Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: A potential route to chemotherapy

Article abstract of DOI:10.1021/jm0002578

We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of the type used in bone resorption therapy have significant activity against parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC₅₀ activity values against parasite replication (e.g. o-risedronate, I₅₀ = 220 nM for T. brucei rhodesiense; risedronate, IC₅₀ = 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating parasitic protozoan diseases.

Full text of DOI:10.1021/jm0002578

J . Med. Chem. 2001, 44, 909-916
909
Bisp h osp h on a tes In h ibit th e Gr ow th of Tr ypa n osom a br u cei, Tr ypa n osom a
cr u zi, Leish m a n ia d on ova n i, Toxopla sm a gon d ii, a n d P la sm od iu m fa lcipa r u m :
A P oten tia l Rou te to Ch em oth er a p y
†
†
†
†
§
Michael B. Martin, J oshua S. Grimley, J ared C. Lewis, Huel T. Heath, III, Brian N. Bailey,
‡
‡
|
|
|
§
Howard Kendrick, Vanessa Yardley, Aura Caldera, Renee Lira, J ulio A. Urbina, Silvia N. J . Moreno,
§
‡
,†,⊥
Roberto Docampo, Simon L. Croft, and Eric Oldfield*
Departments of Chemistry and Biophysics, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue,
Urbana, Illinois 61801, Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at
Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, Illinois 61802, Laboratorio de Qu ´ı mica Biol o´ gica,
Instituto Venezolano de Investigaciones Cientificas, Apartado Postal 21.627, Caracas 1020A, Venezuela, and Department of
Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
Received J une 15, 2000
We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of
Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii,
and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of
the type used in bone resorption therapy have significant activity against parasites, with the
aromatic species having in some cases nanomolar or low-micromolar IC50 activity values against
parasite replication (e.g. o-risedronate, IC50 ) 220 nM for T. brucei rhodesiense; risedronate,
IC50 ) 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate
is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting
farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating
parasitic protozoan diseases.
In tr od u ction
precursor of farnesylated and geranylgeranylated pro-
teins, as well as dolichol and sterol biosynthesis.
Recent studies have shown that nitrogen-containing
bisphosphonates, such as the ones used to treat bone
resorption diseases, are competitive inhibitors of far-
Parasitic protozoan diseases constitute the world’s
most widely spread human health problem. It is esti-
mated that 3 billion individuals suffer from one or more
parasitic infections, with the greatest causes of morbid-
ity being attributed to the trypanosomatid and apicom-
1
1-15
nesylpyrophosphate synthase (FPPS).
Bisphospho-
1
nates are nonhydrolyzable pyrophosphate (P-O-P)
analogues in which the oxygen bridge has been replaced
by a carbon (P-C-P). We have previously proposed that
the potent nitrogen-containing bisphosphonates, such
as pamidronate, alendronate, and risedronate, act as
plexan parasites. The apicomplexan, Plasmodium fal-
ciparum, the causative agent of malaria, infects approxi-
mately 500 million individuals each year, resulting in
-3 million deaths annually.1 Of the trypanosomatid
,2
2
parasites, the combined burden of Trypanosoma brucei,
Trypanosoma cruzi, and Leishmania spp. results in an
(
aza)carbocation pyrophosphate intermediate analogues
1
1
additional 20 million disease cases.3
-6
of the geranylpyrophosphate carbocation.
Since vaccina-
tions against parasitic infections are still in develop-
ment and control of environmental sources of trans-
mission is ineffective and often impractical, the need
for improved chemoprophylactic/chemotherapeutic ap-
proaches in the control of these pathogens is indisput-
able.
In this study we explore the in vitro activity of a series
of bisphosphonates against the parasitic protozoa T.
brucei, T. cruzi, L. donovani, T. gondii, and P. falci-
parum, the causative agents of African sleeping sick-
ness, Chagas’ disease, visceral leishmaniasis, toxoplas-
mosis, and malaria, respectively.
Investigations of the metabolic processes occurring in
a range of parasitic protozoa have demonstrated the
presence of a pathway derived from mevalonic acid,
called “the mevalonate pathway”. This pathway is
responsible for the synthesis of a variety of sterols and
polyisoprenoid compounds which are of vital importance
Resu lts a n d Discu ssion
We show in Table 1 the structures of the 19 bisphos-
phonates tested. 1 is alendronate (Merck’s Fosamax),
and 2 is risedronate (Procter and Gamble/Hoechst
Marion Roussels’ Actonel), while 5 is pamidronate
7
-10
to parasite survival.
The production of farnesylpy-
(Novartis’ Aredia). We tested these and other com-
rophosphate (FPP) marks the branching point in the
mevalonate pathway, with FPP being the primary
pounds (Table 1) against T. brucei rhodesiense, T. cruzi,
L. donovani, T. gondii, and P. falciparum replication
in vitro using the methods described in the Experimen-
tal Section. The experimental concentrations required
to reduce parasite proliferation by 50%, the IC50 values,
were extracted from experimental growth versus inhibi-
tor concentration assays by fitting the rectangular
hyperbolic function:
*
To whom correspondence should be addressed. Tel: (217) 333-
3
374. Fax: (217) 244-0997. E-mail: eo@chad.scs.uiuc.edu.
†
Department of Chemistry, University of Illinois.
Department of Pathobiology, University of Illinois.
Instituto Venezolano de Investigaciones Cientificas.
London School of Hygiene and Tropical Medicine.
§
|
‡
⊥
Department of Biophysics, University of Illinois.
1
0.1021/jm0002578 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/16/2001

9
10 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6
Martin et al.
Ta ble 1. Bisphosphonates Screened for Antiparasitic Activity
Imax
C
In the case of axenically grown T. brucei trypomas-
tigotes, our results indicate the lowest IC50 of any of the
bisphosphonates tested in any organism studied here,
with a value of 220 nM being found for 9, Table 2 and
Figure 1C. We also found some activity against T. brucei
trypomastigotes with 2 (IC50 ∼ 8.6 µM) and its higher
homologue 6, which has an additional methylene group
in its side chain and showed an IC50 of ∼1.7 µM. In
addition, the aminomethylenebisphosphonate 14 showed
very high activity, having an IC50 of 700 nM (Table 2).
These results are all of interest since, on the basis of
previous work, 2 has been found to have an extremely
low IC50 for inhibiting FPPS, with values of 3.9 nM
being reported for a recombinant human enzyme.14
Moreover, 14 (Zeneca’s compound 1 in ref 16) has
been reported to have a 23 nM IC50 in FPPS inhibition
I )
(1)
IC50 + C
where I is the percent (%) inhibition, Imax ) 100%
inhibition, C is the concentration of the inhibitor, and
IC50 is the concentration for 50% growth inhibition. The
regression analyses were performed with Sigma Plot 5.0
(
SPSS Inc., Chicago, IL). By way of example, we show
results for four assays in Figure 1: 2 in T. gondii
tachyzoites (IC50 ) 490 nM, Figure 1A), the ortho-isomer
of risedronate (9) (2-(2-pyridyl)-1-hydroxyethane-1,1-
bisphosphonate) in T. gondii tachyzoites (IC50 ) 9.1 µM,
Figure 1B), 9 in T. brucei bloodstream trypomastigotes
IC50 ) 220 nM, Figure 1C), and 2-(4-imidazolyl)-1-
hydroxyethane-1,1-bisphosphonate (18) in T. cruzi in-
tracellular amastigotes (IC50 ) 35 µM, Figure 1D).
(

A Potential Route to Chemotherapy
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6 911
F igu r e 1. Four growth inhibition results. The percent (%) growth inhibition is plotted as a function of drug concentration. The
experimental results are fit to a rectangular hyperbolic function (see text, eq 1). The drugs/organism tested are as follows: A,
risedronate (2)/T. gondii tachyzoites; B, o-risedronate (9)/T. gondii tachyzoites; C, o-risedronate (9)/T. brucei trypomastigotes; D,
2
-(4-imidazolyl)-1-hydroxyethane-1,1-bisphosphonate (18)/T. cruzi amastigotes.
Ta ble 2. Growth Inhibition Results for Bisphosphonates against Five Parasitic Protozoaa
IC50 (µM)
T. brucei
T. cruzi
L. donovani
T. gondii
P. falciparum
compd
trypomastigotes
amastigotes
amastigotes
tachyzoites
intraerythrocytic stages
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
>200
147 ( 31.2
123 ( 26.4
>200
82.5 ( 14.6
2.3 ( 0.3
>200
25 ( 6.3
0.49 ( 0.05
>200
9.1 ( 1.4
35.6 ( 6.2
>200
>200
68.2 ( 13.2
9.1 ( 1.2
>200
8.6 ( 2.7
>200
123 ( 22.5
>200
92 ( 13.4
177 ( 51.1
1.7 ( 0.3
>200
>200
>200
130 ( 37.6
>200
60 ( 11.4
>200
t/100
>200
>200
>200
>200
129 ( 18.3
>200
>200
40 ( 5.1
>200
0.220 ( 0.09
31.7 ( 8.7
21.3 ( 7.6
19.8 ( 7.2
27.9 ( 13.1
0.7 ( 0.3
7.8 ( 3.0
99.8 ( 21.6
62.4 ( 18.3
8.6 ( 3.6
5.4 ( 0.9
105 ( 20.9
>200
t/100
>200
1
1
1
1
1
1
1
1
1
1
>200
>200
>200
>200
7.7 ( 1.4
158 ( 40.1
>200
>200
>200
>200
82.1 ( 9.0
t/100
147 ( 33.2
>200
>200
>200
>200
>200
>200
>200
56.7 ( 7.1
5.1 ( 1.6
>200
>200
35 ( 8.3
>200
t/+
>200
>200
a
Pentamidine IC50 ) 35 nM (T. brucei); chloroquine IC50 ) 3 nM (P. falciparum); pentostam IC50 ) 4.0 µg/mL (L. donovani); benznidazole
IC50 ) 3.5 µM (T. cruzi); t/100 ) toxic to cells, no parasites presents; t/+ ) toxic to cells, parasites present.

9
12 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6
Martin et al.
in a daffodil chromoplast assay, which strongly suggests
but does not prove) that FPPS is a major target for the
(
bisphosphonate inhibitors in T. brucei trypomastigotes.
For T. cruzi inhibition, we used three assay systems.
In the first, we used γ-irradiated myoblasts to assay for
amastigote proliferation. In the second, we used Vero
cells to assay for both amastigote proliferation and host
cell toxicity induced by the bisphosphonates. And in the
third, we used epimastigotes to investigate sterol bio-
synthesis inhibition induced by 2 alone and in combina-
tion with a sterol biosynthesis inhibitor (SBI), ketocon-
azole.
In the case of T. cruzi amastigote proliferation in
irradiated myoblasts (which do not undergo cell divi-
sion), 1, 2, and 5 have some activity, as does 9, but the
IC50 values are typically on the order of 100 µM,
considerably larger than found in the T. brucei trypo-
mastigote system. Nevertheless, as noted previously, 5
decreases the extent and delays the onset of parasitemia
F igu r e 2. Effects of risedronate (2) and ketoconazole on T.
cruzi epimastigote proliferation: 0, control; ], risedronate (100
µM); 2, ketoconazole (0.3 µM); b, risedronate (100 µM) plus
ketoconazole (0.3 µM).
1
7
in an in vivo murine model of acute Chagas’ disease.
Of greater interest is the observation that the imida-
1
8
zolium compound 18, an isomer of zoledronate, has
the lowest IC50 of any compound tested against T. cruzi
amastigote replication, with an IC50 ) 35 µM. We
believe that this correlates with the observation that
desmethyl sterols and a concomitant accumulation of
di- and trimethylated sterols, particularly 24-methyl-
enedihydrolanosterol, which becomes the most abun-
dant sterol in the cells. When the two drugs are used
in combination, Table 3, there is again a large increase
in the relative proportion of the exogenous sterol,
cholesterol, showing that the blockage of endogenous
sterol synthesis induced by 2 is at a pre-lanosterol level.
Since there is also no accumulation of squalene detected
1
8 has a very low inhibitory concentration for FPPS
1
9
(IC50 ) 1 nM) in the daffodil chromoplast assay and
readily docks into the FPPS active site (data not shown).
The higher IC50 values found with the T. cruzi amastig-
otes are at least in part a reflection of the increased
permeability barriers in this intracellular parasite, and
similar differences between in vitro enzyme inhibition
IC50 values and corresponding cell growth inhibition
IC50 values have previously been reported in bone
(data not shown), these results suggest that 2 inhibition
must be at the pre-squalene level, consistent with the
idea that FPPS is a principal target.
1
4,20,21
resorption work.
These results are clearly of interest since they rep-
resent the first demonstration of the effects of bispho-
sphonates on sterol biosynthesis in T. cruzi, and similar
effects are seen with L. mexicana as well (data not
shown). However, in both cases, the decrease in endog-
enous sterol production alone caused by 2 is insufficient
Thus, in both T. brucei and T. cruzi, some of the most
effective inhibitors of parasite growth have been shown
in other systems to have extremely low IC50 values in
FPP synthesis inhibition: 14 in T. brucei trypomastig-
otes correlating with a 23 nM IC50 in the plant assay
and 18 in T. cruzi amastigotes correlating with a 1 nM
IC50 in the same assay. These results strongly suggest
that FPPS is a major target for the nitrogen-containing
bisphosphonates in these protozoa.
22
to stop parasite growth. On the basis of the correla-
tions with FPPS inhibition activity of 2, 9, and 14 (in
other systems), we therefore expect a significant con-
tribution to parasite growth inhibition due to FPPS
inhibition, resulting in decreases in protein prenylation
and dolichol and ubiquinone formation, as well as the
decreased sterol production.
Since sterol biosynthesis should also be affected by
FPPS inhibition, we investigated the effects of one of
the more potent bisphosphonates, 2, on sterol biosyn-
thesis in T. cruzi, together with the effects of ketocona-
zole, a known 14R-demethylase SBI. We show in Figure
We have also investigated the effects of several
bisphosphonates on T. cruzi amastigote proliferation
inside Vero cells, to assess the general toxicity of such
compounds toward host cells. Vero cells, in contrast to
irradiated myoblasts, proliferate under our experimen-
tal conditions. Results are shown in Table 4. As can be
seen, in this system 2 and its higher homologue 6 at
150 µΜ are very potent inhibitors of replication of the
intracellular amastigote forms of T. cruzi and have no
effect on the host cells. The picolylaminomethylenebi-
sphosphonate ethyl ester 13 was also significantly
active, while compounds 11 and 12 were inactive
against both amastigotes and host cells. All other
compounds (including 1, 2, and 14) were toxic toward
the host cells at the same concentration, leading to
detachment and lysis. Toxicity was clear at 24 h and
cells were dead at 48 h. Further work with lower drug
2
the effects of 2 and ketoconazole on T. cruzi epimas-
tigote proliferation as a function of time. There is clearly
a substantial decrease in epimastigote proliferation in
the presence of 100 µM 2 or 0.3 µM ketoconazole. The
7
drugs were added at a cell density of 1 × 10 epimas-
tigotes/mL (time ) 50 h); 72 h after the addition of the
drugs (time ) 152 h) a marked reduction in the growth
rate was observed, probably due to the reduction of
endogenous sterol and isoprenoid content. The sterol
analyses were carried out 120 h after the addition of
the drugs (time ) 168 h). In Table 3 it can be seen that
2
alone causes a marked reduction in the level of
endogenous sterols, which drop to less than one-third
of total sterols. Treatment with ketoconazole leads, as
expected, to the disappearance of the parasite’s 4,14-

A Potential Route to Chemotherapy
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6 913
Ta ble 3. Effects of Risedronate and Ketoconazole on the Free Sterol Composition of T. cruzi Epimastigotes (EP stock)
Ta ble 4. Effects of Bisphosphonates at 150 µM on the
Proliferation of T. cruzi Amastigotes in Vero Cells
vestigated their effects on the proliferation of the
apicomplexan parasites, T. gondii and P. falciparum.
In the case of T. gondii intracellular tachyzoites, we
found the lowest IC50 with 2: IC50 ) 490 nM, Table 2.
In addition, significant activity was observed with 9,
IC50 ) 9.1 µM, and the simple hydroxybutane bispho-
sphonate 4, IC50 ) 9.1 µM. The significant activity of 2
and its isomer 9 is not unexpected, based on the results
found with these two nitrogen-containing bisphospho-
nates in the trypanosomatid parasites, Table 2. More
surprising is the observation that 4 also has significant
activity, even though it lacks the (aza)carbocation center
found in the (typically) more potent nitrogen-containing
bisphosphonates. This apparently anomalous effect is
even more pronounced in the case of P. falciparum
intraerythrocytic stages, where as shown in Table 2 the
two most effective inhibitors of proliferation are the
unsubstituted aromatic/aliphatic bisphosphonates 11
(IC50 ) 7.7 µM) and 17 (IC50 ) 5.1 µM), which have
benzyl and pentyl substituents, respectively. In the case
of the intraerythrocytic stages of P. falciparum, it seems
%
infected
% inhibition of
Vero cell
compd Vero cells amastigote proliferation
appearence
none
32 ( 4
0
normal
1
2
3
5
6
9
1
1
1
1
1
detached, lysed
normal
detached, lysed
detached, lysed
normal
detached, lysed
normal
normal
0
100
6 ( 2
81
1
2
3
4
8
30 ( 3
33 ( 4
9 ( 2
6
0
69
normal
detached, lysed
detached, lysed
levels is underway. Similarly, in the case of L. donovani
intracellular amastigotes, 2 was once again found to
have the lowest IC50 of any of the compounds investi-
gated, with an IC50 ) 2.3 µM, Table 2.
In addition to investigating the effects of bisphospho-
nates on these trypanosomatid parasites, we also in-

9
14 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6
Martin et al.
possible that drug delivery across the erythrocyte
membrane may be less effective with the polar side
chains found in the nitrogen-containing bisphospho-
nates. Further work with a homologous series of alkyl-
bisphosphonates is underway.
N-(2-(3-P icolyl))a m in om eth ylen ebisp h osp h on a te Tet-
r a eth yl Ester (13) a n d N-(2-(3-P icolyl))a m in om eth yl-
en ebisp h osp h on a te (14). N-(2-(3-Picolyl))aminomethylene-
bisphosphonate (14) and its tetraethyl ester 13 were prepared
by heating 0.05 mol of 2-amino-3-picoline to reflux with 0.10
mol of diethyl phosphite and 0.05 mol of triethyl orthoformate
at 100 °C for 12 h.24 The alcohol formed was distilled off and
the remaining product was purified on a silica gel column
Con clu sion s
(
EtOAc/acetone, 1:1) yielding compound 13. This compound
The results we have obtained above are of interest
for a number of reasons. They represent the first
detailed screening of a library of bisphosphonates
against the major trypanosomatid and apicomplexan
parasites: T. brucei, T. cruzi, L. donovani, T. gondii,
and P. falciparum. The results show that nanomolar to
low-micromolar IC50 values are found in all systems. In
addition, our results indicate that risedronate, 2, one
of the most powerful nitrogen-containing bisphospho-
nates, inhibits sterol biosynthesis in T. cruzi at a pre-
squalene level, most likely at the level of FPPS, based
on similar activity profiles found in plants, mammals,
and D. discoideum. Furthermore, on the basis of results
with T. cruzi amastigotes in cultured Vero cells, risedr-
onate, 2, appears least cytotoxic to host cells but is
highly effective against T. cruzi amastigote proliferation.
Also, with L. donovani, risedronate, 2, again appears
to have a very low IC50 and little toxicity to the host
cells. For the apicomplexan parasites T. gondii and P.
falciparum, our results show that the non-nitrogen-
containing bisphosphonates (4, 11, 17) have surprisingly
high activity (∼5-10 µM). In the case of P. falciparum
these bisphosphonates, which contain simple aryl/alkyl
groups, displayed a 10-20-fold lower IC50 than the
nitrogen-containing bisphosphonates, indicating either
a difference in uptake into the parasite cell and/or a
different site of action. Since none of the compounds
tested above were obtained as the product of any
rational (parasite-directed) drug design program, but
nonetheless exhibit IC50 values in the nanomolar to low-
micromolar range, these results appear very promising
from the perspective of designing more effective bispho-
sphonate chemotherapeutic agents based on compounds
specifically active against parasite enzymes.
was then hydrolyzed in 20 mL of 6 N HCl at 95 °C for 3 h
yielding compound 14 as a crystalline product. The purity of
all 19 samples prepared (Table 1) was verified by microchemi-
cal analysis (H/C/N/P) and via C, P, and H NMR spectros-
copy, the H NMR experiments being performed in triplicate
using an internal maleic acid quantitation standard. Absolute
compound purity as determined from these experiments was
9
13
31
1
1
8.6%, on average. (See Supporting Information for analytical
and spectroscopic data for target compounds.)
T. br u cei. T. brucei rhodesiense (strain STIB900) blood-
stream form trypomastigotes were maintained in HMI-18
medium supplemented with 20% HIFCS at 37 °C in a 5%
CO -air mixture. T. brucei rhodesiense tests were performed
in HMI-18 medium as above. Compounds were tested in
2
5
triplicate. Parasites were diluted to 2 × 10 /mL and added in
equal volumes to the test compounds in 96-well, flat bottom
Microtest III tissue culture plates (Becton Dickinson and Co.,
NJ ) with pentamidine isethionate (Aventis, Frankfort) as the
positive control, set up in parallel. Plates were maintained for
3
2
days at 37 °C in a 5% CO -air mixture. Compound activity
was determined by the use of an Alamar Blue fluorometric
assay25 on day 3.
6
T. cr u zi. γ-Irradiated L
6
E
9
myoblasts (1 × 10 cells/well)
in DMEM medium containing 20% fetal calf serum were plated
in 12-well tissue culture plates (Corning Glass Works, Corning,
NY) and incubated at 35 °C in a 7% CO atmosphere for 24
2
1
7
7
h. Cell monolayers were challenged with 5 × 10 trypomas-
tigotes/well. After 2 h of incubation at 35 °C in a 7% CO
2
atmosphere, cultures were washed twice with Hanks solution
and the culture medium was replaced, so that remaining
extracellular trypomastigotes were removed. At this time, 1.0
µCi of [5,6- H]uracil/well (specific activity, 40-50 Ci/mmol;
NEN Research Products, Boston, MA) and different concentra-
tions of drugs were added, and cultures were incubated for
3
3
an additional 72-h period. Incorporation of the [ H]uracil into
trichloroacetic acid (TCA)-precipitable material was measured
at this time. The supernatants from the monolayers were
removed by aspiration, the cells were washed twice with
Hanks solution and dissolved with 1 mL of 1% sodium dodecyl
sulfate containing 100 µg of cold uracil/mL. The contents of
each well were then pipetted to dissolve the cell layer and then
transferred to tubes. 3.0 mL of 5% TCA (vol/vol) solution was
added to each tube. The resulting precipitates were maintained
for 15 min on ice and collected on glass fiber filters (Whatman
GF/B) using a sampling manifold (Millipore, Bedford, MA). The
filters were washed twice with 3 mL of 5% TCA and once with
Exp er im en ta l Section
Syn th etic Asp ects. We prepared the 19 bisphosphonates
shown in Table 1 using the Merck procedure for the 1-hydroxy-
,1-bisphosphonates (1-12, 15-19):23
1
9
5% ethanol, dried and placed in 5 mL of scintillation cocktail
(BudgetSolve, Research Products International, Mount Pros-
pect, IL). Radioactivity was measured with a Packard (Model
Tri-Carb 2100 TR) liquid scintillation system. To investigate
the effects of antimicrobial agents on the replication of
amastigotes, the drugs were added after the T. cruzi challenge.
Compounds were tested in triplicate. The percent inhibition
The aminomethylenebisphosphonates (13, 14) were prepared
by suitable modification of the procedures of Soloducho
2
4
et al.:
3
of [ H]uracil incorporation (parasite growth) was calculated
with the following formula: (A - B/A) × 100, where A is the
mean counts per minute of infected control-treated myoblasts
and B is the mean counts per minute of infected drug-treated
myoblasts. Subtraction of the minor labeling of the uninfected
controls (typically around 300-400 cpm after 72-h incubation)
yielded the incorporation that could be ascribed to the intra-
cellular parasites (typically around 25 000 cpm after 70-h
incubation).
Ver o Cell Cu ltu r e of T. cr u zi Am a stigotes. Amastigotes
were cultured in Vero cells maintained in minimal essential
medium supplemented with 2% fetal calf serum in a humidi-
as described in detail below.

A Potential Route to Chemotherapy
fied 95% air-5% CO atmosphere at 37 °C. Cells were infected
with 10 tissue culture-derived trypomastigotes/cell for 2 h and
then washed three times with phosphate-buffered saline (PBS)
to remove nonadherent parasites. Fresh medium with or
without drugs was added and the cells were incubated for 96
h with a change of medium at 48 h. Quantitation of the number
of infected cells and the number of parasites per cell were
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 6 915
2
freeze-thawed (3×), harvested onto a Unifilter 96-well plate
(Canberra Packard, Meriden, CT), dried, sealed with 50 mL
of MicroScint40 and read on a Packard Topcount. Drugs were
tested against chloroquine-sensitive P. falciparum 3D7.
Ack n ow led gm en t. This work was supported in part
by the United States Public Health Service (National
Institutes of Health Grants GM-50694 to E.O. and AI-
determined by use of light microscopy. Statistical analysis of
the results was carried out as described previously.26
2
3259 to R.D.) and a Burroughs Wellcome New Initia-
Ster ol An a lyses. Sterols were extracted from T. cruzi
tives in Malaria Research Award to R.D. S.N.J .M. is a
Burroughs Wellcome New Investigator in Molecular
Parasitology. M.B.M. and B.N.B. are American Heart
Association Predoctoral Fellows, Midwest Affiliate. R.D.,
J .A.U., H.K., V.Y., and S.L.C. received financial support
from the UNDP/World Bank/WHO Special Programme
for Research & Training in Tropical Diseases (TDR).
epimastigotes cultured in LIT medium in the presence or
2
7
absence of drugs for 120 h. Drugs were added at a cell density
7
of 1 × 10 epimastigotes/mL. Sterols were separated from polar
lipids by silicic acid column chromatography and analyzed by
quantitative capillary gas-liquid chromatography and mass
spectrometry. Results are expressed as mass percent.
L. d on ova n i. L. donovani (strain MHOM/ET/67/L82) was
maintained in pathogen-free (SPF) female Golden hamsters
(
Wright’s strain, Charles River Ltd.) by passage every 6-8
28
Su p p or tin g In for m a tion Ava ila ble: Analytical data for
target compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
weeks. For in vitro assays, L. donovani peritoneal macroph-
ages were harvested from female CD1 mice (Charles River
Ltd., Margate U.K.) by peritoneal lavage 24 h after induction
by soluble starch (Merck Ltd., Leicester, U.K.). After two
washes in medium the exudate cells were dispensed into 16-
Refer en ces
4
well Lab-tek tissue culture slides (Nunc Inc., IL) at 4 × 10 /
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macrophages were infected at a ratio of 10:1 (4 × 10 /well)
(
3) Division of Parasitic Diseases, National Center for Infectious
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with L. donovani amastigotes freshly isolated from hamster
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presence of drug in a 3-fold dilution series, with quadruplicate
cultures at each concentration, for 5 days. After these periods
of drug exposure slides were fixed by methanol and Giemsa
stained. Drug activity was determined by counting the per-
centage of macrophages cleared of amastigote in treated
cultures in comparison with untreated cultures. Sodium sti-
bogluconate (GlaxoSmithKline) was used as a control.
(
(
(
4) Kirchhoff, L. V. American trypanosomiasis (Chagas’ Disease) -
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human foreskin fibroblast monolayers were grown in DMEM
2
9
medium containing 10% fetal calf serum, plated in 12-well
tissue culture plates and incubated at 37 °C in a 5% CO
atmosphere for 3 days. Cell monolayers were challenged with
6
5
solution and the culture medium replaced so that remaining
extracellular tachyzoites were removed. Different concentra-
tions of drugs were then added and the plates incubated for
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2
protozoa and helminths. Exp. Parasitol. 1996, 82, 76-85.
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× 10 tachyzoites/well. After 4 h of incubation at 37 °C in a
% CO atmosphere, cultures were washed twice with Hanks
2
(
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4
8 h at 37 °C (assay performed in triplicate). At this time, 1.0
3
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µCi of [5,6- H]uracil/well (specific activity, 40-50 Ci/mmol;
New England Nuclear Research Products, Boston, MA) was
added, and cultures were incubated for 4 h. Incorporation of
(
(
(
(
(
11) Martin, M. B.; Arnold, W.; Heath, H. T., 3rd, Urbina, J . A.;
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3
[
H]uracil into TCA-precipitable material was measured at this
time. The cells were dissolved with 1 mL of 1% sodium dodecyl
sulfate containing 100 µg unlabeled uracil/mL. The contents
of each well were then pipetted to dissolve the cell layer and
transferred to tubes. 3.0 mL of 5% TCA (vol/vol) was added to
each tube. The resulting precipitates were maintained at 4 °C
for 15 min and collected on glass fiber filters (Whatman GF/
B) using a sampling manifold (Millipore, Bedford, MA). The
filters were washed twice with 3 mL of 5% TCA and once with
5
60-3.
9
(
5% ethanol, dried and placed in 5 mL of scintillation cocktail
BudgetSolve, Research Products International, Mount Pros-
pect, IL). Radioactivity was measured with a Packard Tri-Carb
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100 TR liquid scintillation system.
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+
in human A erythrocytes in RPMI1640 medium supple-
mented with Albumax II at 37 °C in a 5% CO -air mixture.
P. falciparum intraerythrocytic cultures were set up as above,
with 1% ring stage parasitemia, 2.5% hematocrit, in triplicate
in 100 mL of medium in 96 well, flat-bottomed Microtest III
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added to each well. At the end of the assay, plates were rapidly
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