Synthesis and biological evaluation of a series of aromatic bisphosphonates

Article abstract of DOI:10.1016/j.bmc.2010.08.036

Geminal bisphosphonates display varied biological activity depending on the nature of the substituents on the central carbon atom. For example, the nitrogenous bisphosphonates zoledronate and risedronate inhibit the enzyme farnesyl diphosphate synthase wh

Full text of DOI:10.1016/j.bmc.2010.08.036

Bioorganic & Medicinal Chemistry 18 (2010) 7212–7220
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a series of aromatic bisphosphonates
Rocky J. Barney a, Brian M. Wasko b, Amel Dudakovic c, Raymond J. Hohl b,c,d, David F. Wiemer a,c,
⇑
a Department of Chemistry, University of Iowa, Iowa City, IA 52242-1294, USA
b Program in Molecular and Cell Biology, University of Iowa, Iowa City, IA 52242-1294, USA
c Department of Pharmacology, University of Iowa, Iowa City, IA 52242-1294, USA
d Department of Internal Medicine, University of Iowa, Iowa City, IA 52242-1294, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Geminal bisphosphonates display varied biological activity depending on the nature of the substituents
on the central carbon atom. For example, the nitrogenous bisphosphonates zoledronate and risedronate
inhibit the enzyme farnesyl diphosphate synthase while digeranyl bisphosphonate has been shown to
inhibit the enzyme geranylgeranyl diphosphate synthase. We now have synthesized isoprenoid bisphos-
phonates where an aromatic ring has been used to replace one of the isoprenoid olefins in an isoprenoid
bisphosphonate and investigated the ability of these new compounds to impair protein geranylgeranyla-
tion within cells. Several of these new compounds are potent inhibitors of the enzyme geranylgeranyl
diphosphate synthase.
Received 4 June 2010
Revised 11 August 2010
Accepted 16 August 2010
Available online 19 August 2010
Keywords:
Bisphosphonate
GGDPS
Enzyme inhibitor
Isoprenoid biosynthesis
Protein prenylation
Geranylgeranyl diphosphate
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
isoprenoid metabolism.17 It is now recognized that the enzymatic
target of the clinically used nitrogenous bisphosphonates (NBPs) is
Interest in geminal bisphosphonates (1) as structural analogs of
pyrophosphates (2) is well established (Fig. 1).1 These compounds
are formed by formal replacement of the P–O–P linkage with the
P–C–P bond, but the bisphosphonates are more stabile to metabo-
lism and introduction of the methylene linker allows additional
structural modifications that would be impossible in the pyrophos-
phate structure. Geminal bisphosphonates have demonstrated util-
ity in a variety of applications. Historically, they have been used as
chelating agents2 and water softeners.3 More recently bisphospho-
nates have found application in the clinic as drugs for the treat-
ment of bone related disease. For example, zoledronate (3) has
been used in the treatment of osteoporosis as well as multiple
myeloma and a variety of cancers,1 and risedronate (4) is used
for treatment of osteoporosis.4 In addition, bisphosphonates have
shown great potential as cancer therapeutics5 and as anti-parasitic
agents.6,7 Their ability to inhibit growth of malignant cell lines8,9 as
FDPS.15,16 This enzyme is responsible for production of farnesyl
diphosphate (FPP), the key branch point in the mevalonate pathway.
However the mechanisms leading to the observed cellular effects of
these bisphosphonates are less well understood. Through inhibition
of FDPS, bisphosphonates such as zoledronate cause depletion of FPP
which has a direct impact on protein farnesylation. Depletion of FPP
levels also results in diminished cellular levels of geranylgeranyl
diphosphate (GGPP), and the cellular effects observed may result
from an impact on the downstream process of protein geranylger-
anylation.18–22 Many forms of cancer exhibit abnormalities in
prenylated GTPases,23 which encourages investigation into the
potential of bisphosphonates as direct anti-cancer therapeutics.
H
H
O
P(OH)2
P(OH)2
O
(HO)2P
(HO)2P
O
well as their ability to activate c
d-Tcells10 has been documented.
O
O
There is conjecture that inhibitors can be designed for specific
steps in the mevalonate pathway, and in fact bisphosphonates have
served as inhibitors of squalene synthase,11,12 isopentenyl pyro-
phosphate (IPP, 6, Fig. 2) isomerase,13,14 farnesyl diphosphate
synthase (FDPS, EC 2.5.1.10),15,16 and other enzymes involved in
1
2
N
N
HO
(HO)2P
N
HO
P(OH)2
O
(HO)2P
O
P(OH)2
O
O
3
4
⇑
Corresponding author. Tel.: +1 319 351 5905; fax: +1 319 335 1270.
E-mail address: david-wiemer@uiowa.edu (D.F. Wiemer).
Figure 1. Pyrophosphate and some bisphosphonates.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.036

R. J. Barney et al. / Bioorg. Med. Chem. 18 (2010) 7212–7220
7213
and 14 were synthesized from commercially available m-bromo-
benzyl alcohol (Fig. 3). The alcohol 8 was protected by treatment
with TBSCl and imidazole to provide the silyl ether 9 in 90% yield.
The resulting silyl ether undergoes halogen-metal exchange upon
treatment with n-BuLi and subsequent reaction with prenyl bro-
mide readily affords the alkylated product. Deprotection upon
treatment with TBAF then gives the known alcohol 10 in 72% yield
over three steps. Initial attempts at bromide formation via reaction
of alcohol 10 with PBr3 gave unacceptable results. Instead, bromide
formation was achieved by preparation of the mesylate followed
by treatment with LiBr to form the target benzylic bromide. Be-
cause it was assumed to be reactive, this material was not purified
further but instead was moved forward in the synthesis as first iso-
lated. Thus, tetraethyl methylenebisphosphonate was treated with
NaH and the resulting anion was treated with the intermediate
benzylic bromide to provide the desired bisphosphonate 11 in
47% yield over 3 steps. Hydrolysis of bisphosphonate 11 gave the
tetrasodium salt 12 smoothly. Synthesis of the dialkyl bisphospho-
nate 13 was achieved by alkylation of compound 11 with geranyl
bromide. Ester hydrolysis through reaction with TMSBr and final
treatment with sodium hydroxide provided the bisphosphonate
14 salt in 88% yield over these two steps.
(HO)2P
O
P(OH)2
O
5
O
P
O
P
NBPs
(e.g. 3 or 4)
O
O
OH
OH OH
(DMAPP)
O
P
O
P
FDPS
+
H
O
3
O
OH
O
P
O
OH OH
2
P
7 (FPP)
O
O
OH
GGDPS
+ IPP
OH OH
6 (IPP)
isoprenoid BPs
(e.g. 5)
O
P
O
P
H
O
4
O
OH
OH OH
The isomeric para-substituted compounds were obtained
through an analogous series of reactions as shown in Figure 4.
These reactions proceeded in a fashion parallel to those described
above, and gave the monoalkyl bisphosphonate 19 and the dialkyl
bisphosphonate 21 in good yields.
8 (GGPP)
Figure 2. Some steps in isoprenoid biosynthesis and bisphosphonate inhibitors.
Our labs have reported the synthesis of mono- and dialkyl iso-
prenoid bisphosphonates (e.g., 5, Fig. 2) that selectively target ger-
anylgeranyl diphosphate synthase (GGDPS, EC 2.5.1.29) over FDPS.
24,25,26 The enzyme GGDPS catalyzes elaboration of FPP (7) to GGPP
(8), which is the required precursor for all protein geranylgerany-
lation. To explore further this class of compounds, aromatic ana-
logs of mono- and digeranyl bisphosphonate (5) have been
designed, synthesized, and assayed. These analogues incorporate
both a geranyl chain as found in our GGDPS inhibitors and an aro-
matic ring parallel to that of risedronate.
There are several reasons to include an aromatic moiety in the
structure of potential GGDPS inhibitors. First, many of the most po-
tent inhibitors of FDPS are nitrogenous bisphosphonates with an
aromatic substructure, including zoledronate (3) and risedronate
(4), but it is not yet known whether introduction of an aromatic
moiety will enhance or diminish specificity for GGDPS inhibition
in an isoprenoid bisphosphonate. It would be interesting to deter-
mine if the aromatic compounds will retain their activity as inhib-
itors of GGDPS despite their more sterically demanding profile.
Second, there is evidence that the high charge to mass ratio in salts
of bisphosphonic acids at physiological pH can present some prob-
lem with transversing the cell membrane.27 Aromatic bisphospho-
nates are more lipophilic than the lead compound digeranyl
bisphosphonate (5), which may result in more facile drug delivery
to the cell and relieve the need for use of a prodrug.28 Third, bis-
phosphonates that contain isoprenoid olefin isomers display differ-
ing biological activity, but the potential isomerization or
transposition of an alkene in vivo would be eliminated with an aro-
Compound 24, a bisphosphonate bearing an unsubstituted phe-
nyl ring and one geranyl chain, was prepared from tetraethyl
methylenebisphosphonate (Fig. 5). In this case it was attractive
to add the benzyl substituent to the parent bisphosphonate first,
followed by alkylation with geranyl bromide to obtain the dialkyl
bisphosphonate 23. To prepare the corresponding pyridyl com-
pound, an analogue more similar to risedronate, it was found to
be advantageous to add the geranyl chain first and then alkylate
the geranyl bisphosphonate with bromide 25. In both cases, the
standard hydrolysis was employed to obtain the desired bis-
phosphonate salts 24 and 27. Finally, the known bisphosphonate
28 was prepared to serve as a control.30
3. Biological results and discussion
The aromatic bisphosphonates described above were evaluated
for activity in both enzyme and various whole cell assays. Our lab-
oratory has previously identified numerous mono- and dialkyl bis-
phosphonates as inhibitors of GGDPS, and many of these
compounds also have been shown to decrease protein geranylger-
anylation in cellular assays.25 Based on our previous work, we
hypothesized that the aromatic bisphosphonates synthesized
would inhibit GGDPS. Compounds were first screened in vitro
against recombinantly purified human GGDPS enzyme (Fig. 6A).
Compound 5 was used as a positive control because it was previ-
ously shown to inhibit GGDPS.26 At 10
lM concentrations, the dial-
kyl bisphosphonates 24, 14, 21, and 27 all displayed various
degrees of GGDPS inhibition while the mono alkyl compounds
12, 19, and 28 displayed little or no activity. Concentration–re-
sponse curves were then generated to further characterize the
compounds active in the initial screen. Compounds 14 and 21 were
found to display potent inhibitory activity with IC50 values (the
concentration at which the enzyme is inhibited to 50% maximal
activity) extrapolated to be 250 nM and 800 nM, respectively
(Fig. 6B). As a comparison, the published IC50 value of compound
5 is 200 nM.26 The structure of GGDPS has been solved when com-
plexed with digeranyl bisphosphonate (5), which bound to the
‘inhibitor’ binding site31 in a ‘V-shaped’ conformation occupying
portions of both the FPP and GGPP binding sites.29 Based on the
matic substructure. Finally, the larger
p system of an aromatic ring
may lead to more favorable stacking interactions in the active site
of GGDPS (e.g., with tyrosine 205 or phenylalanine 175).29 For
these reasons, we have pursued the chemical synthesis and biolog-
ical evaluation of aromatic-isoprenoid bisphosphonates, and we
report here these findings.
2. Chemical synthesis
The bisphosphonates described herein were synthesized using
parallel reaction sequences. For example, both compounds 12

7214
R. J. Barney et al. / Bioorg. Med. Chem. 18 (2010) 7212–7220
1) MsCl, TEA, 0 oC
2) LiBr, THF, 0 oC to rt
1) n-BuLi, -78 oC
2) prenyl bromide,
3) NaH, 15-Crown-5, TEMBP
0 oC to rt
47%
OR'
P(O)(OEt)2
P(O)(OEt)2
-78 oC to rt
OH
3) TBAF
72%
R
R
NaH, 15-Crown-5,
geranyl bromide
THF, 0 oC to rt
R
TBSCl
imidazole
90%
8 R = Br; R' = H
9 R = Br; R' =TBS
10 R = CH2CH=C(CH3)2
11 R = CH2CH=C(CH3)2
1) TMSBr, collidine
86%
1) TMSBr, collidine,
2) NaOH
90%
2) NaOH
88%
R''
R''
P(O)(ONa)2
P(O)(OEt)2
P(O)(ONa)2
P(O)(ONa)2
P(O)(OEt)2
P(O)(ONa)2
R
R
R
14 R = CH2CH=C(CH3)2
13 R = CH2CH=C(CH3)2
12 R = CH2CH=C(CH3)2
R'' =
Figure 3. Synthesis of bisphosphonates 12 and 14.
1) MsCl, TEA, 0 oC
2) LiBr, 0 oC to rt
3) NaH, 15-Crown-5
TEMBP, 0 oC to rt
1) n-BuLi, THF, -78 oC
2) prenyl bromide,
P(O)(OEt)2
P(O)(OEt)2
OR'
-78 oC to rt
OH
3) TBAF
77%
61%
R
R
R
NaH, 15-Crown-5,
geranyl bromide
THF, 0 oC to rt
TBSCl
15 R = Br; R' = H
16 R = Br; R' =TBS
17 R = CH2CH=C(CH3)2
imidazole
96%
18 R = CH2CH=C(CH3)2
1) TMSBr, collidine
2) NaOH
83% 59%
1) TMSBr, collidine,
2) NaOH
R''
R''
P(O)(ONa)2
P(O)(OEt)2
P(O)(ONa)2
P(O)(ONa)2
68%
R
P(O)(ONa)2
P(O)(OEt)2
R
R
21 R = CH2CH=C(CH3)2
20 R = CH2CH=C(CH3)2
19 R = CH2CH=C(CH3)2
R'' =
Figure 4. Synthesis of bisphosphonates 19 and 21.
P(O)(OEt)2
1) NaH, 15-crown-5
2) NaH, 15-crown-5
geranyl bromide
1) TMSBr, collidine
2) NaOH
R
R
P(O)(OEt)2
P(O)(ONa)2
P(O)(OEt)2
Br
Br
P(O)(ONa)2
P(O)(OEt)2
88%
40% (2 steps)
R
23
22
24
H
P(O)(OEt)2
NaH
15-crown-5
1) TMSBr, collidine
2) NaOH
R
R
P(O)(OEt)2
P(O)(ONa)2
P(O)(OEt)2
39%
P(O)(OEt)2
P(O)(ONa)2
89%
N
N
N
25
26
27
H
P(O)(ONa)2
R =
P(O)(ONa)2
28
Figure 5. Synthesis of bisphosphonates 24 and 27.

R. J. Barney et al. / Bioorg. Med. Chem. 18 (2010) 7212–7220
7215
esylation is made evident by the appearance of a more slowly
migrating, unmodified band on the gel. In contrast, Rap1a is geranyl-
geranylated in a reaction catalyzed by the enzyme geranylgeranyl
transferase I (GGTase I) and the antibody used here detects only
the unmodified form of the protein; thus accumulation of a detect-
able band represents impairment of geranylgeranylation.
At 48 h, lovastatin (Fig. 7A, lane 2), an inhibitor of HMG-CoA
reductase, depletes mevalonate resulting in a reduction of protein
farnesylation and geranylgeranylation. Compounds 14 and 21
diminish geranylgeranylation of Rap1a, while farnesylation of Ras
appeared unaffected at this level of detection (Fig. 7A). These com-
pounds also decreased geranylgeranylation of Rab6 (data not
shown), a GGTase II substrate. The in vitro data correlated well
with the cellular data, as the two potent in vitro GGDPS inhibitors
were the only compounds to show significantly diminished protein
geranylgeranylation. Compounds 24, 14, 21, and 27, all had similar
enzyme inhibitory effects on GGDPS under initial screening condi-
tions (Fig. 6A) while only compounds 14 and 21 impaired Rap1a
modification in intact cells at 50 lM (Fig. 7A). This implies that
intracellular levels of compounds 14 and 21 were higher than
those of compounds 24 and 27, which may be a consequence of
greater cellular influx or diminished efflux. Were the former to
be the case, then a prodrug approach might further enhance the
potency of these molecules by increasing cellular entry.27 Cellular
concentration–response assays also were performed with both
compounds 14 and 21 (Fig. 7B). Reduction of Rap1a geranylgerany-
Figure 6. Inhibition of GGDPS in vitro by novel bisphosphonates. (A) Compound
screen at 10 M of each compound (mean SD, n = 2). (B) Concentration response
of compounds 14 and 21 (mean SD, n = 2).
lation was apparent at concentrations as low as 12.5
bisphosphonate 14, while both compounds appeared to display
maximal effects at 50 M.
lM with
l
l
To define further the specificity of the compounds for GGDPS in
cells, ‘add-back’ experiments were performed wherein intermedi-
ates of the isoprenoid biosyntheticpathway were added in combina-
tion with the lead compounds (Fig. 8). Lovastatin again was used as a
positive control, where the reduction of protein farnesylation and
geranylgeranylation is prevented by addition of mevalonate. Fur-
thermore, FPP addition prevents lovastatin-induced reduction of
farnesylation, but not geranylgeranylation, and the converse is true
for GGPP. For bisphosphonates 14 and 21, no effects are noticed from
the addition of mevalonate or FPP, while the addition of GGPP pre-
vents the impairment of Rap1a and Rab6 geranylgeranylation
(Rab6 data not shown). This data supports the conclusion that only
GGDPS is inhibited in cells and not FDPS, GGTase I, or GGTase II.
Cell viability was determined in response to compound treat-
ment by determination of the amount of DNA synthesis with a
3H-thymidine incorporation assay (Fig. 9). Bisphosphonates 14
and 21 inhibited DNA synthesis both concentration and time
dependently, with the meta isomer 14 being the more potent
compound. These results suggest that the degree of impairment
of geranylgeranylation correlates with cytotoxicity.
structure of the most potent compounds identified herein, it would
be anticipated that these molecules bind GGDPS in a similar
manner.
This set of compounds then was tested against the K562 human
myelogenous leukemia cell line for inhibition of protein prenylation.
Western blots were performed and prenylation status of a panel of
proteins was determined (Fig. 7A). Because the antibodies available
to monitor protein prenylation have different specificities for mod-
ified and unmodified forms, these analyses must be interpreted with
special care. The Ras protein is farnesylated and reduction of farn-
Figure 8. Impairment of protein geranylgeranylation by compounds 14 and 21 was
prevented by exogenous GGPP. K562 cells were treated with lovastatin (10
novel bisphosphonates (14 and 21 at 50 M) in the presence or absence of
M), FPP (F, 20 M), and GGPP (GG, 20 M) for
lM) and
Figure 7. Impairment of protein prenylation in intact cells. (A) Compound screen.
(B) Concentration response of compounds 14 and 21. K562 cells were treated with
lovastatin (lov) and compounds as indicated for 48 h.
l
exogenous mevalonate (Mev, 500
48 h.
l
l
l

7216
R. J. Barney et al. / Bioorg. Med. Chem. 18 (2010) 7212–7220
1.30 equiv). The reaction mixture was allowed to warm to room
temperature and left to stir overnight. After the solution was
quenched by addition of H2O, it was extracted with CH2Cl2, dried
(MgSO4), and concentrated in vacuo. Final purification by flash
chromatography (8% EtOAc in hexanes) afforded the TBS protected
alcohol 9 (7.26 g, 90%). Both the 1H NMR and 13C NMR data corre-
lated to the literature values.34
5.3. Alcohol 1035
A stirred solution of silyl ether 9 (5.89 g, 19.5 mmol, 1.0 equiv)
in THF was cooled to À78 °C in a dry-ice/acetone bath. Once cool-
ing was complete, a solution of n-BuLi in hexanes (10.2 mL, 2.1 M,
21.5 mmol, 1.1 equiv) was added slowly via syringe, and the solu-
tion was allowed to stir for 15 min. Prenyl bromide (3.82 g,
25.6 mmol, 1.3 equiv) then was added dropwise via syringe. After
the solution was held at À78 °C for 2 h, it was allowed to warm
gradually to room temperature and stirred overnight. The resulting
mixture was quenched by addition of H2O, extracted with diethyl
ether, dried (MgSO4) and concentrated in vacuo. Without further
purification, the initial product was dissolved in THF to make
approximately a 2 M solution, cooled to 0 °C, and a 1 M solution
of TBAF in THF (23.3 mmol, 1.2 equiv) was added dropwise. The
reaction was allowed to stir for 4 h, and then quenched by addition
of H2O. The resulting mixture was extracted with diethyl ether,
dried (MgSO4), and concentrated in vacuo. Final purification by
flash chromatography (10% EtOAc in hexanes) gave compound 10
(2.48 g, 72% over two steps): 1H NMR d 7.20–7.26 (m, 1H), 7.06–
7.13 (m, 3H), 5.26–5.44 (m, 1H), 4.54 (s, 2H), 3.31 (d, J = 7.2 Hz,
2H), 2.57 (s, 1H), 1.73 (s, 3H),1.70 (s, 3H); 13C NMR d 142.0,
140.9, 132.4, 128.5, 127.4, 126.9, 124.3, 122.9, 65.0, 34.2, 25.6,
17.7; HRMS (ESI, m/z) calcd for (M)+ C12H16O: 176.1201. Found
176.1204.
Figure 9. Compounds 14 and 21 inhibited K562 cell proliferation. Cell proliferation
as a percentage of untreated cells at 48 and 72 h was evaluated by [3H]thymidine
incorporation (mean S.E., n = 4). (A) Compound 14. (B) Compound 21.
4. Conclusions
Although there has been some pursuit of multi-enzyme (i.e.,
FDPS and GGDPS) inhibitors within the mevalonate pathway as po-
tential anti-cancer agents,32 specificity remains the ideal for
molecular intervention. Compounds with the ability to inhibit a
single enzyme are very useful tools to study the interrelationships
of this complex system, and may have use in anti-cancer applica-
tions in the clinic.33 The aromatic bisphosphonates reported here,
and especially compounds 14 and 21, demonstrate selective inhibi-
tion of GGDPS over FDPS, and thus expand the list of tools available
for manipulation of isoprenoid biosynthesis. Further studies in this
vein will be reported in due course.
5.4. Tetraethyl bisphosphonate 11
A stirred solution of alcohol 10 (2.00 g, 11.4 mmol, 1.0 equiv) in
CH2Cl2 was cooled to 0 °C in an ice bath, and triethylamine
(2.01 mL, 14.5 mmol, 1.3 equiv) was added followed by dropwise
addition of MsCl (1.05 mL, 13.6 mmol, 1.2 equiv). After the result-
ing solution was allowed to stir for 30 min, a solution of LiBr
(2.52 g, 29.0 mmol, 2.6 equiv) in anhydrous THF was added via syr-
inge. The reaction mixture was allowed to stir for 1.5 h then
quenched by addition of H2O followed by addition of saturated
NaCl solution. The resulting solution was extracted with CH2Cl2,
and the organic extract was dried (Na2SO4), filtered, and concen-
trated in vacuo. The resulting material was utilized directly in
the following reaction without further purification.
To a stirred suspension of NaH (60% in oil, 437 mg, 10.9 mmol,
1.0 equiv) in THF was added 15-crown-5 (0.21 mL, 1.06 mmol,
0.1 equiv), and the resulting solution was cooled to 0 °C in an ice
bath. Tetraethyl methylenebisphosphonate (3.46 g, 12.0 mmol,
1.1 equiv) was added slowly via syringe and the reaction mixture
was allowed to stir for 30 min. After a solution of the benzylic bro-
mide (prepared above) in THF was added slowly via syringe, the
mixture was immediately removed from the ice bath and allowed
to stir overnight. The resulting solution was filtered through a bed
of florasil and concentrated in vacuo. Final purification by flash col-
umn chromatography (6% EtOH in hexanes) afforded the target bis-
phosphonate ester 11 (2.40 g, 47% overall yield from compound
10): 1H NMR d 7.11 (dd, J = 8.1, 7.2 Hz, 1H), 7.04–7.00 (m, 2H),
6.94 (d, J = 7.2 Hz, 1H), 5.25–5.16 (m, 1H), 4.12–3.93 (m, 8H),
3.23 (d, J = 8.1 Hz, 2H), 3.15 (td, JPH = 16.5, J = 6.0 Hz, 2H), 2.69–
2.47 (m, 1H), 1.66 (s, 3H), 1.64 (s, 3H), 1.20 (t, J = 7.2, 6H), 1.18
(t, J = 7.1 Hz, 6H); 13C NMR d 141.3, 139.3 (t, JCP = 7.3 Hz), 131.9,
128.5, 127.9, 126.1, 125.9, 122.9, 62.1 (dd, JCP = 13.4, 6.5 Hz, 4C),
5. Experimental procedures and methods
5.1. General experimental conditions
Tetrahydrofuran was freshly distilled from sodium/benzophe-
none, while methylene chloride was distilled from calcium hydride
prior to use. All other reagents and solvents were purchased from
commercial sources and used without further purification. All reac-
tions in nonaqueous solvents were conducted in flame-dried glass-
ware under a positive pressure of argon and with magnetic stirring.
All NMR spectra were obtained at 300 MHz for 1H, and 75 MHz for
13C with CDCl3 as solvent, and (CH3)4Si (1H, 0.00 ppm) or CDCl3
(
13C, 77.0 ppm) as internal standards unless otherwise noted. The
31P chemical shifts were reported in ppm relative to 85% H3PO4
(external standard). High resolution mass spectra were obtained
at the University of Iowa Mass Spectrometry Facility. Silica gel
(60 Å, 0.040–0.063 mm) was used for flash chromatography.
5.2. Silyl ether 934
To a solution of 3-bromobenzyl alcohol (4.99 g, 26.7 mmol,
1.0 equiv) in CH2Cl2 at 0 °C was added imidazole (8.89 g,
130 mmol, 4.90 equiv) followed by TBSCl (5.24 g, 34.7 mmol,

R. J. Barney et al. / Bioorg. Med. Chem. 18 (2010) 7212–7220
7217
38.7 (t, JCP = 131.5), 33.9, 30.8 (t, JCP = 4.7 Hz), 25.4, 17.4, 15.9 (d,
JCP = 6.4 Hz, 4C); 31P NMR d 23.6; HRMS (ESI, m/z) calcd for
(M)+C21H36O6P2: 446.1987. Found: 446.1991.
5.83–5.78 (m, 1H), 5.41–5.30 (m, 1H), 5.23–5.19 (m, 1H), 3.28 (d,
J = 6.9 Hz, 2H), 3.11 (t, J = 14.1 Hz, 2H), 2.40 (td, JPH = 15.6,
J = 6.0 Hz, 2H), 2.15–1.96 (m, 4H), 1.69 (s, 3H), 1.67 (s, 3H), 1.61 (s,
3H), 1.59 (s, 3H), 1.14 (s, 3H); 13C NMR 141.0, 140.8 (t, JCP = 7.8 Hz),
135.0, 134.2, 133.6, 131.7, 129.8, 128.2, 125.6, 125.2, 124.0 (t,
JCP = 5.9 Hz), 123.8, 45.5 (t, JCP = 109.8 Hz), 39.6, 37.4–37.2 (m),
34.0, 30.9–30.6 (m), 26.3, 25.2, 25.1, 17.4, 17.2, 15.7; 31P NMR
22.7; HRMS (ESI, m/z) calcd for (MÀH)À C23H35O6P2: 469.1909.
Found: 469.1911.
5.5. Bisphosphonate 12
A
stirred solution of bisphosphonate ester 11 (0.767 g,
1.72 mmol, 1.0 equiv) in CH2Cl2 was cooled to 0 °C in an ice bath.
After 2,4,6-collidine (1.14 mL, 8.60 mmol, 5.0 equiv) was added,
TMSBr (1.11 mL, 8.58 mmol, 5.0 equiv) was added slowly and the
reaction mixture was allowed to stir overnight. Toluene was added
and removed in vacuo. This process was repeated three times. After
drying the crude material, the residue was treated with NaOH solu-
tion (5 mL, 1.73 M, 8.65 mmol, 5.0 equiv) and allowed to stir at
room temperature overnight. Acetone was added to the solution
and the resulting material was allowed to cool at 3 °C for 72 h.
The solution was filtered, the solid was washed with several por-
tions of cold acetone, and then dried in vacuo to provide compound
12 (651 mg, 90%): 1H NMR (D20) d 7.26–7.14 (m, 3H), 6.99 (d,
J = 6.3 Hz, 1H), 5.39–5.34 (m, 1H), 3.29 (d, J = 7.2 Hz, 2H), 3.08
(td, JPH = 15.3, J = 6.3 Hz, 2H), 2.07 (tt, JPH = 21.9, J = 5.1 Hz, 1H),
1.68 (s, 3H), 1.67 (s, 3H); 13C NMR 145.5 (t, JCP = 6.6 Hz), 141.8,
134.2, 129.1, 128.6, 126.7, 125.1, 123.7, 42.8 (t, JCP = 117.8 Hz),
33.9, 33.7–33.5 (m), 25.1, 17.3; 31P NMR d 20.8; HRMS (ESI, m/z)
calcd for (MÀH)À C13H19O6P2: 333.0657. Found: 333.0662.
5.8. Silyl ether 1636
Imidazole (4.39 g, 64 mmol, 2.5 equiv) was added to a solution
of 4-bromobenzyl alcohol (4.73 g, 25.7 mmol, 1.0 equiv) in CH2Cl2.
The solution was cooled to 0 °C, TBSCl (4.70 g, 31.2 mmol,
1.2 equiv) was added, and the reaction mixture was allowed to stir
overnight. The solution was quenched by addition of H2O, ex-
tracted with CH2Cl2, dried (MgSO4), and concentrated in vacuo. Fi-
nal purification by flash chromatography (15% EtOAc in hexanes)
afforded the TBS protected alcohol 16 (7.40 g, 96%) with 1H and
13C NMR data corresponding to the literature values.36
5.9. Alcohol 17
A stirred solution of RJB-1-70 (6.61 g, 21.9 mmol, 1.0 equiv) in
THF was cooled to À78 °C in a dry-ice/acetone bath. Once cooling
was complete, n-BuLi (11.5 mL, 2.1 M in THF, 1.1 equiv) was added
slowly via syringe, and the solution was allowed to stir for 15 min.
Prenyl bromide (4.26 g, 28.6 mmol, 1.3 equiv) was added dropwise
via syringe. The reaction solution was held at À78 °C for 2 h, and
then allowed to gradually warm to room temperature and stir
overnight. The resulting mixture was quenched by addition of
H2O, extracted with diethyl ether, dried (MgSO4), and concentrated
in vacuo. The crude mixture was used in the next reaction without
additional purification.
A stirred solution of the above material in THF was cooled to
0 °C in an ice bath and a 1 M solution of TBAF in THF (27.1 mL,
27.1 mmol, 1.0 equiv) was added dropwise to the reaction vessel.
The reaction was allowed to stir for 4 h, then the solution was di-
luted with diethyl ether, and finally H2O was added. The resulting
mixture was extracted with diethyl ether, dried (MgSO4), and con-
centrated in vacuo. Final purification by column chromatography
(10% EtOAc in hexanes) afforded alcohol 17 (2.99 g, 77% over two
steps): 1H NMR d 7.20 (d, J = 8.1 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H),
5.25–5.33 (m, 1H), 4.51 (s, 2H), 3.31 (d, J = 7.5 Hz, 2H), 2.78 (s,
1H), 1.73 (s, 3H), 1.70 (s, 3H); 13C NMR d 141.0, 138.2, 132.4,
128.3 (2C), 127.1 (2C), 123.0, 64.7, 33.9, 25.6, 17.7; HRMS (ESI,
m/z) calcd for (M)+ C12H16O: 176.1201. Found: 176.1199.
5.6. Dialkylbisphosphonate 13
A suspension of NaH (60% in oil, 202 mg, 5.15 mmol, 2.4 equiv)
in THF was cooled to 0 °C in an ice bath. Once the solution was
thoroughly cooled, 15-crown-5 (0.05 mL, 0.3 mmol, 0.1 equiv)
was added followed ester 11 (0.956 g, 2.14 mmol, 1.0 equiv) via
syringe. The solution was allowed to stir for 30 min, then geranyl
bromide (0.816 g, 3.76 mmol, 1.8 equiv) was added, and the solu-
tion was allowed to stir overnight. After the reaction mixture
was filtered through a bed of florasil, the filtrate was concentrated
in vacuo. Final purification via flash chromatography (4% EtOH in
hexanes) afforded the desired compound 13 (1.08 g, 86% yield):
1H NMR d 7.15–7.12 (m, 3H), 7.03–6.97 (m, 1H), 5.69–5.64 (m,
1H), 5.34–5.26 (m, 1H), 5.17–5.12 (m, 1H), 4.18–4.02 (m, 8H),
3.32–3.21 (m, 4H), 2.64 (td, JPH = 16.2, J = 5.7 Hz, 2H), 2.16–2.05
(m, 4H), 1.72 (s, 3H), 1.71 (s, 3H), 1.67 (s, 3H), 1.63 (s, 3H), 1.61
(s, 3H), 1.25 (t, J = 6.6 Hz, 6.9 Hz, 6H), 1.21 (t, J = 6.9 Hz, 6H); 13C
NMR d 140.7, 136.8, 136.3 (t, JCP = 7.1 Hz), 132.0, 131.5, 131.3,
128.8, 127.4, 126.3, 124.2, 123.4, 119.2 (t, JCP = 7.6 Hz), 62.4–62.1
(m, 4C), 47.4 (t, JCP = 129.7 Hz), 39.9, 35.1–34.9 (m), 34.3, 28.2–
28.0 (m), 26.5, 25.6, 25.5, 17.6, 17.5, 16.3, 16.2–15.8 (m, 4C); 31P
NMR
d
26.2; HRMS (ESI, m/z) calcd for (M)+ C31H52O6P2:
582.3239. Found: 582.3235.
5.10. Tetraethyl bisphosphonate 18
5.7. Bisphosphonate 14
A stirred solution of alcohol 17 (4.46 g, 25.3 mmol, 1.0 equiv) in
CH2Cl2 was cooled to 0 °C in an ice bath. To the cooled solution was
added anhydrous triethylamine (4.58 mL, 40.0 mmol, 1.6 equiv)
followed by dropwise addition of MsCl (2.36 mL, 30.5 mmol,
1.2 equiv), and the resulting solution was allowed to stir for
30 min. After LiBr (5.55 g, 63.9 mmol, 2.5 equiv) was dissolved in
THF, the solution was transferred via syringe into the reaction ves-
sel. The reaction mixture was allowed to stir for 1.5 h and then the
solution was quenched by addition of H2O followed by addition of
saturated NaCl solution. The resulting solution was extracted with
CH2Cl2, dried (Na2SO4), and concentrated in vacuo. Without addi-
tional purification, the product was utilized in the following
reaction.
A stirred solution of compound 13 (70 mg, 0.12 mmol, 1.0 equiv)
in CH2Cl2 was cooled to 0 °C in an ice bath. After 2,4,6-collidine
(0.12 mL, 0.91 mmol, 7.6 equiv) was added, slow addition of TMSBr
(0.12 mL, 0.93 mmol, 7.8 equiv) was done and the resulting solution
was allowed to stir overnight. Toluene was added and removed in
vacuo, three times. The resulting residue was treated with NaOH
solution (1.0 M, 0.9 mL, 0.90 mmol, 7.5 equiv) and allowed to stir
at room temperature overnight. Acetone was added to the solution
and the resulting material was stored at 3 °C for 72 h. The suspen-
sion was filtered and the solid material was washed with several
portions of anhydrous acetone. The precipitate then was dissolved
in H2O, filtered, and the aqueous portion was concentrated in vacuo
to give compound 14 as a fine white powder (59 mg, 88%): 1H NMR d
7.36 (d, J = 7.5 Hz, 1H), 7.30–7.12 (m, 2H), 7.02 (d, J = 7.5 Hz, 1H),
To a stirred suspension of NaH (60% in oil, 0.932 g, 23.3 mmol,
1.0 equiv) in THF was added 15-crown-5 (0.51 mL, 2.58 mmol,

7218
R. J. Barney et al. / Bioorg. Med. Chem. 18 (2010) 7212–7220
0.1 equiv) and the resulting solution was cooled to 0 °C in an ice
bath. Tetraethyl methylenebisphosphonate (7.38 g, 25.6 mmol,
1.1 equiv) was added slowly via syringe and the reaction was al-
lowed to stir for 30 min to facilitate complete formation of the an-
ion. After a solution of the benzylic bromide in THF was added
slowly via syringe to the reaction vessel, the resulting mixture
was immediately removed from the ice bath and allowed to stir
overnight. After the solution was filtered through a bed of florasil
and concentrated in vacuo, final purification by flash column chro-
matography (gradient 6–8% EtOH in hexanes) gave the target bis-
phosphonate 18 (6.89 g, 61% over two steps): 1H NMR d 7.11 (dd,
J = 8.3, 2.1 Hz, 2H), 7.01 (dd, J = 7.8, 2.1 Hz, 2H), 5.23–5.16 (m,
5.13. Dialkylbisphosphonate 21
A stirred solution of ester 20 (0.915 g, 1.57 mmol, 1.00 equiv) in
CH2Cl2 was cooled to 0 °C in an ice bath. After 2,4,6-collidine
(1.06 mL, 7.99 mmol, 5.09 equiv) was added and slow addition of
TMSBr (1.01 mL, 7.81 mmol, 4.97 equiv) the solution was allowed
to stir overnight. Toluene was added and removed in vacuo. The
resulting residue was treated with NaOH (1.04 M, 6.4 mL,
6.66 mmol, 4.2 equiv) and allowed to stir at room temperature over-
night. Acetone was added to the solution and the resulting suspen-
sion was stored at 3 °C for 72 h. The suspension was filtered, and
the filtrand was washed with several aliquots of anhydrous acetone.
The resulting solid was dissolved in H2O, filtered, and concentrated
in vacuo to provide compound 21 (598 mg, 68%): 1H NMR d 7.47 (d,
J = 7.8 Hz, 2H), 7.14 (d, J = 7.8 Hz, 2H), 5.88–5.82 (m, 1H), 5.47–5.40
(m, 1H), 5.32–5.25 (m, 1H), 3.35 (d, J = 7.5 Hz, 2H), 3.17
(t, JPH = 14.4 Hz, 2H), 2.47 (td, JPH = 15.9, J = 6.6 Hz, 2H), 2.22–2.03
(m, 4H), 1.76 (s, 6H), 1.69 (s, 3H), 1.67 (s, 3H), 1.54 (s, 3H); 13C
NMR 141.8, 140.7 (t, JCP = 7.3 Hz), 137.4, 136.7, 136.0, 134.9 (2C),
1H), 4.10–3.94 (m, 8H), 3.22 (d, J = 7.2 Hz, 2H), 3.09 (td, JPH
=
16.5, J = 6.3 Hz, 2H), 2.56 (tt, JPH = 23.7, J = 6.3 Hz, 1H), 1.65 (s,
3H), 1.63 (s, 3H), 1.20 (t, J = 7.2, 6H), 1.17 (t, J = 7.2 Hz, 6H), 13C
NMR d 139.8, 136.7 (t, JCP = 7.4 Hz), 132.1, 128.7 (2C), 127.9 (2C),
123.1, 62.2 (dd, JCP = 13.8, 6.8 Hz, 4C), 38.9 (t, JCP = 131.3 Hz),
33.7, 30.6 (t, JCP = 4.9 Hz), 25.5, 17.6, 16.1 (d, JCP = 7.2 Hz, 4C), 31P
NMR
d 23.6; HRMS (ESI, m/z) calcd for (M)+ C21H36O6P2:
446.1987. Found: 446.1989.
130.0 (2C), 127.8, 126.9 (t, JCP = 6.8 Hz), 126.2, 48.3 (t, JCP =
110.6 Hz), 42.1, 39.7–39.5 (m, 1C), 36.0, 33.6–33.4 (m, 1C), 28.7,
27.7, 27.6, 19.8 (2C), 18.0; 31P NMR 23.4; HRMS (ESI, m/z) calcd for
(MÀH)À C23H35O6P2: 469.1909. Found: 469.1912.
5.11. Bisphosphonate 19
A
stirred solution of compound 18 (289 mg, 0.64 mmol,
5.14. Dialkylbisphosphonate 23
1.0 equiv) in CH2Cl2 was cooled to 0 °C in an ice bath. After 2,4,6-col-
lidine (0.70 mL, 5.28 mmol, 8.3 equiv) was added and slow addition
of TMSBr (0.69 mL, 5.34 mmol, 8.2 equiv) the resulting was allowed
to stir overnight. Toluene was added and removed in vacuo three
times. The remaining residue was treated with NaOH solution
(1 M, 3.2 mL, 3.20 mmol, 5.0 equiv) and allowed to stir at room tem-
perature overnight. Acetone was added to the solution and the
resulting suspension was stored at 3 °C for 72 h. The solution was
filtered and the solid was washed with several portions of cold ace-
tone and dried in vacuo giving compound 19 (162 mg, 59%) 1H NMR
(D2O) d 7.38 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 5.46–5.37 (m,
1H), 3.35 (d, J = 7.2 Hz, 2H), 3.11 (td, JPH = 15.6, J = 6.3 Hz, 2H), 2.18
(tt, JPH = 21.6, J = 6.3 Hz, 1H), 1.76 (s, 6H); 13C NMR 143.6 (t,
JCP = 7.3 Hz), 142.0, 136.8, 132.0 (2C), 130.7 (2C), 126.1, 44.6 (t,
JCP = 112.4 Hz), 36.0, 34.0–34.7 (m), 27.6, 19.8; 31P NMR d 20.6;
HRMS (ESI, m/z) calcd for (MÀH)À C13H19O6P2: 333.0657. Found:
333.0677.
To a stirred suspension of NaH (16.7 mmol, 1.4 equiv) in THF at
0 °C was added 15-crown-5 (0.91 mmol, 0.1 equiv) followed by
dropwise addition of the known tetraethyl benzyl bisphospho-
nate.30 The resulting solution was allowed to stir for 30 min and
geranyl bromide (12.1 mmol, 1.0 equiv) was added dropwise as a
neat liquid. The resulting solution was allowed to stir overnight,
dried (MgSO4), filtered, and the filtrate was concentrated in vacuo.
Final purification by flash column chromatography (gradient 2–
10% MeOH in diethyl ether) gave compound 23 (1.97 g, 40% over
two steps): 1H NMR d 7.31 (dd, J = 7.8, 1.8 Hz, 2H), 7.25–7.17 (m,
3H), 5.67–5.60 (m, 1H), 5.15–5.08 (m, 1H), 4.17–3.99 (m, 8H),
3.27 (dd, JPH = 16.5, 12.9 Hz, 2H), 2.61 (td, JPH = 15.3, J = 6.0 Hz,
2H), 2.15–2.03 (m, 4H), 1.66 (s, 3H), 1.60 (s, 6H), 1.23 (t, J = 6.9,
6H), 1.19 (t, J = 6.9 Hz, 6H); 13C NMR d 136.9, 136.4 (t, JCP = 8.6 Hz),
131.5 (2C), 131.4, 127.4 (2C), 126.4, 124.3, 119.2 (t, JCP = 7.7 Hz),
62.5–62.1 (4C, m), 47.4 (t, JCP = 129.6 Hz), 40.0, 35.3–35.1 (m),
28.4–28.2 (m), 26.5, 25.7, 17.7, 16.4, 16.3–16.1 (m, 4C); 31P NMR
d
26.2; HRMS (M+Na)+ calcd C26H44O6P2: 537.2511. Found:
5.12. Dialkylbisphosphonate 20
537.2515.
A suspension of NaH (60% in oil, 215 mg, 5.37 mmol, 2.4 equiv)
in THF was cooled to 0 °C in an ice bath. Once the solution was
thoroughly cooled, 15-crown-5 (0.05 mL, 0.3 mmol, 0.1 equiv)
was added followed by the addition of compound 18 (1.02 g,
2.27 mmol, 1.0 equiv) via syringe. The resulting solution was al-
lowed to stir for 30 min, geranyl bromide (849 mg, 3.91 mmol,
1.7 equiv) was added, and the solution was allowed to stir over-
night. The reaction mixture was filtered through a bed of florasil
and the filtrate was concentrated in vacuo. Final purification via
flash chromatography (4% EtOH in hexanes) afforded the desired
compound (1.09 g, 83%): 1H NMR d 7.23 (d, J = 6.9 Hz, 2H), 7.03
(d, J = 7.2 Hz, 2H), 5.68–5.61 (m, 1H), 5.33–5.24 (m, 1H), 5.18–
5.10 (m, 1H), 4.15–4.02 (m, 8H), 3.30–3.21 (m, 4H), 2.62 (td,
JHP = 15.9, J = 6.3 Hz, 2H), 2.18–2.01 (m, 4H), 1.72 (s, 3H), 1.70 (s,
3H), 1.67 (s, 3H), 1.62 (s, 6H), 1.24 (t, J = 6.9 Hz, 6H), 1.20 (t,
J = 7.2 Hz, 6H); 13C NMR d 139.7, 136.6, 133.4 (t, JCP = 7.5 Hz),
132.0, 131.3 (2C), 131.1, 127.2 (2C), 124.2, 123.3, 119.1 (t,
JCP = 7.7 Hz), 62.3–62.0 (m, 4C), 47.4 (JCP = 129.6 Hz), 39.9, 34.7–
34.5 (m), 33.8, 28.2–27.9 (m), 26.4, 25.6 (2C), 17.6, 17.5, 16.3,
16.2–16.0, (m, 4C); 31P NMR d 25.6; HRMS (ESI, m/z) calcd for
(M)+ C31H52O6P2: 582.3239. Found: 582.3238.
5.15. Bisphosphonate 24
To a solution of 23 (1.66 mmol, 1.0 equiv) in CH2Cl2 at 0 °C,
were added collidine (13.4 mmol, 8.09 equiv) and TMSBr
(13.3 mmol, 8.1 equiv) as neat liquids. After 1 h the solution was
allowed to warm gradually to room temperature and stirred over-
night. After the solvent was removed in vacuo, toluene (15 mL) was
added and removed in vacuo. An aqueous solution of NaOH
(7.4 mL, 0.9 M, 6.66 mmol, 4.0 equiv) was added, and the mixture
was stirred for 1.75 h. The resulting mixture was then poured into
acetone and stored at 3 °C for 72 h. The resulting suspension was
filtered and the filtrand was washed with several portions of anhy-
drous acetone. The remaining solid was dissolved in H2O, filtered,
and the filtrate was concentrated in vacuo to provide the desired
compound 24 (746 mg, 88% yield): 1H NMR (D2O) d 7.48 (dd,
J = 8.1, 1.2, 2H), 7.31–7.16 (m, 3H), 5.79–5.69 (m, 1H), 5.27- 5.18
(m, 1H), 3.15 (t, JPH = 15.0 Hz, 2H), 2.46 (td, JPH = 15.6, J = 6.9 Hz,
2H), 2.15–2.01 (m, 4H), 1.64 (s, 3H), 1.61 (s, 3H), 1.55 (s, 3H) 13C
NMR 139.0 (t, JCP = 7.2 Hz), 137.0, 133.7, 131.8 (2C), 127.8 (2C),
126.3, 125.1, 121.8 (t, JCP = 7.4 Hz), 46.0 (t, JCP = 111.4 Hz), 39.6,

R. J. Barney et al. / Bioorg. Med. Chem. 18 (2010) 7212–7220
7219
36.5 (br s, 1C), 29.9 (br s, 1C), 26.0, 25.2, 17.2, 15.6; 31P NMR d 23.3;
HRMS (MÀH)À calcd C18H27O6P2: 401.1283. Found: 401.1288.
with IPTG. Proteins were purified by batch centrifugation with glu-
tathione agarose. The GGDPS reaction mixtures contained 20
FPP and 40 L buffer (50 mM imidazole pH 7.5,
14C-IPP in 20
lM
lM
l
5.16. Bisphosphonate 26
0.5 mM MgCl2, 0.5 mM ZnCl2). Following a 10-min pre-incubation
with the indicated compounds, reactions were initiated by simul-
taneous addition of 14C-IPP and FPP. Reactions proceeded for 1 h
at 37 °C, and then the longer isoprenoids were extracted with
1 mL saturated butanol and the extracts were washed twice with
Sodium hydride (60% in oil, 588 mg, 14.7 mmol, 2.5 equiv) was
placed in a 3-neck flask fitted with two septa and a solid addition
tube filled with 3-(bromomethyl)pyridine HBr (1.9 g, 7.76 mmol,
1.3 equiv), and the apparatus was purged with argon. Anhydrous
THF was added and the solution was cooled to 0 °C in an ice bath.
After 15-crown-5 (0.31 mL, 1.57 mmol, 0.3 equiv) was added,
monogeranyl bisphosphonate RJB-1–51A was added dropwise as
a neat liquid. The solution was allowed to stir for 30 min, the pyr-
idine salt was added via the solid addition tube, and the reaction
was allowed to stir overnight. After H2O was added, the aqueous
fraction was extracted with diethyl ether, dried (Na2SO4), filtered,
and the filtrate was concentrated in vacuo. Final purification by
flash column chromatography (8% MeOH in Et2O) afforded the de-
sired bisphosphonate 26 (3.01 g, 89%): 1H NMR d 8.46 (s, 1H), 8.36
(d, J = 3.9 Hz, 1H), 7.59 (d, J = 7.5 Hz, 1H), 7.10 (dd, J = 7.5, 5.1 Hz,
1H), 5.59–5.51 (m, 1H), 5.08–5.01 (m, 1H), 4.13–3.95 (m, 8H),
3.18 (t, JPH = 15.3, 2H), 2.54 (td, JPH = 15.6, J = 6.0 Hz, 2H), 2.09–
1.89 (m, 4H), 1.57 (s, 3H), 1.54 (s, 3H), 1.52 (s, 3H), 1.20 (t,
J = 7.2 Hz, 6H), 1.15 (t, J = 7.2 Hz, 6H); 13C NMR d 152.2, 147.5,
138.6, 137.5, 132.2 (t, JCP = 7.7 Hz), 131.3, 124.1, 122.3, 118.6 (t,
JCP = 7.6 Hz), 62.6–62.3 (m, 4C), 47.0 (t, JCP = 130.2 Hz), 39.9, 32.8
(t, JCP = 4.1 Hz), 28.4 (t, JCP = 4.6 Hz), 26.4, 25.6, 17.5, 16.3, 16.2–
16.0 (m, 4C); 31P NMR d 25.7; HRMS (M+Na)+ calcd C25H43NO6P2:
538.2463. Found: 538.2468.
300 lL saturated water. The amount of radioactivity in the butanol
extracts were detected by liquid scintillation counting.
5.20. Western blot analysis
The K562 cells were diluted to
a final concentration of
5 Â 105 cells/mL. After 5 mL of cell suspension was added to 6-well
plates in the presence of compounds, the plates were incubated for
48 h. Cells were harvested by centrifugation and lysed (2% SDS in
66 mM Tris) by passing cells several times through a 27G needle. Ly-
sates were cleared by centrifugation and protein concentration
determined by the bicinchoninic acid (BCA) assay. Proteins were re-
solved by electrophoresis on 12% and 15% gels and transferred to
PVDF membrane. Membranes were blocked in 5% non-fat dry milk
for 40 min at 37 °C. Primary and secondary antibodies were added
sequentially for 1 h at 37 °C and proteins were visualized with ECL
reagents. Anti pan-Ras antibody was obtained from InterBiotechnol-
ogy (Tokyo, Japan). The Rap1a (sc-1482), Rab6 (sc-310), and a-tubu-
lin (sc-8025) antibodies were obtained from Santa Cruz
biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conju-
gated anti-mouse and anti-rabbit were obtained from GE Healthcare
(Buckinghamshire, UK), and horseradish peroxidase-conjugated
anti-goat was obtained from Santa Cruz Biotechnology, Inc.
For the add-back experiments, isoprenoid pathway intermedi-
ates (mevalonate, FPP, or GGPP) were added at indicated concen-
trations simultaneously with isoprenoid pathway inhibitors
(lovastatin, compound 14, or compound 21).
5.17. Bisphosphonate 27
A solution of compound 26 (329 mg, 0.64 mmol, 1.0 equiv) in
CH2Cl2 was cooled to 0 °C in an ice bath. After 2,4,6-collidine
(0.63 mL, 4.75 mmol, 7.4 equiv) and TMSBr (0.62 mL, 4.79 mmol,
7.5 equiv) was added, the reaction mixture was stirred overnight.
Toluene was added and removed in vacuo, three times. The residue
was treated with NaOH solution (1.56 M, 2.45 mL, 6.66 mmol,
6.0 equiv) and allowed to stir at room temperature overnight. Ace-
tone was added to the solution and the resulting material was al-
lowed to stand at 3 °C for 72 h. The resulting suspension was
filtered, and the filtrand was washed with several aliquots of anhy-
drous acetone. The solids were then dissolved in water, filtered,
and the filtrate was concentrated in vacuo to provide the target
compound (121 mg, 39%): 1H NMR (D2O) d 8.44 (d, J = 2.1 Hz,
1H), 8.18 (dd, J = 5.1, 1.5 Hz, 1H), 7.94 (ddd, J = 8.4, 1.8, 1.5 Hz,
1H), 7.21 (dd, J = 7.8, 5.1 Hz, 1H), 5.75–5.70 (m, 1H), 5.20–5.14
(m, 1H), 3.07 (t, JPH = 15.0, Hz, 2H), 2.39 (td, JPH = 14.1, J = 7.5 Hz,
2H), 2.09–1.92 (m, 4H), 1.56 (s, 6H), 1.44 (s, 3H); 13C NMR 153.7,
147.9, 143.0, 139.4 (t, JCP = 6.9 Hz), 138.0, 135.9, 127.5, 126.6 (t,
JCP = 6.7 Hz), 125.6, 48.3 (t, JCP = 112.3 Hz), 42.0, 37.5–37.3 (m),
33.4–33.2 (m), 28.5, 27.5, 19.6, 17.8; 31P NMR d 22.7; HRSM (ESI,
m/z), (MÀH)À calcd C17H26NO6P2: 402.1235. Found: 402.1244.
5.21. DNA synthesis assay
Two hundred microliters of K562 cells were incubated in 96-
well plates and treated with compounds as described previously.25
The 48 h experiments required 2 Â 105 cells/mL while 72 h exper-
iments required 1 Â 105 cells/mL. After 44 or 68 h, 20
lL of
[3H]thymidine (0.1385 TBq/mmol; 3.75 Ci/mmol in media) was
added to each well. At 48 or 72 h, cells were filtered through glass
microfiber paper using a Brandel (Gaithersburg, MD) cell harvester.
[3H]Thymidine incorporated into cellular DNA was quantified by
scintillation counting.
Acknowledgment
This project was supported by the Roy. J. Carver Charitable Trust
as a Research Program of Excellence.
Supplementary data
5.18. Cell culture
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.036.
K562 leukemia cells were obtained from American Type Culture
Collection (Manassas, VA). Cells were maintained in RPMI 1640
medium supplemented with 10% heat-inactivated fetal bovine ser-
um at 37 °C and 5% CO2.
References and notes
1. Licata, A. A. Ann. Pharmacother. 2005, 39, 668.
2. (a) Gumienna-Kontecka, E.; Jezierska, J.; Lecouvey, M.; Leroux, Y.; Kozlowski, H.
J. Inorg. Biochem. 2002, 89, 13; (b) Gumienna-Konteka, E.; Silvagni, R.; Lipinski,
R.; Lecouvey, M.; Marincola, F. C.; Crisponi, G.; Nurchi, V. M.; Leroux, Y.;
Kozlowski, H. Inorg. Chim. Acta 2002, 339, 111; (c) Bailly, T.; Burgada, R.; Prange,
T.; Lecouvey, M. Tetrahedron Lett. 2003, 44, 189.
5.19. GGDPS in vitro assay
The GGDPS in vitro assay was performed as described previ-
ously.26 Briefly, plasmids containing GST-tagged recombinant hu-
man GGDPS were expressed in BL21 gold bacteria by induction
3. Fleisch, H. Der Othopade 2007, 36, 103.

7220
R. J. Barney et al. / Bioorg. Med. Chem. 18 (2010) 7212–7220
20. Coxon, F. P. H. M. H.; Van’t Hof, R.; Sebti, S.; Ralston, S. H.; Hamilton, A.; Rogers,
4. Fleisch, H. Eur. Spine J. 2003, 12, S142.
5. Guise, T. A. Cancer Treat. Rev. 2008, 34, S19.
M. J. J. Bone Miner. Res. 2000, 15, 1467.
6. Martin, M. B.; Grimley, J. S.; Lewis, J. C.; Heath, H. T., 3rd; Bailey, B. N.; Kendrick,
H.; Yardley, V.; Caldera, A.; Lira, R.; Urbina, J. A.; Moreno, S. N.; Docampo, R.;
Croft, S. L.; Oldfield, E. J. Med. Chem. 2001, 44, 909.
21. van Beek, E.; Lowik, C. W.; Papapoulos, S. E. Bone 2002, 30, 64.
22. Goffinet, M.; Thoulouzan, M.; Pradines, A.; Lajoie-Mazene, I.; Weinbaum, C.;
Taye, J. C.; Seronie-Vivien, S. BMC Cancer 2006, 6, 60.
7. Yardley, V.; Khan, A. A.; Martin, M. B.; Slifer, T. R.; Aruajo, F. G.; Moreno, S. N.;
Docampo, R.; Croft, S. L.; Oldfield, E. Antimicrob. Agents Chemother. 2002, 46,
929.
23. Krueger, K. E.; Srivastava, S. Mol. Cell. Proteomics 2006, 5, 1799.
24. Shull, L. W.; Wiemer, A. J.; Hohl, R. J.; Wiemer, D. F. Bioorg. Med. Chem. 2006, 14,
4130.
8. Shipman, C. M.; Rogers, M. J.; Apperley, J. F.; Graham, R.; Russell, G.; Croucher,
P. I. Br. J. Haematol. 1997, 98, 665.
25. Wiemer, A. J.; Yu, J. S.; Lamb, K. M.; Hohl, R. J.; Wiemer, D. F. Bioorg. Med. Chem.
2008, 16, 390.
9. Kunzmann, V.; Bauer, E.; Feurle, J.; Weissinger, F.; Tony, H. P.; Wilhelm, M.
Blood 2000, 96, 384.
26. Wiemer, A. J.; Tong, H.; Swanson, K. M.; Hohl, R. J. Biochem. Biophys. Res.
Commun. 2007, 353, 921.
10. Sanders, J. M.; Ghosh, S.; Chan, J. M.; Meints, G.; Wang, H.; Raker, A. M.; Song,
Y.; Colantino, A.; Burzynska, A.; Kafarski, P.; Morita, C. T.; Oldfield, E. J. Med.
Chem. 2004, 47, 375.
11. Ciosek, C. P., Jr.; Magnin, D. R.; Harrity, T. W.; Logan, J. V. H.; Dickson, J. K., Jr.;
Gordon, E. M.; Hamilton, K. A.; Jolibois, K. G.; Kunselman, L. K.; Lawrence, R. M.
J. Biol. Chem. 1993, 268, 24832.
12. Magnin, D. R.; Dickson, J. K., Jr.; Logan, J. V.; Lawrence, R. M.; Chen, Y.; Sulsky, R.
B., ; Ciosek, C. P., Jr.; Biller, S. A.; Harrity, T. W.; Jolibois, K. G.; Kunselman, L. K.;
Rich, L. C.; Slusarchyk, D. A. J. Med. Chem. 1995, 38, 2596.
13. Thompson, K.; Dunford, J. E.; Ebetino, F. H.; Rogers, M. J. Biochem. Biophys. Res.
Commun. 2002, 290, 869.
27. Wiemer, A. J.; Yu, J. S.; Shull, L. W.; Barney, R. J.; Wasko, B. M.; Lamb, K. M.;
Hohl, R. J.; Wiemer, D. F. Bioorg. Med. Chem. 2008, 16, 3652.
28. Zhang, Y.; Leon, A.; Song, Y.; Studer, D.; Haase, C.; Koscielski, L. A.; Oldfield, E. J.
Med. Chem. 2006, 49, 5804.
29. Chen, C. K. M.; Hudock, M. P.; Zhang, Y.; Guo, R. T.; Cao, R.; No, J. H.; Liang, P. H.;
Ko, T. P.; Chang, T. H.; Chang, S.; Song, Y.; Axelson, J.; Kumar, A.; Wang, A. H. J.;
Oldfield, E. J. Med. Chem. 2008, 51, 5594.
30. (a) Nguyen, L. M.; Niesor, E.; Bentzen, C. L. J. Med. Chem. 1987, 30, 1426; (b)
Lolli, M. L. J. Organomet. Chem. 2002, 650, 77.
31. Kavanagh, K. L.; Dunford, J. E.; Bunkoczi, G.; Russell, R. G.; Oppermann, U. J. Biol.
Chem. 2006, 281, 20004.
14. van Beek, E.; Pieterman, E.; Cohen, L.; Lowik, C.; Papapoulos, S. Biochem.
Biophys. Res. Commun. 1999, 255, 491.
15. van Beek, E.; Pieterman, E.; Cohen, L.; Lowik, C.; Papapoulos, S. Biochem.
Biophys. Res. Commun. 1999, 264, 108.
16. Cheng, F.; Oldfield, E. J. Med. Chem. 2004, 47, 5149.
17. Holstein, S. A.; Cermak, D. M.; Wiemer, D. F.; Lewis, K.; Hohl, R. J. Bioorg. Med.
Chem. 1998, 6, 687.
18. van Beek, E.; Lowik, C.; van der Pluijim, G.; Papapoulos, S. J. Bone Miner. Res.
1999, 14, 722.
32. Zhang, Y.; Cao, R.; Yin, F.; Hudock, M. P.; Guo, R. T.; Krysiak, K.; Mukherjee, S.;
Gao, Y. G.; Robinson, H.; Song, Y.; No, J. H.; Bergan, K.; Leon, A.; Cass, L.;
Goddard, A.; Chang, T. K.; Lin, F. Y.; Van Beek, E.; Papapoulos, S.; Wang, A. H.;
Kubo, T.; Ochi, M.; Mukkamala, D.; Oldfield, E. J. Am. Chem. Soc. 2009, 131, 5153.
33. Wiemer, A. J.; Hohl, R. J.; Wiemer, D. F. Anti-Cancer Agents Med. Chem. 2009, 9,
526.
34. Bonifazi, D.; Accorsi, G.; Armaroli, N.; Song, F. Y.; Palkar, A.; Echegoyen, L.;
Scholl, M.; Seiler, P.; Jaun, B.; Diederich, F. Helv. Chim. Acta 2005, 88, 1839.
35. Matsuda, K.; Hamada, M.; Nishimura, K.; Fujita, T. Pesticide Biochem. Physiol.
1989, 35, 300.
19. Fisher, J. E.; Rogers, M. J.; Halasy, J. M.; Luckman, S. P.; Hughes, D. E.;
Masarachia, P. J.; Wesolowski, G.; Russell, R. G.; Rodan, G. A.; Reszka, A. A. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 133.
36. Bennett, S. M.; Tang, Y.; McMaster, D.; Bright, F. V.; Detty, M. R. J. Org. Chem.
2008, 73, 6849.