Synthesis and biological evaluation of 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase

Article abstract of DOI:10.1016/j.bmc.2007.12.010

The effect of a series of 2-alkylaminoethyl-1,1-bisphosphonic acids against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma g

Full text of DOI:10.1016/j.bmc.2007.12.010

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3283–3290
Synthesis and biological evaluation of 2-alkylaminoethyl-
1,1-bisphosphonic acids against Trypanosoma cruzi and
Toxoplasma gondii targeting farnesyl diphosphate synthase
a,ꢀ
a,b,ꢀ
b
b
Sergio H. Szajnman,b Guadalupe E. Garc ´ı a Li n˜ ares,
Zhu-Hong Li, Cuiying Jiang,
c
b
b
Melina Galizzi, Esteban J. Bontempi, Marcela Ferella, Silvia N. J. Moreno,
b
a,
*
Roberto Docampo and Juan B. Rodriguez
a
Departamento de Qu ´ı mica Org a´ nica and UMYMFOR (CONICET–FCEyN), Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Pabell o´ n 2, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina
b
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology,
University of Georgia, Athens, GA 30602, USA
c
Instituto Nacional de Parasitologia Dr. Mario Fatala Chab e´ n, Administraci o´ n Nacional de Laboratorios e Institutos de Salud,
Ministerio de Salud, Paseo Col o´ n 568, Buenos Aires C1063ACT, Argentina
Received 24 October 2007; revised 5 December 2007; accepted 6 December 2007
Available online 21 December 2007
Abstract—The effect of a series of 2-alkylaminoethyl-1,1-bisphosphonic acids against proliferation of the clinically more relevant
form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas’ disease), and against tachyzoites of Toxo-
plasma gondii has been studied. Most of these drugs exhibited an extremely potent inhibitory action against the intracellular form
of T. cruzi, exhibiting IC50 values at the low micromolar level. This cellular activity was associated with a strong inhibition of the
enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas’ disease chemo-
therapy. Compound 17 was an effective agent against amastigotes exhibiting an IC50 value of 0.84 lM, while this compound showed
an IC50 value of 0.49 lM against the target enzyme TcFPPS. Interestingly, compound 19 was very effective against both T. cruzi and
T. gondii exhibiting IC50 values of 4.1 lM and 2.6 lM, respectively. In this case, 19 inhibited at least two different enzymes of
T. cruzi (TcFPPS and solanesyl diphosphate synthase (TcSPPS); 1.01 lM and 0.25 lM, respectively), while it inhibited TgFPPS
in T. gondii. In general, this family of drugs was less effective against the activity of T. cruzi SPPS and against T. gondii growth
in vitro. As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their
potential low toxicity makes them good candidates to control tropical diseases.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1. Introduction
of the disease, in which 30–40% will develop serious, of-
3
ten lethal, cardiac and gastrointestinal tract lesions.
The hemoflagellated protozoan Trypanosoma cruzi is the
responsible agent of American trypanosomiasis (Chagas
disease), which is the primary source of cardiac death
T. cruzi is transmitted to humans in the feces of its tri-
atomine insect vector (such as Triatoma infestans or
Rhodnius prolixus) and, as other kinetoplastid parasites,
it has a complex life cycle, which includes obligatory
stages in the mammalian host and in the vector. In the
host T. cruzi has two forms: an intracellular dividing
form (amastigote) and a non-dividing highly infective
bloodstream form (trypomastigote) that can invade host
cells. Two forms also occur in the vector: one replicative
1
where this disease is endemic. It has been estimated that
over 18 million people are infected and over 40 million
1
,2
individuals are at risk of infection by this parasite.
Of the infected individuals, most are in the chronic stage
Keywords: Bisphosphonates; Trypanosoma cruzi; Toxoplasma gondii;
Farnesyl diphosphate synthase; Solanesyl diphosphate synthase; Cha-
gas’ disease; Toxoplasmosis.
(epimastigote) and one non-replicative (metacyclic
trypomastigote).
4
,5
*
Corresponding author. Tel.: +54 11 4 576 3346; fax: +54 11 576
385; e-mail: jbr@qo.fcen.uba.ar
These authors contributed equally to this work.
3
Chemotherapy of Chagas’ disease, which is based on old
and fairly non-specific drugs empirically discovered,
ꢀ
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.010

3284
S. H. Szajnman et al. / Bioorg. Med. Chem. 16 (2008) 3283–3290
such as nifurtimox (1) and benznidazole (2), remains
6
O
P
deficient. This illness has also been found in non-ende-
mic countries as a consequence of transfusion of con-
OH
OH
O
P
OH
H N
2
7
NH2
taminated blood from immigrants. For this reason, it
OH
OH
OH
5
HO
P
is very important to have an efficient agent to eradicate
the bloodstream trypomastigotes from blood banks as
well. Crystal violet (3), the only drug employed for
blood sterilization and discovered for that purpose some
4
HO
P
OH
O
OH
O
O
H C
3
8
OH
O
P
decades ago, suffers from some disadvantages, since it
9
was shown to be carcinogenic in in vivo assays (Fig. 1).
P
OH
N
N
OH
OH
OH
OH
HO
P
Studies of T. cruzi biochemistry and physiology have
recognized several crucial enzymes for parasite survival
7
HO
P
OH
6
O
OH
1
0
O
as potential targets for the design of new drugs. One
pathway that has been particularly useful for the identi-
fication of new targets against T. cruzi is the isoprenoid
pathway. Enzymes studied so far that are involved in the
Figure 2. Chemical structure of representative FDA-approved bis-
phosphonates clinically employed for bone disorders.
1
1
12
synthesis of sterols and farnesyl diphosphate, and in
onates has been limited to the isoprenoid pathway and
20
more specifically, to protein prenylation.
1
3
protein prenylation have been reported to be excellent
drug targets against pathogenic parasites. A recent re-
port about the presence of another important prenyl-
1.1. Rationale
transferase in T. cruzi,
a solanesyl diphosphate
synthase (TcSPPS), which is involved in the synthesis
of ubiquinone, suggested that it is another potential tar-
get for chemotherapy. For example, farnesyl diphos-
phate synthase (FPPS) has been demonstrated to be
the target of nitrogen-containing- and nitrogen-free bis-
phosphonates that have activity in vitro and in vivo
Nitrogen-containing bisphosphonates were originally
found to be effective against T. cruzi in vitro and
in vivo without toxicity to the host cells. More
1
4
18
recently, bisphosphonate: derivatives were found to be
effective growth inhibitors of pathogenic trypanosomat-
ids other than T. cruzi (T. brucei rhodesiense, Leishmania
donovani, and L. mexicana) and apicomplexan parasites
6
against trypanosomatids and apicomplexan parasites.
˚
The crystal structure of TcFPPS at 2 A resolution has
2
1–27
(Toxoplasma gondii and Plasmodium falciparum).
In
vivo assays of bisphosphonates have shown that risedr-
1
5
been recently published. In addition, the fact that bis-
phosphonates can target multiple sites of isoprenoid bio-
synthesis reinforces the idea of testing different enzymes
onate can significantly increase survival of T. cruzi-in-
2
8
fected mice.
The above results indicate that
1
6
involved in this biosynthetic pathway.
bisphosphonates are promising candidate drugs to treat
pathogenic parasitic infections by T. cruzi and other
protozoan parasites. In addition, bisphosphonates have
the advantage that they are straightforwardly and inex-
pensively prepared and many compounds bearing the
bisphosphonate moiety are FDA-approved drugs for
the long-term treatment of several bone disorders. A
low toxicity for bisphosphonate-containing drugs might
be predictable.
Several bisphosphonates, which are potent inhibitors of
bone resorption, are currently being used for the treat-
ment of several bone disorders such as osteoporosis, Pa-
get’s disease, hypercalcemia, tumor bone metastases,
1
7
etc. Selective action on bone is based on the binding
1
of the bisphosphonate moiety to the bone mineral.
7
The selective antiparasitic action of bisphosphonates
was proposed to be due to the presence of acidocalci-
somes, which, having an equivalent composition to the
Of special interest are bisphosphonates derived from
fatty acids, which were shown to be potent growth
inhibitors against the clinically more relevant form of
T. cruzi exhibiting IC50 values at the low micromolar
1
8
bone mineral, facilitate their accumulation. Represen-
tative nitrogen-containing bisphosphonates such as
pamidronate (4), alendronate (5), risedronate (6), and
ibandronate (7) act by a mechanism that leads to osteo-
2
3–25
range.
class of bisphosphonates with an IC50 value of
Compound 8 arises as main member of this
1
9
clast apoptosis (Fig. 2). The target of aminobisphosph-
2
3
18 lM. This cellular activity correlates quite properly
with the inhibition of the enzymatic activity toward
-
24
+
TcFPPS, being a competitive inhibitor with an IC
24
5
0
Cl
N
N
value of 1.94 lM and a K of 0.40 lM. Compound 8
i
O N
O
N
N
2
24
also inhibits the enzymatic activity of T. brucei FPPS,
26
and is active in vitro against T. gondii. Besides, SAR
O
S
1
O
studies indicated that the replacement of the hydroxyl
group at C-1 of 8 by a hydrogen atom to yield 9 resulted
in a less effective compound either as inhibitor of the
enzymatic activity of TcFPPS or as inhibitor of T. cruzi
O
O N
2
N
N
H
3
N
N
2
4
growth. The low efficacy observed for 9, in which the
hydroxyl group at C-1 is missing, may be attributable
2
2
to the lack of ability to coordinate Mg present at the
+
Figure 1. Current drugs for the treatment of Chagas’ disease and
blood sterilization.
2
9,30
active site of the target enzyme
or due to poor cell

S. H. Szajnman et al. / Bioorg. Med. Chem. 16 (2008) 3283–3290
3285
3
2
H O P
PO H
H O P
PO H
pound 13) as a Michael acceptor, which in turn was eas-
ily prepared from tetraethyl methylenebisphosphonate
2
3
3
2
2
3
3
2
C
C
R
H
(
compound 12) in two steps according to a slightly modi-
3
3
(
CH2)
CH2
n
fied Degenhart protocol. Therefore, compound 13 was
reacted with the corresponding amine in a Michael-type
reaction to afford the respective Michael adduct. Once
this synthetic intermediate was at hand, it was hydrolyzed
CH3
NH
8
,
n = 5; R = OH
n = 5; R = H
R'
9,
1
34
by treatment with concentrated hydrochloric acid to
11, R' = alkyl group
0, n = 4; R = NH2
afford the title compounds, that is, the corresponding free
bisphosphonic acids (14–27). The preparation of this new
family of2-alkylaminoethyl gem-bisphosphonates is illus-
trated in Scheme 1. Some of these compounds had previ-
Figure 3. Representative bisphosphonic acids derived from fatty acids.
permeability. In contrast, the tridentate 8 has three
coordination sites versus a bidentate structure that
clearly justifies its effectiveness toward TcFPPS. The
presence of the amino group would retain the ability
3
5,36
ously been shown to be herbicidal agents.
Compounds 14–27 were evaluated as growth inhibitors
against the amastigote form of T. cruzi, the clinically more
relevant form of the parasite. WC-9, a well-known anti-
2
+
to coordinate Mg in a tridentate structure similar to
compounds of the type 8. On the other hand, 1-amino-
3
7
parasitic agent, was used as a positive control. In addi-
tion, the correlation of the cellular activity with the
action against its target enzyme (TcFPPS) as well as
TcSPPS was studied. Besides, based on previous studies
on structurally related bisphosphonates against the
1
,1-bisphosphonate derivatives such as 10 were very po-
2
5
tent against TcFPPS at the nanomolar range, and also
were very effective inhibitors of T. cruzi proliferation but
to a lesser extent compared with 1-hydroxy-1,1-bisphos-
2
6,38
2
3,25
opportunistic pathogen T. gondii, this series of new
nitrogen-containing bisphosphonic acids was evaluated
against T. gondii and its putative target enzyme TgFPPS.
phonates.
Moreover, from the X-ray structure of the
complex of risedronate with TcFPPS, it can be observed
that residue Asp250 forms a hydrogen bond with the hy-
droxyl group present at the C-1 position of the molecule
of risedronate. This observation justifies the poor bio-
logical action observed by bisphosphonates where either
the hydroxyl group or the amino group is replaced by a
Most of the title compounds were potent inhibitors of
the enzymatic activity of TcFPPS. The efficacy of several
compounds on this enzyme qualitatively correlated with
their inhibitory action against growth of amastigotes of
T. cruzi. Taken compound 17 as an example, this com-
pound was a potent inhibitor of TcFPPS activity with
an IC50 in the nanomolar range (0.49 lM). These data
correlated completely with the efficacy of this compound
as antiparasitic agent. In fact, compound 17 proved to
be a potent inhibitor of the amastigote form of the par-
asite with an IC50 = 0.84 lM. Remarkably, 17 was >10-
1
5
hydrogen atom. Bearing in mind the above results, it
would seem of interest to carry out structural variations
on compound 10 taken as lead drug. Therefore, com-
pounds where the relative position of the nitrogen atom
with respect to the bisphosphonic unit is such as the
compound of general formula 11 were envisioned. This
family of compounds would retain the ability to coordi-
2
+
nate Mg in a tridentate structure. Since compounds 8
and 9 are nitrogen-free bisphosphonates, they could not
act as carbocation transition state analogues of the sub-
3
7,39
fold more potent than WC-9,
a well-known antipar-
asitic agent employed as positive control, which in turn
proved to be more effective than the currently used
drugs for the treatment of Chagas’ disease such as
nifurtimox and benznidazole under the same assay con-
ditions. The cellular activity exhibited by 17 was >20-
3
1
strate of FPPS as previously postulated. In addition,
nitrogen-containing bisphosphonates of the class 10
and 11 bear the nitrogen atom in such a position that
their protonated form cannot act as an analogue of a
transition state of the target enzyme (Fig. 3).
fold more potent than that exhibited by
2
8
3
(
IC50 = 18.1 lM), taken as lead drug for the present
study. A comparable degree of efficacy as inhibitors of
TcFPPS activity was observed for compounds 14 and
18. Surprisingly, in spite of being one order of magni-
tude more potent than 17 toward TcFPPS, compound
2
. Results and discussion
The title compounds were straightforwardly prepared
employing tetraethyl ethenylidenbisphosphonate (com-
18 exhibited indistinguishable antiproliferative potency
1
1
4, R =
5, R =
n
n
n
n
n
n
-propyl
-butyl
Et O P
PO Et
Et O P
PO Et2
H O P
PO H
2
3
3
2
2
3
3
2
3
3
2
C
C
C
16, R =
-pentyl
-hexyl
-heptyl
-octyl
H
1
1
1
2
2
7, R =
8, R =
9, R =
H
H
a
b,c
CH2
CH2
NH
R
0, R = 3-methyl-but-1-yl
1, R = tert-butyl
13
12
22, R = cyclohexyl
23, R = prop-2-yl
2
2
2
2
4, R = but-2-yl
5, R = 2-methylprop-1-yl
6, R = pyrrolidin-1-yl
7, R = piperidin-1-yl
2 2 2
Scheme 1. Reagents and conditions: (a) Refs. 40–42; (b) RNH , CH Cl (anh.), rt, 30 min; (c) (concd) HCl, reflux, 24 h, 94% for 14, 92% for 15, 98%
for 16, 85% for 17, 91% for 18, 78% for 19, 88% for 20, 71% for 21, 66% for 22, 79% for 23, 80% for 24, 68% for 25, 77% for 26, 84% for 27.

3286
S. H. Szajnman et al. / Bioorg. Med. Chem. 16 (2008) 3283–3290
Table 1. Comparison of IC50S for T. cruzi and T. gondii growth, and TcFPPS, TcSPPS, and TgFPPS inhibition
Compound
IC50 (lM)
T. cruzi amastigotes
IC50 (lM)
T. gondii tachyzoites
IC50 (lM)
TcFPPS
IC50 (lM)
TcSPPS
IC50 (lM)
TgFPPS
1
1
1
1
1
1
2
2
2
2
2
2
2
2
4
5
6
7
8
9
0
1
2
3
4
5
6
7
>50
4.8
6.33
48.67
>50
0.038 ± 0.008
2.28 ± 0.34
1.84 ± 0.25
11.01 ± 1.87
8.96 ± 2.18
0.298 ± 0.035
0.206 ± 0.010
0.080 ± 0.023
0.137 ± 0.087
0.095 ± 0.024
0.087 ± 0.038
0.275 ± 0.041
0.286 ± 0.059
0.337 ± 0.138
0.253 ± 0.069
0.112 ± 0.004
0.181 ± 0.050
0.361 ± 0.203
0.165 ± 0.045
0.54
0.84
16.79 ± 4.37
1.35 ± 0.22
1.69 ± 0.39
9.37
0.49 ± 0.12
10.0
4.1
>50
0.058 ± 0.009
1.014 ± 0.457
0.42 ± 0.18
2.60
0.252 ± 0.050
147.1 ± 77.2
59.8 ± 13.9
10.0
10.0
36.94
42.84
57.3
1.21 ± 0.13
36.3% at 10 lM
0.013 ± 0.003
1.399 ± 0.496
0.944 ± 0.211
0.515 ± 0.104
0.760 ± 0.316
0.287 ± 0.021
77.4 ± 4.09
>50
>50
>50
14
69.81
>100
74.47
13.30
70.60
0.761 ± 0.471
1.980 ± 0.359
3.493 ± 2.678
8.276 ± 6.474
10.034 ± 2.307
6.1
12.0
WC-9
against amastigotes compared to WC-9 and a notably
less potency than that shown by 17. These compounds
have to cross two cell membranes to get to the target
enzyme. Compound 18 is apparently less permeable
than compound 17 despite its lower charge to mass
ratio. For example, compound 14, which was the most
effective compound on TcFPPS (IC50 = 38 nM), was
devoid of activity against the intracellular form of the
parasite. These results were in agreement with our previ-
ous results with the isosteric analogue of 14, compound
Compounds 14 and 17 resulted to be moderately effec-
tive against T. gondii with IC50 values of 6.33 lM and
9.37 lM, respectively. The mode of action of these com-
pounds would also be by affecting protein prenylation at
the level of TgFPPS (IC50 values of 0.30 lM and
0.14 lM, respectively). Atovaquone was used as a posi-
tive control against T. gondii growth (IC = 0.29 lM).
50
4
0
Recent work has indicated that TgFPPS is a bifunc-
tional enzyme, catalyzing the formation of both farnesyl
diphosphate (FPP) and geranyl geranyl diphosphate
(GGPP). In agreement with that work, we found that
the correlation between inhibition of TgFPPS and
TcSPPS (a longer chain prenyl synthase) by 12 of the
bisphosphonates assayed in this work was high
9, in which the amino group was replaced by a methy-
lene group, which exhibited an IC50 value against
TcFPPS of 5.67 lM, and K = 0.47 lM, and was com-
i
pletely devoid of activity against amastigotes. The above
results indicated that the chain length was appropriate
in the case of 14 but its permeability should be opti-
mized. Compound 16 was the most potent compound
against the intracellular form of T. cruzi
2
(R = 0.69; Fig. 4), while there was no correlation
between of TgFPPS and TcFPPS inhibition, or between
TcFPPS and TcSPPS inhibition (data not shown).
(
IC50 = 0.54 lM). This activity correlated quite well with
the effect of this compound against the target enzyme
IC50 = 1.84 lM). Compound 19 was a very interesting
It can be concluded that the isosteric replacement of the
methylene group at the C-3 position of 1-alkyl-1,1-bis-
phosphonates such as compound 9 to give compounds
(
example of a compound effective against growth of both
T. cruzi and T. gondii showing IC50 values of 4.1 lM
and 2.6 lM, respectively. Compound 19 was very potent
against both target enzymes TcFPPS and TcSPPS espe-
cially against the latter one with IC50 of 1.01 lM and
0
.25 lM, respectively. The inhibitory action toward
TcSPPS was not surprising since this compound has
the longest side chain among the designed compounds;
therefore, it was better recognized by TcSPPS due to
substrate similarity. Moreover, 19 also exhibited potent
inhibitory action against TgFPPS at the nanomolar
range (IC50 = 87 nM). Compounds 17 and 18 were also
good inhibitors toward T. cruzi SPPS showing IC50 val-
ues of 1.35 lM and 1.69 lM, respectively. However, a
high selectivity against TcFPPS, except for 19, was ob-
served in all of the designed compounds. It is interesting
to note the lack of trend in inhibition of TcFPPS among
compounds 14–19, where there is a logical stepwise var-
iation in structure, but not in activity, and the lack of
correlation between growth and enzyme inhibition in
other cases (14, 18, 21, 23, 24, 25), suggesting in some
cases (16) off-target effects (Table 1).
Figure 4. Prenyl synthase inhibition correlation. Correlation between
TgFPPS and TcSPPS IC50 values, for the bisphosphonates indicated
with numbers.

S. H. Szajnman et al. / Bioorg. Med. Chem. 16 (2008) 3283–3290
3287
of general formula 11 was of great effectiveness against
both T. cruzi and the target enzyme TcFPPS. Most of
the designed compounds were even much more potent
than the gem-bisphosphonic acid 8, taken as a reference
structure, against both T. cruzi and the target enzyme,
T. cruzi farnesyl diphosphate synthase. Most of these
compounds proved to be inhibitors of the enzymatic
activity of TcSPPS but to a lesser extent than TcFPPS
as previously discussed. Moreover, some of these 1-[2-
3.1.2. 1-[(Prop-1-ylamino)ethyl] 1,1-bisphosphonic acid
1
(14). Yield 94% ; mp 195 ꢁC; H NMR (D O) d 0.86 (t,
2
J = 7.4 Hz, 3H, H-6); 1.60 (sex, J = 7.2 Hz, 2H, H-5);
2.43 (tt, J = 21.3, 7.0 Hz, 2H, H-1), 2.95 (t, J = 7.1 Hz,
1
3
2H, H-4), 3.33 (dt, J = 14.0, 7.1 Hz, 2H, H-2);
C
NMR (D O) d 10.04 (C-6), 19.07 (C-5), 35.98 (t,
2
3
1
J = 121.7 Hz, C-1), 44.58 (C-2), 49.17 (C-4); P NMR
(D O) d 16.14. Anal. (C H NO P ) Calcd C, 24.30;
H, 6.12; N, 5.67. Found: C, 24.01; H, 6.12; N, 5.70.
2
5
15
6 2
(
alkylamino)ethyl] bisphosphonic acids or 1-(3-aza-
alkyl)-1,1-bisphosphonic acids were shown to be moder-
ately effective anti-T. gondii agents indicating the broad
scope that this type of compounds has. Work aimed at
optimizing the chemical structure of 1-(3-azaalkyl)-1,1-
bisphosphonic acids such as compound 17 and other
closely related analogues is currently being pursued in
our laboratory.
3.1.3. 1-[(n-But-1-ylamino)ethyl] 1,1-bisphosphonic acid
(15). Yield 92%; mp 214 ꢁC; IR (BrK, cm ) 3101, 2314,
À1
1
1273, 1180, 956; H NMR (D O) d 0.83 (t, J = 7.4 Hz,
2
3H, H-7), 1.30 (sex, J = 7.5, 2H, H-6), 1.58 (pent,
J = 7.5 Hz, 2H, H-5), 2.42 (tt, J = 21.7, 7.0 Hz, 1H, H-
1), 3.01 (t, J = 7.4 Hz, 2H, H-4), 3.35 (dt, J = 14.3,
1
3
7.3 Hz, 2H, H-2); C NMR (D O) d 12.6 (C-7), 20.0
2
(
2
C-6), 27.5 (C-5), 36.1 (t, J = 120.3 Hz, C-1), 44.7 (C-
P NMR (D O) d 15.99. Anal.
3
1
), 47.4 (C-4);
2
3. Experimental
(C H NO P ) Calcd C, 27.59; H, 6.56; N, 5.36. Found:
6 17 6 2
C, 27.94; H 6.72; N, 5.57.
3
.1. General
3
.1.4. 1-[(n-Pent-1-ylamino)ethyl] 1,1-bisphosphonic acid
À1
The glassware used in air- and/or moisture-sensitive
reactions was flame-dried and reactions were carried
out under dry argon. Unless otherwise noted, chemi-
cals were commercially available and used without
further purification. Solvents were distilled before
use. Dichloromethane was distilled from phosphorus
pentoxide. Nuclear magnetic resonance spectra were
recorded with a Bruker AM-500 MHz spectrometer.
(16). Yield 98%; mp 204 ꢁC; IR (BrK, cm ) 3101, 2310,
1273, 1180, 960; H NMR (D O) d 0.78 (t, J = 6.9 Hz,
1
2
3H, H-8), 1.26 (m, 4H, H-6, H-7), 1.60 (q, J = 7.3 Hz,
2H, H-4), 1.59 (q, J = 7.4 Hz, 2H, H-4), 2.41 (tt,
J = 21.4, 7.3 Hz, 1H, H-1), 3.01 (t, J = 7.3 Hz, 2H, H-
1
3
4), 3.35 (dt, J = 14.3, 7.2 Hz, 2H, H-2); C NMR
(D O) d 13.0 (C-8), 21.4 (C-7), 25.1 (C-6), 27.7 (C-5),
2
3
1
36.1 (t, J = 121.2 Hz, C-1), 44.7 (C-2), 47.6 (C-4);
P
1
The H NMR spectra are referenced with respect to
the residual CHCl3 proton of the solvent CDCl3 at
NMR (D O) d 15.99. Anal. (C H NO P Æ1/2H O)
2
7
19
6
2
2
Calcd C, 29.59; H, 7.09; N, 4.93. Found: C, 29.78; H,
6.86; N, 4.93.
d = 7.26 ppm. Coupling constants are reported in Hz.
C NMR spectra were fully decoupled and are refer-
1
3
enced to the middle peak of the solvent CDCl at
3
d = 77.0 ppm. P NMR spectra are referenced with
3.1.5. 1-[(n-Hex-1-ylamino)ethyl] 1,1-bisphosphonic acid
(17). Yield 85%; mp 212 ꢁC; IR (BrK, cm ) 3105, 2307,
3
1
À1
1
respect to the peak of 85% H PO as external refer-
1712, 1273, 1176, 956; H NMR (D O) d 0.76 (t,
3
4
2
ence. Splitting patterns are designated as s, singlet;
d, doublet; t, triplet; q, quadruplet; dd, double dou-
blet, etc. Melting points were determined with a Fish-
er–Johns apparatus and are uncorrected. IR spectra
were recorded with a Nicolet Magna 550 spectrome-
ter. Analytical TLC was performed on commercial
J = 6.6 Hz, 3H, H-9), 1.21 (m, 4H, H-7, H-8), 1.28 (m,
2H, H-6), 1.59 (pent, J = 7.4 Hz, 2H, H-5), 2.42 (tt,
J = 21.5, 7.2 Hz, 1H, H-1), 3.00 (t, J = 7.4 Hz, 2H, H-
1
3
4), 3.34 (dt, J = 14.3, 7.3 Hz, 2H, H-2); C NMR
(D O) d 13.1 (C-9), 21.6 (C-8), 25.2 (C-7),* 25.4 (C-
2
6),* 30.3 (C-5), 36.1 (t, J = 120.7 Hz, C-1), 44.7 (C-2),
3
1
0.2 mm aluminum-coated silica gel plates (F254) and
visualized by 254 nm UV or immersion in an aqueous
47.7 (C-4);
P
NMR (D O)
d
16.02. Anal.
(C H NO P ) Calcd C, 33.22; H, 7.32; N, 4.84. Found:
2
8
21
6 2
solution of (NH ) Mo O Æ4H O (0.04 M), Ce(SO )
C, 33.39; H, 7.13; N, 4.95.
4
6
7
24
2
4 2
(
0.003 M) in concentrated H SO4 (10%). Elemental
2
analyses were conducted by Atlantic Microlab Inc.,
Norcross, Georgia. The results were within ±0.4% of
the theoretical values.
3.1.6. 1-[(n-Hept-1-ylamino)ethyl] 1,1-bisphosphonic acid
(18). Yield 91%; mp 207 ꢁC; IR (BrK, cm ) 3094, 2310,
À1
1
1269, 1180, 1003; H NMR (D O) d 0.76 (t, J = 6.0 Hz,
2
3H, H-9), 1.18–1.24 (m, 8H, H-8, H-7, H-6, H-5), 1.59
3
.1.1. Synthesis of 1-[2-alkylaminoethyl]-1,1-bisphos-
(q, J = 7.1 Hz, 2H, H-4), 2.43 (tt, J = 21.4, J = 7.0 Hz,
1H, H-1), 3.00 (t, J = 6.9 Hz, 2H, H-3), 3.34 (dt,
phonic acids. General procedure. A solution of com-
pound 13 (10 mmol) in anhydrous methylene chloride
1
3
J = 13.9, 7.1 Hz, 2H, H-2); C NMR (D O) d 13.3 (C-
2
(
(
10 ml) was treated with the corresponding amine
10 mmol) under an argon atmosphere. The reaction
9), 21.8 (C-8), 25.4 (C-7),* 25.5 (C-6),* 27.8 (C-4), 30.7
(C-5), 36.0 (t, J = 120.3 Hz, C-1), 44.7 (C-2), 47.7
3
1
mixture was stirred at room temperature for 30 min.
The solvent was evaporated and the residue was treated
with a concentrated aqueous solution of hydrochloric
acid (2 ml). The resulting mixture was refluxed for
(C-3); P NMR (D O) d 15.61. Anal. (C H NO P )
2 9 13 6 2
Calcd C, 35.65; H, 7.65; N 4.62. Found: C, 35.38; H,
7.59; N, 4.80.
24 h. The solvent was evaporated and the residue was
crystallized from ethanol.
3.1.7. 1-[(n-Oct-1-ylamino)ethyl] 1,1-bisphosphonic acid
1
(19). Yield 78%; mp 202 ꢁC; H NMR (D O) d 0.70 (t,
2

3288
S. H. Szajnman et al. / Bioorg. Med. Chem. 16 (2008) 3283–3290
J = 6.9 Hz, 3H, H-11), 1.13 (m, 10H, H-6, H-7, H-8, H-
, H-10), 1.36 (pent, J = 7.4 Hz, 2H, H-5), 1.73 (tt,
J = 21.0, 6.9 Hz, 1H, H-1), 2.48 (t, J = 7.6 Hz, 2H, H-
(D O) d 0.89 (d, J = 6.9 Hz, 6H, H-6), 1.90 (n,
2
9
J = 6.8 Hz, 1H, H-5), 2.42 (tt, J = 21.3, 7.3 Hz, 1H, H-
1), 2.84 (d, J = 7.0 Hz, 1H, H-4), 3.34 (dt, J = 14.0,
1
3
13
4
), 2.84 (dt, J = 14.3, 6.2 Hz, 2H, H-2); C NMR
7.4 Hz, 2H, H-2); C NMR (D O) d 18.9 (C-6), 25.5
2
(
8
D O) d 13.3 (C-11), 21.9 (C-10), 26.5 (C-9), 28.1 (C-
(C-5), 35.9 (t, J = 121.2 Hz, C-1), 45.1 (C-2), 54.6 (C-
2
3
1
), 28.3 (C-7), 28.5 (C-6), 31.1 (C-5), 40.6 (t,
4); P NMR (D O) d 16.36. Anal. (C H NO P ) Calcd
2 6 17 6 2
3
1
J = 116.7 Hz, C-1), 48.1 (C-2), 48.3 (C-4); P NMR
D O) d 17.99. Anal. (C H NO P ) Calcd C, 37.86;
H, 7.94; N, 4.41. Found: C, 37.69; H, 7.85; N, 4.40.
C, 27.60; H, 6.56; N, 5.36. Found: C, 27.31; H, 6.50; N,
5.20.
(
2
10 25
6 2
3
.1.14. 1-[(2-Pyrrolidin-1-ylamino)ethyl] 1,1-bisphosphon-
1
3
.1.8. 1-[(3-Methyl-but-1-ylamino)ethyl] 1,1-bisphosphon-
ic acid (26). Yield 77%; mp 235–236 ꢁC; H NMR (D O)
2
1
ic acid (20). Yield 88%; mp 231 ꢁC; H NMR (D O) d
d 1.95 (m, 2H, H–), 2.07 (m, 2H, H–), 2.53 (tt, J = 21.6,
7.7 Hz, 1H, H-1), 3.05 (2H, H–), 3.48 (ddd, J = 14.8,
12.6, 7.8 Hz, 2H, H–), 3.65 (p, J = 5.6 Hz, 2H, H–);
2
0
7
.83 (d, J = 6.4 Hz, 6H, H-6), 1.49 (dd, J = 15.1,
.1 Hz, 2H, H-4), 1.57 (hept, J = 6.6 Hz; H, H-5), 2.40
1
3
(
H-3), 3.35 (dt, J = 14.3, 7.2 Hz, 2H, H-2); C NMR
tt, J = 21.5, 7.3 Hz, 1H, H-1), 3.04 (t, J = 7.7 Hz, 2H,
C NMR (D O) d 22.8 (C-5), 36.1 (t, J = 121.0 Hz, C-
2
31
1
3
1), 52.8 (C-2), 54.4 (C-4); P NMR (D O) d 15.51. Anal.
2
(
D O) d 21.29 (C-6), 25.03 (C-5), 34.20 (C-4), 36.18 (t,
(C H NO P Æ2H O) Calcd C, 24.42; H, 6.49; N, 4.75.
Found: C, 24.70; H, 6.47; N, 4.66.
2
6
15
6
2
2
3
1
J = 120.3 Hz, C-1), 44.85 (C-2), 46.09 (C-3); P NMR
D O) d 15.87. Anal. (C H NO P ) Calcd C, 30.55;
H, 6.96; N, 5.09. Found: C, 30.79; H, 6.96; N, 5.08.
(
2
7
19
6 2
3.1.15. 1-[(2-Piperidin-1-ylamino)ethyl] 1,1-bisphosphonic
1
acid (27). Yield 84%; mp 226–228 ꢁC; H NMR (D O) d
2
3
.1.9. 1-[(tert-Butylamino)ethyl] 1,1-bisphosphonic acid
1.38 (m, 1H, H–), 1.58 (m, 2H, H–), 1.69 (m, 1H, H–),
1.85 (m, 2H, H–), 2.61 (tt, J = 21.4, 7.7 Hz, 1H, H-1),
2.86 (t, J = 11.1 Hz, 2H, H–), 3.31 (m, 2H, H–), 3.49
1
(
9
21). Yield 71%; mp 238 ꢁC; H NMR (D O) d 1.28 (s,
2
H, C(CH ) ), 2.31 (tt, J = 21.4, 7.3 Hz, 1H, H-1),
3
3
1
.31 (dt, J = 14.2, 7.3 Hz, 2H, H-2); C NMR (D O)
3
13
3
(t, J = 11.1 Hz, 2H, H–); C NMR (D O) d 21.3 (C-
2
2
d 24.98 C(CH ), 36.34 (t, J = 120.8 Hz, C-1), 38.86
5), 23.4 (C-5), 34.0 (t, J = 121.0 Hz, C-1), 53.4 (C-2),
54.3 (C-4);
3
3
3
1
31
(
C-2), 57.05 (C-3); P NMR (D O) d 16.04. Anal.
P
NMR (D O)
2
d
15.60. Anal.
2
(
C, 27.21; H, 6.55; N, 5.14.
C H NO P ) Calcd C, 27.59; H, 6.56; N, 5.36. Found:
(C H NO P ) Calcd C, 30.78; H, 6.27; N, 5.13. Found:
7 17 6 2
C, 30.33; H, 6.30; N, 4.99.
6
17
6
2
3
.1.10. 1-[(Cyclohexylamino)ethyl] 1,1-bisphosphonic acid
3.2. Drug screening
1
(22). Yield 66%; mp 227 ꢁC; H NMR (D O) d 2.38 (tt,
J = 21.5, 7.3 Hz), 3.07 (m, 1H, H–), 3.35 (dt, J = 14.3,
2
Experiments on the intracellular form of the parasite
were conducted on T. cruzi-infected L E myoblasts
(Y strain) as described before.
1
3
7
2
.2 Hz, 2H, H–); C NMR (D O) d 23.64, 24.42,
2
6 9
3
1
41,42
8.92, 36.01 (t, J = 121.7 Hz, C-1), 41.76, 57.09;
P
Gamma-irradiated
7
NMR (D O) d 16.12. Anal. (C H NO P ) Calcd C,
3
5
L E myoblasts (1 · 10 cells/plate) in DMEM contain-
2
8
19
6
2
6 9
3.46; H, 6.67; N, 4.88. Found: C, 33.38; H, 6.64; N,
.01.
ing 20% FCS were plated in 12-well tissue culture plates
and incubated at 37 ꢁC in a 5% CO atmosphere for
2
2
4 h. After 24 h, wells were washed once and fresh med-
6
3
.1.11. 1-[(Prop-2-ylamino)ethyl] 1,1-bisphosphonic acid
ia were added containing 4.17 · 10 trypomastigotes or
amastigotes/well in DMEM. One well was left without
parasites for control. After 2 h of incubation at 37 ꢁC
1
(
23). Yield 79%; mp 227–228 ꢁC; H NMR (D O) d 1.23
2
(d, J = 6.6 Hz, 6H, H-5), 2.37 (tt, J = 21.5, 7.2 Hz, 1H,
H-1), 3.33 (dt, J = 14.2, 7.1 Hz, 2H, H-2), 3.37 (sept,
in a 7% CO atmosphere, cultures were washed twice
2
1
3
J = 6.5 Hz, 1H, H-4); C NMR (D O) d 18.3 (C-5),
3
NMR (D O) d 16.04. Anal. (C H NO P ) Calcd C,
with Hanks’ solution, and culture medium was replaced
to remove extracellular parasites. At this time, 0.5–1 lCi
of [5,6- H]uracil/well (specific activity, 40–50 Ci/mmol)
2
3
1
6.1 (t, J = 121.7 Hz, C-1), 42.0 (C-2), 50.8 (C-4);
P
3
2
5
15
6 2
2
5
4.30; H, 6.12; N, 5.67. Found: C, 24.50; H, 6.27; N,
.74.
and drug solutions in water were added. Two wells were
left for infection control. Cultures were incubated for
7
3
2 h. Incorporation of the [5,6- H]uracil into trichloro-
3
.1.12. 1-[(But-2-ylamino)ethyl] 1,1-bisphosphonic acid
acetic acid (TCA)-precipitable material was measured
at day 3. The supernatants from the monolayers were
transferred to glass tubes, the cells were dissolved with
1.3 ml of 1% sodium dodecyl sulfate containing 100 lg
of cold uracil per ml, and the suspension was transferred
to the glass tubes. The wells were rinsed with 3 ml of 5%
TCA (ice-cold) which was combined with the previous
suspension. The samples were maintained in ice for
15 min and collected on glass fiber filters (Whatman
GF/B) by using a sampling manifold (Millipore, Bed-
ford, MA). After filtering, the tubes were rinsed twice
with 4 ml of 5% TCA and the filters were rinsed twice
with TCA and once with 95% ethanol. After drying
the filters, they were placed in scintillation vials contain-
1
(
24). Yield 80%; mp 197 ꢁC; H NMR (D O) d 0.83
2
(dist. t, J = 7.4 Hz, 3H, H-6), 1.16 (d, J = 6.6 Hz, 3H,
CH at C-4), 1.49 (m, 1H, H-5 ), 1.60 (m, 1H, H-5 ),
3
a
b
2
3
.38 (tt, J = 21.7, 7.3 Hz, 1H, H-1), 3.15 (m, 1H, H-4),
.26 (m, 1H, H-2 ) 3.37 (m, 1H, H-2 ); C NMR
1
3
a
b
(
J = 122.1 Hz, C-1), 25.9 (C-5), 41.9 (C-2), 56.0 (C-4);
D O) d 8.7 (C-6), 15.1 (CH3 at C-4), 35.8 (t,
2
3
1
P NMR (D O) d 16.14 (d, J = 7.4 Hz, P ), 16.14 (d,
2
a
J = 7.4 Hz, P ). Anal. (C H NO P ) Calcd C, 27.60;
H, 6.56; N, 5.36. Found: C, 27.81; H, 6.65; N, 5.44.
b
6
17
6 2
3
.1.13. 1-[(2-Methylprop-1-ylamino)ethyl] 1,1-bisphos-
1
phonic acid (25). Yield 68%; mp 220–222 ꢁC; H NMR

S. H. Szajnman et al. / Bioorg. Med. Chem. 16 (2008) 3283–3290
3289
ing 4–5 ml of scintillation cocktail Ecolume. Vials were
vortexed for 20 s and counted. The percent inhibition
was calculated by employing the following formula: Per-
cent inhibition = 100 À (DC · 100)/DC , where DC and
was washed with water saturated with NaCl and trans-
ferred to a scintillation vial with 4 ml of scintillation
solution Ecolume for counting. One unit of enzyme
activity was defined as the activity required to incorpo-
d
c
c
1
4
14
DC are the differences in the counts per minute of con-
rate 1 nmol of [4- C]IPP into [4- C]SPP in 1 min.
d
trol cultures and drug-treated cultures, respectively.
Experiments on T. gondii tachyzoites were carried out as
4
Acknowledgments
3
previously published. Briefly, T. gondii tachyzoites of
4
the m2m3 clone expressing yellow fluorescence protein
3
This work was supported by grants from the National
Research Council of Argentina (PIP 5508), ANPCyT
PICT2004 #21897), and the Universidad de Buenos
Aires (X-252) to J.B.R., and the U.S. National Institutes
of Health to R.D. (AI-68647) and S.N.J.M. (AI68467).
G.G.L. thanks the Ellison Medical Foundation for a
Fellowship.
were routinely maintained in vitro in human foreskin
fibroblast monolayers (HFF) in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum, 2 mM glutamine, and 1 mM pyruvate, at
(
3
7 ꢁC in a humid 5% CO atmosphere. Confluent HFF
2
monolayers grown in 96-well black plates with optical
bottoms (Falcon/Becton–Dickinson, Franklin Lakes,
NJ) were used and drugs dissolved in the same medium
and serially diluted in the plates. Freshly isolated tach-
yzoites were filtered through a 3 lm filter and passed
through a 22 gauge needle, before use. The cultures were
Supplementary data
4
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.12.010.
inoculated with 10 tachyzoites/ml in the same media.
The plates were incubated at 37 ꢁC and read daily in a
Molecular Devices fluorescent plate reader. To preserve
sterility the plates were read with covered lids, and both
excitation (510 nm) and emission (540 nm) were read
References and notes
4
1
from the bottom. For the calculation of the IC , the
0
5
percent of growth inhibition was plotted as a function
of drug concentration by fitting the values to the func-
tion: I = ImaxC/(IC50 + C), where I is the percent inhibi-
tion, Imax = 100% inhibition, C is the concentration of
the inhibitor, and IC50 is the concentration for 50%
growth inhibition.
1
. Urbina, J. A.; Docampo, R. Trends Parasitol. 2003, 19,
95–501.
4
2. Moncayo, A. In Eleventh Programme Report of the
UNDP/World Bank/WHO Special Program for Research
and Training in Tropical Diseases (TDR). World Health
Organization: Geneva, 1995, pp 67–75.
3
. Docampo, R. Curr. Pharm. Des. 2001, 7, 1157–1164.
. Brener, Z. Annu. Rev. Microbiol. 1973, 27, 347–382.
4
5. De Souza, W. Int. Rev. Cytol. 1984, 86, 197–283.
3
For TcFPPS
.2.1. TcFPPS and TgFPPS assays and product analysis.
3
1,44,45
100 ll of assay buffer (10 mM Hepes,
6
7
8
. Garc ´ı a Li n˜ ares, G.; Ravaschino, E. L.; Rodriguez, J. B.
Curr. Med. Chem. 2006, 13, 335–360.
. Galel, S. A.; Kirchhoff, L. V. Transfusion 1996, 36, 227–
pH 7.4, 5 mM MgCl , 2 mM dithiothreitol, 100 lM
2
1
4
[
4- C]IPP (10 lCi/lmol)) and 100 lM DMAPP were
prewarmed to 37 ꢁC. The assay was initiated by the
addition of recombinant protein (10–20 ng). The assay
was allowed to proceed for 30 min at 37 ꢁC and was
quenched by the addition of 6 M HCl (10 ll). The reac-
tion mixtures were made alkaline with 6.0 M NaOH
2
31.
. Nussenzweig, V.; Sonntag, R.; Biancalana, A.; Pedreira de
Fleitas, J. L.; Amato Neto, V.; Kloetzel, J. Hospital (Rio
de Janeiro) 1953, 44, 731–744.
9. Docampo, R.; Moreno, S. N. J. Rev. Biochem. Toxicol.
1985, 7, 159–204.
(
15 ll), diluted in water (0.7 ml), and extracted with hex-
10. De Castro, S. L. Acta Trop. 1993, 53, 83–98.
11. Urbina, J. A. Curr. Pharm. Des. 2002, 8, 287–295.
12. Docampo, R.; Moreno, S. N. J. Curr. Drug Targets Infect.
Disord. 2001, 1, 51–61.
ane (1 ml). The hexane solution was washed with water
and transferred to a scintillation vial for counting. One
unit of enzyme activity was defined as the activity re-
1
4
quired to incorporate 1 nmol of [4- C]IPP into
1
3. Gelb, M. H.; Van Voorhis, W. C.; Buckner, F. S.;
Yokoyama, K.; Eastman, R.; Carpenter, E. P.; Panet-
hymitaki, C.; Brown, K. A.; Smith, D. F. Mol. Biochem.
Parasitol. 2003, 126, 155–163.
1
14- C]FPP in 1 min. For TgFPPS the assay conditions
4
[
were as described above except that the buffer contained
1
mM MgCl2.
1
4. Ferella, M.; Montalvetti, A.; Rohloff, P.; Miranda, K.;
Fang, J.; Reina, S.; Kawamukai, M.; B u´ a, J.; Nilsson, D.;
3.2.2. TcSPPS assay. The activity of the enzyme was
determined by a radiometric assay based on that de-
˚
Pravia, C.; Katzin, A.; Cassera, M. B.; Aslund, L.;
1
4,46
Andersson, B.; Docampo, R.; Bontempi, E. J. J. Biol.
Chem. 2006, 281, 39339–39348.
15. Gabelli, S. B.; McLellan, J. S.; Montalvetti, A.; Oldfield,
scribed before.
Briefly, 100 ll of assay buffer
(
(
100 mM Tris–HCl buffer, pH 7.4, 1 mM MgCl , 1%
v/v) Triton X-100, 100 lM [4- C]IPP (10 lCi/lmol))
2
1
4
E.; Docampo, R.; Amzel, L. M. Proteins 2006, 62, 80–88.
and 50 lM GGPP were prewarmed to 37 ꢁC. The assay
was initiated by the addition of 10–20 ng of recombinant
protein. The assay was allowed to proceed for 30 min at
1
6. Guo, R.-T.; Cao, R.; Liang, P.-H.; Ko, T.-P.; Chang, T.-H.;
Hudock, M. P.; Jeng, W.-Y.; Chen, C. K.-M.; Zhang, Y.;
Song, Y.; Kuo, C.-J.; Yin, F.; Oldfield, E.; Wang, A. H.-J.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10022–10027.
3
7 ꢁC and was quenched by chilling quickly in an ice
bath. The reaction products were extracted with 1 ml
of 1-butanol saturated with water. The organic layer
17. Reszka, A. A.; Rodan, G. A. Mini Rev. Med. Chem. 2004,
4, 711–719.

3290
S. H. Szajnman et al. / Bioorg. Med. Chem. 16 (2008) 3283–3290
1
8. Urbina, J. A.; Moreno, B.; Vierkotter, S.; Oldfield, E.;
Payares, G.; Sanoja, C.; Bailey, B. N.; Yan, W.; Scott, D.
30. Tarshis, L. C.; Proteau, P. J.; Kellogg, B. A.; Sacchettini,
J. C.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 15018–15023.
31. Montalvetti, A.; Bailey, B. N.; Martin, M. B.; Severin, G.
W.; Oldfield, E.; Docampo, R. J. Biol. Chem. 2001, 276,
33930–33937.
32. Szajnman, S. H.; Garc ´ı a Li n˜ ares, G. E.; Moro, P.;
Rodr ´ı guez, J. B. Eur. J. Org. Chem. 2005, 3687–
3696.
33. Degenhardt, C. R.; Burdsall, D. C. J. Org. Chem. 1986,
51, 3488–3490.
34. Wasielewski, C.; Antczak, K. Synthesis 1981, 540–541.
35. Cromartie, T. H.; Fisher, K. J. PCT Int. Appl. 1995, WO
95/34207 A1.
36. Fisher, K. J.; Woolard, F. X.; Leadbetter, M. R.; Gerdes,
J. M. 1998, U.S. Patent 5,728,650.
37. Cinque, G. M.; Szajnman, S. H.; Zhong, L.; Docampo,
R.; Rodriguez, J. B.; Gros, E. G. J. Med. Chem. 1998, 41,
1540–1554.
A.; Moreno, S. N. J.; Docampo, R. J. Biol. Chem. 1999,
274, 33609–33615.
1
2
2
9. Hughes, D. E.; Wright, K. R.; Uy, H. L.; Sasaki, A.;
Yoneda, T.; Roodman, G. D.; Mundy, G. R.; Boyce, B. F.
J. Bone Mineral. Res. 1995, 10, 1478–1487.
0. Rogers, M. J.; Frith, J. C.; Luckman, S. P.; Coxon, F. P.;
Benford, H. L.; Monkkonen, J.; Auriola, S.; Chilton, K.
M.; Russell, R. G. Bone 1999, 24, 73S–79S.
1. 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. J. Med. Chem. 2002, 45, 2904–2914.
2. 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, S.; Nozaki, T.; Oldfield, E. J. Med. Chem.
2
2004, 47, 175–187.
2
3. Szajnman, S. H.; Bailey, B. N.; Docampo, R.; Rodriguez,
J. B. Bioorg. Med. Chem. Lett. 2001, 11, 789–792.
4. Szajnman, S. H.; Montalvetti, A.; Wang, Y.; Docampo,
R.; Rodriguez, J. B. Bioorg. Med. Chem. Lett. 2003, 13,
38. Rodrigues, C. O.; Scott, D. A.; Bailey, B.; de Souza, W.;
Benchimol, M.; Moreno, B.; Urbina, J. A.; Oldfield, E.;
Moreno, S. N. J. Biochem. J. 2000, 349, 737–745.
39. Urbina, J. A.; Concepcion, J. L.; Montalvetti, A.; Rodri-
guez, J. B.; Docampo, R. Antimicrob. Agents Chemother.
2003, 47, 2047–2050.
40. Ling, Y.; Li, Z.-H.; Miranda, K.; Oldfield, E.; Moreno, S.
N. J. J. Biol. Chem. 2007, 282, 30804–30816.
41. Ravaschino, E. L.; Docampo, R.; Rodriguez, J. B. J. Med.
Chem. 2006, 49, 426–435.
42. Yan, W.; Moreno, S. N. J. Immunol. Methods 1998, 220,
123–128.
43. Gubbels, M. J.; Li, C.; Striepen, B. Antimicrob. Agents
Chemother. 2003, 47, 309–316.
44. Montalvetti, A.; Fernandez, A.; Sanders, J. M.; Ghosh, S.;
Van Brussel, E.; Oldfield, E.; Docampo, R. J. Biol. Chem.
2003, 278, 17075–17083.
45. Ogura, K.; Nishino, T.; Shinka, T.; Seto, S. Methods
Enzymol. 1985, 110, 167–171.
46. Rilling, H. C. Methods Enzymol. 1985, 110, 145–152.
2
3231–3235.
2
2
2
5. Szajnman, S. H.; Ravaschino, E. L.; Docampo, R.;
Rodriguez, J. B. Bioorg. Med. Chem. Lett. 2005, 15,
4685–4690.
6. Ling, Y.; Sahota, G.; Odeh, S.; Chan, J. M. W.; Araujo, F.
G.; Moreno, S. N. J.; Oldfield, E. J. Med. Chem. 2005, 48,
130–3140.
7. Martin, M. B.; Grimley, J. S.; Lewis, J. C.; Heath, H. T.,
rd; Bailey, B. N.; Kendrick, H.; Yardley, V.; Caldera, A.;
3
3
Lira, R.; Urbina, J. A.; Moreno, S. N.; Docampo, R.;
Croft, S. L.; Oldfield, E. J. Med. Chem. 2001, 44, 909–916.
8. Bouzahzah, B.; Jelicks, L. A.; Morris, S. A.; Weiss, L. M.;
Tanowitz, H. B. Parasitol. Res. 2005, 96, 184–187.
9. Hosfield, D. J.; Zhang, Y.; Dougan, D. R.; Broun, A.;
Tari, L. W.; Swanson, R. V.; Finn, J. J. Biol. Chem. 2003,
2
2
278, 18401–18407.