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

Article abstract of DOI:10.1016/j.bmc.2011.02.037

The effect of long-chain 2-alkylaminoethyl-1,1-bisphosphonates against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii

Full text of DOI:10.1016/j.bmc.2011.02.037

Bioorganic & Medicinal Chemistry 19 (2011) 2211–2217
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of new 2-alkylaminoethyl-1,1-bisphos-
phonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting
farnesyl diphosphate synthase
Valeria S. Rosso a,b, Sergio H. Szajnman a, Leena Malayil b, Melina Galizzi b, Silvia N. J. Moreno b,
Roberto Docampo b, Juan B. Rodriguez a,
⇑
a Departamento de Química Orgánica and UMYMFOR (CONICET–FCEyN), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 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
a r t i c l e i n f o
a b s t r a c t
Article history:
The effect of long-chain 2-alkylaminoethyl-1,1-bisphosphonates against proliferation of the clinically
more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas’
disease), and against tachyzoites of Toxoplasma gondii was investigated. Particularly, compound 26
proved to be an extremely potent inhibitor against the intracellular form of T. cruzi, exhibiting IC50 values
at the nanomolar range. 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 chemotherapy. Compound 26 was an effective agent against T. cruzi (amastigotes) exhibiting
Received 11 January 2011
Revised 16 February 2011
Accepted 18 February 2011
Keywords:
Trypanosoma cruzi
Chagas’ disease
Farnesyl pyrophosphate synthase
Toxoplasma gondii
Toxoplasmosis
an IC50 value of 0.67
enzyme TcFPPS. This drug was less effective against the enzymatic activity of T. cruzi solanesyl diphos-
phate synthase TcSPPS showing an IC50 value of 3.2 M. Interestingly, compound 26 was also very effec-
tive against T. gondii (tachyzoites) exhibiting IC50 values of 6.23 M. This cellular activity was also related
to the inhibition of the enzymatic activity towards the target enzyme TgFPPS (IC50 = 0.093 M) As bis-
lM, while this compound showed an IC50 value of 0.81 lM against the target
l
l
l
phosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disor-
ders, their potential low toxicity makes them good candidates to control different tropical diseases.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
The parasite has a complex life cycle involving blood-sucking
Reduviid insects and mammals.5 It multiplies in the insect gut as
The hemoflagellated protozoan parasite Trypanosoma cruzi is
the etiological agent of Chagas’ disease or American trypanosomi-
asis, which is an endemic disease widespread from southern
United States to southern Argentina. It has been estimated that
close to 18 million people are infected and over 40 million are at
risk of infection by T. cruzi.1 The central nervous system is the most
frequently affected site in patients with AIDS, with meningoen-
cephalitis occurring approximately in 75% of cases. The next nor-
mally affected organ is the heart with myocarditis.
In addition, since people migrate from endemic areas, the
possibility for cases in developed nations will also escalate.
Chemotherapy for Chagas’ disease still remains unsatisfactory
due to limited efficacy and common side effects of the currently
available drugs such as nifurtimox (1), now discontinued, and ben-
znidazole (2), which present toxicity associated with their contin-
ued use.2–4
an epimastigote form and is spread as a non-dividing metacyclic
trypomastigote from the insect feces by contamination of intact
mucosa or wounds produced by the blood-sucking activity of the
vector. In the mammalian host, T. cruzi multiplies intracellularly
as the amastigote form and is subsequently released into the
bloodstream as a non-dividing trypomastigote.5 Transmission of
Chagas’ disease could also occur via the placenta or by blood trans-
fusion.6 This latter mechanism is responsible for the occurrence of
Chagas’ disease in developed countries where the disease is not en-
demic.6,7 For this reason, it 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 decades ago,8
suffers from some disadvantages, since it was shown to be carcin-
ogenic in in vivo assays (Fig. 1).9
Different enzymes involved in the biosynthesis of ergosterol10
and farnesyl diphosphate,11,12 and in protein prenylation,13 have
been reported to be excellent targets against pathogenic parasites.
Farnesyl diphosphate synthase of T. cruzi (TcFPPS), for example, has
been demonstrated to be the target of bisphosphonates that have
⇑
Corresponding author.
E-mail address: jbr@qo.fcen.uba.ar (J.B. Rodriguez).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.037

2212
V. S. Rosso et al. / Bioorg. Med. Chem. 19 (2011) 2211–2217
-
than T. cruzi, such as T. brucei rhodesiense, Leishmania donovani,
+
N
Cl
N
and Leishmania mexicana and Apicomplexan parasites such as
Toxoplasma gondii and Plasmodium falciparum.20,24–30 In vivo assays
of bisphosphonates have shown that risedronate can significantly
increase survival of T. cruzi-infected mice.31 In view of the above
results, it is possible to assume that bisphosphonates are potential
candidates for chemotherapy of neglected diseases. In addition,
bisphosphonates have the advantage that their synthesis is
straightforward and inexpensive. It is reasonable to assume a low
toxicity for bisphosphonate-containing drugs bearing in mind that
many bisphosphonate compounds are FDA-approved drugs for the
long-term treatment of several bone disorders.
O2N
N
O
N
O
S
1
O
O
O2N
N
H
N
3
N
N
2
Figure 1. Current drugs for the treatment of Chagas’ disease and blood sterilization.
Of special interest are 2-alkylaminoethyl-1,1-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 nanomolar range.26 This class of
bisphosphonates has proven to be more efficient than 1-hydroxy-
and 1-amino-bisphosphonates as antiparasitic agents.26 Compound
8 (Fig. 3) appears as the main member of bisphosphonates derived
activity in vitro and in vivo against T. cruzi.14 The gene encoding
this enzyme has been cloned and sequenced and the protein
expressed and biochemically characterized.14,15 In addition, the
crystal structure of TcFPPS at 2 Å resolution have been published.16
Moreover, solanesyl diphosphate synthase, another important pre-
nyltransferase in T. cruzi (TcSPPS), which is involved in the synthe-
sis of ubiquinone, is another potential target for chemotherapy.17
Geminal phosphonates are pyrophosphate analogues in which a
methylene group replaces the oxygen atom bridge between the
two phosphorus atoms of the pyrophosphate moiety. The substitu-
tion of carbon with different side chains has given rise to a large
family of compounds. Unlike pyrophosphate, bisphosphonates
possess better metabolic stability because they are not recognized
by pyrophosphatases and are also stable to hydrolysis under acidic
media. Several bisphosphonates are potent inhibitors of bone
resorption and are in clinical use for the treatment and prevention
of osteoporosis, Paget’s disease, hypercalcemia, tumor bone metas-
tases, and other bone diseases.18,19 Selective action on bone is
based on the binding of the bisphosphonate moiety to the bone
mineral.18,19 It has been postulated that the acidocalcisomes are
equivalent in composition to the bone mineral and that accumula-
tion of bisphosphonates in these organelles, as they do in bone
mineral, assists their antiparasitic action.20 Representative bis-
phosphonates, such as pamidronate (4), alendronate (5), risedro-
nate (6), and ibandronate (7), act by a mechanism that lead to
osteoclast apoptosis (Fig. 2).21 The site of action of amin-
obisphosphonates has been narrowed down to the isoprenoid
pathway and more specifically, to an inhibition of protein
prenylation.22
from fatty acids,26–31 with an IC50 value of 0.84 M.26 This cellular
l
activity is associated with the inhibition of the enzymatic activity
of the target enzyme TcFPPS,26 being a competitive inhibitor28 with
an IC50 value of 0.49
is very broad, because compound 8 also inhibits the enzymatic
activity of T. gondii FPPS (IC50 = 0.14
M),26 and exhibits in vitro
inhibitory action against tachyzoites of T. gondii (IC50 = 9.37
M).26
l
M.26 The scope of this type of bisphosphonates
l
l
As it was mentioned before, the target of 2-alkylaminoethyl-1,1-bis-
phosphonates is FPPS and to a lesser extent, SPPS. Previous studies
have indicated that selectivity towards SPPS increases as the chain
length increases.26 In fact, compound 8 exhibits IC50 = 1.35
l
M to-
wards TcSPPS, while compound 10 presents IC50 values of 1.01
and 0.25
M against FPPS and SPPS, respectively.26
l
M
l
We have demonstrated that 2-alkylaminoethyl-1,1-bisphos-
phonates were by far more potent than the parent compounds
1-aminoalkyl-, 1-hydroxyalkyl-, and 1-alkyl-1,1-bisphospho-
nates.26 The 2-alkylaminoethyl-1,1-bisphosphonate derivatives
are isosteric analogues of 1-alkyl-1,1-bisphosphonates, in which
an amino group replaces the methylene group at the C-3 position.
These aminobisphosphonates were originally designed in order to
maintain the ability to coordinate Mg2+ in a tridentate manner as
1-hydroxy-and 1-amino- derivatives do.26 However, preliminary
studies on the interaction of inhibitor 9 (IC50 = 58 nM) with TcFPPS
based on the X-ray crystallographic structure of 9–TcFPPS have
indicated that the nitrogen atom did not coordinate32 the with
Mg2+ present at the active site of the target enzyme.33,34 The tri-
dentate coordination structure is circumvented to the hydroxyl
groups bonded to the phosphorus atoms either for 2-alkylamino-
ethyl- or 1-hydroxy-1,1-bisphosphonates.35,36 In addition, the
X-ray structure of the complex of risedronate with TcFPPS
indicated that the residue Asp250 forms a hydrogen bond with
the hydroxyl group present at the C-1 position of the molecule of
risedronate, fact not possible with the 2-aminoalkyl deriva-
tives.16,26 Taking into account the above results, it would seem of
2. Rationale
Aminobisphosphonates were initially found to be potent inhib-
itors of T. cruzi proliferation in vitro and in vivo without toxicity to
the host cells.23 Lately, different bisphosphonates were found to be
effective growth inhibitors of pathogenic trypanosomatids other
O
OH
OH
O
P
OH
H2N
P
NH2
OH
OH
OH
HO
5
P
H2O3P
PO3H2
H
4
HO
P
OH
O
C
OH
O
O
CH3
N
CH2
O
OH
OH
P
P
OH
OH
N
NHR
8, R = n-hexyl
OH
OH
HO
P
HO
7
P
OH
9,
R = n-heptyl
6
O
OH
10, R = n-octyl
O
Figure 2. Chemical structure of representative FDA-approved bisphosphonates
Figure 3. Representative members of 1-[2-(alkylamino)ethyl] 1,1-bisphosphonic
clinically employed for different bone disorders.
acid derivatives.

V. S. Rosso et al. / Bioorg. Med. Chem. 19 (2011) 2211–2217
2213
interest to carry out chain length variations on compound 8 taken
as a reference structure (Fig. 3). Base on our previous work,26 the
resulting 1-[2- (alkylamino)ethyl] bisphosphonates were evalu-
ated against both T. cruzi and T. gondii cells and towards their
target enzymes TcFPPS, TcSPPS, and TgFPPS.
was associated with the inhibition of the enzymatic activity to-
wards the target enzyme TcFPPS possessing an IC50 value of
0.81
lM. Compounds 25 and 28 were also potent growth inhibitors
of T. cruzi (amastigotes) with IC50 values of 5.13 and 2.19
lM,
respectively. The inhibition of the enzymatic activity of 25 towards
the target enzyme qualitatively correlated with their inhibitory ac-
tion against growth of amastigotes of T. cruzi as well. Interestingly,
compound 26 did not exhibit superior inhibitory action against
TcSPPS as might be expected by its chain length having an IC50 va-
3. Results and discussion
The title compounds 22–29 were straightforwardly prepared
employing tetraethyl ethenylidenebisphosphonate (compound
13) as a Michael acceptor,37 which in turn was easily prepared
from tetraethyl methylenebisphosphonate (compound 12) in two
steps according to a slightly modified Degenhardt protocol.38,39
Then, compound 13 was reacted with the corresponding n-alkyla-
mine via a 1,4-conjugated addition reaction to afford the respective
Michael adducts (14–21). Once these synthetic precursors were
available, they were hydrolyzed by treatment with bromotrimeth-
ylsilane in methylene chloride40 to afford the free bisphosphonic
acids (22–29). The synthesis of this new 2-alkylaminoethyl gem-
bisphosphonates is presented in Scheme 1. Compounds 22–29
were evaluated as growth inhibitors against the amastigote form
of T. cruzi, the clinically more relevant form of the parasite. Benzni-
dazole, a well-known T. cruzi inhibitor, was used as a positive con-
trol.41 In addition, 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 opportunistic pathogen T. gondii,30,42
this series of new aminobisphosphonic acids was evaluated against
T. gondii and its target enzyme TgFPPS.
lue of 3.18 lM. Compound 26 was not only a potent antiparasitic
agent against T. cruzi, but also against the opportunistic parasite
T. gondii. Certainly, this drug exhibited an extremely potent inhibi-
tion of the enzymatic activity of TgFPPS at the low nanomolar
range (IC50 = 93 nM), efficacy comparable with risedronate
(IC50 = 74 nM). This enzymatic activity correlated well with the
cellular activity exerted by this compound against tachyzoites of
T. gondii (IC50 = 6.23 lM). Compound 24, in spite of being a potent
inhibitor of the enzymatic activity of TgFPPS at the low nanomolar
range (IC50 = 68 nM), was practically devoid of antiparasitic activ-
ity against T. gondii cells (Table 1).
It can be concluded that long-chain analogues of 2-alkylamino-
ethyl-1,1-bisphosphonates such as compound 26 were of the great
effectiveness against both T. cruzi and the target enzyme TcFPPS.
The designed compounds maintained the ability of the lower
members of this family of compounds as antiparasitic agents. Com-
pound 26 arose as the main member of these new set of drugs. Sur-
prisingly, it was not possible to establish a biological activity/chain
length relationship. In fact, compound 8 with a linear 6-carbons in
its structure exhibited similar cellular and enzymatic than 26. Most
of the synthetic drugs proved to be inhibitors of the enzymatic
activity of TcSPPS but to a lesser extent than TcFPPS. Finally, some
of these 1-[2-(alkylamino)ethyl] bisphosphonic acids were shown
to be effective anti-T. gondii agents indicating the broad scope of
Compound 26 resulted to be an extremely potent growth inhib-
itor against the clinically more relevant form of T. cruzi exhibiting
an IC50 value of 0.67
lM, significantly more potent than benznidaz-
ole taken as positive control (IC50 = 2.77
lM). This cellular activity
H2O3P
PO3H2
H
Et2O3P
PO3Et2
H
Et2O3P
PO3Et2
i. (CH2O)n, HNEt2,
MeOH,
Et2O3P
H
PO3Et2
H
RNH2, CH2Cl2, rt
C
C
BrSi(CH3
)
3
C
C
ii. PPTs, MePh,
CH2Cl2, rt
CH2
CH2
CH2
12
NHR
22, R = benzyl
NHR
14, R = benzyl
13
23,
R = n-nonyl
15,
R = n-nonyl
24, R = n-decyl
16, R = n-decyl
25,
R = n-undecyl
17,
R = n-undecyl
26, R = n-dodecyl
18, R = n-dodecyl
27, R = n-tetradecyl
19, R = n-tetradecyl
28,
R = n-hexadecyl
20,
R = n-hexadecyl
29, R = n-octadecyl
21, R = n-octadecyl
Scheme 1.
Table 1
Compd
IC50 (lM)
TcFPPS
>1
0.430 0.088
>1
0.856 0.137
0.811 0.226
>1
—
TgFPPS
TcSPPS
T. cruzi amastigotes
T. gondii tachyzoites
22
23
24
25
26
27
28
29
>10
—
>10
3.081 0.5896
3.182 1.0544
—
>10
>10
>10
5.126 1.915
0.670 0.086
>10
2.189 0.325
>10
>10
>10
>10
>10
6.23
11.27
4.13
5.29
0.440 0.070
0.068 0.036
0.868 0.545
0.093 0.024
0.292 0.170
—
—
—
—
Risedronate
10
Benznidazole
0.027 0.003
—
—
0.074 0.017
—
—
—
0.272 0.037
—
2.768 0.488

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V. S. Rosso et al. / Bioorg. Med. Chem. 19 (2011) 2211–2217
this family of compounds. Work aimed at optimizing the chemical
structure of 1-(3-azaalkyl)-1,1-bisphosphonic acids such as com-
pound 26 and other closely related analogues is currently being
pursued in our laboratory.
4.2.3. Tetraethyl 1-[(n-non-1-ylamino)ethyl] 1,1-bisphospho-
nate (15)
Colorless oil; 1H NMR (500.13 MHz, CDCl3) d 0.85 (t, J = 7.0 Hz,
3H, H-12), 1.22 (m, 12H, –CH2–), 1.32 (t, J = 7.1 Hz, 12H, H-20),
1.44 (p, J = 7.2 Hz, 2H, H-5), 2.54 (t, J = 7.2 Hz, 2H, H-4), 2.62 (tt,
J = 23.6, 6.0 Hz, 1H, H-1), 3.10 (dt, J = 16.7, 5.9 Hz, 2H, H-2), 4.16
(m, 8H, H-10); 13C NMR (125.77 MHz, CDCl3) d 14.0 (C-12), 16.3
(dd, J = 6.4, 2.7 Hz, C-20), 22.6 (C-11), 26.8 (C-6), 27.3 (C-7), 29.2
(C-8), 29.5 (C-10), 29.9 (C-9), 31.8 (C-5), 37.4 (t, J = 132.2 Hz, C-
1), 45.7 (t, J = 4.1 Hz, C-2), 49.2 (C-4), 62.6 (dd, J = 32.3, 6.8 Hz, C-
10); 31P NMR (202.46 MHz, CDCl3) d 22.78.
4. Experimental section
4.1. General
The glassware used in air- and/or moisture-sensitive reactions
was flame-dried and reactions were carried out under an argon
atmosphere. Unless otherwise noted, chemicals were commer-
cially 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. The 1H NMR
spectra are referenced with respect to the residual CHCl3 proton
of the solvent CDCl3 at d = 7.26 ppm. Coupling constants are re-
ported in Hertz. 13C NMR spectra were fully decoupled and are ref-
erenced to the middle peak of the solvent CDCl3 at d = 77.0 ppm.
31P NMR spectra are referenced with respect to the peak of 85%
H3PO4 as external reference. Splitting patterns are designated as
s, singlet; d, doublet; t, triplet; q, quadruplet; dd, double doublet,
etc. Melting points were determined with a Fisher–Johns apparatus
and are uncorrected. IR spectra were recorded with a Nicolet
Magna 550 spectrometer. Analytical TLC was performed on
commercial 0.2 mm aluminum-coated silica gel plates (F254) and
visualized by 254 nm UV or immersion in an aqueous solution of
(NH4)6Mo7O24ꢀ4H2O (0.04 M), Ce(SO4)2 (0.003 M) in concentrated
H2SO4 (10%). Elemental analyses were conducted by UMYMFOR
(CONICET-FCEyN). The results were within 0.4% of the theoretical
values.
4.2.4. Tetraethyl 1-[(n-dec-1-ylamino)ethyl] 1,1-bisphosphonate
(16)
Colorless oil; IR (film, cmꢁ1) 3444, 3332, 2972, 2956, 2925,
2854, 1649, 1569, 1467, 1392, 1369, 1247, 1164, 1026, 970, 798,
532; 1H NMR (500.13 MHz, CDCl3) d 0.88 (t, J = 7.0 Hz, 3H, H-13),
1.23 (m, 14H, –CH2–), 1.35 (t, J = 7.1 Hz, 12H, H-20), 1.47 (p,
J = 7.1 Hz, 2H, H-5), 2.57 (t, J = 7.2 Hz, 2H, H-4), 2.65 (tt, J = 23.5,
5.9 Hz, 1H, H-1), 3.12 (dt, J = 16.5, 5.9 Hz, 2H, H-2), 4.16 (m, 8H,
H-10); 13C NMR (125.77 MHz, CDCl3) d 14.1 (C-13), 16.3 (dd,
J = 6.4, 2.7 Hz, C-20), 22.6 (C-12), 26.8 (C-9), 27.3 (C-6), 29.3
(C-10), 29.5 (C-8), 29.5 (C-7), 29.9 (C-11), 31.9 (C-5), 37.4 (t,
J = 132.2 Hz, C-1), 45.7 (t, J = 4.0 Hz, C-2), 49.2 (C-4), 62.6 (dd,
J = 31.3, 6.8 Hz, C-10); 31P NMR (202.46 MHz, CDCl3) d 22.75. HRMS
(ESI) Calcd for (C20H46NO6P2) [M+H]+: 458.2800; found: 458.2813.
4.2.5. Tetraethyl 1-[(n-undec-1-ylamino)ethyl] 1,1-
bisphosphonate (17)
Colorless oil; IR (film, cmꢁ1) 3433, 2926, 2854, 1639, 1468,
1391, 1219, 1053, 951, 797; 1H NMR (500.13 MHz, CDCl3) d 0.88
(t, J = 6.9 Hz, 3H, H-14), 1.26 (m, 16H, –CH2–), 1.35 (t, J = 7.1 Hz,
12H, H-20), 1.46 (p, J = 7.5 Hz, 2H, H-5), 2.57 (t, J = 7.2 Hz, 2H,
H-4), 2.65 (tt, J = 23.1, 5.7 Hz, 1H, H-1), 3.13 (dt, J = 16.6, 5.9 Hz,
2H, H-2), 4.18 (m, 8H, H-10); 13C NMR (125.77 MHz, CDCl3) d 14.1
(C-14), 16.4 (dd, J = 6.4, 2.8 Hz, C-20), 22.7 (C-13), 26.9 (C-10),
27.3 (C-6), 29.3 (C-11), 29.5 (C-8), 29.6 (C-7), 29.9 (C-12), 31.9
(C-5), 33.5, 37.5 (t, J = 132.6 Hz, C-1), 45.8 (t, J = 4.1 Hz, C-2), 49.3
(C-4), 62.6 (dd, J = 31.8, 7.3 Hz, C-10); 31P NMR (202.46 MHz,
D2O-d6) d 22.79.
4.2. Synthesis of 1-[2-alkylaminoethyl]-1,1-bisphosphonic acids
4.2.1. General procedure
A solution of compound 13 (10 mmol) in anhydrous methylene
chloride (10 mL) was treated with the corresponding amine
(10 mmol) under an argon atmosphere. The reaction mixture was
stirred at room temperature overnight. The solvent was evaporated
and the residue was purified by column chromatography (silica
gel) employing hexane–EtOAc (17:3) as eluent for all compounds.
Then, to a solution of the resulting tetraethyl ester (14–21, 1 equiv)
in anhydrous methylene chloride was added dropwise trimethyl-
silyl bromide (10 equiv) in an argon atmosphere. The reaction mix-
ture was stirred at room temperature for 48 h. After cooling at 0 °C,
anhydrous methanol (10 mL) was added, and the resulting mixture
was allowed to reach room temperature. The solution was then
concentrated under reduced pressure. The residue was dissolved
in dry methanol (10 mL) and subsequently concentrated under re-
duced pressure twice. The solvent was evaporated and the residue
was crystallized from ethanol–water.
4.2.6. Tetraethyl 1-[(n-dodec-1-ylamino)ethyl] 1,1-bisphospho-
nate (18)
Colorless oil; IR (film, cmꢁ1) 3450, 2924, 2854, 1468, 1391, 1221,
1053, 953, 797, 559; 1H NMR (500.13 MHz, CDCl3) d 0.83 (t,
J = 7.0 Hz, 3H, H-15), 1.21 (m, 18H, –CH2–), 1.30 (t, J = 7.1 Hz, 12H,
H-20), 1.42 (p, J = 7.1 Hz, 2H, H-5), 2.53 (t, J = 7.2 Hz, 2H, H-4), 2.64
(tt, J = 23.5, 5.9 Hz, 1H, H-1), 3.10 (dt, J = 16.5, 5.8 Hz, 2H, H-2),
4.16 (m, 8H, H-10); 13C NMR (125.77 MHz, CDCl3) d 14.0 (C-15),
16.3 (dd, J = 6.4, 2.7 Hz, C-20), 22.6 (C-14), 26.8 (C-11), 27.3 (C-6),
29.3 (C-12), 29.5 (C-8), 29.6 (C-7), 29.9 (C-13), 31.8 (C-5), 37.4 (t,
J = 132.2 Hz, C-1), 45.7 (t, J = 4.1 Hz, C-2), 49.2 (C-4), 62.5 (dd,
J = 32.7, 6.3 Hz, C-10); 31P NMR (202.46 MHz, CDCl3) d 22.78.
4.2.2. Tetraethyl 1-[(benzylamino)ethyl] 1,1-bisphosphonate
(14)
4.2.7. Tetraethyl 1-[(n-tetradec-1-ylamino)ethyl] 1,1-bisphos-
phonate (19)
Colorless oil; IR (film, cmꢁ1) 3425, 2982, 2932, 2905, 1639,
1626, 1456, 1391, 1215, 1049, 951, 795, 750, 700; 1H NMR
(500.13 MHz, CDCl3) d 1.32 (t, J = 7.0 Hz, 12H, H-20), 2.67 (tt,
J = 23.6, 5.7 Hz, 1H, H-1), 3.16 (dt, J = 16.9, 5.6 Hz, 2H, H-2), 3.81
(s, 2H, H-4), 4.17 (m, 8H, H-10), 7.34 (m, 5H, aromatic protons);
13C NMR (125.77 MHz, CDCl3) d 16.4 (d, J = 3.8 Hz, C-20), 37.7 (t,
J = 132.5 Hz, C-1), 45.0 (t, J = 4.3 Hz, C-2), 53.2 (C-4), 62.7 (dd,
J = 31.5, 6.6 Hz, C-10), 126.9 (Ph), 128.1 (Ph), 128.4 (Ph), 139.9
(Ph); 31P NMR (202.46 MHz, CDCl3) d 22.66. HRMS (ESI) Calcd for
(C17H32NO6P2) [M+H]+: 408.1705; found 408.1684.
Colorless oil; 1H NMR (500.13 MHz, CDCl3) d 0.85 (t, J = 6.9 Hz,
3H, H-17), 1.22 (m, 22H, –CH2–), 1.32 (t, J = 7.1 Hz, 12H, H-20),
1.44 (p, J = 7.1 Hz, 2H, H-5), 2.55 (t, J = 7.2 Hz, 2H, H-4), 2.62 (tt,
J = 23.4, 5.9 Hz, 1H, H-1), 3.10 (dt, J = 16.5, 5.9 Hz, 2H, H-2), 4.16
(m, 8H, H-10); 13C NMR (125.77 MHz, CDCl3) d 14.1 (C-17), 16.3
(dd, J = 6.4, 2.7 Hz, C-20), 22.6 (C-16), 26.8 (C-13), 27.3 (C-6), 29.2
(C-14), 29.5 (C-7), 29.5 (C-11), 29.6 (C-8) 29.6 (C-14) 29.9 (C-9,
C-10, C-15), 31.9 (C-5), 37.4 (t, J = 132.6 Hz, C-1), 45.7 (t,
J = 4.0 Hz, C-2), 49.2 (C-4), 62.5 (dd, J = 31.8, 6.4 Hz, C-10); 31P
NMR (202.46 MHz, CDCl3) d 22.78.

V. S. Rosso et al. / Bioorg. Med. Chem. 19 (2011) 2211–2217
2215
4.2.8. Tetraethyl 1-[(n-hexadec-1-ylamino)ethyl] 1,1-bisphos-
phonate (20)
1.70 (m, 2H, H-5), 3.21 (m, 2H, H-4), 3.60–3.71 (m, 3H, H-1,
H-2); 13C NMR (50.3 MHz, D2SO4) d 13.9 (C-14), 22.6 (C-13), 26.0
(C-6, C-7), 29.1 (C-8, C-9, C-10, C-11), 29.3 (C-12), 31.8 (C-5),
43.2 (C-2), 51.7 (C-4); 31P NMR (202.46 MHz, D2O) d 14.37. HRMS
(ESI) Calcd for (C13H32O6NP2) [M+H]+: 360.1705; found 360.1691.
Anal. Calcd for C13H31O6NP2: C, 43.45; H, 8.70; N, 3.90. Found: C,
43.69; H, 8.52; N, 4.23.
Colorless oil; IR (film, cmꢁ1) 3450, 2924, 2854, 1639, 1468,
1391, 1221, 1053, 953, 797; 1H NMR (500.13 MHz, CDCl3) d 0.88
(t, J = 7.1 Hz, 3H, H-19), 1.25 (m, 26H, –CH2–), 1.34 (t, J = 7.1 Hz,
12H, H-20), 1.46 (p, J = 6.8 Hz, 2H, H-5), 2.57 (t, J = 7.2 Hz, 2H,
H-4), 2.64 (tt, J = 23.3, 5.9 Hz, 1H, H-1), 3.10 (td, J = 8.3, 6.0 Hz,
2H, H-2), 4.18 (m, 8H, H-10); 13C NMR (125.77 MHz, CDCl3) d 14.1
(C-19), 16.4 (dd, J = 6.4, 2.7 Hz, C-20), 22.7 (C-18), 26.9 (C-15),
27.3 (C-6), 29.3 (C-16), 29.5 (C-8), 29.7 (–CH2–), 29.9 (C-17), 31.9
(C-5), 37.5 (t, J = 132.2 Hz, C-1), 45.8 (t, J = 4.1 Hz, C-2), 49.3
(C-4), 62.6 (dd, J = 32.2, 6.8 Hz, C-10); 31P NMR (202.46 MHz, CDCl3)
d 22.80.
4.2.14. 1-[(n-Dodec-1-ylamino)ethyl] 1,1-bisphosphonic acid
(26)
White solid; mp 177–178 °C; IR (KBr, cmꢁ1) 3092, 2922, 2853,
1470, 1005, 953, 706; 1H NMR (500.13 MHz, DMSO-d6) d 0.84 (t,
J = 6.8 Hz, 3H, H-15), 1.23 (m, 22H, –CH2–), 1.53 (p, J = 6.9 Hz, 2H,
H-5), 2.18 (tt, J = 20.3, 7.4 Hz, 1H, H-1), 2.90 (t, J = 7.2 Hz, 2H,
H-4), 3.16 (dt, J = 14.0, 7.1 Hz, 2H, H-2); 13C NMR (50.3 MHz,
DMSO-d6) d 14.1 (C-15), 22.1 (C-14), 25.7 (C-6), 25.9 (C-7), 28.5
(C-8), 28.7 (C-12), 28.9 (C-11), 29.0 (C-10), 29.0 (C-9), 29.1
(C-13), 31.3 (C-5), 44.4 (C-2), 46.4 (C-4); 31P NMR (202.46 MHz,
DMSO-d6) d 15.28. HRMS (ESI) Calcd for (C14H34O6NP2) [M+H]+:
374.1861; found 374.1844. Anal. Calcd for C14H33O6NP2: C, 45.04;
H, 8.91; N, 3.75. Found: C, 44.80; H, 8.82; N, 3.81.
4.2.9. Tetraethyl 1-[(n-octadec-1-ylamino)ethyl] 1,1-bisphos-
phonate (21)
Colorless oil; IR (film, cmꢁ1) 3435, 2920, 2853, 1639, 1468,
1391, 1221, 1053, 953, 797, 735, 557; 1H NMR (500.13 MHz, CDCl3)
d 0.88 (t, J = 7.0 Hz, 3H, H-21), 1.26 (m, 32H, –CH2–), 1.34 (t,
J = 7.1 Hz, 12H, H-20), 1.47 (p, J = 7.0 Hz, 2H, H-5), 2.57 (t,
J = 7.2 Hz, 2H, H-4), 2.65 (tt, J = 23.4, 5.9 Hz, 1H, H-1), 3.13 (dt,
J = 16.5, 5.9 Hz, 2H, H-2), 4.18 (m, 8H, H-10); 13C NMR
(125.77 MHz, CDCl3) d 14.1 (C-21), 16.4 (dd, J = 5.9, 3.2 Hz, C-20),
22.7 (C-20), 26.8 (C-17), 27.3 (C-6), 29.3 (C-18), 29.5 (C-8), 29.6
(C-7), 29.7 (–CH2–), 29.9 (C-19), 31.9 (C-5), 37.5 (t, J = 132.2 Hz,
C-1), 45.7 (t, J = 4.1 Hz, C-2), 49.2 (C-4), 62.6 (dd, J = 31.8, 6.3 Hz,
C-10); 31P NMR (202.46 MHz, CDCl3) d 22.79.
4.2.15. 1-[(n-Tetradec-1-ylamino)ethyl] 1,1-bisphosphonic acid
(27)
White solid; mp 171–173 °C; IR (KBr, cmꢁ1) 3082, 2920, 2852,
2318, 1469. 1272, 1186, 999, 956, 705; 1H NMR (500.13 MHz,
DMSO-d6) d 0.86 (t, J = 6.5 Hz, 3H, H-16), 1.25 (m, 22H, –CH2–),
1.54 (p, J = 6.8 Hz, 2H, H-5), 2.17 (tt, J = 20.2, 7.1 Hz, 1H, H-1),
2.94 (t, J = 7.2 Hz, 2H, H-4), 3.20 (dt, J = 13.9, 7.1 Hz, 2H, H-2);
4.2.10. 1-[(Benzylamino)ethyl] 1,1-bisphosphonic acid (22)
White solid; mp 210–212 °C; (KBr, cmꢁ1) 3089, 2920, 2856,
2353, 2322, 1604, 1460, 1290, 1265, 1209, 1176, 1006, 956, 811,
773, 750, 711; 1H NMR (500.13 MHz, CDCl3) d 2.41 (m, 1H, H-1),
3.37 (m, 2H, H2), 4.20 (s, 2H, H-4), 7.40 (m, 5H, aromatic protons);
31P NMR (202.46 MHz, CDCl3) d 15.48. HRMS (ESI) Calcd for
(C9H16O6NP2) [M+H]+: 296.0453; found 296.0448. Anal. Calcd for
C9H15O6NP2: C, 36.62; H, 5.12; N, 4.75. Found: C, 36.58; H, 5.07;
N, 4.61.
31P NMR (202.46 MHz, D2O)
d 15.70. HRMS (ESI) Calcd for
(C16H37O6NP2Na) [M+Na]+: 424.1994; found 424.2000. Anal. Calcd
for C16H37O6NP2: C, 47.87; H, 9.29; N, 3.49. Found: C, 47.64; H,
9.34; N, 3.56.
4.2.16. 1-[(n-Hexadec-1-ylamino)ethyl] 1,1-bisphosphonic acid
(28)
White solid; mp 172–174 °C; IR (KBr, cmꢁ1) 3083, 2956, 2918,
2852, 1693, 1469, 1272, 1182, 1002, 952, 914, 806, 705; 1H NMR
(200.13 MHz, D2SO4) d 0.84 (m, 3H, H-19), 1.30 (m, 26H, –CH2–),
1.72 (m, 2H, H-5), 3.21 (m, 2H, H-4), 3.70 (m, 3H, H-1, H-2);
13C NMR (50.3 MHz, D2SO4) d 14.1 (C-19), 22.8 (C-18), 26.1 (C-6,
C-7), 29.6 (C-8, C-9), 29.9 (–CH2–), 32.2 (C-5), 43.4 (C-2), 51.8
(C-4). HRMS (ESI) Calcd for (C18H42NO6P2) [M+H]+: 430.2487;
found: 430.2468. Anal. Calcd for C18H41O6NP2: C, 50.34; H, 9.62;
N, 3.26. Found: C, 50.88; H, 9.54; N, 3.69.
4.2.11. 1-[(n-Non-1-ylamino)ethyl] 1,1-bisphosphonic acid (23)
White solid; mp 193–194 °C; IR (KBr, cmꢁ1) 3082, 2952, 2927,
2856, 1460, 1272, 1184, 999, 972, 954, 709; 1H NMR
(500.13 MHz, DMSO) d 0.85 (t, J = 6.8 Hz, 3H, H-11), 1.25 (m,
12H, –CH2–), 1.54 (m, 2H, H-5), 2.20 (dist t, J = 20.7 Hz, 1H, H-1),
2.91 (m, 2H, H-4), 3.20 (m, 2H, H-2); 13C NMR (125.77 MHz, DMSO)
d 13.9 (C-12), 22.1 (C-11), 25.7 (C-6), 25.9 (C-7), 28.5 (C-8), 28.6
(C-10), 28.8 (C-9), 31.3 (C-5), 44.5 (C-2), 46.5 (C-4); 31P NMR
(202.46 MHz, DMSO) d 15.28; HRMS (ESI) Calcd for (C11H28O6NP2)
[M+H]+: 332.1392; found 332.1378. Anal. Calcd for C11H27O6NP2: C,
39.88; H, 8.21; N, 4.23. Found: C, 39.78; H, 8.30; N, 4.33.
4.2.17. 1-[(n-Octadec-1-ylamino)ethyl] 1,1-bisphosphonic acid
(29)
Mp 121–123 °C; IR (KBr, cmꢁ1) 2920, 2850, 1598, 1469, 1272,
1182, 1006, 954, 705; 1H NMR (200.13 MHz, D2SO4) d 0.84 (m,
3H, H-21), 1.30 (m, 30H, –CH2–), 1.70 (m, 2H, H-5), 3.20 (m, 2H,
H-4), 3.70 (m, 3H, H-1, H-2). HRMS (ESI) Calcd for (C20H27O6P2Na)
[M+Na]+: 354.1211; found 354.1192.
4.2.12. 1-[(n-Dec-1-ylamino)ethyl] 1,1-bisphosphonic acid (24)
White solid; mp 127–128 °C; (KBr, cmꢁ1) 2961, 2921, 2854,
1468, 1192, 1161, 1033, 906, 811, 522; 1H NMR (500.13 MHz,
DMSO-d6) d 0.84 (t, J = 6.7 Hz, 3H, H-13), 1.24 (m, 16H, –CH2–),
1.52 (p, J = 7.5 Hz, 2H, H-5), 2.22 (tt, J = 20.7, 7.3 Hz, 1H, H-1),
2.90 (t, J = 7.3 Hz, 2H, H-4), 3.19 (dt, J = 14.0, 7.4 Hz, 2H, H-2); 13C
NMR (125.77 MHz, DMSO-d6) d 14.0 (C-13), 22.1 (C-12), 25.7
(C-6), 25.9 (C-7), 28.6 (C-8), 28.7 (C-10), 28.9 (C-9), 28.9 (C-11),
31.3 (C-5), 44.7 (C-2), 46.5 (C-4); 31P NMR (202.46 MHz, DMSO-
d6) d 11.51. HRMS (ESI) Calcd for (C11H27O6P2FNa) [M+Na]+:
354.1211; found 354.1192.
4.3. Drug screening
4.3.1. T. cruzi amastigotes assays
Gamma-irradiated (2000 Rads) Vero cells (3.4 ꢂ 104 cells/well)
were seeded in 96-well plates (black, clear bottom plates from
Greiner Bio-One) in 100 lL RPMI media (Sigma) with 10% FBS.
Plates were incubated overnight at 35 °C and 7% CO2. After over-
night incubation, Vero cells were challenged with 3.4 ꢂ 105
trypomastigotes/well (CL strain overexpressing a tdTomato red
4.2.13. 1-[(n-Undec-1-ylamino)ethyl] 1,1-bisphosphonic acid (25)
White solid; mp 170–172 °C; (KBr, cmꢁ1) 3101, 2923, 2854,
2322, 1693, 1467, 1272, 1182, 1002, 950, 705; 1H NMR
(200.13 MHz, D2SO4) d 0.84 (m, 3H, H-14), 1.21 (m, 16H, –CH2–),
fluorescent protein) in 50
35 °C and 7% CO2. After infection, cells were washed once with
Hanks solution (150 L/well) to eliminate any extracellular
lL volume and incubated for 5 h at
l

2216
V. S. Rosso et al. / Bioorg. Med. Chem. 19 (2011) 2211–2217
parasites and compounds were added in serial dilutions in RPMI
media in 150 L volumes. Each dilution was tested in quadrupli-
Acknowledgments
l
cate. Each plate also contained controls with host cells and no par-
asites (for background check), controls with two representative
drug dilutions and no parasites (for cytotoxicity assays), and con-
trols with parasites and no drugs (positive control). For each plate,
We thank Allysa Smith for help with T. gondii cultures. This
work was supported by Grants from the National Research Council
of Argentina (PIP 1888), ANPCyT (PICT 2008 #1690), and the Uni-
versidad de Buenos Aires (X-191) to J.B.R., and the U.S. National
Institutes of Health to R.D. (AI-082542) and S.N.J.M. (AI-068467).
V.S.R. was supported in part by a training grant of the Ellison Med-
ical Foundation to the Center for Tropical and Emerging Global
Diseases.
benznidazole was also used as a positive control at 3.5 and 1.5 lM.
After drug addition, plates were incubated at 35 °C and 7% CO2. At
day 3 post-infection, plates were assayed for fluorescence.43 IC50
values were determined by non-linear regression analysis using
SigmaPlot.
Supplementary data
4.3.2. T. gondii tachyzoites assays
Experiments on T. gondii tachyzoites were carried out as
described previously44 using T. gondii tachyzoites expressing red
fluorescence protein.45 Cells were routinely maintained in hTerT
cells grown in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum, 2 mM glutamine,
1 mM pyruvate, at 37 °C in a humid 5% CO2 atmosphere. Confluent
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.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.02.037.
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4.3.3. TcFPPS and TgFPPS assays and product analysis
For TcFPPS14,15,47 100
l
L of assay buffer (10 mM Hepes, pH 7.4,
MgCl2, dithiothreitol, 100
[4-14C]IPP
mol)), and 100 lM DMAPP were prewarmed to 37 °C.
5 mM
(10 Ci/
2 mM
lM
l
l
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 reactions
were made alkaline with 6.0 M NaOH (15 L), diluted in water
l
(0.7 mL), and extracted with hexane (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-
quired to incorporate 1 nmol of [4-14C]IPP into [14-14C]FPP in 1 min.
For TgFPPS the assay conditions were as described above except that
the buffer contained 1 mM MgCl2.
4.3.4. TcSPPS assay
The activity of the enzyme was determined by a radiometric
assay based on that described before.48 Briefly, 100
l
L of assay
buffer (100 mM Tris–HCl buffer, pH 7.4, 1 mM MgCl2, 1% (v/v)
[4-14C]IPP (10
Triton X-100, 100 Ci/ mol)), and 50 lM
l
M
l
l
GGPP was 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 37 °C and was quenched by
chilling quickly in an ice bath. The reaction products were ex-
tracted with 1 mL of 1-butanol saturated with water. The organic
layer 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 incorporate 1 nmol of [4-14C]IPP into
[4-14C]FPP in 1 min.

V. S. Rosso et al. / Bioorg. Med. Chem. 19 (2011) 2211–2217
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