Pyridinium-1-yl bisphosphonates are potent inhibitors of farnesyl diphosphate synthase and bone resorption

Article abstract of DOI:10.1021/jm040209d

We report the design, synthesis and testing of a series of novel bisphosphonates, pyridinium-1-yl-hydroxy-bisphosphonates, based on the results of comparative molecular similarity indices analysis and pharmacophore modeling studies of farnesyl diphosphate

Full text of DOI:10.1021/jm040209d

J. Med. Chem. 2005, 48, 2957-2963
2957
Pyridinium-1-yl Bisphosphonates Are Potent Inhibitors of Farnesyl
Diphosphate Synthase and Bone Resorption
†
†
†
†
†
John M. Sanders, Yongcheng Song, Julian M. W. Chan, Yonghui Zhang, Samuel Jennings,
†
†
†
†
†
Thomas Kosztowski, Sarah Odeh, Ryan Flessner, Christine Schwerdtfeger, Evangelia Kotsikorou,
†
‡
‡
§
§
Gary A. Meints, Aurora Ortiz G o´ mez, Dolores Gonz a´ lez-Pacanowska, Amy M. Raker, Hong Wang,
|
|
§
,†,⊥
Ermond R. van Beek, Socrates E. Papapoulos, Craig T. Morita, and Eric Oldfield*
Department of Chemistry, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801;
L o´ pez-Neyra Institute of Parasitology and Biomedicine, CSIC, Granada, Spain; Department of Internal Medicine and the
Interdisciplinary Group in Immunology, University of Iowa, EMRB 340F, Iowa City, Iowa 52242; Department of Endocrinology
and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands; and Department of Biophysics,
600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
Received December 3, 2004
We report the design, synthesis and testing of a series of novel bisphosphonates, pyridinium-
-yl-hydroxy-bisphosphonates, based on the results of comparative molecular similarity indices
1
analysis and pharmacophore modeling studies of farnesyl diphosphate synthase (FPPS)
inhibition, human Vγ2Vδ2 T cell activation and bone resorption inhibition. The most potent
molecules have high activity against an expressed FPPS from Leishmania major, in Dictyos-
telium discoideum growth inhibition, in γδ T cell activation and in an in vitro bone resorption
assay. As such, they represent useful new leads for the discovery of new bone resorption,
antiinfective and anticancer drugs.
Introduction
Given their proven clinical utility, there is interest in
their further development.
In recent work, we carried out a series of quantitative
Nitrogen-containing bisphosphonates such as pam-
idronate (Aredia, 1), alendronate (Fosamax, 2), rise-
dronate (Actonel, 3) and zoledronate (Zometa, 4), shown
below in their zwitterionic forms, represent an impor-
structure-activity relationship (QSAR) studies of
1
7
the activity of bisphosphonates in FPPS inhibition,
Vγ2Vδ2 T cell activation30 and bone resorption 3 using
32
3
comparative molecular field analysis (CoMFA), com-
parative molecular similarity indices analysis
(
CoMSIA)34 and pharmacophore modeling approaches.
35
An interesting result emerging from these studies was
that the CoMSIA field maps for the hydrophobic,
electrostatic and steric fields found for FPPS inhibition
by bisphosphonates were similar to the CoMSIA field
maps found for bisphosphonates acting as γδ T cell
stimulators and in bone resorption.1
7,30,32
Likewise, the
pharmacophore models for the inhibition of recombinant
human FPPS were very similar to those found for
stimulation of γδ T cells by bisphosphonates,30 and the
pIC50/pEC50 results for FPPS inhibition and Vγ2Vδ2 T
cell stimulation were highly correlated (R ) 0.89,
p ) 0.0012).30 All of these CoMSIA and pharmacophore
modeling investigations clearly showed the importance
of the presence of a positive charge feature at a
relatively localized position in the bisphosphonate side
chain. This feature can, of course, be related to the
position of the positive charge feature expected in the
pyridinium and imidazolium forms of 3 and 4,
shown above, and is centered close to the bisphospho-
nate backbone. This suggested to us that pyridinium-
tant class of drugs currently used to treat osteoporosis,
Paget’s disease and hypercalcemia due to malignancy.1
These compounds function primarily by adsorbing to
bone and inhibiting the enzyme farnesyl diphosphate
synthase (FPPS) in osteoclasts,5-12 resulting in de-
-4
1
3-15
creased levels of protein prenylation.
Bisphos-
phonates have also been found to have anti-parasitic
1
6-25
activity,
and have been found to stimulate human
where there is currently interest in their
26-30
γδ T cells,
31
use as vaccines for a variety of B cell malignancies.
1
-yl species, such as 5, might have enhanced
*
To whom correspondence should be addressed. Telephone:
(
217) 333-3374. Fax: (217) 244-0997. E-mail: eo@chad.scs.uiuc.edu.
†
Department of Chemistry, University of Illinois at Urbana-
Champaign.
‡
L o´ pez-Neyra Institute of Parasitology and Biomedicine.
§
|
⊥
University of Iowa.
Leiden University Medical Center.
Department of Biophysics, University of Illinois at Urbana-
Champaign.
1
0.1021/jm040209d CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/08/2005

2
958 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Sanders et al.
using the following general scheme:
In the first step, we used (where necessary) coupling
reactions of arylmetallic compounds with bromopy-
ridines, catalyzed by Pd(PPh3)441 or NiCl2(PPh3)2, to
produce substituted pyridines. The substituted py-
ridines were then alkylated by using bromoacetic acid,43
and the resulting pyridinium-1-yl acetic acids were
converted to the corresponding bisphosphonates by
using H3PO3/POCl3.44 Next, we investigated the activity
of each compound in inhibiting the FPPS from L. major,
in Dictyostelium discoideum growth inhibition, in γδ T
cell activation, and, for a subset of compounds, in bone
resorption. We used the FPPS from L. major, since
FPPS is the putative bisphosphonate target in several
trypanosomatid species, and the L. major FPPS was the
enzyme previously used to establish the initial QSAR
models.17 We used D. discoideum to test for cell growth
inhibition, since this organism has been used previously
in the context of the development of bone resorption
42
Figure 1. L. major FPPS CoMSIA fields (from ref 17)
superimposed on 5. A, electrostatic field (blue region ) positive
charge enhances activity, red region ) positive charge detracts
from activity); B, steric field (green regions ) steric feature
enhances activity, yellow regions ) steric feature detracts from
activity); C, hydrophobic field (yellow region ) hydrophobic
feature enhances activity, white region ) hydrophobic feature
detracts from activity).
Figure 2. Charge distributions in the side chains of 3 (A) and
45
drugs. To determine the stimulatory activity of 5 and
3
9,40
5
(B). The numbers represent Merz-Singh-Kollman charges
38
7-18 for Vγ2Vδ2 T cells, we used the TNF-R release
assay described previously,30 and for the bone resorption
assay, we used the 17-day old fetal mouse metatarsal
evaluated using the Gaussian 03 program. The side chains
of 3 and 5 were geometry optimized using the Hartree-Fock
method with a uniform 6-31G* basis set and these optimized
geometries were used in the atomic charge calculations.
45
2+
46
Ca release assay described elsewhere.
In the L. major FPPS inhibition assay, 5 was found
to have a Ki of 18 nM (Table 1) and was thus slightly
less active than the most potent commercially available
bisphosphonates, zoledronate (4, Ki ) 11 nM, in this
assay) and risedronate (3, Ki ) 17 nM, in this assay)
activity, and indeed in previous work several other
groups have reported the synthesis of a variety of
analogues of 5 which lack the 1-OH group, such as 6.36
However, these compounds might not be expected to be
effective in bone resorption, since they lack the “bone
(Table 1). To try to enhance activity, we next inspected
the steric and hydrophobic CoMSIA fields for FPPS
inhibition (Figures 1B and 1C, respectively), which
suggested the desirability of placing an additional steric
and hydrophobic feature at the meta position. We thus
prepared compounds 7-9 (Figure 3) and tested them
in the FPPS assay. The m-methyl analogue of 5 (7) was
not as active as 5 (Ki ) 38 nM versus 18 nM), but
substitution with a m-phenyl group, giving 8, resulted
in a very potent species, having a Ki ) 9 nM, slightly
more active than zoledronate, 4 (Ki ) 11 nM). The
p-phenoxy derivative of 5 (9) was found to be less active
32
hook” feature identified in CoMSIA fields and in other
37
studies. We therefore undertook the synthesis of 5 and
a series of derivatives, all containing the 1-OH feature,
and we discuss here their activities in FPPS inhibition,
γδ T cell activation, Dictyostelium discoideum growth
inhibition (a model for bone resorption) and in an in
vitro bone resorption assay.
Results and Discussion
We show in Figure 1 the CoMSIA fields for L. major
1
7
FPPS inhibition superimposed on the structure of 5.
The pyridinium-1-yl nitrogen in 5 (1-hydroxy-2-(pyri-
dinium-1-yl)ethylidene-1,1-bisphosphonic acid) can be
seen to be located in the blue CoMSIA field region,
where positive charge enhances activity. The results of
(Ki ) 75 nM), possibly due to unfavorable electrostatic
interactions of the OH group in the FPPS active site.
Since the phenylpyridinium species, 8, displayed good
activity, we next synthesized 10 and 11. Both of these
compounds contain a methylene linker between the two
aromatic groups, and it seemed possible that they might
better mimic the putative geranyl diphosphate reactive
intermediate (19), but each of these compounds was less
3
8
quantum chemical calculations of the truncated side
chains of 3 and 5 show, as expected, that there are
relatively large positive charges on the nitrogen and
adjacent C, H atoms in this region, as can be seen in
39,40
the Merz-Singh-Kollman charges
(Figure 2). These
results indicate that 5 should contain considerably more
positive charge than does 3 in the region identified by
the CoMSIA electrostatic field (Figure 1A) and might
contribute to enhanced activity. We therefore synthe-
sized the hydroxy species, 5, and a series of derivatives
(7-18, Figure 3), all of which contain the 1-OH feature,
active than 8, with Kis in the 70-160 nM range, Table

Pyridinium-1-yl Bisphosphonates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2959
Figure 3. Structures of pyridinium-1-yl bisphosphonates.
Table 1. Activities of Bisphosphonates as L. Major FPPS Inhibitors, D. Discoideum Cell Growth Inhibitors, γδ T Cell Simulators and
Bone Resorption Agents
L. major FPPS
Ki (nM)
D. discoideum
IC50 (µM)
γδ T cell stimulation
EC50 (µM)
in vitro bone resorption
compound
IC50 (µM)
a,b
1
2
3
4
5
7
8
9
1
1
1
1
1
1
1
1
1
2
(pamidronate)
190
95
167
32
43
1.6
a,b
(alendronate)
32
0.29
0.30
0.034
0.67
0.22
0.41
a,b
(risedronate)
(zoledronate)
17
3.0
2.9
2.1
1.5
3.8
2.9
11
5.0
5.4
4.8
4.5
4.1
2.5
91
a,b
11
18
38
9
c
75
N.D.
0
160
70
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.37
1
2
3
4
5
6
7
8
0
9.2
2.3
20
44
950
80
380
20
20
30
110
50
inactive
30
72
56
5.6
2.9
6.0
12
4.4
4.3
9.3
150
2.7
N.D.
0.075
4.6
a
b
L. major FPPS inhibition data from ref 17. Data averaged over unconstrained γδ T cell stimulation data from ref 30 and data
c
obtained since publication of ref 30. N.D. ) not determined.
1
1
. We also prepared the biphenylpyridinium compound,
2, since the added hydrophobicity of 8 appeared
(3, IC50 ) 3.0 µM) and zoledronate (4, IC50 ) 2.9 µM).
As with the FPPS inhibition results, the benzylpyri-
dinium bisphosphonates (10, 11, 18) were less active
than the pyridinium and phenylpyridinium species
(5, 7-9). Surprisingly, 12 showed high activity
(IC50 ) 2.3 µM), due perhaps to the possibility of an
additional target in D. discoideum or the possibility of
structural differences between L. major and D. discoi-
deum FPPSs. With the exception of 12, the activity
results for the 17 bisphosphonates were found to be
encouraging, but 12 proved to be relatively inactive,
having a Ki of 950 nM. Enhancing the size of the
hydrophobic/steric feature by use of isoquinoline and
quinoline side chains, 13 and 14, resulted in modest
activity (80 and 380 nM, respectively, for 13 and 14),
but the m-ethyl (15), -butyl (16), -methoxy (17) and
p-benzyl (18) pyridinium species were generally more
active (20, 20, 30 and 110 nM, respectively), although
they were less active than 3-5, 8, Table 1.
2
correlated (R ) 0.61, p ) 0.0002) with the L. major
We next investigated D. discoideum growth inhibition
by 1-5 and 7-18 (Table 1). The most active compound
found was the meta-methyl pyridinium compound, 7,
which had an IC50 of 1.5 µM, followed by the unsub-
stituted pyridinium bisphosphonate, 5, which was slightly
more active (IC50 ) 2.1 µM) than was risedronate
FPPS inhibition activities, as shown in Figure 4A.
Next, we investigated the ability of 5 and 7-18 to
stimulate γδ T cells, using the TNF-R release assay and
the unconstrained fitting procedure described previ-
ously.30 The most active compound was found to be
8 (EC50 ) 4.1 µM), followed by 16 (EC50 ) 4.3 µM),

2
960 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Sanders et al.
pyridinium-1-yl hydroxy-bisphosphonates for activity
against a farnesyl diphosphate synthase, D. discoideum
growth inhibition, human γδ T cell activation, and in
bone resorption. In each of the four assays, pyridinium-
1
-yl bisphosphonates having high activity have been
found, suggesting that the further development of this
class of compounds may be of interest in the context of
the chemotherapy of infectious diseases, in bone resorp-
tion, and in immunotherapy.
Experimental Section
Synthetic Aspects. There are two basic steps to produce
the pyridinium-1-yl bisphosphonates: (1) synthesis of the
parent pyridine (when not commercially available), using
Figure 4. Graphs showing correlations between FPPS inhibi-
tion and A, D. discoideum growth inhibition and B, γδ T cell
2
activation (as determined by TNF-R release). The R and p
2
2
either Pd(PPh
3 4
) catalyzed (Suzuki) coupling of an arylboronic
values are R ) 0.61 and p ) 0.0002 in A and R ) 0.56 and
p ) 0.0005 in B.
4
1
acid and bromopyridine or, in the case of 11, NiCl
2 3 2
(PPh ) -
catalyzed coupling of a benzyl zinc compound and bromo-
42
pyridine; (2) carboxymethylation of the (substituted) pyridine
1
5 (EC50 ) 4.4 µM), 7 (EC50 ) 4.5 µM) and 5
43
with bromoacetic acid followed by phosphorylation of the
(
EC50 ) 4.8 µM), with these compounds having more
44
3 3 3
resulting pyridinium-1-yl acetic acids with POCl /H PO . Full
activity than risedronate (3, EC50 ) 5.0 µM) or zoledr-
onate (4, EC50 ) 5.4 µM) in this TNF-R release assay.
Addition of the p-hydroxyl group (8 f 9) decreased the
EC50 slightly (Table 1), but 9 did not display the
maximal TNF-R release achieved with risedronate. All
three methylene-bridged compounds (10, 11, 18) had
poor activity, as in the other assays investigated. The
activity results for FPPS inhibition were found to be
details of the synthesis of each compound are given below, and,
in most cases (i.e. except for 11), the syntheses followed both
of the following two general procedures. Purity of products was
1
established via C/H/N microanalysis and 500 MHz H NMR
spectroscopy.
General Procedure 1: Preparation of Aryl-Substituted
Pyridines. A mixture of an aryl boronic acid or its ester
(3 mmol), 3-bromopyridine (3 mmol), Pd(PPh ) (0.075 mmol),
3
4
2 3 2
K CO (1.2 g, 9 mmol), toluene (12 mL) and H O (3 mL) was
4
1
refluxed for 10 h. The product was extracted with Et
purified by using silica gel chromatography.
2
O and
correlated with the γδ T cell TNF-R release results
2
(R
) 0.56, p ) 0.0005), as shown in Figure 4B,
General Procedure 2: Preparation of 1-Hydroxy-2-
substituted-pyridinium-1-yl)ethylidene-1,1-bisphospho-
suggesting once again the likely importance of FPPS
inhibition in γδ T cell activation.
(
2
9,30
nic Acids. Bromoacetic acid (2 mmol) was added to a solution
of substituted pyridine (2 mmol) in ethyl acetate (3 mL) and
the reaction mixture stirred at room temperature for 2 days,
giving the substituted 1-carboxymethylpyridinium bromide as
a white precipitate. This was then filtered, washed with ethyl
acetate (2 × 3 mL) and dried in vacuo. The resulting white
Finally, we tested a subset of these compounds (1, 2,
, 4, 5, 7, 8 and 17) in a bone resorption assay: 45Ca
2+
3
46
release from 17-day old fetal mouse metatarsals.
Results for pamidronate (1), alendronate (2), risedonate
3), zoledronate (4), 5, 7, 8 and 17 are shown in Figure
(
5
3
3 3
powder was added to a mixture of H PO (5 equivalents) and
A and yielded IC50 values of 1.6 µM (1), 290 nM (2),
toluene (6 mL) and heated to 80 °C, with stirring. After all
solids had melted, POCl (5 equiv) was added dropwise and
00 nM (3), 34 nM (4), 670 nM (5), 220 nM (6), 410 nM
3
(
8) and 370 nM (17), Figure 5B. So the pyridinium
the reaction mixture vigorously stirred at 80 °C for 5 h. Upon
cooling, the supernatant was decanted and 6 N HCl (3 mL)
added to the residue. The resulting solution was refluxed for
bisphosphonates studied are all more active in this bone
resorption assay than is pamidronate, with 7 showing
slightly higher activity than alendronate and rise-
dronate.
1
h, then most of the solvent was removed in vacuo. 2-Propanol
25 mL) was added to precipitate the title compounds as
white powders. These were filtered, washed with 2-propanol
(
In an effort to improve upon these promising bone
resorption results, we synthesized the 3-fluoro analogue
of 5, 20, since the electron-withdrawing nature of this
fluorine would be expected to increase the amount of
positive charge on the ring. When tested in the L. major
FPPS assay, 20 was found to have a Ki of 50 nM, and
in the D. discoideum inhibition assay, we found an
IC50 ) 4.6 µM. In both cases, then, there was a decrease
in activity. However, when tested in the human γδ T
cell activation assay, we found a slight increase in
activity on fluorine substitution (from 4.8 µM with 5 to
(
5 × 5 mL) then dried and were further purified by recrystal-
lization from H O/2-PrOH. In some cases, oily products were
2
neutralized with NaOH, then crystallized as the sodium salts
from H O/2-PrOH.
2
1
-Hydroxy-2-(pyridinium-1-yl)ethylidene-1,1-bisphos-
phonic Acid (5). 5 was prepared from pyridine (395 mg,
mmol) following general procedure 2 (960 mg, 68% overall
5
1
yield). Anal. (C
δ 4.87 (t, JPH ) 7.2 Hz, 2H, CH
aromatics), 8.38 (t, JHH ) 5.6 Hz, 1H, aromatics), 8.71 (d, JHH
7
H
11NP
2
O
7
) C, H, N. H NMR (400 MHz, D
2
O):
2
), 7.84 (t, JHH ) 5.6 Hz, 2H,
3
1
) 5.6 Hz, 2H, aromatics); P NMR (162 MHz, D
s).
2
O): δ 14.3
(
1-Hydroxy-2-(3-methylpyridinium-1-yl)ethylidene-1,1-
2
.7 µM with 20), and in the bone resorption assay the
effect was much larger, with a 75 nM IC50 for 20 (Figure
) to be compared with a 670 nM IC50 for 5, Table 1.
bisphosphonic Acid (7). 7 was prepared from 3-methyl-
pyridine (465 mg, 5 mmol) following general procedure 2
5
1
(
8 2 7
850 mg, 57% overall yield). Anal. (C H13NP O ) C, H, N. H
These results show, therefore, that 20 is more active in
this bone resorption assay than the drugs currently used
to treat osteoporosis, alendronate (2, IC50 ) 290 nM)
and risedronate (3, IC50 ) 300 nM), although it is less
active than zoledronate (4, IC50 ) 34 nM).
2 3 PH
NMR (400 MHz, D O): δ 2.36 (s, 3H, CH ), 4.67 (t, J ) 8.0
2
Hz, 2H, CH ), 7.68 (t, JHH ) 6.4 Hz, 1H, aromatics), 8.16
(
d, JHH ) 6.4 Hz, 1H, aromatics), 8.50 (d, JHH ) 6.4 Hz, 1H,
31
2
aromatics), 8.53 (s, 1H, aromatics); P NMR (162 MHz, D O):
δ 14.1 (s).
1-Hydroxy-2-(3-phenylpyridinium-1-yl)ethylidene-1,1-
Overall, the results presented above are of interest
since they represent the first report of the QSAR-based
bisphosphonic Acid (8). 8 was prepared from 3-phenyl-
pyridine (310 mg, 2 mmol) following general procedure 2 as
the disodium salt (570 mg, 65% overall yield). Anal.
1
7,30
design,
synthesis and testing of a series of novel

Pyridinium-1-yl Bisphosphonates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2961
Figure 5. A, 45Ca2+ release from 17-day old fetal mouse metatarsals46 for control and compounds 1, 2, 3, 4, 5, 7, 8, 17 and 20.
The braces delineate bone resorption data sets and compound numbers. B, Dose-response curves for 1 (b), 2 (O), 3 (2), 4 (×) and
2
0 ([).
1
1
(
C
13
H
13NP
2
O
7
Na
δ 4.75 (t, JPH ) 8.0 Hz, 2H, CH
.50-7.60 (m, 2H, aromatics), 7.68 (t, JHH ) 5.6 Hz, 1H,
aromatics), 8.39 (d, JHH ) 5.6 Hz, 1H, aromatics), 8.60 (d, JHH
2
‚2 H
2
O) C, H, N. H NMR (400 MHz, D
2
O):
NP
2 7 2
O ‚0.5 H O) C, H, N; C: calcd, 51.36; found, 50.94. H NMR
2
), 7.30-7.40 (m, 3H, aromatics),
(400 MHz, D O): δ 4.84 (t, JPH ) 8.8 Hz, 2H, CH ), 7.20-7.80
2
2
7
(m, 10H, aromatics), 8.44 (d, JHH ) 6.0 Hz, 1H, aromatics),
8.68 (d, JHH ) 6.0 Hz, 1H, aromatics), 8.98 (s, 1H, aromatics);
3
1
31
)
(
5.6 Hz, 1H, aromatics), 8.91 (s, 1H, aromatics); P NMR
162 MHz, D O): δ 14.3 (s).
-Hydroxy-2-[3-(4-hydroxyphenyl)pyridinium-1-yl]eth-
ylidene-1,1-bisphosphonic Acid (9). 9 was prepared from
-(4,4,5,5-tetramethyl-1,3-dioxaborolan-2-yl)phenyl acetate
786 mg, 3 mmol) following general procedures 1 and 2 as a
tetrasodium salt (620 mg, 40% overall yield). Anal.
2
P NMR (162 MHz, D O): δ 14.0 (s).
2
1-Hydroxy-2-[isoquinolinium-2-yl]ethylidene-1,1-bis-
1
phosphonic Acid (13). 13 was prepared from isoquinoline
(400 mg, 3 mmol) following general procedure 2 (486 mg, 47%
1
4
(
overall yield). Anal. (C11
(400 MHz, D
H13NP
2
O
7
‚0.25 H
2
O) C, H, N. H NMR
2
O): δ 4.99 (t, JPH ) 8.0 Hz, 2H, CH
2
), 7.84
(t, JHH ) 5.6 Hz, 1H, aromatics), 8.00-8.10 (m, 2H, aromatics),
8.15 (d, JHH ) 5.6 Hz, 1H, aromatics), 8.27 (d, JHH ) 5.6 Hz,
1H, aromatics), 8.39 (d, JHH ) 5.6 Hz, 1H, aromatics), 9.56
1
(
C
13
H
11NP
2
O
8
Na
δ 4.83 (t, JPH ) 8.0 Hz, 2H, CH
aromatics), 7.49 (d, JHH ) 6.8 Hz, 2H, aromatics), 7.77 (t, JHH
6.0 Hz, 1H, aromatics), 8.45 (d, JHH ) 6.0 Hz, 1H, aromatics),
.55 (d, JHH ) 6.0 Hz, 1H, aromatics), 8.89 (s, 1H, aromatics);
4
‚3 H
2
O) C, H, N. H NMR (400 MHz, D
2
O):
2
), 6.74 (d, JHH ) 6.8 Hz, 2H,
3
1
2
(s, 1H, aromatics); P NMR (162 MHz, D O): δ 14.2 (s).
)
1-Hydroxy-2-[6-chloroquinolinium-1-yl]ethylidene-1,1-
bisphosphonic Acid (14). 14 was prepared from 6-chloro-
quinoline (1 g, 6.1 mmol) following general procedure 2
8
3
1
2
P NMR (162 MHz, D O): δ 14.6 (s).
1
-Hydroxy-2-(3-benzylpyridinium-1-yl)ethylidene-1,1-
(243 mg, 10% overall yield). Anal. (C11
N. H NMR (400 MHz, D
CH
H
12NP
2
O
7
Cl‚H
O): δ 5.33 (t, JPH ) 10.0 Hz, 2H,
2
), 7.80-7.85 (m, 1H, aromatics), 7.95-8.00 (m, 1H, aromat-
2
O) C, H,
1
bisphosphonic Acid (10). 10 was prepared from 3-benzyl-
2
pyridine (340 mg, 2 mmol) following general procedure 2 as a
trisodium salt (560 mg, 60% overall yield). Anal. (C14
H
14NP
2
O
7
-
ics), 8.15 (s, 1H, aromatics), 8.50 (d, JHH ) 5.6 Hz, 1H, aro-
1
Na
3
‚1.5 H
2
O) C, H, N. H NMR (400 MHz, D
2
O): δ 4.07
matics), 8.84 (d, JHH ) 5.6 Hz, 1H, aromatics), 9.18 (d, JHH
)
3
1
(
(
(
s, 2H, PhCH
2
), 4.76 (t, JPH ) 9.2 Hz, 2H, NCH ), 7.10-7.25
2
5.6 Hz, 1H, aromatics); P NMR (162 MHz, D
2
O): δ 14.3 (s).
m, 5H, aromatics), 7.69 (t, JHH ) 6.8 Hz, 1H, aromatics), 8.14
d, JHH ) 6.8 Hz, 1H, aromatics), 8.54 (d, JHH ) 6.8 Hz, 1H,
1-Hydroxy-2-[3-ethylpyridinium-1-yl]ethylidene-1,1-
bisphosphonic Acid (15). 15 was prepared from 3-ethyl-
3
1
aromatics), 8.58 (s, 1H, aromatics); P NMR (162 MHz, D
2
O):
pyridine (430 mg, 4 mmol) following general procedure 2
1
δ 13.9 (s).
(750 mg, 60% overall yield). Anal. (C
NMR (400 MHz, D
(q, JHH ) 7.6 Hz, 2H, CH
7.68 (t, JHH ) 6.4 Hz, 1H, aromatics), 8.17 (d, JHH ) 6.4 Hz,
9
H
15NP
2
O
7
) C, H, N. H
1
-Hydroxy-2-[3-(3-methylbenzyl)pyridinium-1-yl]eth-
ylidene-1,1-bisphosphonic Acid (11). 3-(3-Methylbenzyl)-
pyridine was prepared by a published method. In brief,
-methylbenzyl bromide (0.88 mL, 6.5 mmol) and Zn powder
0.55 g, 8.5 mmol) in THF (6 mL) were stirred for 2 h at room
2
O): δ 1.11 (t, JHH ) 7.6 Hz, 3H, CH
3
), 2.69
),
2
), 4.74 (t, JPH ) 8.0 Hz, 2H, NCH
2
4
2
3
1
3
(
1H, aromatics), 8.50-8.60 (m, 2H, aromatics); P NMR
(162 MHz, D O): δ 14.3 (s).
2
temperature, and the supernatant solution transferred to a
THF solution (6 mL) containing 3-bromopyridine (0.49 mL,
1-Hydroxy-2-[3-butylpyridinium-1-yl]ethylidene-1,1-
bisphosphonic Acid (16). 16 was prepared from 3-butyl-
pyridine (540 mg, 4 mmol) following general procedure 2
5
2 3 2
mmol) and NiCl (PPh ) (654 mg, 1 mmol). The reaction
mixture was stirred at room-temperature overnight before
quenching with 10% ammonia. The product was extracted with
(690 mg, 51% overall yield). Anal. (C11
H
19NP
2
O
7
‚0.25 H
O): δ 0.72 (t, JHH ) 7.2 Hz, 3H,
), 1.45-1.55 (m, 2H, CH ), 2.67
), 4.79 (t, JPH ) 8.0 Hz, 2H, NCH ),
7.70 (t, JHH ) 6.8 Hz, 1H, aromatics), 8.20 (d, JHH ) 6.8 Hz,
2
O) C,
1
H, N. H NMR (400 MHz, D
CH ), 1.10-1.20 (m, 2H, CH
(q, JHH ) 7.2 Hz, 2H, CH
2
Et
2
O and purified by column chromatography (silica gel, Et
2
O/
3
2
2
hexane 1:1, 86% yield). The title compound 11 was prepared
2
2
from 3-(3-methylbenzyl)pyridine (370 mg, 2 mmol) following
3
1
general procedure 2 as a trisodium salt (700 mg, 70% overall
1H, aromatics), 8.50-8.60 (m, 2H, aromatics); P NMR
(162 MHz, D O): δ 14.3 (s).
1
yield). Anal. (C15
H
16NP
2
O
7
Na
O): δ 2.15 (s, 3H, CH
t, JPH ) 7.6 Hz, 2H, NCH ), 6.90-7.20 (m, 4H, aromatics),
.70 (t, JHH ) 6.0 Hz, 1H, aromatics), 8.15 (d, JHH ) 6.0 Hz,
H, aromatics), 8.55 (d, JHH ) 6.0 Hz, 1H, aromatics), 8.59
3
‚2.5 H
2
O) C, H, N. H NMR
2
(
400 MHz, D
2
3
), 4.04 (s, 2H, PhCH
2
), 4.78
1-Hydroxy-2-[3-methoxylpyridinium-1-yl]ethylidene-
1,1-bisphosphonic Acid (17). 17 was prepared from 3-meth-
oxypyridine (440 mg, 4 mmol) following general procedure 2
(
2
7
1
(
(900 mg, 72% overall yield). Anal. (C
8
H
13NP
2
O
3
7
‚H
), 4.75 (t, JPH
2
), 7.58 (t, JHH ) 6.8 Hz, 1H, aromatics), 7.78
2
O) C, H, N.
3
1
1
s, 1H, aromatics); P NMR (162 MHz, D
-Hydroxy-2-[3-(3-biphenyl)pyridinium-1-yl]ethylidene-
,1-bisphosphonic Acid (12). 12 was prepared from 3-bi-
2
O): δ 14.3 (s).
H NMR (400 MHz, D
8.4 Hz, 2H, CH
2
O): δ 3.80 (s, 3H, CH
)
1
1
(d, JHH ) 6.8 Hz, 1H, aromatics), 8.17 (d, JHH ) 6.8 Hz, 1H,
31
phenyl boronic acid (600 mg, 3 mmol) following general
2
aromatics), 8.45 (s, 1H, aromatics); P NMR (162 MHz, D O):
procedures 1 and 2 (750 mg, 56% overall yield). Anal. (C19
H
19
-
δ 14.2 (s).

2
962 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Sanders et al.
1
-Hydroxy-2-[4-benzylpyridinium-1-yl]ethylidene-1,1-
(10) Bergstrom, J. D.; Bostedor, R. G.; Masarachia, P. J.; Reszka, A.
A.; Rodan, G. Mechanism of aminobisphosphonate action: char-
acterization of alendronate inhibition of the isoprenoid pathway.
Arch. Biochem. Biophys. 2000, 373, 231-241.
11) Grove, J. E.; Brown, R. J.; Watts, D. J. The intracellular target
for the antiresorptive aminobisphosphonate drugs in Dictyo-
stelium discoideum is the enzyme farnesyl diphosphate synthase.
J. Bone Miner. Res. 2000, 15, 971-981.
(12) Dunford, J. E.; Thompson, K.; Coxon, F. P.; Luckman, S. P.;
Hahan, F. M.; Poulter, C. D.; Ebetino, F. H.; Rogers, M. J.
Structure-activity relationships for inhibition of farnesyl diphos-
phate synthase in vitro and inhibition of bone resorption in vivo
by nitrogen-containing bisphosphonates. J. Pharmacol. Exp.
Ther. 2001, 296, 235-242.
bisphosphonic Acid (18). 18 was prepared from 4-benzyl-
pyridine (380 mg, 2.2 mmol) following general procedure 2
(
337 mg, 40% overall yield). Anal. (C14
H
16NP
2
O
7
Na‚0.5 H
O): δ 4.08 (s, 2H, PhCH
), 7.10-7.30 (m, 5H, aromatics),
.61 (d, JHH ) 6.8 Hz, 2H, aromatics), 8.46 (d, JHH ) 6.8 Hz,
2
O)
2
(
1
C, H, N. H NMR (400 MHz, D
2
),
4
7
2
.76 (t, JPH ) 9.2 Hz, 2H, NCH
2
3
1
H, aromatics); P NMR (162 MHz, D
2
O): δ 14.0 (s).
1
-Hydroxy-2-[3-fluoropyridinium-1-yl]ethylidene-1,1-
bisphosphonic Acid (20). 20 was prepared from 3-fluoro-
pyridine (214 mg, 2.2 mmol) following general procedure 2
(
304 mg, 46% overall yield). Anal. (C
7
H
10FNP
2
O
7
) C, H, N; C:
O): δ 4.80
), 7.80-7.88 (m, 1H, aromatics), 8.15
t, JHH ) 8.0 Hz, 1H, aromatics), 8.70 (d, JHH ) 8.0 Hz, 1H,
1
calcd, 28.41; found, 27.92. H NMR (400 MHz, D
2
(
13) Luckman, S. P.; Hughes, D. E.; Coxon, F. P.; Graham, R.; Russell,
G.; Rogers, M. J. Nitrogen-containing bisphosphonates inhibit
the mevalonate pathway and prevent post-translational prenyl-
ation of GTP-binding proteins, including Ras. J. Bone Miner.
Res. 1998, 13, 581-589.
(
(
t, JPH ) 8.4 Hz, 2H, CH
2
3
1
2
aromatics); P NMR (162 MHz, D O): δ 14.0 (s).
Acknowledgment. This work was supported by the
US National Institutes of Health (grants GM65307 to
E.O. and AR45504 to C.T.M.), the European Commis-
sion (INCO program), the Plan Nacional and by the Plan
Andaluz de Investigacion (Cod. CVI-199). J.M.S. is an
American Heart Association, Midwest Affiliate, Pre-
doctoral Fellow. J.M.W.C. is a Jean Dreyfus Boissevain
Undergraduate Scholar. G.A.M. was supported by the
U.S. National Institutes of Health under a Ruth L.
Kirschstein National Research Service Award (grant
GM-65782). A.O. is a Spanish Ministry of Science and
Technology Predoctoral Fellow. We thank Carmen Te-
jero Extremera for technical assistance, and Roberto
Docampo and Felix Ruiz, for advice on D. discoideum
culture.
(14) 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. Alendronate mechanism of
action: geranylgeraniol, an intermediate in the mevalonate
pathway, prevents inhibition of osteoclast formation, bone
resorption, and kinase activation in vitro. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 133-138.
15) van Beek, E.; L o¨ wik, C.; van der Pluijm, G.; Papapoulos, S. The
role of geranylgeranylation in bone resorption and its suppres-
sion by bisphosphonates in fetal bone explants in vitro: A clue
to the mechanism of action of nitrogen-containing bisphos-
phonates. J. Bone Miner. Res. 1999, 14, 722-729.
(16) Montalvetti, A.; Bailey, B. N.; Martin, M. B.; Severin, G. W.;
Oldfield, E.; Docampo, R. Bisphosphonates are potent inhibitors
of Trypanosoma cruzi farnesyl pyrophosphate synthase. J. Biol.
Chem. 2001, 276, 33930-33937.
(17) Sanders, J. M.; G o´ mez, A. O.; Mao, J.; Meints, G. A.; van Brussel,
E. M.; Burzynska, A.; Kafarski, P.; Gonz a´ lez-Pacanowska, D.;
Oldfield, E. 3-D QSAR investigations of the inhibition of Leish-
mania major farnesyl pyrophosphate synthase by bisphos-
phonates. J. Med. Chem. 2003, 46, 5171-5183.
(
Supporting Information Available: Microanalytical and
NMR data are available as Supporting Information. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(
18) Martin, M. B.; Grimley, J. S.; Lewis, J. C.; Heath, H. T. III;
Bailey, B. N.; Kendrick, H.; Yardley, V.; Caldera, A.; Lira, R.;
Urbina, J. A.; Moreno, S. N.; Docampo, R.; Croft, S. L.; Oldfield,
E. Bisphosphonates inhibit the growth of Trypanosoma brucei,
Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii,
and Plasmodium falciparum: A potential route to chemo-
therapy. J. Med. Chem. 2001, 44, 909-916.
References
(
1) Sambrook, P. N.; Geusens, P.; Ribot, C.; Solimano, J. A.; Ferrer-
Barriendos, J.; Gaines, K.; Verbruggen, N.; Melton, M. E.
Alendronate produces greater effects than raloxifene on bone
density and bone turnover in postmenopausal women with low
bone density: results of EFFECT (EFficacy of FOSAMAX versus
EVISTA Comparison Trial) International. J. Intern. Med. 2004,
(
(
(
19) Martin, M. B.; Sanders, J. M.; Kendrick, H.; de Luca-Fradley,
K.; Lewis, J. C.; Grimley, J. S.; van Brussel, E. M.; Olsen, J. R.;
Meints, G. A.; Burzynska, A.; Kafarski, P.; Croft, S. L.; Oldfield,
E. Activity of bisphosphonates against Trypanosoma brucei
rhodesiense. J. Med. Chem. 2002, 45, 2904-2914.
20) Moreno, B.; Bailey, B. N.; Luo, S.; Martin, M. B.; Kuhlenschmidt,
M.; Moreno, S. N.; Docampo, R.; Oldfield, E. 31P NMR of
apicomplexans and the effects of risedronate on Cryptosporidium
parvum growth. Biochem. Biophys. Res. Commun. 2001, 284,
255, 503-511.
(
2) Vasireddy, S.; Talwakar, A.; Miller, H.; Mehan, R.; Swinson, D.
R. Patterns of pain in Paget’s disease of bone and their outcomes
on treatment with pamidronate. Clin. Rheumatol. 2003, 22,
6
32-637.
376-380.
21) Ghosh, S.; Chan, J. M.; Lea, C. R.; Meints, G. A.; Lewis, J. C.;
Tovian, Z. S.; Flessner, R. M.; Loftus, T. C.; Bruchhaus, I.;
Kendrick, H.; Croft, S. L.; Kemp, R. G.; Kobayashi, E. Effects of
bisphosphonates on the growth of Entamoeba histolytica and
Plasmodium species in vitro and in vivo. J. Med. Chem. 2004,
47, 175-187.
(
3) Dawson, N. A. Therapeutic benefit of bisphosphonates in the
management of prostate cancer-related bone disease. Expert.
Opin. Pharmacother. 2003, 4, 705-716.
(
4) Rosen, L. S.; Gordon, D. H.; Dugan, W. Jr.; Major, P.; Eisenberg,
P. D.; Provencher, L.; Kaminski, M.; Simeone, J.; Seaman, J.;
Chen, B. L.; Coleman, R. E. Zoledronic acid is superior to
pamidronate for the treatment of bone metastases in breast
carcinoma patients with at least one osteolytic lesion. Cancer
(22) Yardley, V.; Khan, A. A.; Martin, M. B.; Slifer, T. R.; Araujo, F.
G.; Moreno, S. N.; Docampo, R.; Croft, S. L.; Oldfield, E. In vivo
activities of farnesyl pyrophosphate synthase inhibitors against
Leishmania donovani and Toxoplasma gondii. Antimicrob.
Agents Chemother. 2002, 46, 929-931.
2
004, 100, 36-43.
(
(
(
5) Cromartie, T. H.; Fisher, K. J.; Grossman, J. N. The discovery
of a novel site of action for herbicidal bisphosphonates. Pesticide
Biochem. Phys. 1999, 63, 114-126.
(
23) Rodriguez, N.; Bailey, B. N.; Martin, M. B.; Oldfield, E.; Urbina,
J. A.; Docampo, R. Radical cure of experimental cutaneous
leishmaniasis by the bisphosphonate pamidronate. J. Infect. Dis.
6) Cromartie, T. H.; Fisher, K. J. Method of controlling plants by
inhibition of farnesyl pyrophosphate synthase. U.S. Patent 5,-
756,423, May 26, 1998.
2002, 186, 138-140.
7) van Beek, E.; Pieterman, E.; Cohen, L.; L o¨ wik, C.; Papapoulos,
S. Nitrogen-containing bisphosphonates inhibit isopentenyl pyro-
phosphate isomerase/farnesyl pyrophosphate synthase activity
with relative potencies corresponding to their antiresorptive
potencies in vitro and in vivo. Biochem. Biophys. Res. Commun.
(
24) Garzoni, L. R.; Caldera, A.; Meirelles, M. N. L.; de Castro, S. L.;
Meints, G.; Docampo, R.; Oldfield, E.; Urbina, J. A. Selective in
vitro effects of the farnesyl pyrophosphate synthase inhibitor
risedronate on Trypanosoma cruzi. Intl. J. Antimicrob. Agents
2004, 23, 273-285.
1
999, 255, 491-494.
(
25) Garzoni, L. R.; Waghabi, M. C.; Baptista, M. M.; de Castro, S.
L.; Meirelles, M. N. L.; Britto, C.; Docampo, R.; Oldfield, E.;
Urbina, J. A. Antiparasitic activity of risedronate in a murine
model of acute Chagas' disease. Int. J. Antimicrob. Agents 2004,
23, 286-290.
(
8) van Beek, E.; Pieterman, E.; Cohen, L.; L o¨ wik, C.; Papapoulos,
S. Farnesyl pyrophosphate synthase is the molecular target of
nitrogen-containing bisphosphonates. Biochem. Biophys. Res.
Commun. 1999, 264, 108-111.
(
9) Keller, R. K.; Fliesler, S. J. Mechanism of aminobisphosphonate
action: characterization of alendronate inhibition of the iso-
prenoid pathway. Biochem. Biophys. Res. Commun. 1999, 266,
(26) Wang, L.; Kamath, A.; Das, H.; Li, L.; Bukowski, J. F. Antibacte-
rial effect of human Vγ2Vδ2 T cells in vivo. J. Clin. Invest. 2001,
108, 1349-1357.
560-563.

Pyridinium-1-yl Bisphosphonates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2963
(
27) Kunzmann, V.; Bauer, E.; Feurle, J.; Weissinger, F.; Tony, H.
P.; Wilhelm, M. Stimulation of γδ T cells by aminobisphos-
phonates and induction of antiplasma cell activity in multiple
myeloma. Blood 2000, 96, 384-392.
Golomb, G. Synthesis and evaluation of nitrogen-containing
novel bisphosphonates for calcium-related disorders. Pharm. Res.
1996, 13, s-139. (k) Alfer’ev, I. S.; Mikhalin, N. V. Reactions of
vinylidenediphosphonic acid with nucleophiles 5. Addition of
heterocyclic amines and trimethylamine to vinylidenediphos-
phonic acid. Russ. Chem. Bull. 1995, 44, 1528-1530. (l) Alfer’ev,
I. S.; Mikhalin, N. V.; Novikova, V. M.; Kozlov, V. A. Substituted
ammonioethylidene-1,1-diphosphonic acids with immunosup-
pressant activity. Russian Patent SU1089953, 1995.
(
(
(
28) Kato, Y.; Tanaka, Y.; Miyagawa, F.; Yamashita, S.; Minato, N.
Targeting of tumor cells for human γδ T cells by nonpeptide
antigens. J. Immunol. 2001, 167, 5092-5098.
29) Thompson, K.; Rogers, M. J. Statins prevent bisphosphonate-
induced γδ-T-cell proliferation and activation in vitro. J. Bone
Miner. Res. 2004, 19, 278-288.
(37) van Beek, E. R.; L o¨ wik, C. W. G. M.; Ebetino, F. H.; Papapoulos,
S. E. Binding and antiresorptive properties of heterocycle-
containing bisphosphonate analogs: Structure-activity relation-
ships. Bone 1998, 23, 437-442.
30) Sanders, J. M.; Ghosh, S.; Chan, J. M. W.; Meints, G.; Wang,
H.; Raker, A. M.; Song, Y.; Colantino, A.; Burzynska, A.;
Kafarski, P.; Morita, C. T.; Oldfield, E. Quantitative structure-
activity relationships for γδ T cell activation by bisphosphonates.
J. Med. Chem. 2004, 47, 375-384.
(38) Gaussian 03, Revision C.02, Frisch, M. J.; Trucks, G. W.;
Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.;
Montgomery, Jr., J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci,
B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji,
H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene,
M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;
Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J.
W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.;
Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A.
D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul,
A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D.
J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong,
M. W.; Gonzalez, C.; Pople, J. A.; Gaussian, Inc., Wallingford,
CT, 2004.
(
31) Wilhelm, M.; Kunzmann, V.; Eckstein, S.; Reimer, P.; Weis-
singer, F.; Ruediger, T.; Tony, H. P. γδ T cells for immune
therapy of patients with lymphoid malignancies. Blood 2003,
102, 200-206.
(
32) Kotsikorou. E.; Oldfield, E. A quantitative structure-activity
relationship and pharmacophore modeling investigation of aryl-X
and heterocyclic bisphosphonates as bone resorption agents. J.
Med. Chem. 2003, 46, 2932-2944.
(
33) Cramer, R. D. III; Patterson, D. E.; Bunce, J. D. Recent advances
in comparative molecular field analysis (CoMFA). Prog. Clin.
Biol. Res. 1989, 291, 161-165.
(
34) Klebe, G.; Abraham, U.; Mietzner, T. Molecular similarity indices
in a comparative analysis (CoMSIA) of drug molecules to
correlate and predict their biological activity. J. Med. Chem.
1
994, 37, 4130-4146.
(
(
35) Catalyst, version 4.8; Accelrys Inc., 9685 Scranton Rd., San
Diego, CA 92121.
36) (a) Golomb, G.; Danenberg, H. Treatment of restenosis. US
Patent 6,719,998, 2004. (b) Danenberg, H.; Golomb, G. Treat-
ment of restenosis. World Patent WO0003677, 2000. (c) Gross-
mann, G.; Grossmann, A.; Ohms, G.; Breuer, E.; Chen, R.;
Golomb, G.; Cohen, H.; H a¨ gele, G.; Classen, R. Solid-state NMR
of bisphosphonates adsorbed on hydroxyapatite. Magn. Reson.
Chem. 2000, 38, 11-16. (d) Cohen, H.; Alfer’ev, I. S.; Monkkonen,
J.; Seibel, M. J.; Pinto, T.; Ezra, A.; Solomon, V.; Stepensky, D.;
Sagi, H.; Ornoy, A.; Patlas, N.; Hagele, G.; Hoffman, A.; Breuer,
E.; Golomb G. Synthesis and preclinical pharmacology of 2-(2-
aminopyrimidinio) ethylidene-1,1-bisphosphonic acid betaine
(39) Besler, B. H.; Merz, K. M.; Kollman, P. A. Atomic charges derived
from semiempirical methods. J. Comput. Chem. 1990, 11, 431-
439.
(40) Singh, U. C.; Kollman, P. A. An approach to computing electro-
static charges for molecules. J. Comput. Chem. 1984, 5, 129-
145
(41) Miyaura, N.; Yanagi, T.; Suzuki, A. The palladium-catalyzed
cross-coupling reaction of phenylboronic acid with haloarenes
in the presence of bases. Synth. Commun. 1981, 11, 513-519.
(42) Krapcho, A. P.; Ellis, M. Synthesis of regioisomeric difluoro- and
(
ISA-13-1)-a novel bisphosphonate. Pharm. Res. 1999, 16,
8-chloro-9-fluorobenz[g]isoquinoline-5,10-diones and S Ar dis-
N
1
399-1406. (e) Patlas, N.; Golomb, G.; Yaffe, P.; Pinto, T.;
placements studies by diamines: bis(aminoalkyl)aminobenz[g]-
Breuer, E.; Ornoy, A. Transplacental effects of bisphosphonates
on fetal skeletal ossification and mineralization in rats. Tera-
tology 1999, 60, 68-73. (f) Cohen, H.; Solomon, V.; Alfer’ev, I.
S.; Breuer, E.; Ornoy, A.; Patlas, N.; Eidelman, N.; Hagele, G.;
Golomb G. Bisphosphonates and tetracycline: experimental
models for their evaluation in calcium-related disorders. Pharm.
Res. 1998, 15, 606-613. (g) Klein, B. Y.; Ben-Bassat, H.; Breuer,
E.; Solomon, V.; Golomb, G. Structurally different bisphospho-
nates exert opposing effects on alkaline phosphatase and
mineralization in marrow osteoprogenitors. J. Cell. Biochem.
isoquinoline-5, 10-diones. J. Fluorine Chem. 1998, 90, 139-147.
(43) Zhang, L.; Liang, F.; Sun, L.; Hu, Y.; Hu, H. A novel and practical
synthesis of 3-unsubstituted indolizines. Synthesis 2000, 1733-
1737.
(44) Harel, Z.; Kovalevski-Liron, E.; Lidor-Hadas, R.; Lifshitz-Liron,
R. Use of certain diluents for making bisphosphonic acids. World
Patent WO03097655, November 27, 2003.
(45) Rogers, M. J.; Watts, D. J.; Russell, R. G.; Ji, X.; Xiong, X.;
Blackburn, G. M.; Bayless, A. V.; Ebetino, F. H. Inhibitory effects
of bisphosphonates on growth of amoebae of the cellular slime
mold Dictyostelium discoideum. J. Bone Miner. Res. 1994, 9,
1029-1039.
(46) van Beek, E. R.; Cohen, L. H.; Leroy, I. M.; Ebetino, F. H.; L o¨ wik,
C. W.; Papapoulos, S. E. Differentiating the mechanisms of
antiresorptive action of nitrogen containing bisphosphonates.
Bone 2003, 33, 805-11.
1
998, 68, 186-194. (h) Alfer’ev, I. S.; Breuer, E.; Golomb, G.;
Ornoy, A. Novel bisphosphonates, process for their preparation
and pharmaceutical compositions containing them. World Patent
WO9708178, 1997. (i) Breuer, E.; Golomb, G.; Amidon, G. L.;
Alfer’ev, I. S.; Rozen, N. E.-H.; Friedman-Ezra, A. Phosphonates,
bisphosphonates and pharmaceutical compositions containing
them. World Patent WO9631227, 1996. (j) Cohen, H.; Breuer,
E.; Chen, R.; Solomon, V.; Ornoy, A.; Patlas, N.; Pinto, T.;
JM040209D