Synthesis of fluorescently tagged isoprenoid bisphosphonates that inhibit protein geranylgeranylation

Article abstract of DOI:10.1016/j.bmc.2007.01.002

Geminal bisphosphonates can be used for a variety of purposes in human disease including reduction of bone resorption in osteoporosis, treatment of fractures associated with malignancies of the prostate, breast, and lung, and direct anticancer activity ag

Full text of DOI:10.1016/j.bmc.2007.01.002

Bioorganic & Medicinal Chemistry 15 (2007) 1959–1966
Synthesis of fluorescently tagged isoprenoid bisphosphonates that
inhibit protein geranylgeranylation
Mona A. Maalouf,a, Andrew J. Wiemer,b, Craig H. Kuder,c
Raymond J. Hohld,c,b and David F. Wiemera,c,
*
aDepartment of Chemistry, University of Iowa, Iowa City, IA 52242-1294, USA
bProgram in Molecular & Cellular Biology, University of Iowa, Iowa City, IA 52242-1294, USA
cDepartment of Pharmacology, University of Iowa, Iowa City, IA 52242-1294, USA
dDepartment of Internal Medicine, University of Iowa, Iowa City, IA 52242-1294, USA
Received 13 July 2006; revised 21 November 2006; accepted 1 January 2007
Available online 4 January 2007
Abstract—Geminal bisphosphonates can be used for a variety of purposes in human disease including reduction of bone resorption
in osteoporosis, treatment of fractures associated with malignancies of the prostate, breast, and lung, and direct anticancer activity
against bone marrow derived malignancies. Previous research led to identification of some novel isoprenoid bisphosphonates that
inhibit geranylgeranyl pyrophosphate (GGPP) synthesis and diminish protein geranylgeranylation. Described here is the synthesis of
fluorescent anthranilate analogues of the most active isoprenoid bisphosphonates and examine their ability to impact post-transla-
tional processing of the small GTPases Ras, Rap1a, and Rab6. Similar to their non-fluorescent counterparts, some of these fluores-
cent isoprenoid bisphosphonates diminish protein geranylgeranylation. Their biological activity and fluorescent character suggest
that they may be useful in studies of bisphosphonate localization both in cultured cells and in whole organisms.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
metabolism. Previous studies have focused on geminal
bisphosphonates bearing one4–6 or more isoprenoid7
substituents, and recently some were prepared (e.g., 4)
that diminish protein geranylgeranylation.8 The struc-
tures of these isoprenoid bisphosphonates differ signifi-
cantly from current clinical compounds because they
lack the –OH group on the geminal carbon that has been
identified as a key part of the molecule that enhances
localization to bone. These novel compounds also are
much more hydrophobic than the clinical compounds
due to the presence of the isoprenoid chains. The impact
of these structural differences on intracellular and tissue
localization is not yet known. Fluorescent analogues, as
reported herein, will afford the opportunity to elucidate
these characteristics.
Several geminal bisphosphonates are used clinically for
treatment of bone-related human diseases.1 For exam-
ple, risedronate (1, Fig. 1) is used for treatment of
post-menopausal osteoporosis.2 Zoledronate (2) is used
to treat excess bone resorption caused by bone metasta-
ses from diseases such as multiple myeloma and pros-
tate, breast, and lung cancers.3 These bisphosphonates
can be viewed as structural analogues of pyrophosphate
(3) that contain a carbon in place of the central oxygen
atom. The central carbon results in greater metabolic
stability and also provides a scaffold that can be modi-
fied with additional substituents, such as the hydroxyl
group and heteroaromatic rings found in risedronate
and zoledronate. It has been postulated that through
modification of their substituents, bisphosphonates can
be targeted to specific enzymes involved in isoprenoid
Despite extensive studies,9 the mechanisms of bis-
phosphonate action are not completely defined. On an
organismal level, the clinically used bisphosphonates
are known to localize to the bone based on studies with
radiolabeled compounds. On a cellular level bisphos-
phonates have been shown to inhibit growth of a variety
of cancer cell lines.10 On a molecular level, it is known
that the nitrogen-containing bisphosphonates target
the isoprenoid biosynthetic pathway and impact
Keywords: Bisphosphonate; Isoprenoid; Protein prenylation; Anthra-
nilate; Fluorescence.
*
Corresponding author. Tel.: +1 319 335 1365; fax: +1 319 335
1270; e-mail: david-wiemer@uiowa.edu
These authors contributed equally to this work, in chemistry and
biology, respectively.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.002

1960
M. A. Maalouf et al. / Bioorg. Med. Chem. 15 (2007) 1959–1966
isoprenylation of small GTPases.11 Even some of the
O
O
earliest studies suggested that there may be multiple tar-
get sites in living systems, including squalene synthase.12
The clinically utilized nitrogenous bisphosphonates,
such as risedronate, alendronate, and zoledronate, have
been reported to inhibit farnesyl pyrophosphate (FPP)
synthase,13,14 and indeed the structure of FPP synthase
complexed with zoledronate recently has been
revealed.15 Other bisphosphonates have been demon-
strated to inhibit IPP isomerase.16 Yet others,17,18 includ-
ing the digeranyl bisphosphonate 4,7 inhibit protein
geranylgeranylation with little or no effect upon protein
farnesylation, indicating that they target the isoprenoid
pathways more selectively and further downstream.
P(OH)2
P(OH)2
N
N
N
HO
HO
P(OH)2
O
P(OH)2
O
1
2
(risedronate)
(zoledronate)
O
O
P
(HO)2P
(OH)2
O
3
(pyrophosphoric acid)
One approach to further characterize the mechanism of
bisphosphonate action is to study their organismal and/
or cellular localization. Fluorescent bisphosphonates
such as YM529 (5)18,19 have been reported to be readily
detectable at very low levels in plasma, urine, and
bone,19 and this can simplify the study of their pharma-
cokinetics. Additionally, a fluorescent pamidronate ana-
logue, Pam78, has been shown to allow for visualization
of microcalcifications in a mouse breast cancer model
system.20 As a tool for further study of localization of
isoprenoid bisphosphonates, and to facilitate studies of
the metabolism of isoprenoid bisphosphonates, fluores-
cent analogues of digeranyl bisphosphonate were
designed (4). Despite incorporation of an anthranilate
P(OH)2
O
(HO)2P
O
4
N
O
N
P(OH)2
HO
P(OH)2
O
5
(YM529)
Figure 1. Representative bisphosphonates and pyrophosphoric acid.
CO Me
2
1)
8
NH
2
H
N
MeO2C
1) t-BuOOH, SeO2
NaBH(OAc) , AcOH
3
2) MnO2, hexanes
H
2) MeOH, K2CO3
OAc
X
OAc
O
40%
87%
7
MsC l
Et N
3
LiBr
6
9
X = OH
10 X = Br
O
P(OEt)
NaH, 15-crown-5
2
TH F
28%
P(OEt)
11
2
O
H
N
R
H
N
NH4O2C
MeO2C
1) TMSBr, collidine
2) NaOH
3) ion exchange
O
P(ONH )
O
P(OEt)
2
4 2
R
P(ONH )
P(OEt)
2
4 2
O
O
27%
13 R =
12 R =
O
)
(P(OEt)
14
CH2
2 2
1) TMSBr
collidine
2) NaOH
H
N
H
N
MeO2C
NaO2C
O
O
NaH, THF,
15-crown-5
P(OEt)
P(ONa)
2
2
10
P(OEt)
O
P(ONa)
O
2
2
2
47%
98%
2
15
Scheme 1. Preparation of bisphosphonates 13 and 16.
16

M. A. Maalouf et al. / Bioorg. Med. Chem. 15 (2007) 1959–1966
1961
fluorophore, some of these compounds still show a
significant impact on protein geranylgeranylation but
not upon protein farnesylation. In this manuscript, the
synthesis and biological activity of the new isoprenoid
bisphosphonates that incorporate a fluorescent label in
a modified isoprenoid chain is reported.
bromide 18 with the anion of geranyl bisphosphonate
11. Standard hydrolysis afforded the sodium salt 22 in
87% yield.
3. Biological assays
In order to gauge their impact on cell growth, com-
pounds 13, 16, 20, and 22 were tested for their ability
to inhibit DNA synthesis in K562 leukemia cells. Similar
to the digeranyl bisphosphonate 4,7 24 h IC50 values
for all of the compounds toward inhibition of [3H]thy-
midine incorporation were greater than 100 lM (data
not shown). To determine the internalization of the
fluorescent compounds, treated cells were examined by
fluorescent microscopy. As shown in Figure 2, the bis-
phosphonates 13, 22, 20, and 16 are internalized, even
though all are highly charged compounds. The com-
pounds do not co-localize with the nuclear stain DRAQ5,
and instead appear to be present in the cytoplasm.
2. Chemical synthesis
Synthesis of the allylic alcohol 9 begins with com-
mercially available prenyl acetate 6 (Scheme 1). Reac-
tion with selenium dioxide followed by oxidation with
MnO2 afforded the aldehyde 7.21 Treatment of the alde-
hyde with methyl anthranilate (8) under reductive ami-
nation conditions afforded allylic alcohol 9.22 The
alcohol was then converted to the corresponding bro-
mide (10) via the mesylate, providing entry to the fluo-
rescent bisphosphonates with one isoprene unit
between the fluorescent group and the bisphosphonate
head. Treatment of bromide 10 with the anion of either
geranyl bisphosphonate (11)4 or tetraethyl meth-
ylenebisphosphonate (14) afforded bisphosphonate es-
ters 12 and 15, respectively. Finally, the corresponding
salts 13 and 16 were obtained employing standard
hydrolysis conditions.23
The compounds were subsequently tested for their abil-
ity to impair protein prenylation in this cell line (Fig. 3).
Ras protein was studied as a model of farnesylation,
while Rap1a and Rab6 were studied as models for gera-
nylgeranylation by GGPTase I and II, respectively. As
positive controls, cells also were treated with lovastatin
or digeranyl bisphosphonate (4).7 Lovastatin has been
shown to inhibit HMG-CoA reductase and to deplete
cells of mevalonate and its derivatives FPP and
GGPP,26 which in turn diminishes both protein farnesy-
lation and geranylgeranylation. This effect can be visual-
ized by the appearance of slower migrating, double
bands in the Ras and Rab6 Western blots. Because the
Rap1a antibody only detects unmodified Rap1a and
not modified Rap1a, lovastatin causes the appearance
of a single band which is not seen in control cells. Diger-
anyl bisphosphonate (4) has been shown to inhibit gera-
nylgeranylation7 which occurs through depletion of
The second set of compounds (Scheme 2) was prepared
from the known farnesol analogue 17.24 The alcohol 17
was first converted to the bromide 18 through reaction
with MsCl followed by LiBr.25 The allylic bromide 18
was treated with the anion of tetraethyl meth-
ylenebisphosphonate (14) to afford the bisphosphonate
ester 19 in modest yield. Standard hydrolysis conditions
resulted in the sodium salt which was passed through an
ion-exchange column (ammonium form) to yield the
fluorescently tagged bisphosphonate 20 as the ammoni-
um salt in 58% yield. The dialkyl bisphosphonate ester
21 was prepared through treatment of the allylic
14
NaH, 15-crown-5
THF
H
N
MeO2C
H
N
MeO2C
O
P(OEt)
2
X
P(OEt)
2
47%
17 X = OH
18 X = Br
19
MsCl, LiBr,
Et N
3
O
1) TMSBr, collidine
2) NaOH
58%
3) ion exchange
11
NH4O2C
H
N
O
NaH, 15-crown-5
60%
THF
P(ONH )
4 2
P(ONH )
4 2
20
O
1) TMSBr
collidine
2) NaOH
H
N
MeO2C
NaO2C
H
N
O
O
P(OEt)
P(ONa)
2
2
R
R
P(OEt)
P(ONa)
2
87%
2
O
O
21 R =
22 R =
Scheme 2. Synthesis of bisphosphonates 20 and 22.

1962
M. A. Maalouf et al. / Bioorg. Med. Chem. 15 (2007) 1959–1966
reported. The key feature of the synthetic path is the use
of the geraniol and farnesol analogues as building blocks
for a diverse family of compounds. These data provide the
first examples of visualization of isoprenoid bisphospho-
nates in cells. The ability of compounds 13 and 22, but not
compounds 16 or 20, to inhibit geranylgeranylation fur-
ther supports the hypothesis that at least one isoprenoid
chain is required on the bisphosphonate template for inhi-
bition of geranylgeranylation.7 The ability of fluorescent
bisphosphonates containing either a five- or ten-carbon
linker to inhibit geranylgeranylation could indicate a
GGPP synthase binding pocket27 with sufficient size to
accommodate further modifications, or a region of the
molecule that is not directly involved in binding to the
enzyme. Distinguishing between possibilities such as
these, as well as investigations of the localization and
activity in animal models, will form the basis for further
studies of these intriguing compounds.
5. Experimental
5.1. General experimental conditions
Tetrahydrofuran (THF) was freshly distilled from sodi-
um/benzophenone, and CH2Cl2 and Et3N were freshly
distilled from CaH2; other solvents were purchased from
commercial sources and used as provided. All reactions
in non-aqueous solvents were conducted in oven-dried
glassware under positive pressure of argon with magnet-
ic stirring. Oil-free NaH was prepared by washing min-
eral oil dispersions with hexanes. NMR spectra were
1
recorded at 300 MHz for H, and 75 MHz for 13C with
CDCl3 as solvent and (CH3)4Si (1H) or CDCl3 (13C,
77.2 ppm) as internal standards unless otherwise noted.
The 31P chemical shifts are reported in ppm relative to
85% H3PO4 (external standard). High resolution mass
spectra were obtained at the University of Iowa Mass
Spectrometry Facility or at the Washington University
Resource for Biomedical and Bioorganic Mass Spec-
trometry. Elemental analyses were performed by Atlan-
tic Microlab, Inc. (Norcross, GA).
Figure 2. Compound cellular internalization. Fluorescent images of
K562 cells treated for 24 h with 50 lM of active compounds 13 and 22
and inactive compounds 20 and 16. Cells were stained with DRAQ5
nuclear stain as described in methods. Nucleus, compound, and
merged images are displayed for each test compound.
5.2. 4-Acetoxy-2-methyl-2-E-butenal (7)
To a stirred suspension of 4-hydroxybenzoic acid
(0.64 g, 4.7 mmol) and selenium dioxide (2.1 g,
19 mmol) in CH2Cl2 was added 70% tert-butyl hydro-
peroxide (20 mL, 140 mmol). After 1 h, the mixture
was cooled to 0 ꢁC, stirred for 10 min, and prenyl ace-
tate (5.7 g, 45 mmol) was added. The reaction mixture
was allowed to warm to rt over a period of 24 h after
which saturated NaHCO3 was added. The mixture was
extracted with CH2Cl2 and the organic extract was dried
(MgSO4) and filtered. The filtrate was concentrated to
afford a yellow oil that was dissolved in hexanes and
cooled to À10 ꢁC. Solid MnO2 (57 g, 560 mmol) was
added to the vigorously stirred reaction, and after 24 h
at À10 ꢁC the mixture was filtered through a pad of Cel-
ite. The filtrate was concentrated in vacuo and purified
by flash chromatography (hexanes/EtOAc; 80:20) to
afford aldehyde 7 (5.0 g, 40%) with 1H, 13C spectra
identical with literature data.28
GGPP. This can be visualized by both the appearance of
the unmodified Rap1a band and the appearance of the
upper band or widening of the band on the Rab6 blot.
Digeranyl bisphosphonate (4) does not affect farnesyla-
tion, and as such no upper band appears on the Ras
blot. Compounds 13 and 22 inhibited Rap1a and
Rab6 geranylgeranylation at concentrations similar to
digeranyl bisphosphonate and like digeranyl bisphosph-
onate had no effect on Ras farnesylation. Compounds
16 and 20 did not inhibit prenylation of Ras, Rap1a,
or Rab6 at the concentrations tested.
4. Conclusions
In conclusion, the synthesis of four novel isoprenoid bis-
phosphonates incorporating an anthranilate moiety is

M. A. Maalouf et al. / Bioorg. Med. Chem. 15 (2007) 1959–1966
1963
Figure 3. Western blot analysis of K652 cells treated for 24 h with indicated concentrations of compounds 4, 13, and 22. Samples were compared to
untreated K562 cells as a negative control (Cont), and to K562 cells treated with 10 lM lovastatin (Lov) as a positive control for inhibition of protein
prenylation. Antibodies for total Ras and Rab6, and for unmodified Rap1a, were used for visualization of proteins.
5.3. Alcohol 9
used without further purification. To a suspension of
NaH (0.17 g, 60% dispersion in mineral oil, 4.3 mmol)
in anhydrous THF at 0 ꢁC was added 15-crown-5
(0.04 mL, 0.22 mmol) followed by geranyl bisphospho-
nate 11 (1.8 g, 4.3 mmol). After 1 h, the allylic bromide
(0.81 g, 2.7 mmol) in THF was added via cannula. The
reaction mixture was allowed to warm to rt over 2 h
and then filtered. The filtrate was concentrated in vacuo
and the resulting residue was purified by flash chroma-
tography (4% MeOH in ether) to give bisphosphonate
To a solution of aldehyde 7 (1.9 g, 13 mmol) and methyl
anthranilate (8, 1.9 mL, 15 mmol) in anhydrous dichlo-
˚
roethane were added molecular sieves (4 A) and glacial
acetic acid (0.94 mL, 16 mmol). The resulting mixture
was allowed to stir at rt for 5 min, and then sodium tri-
acetoxyborohydride (4.2 g, 19 mmol) was added at 0 ꢁC.
The reaction mixture was allowed to warm to rt over
2.5 h after which saturated NaHCO3 was added drop-
wise at 0 ꢁC. The aqueous layer was extracted with ether
and the combined organic extract was dried (MgSO4)
and filtered. The filtrate was concentrated in vacuo and
the residue was dissolved in methanol at rt. After
potassium carbonate (4.6 g, 33 mmol) was added, the
reaction mixture was left to stir for 2 h. Saturated
NH4Cl was added and the mixture was extracted with
ether. The combined organic extract was dried (MgSO4),
filtered, and the filtrate was concentrated in vacuo and
purified by flash chromatography (hexanes/EtOAc;
85:15) to give allylic alcohol 9 (2.8 g, 87%) as a yellow
1
12 (0.39 g, 28%) as a yellow oil: H NMR d 7.87 (dd,
J = 8.0, 1.5 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 6.65 (d,
J = 8.5 Hz, 1H), 6.55 (t, J = 7.6 Hz, 1H), 5.72 (t,
J = 6.5 Hz, 1H), 5.40 (t, J = 6.8 Hz, 1H), 5.09 (t,
J = 6.4 Hz, 1H), 4.15 (t, J = 7.0 Hz, 8H), 3.84 (s, 3H),
3.77 (d, J = 5.4 Hz, 2H), 2.64 (dd, J = 15.6, 6.7, 4H),
2.07–1.97 (m, 4H), 1.68 (s, 3H), 1.67 (s, 3H), 1.59 (s,
3H), 1.56 (s, 3H), 1.29 (dt, J = 7.1, 1.5 Hz, 12H); 13C
NMR d 169.1, 151.5, 137.4, 134.6, 133.6, 131.6, 131.4,
124.4, 121.6 (t, JCP = 7.3 Hz), 119.2 (t, JCP = 7.2 Hz),
114.5, 111.9, 109.9, 62.6 (t, JCP = 3.2 Hz, 4C), 51.5,
51.0, 45.8 (t, JCP = 131.4 Hz), 40.2, 29.3 (t,
JCP = 4.6 Hz), 29.1 (t, JCP = 4.4 Hz), 26.8, 25.8, 17.8,
16.6 (t, JCP = 3.0 Hz, 4C), 16.3, 14.7; 31P NMR d
27.1 ppm; HRMS (ESI, m/z): calcd C32H53NO8P2Na
[M+Na]+, 664.3144; found 664.3147.
1
oil: H NMR d 7.92 (br s, 1H, exchanges with D2O),
7.89 (dd, J = 8.0, 1.5 Hz, 1H), 7.30 (dd, J = 8.0,
1.5 Hz, 1H), 6.61–6.54 (m, 2H), 5.63 (t, J = 6.4 Hz,
1H), 4.16 (d, J = 6.7 Hz, 2H), 3.83 (s, 3H), 3.75 (d,
J = 5.6 Hz, 2H; br s upon addition of D2O), 1.99 (br s,
1H, exchanges with D2O), 1.70 (s, 3H); 13C NMR d
169.2, 151.2, 135.2, 134.6, 131.7, 124.6, 114.8, 111.6,
110.0, 59.0, 51.5, 49.9, 14.7; HRMS (ESI, m/z): calcd
C13H18NO8 [M+H]+, 236.1287; found 236.1283.
5.5. Bisphosphonate salt 13
To a solution of bisphosphonate ester 12 (0.39 g,
0.61 mmol) in anhydrous CH2Cl2 at 0 ꢁC were added
2,4,6-collidine (0.81 mL, 6.1 mmol) and TMSBr
(0.80 mL, 6.1 mmol). The reaction mixture was allowed
to warm to rt for 24 h, and toluene was added. The vol-
atiles were removed in vacuo to afford a white solid that
was dissolved in aqueous NaOH (10 mL, 1 N) at rt.
After 24 h, the mixture was lyophilized to afford a gray
solid. This solid was dissolved in a buffer solution (1:49
v/v isopropyl alcohol: 25 mM aqueous NH4HCO3),
passed through an ion-exchange column using Dowex
resin (50WX8-200) hydrogen form, washed with ammo-
nium hydroxide solution (1 N), and then allowed
to equilibrate with ion-exchange buffer solution. The
5.4. Bisphosphonate ester 12
To a solution of allylic alcohol 9 (0.51 g, 2.2 mmol) in
CH2Cl2 at À50 ꢁC were added triethylamine (0.39 mL,
2.8 mmol) and MsCl (0.20 mL, 2.6 mmol) consecutively.
After 30 min, a solution of LiBr (0.47 g, 5.4 mmol) in
THF was added to the cooled reaction mixture via can-
nula. The reaction flask was transferred to an ice bath
for 1.5 h, and then water was added. The mixture was
extracted with CH2Cl2, washed with ice-cold brine, dried
(MgSO4), and filtered. The solvent was removed in vac-
uo to obtain the allylic bromide as a yellow oil that was

1964
M. A. Maalouf et al. / Bioorg. Med. Chem. 15 (2007) 1959–1966
sample was eluted with the buffer solution and the
eluant was lyophilized to give bisphosphonate salt 13
at 0 ꢁC for 24 h. The mixture was filtered and the residue
was washed with cold hexanes and dried in vacuo to af-
ford bisphosphonate 16 (69 mg, 98%) as a white solid:
1H NMR (400 MHz, D2O-dioxane) d 7.76 (dd, J = 7.8,
1.6 Hz, 2H), 7.32 (dt, J = 7.7, 1.6 Hz, 2H), 6.87 (d,
J = 8.3, 2H), 6.72 (dt, J = 7.7, 1.1 Hz, 2H), 5.90 (t,
J = 6.4 Hz, 2H), 3.73 (s, 4H), 2.55 (dt, J = 15, 6.4 Hz,
4H), 1.61 (s, 6H); 13C NMR (100 MHz, 67.4 ppm with
respect to dioxane) d 177.0 (2C), 150.5 (2C), 133.3
(2C), 132.6 (2C), 132.2 (2C), 127.0 (t, JCP = 6.4 Hz,
2C), 120.7 (2C), 117.0 (2C), 114.4 (2C), 52.6 (2C), 44.4
(t, JCP = 111.3 Hz), 31.1 (2C), 14.6 (2C); 31P (D2O)
24.8 ppm; HRMS (ESI, m/z): calcd. C25H31N2O10 P2
[MÀH]À 581.1454; found 581.1439.
1
(0.36 g, 27%) as a white solid: H NMR (25% ND4OD
in D2O) d 7.80 (d, J = 7.8 Hz, 1H), 7.55 (t, J = 7.1 Hz,
1H), 7.09–7.01 (br m, 1H), 6.93 (t, J = 7.8 Hz, 1H),
6.13 (br s, 1H), 5.82 (br s, 1H), 5.41 (br s, 1H), 3.97
(br s, 2H), 2.88–2.66 (m, 4H), 2.35–2.18 (m, 4H), 1.89
(s, 6H), 1.83 (s, 3H), 1.80 (s, 3H); 13C NMR
(100 MHz) d 176.4, 150.5, 135.6, 133.6, 132.9, 132.1
(2C), 126.8, 125.5, 123.9, 120.2, 116.4, 113.7, 52.3, 44.4
(t, JCP = 110.6 Hz), 40.2, 30.8, 26.8, 25.5, 17.6, 16.0,
14.5 (2C); 31P (D2O) d 25.0 ppm; HRMS (ESI, m/z):
calcd C23H34NO8P2 [MÀH]À, 514.1756; found 514.1781.
5.6. Ester 15
5.8. Bisphosphonate ester 19
To a solution of allylic alcohol 9 (2.8 g, 12 mmol) in
CH2Cl2 at À50 ꢁC were added triethylamine (2.1 mL,
15 mmol) and MsCl (1.1 mL, 14 mmol) consecutively.
After 30 min, a solution of LiBr (2.5 g, 29 mmol) in
THF was added to the cooled reaction mixture via can-
nula. The reaction flask was transferred to an ice bath
and after 1.5 h water was added. The mixture was
extracted with CH2Cl2, washed with ice cold brine, dried
(MgSO4), and filtered. The solvent was removed in vac-
uo to obtain the allylic bromide 10 as a yellow oil that
was used without further purification. To a suspension
of NaH (0.94 g, 60% dispersion in mineral oil, 24 mmol)
in anhydrous THF at 0 ꢁC was added 15-crown-5
(0.23 mL, 1.2 mmol) followed by tetraethyl meth-
ylenebisphosphonate (1.7 g, 5.8 mmol). After 1 h, the
allylic bromide was added via cannula as a solution in
THF. The reaction mixture was allowed to warm to rt
overnight and then quenched by addition of water.
The mixture was extracted with ether and the combined
organic extract was dried (MgSO4) and filtered. The fil-
trate was concentrated in vacuo and the resulting resi-
due was purified by flash chromatography (4% MeOH
in ether) to give bisphosphonate 15 (2.0 g, 47%) as a yel-
To a solution of allylic alcohol 17 (2.8 g, 12 mmol) in
CH2Cl2 at À50 ꢁC were added triethylamine (2.1 mL,
15 mmol) and MsCl (1.1 mL, 14 mmol) consecutively.
After 30 min, a solution of LiBr (2.5 g, 29 mmol) in
THF was added to the cooled reaction mixture via can-
nula. The reaction flask was transferred to an ice bath
and allowed to stir for 1.5 h after which water was add-
ed. The mixture was extracted with CH2Cl2, washed
with ice cold brine, dried (MgSO4), and filtered. The sol-
vent was removed in vacuo to obtain the allylic bromide
18 as a yellow oil that was used without further purifica-
tion. To a suspension of NaH (0.94 g, 60% dispersion in
mineral oil, 24 mmol) in anhydrous THF at 0 ꢁC was
added 15-crown-5 (0.23 mL, 1.2 mmol) followed by tet-
raethyl methylenebisphosphonate (1.7 g, 5.8 mmol).
After 1 h, the allylic bromide 18 was added via cannula
as a solution in THF. The reaction mixture was allowed
to warm to rt overnight and then quenched by addition
of water. The mixture was extracted with ether and the
combined organic extract was dried (MgSO4) and fil-
tered. The filtrate was concentrated in vacuo and the
resulting residue was purified by flash chromatography
(4% MeOH in ether) to give bisphosphonate 19 (2.0 g,
47%) as a yellow oil: 1H NMR d 7.89 (dd, J = 8.1,
1.7 Hz, 1H), 7.35–7.28 (m, 1H), 6.63 (d, J = 8.4 Hz,
1H), 6.56 (t, J = 7.3 Hz, 1H), 5.41 (t, J = 6.8 Hz, 1H),
5.31 (t, J = 7.2 Hz, 1H), 4.18–4.05 (m, 8H), 3.86 (s,
3H), 3.73 (d, J = 4.6 Hz, 2H), 2.73–2.54 (m, 2H), 2.31
(tt, J = 23.8, 5.7 Hz, 1H), 2.19–2.08 (m, 2H), 2.05–1.97
(m, 2H), 1.67 (s, 3H), 1.64 (s, 3H), 1.33 (t, J = 7.1 Hz,
12H); 13C NMR d 169.3, 151.5, 136.6, 133.7, 134.6,
131.7, 126.1, 122.2 (d, JCP = 7.3 Hz), 114.5, 111.8,
109.9, 62.6 (t, JCP = 7.6 Hz, 4C), 51.6, 50.7, 39.5, 37.7
(t, JCP = 132.7 Hz), 30.5, 26.5, 24.2 (t, JCP = 5.1 Hz,
2C), 16.6 (t, JCP = 5.3 Hz, 2C), 16.3, 14.7; 31P NMR d
24.9 ppm. Anal. Calcd for C27H45NO8P2: C, 56.54; H,
7.84. Found: C, 56.70; H, 8.03.
1
low oil: H NMR d 7.87 (dd, J = 8.0, 1.7 Hz, 2H), 7.28
(dt, J = 8.7, 1.6 Hz, 2H), 6.62 (d, J = 8.5 Hz, 2H), 6.54
(dt, J = 7.5, 1.1 Hz, 2H), 5.69 (t, J = 6.7 Hz, 2H), 4.12
(t, J = 7.8 Hz, 8H), 3.83 (s, 6H), 3.74 (d, J = 5.6 Hz,
4H), 2.65 (dt, J = 15.9, 7.1 Hz, 4H), 1.62 (s, 6H), 1.26
(t, J = 7.1 Hz, 12H); 13C NMR d 169.1 (2C), 151.4
(2C), 134.5 (2C), 133.7 (2C), 131.5 (2C), 121.2 (t,
JCP = 7.3 Hz, 2C), 114.5 (2C), 111.8 (2C), 109.9 (2C),
62.6 (t, JCP = 3.3 Hz, 4C), 51.4 (2C), 50.8 (2C), 45.6 (t,
JCP = 131.6 Hz), 29.1 (t, JCP = 4.4 Hz, 2C), 16.5 (t,
JCP = 2.8 Hz, 4C), 14.7 (2C); 31P NMR d 27.5 ppm;
HRMS (ESI, m/z): calcd C35H53N2O10 P2 [M+H]+
723.3175; found 723.3162.
5.7. Bisphosphonate 16
To a solution of bisphosphonate 15 (71 mg, 0.01 mmol)
in anhydrous CH2Cl2 at 0 ꢁC were added 2,4,6-collidine
(0.19 mL, 15 mmol) and TMSBr (0.19 mL, 15 mmol).
The reaction mixture was allowed to warm to rt over a
period of 24 h, and toluene was then added. The vola-
tiles were removed in vacuo to afford a white solid that
was dissolved in aqueous NaOH (5 mL, 1 N). After
24 h, the mixture was poured into acetone and stored
5.9. Bisphosphonate salt 20
To a solution of bisphosphonate 19 (0.26 g, 0.45 mmol)
in anhydrous CH2Cl2 at 0 ꢁC were added 2,4,6-collidine
(0.59 mL, 4.5 mmol) and TMSBr (0.58 mL, 4.5 mmol),
and the reaction mixture was allowed to warm to rt over
a period of 22 h. Toluene was then added, and the vol-
atiles were removed in vacuo to afford a white solid.

M. A. Maalouf et al. / Bioorg. Med. Chem. 15 (2007) 1959–1966
1965
This material was dissolved in aqueous NaOH (5 mL,
5.11. Bisphosphonate salt 22
1 N) at rt. After 24 h, the mixture was lyophilized to af-
ford a gray solid. This solid was dissolved in a buffer
solution (1:49 v/v isopropyl alcohol: 25 mM aqueous
NH4HCO3), passed through an ion-exchange column
using Dowex resin (50WX8-200) hydrogen form,
washed with ammonium hydroxide solution (1 N), and
then allowed to equilibrate with ion-exchange buffer
solution. The sample was eluted with the buffer solution
and the eluant was lyophilized to give bisphosphonate
To a solution of the bisphosphonate ester 21 (0.20 g,
0.28 mmol) in anhydrous CH2Cl2 at 0 ꢁC were added
2,4,6-collidine (0.37 mL, 2.8 mmol) and TMSBr
(0.36 mL, 2.8 mmol). The reaction mixture was allowed
to warm to rt over a period of 19 h and then toluene was
added. The volatiles were removed in vacuo to afford a
white solid that was dissolved in aqueous NaOH (5 mL,
1 N) at rt. After 3.5 h, the mixture was poured into ace-
tone and stored at 0 ꢁC for 14 h. The mixture was fil-
tered and the residue was washed with cold hexanes
and dried in vacuo to afford the bisphosphonate salt
1
salt 20 as a white solid (0.14 g, 58%): H NMR (D2O)
d 7.80 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H),
6.86 (d, J = 7.9 Hz, 1H), 6.78 (t, J = 7.8 Hz, 1H), 5.60
(t, J = 5.9 Hz, 1H), 5.55–5.47 (m, 1H), 3.76 (s, 2H),
2.61–2.39 (m, 3H), 2.17 (t, J = 6.9 Hz, 2H), 2.09–2.01
(m, 2H), 1.68 (s, 6H); 13C d 176.9, 150.3, 135.2, 133.2,
132.2, 131.6, 128.5 (t, JCP = 7.0 Hz), 127.7, 120.7,
117.1, 114.3, 51.5, 42.6 (t, JCP = 119.4 Hz), 39.8, 27.3,
27.1, 16.4, 14.6; 31P NMR d 22.7 ppm; HRMS (ESI,
m/z): calcd C18H23NO8P2Na5 [M+H]+, 558.0385; found
558.0387.
1
22 (0.17 g, 87%) as a white solid: H NMR (D2O) d
7.84–7.79 (m, 1H), 7.32–7.24 (m, 1H), 6.65 (d,
J = 8.2 Hz, 1H), 6.52 (t, J = 7.5 Hz, 1H), 5.90–5.79 (br
m, 2H), 5.44–5.39 (br m, 1H), 5.11–5.02 (br m, 1H),
3.70 (bs, 2H), 2.62–2.43 (br m, 4H), 2.15–1.90 (m,
8H), 1.60 (s, 6H), 1.54 (s, 6H); 13C NMR d 176.7,
150.8, 135.2, 135.1, 132.8, 132.5, 132.4, 131.5, 126.5,
125.7, 124.3, 119.3, 116.0, 112.7, 110.2, 51.2, 44.3 (t,
JCP = 113.5 Hz), 40.6, 40.4, 29.6, 27.5, 27.3, 26.0 (2C),
18.0, 16.5, 16.3, 14.9; 31P NMR d 27.2 ppm; HRMS
(ESI, m/z): calcd C28H39NO8P2Na5 [M+H]+, 694.1638;
found 694.1632.
5.10. Bisphosphonate 21
To a solution of allylic alcohol 17 (2.4 g, 7.8 mmol) in
CH2Cl2 at À50 ꢁC were added triethylamine (1.4 mL,
10 mmol) and MsCl (0.72 mL, 9.3 mmol) consecutively.
After 30 min, a solution of LiBr (1.7 g, 19 mmol) in
THF was added to the reaction mixture via cannula.
The reaction flask was transferred to an ice bath, al-
lowed to stir for 1.5 h, and then water was added. The
mixture was extracted with CH2Cl2, washed with ice-
cold brine, dried (MgSO4), and filtered. The solvent
was removed in vacuo and the resulting residue was
purified by flash chromatography (hexanes/EtOAc;
95:5) to afford the allylic bromide 18 (2.0 g, 70%) as a
yellow oil. To a suspension of NaH (0.26 g, 60% disper-
sion in mineral oil, 6.5 mmol) in anhydrous THF at 0 ꢁC
was added 15-crown-5 (0.07 mL, 0.33 mmol) followed
by the geranyl bisphosphonate 11 (1.4 g, 3.3 mmol).
After 1 h, the allylic bromide 18 (1.2 g, 3.3 mmol) was
added via cannula as a solution in THF. The reaction
mixture was allowed to warm to rt overnight and then
quenched by addition of water. The mixture was extract-
ed with ether and the combined organic extract was
dried (MgSO4) and filtered. The filtrate was concentrat-
ed in vacuo and the resulting residue was purified by
flash chromatography (4% MeOH in ether) to give bis-
5.12. Cell culture
K562 cells29 were cultured at 37 ꢁC in the presence of 5%
CO2 in RPMI-1640 media containing 10% heat-inacti-
vated fetal bovine serum. Bisphosphonates were diluted
in water and added at the concentrations indicated.
5.13. [3H]Thymidine assay
K562 cells (2 · 105 cells/200 lL) were incubated in a 96-
well plate with the test compounds for 24 h. During the
last 2 h, cells were labeled with [3H]thymidine (0.04 lCi/
well). Cells were collected and [3H]thymidine incorpo-
rated into cellular DNA was determined by liquid
scintillation counting as described previously.30
5.14. Western blot analysis
K562 cells (5 · 106 cells/5 mL) were incubated for 24 h
in the presence or absence of test compounds and lova-
statin. Western blotting was performed as previously
described. Pan-Ras antibody was obtained from Inter-
Biotech Corporation (Tokyo, Japan). Rab6, unmodified
Rap1a, and HRP-conjugated anti-goat antibodies were
purchased from Santa Cruz Biotech (CA). The antibody
for Rap1a detects only the unmodified form of Rap1a,
and not the isoprenylated form. The HRP-conjugated
anti-mouse and anti-rabbit antibodies were purchased
from Amersham/GE Healthcare.
1
phosphonate 21 (1.4 g, 60%) as a yellow oil: H NMR
d 7.81 (dd, J = 8.1, 1.4 Hz, 1H), 7.24–7.21 (m, 1H),
6.56 (d, J = 8.5 Hz, 1H), 6.48 (dt, J = 7.6, 1.0 Hz, 1H),
5.40–5.31 (m, 3H), 5.03 (t, J = 6.4 Hz, 1H), 4.09 (t,
J = 6.9 Hz, 8H), 3.78 (s, 3H), 3.65 (d, J = 5.3 Hz, 2H),
2.55 (dt, J = 16.0, 7.5 Hz, 4H), 2.10–1.89 (m, 8H), 1.59
(s, 6H), 1.54 (s, 6H), 1.52 (s, 3H), 1.24 (t, J = 7.1,
12H); 13C NMR d 169.2, 151.5, 137.1, 136.9, 134.6,
131.6, 131.5, 131.4, 126.4, 124.5, 119.3 (m, 2C), 114.5,
111.8, 109.9, 62.5 (t, JCP = 3.0 Hz, 4C), 51.3, 50.8, 46.0
(t, JCP = 131.3 Hz), 40.2, 39.9, 30.4, 29.2 (d,
JCP = 2.6 Hz), 26.8, 26.7, 25.8, 17.8, 16.6 (t,
JCP = 3.0 Hz, 4C), 16.4 (2C), 14.6; 31P NMR
27.9 ppm; HRMS (ESI, m/z): calcd C37H62NO8P2
[M+H]+, 710.3951; found 710.3937.
5.15. Fluorescence microscopy
K562 cells were incubated with the test compounds at
indicated concentrations for 24 h. Following three wash-
es, nuclei were stained with 10 lM DRAQ5, which binds
to cellular DNA, and imaged with a Bio-Rad Radiance
2100 MP multi-photon LSCM at the University of Iowa

1966
M. A. Maalouf et al. / Bioorg. Med. Chem. 15 (2007) 1959–1966
Jolibois, K. G.; Kunselman, L. K.; Lawrence, R. M.;
Mookhtiar, K. A.; Rich, L. C.; Slusarchyk, D. A.; Sulsky,
R. B.; Biller, S. A. J. Biol. Chem. 1993, 268, 24832–24837.
13. Bergsrtom, J. D.; Bostedor, R. G.; Masarachia, P. J.;
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373, 231–241.
14. van Beek, E.; Pieterman, E.; Cohen, L.; Lowik, C.;
Papapoulos, S. Biochem. Biophys. Res. Commun. 1999,
263, 108–111.
Central Microscopy Research Facility. Test compounds
containing the anthranilate fluorophore were excited
with a Mai Tai multi-photon laser and their emission
was detected between 400 and 450 nm. Nuclei were visu-
alized via DRAQ5 excitation with a red diode laser and
emission was detected between 650 and 700 nm.
Acknowledgments
15. Rondeau, J. M.; Bitsch, F.; Bourgier, E.; Geiser, M.;
Hemming, R.; Kroemer, M.; Lehmann, S.; Ramage, P.;
Rieffel, S.; Strauss, A.; Green, J. R.; Jahnke, W. Chem.
Med. Chem. 2006, 1, 267–273.
16. Thompson, K.; Dunford, J. E.; Ebetino, F. H.; Rogers,
M. J. Biochem. Biophys. Res. Commun. 2002, 290, 869–
873.
17. Szabo, C. M.; Matsumura, Y.; Fukura, S.; Martin, M. B.;
Sanders, J. M.; Sengupta, S.; Cieslak, J. A.; Loftus, T. C.;
Lea, C. R.; Lee, H. J.; Koohang, A.; Coates, R. M.;
Sagami, H.; Oldfield, E. J. Med. Chem. 2002, 45, 2185–
2196.
We thank the University of Iowa Central Microscopy
Research Facility for their assistance with the fluores-
cence microscopy. Financial support by the Roy J.
Carver Charitable Trust as a Research Program of
Excellence and the Roland W. Holden Family Program
for Experimental Cancer Therapeutics is gratefully
acknowledged.
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