Bisphosphonamidate clodronate prodrug exhibits potent anticancer activity in non-small-cell lung cancer cells

Article abstract of DOI:10.1021/jm200521a

Bisphoshonates are used clinically to treat disorders of calcium metabolism, hypercalcemia and osteoporosis, and malignant bone disease. Although these agents are commonly used in cancer patients and have potential direct anticancer effects, their use for

Full text of DOI:10.1021/jm200521a

ARTICLE
pubs.acs.org/jmc
Bisphosphonamidate Clodronate Prodrug Exhibits Potent Anticancer
Activity in Non-Small-Cell Lung Cancer Cells
Marie R. Webster,† Ming Zhao,‡ Michelle A. Rudek,‡ Christine L. Hann,§ and Caren L. Freel Meyers*,†
†Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, WBSB 305,
Baltimore, Maryland 21201, United States
‡Analytical Pharmacology Core of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine,
Baltimore, Maryland, United States
§Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
S Supporting Information
b
ABSTRACT:
Bisphoshonates are used clinically to treat disorders of calcium metabolism, hypercalcemia and osteoporosis, and malignant bone
disease. Although these agents are commonly used in cancer patients and have potential direct anticancer effects, their use for the
treatment of extraskeletal disease is limited as a result of poor cellular uptake. We have designed and synthesized bispho-
sphonamidate prodrugs that undergo intracellular activation to release the corresponding bisphosphonate and require only two
enzymatic activation events to unmask multiple negative charges. We demonstrate efficient bisphosphonamidate activation and
significant enhancement in anticancer activity of two bisphosphonamidate prodrugs in vitro compared to the parent bispho-
sphonate. These data suggest a novel approach to optimizing the anticancer activities of commonly used bisphosphonates.
’ INTRODUCTION
Clinically used bisphosphonates (BPs) are stable analogues of
naturally occurring pyrophosphate.1,2 BPs are known to inhibit
cancer cell adhesion and invasion and to inhibit the growth of
cancer cells in the bone microenvironment.3,4 The two bispho-
sphonate classes, nitrogen-containing (NBP) and non-nitrogen-
containing (NNBP), are distinguished structurally by the sub-
stitution pattern at the bridging methylene of the PꢀCꢀP
linkage (Figure 1). The NBP class incorporates a nitrogen-contain-
ing substituent at the bridging methylene (e.g., zoledronate,
aledronate, pamidronate), whereas the NNBP class lacks this
nitrogen-containing substituent (e.g., clodronate, etidronate).
These BP classes are further distinguished by differences in
mechanism of action. The NBP class inhibits an essential enzyme
in isoprenoid biosynthesis, farnesyl pyrophosphate synthase
(FPPS), leading to lower FPP levels and subsequent reduction
in downstream protein prenylation events in osteoclasts and
malignant bone cells. Recent reports suggest that increased levels
of the FPPS substrate IPP, caused by inhibition of FPPS by
NBPs, promote formation of AppIPP (triphosphoric acid 1-ade-
nosine-50-yl ester 3-(3-methylbut-3-enyl) ester), which is be-
lieved to induce apoptosis.5 In contrast, NNBPs undergo conversion
to the corresponding nonhydrolyzable ATP analogues. Clodronate is
Figure 1. Structures of nitrogen-containing bisphosphonates (NBPs)
and non-nitrogen-containing bisphosphonates (NNBPs).
metabolized to the ATP analogue AppCCl2p (adenosine 50-β-γ-
dichloromethylene triphosphosphate), which is believed to be
the active metabolite responsible for the apoptotic activity of
clodronate observed in osteoclasts and malignant bone cells.6,7
Further, AppCCl2p was shown to inhibit mitochondrial meta-
bolism through inhibition of ADP/ATP translocase, and it is
Received: April 28, 2011
Published: August 24, 2011
r
2011 American Chemical Society
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Scheme 1. Proposed Intracellular Bioreductive Activation of Bisphosphonamidate Prodrugs (R = H or Cl)
conceivable that additional targets are susceptible to inhibition
by AppCCl2p.8
to partially unmasked, impermeable intermediates, which are
often inefficiently converted to the fully unmasked BP.20 There
are no such prodrug strategies reported for BPs bearing the
tertiary hydroxyl group at the bridging methylene position, includ-
ing the NBP class, owing to the intrinsic instability of these
compounds when masked as tetraesters.23 Other strategies to
increase BP cell permeability have focused on introducing modi-
fications at the bridging methylene of the PꢀCꢀP linkage to
increase hydrophobicity. Such modifications have also been shown
to impart changes in target specificity.24ꢀ26 Increasing hydro-
phobicity of substituents at the bridging methylene group does
not overcome low membrane permeability entirely, as phospho-
nate masking strategies have been employed in these cases as
well.22,27
Here we report a novel bisphosphonamidate prodrug strategy
for the intracellular release of bisphosphonates and demonstrate
its application to bisphosphonic acid and the clinically used
NNBP clodronate. Our results suggest bisphosphonamidate pro-
drugs undergo rapid activation to release the corresponding BP
following reductive activation of nitroaryl delivery groups.
Further, we demonstrate intracellular conversion of a bispho-
sphonamidate prodrug to the corresponding bisphosphonate
and a remarkable enhancement in anticancer activity of two
bisphosphonamidate prodrugs compared to the parent bispho-
sphonates in A549 cells.
Skeletal-related events (SKE) such as fracture, spinal cord
compression, and hypercalcemia are common and cause sig-
nificant morbidity in cancer patients with bone metastases.9
Bisphosphonate therapy has been shown to reduce the rate of
SKE in several clinical trials, leading to its use as a standard
adjunct therapy in patients with bone metastases. The clinical
success of NBPs in the prevention and management of bone
metastatases has led to the evaluation of BPs as potential
therapeutic agents for the treatment of cancer in soft tissues.1
The NBP zoledronate (6, Figure 1) is a commonly used BP in
metastatic bone disease and exhibits varying anticancer activities
with IC50 ranging from 3 to >100 μM in several cancer cell
lines.1,10,11 The cytotoxic effects of zoledronate in cancer cells are
believed to be exerted through a variety of mechanisms, includ-
ing blockage of cell cycle in models of non-small-cell lung
cancer,12 inhibition of angiogenesis,13ꢀ16 and induction of apo-
ptosis in small-cell lung cancer cell lines,11 although the molecular
mechanisms beyond inhibition of FPPS are not well-understood.
NNBPs, including clodronate (2, Figure 1), are significantly
less potent anticancer agents,17 exhibiting growth inhibition in
the high micromolar or low millimolar range in breast and ovarian
cancers10 and minimal activity against lung cancer cell lines.10
The anticancer activity of clodronate is thought to correlate with
formation of AppCCl2p in breast, prostate, and myeloma cells;18
however, the molecular mechanisms underlying the anticancer
effects of clodronate are not well-understood.
’ RESULTS
BPs are polyanionic at physiologic pH and are consequently
concentrated in the mineralized bone matrix.19 While beneficial
for the treatment of bone disorders, this structural characteristic
of BPs precludes efficient uptake into extraskeletal tumor cells.
The low cellular uptake of BPs presents a critical barrier both for
the development of these agents to treat tumors in soft tissues
and for studies to elucidate the intracellular mechanisms by
which BPs exert antitumor effects.
Existing strategies to increase bioavailability of NNBPs such as
clodronate have involved masking of the BP scaffold with
biodegradable or chemically labile groups designed to release
the corresponding BP through nonspecific esterase activation or
chemical hydrolysis postintestinal absorption.20ꢀ22 These prodrugs
generally undergo rapid extracellular bioactivation in serum, leading
Design and Synthesis of Bisphosphonamidate Prodrugs.
We have designed a prodrug strategy to enhance membrane
permeability of bisphosphonates through incorporation of two
biodegradable nitroaryl delivery groups and two halobutylamine
masking groups, which effectively mask the polyanionic charges
(Scheme 1). The bisphosphonate design shown here extends the
halobutyl phosphoramidate prodrug strategy developed for the
intracellular delivery of nucleotides.28
Membrane permeable bisphosphonamidate prodrugs (7) are
designed to undergo rapid intracellular bioreduction to produce
the corresponding hydroxylamine 8 (Scheme 1), which under-
goes elimination through the aromatic ring and expulsion of pho-
sphonamidate anion 9. The resulting increase in electron density
of the phosphonamide nitrogen atom facilitates a cyclization
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Scheme 2. Synthesis of Prodrugs 14 and 15a
a Reaction conditions: (a) N-methyl-N-(4-chlorobutyl)amine hydro-
chloride, DIPEA, CH2Cl2, 0 °C; (b) nitrobenzyl alochol, DIPEA,
DMAP, room temp; (c) NaOCl, benzyltriethyl ammonium chloride,
CCl4, MeOH.
reaction to produce the corresponding zwitterionic intermediate
10. Subsequent rapid PꢀN bond hydrolysis affords the un-
masked phosphonate 11 in a manner similar to nucleotide
release.28,29 The release of the second phosphonyl group is
achieved in the same manner to give the fully unmasked bis-
phosphate 12. Although it is difficult to predict the kinetics of
activation, efficient release of the fully unmasked bisphosphonate
is expected. An advantage of this strategy over existing bispho-
sphonate prodrug strategies is the requirement of a minimal
number of enzymatic bioactivation steps to unmask multiple
negative charges. As with the chemical deprotection of phos-
phoryl ester groups, removal of the first protective group is most
often rapid while removal of the second masking group is
considerably slower as a result of increased electron density at
the phosphoryl leaving group and a slower rate of elimination.30
The prodrug design incorporates a single nitroaryl delivery group
at each phosphonyl group that is susceptible to rapid intracellular
enzymatic activation by nitroreduction. The subsequent activa-
tion steps to release the fully unmasked bisphosphonate rely only
upon the intrinsic chemical reactivity of the enzymatically reduced
bisphosphonamidate 8 rather than subsequent, inefficient enzy-
matic activation events.
Bisphosphonamidate prodrugs were synthesized as diastereo-
meric mixtures (Scheme 2). Des-chloro bisphosphonamidate 14
was prepared via a two-step, one-pot synthesis. N-Methyl-N-
chlorobutylamine hydrochloride was prepared as previously
described.31 Coupling of N-methyl-N-chlorobutylamine substit-
uents to methylenebis(phosphonic dichloride) in the presence of
DIPEA was monitored using 31P NMR. In a representative
synthesis, complete conversion to intermediate 13 was confirmed
by the disappearance of the starting methylenebis(phosphonic
dichloride) at δ ꢀ1.53 ppm (relative to TPPO) and appearance
of two 31P resonances at δ 6.17 and 6.10 ppm (1.7:1), indicating
the formation of phosphonamidate dichloride 13 as a diastere-
omeric mixture. Treatment of 13 with nitrobenzyl alcohol in the
presence of DIPEA and a nucleophilic catalyst (DMAP) afforded
bisphosphonamidate prodrug 14 as a diastereomeric mixture (δ 0.84
and 0.81 ppm, 1.3:1), along with apparent hydrolysis product
(∼20%). Clodronate prodrug 15 was prepared by chlorination of
Figure 2. Reductive activation of BP prodrug 14: (a) prodrug 14 prior
to reduction; (b) following addition of dithionite to 14; (c) at 4 h;
(d) following addition of authentic 1 to reaction mixture shown in (c).
bisphosphonamidate 14. In a representative synthesis, bispho-
sphonamidate 14 (1.5:1 mixture of diastereomers) was treated
with sodium hypochlorite, resulting in a new set of 31P reso-
nances corresponding to a diaseteromeric mixture of clodronate
prodrug 15 (ꢀ9.80 and ꢀ9.98 ppm, 1:1.6).
Confirmation of Bisphosphonamidate Activation. To con-
firm the release of a fully unmasked bisphosphonate following
reduction of the bisphosphonamidate nitrobenzyl ester, bispho-
sphonamidate prodrug 14 was subjected to chemical reduction
by dithionite under model physiologic conditions, and the
subsequent reactions were monitored using 31P NMR (Figure 2).
Upon solubilization and reduction of 14, a resonance appearing
at +2 ppm (relative to TPPO) was observed in the 31P NMR
spectrum (Figure 2b). Conversion of this peak to a new reso-
nance at ꢀ8.5 ppm (Figure 2c) was observed and is consistent
with elimination and release of the bisphosphonate 1. Confirma-
tion of bisphosphonate release was accomplished by comparison
to authentic bisphosphonate 1 (Figure 2d).
Inhibition of Cell Proliferation by Bisphosphonate Pro-
drugs. Clodronate has shown little or no antiproliferative effects
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Figure 3. Effect of bisphosphonate prodrug 14 and clodronate prodrug 15 on proliferation of A549 cells, determined using the MTS assay. Results are
displayed as % of control. (A) Bisphosphonate prodrug 14 (9) shows an enhanced antiproliferative effect compared to bisphosphate (b) at 72 h. (B)
Clodronate prodrug 15 (9) shows an enhanced antiproliferative effect compared to clodronate (b). (C) Effect of 14 at 24 h (0), 48 h (2), and 72 h (O).
(D) Effect of 15 at 24 h (0), 48 h (2), and 72 h (O).
Table 1. In Vitro Effects of 14 and 15 on A549 NSCLC Cells
Permeability and Intracellular Activation of Prodrug 14.
The enhanced activity of prodrugs 14 and 15 against A549 cells
(Figure 3) is consistent with increased cell permeability of these
analogues. To provide further evidence that bisphosphonami-
dates are cell permeable, intracellular levels of prodrug 14 were
measured in A549 cells treated with prodrug at varying concen-
trations (0, 10, or 30 μM) for 8 h. As expected, LCꢀMSꢀMS
analysis of cell lysate in each case indicates that reasonably high
intracellular prodrug levels are achieved in a concentration-
dependent manner, with 68.6 and 169.3 ng/104 cells 14 detected
in cells treated with 10 and 30 μM 14, respectively (Figure 4A).
Bisphosphonamidate 14 is designed to undergo intracellular
activation to release the corresponding bisphosphonate 1. Direct
detection of intracellular bisphosphonate by mass spectrometry
is difficult, as these highly polar compounds have low ionization
efficiency. Thus, chemicalderivatizationofbisphosphonate released
intracellularly was carried out in an effort to provide evidence for
intracellular prodrug activation. N-tert-Butyldimethylsilyl-N-
methyltrifluoroacetamide (MTBSTFA) has been used success-
fully to derivatize polar functional groups, including carboxylic
acids, for mass spectrometry analysis.32,33 MTBSTFA has also
been used as a derivatizing agent for the detection of phospho-
nate-bearing 2-(phosphonomethyl)pentanedioic acid (2-PMPA).34
Thus, we have used MTBSTFA as derivatization agent for the
qualitative detection of 1 released from prodrug 14 intracel-
lularly. The presence of intracellular bisphosphonate 1 in
A549 cells treated for 8 h with prodrug 14 (10 or 30 μM) or
bisphosphonate 1 (30 μM) was confirmed by detection of the
corresponding tetrasilylated derivative 18 formed following
treatment of cell lysate with MTBSTFA. Indeed, tetrasilyl
bisphosphonate 18 was detected in A549 cells treated with
14
EC50 (μM)b
15
EC50 (μM)b
time (h)
IC50 (μM)a
IC50 (μM)a
24
48
72
17 ( 4
15 ( 0.4
13 ( 1
ndc
7.6 ( 3
5.2 ( 1
4.4 ( 2
ndc
24 ( 4
25 ( 4
19 ( 4
16 ( 1
a IC50 is concentration of 14 or 15 that decreased cell proliferation by
50% compared to control. b EC50 is the concentration of 14 or 15 that
decreased cell number by 50% compared to control. The viability of
c
A549 cells did not decrease by 50% after 24 h of treatment with 14 or 15.
against multiple cells lines, in part because of low membrane
permeability. Bisphosphonates 14 and 15 are fully masked and
are expected to have increased cell permeability. As predicted,
both 14 and 15 exhibit potent activity against A549 NSCLC cells
in vitro (Figure 3) as determined by the MTS cell proliferation
assay. Doseꢀresponse curves were generated using drug con-
centrations ranging from 0.2 to 300 μM, and cell proliferation
was measured at 24, 48, and 72 h after drug treatment (Figure 3,
Table 1). Consistent with published reports,10 clodronate does
not exhibit a detectable growth inhibitory effect at concentra-
tions up to 1 mM against the growth of A549 cells. In contrast,
clodronate prodrug 15 and bisphosphonate prodrug 14 exhibit
remarkably enhanced activity with IC50 values of 4.4 ( 2 and
13 ( 1 μM, respectively, at 72 h (Figure 3A,B). While it is possible
the diastereomers of 14 and 15 may be activated at different rates,
they release the same achiral bisphosphonate, and comparable
antiproliferative activities are observed with different diastereo-
meric mixtures (data not shown).
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Figure 5. Effect of 15 on cell viability of NSCLC cells. Cell number was
determined using trypan blue. (A) Decrease in number of NSCLC cells
by 15 at 24 (0), 48 (2), 72 (O) h. (B) Crystal violet staining of A549
NSCLC cells treated with 15 for 72 h.
determined at 0, 24, 48, and 72 h following drug treatment and
plotted as a percent of control (Figure 5A). The EC50 of 15
determined at 48 h is 19 ( 4 μM, ∼3.6-fold higher than its IC50
determined by the MTS assay (Table 1). A similar difference in
the effects of 15 on cell viability versus inhibition of proliferation
was observed after a 72 h drug treatment. Interestingly, at 24 h,
cell viability was reduced by only 57% at the highest drug con-
centration tested; therefore, an EC50 was not determined. In
parallel, viable cells were stained with crystal violet at 72 h
following drug treatment to further demonstrate the marked
reduction in cell viability upon treatment with clodronate
prodrug 15 (Figure 5B). In order to confirm this reduction in
cell viability, cells treated with 15 for 72 h (at 10, 30, 100, and
300 μM) were washed to remove the prodrug and fresh
medium without drug was added. Cells were then allowed
to grow for an additional 48 h. No new cell growth was
observed in wells containing cells treated with 15 (data not
shown). Prodrug 14 affects cell viability in a similar manner
with an EC50 at 48 h that is ∼1.6-fold greater than its IC50
determined using the MTS assay.
Cell Cycle. PI and flow cytometry analysis were performed to
study the effects of prodrug 15 on cell cycle. Cell cycle analysis of
cells treated with 0, 3, 10, or 30 μM 15 for 72 h suggested cell
cycle arrest with low micromolar concentrations of 15. Unequi-
vocal support of G1 cell cycle arrest was obtained through flow
cytometry analysis of cells treated with 0, 3, or 10 μM 15 for 72 h
and with nocodazole treatment for the last 24 h of prodrug
exposure. Nocodazole is a microtubulin polymerization inhibi-
tor, known to cause G2 cell cycle arrest. In the absence of prodrug
15, nocadazole-induced G2 arrest is evident (Figure 6B). How-
ever, in the presence of prodrug, a marked accumulation of cells
in G1 is observed as low as 3 μM, indicating G1 cell cycle arrest
caused by prodrug 15.
Figure 4. Prodrug 14 is cell permeable (A) and undergoes intracellular
activation in A549 cells to release bisphosphonate 1 (B). (A) Ion
chromatograms showing absence of prodrug 14 in untreated cells
(a) and detection of 14 (68.6 ng/104 cells) in cells treated with 10 μM
14 (b) and cells treated with 30 μM 14 (169.3 ng/104 cells) (c). (B) Ion
chromatograms showing absence of tetrasilyl bisphosphonate 18 follow-
ing MTBSTFA reaction of lysate from untreated cells (d) and detection
of 18 in cells treated with 30 μM bisphosphonate 1 (e), cells treated with
10 μM prodrug 14 (f), and cells treated with 30 μM prodrug 14 (g). Ion
chromatograms are offset in the y-dimension for clarity.
bisphosphonamidate 14 and at considerably lower levels in
cells treated with free bisphosphonate 1 at a comparable con-
centration (Figure 4B).
Cell Viability. In order to correlate the growth inhibitory
effects of 14 and 15 with cell viability, absolute cell number at
varying drug concentration was determined using a trypan blue
exclusion assay. The number of viable cells at each dose was
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Figure 6. Effect of clodronate prodrug 15 on cell cycle, determined using the PI and FACS analysis. (A) A549 cells treated with 0, 3, and 10 μM 15 for
72 h. (B) A549 cells treated with 0, 3, and 10 μM 15 for 72 h and nocodazole for the final 24 h. In the nocodazole-treated cells, accumulation of cells in the
G1 phase is evident at 3 and 10 μM 15.
’ DISCUSSION AND CONCLUSIONS
is presumably due to increased cell permeability of the prodrug,
which allows for a substantial increase in intracellular bispho-
sphonate concentration. The LCꢀMSꢀMS detection of reason-
ably high intracellular prodrug concentrations and the observation
that bisphosphonate is released intracellularly support this idea.
However, cytotoxicity of the prodrug itself cannot be ruled out as a
contributing factor in the observed activity of these prodrugs. A
partial growth inhibitory effect of the clodronate prodrug on A549
cells is observed as early as 24 h, and complete growth inhibition by
the prodrug is observed by 48 and 72 h. The observed partial growth
inhibitory effect at 24 h may reflect the dependence upon prodrug
activation to release the corresponding bisphosphonate, the con-
version of bisphosphonate to its active metabolite (AppCH2p or
AppCCl2p), and subsequent action of these metabolites on cellular
target(s). T4 RNA ligase has been shown to effectively convert
NNBPs to their ATP analogues.35 Both bisphosphonate and
clodronate are known to be substrates of T4 RNA ligase with
clodronate being converted to AppCCl2p more efficiently than the
corresponding conversion of the nonchlorinated bisphosphonate to
AppCH2p. On this basis, we anticipate that intracellular conversion
of bisphosphonate to AppCH2p is also less efficient than the
conversion of clodronate to AppCCl2p. The observed 3-fold
decrease in IC50 of 14 compared to 15 is consistent with this idea.
Detailed studies to determine the kinetics of intracellular prodrug
activation and clodronate metabolism are required to correlate these
events with observed growth in inhibitory activity.
The poor cellular uptake of bisphosphonates into soft tissues
has limited their use in the treatment of extraskeletal diseases and
detailed studies to elucidate the molecular mechanisms under-
lying the anticancer activity of this compound class. We sought to
develop a more efficient strategy for the intracellular delivery of
bisphosphonates in order to realize the potential of this clinically
used compound class for the treatment of extraskeletal tumors.
The bisphosphonamidate prodrugs described here are designed
to be more membrane permeable than the corresponding free
bisphosphonates and undergo efficient bioreductive activation to
release either bisphosphonate or clodronate intracellularly. A
bisphosphonamidate prodrug was rapidly activated to the corre-
sponding bisphosphonate under model physiological conditions
following chemical reduction of the nitroaryl delivery groups.
Formation of free bisphosphonate as the only product is con-
sistent with an activation mechanism that takes place via elim-
ination, cyclization, and PꢀN bond hydrolysis (Figure 2). As no
other bisphosphonate ester intermediates were observed in the
31P NMR spectrum, PꢀN bond hydrolysis prior to CꢀO cleavage
is an unlikely activation pathway.
In this study, clodronate and bisphosphonate exhibited mini-
mal activity against A549 cells up to 1 mM. The bisphosphona-
midate prodrugs described here exhibit >250-fold increase in
growth inhibitory activity against A549 cells, compared to the
free bisphosphonates. This remarkable enhancement in potency
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To provide further evidence for the anticancer activity of
bisphosphonamidate prodrugs 14 and 15, a complementary assay
was performed to evaluate the effects of the bisphosphonamidate
prodrugs on cell viability. Cell number was determined at each
dose at 24, 48, and 72 h. Interestingly, 15 decreases the viability of
A549 cells to just above 50% of control at higher doses over 24 h.
However, a more pronounced effect on cell proliferation was
observed at 24 h in the MTS assay. The difference between
antiproliferative activity and effects on cell viability at 24 h
suggests that 14 and 15 exert effects on metabolic activity, which
leads to cell death over time. Taken together with the observation
that somewhat higher prodrug concentrations are required for
effects oncellviability, theseresultsareconsistentwithamechanism
of action involving prodrug activation to the corresponding
bisphosphonate and subsequent conversion to the nonhydrolyz-
able ATP analogue.
The more potent clodronate prodrug 15 was selected for cell
cycle analysis studies. PI and flow cytometry analysis of A549
cells treated with the clodronate prodrug over 72 h suggested G1
cell cycle arrest of A549 cells at low concentrations of prodrug 15.
G1 cell cycle arrest was unequivocally confirmed in nocodazole-
treated cells where an obvious shift from nocodazole-induced G2
arrest to G1 arrest occurred in the low micromolar range of
prodrug 15. G1 cell cycle arrest correlates with the antiprolifera-
tive activity of this compound, suggesting that prodrug 15 affects
mitochondrial function, which leads to cell cycle arrest and
eventually cell death.
The low membrane permeability of bisphosphonates imposes
a significant barrier to the development of these agents for the
treatment of extraskeletal tumors. Clodronate displays varying
effects in different cancer cell types,10 with minimal activity against
lung cancer cells. Studies to investigate differences in the
mechanism of clodronate action that could account for these
differences are also impeded by poor cellular uptake. We have
developed a bisphosphoamidate prodrug strategy that signifi-
cantly enhances the membrane permeability of bisphosphonates
through incorporation of two biodegradable nitroaryl delivery
groups and two halobutyl amine masking groups. The use of only
two biodegradable delivery groups takes advantage of the most
efficient enzymatic activation steps and exploits the exquisite
reactivity of chemically labile halobutyl phosphonamidate anion
intermediates along the prodrug activation pathway for rapid
intracellular activation and release of the fully unmasked bispho-
sphonate. The remarkable enhancement of activity of bispho-
sphonamidate prodrugs in A549 cells compared to the parent
BPs highlights the potential utility of this approach to extend the
use of bisphosphonates beyond the treatment of skeletal diseases
and presents a potential tool for investigating bisphosphonate
mechanism of action.
and 1% sodium pyruvate. N-Methyl-N-(4-chlorobutyl)amine hydro-
chloride was synthesized as previously described.31 Methylenedipho-
sphonic acid, dichloromethylenediphosphonic acid disodium salt, and
nocodazole used in biological assays were obtained from commercial
sources. Methylenediphosphonic acid was determined to be >95% pure by
sodium hydroxide titration, determined by vendor. Dichloromethylenedi-
phosphonic acid was submitted, by the vendor, to elemental analysis:
calculated C (4.95%), found C (4.2%); the amount of water was deter-
mined to be 2% by the Karl Fischer method. Nocodazole was determined to
be g95% pure by reverse-phase HPLC. LCꢀMSꢀMS experiments were
conducted using an AB Sciex triple quadrapole TM 5500 mass spectro-
metric detector (Applied Biosystems, Foster City, CA, U.S.). The instru-
ment was equipped with an electrospray interface in positive ion mode and
controlled by the Analyst, version 1.2, software (Applied Biosystems).
4-Nitrobenzyl Methylenebis(N-4-chlorobutyl-N-methylp-
hosphonamidate) 14. Methylenebis(phosphonic dichloride) (0.466
g, 1.87 mmol) and N-methyl-N-(4-chlorobutyl)amine hydrochloride
(0.589 g, 3.73 mmol) were dissolved in CH2Cl2 (7.48 mL) and cooled to
0 °C with stirring under an Ar atmosphere. DIPEA (1.56 mL, 8.98
mmol) was added dropwise. The reaction mixture was allowed to warm
to room temperature, and stirring was continued for 2 h. Nitrobenzyl
alcohol (1.43 g, 9.35 mmol) was added to the reaction mixture in one
portion. In a separate flask under an Ar atmosphere, DMAP (0.228 mg,
1.87 mmol) was dissolved in CH2Cl2 (0.2 mL) and DIPEA (0.782 mL,
4.49 mmol), and the resulting mixture was added dropwise to the
reaction mixture at room temperature. The reaction mixture was stirred
at room temperature for 4 h. 31P NMR analysis of the crude reaction
mixture showed a 1.3:1 diastereomeric mixture. The mixture was washed
with saturated NH4Cl (1 ꢁ 2 mL). The organic layers were combined,
dried over NaSO4, and concentrated under reduced pressure. Removal
of impurities by flash chromatography (1:99, MeOH/ethyl acetate)
resulted in the isolation of a 1.9:1 diastereomeric mixture of 14 as a pale
yellow oil, in 32% yield. The purity of 14 was determined to be >95% by
HPLC (C18 Rocket column). The method was as follows: 100% water to
100% acetonitrile over 3 min, then 5 min at 100% acetonitrile; retention
time was 3.3 min. 31P NMR (CDCl3) δ 1.00 and 0.88 ppm (1.9:1
1
mixture); H NMR (CDCl3) δ 8.23 and 8.19 (d, 4H, 1:1.9 mixture);
7.61 and 7.54 (d, 4H, 1:1.9 mixture); 5.21 and 5.19 (m, 4H, 1:1.9
mixture); 4.94 (m, 2H); 3.56 (m, 4H); 3.25 and 3.15 (m, 2H, 1.9:1
mixture); 2.97 (m, 2H); 2.68 and 2.65 (d, 6H, 1.9:1 mixture); 2.51 and
2.41 (t, 2H, diastereotopic), 1.73 (m, 8H). HRMS: calcd for C25H37N4-
O8P2Cl2, m/z 653.1464 [M + H]+; found, 653.1467.
4-Nitrobenzyl Dichloromethylenebis(N-4-chlorobutyl-N-
methylphosphonamidate) 15. Bisphosphonamidate 14 (0.350 g,
0.536 mmol) was dissolved in CCl4 (1.2 mL) and MeOH (0.6 mL).
Benzyltriethylammonium chloride (0.054 g, 0.236 mmol) was added in
one portion. Then 10% NaOCl solution (1.8 mL) was added with
stirring. The reaction was monitored by 31P NMR over 4 h until
completion. 31P NMR analysis of the crude reaction mixture showed a
1:1.6 diastereomeric mixture of 15. The reaction mixture was quenched
with saturated NH4Cl solution (2 mL), and the product was extracted
using CH2Cl2 (2 ꢁ 0.5 mL). Removal of impurities by flash chroma-
tography (100% ethyl acetate to 5:95 MeOH/ethyl acetate) resulted in
the isolation of a 1:1 diastereomeric mixture of 15 as a pale yellow oil, in
60% yield. Purity of 15 was determined to be >95% by HPLC (Altima
C18 column, 250 mm ꢁ 4.6 mm i.d.). Method was as follows: 5:95
water/acetonitrile to 75% acetonitrile over 5 min, 75% acetonitrile for
5 min, 75ꢀ100% acetonitrile over5 min, then 100%acetonitrile for 5 min;
retention time was 15.2 min. Although a diastereomeric mixture of 15 was
observed by 31P NMR during reaction monitoring in methylene chloride as
the reaction solvent, the diastereomers of purified 15 appear identical by 31P
NMR in CDCl3. 31PNMR(CDCl3) δꢀ9.46 (s); 1HNMR(CDCl3) δ8.25
and 8.21 (d, 4H, 1:1 mixture); 7.65 and 7.62 (d, 4H, 1:1 mixture); 5.41 (m,
2H); 5.26 (m, 2H); 3.53 (m, 4H); 3.42 (m, 2H); 3.13 (m, 2H); 2.90 and 2.86
’ EXPERIMENTAL PROCEDURES
All 31P and 1H NMR spectra were acquired on a 400 MHz Varian or
Br€uker NMR. 31P shifts were recorded in parts per million and
referenced to triphenylphosphine oxide (TPPO) as the coaxial reference
in either benzene or benzene-d6. 1H chemical shifts are reported in parts
per million from tetramethylsilane. HMRS characterization of prodrugs
was carried out at the University of Illinois Mass Spectrometry Lab using
ES ionization. All reactions were carried out under argon unless
otherwise noted. Methylene chloride and diisopropylethylamine were
obtained from commercial sources and distilled prior to use. A549 cells
were maintainedin 1640 RMPIwith10%FBS, 1%pen/strep, 1%glutamate,
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Journal of Medicinal Chemistry
ARTICLE
(d, 6H, 1:1 mixture); 1.72(m, 8H). HRMS: calcd for C25H35N4O8P2Cl4, m/
z 721.0684 [M + H]+; found, 721.0682.
internal standard. The suspension was mixed vigorously for 1 min on a
vortex mixer and then subjected to centrifugation at 1200g for 10 min at
ambient temperature. A volume of 300 μL of the supernatant was
transferred to a glass test tube and evaporated to dryness under nitrogen
at 40 °C. Acetonitrile (100 μL) was added to the residue along with 100 μL
of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA).
The mixture was subjected to vortex-mixing prior to and every 15 min
during a 1 h incubation at 80 °C. After 1 h, the solution was cooled to
room temperature and diluted in acetonitrile (1:5, v/v) prior to injection
(10 μL) into the LCꢀMSꢀMS instrument using an autosampling
device operating at room temperature. Separation was achieved on a
Waters X-Terra C18 (50 mm ꢁ 2.1 mm i.d., 3 μm) at room temperature
using isocratic elution with acetonitrile/water mobile phase (70:30, v/v)
containing 0.1% formic acid at a flow rate of 0.2 mL/min. Detection of
tetrasilyl bisphosphonate 18 was performed using electrospray tandem
mass spectrometry operating in positive ion mode by monitoring the ion
transitions from m/z 633.4 to 617.0 (derivatized bisphosphonate 18)
and from m/z 683.0 to 551.4 (derivatized 2-PMPA).
Cell Cycle Analysis. Cell cycle distribution was determined using
flow cytometry. A549 NSCLC cells were plated at 6.7 ꢁ 104 cells per
well in flat bottom six-well plates. Cells were dosed as described above.
At 24, 48, or 72 h following drug treatment, the medium was collected
and the cells were washed with 1 mL of PBS. Each well was trypsanized
with 600 μL of trypsin for 3ꢀ5 min. The trypsin reaction was quenched
with an equal volume of medium. All supernatants and washes were
combined and centrifuged at 1500 rpm for 5 min. Supernantant was
decanted, and the cells were washed with 2 mL of 1% FBS/PBS. Cells
were centrifuged at 1500 rpm for 5 min, and the supernantant was
decanted. Cells were resuspended in 1 mL of cold PBS, fixed in 9 mL of
cold 70% ethanol, and incubated at 4 °C for at least 30 min. Cells were
centrifuged at 1500 rpm for 5 min, washed with 1% FBS/PBS, and
resuspended in 1 mL of 2:1 1% FBS in PBS/phosphate citric acid buffer
(pH 7.8). Cells were incubated at room temperature for 5 min, then
spun at 1500 rpm for 5 min. The supernatant was decanted, and the cells
were resuspended and incubated in 300 μL of PBS/FBS/PI/RNase
solution (10 μg/mL propidium iodide and 3 KU of RNase A) for 30 min
at 37 °C. Flow cytometry was performed to analyze DNA content,
collecting 10 000 PI positive gated events per sample.
31P NMR Detection of Prodrug Activation. A suspension of
BP prodrug 14 (22 mM) was prepared with sonication in a 1:2:8 mixture
of DMF/acetonitrile/cacodylate buffer (200 mM, pH 7.4) at 37 °C, and
the 31P chemical shift was recorded. Under these conditions, 14
appeared as a single resonance, and low signal intensity was observed
as a result of low solubility of the prodrug under these conditions. After
addition of dithionite (8 molar equiv), signal intensity increased as a
consequence of increased solubility of reduced and/or eliminated
species. The reaction mixture was incubated at 37 °C for the duration
of the experiment.
In Vitro Cell Proliferation Assays. Cell proliferation was deter-
mined using the CellTiter 96 Aqueous One Solution Cell Proliferation
Assay MTS assay. A549 NSCLC cells were plated at 1.5 ꢁ 103 cells per
well in flat bottom 96-well plates in 99 μL of medium and allowed to
adhere overnight. The drugs were serially diluted in 100% DMSO. For
each drug treatment group, 1 μL of a 100ꢁ stock solution was added to
each well for a final DMSO concentration of 1%. Cells were treated for
24, 48, or 72 h. Cells were incubated with MTS dye (20 μL wellꢀ1) for
40 min to 2 h. Absorbance at 490 nm was determined using a SpectraMax
M2 (Molecular Devices) plate reader. The percent cell proliferation was
calculated by converting the experimental absorbance to percentage of
control, which was then plotted vs drug concentration. The IC50 values were
determined using a nonlinear doseꢀresponse analysis in GraphPad Prism,
version 4.0. The IC50 is defined as the concentration of drug needed to cause
a 50% decrease in proliferation compared to vehicle control.
Detection of Intracellular Prodrug and Bisphosphonate.
A549 NSCLC cells were plated at 1.3 ꢁ 105 cells per well in flat bottom
six-well plates. Cells were dosed as described above. At 8 h following
drug treatment, the medium was removed, and the cells were washed
with 1 mL of PBS. Each well was trypsanized with 600 μL of trypsin for
3ꢀ5 min. The trypsin reaction was quenched with an equal volume of
medium. The cells were transferred to a 15 mL conical and centrifuged at
1100 rpm for 5 min. Supernatant was removed, and the cells were
resuspended in 200 μL of medium. The cells were diluted 1:1 in 0.04%
trypan blue and counted using a cytometer. The number of cells was
determined at each drug concentration and time. The cells were then
centrifuged at 1100 rpm for 5 min. The supernatant was removed, and
the cell pellets were washed 2ꢁ with PBS. The supernatant was
removed, and the pellets were frozen at ꢀ80 °C until analysis.
In Vitro Cell Count. A549 NSCLC cells were plated at 1.7 ꢁ 104
cells per well in flat bottom 12-well plates. Cells were dosed as described
above. At 24, 48, or 72 h following drug treatment, the medium was
collected, and the cells were washed with 200 μL of PBS. Each well
was trypsinized with 200 μL of trypsin for 3ꢀ5 min. The trypsin reaction
was quenched with an equal volume of medium. All supernatants and
washes were combined and spun at 1500 rpm for 5 min. Supernatant was
decanted, and the cells were resuspended in 200 μL of medium. The cells
were diluted 1:1 in 0.04% trypan blue and counted using a cytometer.
The absolute number of cells was determined at each drug concentra-
tion. The cell number for each concentration was converted to percent
of control for each time point and plotted using GraphPad Prism 4.0.
The EC50 was calculated as the concentration of drug that caused a 50%
decrease in number of cells compared to control.
For detection of bisphosphonamidate prodrug 14 in A549 cells
treated for 8 h, the cell pellet was suspended in 0.5 mL of deionized
water, and 100 μL of the suspension was lysed using 300 μL of
acetonitrile containing temazepam (100 ng/mL) as an internal standard.
The suspension was mixed vigorously for 1 min on a vortex mixer and
then subjected to centrifugation at 1200g for 10 min at ambient
temperature. Thesupernatant(10μL) was injected into the LCꢀMSꢀMS
instrument using an autosampling device operating at room tempera-
ture. Separation was achieved on a Waters X-Terra C18 (50 mm ꢁ 2.1
mm i.d., 3 μm) at room temperature using isocratic elution with
acetonitrile/water mobile phase (60:40, v/v) containing 0.1% formic
acid at a flow rate of 0.2 mL/min. Prodrug detection was performed
using electrospray tandem mass spectrometry operating in positive ion
mode by monitoring the ion transitions from m/z 653.0 f 532.0 (BP
prodrug) and m/z 301.2 f 255.1 (temazepam). Samples were quanti-
tated over the assay range of 2ꢀ1000 ng/mL. Samples were then
quantitated in ng/104 cells as [nominal concentration (ng/mL)][5
(standardized dilution)]/[total number of cells (expressed as 104)].
For qualitative detection of intracellular bisphosphonate 1 in A549
cells treated for 8 h with prodrug 14 or bisphosphonate 1, the cell pellet
suspension was prepared as described above. Cell lysis was achieved by
addition of 300 μL of acetonitrile to 100 μL of the cell suspension and
1000 ng/mL 2-(phosphonomethyl)pentanedioic acid (2-PMPA) as an
Crystal Violet Assay. A549 NSCLC cells were plated at 1.7 ꢁ 104
cells per well in flat bottom six-well plates. Cells were dosed as described
above. Cells were analyzed at 24, 48, and 72 h. The medium was
removed, and the cells were washed twice with PBS. The cells were then
stained with crystal violet solution (1 mL wellꢀ1, 0.5% crystal violet in
95% EtOH) for 5ꢀ15 min. The stain was removed, and the plates were
rinsed with cold water and dried at room temperature.
’ ASSOCIATED CONTENT
S
Supporting Information. Characterization data for 14
b
and 15, including NMR and HPLC results; stability of 14 and 15
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in media; control experiment assessing chemical conversion of
prodrug 14 to derivatized bisphosphonate 18. This material is
available free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: 410-502-4807. Fax: 410-955-3023. E-mail: cmeyers@
jhmi.edu.
’ DISCLOSURE
The contents of the studies are solely the responsibility of the
authors and do not necessarily represent the official view of
NCRR or NIH.
’ ACKNOWLEDGMENT
This research was supported by the Flight Attendants Medical
Research Institution (FAMRI) Center for Excellence at the
Johns Hopkins University School of Medicine. Mass spectro-
metry studies were supported by the Analytical Pharmacology
Core of the Sidney Kimmel Comprehensive Cancer Center at
Johns Hopkins (NIH Grants P30 CA006973 and UL1 RR025005
and the Shared Instrument Grant 1S10RR026824-01). Mass spec-
trometry studies were supported in part by Grant UL1 RR 025005
from the National Center for Research Resources (NCRR), a
component of the National Institutes of Health (NIH) and NIH
Roadmap for Medical Research. We thank Dr. Charles Rudin and
Dr. Richard Borch for critical evaluation of the manuscript. We also
thank Dr. Timothy Burns for his contribution to the design and
analysis of flow cytometery experiments.
(14) Croucher, P. I.; De Raeve, H.; Perry, M. J.; Hijzen, A.; Shipman,
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K. Zoledronic Acid Treatment of 5T2MM-Bearing Mice Inhibits the
Development of Myeloma Bone Disease: Evidence for Decreased
Osteolysis, Tumor Burden and Angiogenesis, and Increased Survival.
J. Bone Miner. Res. 2003, 18 (3), 482–492.
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Dicuonzo, G.; Tonini, G. Repeated Intermittent Low-Dose Therapy
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BP, bisphosphonates; NBP, nitrogen-containing bisphospho-
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farnesyl pyrosphosphate;FPPS, farnesyl pyrophosphate synthase;
SKE, skeletal related events
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