀
Jiaqi Yao,∥ Tadahiko Kubo,§ Taisuke Furuta,§ Eric Oldfield,*,‡,∥ and Anne Dolbecq*,†
†Institut Lavoisier de Versailles, UMR 8180, Universite
des Etats-Unis, 78035 Versailles Cedex, France
́ ́
de Versailles Saint-Quentin en Yvelines, Universite Paris-Saclay, 45 Avenue
∥Department of Chemistry, University of Illinois at Urbana−Champaign, 600 South Mathews Avenue, Urbana, Illinois 6180, United
States
‡Center for Biophysics and Quantitative Biology, University of Illinois at Urbana−Champaign, 1110 W Green Street, Urbana, Illinois
61801, United States
§Department of Orthopedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
⊥Institut de Chimie Molec
́ ́ ́ ́
ulaire et des Materiaux d’Orsay, UMR 8182, Universite Paris-Sud, Universite Paris-Saclay, 91405 Orsay,
France
S
* Supporting Information
ABSTRACT: We synthesized a series of polyoxometalate-
bisphosphonate complexes containing MoVIO6 octahedra, zoledr-
onate, or an N-alkyl (n-C6 or n-C8) zoledronate analogue, and in
two cases, Mn as a heterometal. Mo6L2 (L = Zol, ZolC6, ZolC8)
and Mo4L2Mn (L = Zol, ZolC8) were characterized by using
single-crystal X-ray crystallography and/or IR spectroscopy,
elemental and energy dispersive X-ray analysis and 31P NMR.
We found promising activity against human nonsmall cell lung
cancer (NCI-H460) cells with IC50 values for growth inhibition of
∼5 μM per bisphosphonate ligand. The effects of bisphosphonate
complexation on IC50 decreased with increasing bisphosphonate
chain length: C0 ≈ 6.1×, C6 ≈ 3.4×, and C8 ≈ 1.1×. We then
determined the activity of one of the most potent compounds in
the series, Mo4Zol2Mn(III), against SK-ES-1 sarcoma cells in a mouse xenograft system finding a ∼5× decrease in tumor volume
than found with the parent compound zoledronate at the same compound dosing (5 μg/mouse). Overall, the results are of
interest since we show for the first time that heteropolyoxomolybdate-bisphosphonate hybrids kill tumor cells in vitro and
significantly decrease tumor growth, in vivo, opening up new possibilities for targeting both Ras as well as epidermal growth
factor receptor driven cancers.
groups appear to be the most efficient. Recently, the cobalt
derivative {CoSb6O4(H2O)3[Co(hmta)SbW8O31]3}15− (hmta =
hexamethylenetetramine) was also shown to kill various cancer
cells.13 Polyoxometalates, in particular, Dawson-type
[P2Mo18O62]6− anions, were also identified as inhibitors of
protein kinase CK2, a multifunctional kinase that is deregulated
in many cancers.14
In order to try to improve activity, one approach is to graft
bioactive organic ligands on the inorganic POM core. However,
examples of this approach are rare. Organoimido- derivatives of
hexamolybdate based on amantadine, an antiviral and anti-
Parkinson drug, have been tested in vitro against MCF-7 cells
and exhibit better activity than do unfunctionalized hexamo-
lybdate and amantadine.15 Cholic acid was also grafted onto the
INTRODUCTION
■
Polyoxometalates (POMs) constitute a class of discrete,
anionic, metal−oxygen clusters which can be considered as
soluble oxide fragments. They are built from the connection of
{MOx} polyhedra, M being a d-block transition metal ion in a
high oxidation state, usually WVI, MoV,VI, or VIV,V.1 They can
incorporate a large range of organic or inorganic species into
their structures. This results in a great variety of sizes,
nuclearities, and shapes2 and a vast range of photochemical,3
catalytic,4,5 magnetic6,7 as well as biological properties including
antibacterial,8 antiviral, and antitumoral activities.9 POMs can
also have activity in tumor cell growth inhibition; e.g.,
[NH3Pri]6[Mo7O24]·3H2O exhibits potent antitumor activity
against breast, sarcoma, adenocarcinoma, and pancreatic cancer
cells.10 The antitumor activities in vitro of a variety of
polyoxotungstates have also been reported, and organometallic
derivatives with RSn11 or CpTi12 (Cp = cyclopentadienyl)
Received: May 3, 2017
© XXXX American Chemical Society
A
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tris-modified Anderson-type POM [MnMo6O18{(OCH2)3-
survival.24 Finally, we report the first results of an in vivo
xenograft tumor experiment with a POM/BP/Mn complex.
CNH2}2]3−, resulting in enhanced activity over the non-
functionalized POM against breast cancer cells (MCF-7, IC50
≈
56 μM and MDA-MB-231, IC50 ≈ 38 μM).16 A variant of this
approach consists of grafting a cleavable group onto a
hexamolybdate POM in order to improve degradability with
one such hybrid POM being reported to exhibit good activity
against human malignant glioma cells (U251, IC50 ≈ 25 μM),
the ability to penetrate the blood brain barrier, as well as low
toxicity toward rat pheochromocytoma cells (PC12).17
RESULTS AND DISCUSSION
■
In order to test the effect of the introduction of a lipophilic
chain onto the BP-ligand on tumor cell growth inhibition, two
zoledronate-based bisphosphonates, ZolC6 and ZolC8 (Figure
1), were selected for synthesis of Mo/BP complexes. ZolC6 and
ZolC8 are zoledronate analogues that have N-alkyl chains (n-C6
or n-C8) on the unsubstituted nitrogen in the parent
zoledronate. These ligands were chosen because they were
previously found to be the most active (of Zol analogues
containing between 0 and 15 carbon substituents) against a
range of tumor cell lines.24 Two families of complexes were
investigated: Mo6L2 (L = Zol, ZolC6, ZolC8) or with Mn(III) as
the heterometal and with general formulas Mo4L2Mn(III) (L =
Zol, ZolC8). The structures and ligand abbreviations as well as
the structures of all of the complexes reported in this study are
shown in Figure 1.
In our group, we have been investigating polyoxometalate-
bisphosphonate complexes. Bisphosphonates (BPs) having the
general formula H2O3PC(OH)(R)PO3H2 have been used
clinically for decades to treat bone resorption diseases, and
some of them exhibit antitumor activity.18 Once deprotonated,
BPs can form complexes with Mo, W, or V ions, with
nuclearities ranging from 1 to 12.19 In our first investigation, we
reported the anticancer activity of hybrid POM/alendronate
(Ale) complexes.20 The dodecanuclear complex [(MoVI3O8)4-
(Ale)4]8−, noted here as Mo12Ale4, had IC50 values of ∼10 μM
against human breast cancer cells (MCF-7) in vitro. This value
represents about 4× the activity of the parent alendronate
molecule (i.e., on a per-alendronate basis), indicating a
potentiating or synergistic effect. We then extended our study
to include zoledronate (Zol; Figure 1), the most potent
Synthesis and Characterization. Mo6Zol2 was synthe-
sized as a reference according to a previously reported
procedure.21 Mo(VI) complexes with lipophilic bisphospho-
nates were synthesized by reaction of the BP precursor with
Mo(VI) ions in aqueous solution at pH ≈ 5. While it was
possible to isolate single crystals suitable for X-ray diffraction by
slow evaporation of the reaction mixture for Mo6(ZolC6)2, only
a poorly crystalline powder could be obtained for the
Mo6(ZolC8)2 complex. However, the similarity of the IR
spectra (Figure S1a) and of the 31P NMR spectra (Figure S2)
of both compounds, as well as the results of elemental analysis,
confirm formation of Mo6(ZolC8)2. Mo4Zol2Mn(III) was
synthesized following a slight modification of the procedure
reported previously.22 Mo4Zol2Mn(II) was synthesized by
reduction of Mo4Zol2Mn(III). Attempts to reduce this complex
in solution failed, so its reduction was performed by adding
ascorbic acid to a suspension of crystals in methanol, as
described for a Mn(III)-containing polyoxotungstate.25 A color
change of the crystals from gray to yellow was observed. This
implies that the Mo(VI) centers have not been reduced,
because if they were, a blue coloration would be expected. The
χMT vs T curve for this compound is shown in Figure S3 where
it can be seen that the χMT product is nearly constant between
300 to 50 K (χMT = 4.15 cm3 mol−1 K at 300 K), with a
decrease at low temperature (χMT = 2.68 cm3 mol−1 K at 2 K)
which can be attributed to the zero-field splitting effect. This
indicates that Mo4Zol2Mn(III) has been fully reduced to
Mo4Zol2Mn(II) (at room temperature, the calculated χMT
values are 2.94 cm3 mol−1 K for a Mn(III) monomer and 4.29
cm3 mol−1 K for a Mn(II) monomer assuming g = 1.98). As
expected, the IR spectra of the oxidized and reduced POMs are
very similar (Figure S1b). The synthesis of Mo4(ZolC8)2Mn-
(III) was performed by reacting Mn(OAc)3 and ZolC8 with a
large excess of Na2MoO4 in acetate buffer. The reaction with a
stoichiometric amount of Mo(VI) ions gave only an insoluble
unidentified powder. Mo4(ZolC8)2Mn(III) was characterized
by IR spectroscopy (Figure S1c), elemental analysis, and energy
dispersive X-ray (EDX) analysis measurements. Despite many
attempts, we were unable to isolate the analogous compound
with the ZolC6 bisphosphonate.
Figure 1. Abbreviations and formulas of the BPs used in this study
together with schematic structures of some complexes. Gray polyhedra
= MoO6, purple polyhedron = MnO6, green spheres = P, black spheres
= C, red spheres = O.
commercially available bisphosphonate, and synthesized the
hexanuclear complex [(MoVI3O8)2O(Zol)2]6−.21 We also
investigated the incorporation of a heterometal finding that
the most active compound was the Mn(III)-containing POM
[(MoVI2O6)2(Zol)2Mn]5−, also known as Mo4Zol2Mn(III),
suggesting a possible role for both the heterometal and the
BP in growth inhibition.22
Here, we describe the synthesis and characterization of a new
series of POM/BP complexes and investigate the influence of
the oxidation state of the Mn, and the introduction of alkyl
chains grafted onto the zoledronate core, on the in vitro activity
of these compounds against human nonsmall cell lung cancer
(NCI-H460) cells. Lipophilic chains were expected to improve
cell uptake of the hybrid POMs since it is known that lipophilic
BPs have activities far greater than do nonlipophilic BPs in
inhibiting tumor cell growth and invasiveness, both in vitro and
in vivo.23 Furthermore, a recent study revealed that lipophilic
zoledronates inhibit both farnesyl and geranylgeranyldiphos-
phate synthases, effectively blocking prenylation of KRAS and
other small G-proteins critical for tumor growth and cell
31P NMR spectroscopy was used to determine whether
structures characterized in the solid state were maintained in
solution. The 31P NMR spectrum of Mo6(ZolC6)2 dissolved in
water at room temperature exhibits two singlets at 17.12 and
B
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16.82 ppm with relative intensities 0.63:1.37 (Figures 2 and
S2a). The same behavior is observed for Mo6(ZolC8)2 with two
expected, dynamic exchange between the two conformers
increases on heating.
We also studied the stability of Mo6(ZolC6)2 and
Mo6(ZolC8)2 under more physiological conditions (0.1 M
KH2PO4 buffer, pH = 7.4, 0.12 M NaCl, 37 °C) (Figure S2).
The 31P NMR spectrum of Mo6(ZolC6)2 exhibits two peaks, as
observed for the aqueous solution, confirming the stability of
the complex. In the spectrum of Mo6(ZolC8)2, one of the peaks
is split, which may be attributable to the existence of several
conformers which could result from different positions of the
alkyl chains, as seen in the crystal structure of Mo6(ZolC6)2
1
(vide infra). It can also be seen that the H NMR spectra of
Mo6(ZolC8)2 in water and in the more physiological conditions
are identical (Figure S5).
Structures. The structure of Mo6(ZolC6)2 was determined
by means of single-crystal X-ray diffraction. There are two
crystallographically independent molecules (Figure 3), and the
structure of the inorganic core is identical in both molecules.
Two {Mo3O8} trimeric units are connected by a central oxygen
atom, and in each trimer Mo(VI) ions are connected to a
pentadentate BP ligand by P−O−Mo and C−O−Mo bonds.
The six Mo(VI) ions are coplanar, as usually observed for
hexanuclear Mo(VI) complexes with BP ligands.19b The
differences between both complexes lie in the position of the
C6 alkyl chain on the imidazole ring. In one of the molecules
(Molecule A, Figure 3a) the chain is bent, and there are short
C−H···O distances (dC−H...O in the 2.33−2.80 Å range).26 In
the other molecule (Molecule B, Figure 3b), the alkyl chain is
extended away from the cluster. Molecules A and B stack into
columns that align in the unit-cell (Figure 3c).
Figure 2. 31P{1H} NMR spectra of Mo6(ZolC6)2 dissolved in D2O at
two different temperatures and of ZolC6 at room temperature (RT).
singlets at 17.46 and 16.82 ppm with relative intensities 0.89:1.1
(Figure S2b). Only one singlet was expected if the structure
observed in the solid state (Figure 3) was retained in solution,
In the Mo4L2Mn complexes, IR, elemental analysis, and EDX
measurements indicate that the Mo4L2Mn(III) (L = Zol,
ZolC8) and Mo4Zol2Mn(II) complexes have structures that are
similar to Mo4Zol2Fe.22 That is, two {Mo2O6} dimeric units
with faced-shared Mo(VI) octahedra are bound to an
octahedrally coordinated central Mn ion (Figure 1), and two
BP ligands are connected to the Mo and Mn ions through P−
O−M and C−O−M (M = Mo, Mn) bonds.
In Vitro and in Vivo Results. We next investigated the
effects of the new compounds (together with some compounds
reported previously) on tumor cell growth inhibition using the
nonsmall cell human lung cancer cell line NCI-H460.
Representative dose−response curves are shown in Figure 4
for zoledronate (Figure 4a), Mo4Zol2Mn(II) (Figure 4b),
Mo6(ZolC6)2 (Figure 4c), and Mo4(ZolC8)2Mn(III) (Figure
4d).
IC50 values for all compounds are reported in Table 1 on
both a molecular weight as well as on a per-bisphosphonate
basis. We anticipated that there would be a large increase in
activity of the POM/BP complexes containing the more
lipophilic bisphosphonates since the lipophilic bisphosphonates
acting alone are more active than is zoledronate. With the
zoledronate complexes we find that the new Mo4Zol2Mn(II)
complex has potent activity (on a per bisphosphonate basis),
Figure 4b, similar to that of Mo4Zol2Mn(III), on average ∼6×
the activity of zoledronate acting alone and considerably more
activity than the Mo6Zol2 complex, Table 1. This suggests that
the oxidation state of Mn has little influence on the activity of
the complex. It can also be seen that zoledronate and the
Mo4Zol2Mn(III) complex are about 2× more potent in MCF-7
than in NCI-H460 cell lines, as reported previously,22 reflecting
not unexpected cell-to-cell variability. With the ZolC6 system
we obtained more activity with the Mo6(ZolC6)2 system than
Figure 3. Representation of the two crystallographically independent
molecules (a) A and (b) B in the structure of Mo6(ZolC6)2. (c) View
of the crystal packing. Purple octahedra = MoO6 octahedra in
molecule A, gray octahedra = MoO6 octahedra in molecule B, green
spheres = P, black spheres = C, small black spheres = H, blue spheres
= N, red spheres = O, hydrogen bonds are represented as dotted lines.
corresponding to four equivalent phosphorus nuclei. The
presence of another singlet close to the first one cannot be
attributed to free ZolC6 ligands as the chemical shift of this
ligand is equal to 14.45 ppm (Figure 2) and is attributed to an
equilibrium between two conformers which differ by the
rotation of the two {Mo3O8} groups around the central oxo
bridge (Figure S4).21 The two resonances are relatively sharp at
room temperature and broaden when the temperature increases
and finally coalesce at 70 °C (Figure 2), indicating that, as
C
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very lipophilic pyridinium analogue of zoledronate (BPH-715,
containing an n-C10 alkyloxy side-chain) resulted in a ∼5×
decrease in tumor volume versus zoledronate.23 Representative
results for PBS control, zoledronate, and Mo4Zol2Mn(III)
treated mice as a function of time are shown in Figures S6a−c,
tumor cell volumes as a function of time are shown in Figure
S6d, and tumor cell volumes in each of the individual mice after
a 4 week treatment are shown in Figure 5a. As can be seen in
Figure 5. Effects of zoledronate and Mo4Zol2Mn(III) on SK-ES-1
tumor volume in 6-week old athymic nude mice treated daily for 28
days with 5 μg compound or phosphate-buffered saline (PBS); 6 mice
per group. (a) Tumor volumes in each of the individual mice after a 4
week treatment. (b) Body weights as a function of time for different
treatments.
Figure 4. Dose response curves for NCI-H460 cell growth inhibition.
(a) Zoledronate. (b) Mo4Zol2Mn(II). (c) Mo6(ZolC6)2. (d)
Mo4(ZolC8)2Mn(III). Three replicates of duplicates were determined,
and representative data from one duplicate set are shown.
Table 1. Growth Inhibition of Human Cancer Cell Line
NCI-H460 by Bisphosphonates and POM/BP Complexes
IC50 [μM] per
IC50 decrease vs
BP
Figures 5a and S6d, there is a very significant (p = 0.045)
decrease in tumor cell volume with Mo4Zol2Mn(III) dosed at 5
μg/mouse via intratumoral injection once a day for 28 days,
corresponding to a ∼5× decrease in tumor cell growth versus
zoledronate, even though there was only ∼1/3 the amount of
zoledronate present in the Mo4Zol2Mn(III) treatment (due to
the high mass of the POM cluster). There is, however, an
overlap in the “error bars” between the different treatments.
But, as can be seen in Figure 5a, this is due to the presence of
just a single “outlier” mouse in each of the three treatments,
with 5/6 mice having tumor volumes that are very tightly
clustered in each treatment and where, for these points, the
reduction in tumor volumes on treatment with Mo4Zol2Mn-
(III) versus zoledronate is rather unambiguous, Figure 5a.
There was also only a small difference in mice body weight on
zoledronate or Mo4Zol2Mn(III) treatment, Figure 5b, showing
the absence of any increased toxicity of the complex versus
zoledronate alone therapy. These relatively low bisphosphonate
concentrations (compared with zoledronate or BPH-715 levels)
may make such hybrids in general less toxic than bi-
sphosphonates acting alone, although further work will be
needed to investigate this possibility. In the future, it may be
possible to carry out detailed structure−activity relationships
investigations of these types of complex, although this will be
very challenging since it will be necessary to know cell
concentrations of the complexes, their stability, as well as the
targets for the Mo-moiety as well as the Mn. Plus, as briefly
discussed below, both protein prenylation as well as kinase
inhibition can be targeted by bisphosphonates.
formula
Mo6Zol2
IC50 [μM]
BP
10 0.9
2.6 0.3
2.0 0.1
20 1.8
5.2 0.6
4.0 0.2
1.4×
5.4×
7.0×
1.0×
3.4×
1.0×
1.3×
0.9×
1.0×
Mo4Zol2Mn(III)
Mo4Zol2Mn(II)
Zol
28
3
28
3
Mo6(ZolC6)2
ZolC6
3.5 0.1
24 0.8
1.9 0.2
2.9 0.3
5.1 0.2
7.0 0.2
24 0.8
3.8 0.4
5.8 0.6
5.1 0.2
Mo6(ZolC8)2
Mo4(ZolC8)2Mn(III)
ZolC8
with ZolC6 alone. However, with the more lipophilic ZolC8
species, the activities of all three species: ZolC8, Mo6(ZolC8)2,
and Mo4(ZolC8)2Mn(III) (Figure 4d) are quite similar to each
other as well as to the Mo4Zol2Mn complexes, Table 1.
We thus next moved to in vivo studies which since the
pioneering work of Yamase and co-workers10a have been quite
rare.27 Since there were only small differences in IC50 values on
a per-bisphosphonate basis between the most active species, we
chose to investigate the Mo4Zol2Mn(III) species reported
earlier in our in vivo study, for the following reasons: (i) it has
good activity in both the H460 (lung cancer) cell line described
here, as well as against the MCF-7 (breast cancer) cell line
reported previously;22 (ii) zoledronate is commercially available
while the alkyl zoledronates require several additional synthesis
steps; (iii) zoledronate is already used clinically while the alkyl
zoledronates are not; (iv) synthesis of the Mo4Zol2Mn(II)
complex requires formation of the Mn(III) complex followed
by the reduction of the Mn(III) crystals by ascorbate-again an
additional synthetic step, and (v) there is little difference in
activity between the Mn(II) and Mn(III) complexes, due
perhaps to reduction of the latter in cells.
CONCLUSIONS
■
We synthesized and characterized four new POM/BP
complexes: Mo6L2 (L = ZolC6, ZolC8), Mo4(ZolC8)2Mn(III),
and Mo4Zol2Mn(II), and tested them, together with previously
reported analogues, in vitro against a human nonsmall cell lung
cancer cell line NCI-H460. The most active compounds had
IC50 values of ∼4−5 μM (on a per-bisphosphonate basis),
We used the mouse xenograft (SK-ES-1 sarcoma tumor cell
growth) model because in previous work we used this system
to investigate the effects of lipophilic bisphosphonate chain-
length variation on tumor cell growth where we found that a
D
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1 M HCl was added dropwise to pH = 5. NH4Cl (0.600 g, 11.3 mmol)
was added, resulting in the precipitation of a fine white powder. The
mixture was allowed to stir at room temperature for 20 min. The
powder was filtered and dried with ethanol and diethyl ether. Yield:
0.061 g (48% based on Mo). 31P NMR (300 MHz, D2O, 25 °C): δ
17.46 (s), 16.82 (s). 1H NMR (300 MHz, D2O, 60 °C, water
saturation): 9.08 (d, 1H, NCHCHN), 7.86 (d, 1H, NCHCHN), 7.65
(s, 1H, NCHN), 5.05 and 4.96 (m, 2H, NCH2C), 4.46 (t, 2H,
NCH2CH2), 2.17 (m, 2H, NCH2CH2CH2), 1.61 and 1.56 (m, 10H,
CH2), 1.13 (m, 3H, CH3). IR (FTR): ν (cm−1) = 1560 (w), 1414 (vs),
1153 (sh), 1134 (s), 1083 (m), 1058 (sh), 1048 (s), 980 (m), 917 (s),
876 (vs), 792 (w), 732 (m), 690 (s), 619 (s), 578 (m), 556 (m), 533
(m), 487 (w), 458 (w). Anal. Calc. (found) for C26H88N10Mo6O41P4
(1896.5 g mol−1): C 16.47 (16.69), H 4.68 (4.51), N 7.39 (7.36). EDX
measurements confirm the Mo/P ratio.
about 6× more active than zoledronate itself (IC50 = 28 μM).
One of the most active compounds tested here in vitro was also
the most active compound against the MCF-7 breast cancer cell
line reported previously, so was investigated in vivo against SK-
ES-1 sarcoma cells in a mouse xenograft system. There was a 5-
fold decrease in tumor cell volume versus that found with
zoledronate after a 28 day treatment period at 5 μg/mouse
dosing (∼0.2 mg/kg). At present, the molecular basis for the
enhanced activity (over that seen with bisphosphonates alone)
remains to be determined since both masking of bi-
sphosphonate charge, increasing cell permeability, as well as
potentiation of bisphosphonate activity by the (hetero)-
polymolybdate moiety may be involved. Nevertheless, the
results are of considerable interest because they show for the
first time that POM/BP/Mn complexes have potent in vivo
activity.
What is particularly attractive about the use of bi-
sphosphonates as ligands in POM complexes is that
zoledronate, for example, has the potential for targeting both
Ras as well as epidermal growth factor (EGF)-driven tumors.
Zoledronate inhibits farnesyl diphosphate synthase and this
leads to inhibition of Ras prenylation. Moreover, in two recent
articles28 zoledronate has also been shown to lead to inhibition
of EGFR signaling/phosphorylation. It may thus be possible to
develop POM-bisphosphonate compounds that target both
EGFR as well as Ras-driven cancers. Zoledronate is also an
important POM-ligand since zoledronate itself activates
gamma-delta T cells (containing the Vγ2 Vδ2 T cell receptor)
to kill tumor cells,29 it switches macrophages from an M2
(tumor promoting) to an M1 (tumor killing) phenotype30 and
inhibits angiogenesis/invasiveness. There are thus good reasons
to believe that POM/bisphosphonate/Mn complexes may find
utility as drug leads for combination therapies against both Ras
as well as EGFR-driven cancers, that also target innate
immunity.
Synthesis of (NH4)5[(MoVI2O6)2(O3PC(C4H6N2)OPO3)2MnIII]·
10H2O (Mo4Zol2Mn(III)). To a solution of Na2MoO4·2H2O (0.242
g, 1 mmol) in 10 mL of 1 M CH3COONH4/CH3COOH buffer was
added Mn(OAc)3·2H2O (0.070 g, 0.26 mmol) and zoledronic acid
(0.137 g, 0.5 mmol). The solution was stirred for 5 min then NH3
(33% in water) was added dropwise to pH = 7.5. The solution was left
to evaporate and was filtered after 24 h in order to remove a pink
precipitate. EDX measurements and IR spectroscopy indicated that the
precipitate was a BP complex that did not contain molybdenum. An
homogeneous gray crystalline phase of the title compound appeared
after 3 days (see picture in Figure S7). Yield: 0.080 g (21% based on
Mo). Anal. Calc. (found) for C10H52MnMo4N9O36P4 (1437.2 g
mol−1): C 8.36 (8.88), H 3.65 (3.90), N 8.77 (8.78). IR (FTR): ν
(cm−1) = 1575(m), 1546 (w), 1421(s), 1288 (w), 1136(s), 1112(sh),
1045(s), 1018(sh), 973(m), 915(s), 888(s), 790(s), 699(m), 656(m),
619(w), 560(w), 529(m). EDX measurements confirm the Mo/P and
Mo/Mn ratios.
Synthesis of (NH4)5(H3O)[(MoVI2O6)2(O3PC(C4H6N2)-
OPO3)2MnII]·6H2O·4CH3OH (Mo4Zol2Mn(II)). Ascorbic acid (0.150
g, 0.85 mmol) was added to a suspension of Mo4Zol2Mn(III) (0.190 g,
0.13 mmol) in 30 mL of MeOH. The solution was stirred for 6 h and
was filtered. The resulting pale yellow solid was washed with MeOH
and dried. Yield: 0.129 g (68%). IR (FTIR): ν (cm−1) = 1579(m),
1546 (w), 1418(s), 1286 (w), 1137(s), 1112(sh), 1047(s), 1018(sh),
975(m), 911(s), 887(s), 786(s), 699(m), 659(m), 619(w), 561(w),
529(m), 485(w). Anal. Calc. (found) for C14H63N9MnMo4O37P4
(1512.3 g mol−1): C 11.12 (11.02), H 4.20 (3.47), N 8.34 (7.98).
EDX measurements confirm the Mo/P and Mo/Mn ratios.
EXPERIMENTAL SECTION
■
Experimental Procedures and Characterization. Zoledronic
acid H2O3PC(C4H5N2)(OH)PO3H2 (H5Zol)31 and (NH4)6-
[(Mo3O8)2O(O3PC(C4H6N2)OPO3)2]·9H2O (Mo6Zol2)21 were syn-
thesized according to reported procedures. Li2H2(ZolC6) and
Li2H2(ZolC8) were synthesized with a protocol adapted from literature
procedures24 (see Supporting Information).
Synthesis of Li2(NH4)3[(MoVI2O6)2(O3PC(C4H5N2)(C8H17)-
OPO3)2MnIII]·8H2O (Mo4(ZolC8)2Mn(III)). Na2MoO4·2H2O (0.197
g, 0.82 mmol) was dissolved in 5 mL of 1 M NH4OAc/HOAc and
Mn(OAc)3.2H2O (0.018 g, 0.048 mmol) was added. A solution of
Li2H2(ZolC8) (0.027 g, 0.060 mmol) in 3 mL of H2O acidified with a
few drops of 1 M HCl until dissolution was slowly added. The pH was
adjusted to 5.8 with 33% NH3. The solution was stirred for 2 h and an
orange crystalline precipitate which did not contain ZolC8 was
removed by filtration. The pH of the solution was equal to 6.1. The
solution was left at room temperature for 5 days, and the thin pinkish
platelets (see picture in Figure S7) of the title compound were
removed by filtration. Yield 0.011 g (23% based on ZolC8). IR
(FTIR): ν (cm−1) = 1639(w), 1661(m), 1420(s), 1123(m), 1046(s),
990(w), 912(m), 871(s), 796(w), 743(w), 691(m), 620(m), 661(m).
Anal. Calc. (found) for C26H64Li2N7MnMo4O36P4 (1627.3 g mol−1):
C 19.19 (19.97), H 3.96 (4.18), N 6.03 (5.80). EDX measurements
confirm the Mo/P and Mo/Mn ratios.
Synthesis of (NH4)6[(Mo3O8)2O(O3PC(C4H5N2)(C6H13)(O)-
PO3)2]·8H2O (Mo6(ZolC6)2). (NH4)6Mo7O24·4H2O (0.120 g, 0.10
mmol) was dissolved in 5 mL of 1 M NH4OAc/HOAc buffer before
addition of Li2H2(ZolC6) (0.106 g, 0.24 mmol). The mixture was
sealed in a 23 mL Teflon-lined stainless steel reactor before heating to
130 °C over a period of 1 h, kept at this temperature for 20 h, then
cooled to room temperature over a period of 36 h. The resulting
colorless crystals were collected by filtration. Yield: 0.144 g (68%
based on Mo). 31P NMR (300 MHz, D2O, 25 °C): δ 17.12 (s), 16.76
(s). 1H NMR (300 MHz, D2O, 60 °C, water saturation): 9.07 (d, 1H,
NCHCHN), 7.83 (d, 1H, NCHCHN), 7.63 (s, 1H, NCHN), 5.07 and
4.94 (2m, 2H, NCH2C), 4.44 (t, 2H, NCH2CH2), 2.14 (m, 2H,
NCH2CH2CH2), 1.59 (m, 6H, CH2), 1.14 (m, 3H, CH3). IR (FTR): ν
(cm−1) = 1640 (w), 1566 (w), 1433 (vs), 1152 (s), 1127 (s), 1086 (s),
1062 (sh), 1048 (s), 978 (m), 928 (sh), 912 (s), 875 (vs), 730 (vs),
698 (s), 629 (s), 560 (m), 518 (m), 482 (m). Anal. Calc. (found) for
C22H76N10Mo6O39P4 (1804.4 g mol−1): C 14.64 (14.40), H 4.25
(3.99), N 7.76 (7.69). EDX measurements confirm the Mo/P ratio.
Synthesis of (NH4)6[(Mo3O8)2O(O3PC(C4H5N2)(C8H17)(O)-
PO3)2]·10H2O (Mo6(ZolC8)2). Na2MoO4·2H2O (0.096 g, 0.40
mmol) was dissolved in 1.5 mL of water (sol. A); Li2H2(ZolC8)
(0.055 g, 0.12 mmol) was dissolved in 1.5 mL of water acidified with 2
drops of 1 M hydrochloric acid (sol. B). Sol B was added to sol. A and
Infrared spectra were recorded on a Nicolet 30 ATR 6700 FTIR
spectrometer.
Crystal Structure Determination. Single crystal X-ray diffraction
data for Mo6(ZolC6)2 were collected by using a Bruker Nonius X8
APEX 2 instrument equipped with a CCD bidimensional detector
using the monochromatised wavelength λ(Mo Kα) = 0.71073 Å.
Absorption corrections were based on multiple and symmetry-
equivalent reflections in the data set using the SADABS program32
based on the method of Blessing.33 The structure was solved by direct
methods and refined by full-matrix least-squares using the SHELX-TL
E
DOI: 10.1021/acs.inorgchem.7b01114
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
package.34 NH4 and H2O could not be distinguished based on the
+
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b01114.
■
observed electron densities, therefore, all positions were labeled as O
and assigned the oxygen atomic diffusion factor. Crystallographic data
are given below and the complete data can be found in the cif file
CCDC 1547689.
S
Crystal data for compound Mo6(ZolC6)2: C22H36Mo6N4O41P4, M =
1712.07 g mol−1, triclinic, space group P1, a = 8.8527(6) Å, b =
Synthesis of ZolC6 and ZolC8, IR and NMR spectra, χMT
= f(T) curve for Mo4Zol2Mn(II), representation of the A
and B conformers of the Mo6L2 compounds, pictures of
mice treated with PBS, zoledronate or Mo4Zol2Mn(III),
SEM images of crystals of Mo4Zol2Mn(III) and platelets
of Mo4(ZolC8)2Mn(III), powder X-ray diffraction
pattern of Mo6(ZolC6)2 (PDF)
̅
16.8404(11) Å, c = 19.6929(13) Å, α = 81.370(2)°, β = 83.028(2)°, γ
= 76.955(2)°, V = 2816.0(3) Å3, T = 296 K, Z = 2, Dc = 2.019 g cm−3,
μ = 1.515 mm−1, GOF = 1.145, final R indices (I ≥ 2σ(I)) R1 =
0.0406, wR2 = 0.1191.
Powder X-ray. Powder X-ray diffraction data were obtained on a
Bruker D5000 diffractometer using Cu radiation (1.54059 Å). The
comparison of the experimental X-ray powder pattern with the powder
pattern calculated from the structure solved from single-crystal X-ray
diffraction data confirm the homogeneity of Mo6(ZolC6)2 (Figure S8).
EDX Measurements. EDX measurements were performed on a
JEOL JSM 5800LV instrument.
Accession Codes
CCDC 1547689 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_
request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2
1EZ, UK; fax: +44 1223 336033.
NMR Measurements. 31P NMR spectra were recorded in 5 mm
tubes with H decoupling by using a Bruker AC-300 spectrometer
1
operating at 121.5 MHz. 31P chemical shifts were referenced with
respect to an external standard, 85% H3PO4, using the convention that
low-field, paramagnetic, deshielded values are positive (IUPAC δ-
scale). For all compounds 15−20 mg of sample was dissolved in D2O
(500 μL). The concentrations thus varied in the 10−30 mM range.
Magnetic Measurements. Magnetic susceptibility measurements
were carried out on polycrystalline samples using a Quantum Design
MPMS SQUID magnetometer operating in the 300−2 K temperature
range and 0−5.5 T. Susceptibility measurements were performed with
an applied field of 1000 Oe. Pascal’s constants were utilized to estimate
diamagnetic corrections, the value in each case being subtracted from
the experimental susceptibility data to give the molar magnetic
susceptibility (χM).
AUTHOR INFORMATION
Corresponding Authors
*(E.O.) E-mail: eo@chad.scs.uiuc.edu.
*(A.D.) E-mail: anne.dolbecq@uvsq.fr.
ORCID
Anne Dolbecq: 0000-0001-7940-4152
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by Centre National de la Recherche
Cell-Growth Inhibition Assays. The human tumor cell line NCI-
H460 (nonsmall cell lung cancer) was obtained from the National
Cancer Institute and maintained at 100% humidity and 5% CO2 at 37
°C. Cell growth inhibition assays were carried out as described
previously.20 A broth microdilution method was used to determine the
growth inhibition IC50 values. Briefly, ∼5 × 104 cells suspended in 100
μL of DMEM supplemented with 10% fetal bovine serum (FBS), 4.5
g/L glucose and L-glutamine and preserved with 1% penicillin−
streptomycin were seeded in 96-well plates (Corning Inc., Corning,
NY) and incubated at 37 °C in a 5% CO2 atmosphere. The cells were
incubated with 1000 μM, 333 μM, 111 μM, 37 μM, 12 μM, 4.1 μM,
1.4 μM, 0.46 μM, 0.15 μM, and 0.051 μM of compounds and H2O as a
control for 4 days.20 Then, an MTT ((3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) cell proliferation assay (ATCC,
Manassas, VA) was performed to obtain dose−response curves. The
IC50 values of the free BP, Mo6Zol2 and Mo4Zol2Mn(III), which had
been measured in MCF-7 cells in our first studies21,22 were
redetermined here using the same conditions as for the other
compounds (Table 1).
■
Scientifique, Universite
́
de Versailles-Saint-Quentin-en-Yve-
lines, L’Agence Nationale de la Recherche (Grant ANR-11-
BS07-011-01-BIOOPOM), and by the United States Public
Health Service (National Institutes of Health Grants
́
GM065307 and CA158191). Flavien Bourdreux, Gregoire
Paille, and Tarik Benali are gratefully acknowledged for the
NMR measurements, the SEM images and the synthesis of
lipophilic bisphosphonates, respectively.
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DOI: 10.1021/acs.inorgchem.7b01114
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DOI: 10.1021/acs.inorgchem.7b01114
Inorg. Chem. XXXX, XXX, XXX−XXX