Bisphosphonate prodrugs: Synthesis and biological evaluation in HuH7 hepatocarcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2014.02.054

We investigated the biological effects of new synthesized bisphosphonates (BPs) on HuH7 hepatocarcinoma cells. BPs containing p-bromophenyl (R₁ = p-Br, Ph, 2) in their side chain were the more potent to inhibit HuH7 cell viability. In addition, phenyl diesterified analogues (R₂ = R ₃ = Ph, 2a) were more potent than methyl (R₂ = R ₃ = Me, 2b) or non-esterified BPs (2) inducing more necrosis suggesting that they better entered into cells. Phosphodiesterase inhibitor (IBMX) reversed the effect of the esterified BPs and not that of non-esterified ones suggesting role of cell phosphodiesterases to release active BPs. BP analogues inhibited HuH7 cell migration but esterified ones had no effect on invasion due to the hiding of phosphonic groups. All together, these results indicated the therapeutic interest of these new BP prodrugs.

Full text of DOI:10.1016/j.ejmech.2014.02.054

European Journal of Medicinal Chemistry 77 (2014) 56e64
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Bisphosphonate prodrugs: Synthesis and biological evaluation in
HuH7 hepatocarcinoma cells
Maelle Monteil, Evelyne Migianu-Griffoni, Odile Sainte-Catherine, Mélanie Di Benedetto,
*
Marc Lecouvey
Université Paris 13, Sorbonne Paris Cité, Laboratoire de Chimie, Structure, Propriétés de Biomatériaux et d’Agents Thérapeutiques (CSPBAT), CNRS UMR
7244, 74, Rue Marcel Cachin, F-93017 Bobigny, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We investigated the biological effects of new synthesized bisphosphonates (BPs) on HuH7 hep-
atocarcinoma cells. BPs containing p-bromophenyl (R1 ¼ p-Br, Ph, 2) in their side chain were the more
potent to inhibit HuH7 cell viability. In addition, phenyl diesterified analogues (R2 ¼ R3 ¼ Ph, 2a) were
more potent than methyl (R2 ¼ R3 ¼ Me, 2b) or non-esterified BPs (2) inducing more necrosis suggesting
that they better entered into cells. Phosphodiesterase inhibitor (IBMX) reversed the effect of the ester-
ified BPs and not that of non-esterified ones suggesting role of cell phosphodiesterases to release active
BPs. BP analogues inhibited HuH7 cell migration but esterified ones had no effect on invasion due to the
hiding of phosphonic groups. All together, these results indicated the therapeutic interest of these new
BP prodrugs.
Received 13 November 2013
Received in revised form
19 February 2014
Accepted 21 February 2014
Available online 23 February 2014
Keywords:
Bisphosphonates
Prodrugs
Phosphodiesterase inhibitor (IBMX)
Hepatocarcinoma
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
implicated in several important normal and tumour cellular path-
ways, via small G proteins like Rho or Ras. The inhibition of FPPS
Bisphosphonates (BPs) are currently the most widely used
treatment of common skeletal disorders. They are used as in-
hibitors of bone resorption, in particular osteoporosis, solid tumour
bone metastases and myeloma bone disease [1]. Most BPs contain a
hydroxyl group on their PeCeP structure that confers high affinity
binding to calcium phosphate (hydroxyapatite (Ca10(PO4)6(OH)2))
and is the basis for the bone-targeting properties of bisphospho-
nates [2,3]. The nature of their side chain (R1, Fig. 1) gives rise to a
variety of possible structures determining their different potencies.
Two classes of bisphosphonates depending on chemical struc-
ture can be distinguished: nitrogen-containing bisphosphonates,
where the side chain possesses an amino function (i.e. alendronate)
or a nitrogen heterocycle (i.e. zoledronate) and non-nitrogen-
containing bisphosphonates corresponding to the others (i.e.
clodronate, etidronate). It is clearly established that the mechanism
of action on osteoclasts depends on the nature of bisphosphonates
[4]. N-BPs in their side chain such as zoledronate, act on the
mevalonate pathway inhibiting the farnesyl diphosphate synthase
(FPPs) thereby depleting the cells of the farnesyl (FPP) or ger-
anylgeranyl (GGPP) diphosphate isoprenoids. Isoprenoids are
also results in the accumulation of its substrate, isopentenyl
diphosphate (IPP), which can be converted to the isopentenyl ester
of ATP (APPI), which is strongly pro-apoptotic and is thought to
contribute to the overall efficacy of bisphosphonates, both in vitro
and in vivo ([2,5,6]).
Non-N-BPs (e.g. etidronate or clodronate) cause the intracellular
accumulation of non-hydrolysable cytotoxic ATP analogues that
subsequently induce osteoclast apoptosis. The direct growth
inhibitory effects have been attributed to cell cycle arrest and/or
induction of apoptosis [4]. BPs also exhibit direct and indirect
antitumour effects in vitro against a broad variety of tumour cell
lines, including melanoma, mesothelioma, prostate, breast, lung
and myeloma cancer cells [7e9]. Numerous studies have also
described the ability of BPs to inhibit tumour cell adhesion and
invasion, but the mechanisms of these effects are still unclear
[8,10,11]. We have shown that some non-N-BPs had similar prop-
erties to N-BPs in particular in aromatic series [12,13]. However,
in vivo efficacy of BPs on extra-osseous sites is still being debating.
Only a small number of studies have demonstrated activity on soft
tissues in vivo [14]. The reasons are the poor oral bioavailability
(0.3e7% in humans) due to chelation of metal ions by phosphonic
acid groups, poor membrane permeability due to poor BP lip-
ophilicity as well as strong uptake by bone tissue [15]. In order to
increase the pharmacological properties of bisphosphonates,
* Corresponding author.
E-mail address: marc.lecouvey@univ-paris13.fr (M. Lecouvey).
http://dx.doi.org/10.1016/j.ejmech.2014.02.054
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

M. Monteil et al. / European Journal of Medicinal Chemistry 77 (2014) 56e64
57
O
P
O
P
O
P
O
P
O
P
O
P
O
P
O
P
HO
HO
R2O
HO
R3O
HO
OH
OR2
OH
OH
OH
OH
R3O
HO
OH
OR2
OH
OH
OR3
OH
R1
R1
2
R2=R3= H
1
R2=R3= H
2a R2=R3= C6H5
2b R2=R3= CH3
B
A
1a
R2=R3= C6H5
Br
2c R2=C6H5; R3=H
Fig. 1. General structures of BPs (A) or esterified BPs (B).
Fig. 2. General structures of BPs (1, 2, 1a, 2a, 2b, 2c).
several drug delivery systems can be used, like nanoparticles [16] or
liposomes [17]. Another strategy has been considered: the use of
bisphosphonate prodrugs [18e21]. Few studies about the design of
phosphonoester prodrugs have been reported in the literature
[22,23], masking the phosphonic acid with organic functions
[24,25]. We have demonstrated that partly esterified bisphospho-
nates display antitumour growth and antiangiogenic activities on
A431 tumours and MDA-MB-231 tumours being more effective
in vivo than in vitro [26,27]. In this last work, we have suggested
that esterified BPs act like prodrug molecules being hydrolysed by
cellular or serum environment containing phosphoesterase en-
zymes, leading to the release of the tetraacid active molecule.
Hepatocellular carcinoma (HCC) is one of the most common
aggressive malignancies in the world [28]. More than 80% of pa-
tients have advanced or unresectable disease and when they do
undergo resection, 50% of the patients present recurrence at the
two years. Systemic chemical cytotoxic chemotherapy is used
without satisfactory results and these therapies can induce signif-
icant toxicity due to the liver dysfunction [29]. Thus, new agents
that target molecular abnormalities in HCC will improve the
treatment of the patients. BPs could be effective in the treatment of
HCC since FPP or GPP are crucial for the signalisation of small
proteins G as Ras which can be mutated in HCC [30]. Accordingly,
we have already demonstrated that BPs synthesized in our labo-
ratory can inhibit the Ras processing [13]. Furthermore, another
small G protein, Rho, has been proposed to play a crucial role in
intrahepatic metastasis [31,32]. Only one work has demonstrated
that N-BP (pamidronate) can inhibit several HCC cell line tumour
growth and motility acting via the Ras and the Rho A pathway,
respectively [33].
bisphosphonate diesters varying both on the lateral chain and ester
functions [24,25]. In this study, we chose to work with two aro-
matic bisphosphonates. Both compounds 1 (R1 ¼ Ph) and 2 (R1 ¼ p-
BrPh) have already proved their biological activity on various
tumour cell lines in vitro and/or in vivo [12,13]. The synthesis of 1
and 2 has been fully described earlier [36]. Products were obtained
by reacting for 2 h, at room temperature, two equivalents of tris(-
trimethylsilyl) phosphite or methyl (or phenyl) bis(trimethylsilyl)
phosphite with the appropriate acid chloride (Scheme 1). After
evaporation of the volatile fractions and treatment with methanol,
products were purified by precipitation in a mixture of appropriate
solvent. Products were obtained as powder in good yields from 70
to 95%. Product 2c was obtained as described for BP monomethyl
esters [37]. The successive addition of one equivalent of phenyl
bis(trimethylsilyl) phosphite followed by another equivalent of
tris(trimethylsilyl) phosphite on acid chloride only gave a mixture
of BPs (non-esterified, mono and diesterified). Consequently the
two step procedure going through the formation of the p-bromo-
phenyl-a-ketophosphonate dimethyl ester was used. It was ob-
tained by condensing at ꢀ10 ꢁC one equivalent of
trimethylphosphite with p-bromobenzoyl chloride. After the end of
the addition, the reaction was kept 2 h at room temperature and
evaporation under reduced pressure (0.1 Torr) gave quantitatively
the p-bromophenyl-
powder. The -ketophosphonate was then converted to its di(tri-
methylsilyl) ester in with 2.5 equivalents of bromo-
a-ketophosphonate dimethyl ester as a white
a
5
h
trimethylsilane. After vacuum evaporation of the volatile fractions,
one equivalent of phenyl bis(trimethylsilyl) phosphite was added at
0
ꢁC, under argon. The reaction was then let stirred 2 h at room
temperature. The resulting mixture was evaporated and methanol
treatment was done. Compound 2c was then obtained by a simple
wash with ether and was isolated as a white powder in 90% yield.
All the different steps were followed by 31P {1H} NMR. 2c shows the
expected presence of two doublets in 31P {1H} NMR corresponding
to the signal obtained for the two different phosphorus atom with a
2JPeP coupling constant of 28.6 Hz. Moreover the 13C {1H} NMR
spectrum shows the characteristic signal at 77.6 ppm (dd) with a
1JCeP coupling constant of 142 Hz.
In this work, we study the efficacy of our non-nitrogen-
containing BPs, the BPs containing phenyl ring without or with
bromine (BP 1 and 2, respectively) and esterified with different
organic functions (1a, 2a, 2b, 2c) on the viability, the migration and
the invasion of the HuH7 HCC cell line (Fig. 2). We further evaluate
the mechanism of action of these compounds by studying the effect
of
IBMX
phosphodiesterase
inhibitor
(3-isobutyl-1-
methylxanthine) on the antiproliferative effect of esterified BPs.
For biological evaluation, BP stock solution (1 mM) was pre-
pared in sterilised distilled water using acid form. All the com-
pounds were readily soluble at this concentration. The pHs were
adjusted to 7.2 with a diluted potassium hydroxide solution.
2. Chemistry
Synthesis of esterified bisphosphonates is not possible using
standard procedure used for non esterified BPs. Most of the syn-
thesis described up to nowadays usually goes through the making
of the bisphosphonate tetraester which is then dealkylated [23].
The main drawback of this pathway is the thermal and basic
instability of bisphosphonate tetraesters prone to isomerisation in
phosphonate-phosphate [34]. Moreover the regioselective deal-
kylation to obtain partial esters is difficult and does not occur in
good yields [23]. Our group has proposed a very mild and one-pot
synthesis to obtain bisphosphonate methyl esters from methyl
bis(trimethylsilyl) phosphite and acyl chloride [35]. We have
further exemplified this synthesis to the obtaining of numerous
3. Biological evaluation
3.1. BP effects on HuH7 cell viability
Two different classes of BPs regarding to their side chain
(R1 ¼ Ph, compounds 1 or R1 ¼ p-BrPh, compounds 2) were eval-
uated on the HuH7 cell viability (Fig. 3, Table 1). Cells were treated
with esterified (1a, 2aec) or non-esterified BP (1 and 2) at con-
centrations ranging from 10 mM to 1 mM.

58
M. Monteil et al. / European Journal of Medicinal Chemistry 77 (2014) 56e64
O
O
P
HO
OH
OH
OH
P
HO
1) 2 eq. P(OSi(CH3)3)3
2) CH3OH
X=H 1
X=Br 2
X
O
O
P
HO
OH
OH
OH
1) 1 eq. P(OCH3)3
O
C
P
2) 2.5 eq. (CH3)3SiBr
2c
X
Cl
O
3) 1 eq. P(OSi(CH3)3)2(OC6H5)
R= H, Br
O
P
R2O
R2O
H
X
H4N O
H4N O
(H3C)3SiO
1) 2 eq.
NH3 excess
HMDS excess
O
O
P
OR2
P
OR2
HO
OH
OH
(H3C)3SiO
P
P
R2O
OR2
R2O
R2O
2) CH3OH
P
OH
X=H, R2=C6H5 1a
X=Br, R2=Me 2a
X=Br, R2=C6H5 2b
R2=CH3; C6H5
X
Scheme 1. Synthesis of Bisphosphonates (1, 2, 1a, 2a, 2b, 2c).
As a positive control, the N-BP zoledronate was used. Zoledro-
nate exhibited an IC50 of 60 M on HuH7 cell viability. While the
BPs 1 and 2b only inhibited the HuH7 cell viability about 15% at
each used concentration, the BPs 1a, 2, 2a and 2c inhibited the
HuH7 cell viability in a dose-dependent manner. The IC50 were
(3-isobutyl-1-methylxanthine, IBMX) (Fig. 4). We evaluated the
effect of IBMX on the BP 1 and its esterified analogue 1a as well as
the compounds 2 and 2a.
When HuH7 cells were treated with IBMX alone at 1 mM for
144 h, no significant inhibition of HuH7 cell viability was observed.
Addition of IBMX to non-esterified BPs (1 or 2) increased their
antiproliferative effects. In contrast, the addition of IBMX to ester-
ified BPs (1a and 2a) totally reversed the inhibitory effect of each
compound suggesting that cell phosphodiesterase activities are
essential for esterified BP activity on HuH7 viability.
m
550 mM for 1a, 440 mM for 2, 125 mM for 2a and 100 mM for 2c
showing a better effect of PhBr-BPs esterified with Ph functions.
Although zoledronate IC50 was better than phenyl esterified 2a and
2c BPs, it was interesting to note that the maximal effect (about
80%) of the three compounds was reached with the same concen-
tration (250
m
M).
To confirm that phosphodiesterase allowed the release of non-
esterified BP into the cells, we tested the stability of diesterified
bisphosphonates in different media over time (Table 2).
As shown in Table 2, diesterified bisphosphonate 2a is very
stable over time. After five days, no hydrolysis product has been
observed in water, in cell culture medium and in human serum. The
same stability study was carried out in presence of cellular extract.
After five days, the 31P NMR spectrum showed the presence of
several phosphorylated species (Fig. 5).
3.2. BP and phosphodiesterase inhibitor (IBMX) combination effect
on HuH7 cell viability
In order to state if the activity of phenyl esterified BP was related
to cell phosphodiesterase activity on esterified phosphonic func-
tions, we pretreated the cells with a phosphodiesterase inhibitor
Three major products have been identified. The structure of
compound 2a (12.09 ppm) is the main product. We also observed
two other phosphorylated products, one singlet resonating at
15.84 ppm and one compound corresponding to two doublets
(12.66 and 16.77 ppm). The structure of these compounds has been
determined thanks to the synthesis of the pure compounds and the
comparison of 31P NMR data. The first product corresponds to the
hydrolysis of the two phosphonic phenyl esters 2a and the second
was identified as the monodealkylation product 2c. The presence of
intracellular phosphodiesterases could explain this hydrolysis
reaction.
3.3. BP effects on HuH7 cell apoptosis and necrosis
Fig. 3. Comparative effects of BPs on HuH7 cell proliferation. HuH7 cells were incu-
bated with different concentrations of each compound. After 144 h, HuH7 cell prolif-
eration was assessed as described in “Experimental protocols”. Data represent the
mean value ꢂ SD of three independent experiments.
As the esterified BPs containing p-BrPh are more efficient that
Ph analogues, we compared the effects of BP 2 and 2a on HuH7 cell

M. Monteil et al. / European Journal of Medicinal Chemistry 77 (2014) 56e64
59
Table 1
Table 2
Structures and IC50 of BPs analogues.
Stability study of diesterified bisphosphonate 2a in different media.
31P NMR chemical shift
(ppm)
NMR signal
multiplicity
Observation
b
Name
R1
R2
R3
IC50
(m
M)
a
1
C6H5
H
H
1a
2
2a
2b
2c
C6H5
C6H5
H
C6H5
CH3
C6H5
C6H5
H
C6H5
CH3
H
555 ꢂ 0.008
440 ꢂ 0.012
1 day
3 days
5 days
p-BrC6H4
p-BrC6H4
p-BrC6H4
p-BrC6H4
D2O
Cell culture
medium
12.80
12.85
12.78
12.83
12.80
12.83
Singlet
Singlet
No degradation
No degradation
125 ꢂ 0.04
a
100 ꢂ 0.03
Human serum
12.85
12.65
12.80
Broad
singlet
No degradation
a
Major than the maximum concentration tested.
Mean values of three independent experiments and standard deviation is given.
b
4. Discussion
death (Fig. 6). Both 2 and 2a induced apoptosis effect on the HuH7
cells. Results did not show significant differences between the BPs 2
and 2a to induce the early or late HuH7 apoptosis phases. The only
difference was observed regarding necrosis where the esterified BP
2a increased about 10% the number of cells in the necrosis phase as
compared to 2. In addition, we found that compounds 2 and 2a
arrested the cells in the G1 phase of cell cycle with the same efficacy
(58%, data not shown).
In this work, we demonstrated for the first time, the anti-
proliferative activity of BPs on HuH7 HCC cell line. Results showed
the importance of esterification as well as the presence of p-bro-
mophenyl (p-BrPh) as R1 side chain to inhibit HuH7 cancer cell
viability. Indeed the p-bromophenyl (p-BrPh) containing BPs were
more efficient than the non-bromine-containing ones (2 versus 1,
respectively). These results were in agreement with previous data
of our laboratory which showed that addition of bromine on the
para position of the phenyl group in side chain exhibits an IC50 10-
fold better than non-substituted ones on A431 cell proliferation
[12]. Herein, the antiproliferative improvement provided by
bromine substituted phenyl ring is very important since the IC50
3.4. BP effects on HuH7 cell migration
As the hepatocarcinoma cells are highly invasive, we tested the
effect of these new BPs on the motility of the HuH7 cells (Fig. 7). In
the presence of HGF in the lower part of the Boyden migration
chamber, the HuH7 cells migrated through the pores to the lower
was about 440 mM for the compound 2 and whereas not reached for
the compound 1 to inhibit HuH7 cells. Further, we demonstrated
that bromine substituted compounds were non-toxic acting by
inducing aponecrosis [38]. Accordingly, a crystallographic and
computational investigation demonstrated that non nitrogen-
containing BPs could interact with farnesyl enzyme thanks to the
presence of benzyl ring in the side chain [39]. We could suppose
that the presence of bromine on phenyl also let to a better
arrangement of the BPs in the farnesyl enzyme pocket despite of
the absence of benzyl. In another hand, the major improvement of
our molecules was obtained by esterification of BPs. This result
could be due to the higher lipophilicity of the phenyl group that
could enhance entry into the cells. This observation was consistent
with our hypothesis that the esterification of the phosphonic acid
could help to increase lipophilicity and membrane permeability to
BPs. Also, we thought that cell phosphodiesterases could liberate
active BP into cells as well as in extracellular compartment. Indeed,
to further evaluate the implication of phosphodiesterase in the
activity of esterified BPs, we used IBMX which has been demon-
strated to inhibit all phosphodiesterases [40]. Phosphodiesterases
and more specifically the cyclic nucleotide phosphodiesterase 3
(PDE3) have been detected in HepG2, Hep3b and HuH7 HCC lines
[41]. Only the esterified forms of BPs were reversed by IBMX
showing the involvement of cell phosphodiesterases in the anti-
proliferative effect of esterified BPs. In contrast, the anti-
proliferative of non-esterified BPs was increased by IBMX in an
additive manner. This additive effect is probably due to the asso-
ciation of the cell alteration induced by IBMX hydrolysing AMPc or
GMPc [39] with the effect of BPs on small G proteins. Accordingly,
we had previously described the inhibition of Ras processing by BPs
[13]. It is noteworthy that non-N-BPs (as BPs) could also induce
non-hydrolysable analogues of ATP [14]. Thus, this last effect could
induce additive effects with that of IBMX. In contrast, reversion of
esterified BP effects strongly suggested that these molecules acted
like prodrugs by releasing the tetra-acid active form into the cells.
Furthermore, we demonstrated that esterified BPs inhibited HuH7
cell migration but not invasion where metalloproteinases are
important for the proteolytic degradation of the extracellular ma-
trix. BPs could inhibit invasion by chelating zinc from the active site
surface of the membrane (Fig. 7A). Compounds 2 and 2a (100 mM)
significantly reduced migration by 95% and 68% respectively
(p < 0.05, Fig. 7B) while zoledronate at the same concentration
inhibited the HuH7 cell migration by 72%.
3.5. BP effects on HuH7 cell invasion
In the presence of HGF in the lower part of the Boyden migration
chamber, the HuH7 cells invaded the Matrigel and were visualised
at lower surface of the membrane (Fig. 8A). BP 2 (100 mM) reduced
the invasion of the cells by 40% as compared with untreated control
cells (Fig. 8B). Zoledronate at this concentration inhibited the HuH7
cell invasion by 54%. In contrast, BP 2a did not reduce HuH7 inva-
sion (Fig. 8A, B).
Fig. 4. Comparative effects of BPs in association with IBMX on HuH7 cell proliferation.
HuH7 cells were pretreated with IBMX for 1 h prior to incubation with BPs. After 144 h,
HuH7 cell proliferation was assessed as described in “Experimental protocols”. Data
represents the mean value ꢂ SD of three independent experiments.

60
M. Monteil et al. / European Journal of Medicinal Chemistry 77 (2014) 56e64
Fig. 5. 31P NMR spectra of HuH7 cellular extract in the presence of 2a (0.5 mM) during 5 days. The cellular extract concentrations are comparable (5 ꢃ 106 cells/mL) for each sample.
The peak assignments are shown in figure. 50000 transients were accumulated for the spectrum.
of MMPs [7]. We confirmed this hypothesis with our non-esterified
BPs which inhibited the activity of MMP-2 in MDA-MB-231 cancer
cells (data not shown). In this work, the ineffective effect of ester-
ified BPs suggested that the masking of phosphonic groups
rendered the molecules enable to inhibit the active site of MMPs in
our experimental condition. In the present study, we showed that
all BPs were able to inhibit the motility of HuH7 HCC cells probably
by inhibiting the anchorage of small G proteins such as Rho mol-
ecules like yet demonstrated for zoledronate [42]. In this work, the
ineffective effect of esterified BPs suggested that the masking of
phosphonic groups rendered the molecules enable to inhibit the
active site of MMPs in our experimental conditions. Furthermore,
esterified BPs were expected to give better results in vivo as
compared to in vitro studies as well as to N-BPs since these com-
pounds are chemically studied to improve their bioavailability to
soft tissue as compared to bone.
6. Experimental
6.1. BPs synthesis
6.1.1. General methods
Unless otherwise noted, all solvents and reagents were high-
purity-grade materials and used without further purification. THF
was distilled from benzophenone sodium. Diethylether was
distilled from sodium. Trimethylphosphite and bromo-
trimethylsilane were distilled prior use. Solid acyl chlorides were
used directly and liquid acid chlorides were distilled under reduced
pressure. Boiling points are given in Torr (B.p.value). NMR spectra
were recorded with
a VARIAN Unity Inova 500 MHz (
13C:
125.9 MHz, 1H: 500.6 MHz, 31P: 200.7 MHz) spectrometer, a VAR-
IAN Gemini 200 MHz (13C: 50.3 MHz, 1H: 200 MHz, 31P: 80.9 MHz)
spectrometer or a BRUKER Avance 400 MHz (13C: 101 MHz, 1H:
400 MHz, 31P: 161.9 MHz) spectrometer in D2O. Chemical shifts (
d)
are given in ppm. 31P and 13C NMR spectra were recorded with
phosphoric acid and methanol as external references, respectively.
1H NMR spectra were recorded using HOD or trimethylsilane as
internal standard in D2O. Attribution of aromatic carbons and
protons is given in the text by adding o for ortho, m for meta and p
for para. IR spectra were recorded with a Perkin Elmer FT-IR model
2000 spectrophotometer in the 4000e500 cmꢀ1 spectral domain.
Spectral resolution was 2 cmꢀ1 and 5 scans were usually accumu-
lated. Multiple point baseline correction was performed using the
PE Spectrum software. Samples were studied in D2O solutions
placed in cells closed by ZnSe windows.
5. Conclusion
In the present work, we demonstrated that diesterified BPs by
phenyl groups, are better inhibitors of HuH7 HCC cell proliferation
and migration as compared to non-esterified molecules. This
improvement could be related to an increase of their lipophilicity
inducing a better penetration into cells without toxicity. Further-
more, we confirmed the prodrug strategy of these compounds since
cell phosphodiesterases can release active drug into cells.
All these results showed that p-bromophenyl and partly ester-
ified BPs can be considered as lead compounds for the design of
new BPs.
Mass spectra were recorded in positive reflectron mode with
DHB as a matrix on a MALDI-TOF-MS (Bruker). Microanalyses were

M. Monteil et al. / European Journal of Medicinal Chemistry 77 (2014) 56e64
61
THF was added under N2 to tris(trimethylsilyl) phosphite or methyl
(or phenyl) bis(trimethylsilyl) phosphite (5 mmol). When addition
was completed, reaction mixture was allowed to stand at room
temperature for 2 h. The end of the reaction was monitored by 31
P
{1H} NMR. Then, volatile fractions were evaporated under reduced
pressure (0.1 Torr) before being hydrolysed with methanol. After
evaporation, crude products were washed with diethylether and
precipitated in an appropriate mixture of solvents. (tetrahydro-
furan/dichloromethane 95/5).
6.1.2.1. (Hydroxy-phenyl-phosphono-methyl)-phosphonic acid (1).
Precipitation in diethylether. Yield: 90%. M.p.186 ꢁC. IR (D2O): 1645,
1598, 1495, 1450 (C]C), 1185, 1031, 1015, 970, 923, 775, 704,
Fig. 6. Comparative effect of BPs on HuH7 cell death. HuH7 cells were treated with BPs
for 144 h. Cells were stained with Annexin V and PI. Early and late apoptosis were
determined by Ann Vþ/PIꢀ and Ann Vþ/PIþ staining by flow cytometric analysis. Data
represent the mean value ꢂ SD of three independent experiments.
667 cmꢀ1
. d
31P NMR {1H} (200.7 MHz, D2O) 15.9. 1H NMR
(500 MHz, D2O)
d
7.33 (m, 1H, p-C6H5), 7.38 (dd, 2H, 3JHeH ¼ 7.3 Hz,
3
3JHeH ¼ 7.3 Hz, m-C6H5), 7.71 (d, 2H, JHeH ¼ 7.3 Hz, o-C6H5). 13C
performed by the Service Central d’Analyse, CNRS, F-69390, Ver-
naison. France.
NMR {1H} (125.9 MHz, D2O)
d
56.9 (OCH3), 78.8 (t, 1JPeC ¼ 145.7 Hz,
PeC(OH)eP), 129.2 (o-C6H5), 131.0 (p-C6H5), 131.4 (m-C6H5), 140.0
(C6H5C(OH)).
6.1.2. General procedure for synthesis of bisphosphonic acid 1, 2
and dialkyl or diaryl bisphosphonates 1a, 2a, 2b
In a 50 mL round-bottom three-neck flask equipped with a
thermometer and a condenser, acid chloride (2.5 mmol) in 5 mL
6.1.2.2. Disodium salt of [1-Hydroxy-(1-hydroxy-phenoxy-phos-
phoryl)-2-phenyl-methyl]-phosphonic acid monophenyl ester (1a).
Precipitation in diethylether. Yield: 78%. M.p. < 50 ꢁC. 31P NMR {1H}
Fig. 7. Comparative effect of BPs on HuH7 cell migration. HuH7 cells treated with 100
mM of BPs or zoledronate were added to the upper chamber of fibronectin-coated inserts
containing 8
mm diameter pore size. HGF (20 ng/mL) was used as a chemoattractant and placed in the lower chamber. After 24 h of incubation, cells at the lower side of the insert
were fixed, stained and counted. Data represents the mean value ꢂ SD of three independent experiments.
Fig. 8. Comparative effect of BPs on HuH7 cell invasion. HuH7 cells treated with 100
containing 8 m diameter pore size. HGF (20 ng/mL) was used as a chemoattractant and placed in the lower chamber. After 24 h of incubation, cells at the lower side of the insert
were fixed, stained and counted. Data represent the mean value ꢂ SD of three independent experiments.
mM of BPs or Zoledronate were added to the upper chamber of Matrigel-coated inserts
m

62
M. Monteil et al. / European Journal of Medicinal Chemistry 77 (2014) 56e64
3
(200.7 MHz, D2O)
d
11.6. 1H NMR (500 MHz, D2O)
d
6.75e7.89 (m,
C6H4), 7.71 (d, 2H, JHeH ¼ 8.5 Hz, o-C6H4). 13C NMR {1H}
15H, C6H5). 13C NMR {1H} (50.3 MHz, D2O)
d
76.9 (t, 1JPeC ¼ 143.1 Hz,
(125.9 MHz, D2O)
d
77.6 (dd, 1JPeC ¼ 142.3 Hz, 1JPeC ¼ 142.3 Hz, Pe
PeC(OH)eP), 119.9e128.6 (C6H5; C6H5C(OH)), 136.8 (C6H5C(OH)),
150.7 (C6H5O). Anal. Calcd for C19H16Na2O7P2: C, 49.15; H, 3.47; P,
13.34; Found: C, 49.22; H, 3.48; P, 13.39.
C(OH)eP), 122.1 (C6H5), 122.4 (C6H5), 125.4 (p-C6H4), 129.4 (C6H5),
130.7 (m-C6H4), 132.2 (o-C6H4), 136.9 (C6H4C(OH)), 152.5 (OeC6H5).
Anal. Calcd for C13H13BrO7P2: C, 36.90; H, 3.10; P, 14.64; Found: C,
37.00; H, 3.12; P, 14.58.
6.1.2.3. [(4-Bromo-phenyl)-hydroxy-phosphono-methyl]-phosphonic
acid (2). Precipitation in diethylether. Yield: 90%. M.p. 210 ꢁC. 31P
6.2. Chemical material
NMR {1H} (200.7 MHz, D2O)
d d 7.54
16.4. 1H NMR (500 MHz, D2O)
(d, 2H, 3JHeH ¼ 8.6 Hz, m-C6H4), 7.65 (d, 2H, 3JHeH ¼ 8.6 Hz, o-C6H4).
3-isobutyl-1-methylxanthine (IBMX) from Sigma (France) was
dissolved in DMSO at a 500-fold concentration of the final con-
centration. The concentration of IBMX used was always kept at
0.2%.
1
13C NMR {1H} (125.9 MHz, D2O)
d
75.6 (t, JPeC ¼ 145.3 Hz, Pe
C(OH)eP), 123.3 (p-C6H4), 130.5 (m-C6H4), 132.9 (o-C6H4), 140.7
(C6H4C(OH)).
6.1.2.4. [(4-Bromo-phenyl)-hydroxy-(hydroxy-phenoxy-phosphoryl)-
methyl]-phosphonic acid monophenyl ester (2a). Precipitation in
diethylether. Yield: 70%. M.p. 92 ꢁC. 31P NMR {1H} (200.7 MHz, D2O)
6.3. NMR spectra of 2a in different media
31P NMR spectra of the diesterified bisphosphonate 2a in the
different media were obtained on a Bruker Avance 400 spectrom-
eter operating at 161.943 MHz phosphorus frequencies at 20 ꢁC. The
data were recorded with a spectral width of 50 ppm and 8 K data
point 50,000 scans were collected for each FID. The 31P signals were
d
12.8. 1H NMR (500 MHz, D2O)
NMR (50.3 MHz, DMSO-d6)
d C
6.93e7.80 (m,14H, C6H4, C6H5). 13
d
77.1 (t, 1JPeC ¼ 145.4 Hz, PeC(OH)eP),
116.1 (C6H5), 121.5 (C6H5), 124.6 (p-C6H4), 129.9 (C6H5), 130.7 (m-
C6H4), 130.8 (o-C6H4), 137.5 (C6H4C(OH)), 152.3 (OeC6H5). Anal.
Calcd for C19H17BrO7P2: C, 45.72; H, 3.43; P, 12.41; Found: C, 45.62;
H, 3.42; P, 12.36.
referenced to an external standard of 85% phosphoric acid. For 31
P
NMR experiment, the cellular extracts were prepared. HuH7 cells
(5 ꢃ 106 cells/mL) were rinsed, trypsinized. The lysate was centri-
fuged at 1500 rpm for 5 min at 4 ꢁC, and the pellet was resuspended
in cold isotonic mixture of HEPES buffer. The sample vial was kept
in an ice-water bath. This step is necessary to prevent significant
heating in the sample during sonication (3 ꢃ 30 s). Bisphosphonate
2a was added to the solution. After 5 days, 0.6 mL of solution was
transferred to a 5 mm NMR tube, which was then placed in the
magnet.
6.1.2.5. [(4-Bromo-phenyl)-hydroxy-(hydroxy-methoxy-phosphoryl)-
methyl]-phosphonic acid monomethyl ester (2b). Precipitation in
diethylether. Yield: 90%. M.p. 165 ꢁC. 31P NMR {1H} (200.7 MHz,
3
D2O)
d
17.3. 1H NMR (500 MHz, D2O)
d
3.79 (d, 3H, JPeH ¼ 3.0 Hz,
OCH3), 3.81 (d, 3H, 3JPeH ¼ 3.0 Hz, OCH3), 7.59 (d, 2H, 3JHeH ¼ 8.0 Hz,
m-C6H4), 7.66 (d, 2H, JHeH ¼ 8.0 Hz, o-C6H4). 13C NMR {1H}
3
(125.9 MHz, D2O)
d
56.8 (OCH3), 74.6 (t, 1JPeC ¼ 146.2 Hz, PeC(OH)e
P), 124.4 (p-C6H4), 131.1 (m-C6H4), 134.3 (o-C6H4), 138.9
(C6H4C(OH)). Anal. Calcd for C9H13BrO7P2: C, 28.82; H, 3.49; P,
16.52; Found: C, 28.92; H, 3.50; P, 16.57.
6.4. Cell line and cell culture
HuH7 hepatocarcinoma cell lines were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% calf
serum at 37 ꢁC in 5% CO2 and 95% room air. Prior to the experi-
ments, cells were cultured in the same seeding and growth con-
ditions so as to achieve comparable growth behaviour.
6.1.3. General procedure for synthesis of bisphosphonate 2c
p-Bromobenzoyl acid chloride (50 mmol) was added dropwise
at ꢀ10 ꢁC under argon to trimethylphosphite (5.9 mL, 50 mmol).
The reaction mixture was then stirred at room temperature for 2 h
(the end of the reaction was controlled by 31P {1H} NMR or IR
spectroscopy). The crude product was purified by extraction with
6.5. Cell viability experiments
diethylether to furnish the corresponding
a-ketophosphonate
dimethyl ester (31P {1H} NMR (CDCl3) ¼ 0.4 ppm).
Cell viability was evaluated using the MTT microculture tetra-
zolium assay. HuH7 cells were seeded at a density of 5x104 cells/
well in 96-well flat-bottom plates (Falcon, Strasbourg, France) and
incubated in complete culture medium for 24 h. Then, medium was
removed and replaced by 2% FCS (Feotal Calf Serum)-medium
containing increasing concentrations of BP (10ꢀ6e10ꢀ3 M). After
144 h incubation, HuH7 cells were washed with phosphate buff-
ered saline (PBS, Life Technologies) and incubated with 0.1 mL of
MTT (2 mg/mL, SigmaeAldrich) for additional 4 h at 37 ꢁC. The
insoluble product was then dissolved by addition of DMSO (Sigmae
Aldrich). Optical density was measured at 570 nm using a Labsys-
tems Multiskan MS microplate reader.
To
a-ketophosphonate dimethyl ester (5 mmol) in 4 mL of
distilled THF or dichloromethane at 0 ꢁC under argon was added
dropwise trimethylsilyl bromide (1.65 mL, 12.5 mmol). The reaction
was exothermic and the temperature had to be maintained below
10 ꢁC during the addition. The reaction mixture was stirred at room
temperature for 5e6 h (the end of the reaction was controlled by
31P {1H} NMR) and evaporation of volatile fractions (0.01 Torr) at
50 ꢁC gave bis(silylated)
trimethylsilyl) phosphite (5 mmol) was then added dropwise at
ꢁC under argon. The reaction mixture was stirred overnight at
a-ketophosphonate. Phenyl bis(-
0
room temperature and methanolysis for 2 h led to bisphosphonate
monophenyl ester 2c. After reduced pressure evaporation of vola-
tile fractions, 2c was purified by precipitation in a mixture of
diethylether and hexane.
For IBMX experiments, a pretreatment (1 mM) of the cells was
carried out for 30 min. The cells were then incubated with IBMX
(1 mM) and the BPs, for 144 h at 37 ꢁC in a 5% CO2-incubator.
[(4-Bromo-phenyl)-hydroxy-(hydroxy-phenoxy-phosphoryl)-
methyl]-phosphonic acid (2c). Precipitation in diethylether/hexane:
6.6. Cell death experiment
90/10. Yield: 64%. Decomposition 78 ꢁC. 31P NMR {1H} (200.7 MHz,
2
D2O)
d
12.1 (d, 1P, JPeP ¼ 28.6 Hz, P(O)(OH)(OeC6H5), 17.3 (d, 1P,
Apoptosis was analysed using the Annexin V-PI kit (R&D)
following the manufacturer’s instructions. HuH7 cells were treated
with BPs for 144 h. Briefly, both BP-treated and untreated cells were
stained with Annexin V and PI. Early and late apoptosis were
2JPeP ¼ 28.6 Hz, P(O)(OH)2). 1H NMR (500 MHz, D2O)
d 6.91 (d, 2H,
3JHeH ¼ 7.5 Hz, o-C6H5), 7.12 (dd, 1H, JHeH ¼ 7.5 Hz, p-C6H5), 7.27
3
3
3
(dd, 2H, JHeH ¼ 7.5 Hz, m-C6H5), 7.56 (d, 2H, JHeH ¼ 8.5 Hz, m-

M. Monteil et al. / European Journal of Medicinal Chemistry 77 (2014) 56e64
63
determined by Ann Vþ/PIꢀ and Ann Vþ/PIþ staining, respectively, as
determined by flow cytometric analysis with FACS.
[12] E. Guenin, D. Ledoux, O. Oudar, M. Lecouvey, M. Kraemer, Structure-ac-
tivity relationships of a new class of aromatic bisphosphonates that inhibit
tumor cell proliferation in vitro, Anticancer Research 25 (2005) 1139e
1145.
[13] Y. Hamma-Kourbali, M. Di Benedetto, D. Ledoux, O. Oudar, Y. Leroux,
M. Lecouvey, M. Kraemer, A novel non-containing-nitrogen bisphosphonate
inhibits both in vitro and in vivo angiogenesis, Biochemical and Biophysical
Research Communications 310 (2003) 816e823.
[14] V. Stresing, F. Daubiné, I. Benzaid, H. Mönkkönen, P. Clézardin, Bisphospho-
nates in cancer therapy, Cancer Letters 257 (2007) 16e35.
[15] A. Ezra, G. Golomb, Administration routes and delivery systems of
bisphosphonates for the treatment of bone resorption, Advanced Drug De-
livery Reviews 42 (2000) 175e195.
[16] F. Benyettou, Y. Lalatonne, I. Chebbi, M. Di Benedetto, J.M. Serfaty,
M. Lecouvey, L. Motte, A multimodal magnetic resonance imaging nanoplat-
form for cancer theranostics, Physical Chemistry Chemical Physics 13 (2011)
10020e10027.
[17] I. Chebbi, E. Migianu-Griffoni, O. Sainte-Catherine, M. Lecouvey, O. Seksek,
In vitro assessment of liposomal neridronate on MDA-MB-231 human
breast cancer cells, International Journal of Pharmaceutics 383 (2010) 116e
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[18] K. Hochdorffer, K. Abu Ajaj, C. Schafer-Obodozie, F. Kratz, Development of
novel bisphosphonate prodrugs of doxorubicin for targeting bone metastases
that are cleaved pH dependently or by cathepsin B: synthesis, cleavage
properties, and binding properties to hydroxyapatite as well as bone matrix,
Journal of Medicinal Chemistry 55 (2012) 7502e7515.
6.7. Cell migration assay
Cell migration experiments were performed using migration
chambers (Becton Dickinson), as described above. The undersides
of the inserts were coated with 100 mL of fibronectin (100 mg/mL,
Santa Cruz Biotechnology, Santa Cruz, CA, USA) and were allowed
to stand overnight at 4 ꢁC. HGF (R&D) chemoattractant for HuH7
cells was added (20 ng/mL) to the lower chamber. HuH7 cells
(2.5 ꢃ 105) with 100
mM of BPs or zoledronate were added to each
insert (upper chamber). After 24 h incubation, non-migrated cells
were removed by scraping and cells on the lower filter face were
fixed, stained and counted. Results were expressed as a percentage,
relative to controls normalised to 100%. Experiments were per-
formed in triplicate.
6.8. Cell invasion assay
[19] M.R. Webster, M. Zhao, M.A. Rudek, C.L. Hann, C.L. Freel Meyers, Bisphos-
phonamidate clodronate prodrug exhibits potent anticancer activity in non-
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G. Weiss, D. Stepensky, I. Gati, H. Cohen, S. Tormalehto, G.L. Amidon,
G. Golomb, A peptide prodrug approach for improving bisphosphonate oral
absorption, Journal of Medicinal Chemistry 43 (2000) 3641e3652.
[21] J.R. Atack, A.M. Prior, S.R. Fletcher, K. Quirk, R. McKernan, C.I. Ragan, Effects of
L-690,488, a prodrug of the bisphosphonate inositol monophosphatase in-
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(2002) 1201e1208.
HuH7 cell invasion was investigated using Boyden invasion
chambers with 8 mm pore size filters coated with Matrigel mem-
brane matrix (Falcon, Becton Dickinson Labware, Bedford, MA,
USA). 2.5 ꢃ 105 HuH7 cells with 100
mM of BPs or zoledronate were
seeded in the upper well of the Boyden chamber. HGF (R&D) was
added (20 ng/mL) to the lower chamber. After 24 h, non-migrated
cells in the upper chamber were wiped with a cotton swab and
the filters were fixed in methanol and stained with haematoxylin.
Cells invading the lower surface of the filter were counted in 10-
fields using a Zeiss microscope. Results were expressed as a per-
centage relative to controls and therefore normalised to 100%. Ex-
periments were performed in triplicate.
[24] M. Monteil, E. Guenin, E. Migianu, D. Lutomski, M. Lecouvey, Bisphosphonate
prodrugs: synthesis of new aromatic and aliphatic 1-hydroxy-1,1-
bisphosphonate partial esters, Tetrahedron 61 (2005) 7528e7537.
[25] E. Guenin, M. Monteil, N. Bouchemal, T. Prange, M. Lecouvey, Syntheses of
phosphonic esters of alendronate, pamidronate and neridronate, European
Journal of Organic Chemistry (2007) 3380e3391.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.054.
[26] D. Ledoux, Y. Hamma-Kourbali, M. Di Benedetto, A. Foucault-Bertaud,
O. Oudar, O. Sainte-Catherine, M. Lecouvey, M. Kraemer, A new dimethyl ester
bisphosphonate inhibits angiogenesis and growth of human epidermoid
carcinoma xenograft in nude mice, Anticancer Drugs 17 (2006) 479e485.
[27] M. Abdelkarim, E. Guenin, O. Sainte-Catherine, N. Vintonenko, N. Peyri,
G.Y. Perret, M. Crepin, A.M. Khatib, M. Lecouvey, M. Di Benedetto, New
symmetrically esterified m-bromobenzyl non-aminobisphosphonates inhibi-
ted breast cancer growth and metastases, PLoS One 4 (2009) e4685.
[28] R.G. Simonetti, C. Camma, F. Fiorello, F. Politi, G. D’Amico, L. Pagliaro, Hepa-
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