Synthesis and biological evaluation of 1-alkylaminomethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii

Article abstract of DOI:10.1016/j.bmc.2019.07.004

As an extension of our project aimed at the search for new chemotherapeutic agents against Chagas disease and toxoplasmosis, several 1,1-bisphosphonates were designed, synthesized and biologically evaluated against Trypanosoma cruzi and Toxoplasma gondii,

Full text of DOI:10.1016/j.bmc.2019.07.004

Accepted Manuscript
Synthesis and Biological Evaluation of 1-Alkylaminomethyl-1,1-Bisphosphon-
ic Acids against Trypanosoma cruzi and Toxoplasma gondii
Tamila Galaka, Bruno N. Falcone, Catherine Li, Sergio H. Szajnman, Silvia N.J.
Moreno, Roberto Docampo, Juan B. Rodriguez
PII:
S0968-0896(19)30740-0
https://doi.org/10.1016/j.bmc.2019.07.004
BMC 14988
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 May 2019
23 June 2019
3 July 2019
Please cite this article as: Galaka, T., Falcone, B.N., Li, C., Szajnman, S.H., Moreno, S.N.J., Docampo, R.,
Rodriguez, J.B., Synthesis and Biological Evaluation of 1-Alkylaminomethyl-1,1-Bisphosphonic Acids against
Trypanosoma cruzi and Toxoplasma gondii, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
10.1016/j.bmc.2019.07.004
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Synthesis and Biological Evaluation of 1-Alkylaminomethyl-1,1-
Bisphosphonic Acids against Trypanosoma cruzi and Toxoplasma
gondii
Tamila Galaka,a Bruno N. Falcone,a Catherine Li,b Sergio H. Szajnman,a Silvia N. J.
Moreno,b Roberto Docampo,b and Juan B. Rodrigueza,*
aDepartamento 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, bCenter for Tropical and
Emerging Global Diseases and Department of Cellular Biology, University of Georgia,
Athens, Georgia, 30602, USA
Abstract – As an extension of our project aimed at the search for new chemotherapeutic
agents against Chagas disease and toxoplasmosis, several 1,1-bisphosphonates were
designed, synthesized and biologically evaluated against Trypanosoma cruzi and
Toxoplasma gondii, the etiologic agents of these diseases, respectively. In particular,
and based on the antiparasitic activity exhibited by 2-alkylaminoethyl-1,1-
bisphosphonates targeting farnesyl diphosphate synthase, a series of linear 2-
alkylaminomethyl-1,1-bisphosphonic acids (compounds 21−33), that is, the position of
the amino group was one carbon closer to the gem-phosphonate moiety, were evaluated
as growth inhibitors against the clinically more relevant dividing form (amastigotes) of
T. cruzi. Although all of these compounds resulted to be devoid of antiparasitic activity,
these results were valuable for a rigorous SAR study. In addition, unexpectedly, the
synthetic designed 2-cycloalkylaminoethyl-1,1-bisphosphonic acids 47−49 were free of
antiparasitic activity. Moreover, long chain sulfur-containing 1,1-bisphosphonic acids,
such as compounds 54−56, 59, turned out to be nanomolar growth inhibitors of
tachyzoites of T. gondii. As many bisphosphonate-containing molecules are FDA-
approved drugs for the treatment of bone resorption disorders, their potential
nontoxicity makes them good candidates to control American trypanosomiasis and
toxoplasmosis.

Introduction
American Trypanosomiasis (Chagas disease) is a chronic zoonosis produced by
the kinetoplastid parasite Trypanosoma cruzi, is the major parasitic disease burden of
the American continent, and can be considered as one of the most important diseases in
the world together with malaria and schistosomiasis.1 The commonly affected organ is
the heart with myocarditis and the central nervous system is also commonly attacked.2–4
In addition, the second most common symptoms are the so-called megavisceras;
therefore, the most commonly affected system is the digestive tract. The current
chemotherapy is still deficient and based on two empirically-discovered drugs:
nifurtimox (Lampit®, Bayer - El Salvador), which is available under a CDC
investigational protocols5 and benznidazole (Abarax®, Elea - Argentina), which was a
recently
FDA-approved
drug
but
for
pediatric
use
only.5
(https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm573942.htm ). In
most of endemic countries benznidazole is a drug of choice to treat the acute phase of
the infection and is indicated to treat the chronic phase even with low efficiency.6 As
both of these drugs are associated to long term treatment and severe side effects there is
a serious necessity to develop new safe drugs based on the knowledge of the
biochemistry and physiology of these microorganisms. In AIDS-infected patients,
chronic suppressive therapy is required and this is associated with neurotoxicity due to
the drugs.
On the other hand, T. gondii is an opportunistic protozoan parasite that is
responsible for toxoplasmosis.7 T. gondii is able to infect a wide range of hosts,
particularly humans and warm-blooded animals.8 Toxoplasmosis is positively one of the
most prevalent parasitic diseases affecting close to one billion people worldwide.9
Particularly, this parasite can cause mortality among immunocompromised individuals
such as AIDS patients, organ transplant recipients, as well as congenitally infected
children. Toxoplasmosis may lead to severe central nervous system disease. As occurs
with Chagas disease treatment, the current chemotherapy for toxoplasmosis is still
deficient as well.10
Several enzymes of the isoprenoid pathway, involved in the synthesis of sterols
and farnesyl diphosphate, have been reported to be excellent drug targets against
trypanosomatids.11,12 Regardless of their different structures and functional diversity, all

isoprenoids have a common precursor: isopentenyl diphosphate, and its isomer,
dimethylallyl diphosphate. In T. cruzi, isopentenyl diphosphate is biosynthesized
exclusively via the so-called mevalonate pathway, which has the 3-hydroxy-3-
methylglutaryl-CoA (HMG-CoA) reductase as the fundamental directing enzyme.
Although T. cruzi takes up sterols from its mammalian host (largely cholesterol), it has
an essential requirement for de novo sterol biosynthesis in all stages of its life cycle and
is extremely susceptible to sterol biosynthesis inhibitors. On the other hand, T. gondii
lacks the mevalonate pathway and employs an apicoplast localized biosynthesis,
prokaryotic-type, 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway, instead.13,14 The
DOXP pathway is not present in the host. In addition, as T. gondii does not synthesize
cholesterol, which is taken from the host indicating that inhibitors of the host isoprenoid
biosynthesis would be putative growth inhibitors of T. gondii.15 Certainly, mevalonate
pathway inhibitors are able to control proliferation of a number of Apicomplexan
parasites such as Babesia divergens,16 Plasmodium falciparum,17 Cryptosporidium
parvum,18 and T. gondii,19 indicating that parasites lacking the mevalonate pathway are
reliant on host precursors of isoprenoid biosynthesis. Interestingly, the synergistic effect
of host and parasite isoprenoid pathway inhibitors has been reported.20 For example,
the used of two commonly used drugs, zoledronic acid and atorvastatin, exhibited a
strong synergism in modulating T. gondii multiplication.20 It would be possible to take
advantage the fact of T. cruzi and T. gondii proliferate intracellularly to be used as a
model for two major groups of parasites: trypanosomatids (T. cruzi, African
trypanosomes, Leishmania spp) and Apicomplexan (P. falciparum, C. parvum, etc.). T.
gondii main proliferative stage in the mammalian host is the rapidly dividing or
tachyzoite stage, which is the form used in this study. Sometimes they are described as
cells because these are unicellular parasites. We now use tachyzoites all the time to
avoid confusion.9 Scheme 1 illustrates ergosterol biosynthesis for trypanosomatids.
Farnesyl diphosphate synthase (FPPS) is a key enzyme of the isoprenoid biosynthesis
that catalyzes the consecutive condensation of isopentenyl diphosphate with
dimethylallyl diphosphate and with geranyl diphosphate to form farnesyl diphosphate,
which is the common substrate for enzymes to produce sterols, ubiquinones, dolichols,
heme a, and prenylated proteins. Farnesyl diphosphate is able to be condensed with an
additional molecule of isopentenyl diphosphate by the geranylgeranyl diphosphate
synthase (GGPPS) to form the 20-carbon isoprenoid geranylgeranyl diphosphate.21

O
O
Statins
O
O
O
O
HO
HO
HO
-O2C
-O2C
Mevalonate
P
O-
P
O-
-O2C
CoA
2
CoA
CoA
OH
O
O
O-
OPP
Isopentenyl-PP
OPP
Dimethylallyl-PP
Isopentenyl-PP
isomerase
S
S
S
-Hydroxy--metylglutaryl-
Mevalonate
Acetoacetyl-CoA
2-Acetoacetyl-CoA
thiolase
Acetyl-CoA
Mevalonate-PP
kinase
CoA
HMG-CoA
HMG-CoA
reductase
Phosphomevalonate
decarboxylase
synthase
Dimethylallyl-PP
Farnesyl-PP synthase
BP
OPP
Geranyl-PP
Isopentenyl-PP
Farnesyl-PP synthase
BP
WC-9
Phosphomevalonate
kinase
Terbinafine
OPP
Squalene
2
Oxidosqualene
cyclase
HO
Farnesyl-PP
Squalene synthase
HO
Squalene
epoxidase
Eburicol
Farnesyl-PP
Lanosterol
O
Squ
alen
e-
2,3-
epo
xide
Farnesyl
protein transferase
14-demethylase
Azoles
Isopentenyl-PP
Geranylgeranyl-PP
synthase
Farnesylated Proteins
Decaprenyl-PP
synthase
Farnesyl:
hemo Transferasa
OPP
3
Geranylgeranyl-PP
Geranylgeranyl protein
transferase
Hemo-a
Ubiquinones
H
H
Dolichyl-PP
synthase
H
HO
HO
Ergosterol
14-demethyl
14-dihydroeburicol
Geranylgeranylated Proteins
Fecosterol
HO
Dolichyl-PP
Scheme 1. Ergosterol biosynthesis for trypanosomatids.
On the other hand, Scheme 2 shows the alternate pathway found in Apicomplexan
parasites to biosynthesize isopentenyl and dimethylallyl diphosphate. It is well-
documented that FPPS is essential for trypanosomatids.22 TgFPPS can be considered a
key enzyme only under certain circumstances, to be precise, when the isoprenoid
biosynthesis of the host is inhibited, process known as genetic validation.20 The
essentiality of FPPS was demonstrated in T. brucei rather than T. cruzi because of the
availability of better genetic tools for work with T. brucei.22
O
OH
O
P
2-
H3C OH
OH OH
H
OPO3
H3C OH
H3C
2-
OH
OPO3
O
CDP
O-
2-
OPO3
DXP
D-glyceraldehyde
3-phosphate
DXP
synthase
MEP
reductoisomerase
O
OH
cytidylyltransferase
OH OH
MEP
DXP
D-xylulose-phosphate
CDP-MEP
+
O
CO2H
pyruvate
O
P
CH3
O
P
O
P
2-
O
O
H3C OPO3
H3C
O
-O
O
O
O
O
O-
P
P
O-
-O -O
O
CMP
O-
HMBDP
synthase
CDP-ME
kinase
MECDP
synthase
OH
OH OH
CDP-ME2P
OH OH
MECDP
HMBDP
HMBDP reductase
OPP
isopentenyl-PP
isopentenyl-PP
isomerase
OPP
dimethylallyl-PP
Scheme 2. The 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway to biosynthesize
isopentenyl diphosphate and dimethylallyl diphosphate in Apicomplexan parasites.
The recent report of the crystal structure of some complexes of lineal 1,1-
bisphosphonates, developed in our laboratory such as 2-alkylaminoethyl-1,1-
bisphosphonates, with the target enzyme T. cruzi farnesyl diphosphate synthase
(TcFPPS) provided conclusive data in determining the precise mechanism of action of
these antiparasitic agents.23 These results, together with previous findings by other

groups24,25 indicated that these compounds behave as competitive inhibitors of the
substrate by binding to the allylic site of the enzyme, with the phosphonic moieties
coordinating three magnesium ions at the active site.23 It is worth mentioning that
predictions of the inhibitors optimal chain length is not straightforward bearing in mind
that small changes in the number of carbons of the alkyl chain brings about a dramatic
influence on biological action.26 In addition, FPPS of T. gondii is a bifunctional enzyme
that catalyzes the condensation of isopentenyl diphosphate with three allylic substrates:
dimethylallyl diphosphate, geranyl diphosphate, and farnesyl disphosphate.27
Interestingly, T. gondii FPPS has less than 50% identity of TcFPPS; therefore, it will be
possible to access extremely selective inhibitors for both enzymes.9
We were able to develop effective inhibitors against either TcFPPS or TgFPPS.21
Until now all the FPPSs are homodimeric enzymes that involve Mg2+ or Mn2+ for
activity.28 TcFPPS is localized to the cytosol.29
Bisphosphonates (2) are diphosphate (1) analogues in which a substituted
methylene group replaces the oxygen atom bridge between the two phosphorus atoms of
the pyrophosphate moiety giving rise to a large family of compounds.30 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
metastases, and other bone diseases.30–32 Aminobisphosphonates became putative
antiparasitic drugs when these molecules were first found to be effective inhibitors T.
cruzi proliferation in in vitro and in vivo assays without toxicity to the host cells.33 In
addition, this behavior was not limited exclusively to T. cruzi but was also observed in
other trypanosomatids (T. brucei rhodesiense and Leishmania donovani) and
Apicomplexan parasites, such as T. gondii and P. falciparum34–37, indicating that
bisphosphonates are promising molecules to control parasitic infections. Selective
action on bone is based on the binding of the bisphosphonate moiety to the bone
mineral.30 Interestingly, the acidocalcisomes are equivalent in composition to the bone
mineral and that accumulation of bisphosphonates in these organelles, as they do in
bone mineral, aids their antiparasitic activity.4,38–41 The isoprenoid pathway is the target
of bisphosphonates through the inhibition of the enzymatic activity of FPPS.21,42

O
O
O
O
O
O
O
OH
OH
OH
OH
OH
P
P
R1
P
P
H2N
P
P
P
P
P
P
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H2N
N
O
HO
HO
HO
R2
OH
OH
OH
OH
OH
O
O
O
1
2
3
4
5
Figure 1. Chemical structures of pyrophosphoric acid, general formula of
bisphosphonic acids and representative FDA-approved bisphosphonates clinically
employed for the treatment of different bone disorders.
A relevant achievement of our laboratory was the finding that linear 1,1-
bisphosphonates turned out to be efficient antiparasitic agents43–45 where, in most of
them, the hydroxyl group at the C-1 position, usually found in those bisphosphonates
currently employed in the treatment of bone disorders, was absent.42 Compounds 6−9
were the first examples of linear bisphosphonates that exhibited antiparasitic activity
against trypanosomatids and Apicomplexan parasites targeting parasitic FPPS.27,43–46
For example, 6 was a moderate growth inhibitor of intracellular T. cruzi43 and also
against tachyzoites of T. gondii,27,46 whereas 7 was effective against P. falciparum.27 8
was practically devoid of activity,44 whereas 9 showed similar cellular activity against
T. cruzi.45 In addition, -fluoro-1,1-bisphosphonates such as 10 and 11 were neither
effective against amastigotes of T. cruzi nor the target enzyme TcFPPS; nevertheless,
they were extremely effective inhibitors of the enzymatic activity of TgFPPS.47 Of
paramount concern were the 2-alkyl(amino)ethyl-1,1-bisphosphonate derivatives, which
were potent inhibitors of T. cruzi proliferation targeting TcFPPS with IC50 values in the
low nanomolar concentrations.48,49 Unquestionable, compounds 12−14 emerge as
pertinent members of this type of bisphosphonates. For example, 12 was significantly
more potent than the well-known antiparasitic agent WC-9 against T. cruzi
(amastigotes),48 under the same assays conditions;50 while 13 was a potent inhibitor of
the enzymatic activities of TcFPPS.48 Moreover, 14, the bisphosphonate bearing a long
aliphatic chain, was an effective growth inhibitor of T. cruzi.49 15 was a motivating
example of a linear bisphosphonate that was designed and synthesized in order to
optimize structures 12–14. 15 had been designed based on the fact that the presence of
electron withdrawing group (HO-) at C-1 would enhance the ability to coordinate Mg2+,
would increase pKa and also by the fact that most bisphosphonates clinically in use have
this functionality at C-1.51 Unfortunately, 15 is devoid of activity against T. cruzi

growth and TcFPPS, but exhibited a potent and selective inhibition of the enzymatic
activity towards TgFPPS.51 Linear sulfur-containing bisphosphonates are interesting
examples of selective anti-Toxoplasma agents as it is the case of 16 and 17.52 Certainly,
16 is a potent inhibitor of T. gondii proliferation. This cellular activity was associated
with a potent action against the target enzyme TgFPPS,52 whereas 17 is an unusually
potent inhibitor towards TgFPPS.52 A strong synergistic effect is observed when the
sulfur-containing 1,1-bisphosphonate 16 was used in combination with statins against
the hypervirulent RH strain of T. gondii in in vivo assays.53 Statins, which block the
mevalonate pathway within the mammalian cells, had exhibited a rather modest
inhibitory effect against T. gondii cells when tested alone.53 Moreover, the sulfone-
containing derivative 18 exhibited a very potent in vitro activity against T. gondii (ED50
= 0.1 µM).54 This compound also showed high activity in vivo (ED50 of 0.02 mg/kg) in
a toxoplasmosis mouse model employing the hypervirulent RH strain of T. gondii.54 It is
worth mentioning that 18 did not behave as an effective inhibitor of the enzymatic
activity of TgFPPS (IC50 = 0.27 µM) indicating that TgFPPS was not the primary target
of this molecule.55 The methylsulfonium derivative 19 has proven to be a moderate
growth inhibitor against both T. cruzi and T. gondii cells, but a very potent inhibitor of
the enzymatic activity towards the target enzymes TcFPPS and TgFPPS.52 Finally, -
fluoro-2-alkyl(amino)ethyl derivatives such as 20a20h were unpredictably free of
cellular activity.55 The structures of these representative compounds are illustrated in
Figure 2, whereas relevant already published antiparasitic data are listed in Table 1.
H2O3P
PO3H2
H2O3P
PO3H2
C
C
Y
R
CH2
XR
(CH2)n
CH3
6,
12,
13,
14,
15,
16,
17,
18,
19,
20,
n = 5; R = OH
X = NH; Y = H;
X = NH; Y = H;
X = NH; Y = H;
X = NH; Y = OH; R = n-octyl
X = S; Y = H;
X = S; Y = H;
X = S(O)2; Y = H; R = n-decyl
X = S+(CH3); Y = H; R = n-octyl
X = NH; Y = F;
R = n-hexyl
R = n-heptyl
R = n-dodecyl
7
, n = 9; R = OH
8,
9,
10,
11,
n = 5; R = H
n = 4; R = NH2
n = 7; R = F
n = 8; R = F
R = n-heptyl
R = n-decyl
R = alkyl
20a,
20e,
20b,
20f,
20c,
R = n-butyl;
20g,
R = n-octyl;
20d,
R = n-propyl;
R = n-heptyl;
R = n-pentyl;
20h,
R = n-hexyl;
R = n-decyl;
R = n-nonyl;
Figure 2. Chemical structures of representative lineal bisphosphonic acids developed in
our laboratory as putative antiparasitic agents targeting FPPS.

Table 1. Biological activity of bisphosphonates previously prepared in our laboratory
against T. cruzi (amastigotes), T. gondii (tachyzoites), TcFPPS, and TgFPPS.
Compound
T. cruzi growth
ED50 (µM)
> 10.0
T. gondii growth
ED50 (µM)
2.67
TcFPPS IC50 (µM)
TgFPPS IC50 (µM)
10
11
12
13
14
15
16
17
18
19
> 1.0
0.035
> 10.0
0.84
> 1.0
0.49
> 10.0
9.4
0.060
0.14
10.0
0.058
0.81
> 50.0
6.23
2.0
0.095
0.093
0.039
0.069
0.021
0.27
0.67
> 10.0
> 20.0
> 20.0
NT
> 10.0
> 10.0
0.097
NT
0.97
1.8
0.11
7.0
>20.0
0.040
0.013
Rationale
The lack of antiparasitic activity found in the -fluoro-2-alkyl(amino)ethyl
derivatives was rather unexpected. In fact we further evaluated compounds 20a20h as
inhibitors of the enzymatic activity against the target enzymes TcFPPS and TgFPPS,
respectively.
On the other hand, in order to study the influence of the position of the nitrogen
atom in our linear 1,1-bisphosphonates, 2-alkylaminomethyl-1,1-bisphosphonates such
as 21−33 could be considered as very exciting structural variations taking into account
the selective and potent antiparasitic activity exhibited by 2-alkylaminoethyl-1,1-
bisphosphonates such as 12−14 towards both T. cruzi cells and TcFPPS.23,48,49 Although
some of the title compounds 21−33 had been previously described as growth inhibitors
of Entamoeba histolytica and Plasmodium species in in vitro and in vivo studies, there
were not spectroscopic data available for these compounds.56 Therefore, the influence of
the position of the nitrogen atom (C-3 versus C-2) seemed to be relevant from the
pharmacological point of view.
Taking into account that the 2-alkylaminoethyl-1,1-bisphosphonates of type
1214 were extremely effective growth inhibitors either of T. cruzi cells or of the
enzymatic activity of the target enzyme TcFPPS,23,48,49 based on the biological activity
exhibited by the cyclohexylamine-containing bisphosphonate,48 which showed an IC50
value of 0.013 µM against TcFPPS but limited biological activity against intracellular T.

cruzi, it was decided to prepare some 1,1-bisphosphonate derivatives bearing a
cycloalkylamino group in their structure such as 4749.
Results and discussion
Preparation of the respective 2-alkylaminomethyl-1,1-bisphosphonic acids
21−33 was successfully carried out according to already published methods.57,58 Thus,
following a slightly modified published protocol,59 a suitable alkyl or cycloalkyl amine
treated with triethyl orthoformate and diethyl phosphite at 135 ºC for 2 hours gave rise
to the corresponding tetraethyl ester derivatives 34−46 in a relatively modest but
reproducible yields. Then, on treatment with concentrated hydrochloric acid at 100 ºC
overnight the esters were converted into the title molecules 21−33 in very good yields
as shown in Scheme 3.
PO3Et2
PO3H2
O
OEt
OEt
135 ºC
2 h
HCl (conc.)
100 ºC, 24 h
R
R
HP
R-NH2
OEt
OEt
N
PO3OEt2
HN
21,
PO3H2
EtO
H
34,
R = CH2CH2CH3
CH2(CH2)2CH3
CH2(CH2)3CH3
CH2(CH2)4CH3
CH2(CH2)5CH3
CH2(CH2)6CH3
CH2(CH2)7CH3
CH2(CH2)8CH3
CH2(CH2)9CH3
cyclopentyl
(16%)
(15%)
(11%)
(27%)
(11%)
(23%)
(22%)
(26%)
(19%)
(28%)
(11%)
(15%)
(37%)
(73%)
35,
36,
37,
38,
39,
40,
41,
42,
43,
44,
45,
46,
22,
23,
24,
25,
26,
27,
28,
29,
30,
31,
(100%)
(70%)
(81%)
(100%)
(97%)
(82%)
(100%)
(100%)
(99%)
(94%)
(83%)
(82%)
cyclohexyl
cycloheptyl
piperidyl
32,
33,
Scheme 3. Synthetic approach for the preparation of 2-alkylaminomethyl-1,1-
bisphosphonic acids.
The 2-cycloalkylaminoethyl-1,1-bisphosphonates 4749 were easily synthesized
starting from the corresponding cycloalkylamine and the well-known Michael-type
acceptor 50.60–62 Then, conjugate addition of the cycloalkylamine to form the respective
tetraethyl esters 4749 with excellent yields followed by hydrolysis by treatment with
concentrated hydrochlroric acid yielded the desired molecules 4749 as presented in
Scheme 4.
PO3Et2
PO3H2
CH2Cl2, rt,
24 h
HCl (conc.)
Et2O3P
PO3Et2
R-NH2
PO3Et2
PO3H2
100 ºC, 24 h
NHR
NHR
50
51,
52,
53,
47,
48,
49,
R = cyclopentyl
cycloheptyl
(95%)
(99%)
(94%)
(88%)
(73%)
(76%)
4-methylcyclohexyl
Scheme 4. Synthetic approach for the synthesis of cycloalkyl 2-aminoethyl-1,1-

bisphosphonates.
Long chain sulfur-containing 1,1-bisphosphonates such as 5459 were
synthesized according to our previous results.52 Therefore, the Michael-type acceptor 50
was treated with undecylmercaptan and dodecylmercaptan to produce the respective
tetraethyl esters 60 and 61, respectively. On treatment with bromotrimethylsilane in
methylane chloride, which is the protocol developed by McKenna,63,64 followed by
digestion with methanol, 60 and 61 were converted into the free bisphosphonic acids 54
and 57. Sulfur-containing 1,1-bisphosphonate esters are quite sensitive to hydrolysis by
treatment with concentrated hydrochlroric acid.52 Each compound, in independent
experiments, was oxidized by treatment with hydrogen peroxide (one equivalent) to
yield the corresponding sulfoxides 55 and 58, respectively or with two equivalents of
hydrogen peroxide to produce the respective sulfones 56 and 59, respectively as shown
in Scheme 5.
PO(OEt)2
PO(OH)2
i. BrSi(CH3
CH2Cl2, rt
ii. methanol,
rt, 45%
)
3,
CH2Cl2, Et3N, rt
S
S
50
50
undecylmercaptan +
dodecylmercaptan +
PO(OEt)2
PO(OH)2
60
54
H2O2 (1 equiv.)
O
PO(OH)2
PO(OH)2
CH2Cl2, Et3N, rt
S
PO(OH)2
PO(OH)2
PO(OEt)2
PO(OEt)2
i. BrSi(CH3)3,
55,
(81%)
H2O2 (2 equiv.) from
S
S
S
CH2Cl2, rt
ii. methanol,
rt, 80%
54
61
57
PO(OH)2
O
O
S
H2O2 (1 equiv.)
H2O2 (2 equiv.)
PO(OH)2
PO(OH)2
O
O
O
S
PO(OH)2
PO(OH)2
56,
(58%)
PO(OH)2
59,
(81%)
58,
(67%)
Scheme 5. Synthetic approach for the preparation of long chain sulfur-containing-1,1-
bisphosphonates.
Biological data are shown in Table 2 and 3. As previously mentioned, the -
fluoro-2-alkyl(amino)ethyl derivatives 20a20h resulted to be almost free of
antiparasitic activity. In spite of not having a significant cellular activity compounds
20a20h were further evaluated against the main target of the linear bisphosphonates,
FPPS. All of these fluorine-containing-1,1-bisphosphonates exhibited a potent
inhibitory action towards the enzymatic activity of either TcFPPS or of TgFPPS.
Certainly, most of these compounds showed to be nanomolar inhibitors of these key
enzymes. Long chain derivatives such as 20f and 20g were representative examples of
potent inhibitors showing IC50 values of 0.085 µM and 0.079 µM against TcFPPS and
TgFPPS, respectively for 20f and 0.034 µM and 0.051 µM, respectively for 20g.
Unfortunately, it is difficult to rationalize the lack of cellular activity of this family of

compounds. Evidently, -fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates do not have
the appropriate physicochemical parameters to cross two cell membranes (the
mammalian one and the parasite cell membrane) to reach the target enzyme. Moreover,
the long chain -fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates 20f20h were
cytotoxic molecules making these derivatives of low interest in Medicinal Chemistry.
This decrease in the antiparasitic activity by making small structural variations on
nitrogen-containing bisphosphonates such as such 121448,49 to give 1551 has also been
observed in other bisphosphonates acting on bone mineral affinity targeting the
mevalonate pathway between -hydroxy- and -fluorobisphosphonates.65
Biological evaluation of 1-alkylaminomethyl-1,1-bisphosphonic acids 2129
indicated that this structural variation was not beneficial for the biological activity. With
the exception of compound 28, which resulted to be a fairly modest growth inhibitor of
intracellular T. cruzi (ED50 5.3 µM), all the title compounds were devoid of cellular
activity and, for that reasons, they were not further analyzed.
Unexpectedly, the designed 2-cycloalkyl(amino)ethyl-1,1-bisphosphonates
4749 were devoid of activity against T. cruzi cells and T. gondii cells. Interestingly, the
long chain sulfur-containing-1,1-bisphosphonates were effective inhibitors of
tachyzoites of T. gondii as anticipated according to our previous data in closely related
sulfur-containing analogues.52–54 All of them 5456, 59 were nanomolar inhibitors of
tachyzoites of T. gondii being 56 the most effective growth inhibitor with an ED50 value
of 0.53 µM. In this regard, it is important to note that the reference structure 18 (Figure
2), which behaved as a potent inhibitor of tachyzoites of T. gondii proliferation (ED50 =
0.1 µM), did not have TgFPPS as a primary target exhibiting a modest a fairly modest
inhibitory action towards this target enzyme (IC50 value of 0.8 M).54 Indeed, these long
chain sulfur-containing -1,1-bisphosphonates will require further efforts to investigate
their precise mode of action.

Table 2. Biological activity of bisphosphonates against T. cruzi (amastigotes), T. gondii
(tachyzoites), TcFPPS, TgFPPS, and Vero cells
Compound
T. cruzi cells T. gondii cells
TcFPPS IC50 TgFPPS IC50 Cytotoxicity
ED50 µM
ED50 µM
(µM)
(µM)
ED50 (µM)
NT
> 10.055
0.060 ± 0.03
0.173 ± 0.04
0.611 ± 0.37
0.915 ± 0.18
0.071 ± 0.02
0.085 ± 0.03
0.034 ± 0.023
0.136 ± 0.047
0.245 ± 0.081
0.286 ± 0.087
0.299 ± 0.028
0.131 ± 0.017
0.088 ± 0.005
0.079 ± 0.013
0.051 ± 0.006
0.080 ± 0.004
20a
20b
20c
20d
20e
20f
> 10.055
> 20055
> 10.055
> 10.055
NT
NT
NT
161.755
15.0
20.0
> 10.055
>10.055
> 10.055
> 10.055
>10.055
> 10.055
4.008 ± 1.19155
2.24 ± 0.31
2.26 ± 0.75
cytotoxic55
cytotoxic55
20g
20h
Table 3. Biological activity of bisphosphonates against T. cruzi (amastigotes), T. gondii
(tachyzoites), and Vero cells
Compound
T. cruzi cells T. gondii cells
Cytotoxicity
ED50 (µM)
ED50 µM
> 10.0
ED50 µM
21
> 10.0
22
> 10.0
23
> 10.0
24
> 10.0
25
> 10.0
26
> 10.0
27
5.26 ± 0.45
28
29
30
31
32
33
47
48
49
cytotoxica
> 10.0
> 10.0
> 10.0
> 10.0
> 10.0
> 10.0
> 10.0
10.0
> 20.0
> 20.0
> 20.0
0.54 ± 0.10
0.53 ± 0.096
0.38 ± 0.082
0.93 ± 0.24
54
55
56
59
benznidazole
2.58 ± 0.50
a cytotoxic at 10 µM, 5 µM, no inhibition at 2.5 µM
In order to account for the lack of activity of compounds bearing the amino
group at position 2, Molecular Dynamics (MD) and Quantum Mechanical (QM)
optimizations were carried out. The protein ligand complex for the compound with the

amino group at position 2 (compound 21) was generated from the crystal structure for
compound 62 (pdb id 4DXJ). Although no specific interactions between the amino
groups and the receptor were observed in the 10 ns MD simulations, the side chain is
fixed in one orientation, directed towards the unsymmetrical magnesium ion (Figure 3).
A plot for the dihedral angle P-C1-C2-N for 21 and P-C1-N-C3 for 62 (Figure 4) shows
that this angle remains constant throughout the simulation.
Figure 3. The side chain is fixed in one orientation, directed towards the unsymmetrical
magnesium ion present at the allylic site of the active site of TcFPPS.
Figure 4. Dihedral angle P-C1-C2-N for 21 (red) and P-C1-N-C3 for 62 (black)
throughout the MD simulation

QM optimizations at the B3LYP/6-311+G(d,p) level were carried out for the
main conformers of each model compound (63 and 64), simulating the aqueous solvent
with the polarizable continuum method (PCM). Consistent with previous studies,51 the
phosphate groups were considered to be doubly protonated, as it is expected for a
physiological pH of 6.5. We have considered both protonation states for the amino
group, yielding equivalent conclusions. The molecules were completed by the addition
of one magnesium atom. The lowest energy conformations (Figure 5) show a strong
intramolecular hydrogen bond between the phosphate groups for both compounds.
However, compound 64 shows a weak H-bond between the amino and phosphate
groups (bond length 2.32 Å and N-H...O angle 120º) while the equivalent H-bond is
relatively strong for compound 63 (1.77 Å, 151º). The formation of this strong H-bond
is expected to decrease the conformational entropy in solution, therefore decreasing the
entropy penalty upon complex formation, and yielding a higher inhibition activity. It is
worth mentioning that, consistently with our MD and QM studies, ITC measurements23
found that bisphosphonate compounds bind to the active site due to a combination of a
favorable entropic driving force and an unfavorable enthalpic energy.
Figure 5. A strong intramolecular hydrogen bond between the phosphate groups for
both compounds.
In conclusion, the lack of antiparasitic activity shown by the regioisomers of the
nitrogen-containing bisphosphonates 1214, that is, amino derivatives 2129 provided
significant insights concerning chemical structure-biological activity relationship. The
simple moving of the nitrogen atom in representative compounds 1214 had a marked

outcome on the biological activity giving rise to inactive compounds. Efforts in
optimizing linear bisphosphonates are currently being pursued in our laboratory.
Experimental Section
The glassware used in air- and/or moisture-sensitive reactions was flame-dried and
reactions were carried out under dry argon. Unless otherwise noted, chemicals were
commercially available and used without further purification. Solvents were distilled
before use. Dichloromethane was distilled from phosphorus pentoxide. Nuclear
magnetic resonance spectra were performed with a Bruker AVANCE NEO 500 or with
a Bruker Fourier 300 spectrometers. The 1H NMR spectra are referenced with respect to
the residual CHCl3 proton of the solvent CDCl3 at δ = 7.26 ppm. Coupling constants are
reported in Hz. 13C NMR spectra were fully decoupled and are referenced to the middle
peak of the solvent CDCl3 at δ = 77.0 ppm. 31P NMR spectra are referenced with respect
to the peak of 85% H3PO4 as external reference. For comparative purposes, all NMR
spectra acquired in D2O for free bisphosphonic acids were recorded at the same
conditions. 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. Elemental analyses were performed with an Exeter CE-440
Elemental Analyzer. 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)6Mo7O24·4H2O (0.04 M), Ce(SO4)2 (0.003 M) in concentrated H2SO4
(10%).
1
As judged from the homogeneity of the H, 13C, 31P NMR spectra and HPLC
analyses of the title compounds employing a Beckmann Ultrasphere ODS-2 column 5
M, 250 × 10 mm eluting with water–acetonitrile (9:1) at 3.00 mL/min with a refractive
index detector indicated a purity >97%.
General procedure for the preparation of tetraethyl 1-n-alkylaminomethyl-1,1-
bisphosphonates
A mixture of the corresponding alkyl or cycloalkyl amine (30 mmol, 4.14 g), triethyl
orthoformate (30 mmol, 5.0 mL) and diethyl phosphite (60 mmol, 7.7 mL) was heated
at 135 ºC for 2 h. Then, volatile components were evaporated and the respective

residues were purified by column chromatography (silica gel) eluting with mixtures of
hexaneacetone giving rise to the desired tetraethyl esters.
General procedure for hydrolysis of tetraethyl 1-n-alkylaminomethyl-1,1-
bisphosphonates
The solvent was evaporated and the residue was treated with a concentrated aqueous
solution of hydrochloric acid (2 mL). The resulting mixture was heated to reflux for 24
h. The solvent was evaporated and the residue was crystallized from ethanolwater.
Synthesis of tetraethyl 2-[(alkylthio)ethyl] 1,1-bisphosphonates. General procedure
To a solution of tetraethyl ethenylidenbisphosphonate (50; 300 mg, 1.0 mmol) in
anhydrous dichloromethane (10 mL) was added triethylamine (1 mmol) and the
corresponding alkylmercaptan (1 mmol). The reaction mixture was stirred at room
temperature for 1 h. Water (20 mL) was added, and the mixture was extracted with
dichloromethane (3  10 mL). The combined organic layers were washed with brine (20
mL), dried (MgSO4), and the solvent was evaporated.
Synthesis of 2-(alkylthio)ethyl-1,1-bisphosphonic acids. General procedure. A
solution of the corresponding tetraethyl 2-[(alkylthio)ethyl] 1,1-biphosphonate (1 mmol)
in anhydrous methylene chloride (10 mL) was treated with trimethylsilyl bromide (10
equiv.) under an argon atmosphere. The reaction mixture was stirred at room
temperature for 48 h. Then, methanol (1.0 mL) was added and the solvent was
evaporated. The residue was dissolved in methanol (8 mL) and the mixture was stirred
at room temperature for 24 h. The solvent was evaporated and the residue
redissolved/evaporated in methanol four times, to complete the hydrolysis of remaining
trimethylsilyl bromide and eliminate the hydrobromic acid created. The residue was
purified by column chromatography on reverse phase with a mixture of watermethanol
as eluent and the pure compound was obtained after lyophilization.
Tetraethyl 1-n-propylaminomethyl-1,1-bisphosphonate (34). Colorless oil; 16%
yield; 1H NMR (500.13 MHz, CDCl3) δ 0.92 (t, J = 7.4 Hz, 3H, H-5), 1.35 (dt, J = 7.1,
0.9 Hz, 12H, CH2CH3), 1.49 (sext, J = 7.2 Hz, 2H, H-4), 2.80 (t, J = 7.2 Hz, 2H, H-3),
3.25 (t, J = 21.7 Hz, 1H, H-1), 4.22 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)

δ 11.5 (C-5), 16.39 (t, J = 3.1 Hz, CH2CH3), 16.44 (t, J = 3.1 Hz, CH2C′H3), 23.1 (C-4),
52.4 (t, J = 6.0 Hz, C-3), 54.3 (t, J = 145.5 Hz, C-1), 62.8 (t, J = 3.3 Hz, CH2CH3), 63.2
(t, J = 3.3 Hz, C′H2CH3); 31P NMR (202.46 MHz, CDCl3) δ 19.74 ppm.
Tetraethyl 1-n-butylaminomethyl-1,1-bisphosphonate (35). Colorless oil; 15% yield;
1H NMR (500.13 MHz, CDCl3) δ 0.90 (t, J = 7.3 Hz, 3H, H-6), 1.35 (dt, J = 7.1, 1.0 Hz,
12H, CH2CH3), 1.37 (m, J = 7.2 Hz, 2H, H-4), 1.45 (m, J = 7.2 Hz, 2H, H-5), 2.83 (t, J
13
= 7.1 Hz, 2H, H-3), 3.24 (t, J = 21.7 Hz, 1H, H-1), 4.21 (m, 8H, CH2CH3); C NMR
(125.77 MHz, CDCl3) δ 13.9 (C-6), 16.4 (t, J = 3.0 Hz, CH2CH3), 16.5 (t, J = 3.1 Hz,
CH2C′H3), 20.1 (C-5), 32.1 (C-4), 50.3 (t, J = 6.1 Hz, C-3), 54.4 (t, J = 97.4 Hz, C-1),
31
62.8 (t, J = 3.4 Hz, CH2CH3), 63.3 (t, J = 3.2 Hz, C′H2CH3); P NMR (202.46 MHz,
CDCl3) δ 19.77 ppm.
Tetraethyl 1-n-pentylaminomethyl-1,1-bisphosphonate (36). Colorless oil; 11%
yield; 1H NMR (500.13 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H, H-7), 1.30 (m, 4H, H-5,
H-6), 1.35 (dt, J = 7.1, 1.0 Hz, 12H, CH2CH3), 1.47 (m, 2H, H-5), 2.82 (t, J = 7.2 Hz,
13
2H, H-3), 3.25 (t, J = 21.8 Hz, 1H, H-1), 4.22 (m, 8H, CH2CH3); C NMR (125.77
MHz, CDCl3) δ 14.0 (C-7), 16.39 (t, J = 3.1 Hz, CH2CH3), 16.44 (t, J = 3.1 Hz,
CH2C′H3), 22.5 (C-6), 29.1 (C-5), 29.7 (C-4), 50.6 (t, J = 6.0 Hz, C-3), 54.3 (t, J =
145.4 Hz, C-1), 62.8 (t, J = 3.3 Hz, CH2CH3), 63.2 (t, J = 3.2 Hz, C′H2CH3); 31P NMR
(202.46 MHz, CDCl3) δ 19.74 ppm.
Tetraethyl 1-n-hexylaminomethyl-1,1-bisphosphonate (37). Colorless oil; 27% yield;
1H NMR (500.13 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H, H-8), 1.30 (m, 6H, H-5, H-6,
H-7), 1.35 (dt, J = 7.1, 1.1 Hz, 12H, CH2CH3), 1.46 (p, J = 7.3 Hz, 2H, H-4), 2.82 (t, J =
13
7.2 Hz, 2H, H-3), 3.25 (t, J = 21.7 Hz, 1H, H-1), 4.22 (m, 8H, CH2CH3); C NMR
(125.77 MHz, CDCl3) δ 14.0 (C-8), 16.42 (t, J = 3.1 Hz, CH2CH3), 16.46 (t, J = 3.0 Hz,
CH2C′H3), 22.6 (C-7), 26.7 (C-5), 30.0 (C-4), 31.5 (C-6), 50.6 (t, J = 6.0 Hz, C-3), 54.3
(t, J = 145.4 Hz, C-1), 62.8 (t, J = 3.3 Hz, CH2CH3), 63.2 (t, J = 3.0 Hz, C′H2CH3); 31P
NMR (202.46 MHz, CDCl3) δ 19.76 ppm.
Tetraethyl 1-n-hepylaminomethyl-1,1-bisphosphonate (38). Colorless oil; 11% yield;
1H NMR (500.13 MHz, CDCl3) δ 0.86 (t, J = 6.8 Hz, 3H, H-9), 1.26 (m, 8H, H-5, H-6,
H-7, H-8), 1.33 (t, J = 7.1 Hz, 12H, CH2CH3), 1.44 (p, J = 7.1 Hz, 2H, H-4), 2.80 (t, J =

13
7.1 Hz, 2H, H-3), 3.23 (t, J = 21.7 Hz, 1H, H-1), 4.22 (m, 8H, CH2CH3); C NMR
(125.77 MHz, CDCl3) δ 14.0 (C-9), 16.40 (t, J = 3.1 Hz, CH2CH3), 16.44 (t, J = 3.0 Hz,
CH2C′H3), 22.6 (C-8), 27.0 (C-5), 29.1 (C-6), 30.0 (C-4), 31.8 (C-7), 50.6 (t, J = 6.0 Hz,
C-3), 54.3 (t, J = 145.4 Hz, C-1), 62.8 (t, J = 3.2 Hz, CH2CH3), 63.2 (t, J = 3.2 Hz,
C′H2CH3); 31P NMR (202.46 MHz, CDCl3) δ 19.74 ppm.
Tetraethyl 1-n-octylaminomethyl-1,1-bisphosphonate (39). Colorless oil; 23% yield;
1H NMR (500.13 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H, H-10), 1.26 (m, 10H, -CH2-),
1.35 (t, J = 7.1 Hz, 12H, CH2CH3), 1.46 (p, J = 7.21 Hz, 2H, H-4), 2.82 (dt, J = 7.1, 1.1
Hz, 2H, H-3), 3.24 (t, J = 21.7 Hz, 1H, H-1), 4.21 (m, 8H, CH2CH3); 13C NMR (125.77
MHz, CDCl3) δ 14.1 (C-10), 16.4 (t, J = 3.1 Hz, CH2CH3), 16.5 (t, J = 3.0 Hz,
CH2C′H3), 22.6 (C-9), 27.0 (C-5), 29.2 (C-6), 29.4 (C-7), 30.0 (C-4), 31.8 (C-8), 50.6 (t,
J = 6.0 Hz, C-3), 54.4 (t, J = 145.4 Hz, C-1), 62.8 (t, J = 3.3 Hz, CH2CH3), 63.2 (t, J =
3.2 Hz, C′H2CH3); 31P NMR (202.46 MHz, CDCl3) δ 19.76 ppm.
Tetraethyl 1-n-nonylaminomethyl-1,1-bisphosphonate (40). Colorless oil; 22%
yield; 1H NMR (500.13 MHz, CDCl3) δ 0.88 (t, J = 7.0 Hz, 3H, H-11), 1.25 (m, 12H, -
CH2-), 1.35 (dt, J = 7.0, 1.0 Hz, 12H, CH2CH3), 1.45 (p, J = 7.2 Hz, 2H, H-4), 2.81 (t, J
13
= 7.2, 1.1 Hz, 2H, H-3), 3.25 (t, J = 21.7 Hz, 1H, H-1), 4.20 (m, 8H, CH2CH3); C
NMR (125.77 MHz, CDCl3) δ 14.0 (C-11), 16.37 (t, J = 3.1 Hz, CH2CH3), 16.42 (t, J =
3.1 Hz, CH2C′H3), 22.6 (C-10), 27.0 (C-5), 29.2 (C-6), 29.4 (C-7), 29.4 (C-8), 30.0 (C-
4), 31.8 (C-9), 50.8 (t, J = 5.7 Hz, C-3), 54.3 (t, J = 145.4 Hz, C-1), 62.8 (t, J = 3.3 Hz,
CH2CH3), 63.2 (t, J = 2.9 Hz, C′H2CH3); 31P NMR (202.46 MHz, CDCl3) δ 19.73 ppm.
Tetraethyl 1-n-decylaminomethyl-1,1-bisphosphonate (41). Colorless oil; 26% yield;
1H NMR (500.13 MHz, CDCl3) δ 0.86 (t, J = 6.8 Hz, 3H, H-12), 1.26 (m, 14H, -CH2-),
1.33 (t, J = 6.7 Hz, 12H, CH2CH3), 1.43 (p, J = 7.2 Hz, 2H, H-4), 2.79 (t, J = 7.0, 1.1
Hz, 2H, H-3), 3.23 (t, J = 21.7 Hz, 1H, H-1), 4.19 (m, 8H, CH2CH3); 13C NMR (125.77
MHz, CDCl3) δ 14.1 (C-12), 16.39 (t, J = 3.0 Hz, CH2CH3), 16.44 (t, J = 3.1 Hz,
CH2C′H3), 22.6 (C-11), 27.0 (C-5), 29.3 (C-6), 29.4 (C-7), 29.5 (C-8), 29.6 (C-9), 30.0
(C-4), 31.8 (C-10), 50.6 (t, J = 6.0 Hz, C-3), 54.3 (t, J = 145.4 Hz, C-1), 62.8 (t, J = 3.3
31
Hz, CH2CH3), 63.2 (t, J = 3.0 Hz, C′H2CH3); P NMR (202.46 MHz, CDCl3) δ 19.74
ppm.

Tetraethyl 1-n-Undecylaminomethyl-1,1-bisphosphonate (42). Colorless oil; 19%
yield; 1H NMR (500.13 MHz, CDCl3) δ 0.86 (t, J = 6.8 Hz, 3H, H-13), 1.23 (m, 16H, -
CH2-), 1.33 (t, J = 6.7 Hz, 12H, CH2CH3), 1.43 (p, J = 7.2 Hz, 2H, H-4), 2.79 (t, J = 7.0
Hz, 2H, H-3), 3.22 (t, J = 21.7 Hz, 1H, H-1), 4.19 (m, 8H, CH2CH3); 13C NMR (125.77
MHz, CDCl3) δ 14.1 (C-13), 16.39 (t, J = 3.0 Hz, CH2CH3), 16.44 (t, J = 3.1 Hz,
CH2C′H3), 22.6 (C-12), 27.0 (C-5), 29.3 (C-6), 29.47 (C-7), 29.57 (C-8), 29.59 (C-9, C-
10), 30.0 (C-4), 31.9 (C-11), 50.6 (t, J = 6.1 Hz, C-3), 54.3 (t, J = 145.6 Hz, C-1), 62.8
(t, J = 3.3 Hz, CH2CH3), 63.3 (t, J = 3.2 Hz, C′H2CH3); 31P NMR (202.46 MHz, CDCl3)
δ 19.75 ppm.
Tetraethyl (Cyclopentylamino)methyl-1,1-bisphosphonate (43). Colorless oil; 28%
1
yield; H NMR (500.13 MHz, CDCl3) δ 1.35 (dt, J = 7.1, 1.6 Hz, 12H, CH2CH3), 1.52
(m, 4H, -CH2-), 1.72 (m, 4H, -CH2-), 3.32 (t, J = 22.0 Hz, 1H, H-1), 3.48 (t, J = 5.8 Hz,
2H, H-3), 4.19 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3) δ 16.4 (t, J = 3.1 Hz,
CH2CH3), 16.5 (t, J = 3.1 Hz, CH2C′H3), 23.9 (C-5), 32.8 (C-4), 52.6 (t, J = 145.4 Hz,
C-1), 59.2 (t, J = 5.9 Hz, C-3), 62.8 (t, J = 3.4 Hz, CH2CH3), 63.4 (t, J = 3.4 Hz,
C′H2CH3); 31P NMR (202.46 MHz, CDCl3) δ 19.93 ppm.
Tetraethyl (Cyclohexylamino)methyl-1,1-bisphosphonate (44). Colorless oil; 11%
yield; 1H NMR (500.13 MHz, CDCl3) δ 1.061.25 (m, 5H, -CH2-), 1.35 (dt, J = 7.1, 1.3
Hz, 12H, CH2CH3), 1.49 (m, 1H, -CH2-), 1.59 (m, 1H, -CH2-), 1.72 (m, 2H, -CH2-),
1.83 (m, 3H, -CH2-), 2.76 (tt, J = 9.9, 3.7 Hz, 2H, H-3), 3.42 (t, J = 22.0 Hz, 1H, H-1),
4.19 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3) δ 16.4 (t, J = 3.1 Hz, CH2CH3),
16.5 (t, J = 2.9 Hz, CH2C′H3), 24.7 (C-5), 26.0 (C-6), 33.1 (C-4), 51.0 (t, J = 145.4 Hz,
C-1), 56.2 (t, J = 5.8 Hz, C-3), 62.8 (t, J = 3.3 Hz, CH2CH3), 63.3 (t, J = 3.2 Hz,
C′H2CH3); 31P NMR (202.46 MHz, CDCl3) δ 19.94 ppm.
Tetraethyl (Cycloheptylamino)methyl-1,1-bisphosphonate (45). Colorless oil; 15%
1
yield; H NMR (500.13 MHz, CDCl3) δ 1.34 (dt, J = 7.1, 1.2 Hz, 12H, CH2CH3), 1.39
(m, 2H, -CH2-), 1.52 (m, 4H, -CH2-), 1.64 (m, 2H, -CH2-), 1.78 (m, 4H, -CH2-), 2.98 (p,
J = 4.1 Hz, 2H, H-3), 3.36 (t, J = 22.1 Hz, 1H, H-1), 4.21 (m, 8H, CH2CH3); 13C NMR
(125.77 MHz, CDCl3) δ 16.4 (t, J = 2.9 Hz, CH2CH3), 16.5 (t, J = 3.3 Hz, CH2C′H3),
24.0 (C-5), 28.4 (C-6), 34.3 (C-4), 51.7 (t, J = 145.3 Hz, C-1), 58.4 (t, J = 5.7 Hz, C-3),

31
62.7 (t, J = 3.3 Hz, CH2CH3), 63.4 (t, J = 3.2 Hz, C′H2CH3); P NMR (202.46 MHz,
CDCl3) δ 19.99 ppm.
Tetraethyl (Piperidin-1-yl)methyl-1,1-bisphosphonate (46). Colorless oil; 37% yield;
1H NMR (500.13 MHz, CDCl3) δ 1.35 (dt, J = 7.1, 1.2 Hz, 12H, CH2CH3), 1.42 (m, 2H,
-CH2-), 1.54 (p, J = 5.4 Hz, 4H, -CH2-), 1.64 (m, 2H, -CH2-), 1.78 (m, 4H, -CH2-), 2.96
(m, 4H, H-3), 3.34 (t, J = 25.0 Hz, 1H, H-1), 4.20 (m, 8H, CH2CH3); 13C NMR (125.77
MHz, CDCl3) δ 16.4 (t, J = 2.9 Hz, CH2CH3), 16.5 (t, J = 3.1 Hz, CH2C′H3), 23.9 (C-5),
26.8 (C-4), 53.0 (t, J = 4.5 Hz, C-3), 62.4 (t, J = 140.8 Hz, C-1), 62.7 (t, J = 3.3 Hz,
CH2CH3), 63.4 (t, J = 3.2 Hz, C′H2CH3); 31P NMR (202.46 MHz, CDCl3) δ 18.56 ppm.
Tetraethyl 1-[(Cyclopentylamino)ethyl] 1,1-bisphosphonate (51). Colorless oil; 95%
1
yield; H NMR (500.13 MHz, CDCl3) δ 1.32 (t, J = 7.1 Hz, 12H, CH2CH3), 1.36 (m,
1H, -CH2-), 1.51 (m, 2H, -CH2-), 1.66 (m, 2H, -CH2-), 1.80 (m, 2H, -CH2-), 2.70 (tt, J =
23.5, 5.7 Hz, 1H, H-1), 3.12 (dt, J = 16.7, 6.0 Hz, 2H, H-2), 3.06 (q, J = 6.9 Hz, 1H, H-
4), 4.16 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3) δ 16.3 (t, J = 3.2 Hz,
CH2CH3), 16.4 (t, J = 3.1 Hz, CH2C′H3), 24.0 (C-5), 32.6 (C-4), 37.2 (t, J = 132.9 Hz,
C-1), 44.2 (t, J = 4.2 Hz, C-2), 58.9 (C-4), 62.5 (t, J = 6.8 Hz, CH2CH3), 62.8 (t, J = 6.8
Hz, C′H2CH3); 31P NMR (202.46 MHz, CDCl3) δ 22.60 ppm.
Tetraethyl 1-[(Cycloheptylamino)ethyl] 1,1-bisphosphonate (52). Colorless oil; 99%
yield; 1H NMR (500.13 MHz, CDCl3) δ 1.35 (t, J = 7.1 Hz, 12H, CH2CH3), 2.63 (tt, J =
23.6, 5.8 Hz, 1H, H-1), 3.15 (dt, J = 16.7, 5.9 Hz, 2H, H-2), 4.18 (m, 8H, CH2CH3); 13C
NMR (125.77 MHz, CDCl3) δ 16.39 (t, J = 2.9 Hz, CH2CH3), 16.43 (t, J = 3.1 Hz,
CH2C′H3), 24.3 (C-6), 28.3 (C-7), 34.5 (C-5), 37.4 (t, J = 132.2 Hz, C-1), 43.2 (t, J =
4.3 Hz, C-2), 57.9 (C-4), 62.5 (t, J = 6.6 Hz, CH2CH3), 62.8 (t, J = 6.8 Hz, C′H2CH3);
31P NMR (202.46 MHz, CDCl3) δ 22.87 ppm.
Tetraethyl
1-[(4-methylcyclohexylamino)ethyl] 1,1-bisphosphonic acid (53).
Colorless oil, 94% yield; 1H NMR (500.13 MHz, CDCl3) δ 0.87, 0.90 (d, J = 6.5 Hz, 3H
CH3), 1.342, 1.347 (t, J = 7.0 Hz, 12H, CH2CH3), 2.63 (tt, J = 22.9, 5.6 Hz, 1H, H-1),
31
3.15, 3.19 (dt, J = 16.5, 5.6 Hz, 2H, H-2), 4.19 (m, 8H, CH2CH3); P NMR (202.46
MHz, CDCl3) δ 22.78, 22.90 ppm.

1-n-Propylaminomethyl-1,1-bisphosphonic acid (21). White solid, 73% yield; mp
207 ºC; 1H NMR (500.13 MHz, D2O) δ 0.91 (t, J = 7.5 Hz, 3H, H-5), 1.64 (sext, J = 7.5
13
Hz, 2H, H-4), 2.90 (t, J = 16.4 Hz, 1H, H-1), 3.22 (t, J = 7.5 Hz, 2H, H-3); C NMR
(125.77 MHz, D2O) δ 10.1 (C-5), 19.8 (C-4), 51.1 (t, J = 2.8 Hz, C-3), 58.3 (t, J = 115.8
Hz, C-1); 31P NMR (202.46 MHz, D2O) δ 8.40 ppm. HRMS (ESI) calcd. for
(C4H13O6NP2Na) [M+Na]+ 256.0110; found 256.0110. Anal. calcd. for
(C4H13O6NP2.½H2O): C, 19.84; H, 5.83; N, 5.79. Found C, 19.34; H, 6.08; N, 5.62.
1-n-Butylaminomethyl-1,1-bisphosphonic acid (22). White solid; 100% yield; mp
215 ºC; 1H NMR (500.13 MHz, D2O) δ 0.85 (t, J = 7.4 Hz, 3H, H-6), 1.32 (sext, J = 7.5
Hz, 2H, H-5), 1.61 (q, J = 7.6 Hz, 2H, H-4), 2.90 (t, J = 16.4 Hz, 1H, H-1), 3.25 (t, J =
13
7.5 Hz, 2H, H-3); C NMR (125.77 MHz, D2O) δ 12.9 (C-6), 19.0 (C-5), 28.3 (C-4),
31
49.5 (t, J = 2.9 Hz, C-3), 58.4 (t, J = 115.2 Hz, C-1); P NMR (202.46 MHz, D2O) δ
8.39 ppm. HRMS (ESI) calcd. for (C5H15O6NP2Na) [M + Na]+ 270.0267; found
270.0262. Anal. calcd. for (C5H15O6NP2): C, 24.30; H, 6.12; N, 5.67. Found C, 24.69; H,
6.60; N, 5.39.
1-n-Pentylaminomethyl-1,1-bisphosphonic acid (23). white solid; 70% yield; mp =
201 ºC; 1H NMR (500.13 MHz, D2O) δ 0.79 (t, J = 7.1 Hz, 3H, H-7), 1.25 (m, 4H, H-5,
H-6), 1.62 (q, J = 7.5 Hz, 2H, H-4), 2.88 (t, J = 16.3 Hz, 1H, H-1), 3.24 (t, J = 7.8 Hz,
13
2H, H-3); C NMR (125.77 MHz, D2O) δ 13.1 (C-7), 21.6 (C-6), 26.0 (C-5), 27.7 (C-
4), 49.8 (t, J = 2.6 Hz, C-3), 58.5 (t, J = 115.4 Hz, C-1); 31P NMR (202.46 MHz, D2O) δ
8.45 ppm. HRMS (ESI) calcd. for (C6H18O6NP2) [M+H]+ 262.0604; found 262.0606.
1-n-Hexylaminomethyl-1,1-bisphosphonic acid (24). White solid, 81% yield; mp =
210 ºC; 1H NMR (500.13 MHz, D2O) δ 0.77 (t, J = 7.1 Hz, 3H, H-8), 1.23 (m, 4H, H-6,
H-7), 1.31 (m, 2H, H-5), 1.61 (q, J = 7.6 Hz, 2H, H-4), 2.87 (t, J = 16.3 Hz, 1H, H-1),
13
3.23 (t, J = 7.6 Hz, 2H, H-3); C NMR (125.77 MHz, D2O) δ 13.3 (C-8), 21.7 (C-7),
25.2 (C-5), 26.3 (C-4), 30.3 (C-6), 49.8 (t, J = 3.2 Hz, C-3), 58.5 (t, J = 116.3 Hz, C-1);
31P NMR (202.46 MHz, D2O) δ 8.46 ppm. HRMS (ESI) calcd. for (C7H20O6NP2) [M +
H]+ 276.0760; found 277.0767. Anal. calcd. for (C7H19O6NP2): C, 30.55; H, 6.96; N,
5.09. Found C, 30.17; H, 7.06; N, 4.97.

1
1-n-Heptylaminomethyl-1,1-bisphosphonic acid (25). White solid; mp 198 ºC. H
NMR (500.13 MHz, D2O) δ 0.78 (t, J = 6.9 Hz, 3H, H-9), 1.21 (m, 4H, H-7, H-8), 1.29
(m, 2H, H-5, H-6), 1.63 (p, J = 7.7 Hz, 2H, H-4), 2.90 (t, J = 16.4 Hz, 1H, H-1), 3.25 (t,
13
J = 7.6 Hz, 2H, H-3); C NMR (125.77 MHz, D2O) δ 13.3 (C-9), 21.9 (C-8), 25.5 (C-
5), 26.3 (C-4), 28.0 (C-6), 30.8 (C-7), 49.8 (t, J = 3.1 Hz, C-3), 58.4 (t, J = 116.0 Hz, C-
1); 31P NMR (202.46 MHz, D2O) δ 8.36 ppm. HRMS (ESI) calcd. for (C8H21O6NP2Na)
[M + Na]+ 312.0736; found 312.0735. Anal. calcd. for (C8H21O6NP2): C, 33.22; H, 7.32;
N, 4.84. Found C, 33.65; H, 7.01; N, 5.11.
1-n-Octylaminomethyl-1,1-bisphosphonic acid (26). White solid; 97% yield; mp =
1
201 ºC. H NMR (500.13 MHz, D2O) δ 0.75 (m, 3H, H-10), 1.17 (m, 6H, -CH2-), 1.28
(m, 4H, -CH2-), 1.61 (m, 2H, H-4), 2.87 (t, J = 16.2 Hz, 1H, H-1), 3.22 (t, J = 7.0 Hz,
2H, H-3); 13C NMR (125.77 MHz, D2O) δ 13.4 (C-10), 22.0 (C-9), 25.6 (C-5), 26.3 (C-
4), 28.1 (C-6), 28.2 (C-8), 31.0 (C-7), 49.8 (t, J = 2.8 Hz, C-3), 58.5 (t, J = 115.5 Hz, C-
1); 31P NMR (202.46 MHz, D2O) δ 8.51 ppm. HRMS (ESI) calcd. for (C9H23O6NP2Na)
[M+Na]+ 326.0893; found 326.0850. Anal. calcd. for (C9H23O6NP2): C, 35.65; H, 7.65;
N, 4.62. Found C, 35.48; H, 8.01; N, 3.86.
1-n-Nonylaminomethyl-1,1-bisphosphonic acid (27). White solid; 82% yield; mp =
1
195 ºC. H NMR (500.13 MHz, D2O) δ 0.77 (t, J = 6.8 Hz, 3H, H-11), 1.20 (m, 8H, -
CH2-), 1.28 (m, 4H, -CH2-), 1.64 (p, J = 7.5 Hz, 2H, H-4), 3.00 (t, J = 16.3 Hz, 1H, H-
1), 3.25 (t, J = 7.6 Hz, 2H, H-3); 13C NMR (125.77 MHz, D2O) δ 13.4 (C-11), 22.0 (C-
10), 25.6 (C-5), 26.2 (C-4), 28.26 (C-6), 28.31 (C-9), 28.4 (C-8), 31.1 (C-7), 49.7 (t, J =
31
3.1 Hz, C-3), 57.0 (t, J = 113.3 Hz, C-1); P NMR (202.46 MHz, D2O) δ 8.34 ppm.
HRMS (ESI) calcd. for (C10H26O6NP2) [M+H]+ 318.1230; found 318.1220. Anal. calcd.
for (C10H25O6NP2): C, 37.85; H, 7.94; N, 4.41. Found C, 37.82; H, 8.07; N, 4.44.
1-n-Decylaminomethyl-1,1-bisphosphonic acid (28). White solid; 100% yield; mp =
197 ºC; 1H NMR (500.13 MHz, D2O) δ 0.76 (t, J = 6.9 Hz, 3H, H-12), 1.18 (m, 10H, -
CH2-), 1.28 (m, 4H, -CH2-), 1.62 (p, J = 7.4 Hz, 2H, H-4), 2.88 (t, J = 16.8 Hz, 1H, H-
1), 3.23 (t, J = 7.6 Hz, 2H, H-3); 13C NMR (125.77 MHz, D2O) δ 13.4 (C-12), 22.0 (C-
11), 25.6 (C-5), 26.4 (C-4), 28.26 (C-6), 28.41 (C-10), 28.45 (C-9), 28.6 (C-8), 31.1 (C-
7), 49.9 (t, J = 2.9 Hz, C-3), 58.5 (t, J = 115.6 Hz, C-1); 31P NMR (202.46 MHz, D2O) δ
8.44 ppm. HRMS (ESI) calcd. for (C11H27O6NP2Na) [M+Na]+ 354.1206; found

354.1208. Anal. calcd. for (C11H27O6NP2): C, 39.88; H, 8.21; N, 4.23. Found C, 39.51;
H, 8.01; N, 3.98.
1-n-Undecylaminomethyl-1,1-bisphosphonic acid (29). White solid; 100% yield; mp
= 203 ºC; 1H NMR (500.13 MHz, D2O) δ 0.77 (m, 3H, H-13), 1.19 (m, 16H, -CH2-),
1.64 (m, 2H, H-4), 3.11 (t, J = 15.7 Hz, 1H, H-1), 3.25 (m, 2H, H-3); 13C NMR (125.77
MHz, D2O) δ 13.4 (C-13), 22.0 (C-12), 25.6 (C-5), 26.1 (C-4), 28.25 (C-6), 28.46 (C-
10, C-11), 28.6 (C-9), 28.7 (C-8), 31.2 (C-7), 49.5 (t, J = 3.3 Hz, C-3), 55.5 (t, J = 111.8
Hz, C-1); 31P NMR (202.46 MHz, D2O) δ 8.33 ppm. HRMS (ESI) calcd. for
(C12H29O6NP2Na) [M+Na]+ 368.1362; found 368.1361. Anal. calcd. for (C12H29O6NP2):
C, 41.74; H, 8.46; N, 4.06. Found C, 41.21; H, 8.32; N, 3.93.
(Cyclopentylamino)methyl-1,1-bisphosphonic acid (30). White solid; 99% yield; mp
210 ºC; 1H NMR (500.13 MHz, CDCl3) δ 1.62 (m, 6H, H4a, H-5), 2.05 (m, 2H, H-4b),
13
3.43 (t, J = 18.1 Hz, 1H, H-1), 3.98 (p, J = 7.2 Hz, 1H, H-3); C NMR (125.77 MHz,
CDCl3) δ 23.6 (C-5), 29.2 (C-4), 54.0 (t, J = 124.3 Hz, C-1), 61.3 (t, J = 3.2 Hz, C-3);
31P NMR (202.46 MHz, D2O) δ 8.02 ppm. HRMS (ESI) calcd. for C6H16NO6P2 [M +
H]+ 260.0447; found 260.0454. Anal. calcd. for (C6H15O6NP2): C, 27.81; H, 5.83; N,
5.41. Found C, 28.17; H, 5.72; N, 5.08.
(Cyclohexylamino)methyl-1,1-bisphosphonic acid (31). white solid; 94% yield; mp
1
213 ºC; H NMR (500.13 MHz, D2O) δ 1.14 (m, 2H, H-6ax.), 1.28 (sext, J = 11.5 Hz,
4H, H-5), 1.54 (m, 2H, H-6eq.), 1.71 (m, 2H, H-4ax.), 2.04 (m, 2H, H-4eq.), 3.06 (t, J =
13
16.2 Hz, 1H, H-1), 3.46 (m, 1H, H-3); C NMR (125.77 MHz, CDCl3) δ 23.7 (C-6),
24.6 (C-5), 29.5 (C-4), 54.9 (t, J = 124.3 Hz, C-1), 57.9 (C-3); 31P NMR (202.46 MHz,
D2O) δ 8.72 ppm. HRMS (ESI) calcd. for C7H18NO6P2 [M + H]+ 274.0604; found
274.0606. Anal. calcd. for (C7H17O6NP2): C, 30.78; H, 6.27; N, 5.13. Found C, 30.43;
H, 6.18; N, 4.99.
(Cycloheptylamino)methyl-1,1-bisphosphonic acid (32). White solid; 83% yield; mp
225 ºC; 1H NMR (500.13 MHz, D2O) δ 1.47 (m, 6H, -CH2-), 1.62 (m, 6H, -CH2-), 2.04
(p, J = 6.5 Hz, 2H, H-5b), 3.12 (t, J = 16.3 Hz, 1H, H-1), 3.62 (sept, J = 4.4 Hz, 1H, H-
3); 13C NMR (125.77 MHz, D2O) δ 23.1 (C-6), 27.4 (C-5), 30.8 (C-4), 54.4 (t, J = 112.4
31
Hz, C-1), 61.0 (t, J = 3.1 Hz, C-3); P NMR (202.46 MHz, D2O) δ 8.74 ppm. HRMS

(ESI) calcd. for C8H20NO6P2 [M+H]+ 288.0760; found 288.0758. Anal. calcd. for
(C8H19O6NP2): C, 33.46; H, 6.67; N, 4.88. Found C, 33.70; H, 6.58; N, 4.65.
(Piperidin-1-yl)methyl-1,1-bisphosphonic acid (33). White solid; 82% yield; mp 238
1
ºC; H NMR (500.13 MHz, D2O) δ 1.69 (m, 2H, H-5), 1.87 (m, 4H, H-4), 3.15 (t, J =
17.6 Hz, 1H, H-1), 3.52 (m, 4H, H-3); 13C NMR (125.77 MHz, D2O) δ 21.0 (C-5), 24.3
(C-4), 53.8 (C-3), 63.8 (t, J = 106.8 Hz, C-1); 31P NMR (202.46 MHz, D2O) δ 6.77
ppm. HRMS (ESI) calcd. for C6H15NO6P2Na [M+Na]+ 282.0267; found 282.0258.
Anal. calcd. for (C6H15O6NP2): C, 27.81; H, 5.83; N, 5.41. Found C, 27.44; H, 5.58; N,
5.06.
1-[(Cyclopentylamino)ethyl] 1,1-bisphosphonic acid (47). White solid; 88% yield;
1
mp 228 ºC; H NMR (500.13 MHz, D2O) δ 1.62 (m, 6H, H-5a, H-6), 1.95 (m, 2H, H-
5b), 2.39 (tt, J = 21.5, 7.3 Hz, 1H, H-1), 3.35 (dt, J = 14.3, 7.3 Hz, 2H, H-2), 3.55 (m.
1H, H-4); 13C NMR (125.77 MHz, D2O) δ 23.4 (C-6), 29.4 (C-5), 36.3 (t, J = 120.9 Hz,
C-1), 43.6 (t, J = 2.4 Hz, C-2), 59.5 (C-4); 31P NMR (202.46 MHz, D2O) δ 15.58 ppm.
HRMS (ESI) calcd. for C7H18O6NP2 [M+H]+ 274.0604; found 274.0610.
1-[(Cycloheptylamino)ethyl] 1,1-bisphosphonic acid (48). 73% yield; white solid;
mp 220 ºC; 1H NMR (500.13 MHz, D2O) δ 1.45 (m, 6H, H-6a, H-7), 1.60 (m, 4H, H-5a,
H-6b), 1.97 (m, 2H, H-5b), 2.37 (tt, J = 21.4, 7.3 Hz, 1H, H-1), 3.28 (hept, J = 4.4 Hz,
1H, H-4), 3.36 (dt, J = 14.3, 7.3 Hz, 2H, H-2); 13C NMR (125.77 MHz, D2O) δ 23.0 (C-
7), 27.3 (C-6), 30.6 (C-5), 36.2 (t, J = 120.8 Hz, C-1), 42.3 (t, J = 2.3 Hz, C-2), 59.4 (C-
4); 31P NMR (202.46 MHz, D2O) δ 15.59 ppm. HRMS (ESI) calcd. for C9H22O6NP2
[M+H]+ 302.0917; found 302.0905.
1-[(4-methylcyclohexylamino)ethyl] 1,1-bisphosphonic acid (49). 76% yield; white
solid; mp = 175 ºC; 1H NMR (125.77 MHz, D2O) δ 0.80 (d, J = 6.6 Hz, 6H, CH(CH3)2),
0.84 (d, J = 6.6 Hz, 6H, CH(CH′3)2), 2.41 (tt, J = 21.4, 7.3 Hz, 1H, H-1), 3.25 (m. 1H,
13
H-4), 3.36 (dt, J = 14.0, 7.1 Hz, 2H, H-2); C NMR (125.77 MHz, D2O) δ 19.9 21.0
(CH3), 25.4 (C-5), 28.4 28.9 (C-5), 30.9 32.2 (C-6), 36.1 (t, J = 121.1 Hz, C-1), 36.2 (t,
J = 120.8 Hz, C-1), 42.0 (t, J = 2.2 Hz, C-2), 42.5 (t, J = 2.6 Hz, C-2), 55.5 57.1 (C-4);
31P NMR (202.46 MHz, D2O) δ 15.59 ppm. HRMS (ESI) calcd. for C9H22O6NP2
[M+H]+ 302.0917; found 302.0931.

Tetraethyl 1-[(n-Undec-1-ylthio)ethyl]-1,1-bisphosphonate (60). 74% yield, colorless
1
oil; H NMR (500.13 MHz, CDCl3)  0.88 (t, J = 7.0 Hz, 3H, H-14), 1.26 (m,16H, -
CH2-), 1.35 (t, J = 7.1 Hz, 12H, CH2CH3), 1.59 (p, J = 7.3 Hz, 2H, H-5), 2.56 (t, J = 7.4
Hz, 2H, H-4), 2.59 (tt, J = 23.6, 6.0 Hz, 1H, H-1), 3.04 (dt, J = 16.3, 5.9 Hz, 2H, H-2),
4.21 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)  14.1 (C-14), 16.3 (d, J = 6.4
Hz, CH2CH3), 22.6 (C-13), 27.7 (t, J = 5.2 Hz, C-2), 28.8 (C-12), 29.2 (C-11), 29.3 (C-
10), 29.4 (C-9), 29.47 (C-8), 29.53 (C-6, C-7), 31.8 (C-5), 33.1 (C-4), 39.0 (t, J = 131.4
Hz, C-1), 62.8 (dd, J = 18.8, 6.8 Hz, CH2CH3); 31P NMR (202.46 MHz, CDCl3) 
21.75.
Tetraethyl 1-[(n-Dodec-1-ylthio)ethyl]-1,1-bisphosphonate (61). Colorless oil; 55%
1
yield; H NMR (500.13 MHz, CDCl3)  0.88 (t, J = 7.0 Hz, 3H, H-15), 1.26 (m,16H, -
CH2-), 1.35 (t, J = 7.1 Hz,12H, CH2CH3), 1.59 (p, J = 7.5 Hz, 2H, H-5), 2.56 (t, J = 7.5
Hz, 2H, H-4), 2.59 (tt, J = 23.9, 5.9 Hz, 1H, H-1), 3.04 (dt, J = 16.3, 5.9 Hz, 2H, H-2),
4.21 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)  14.1 (C-15), 16.4 (d, J = 6.3
Hz, CH2CH3), 22.7 (C-14), 27.7 (t, J = 4.9 Hz, C-2), 28.9 (C-13), 29.2 (C-12), 29.3 C-
11), 29.4 (C-10), 29.51 (C-9), 29.57 (C-8), 29.59 (C-7), 29.62 (C-6), 31.9 (C-5), 33.1
(C-4), 39.1 (t, J = 131.4 Hz, C-1), 62.8 (dd, J = 18.7, 6.6 Hz, CH2CH3); 31P NMR
(202.46 MHz, CDCl3)  21.76.
1-[(n-Undecylthio)ethyl]-1,1-bisphosphonic acid (54). 45% yield; white solid; mp =
1
95 ºC; H NMR (500.13 MHz, DMSO-d6) δ 0.84 (t, J = 6.9 Hz, 3H, H-14), 1.23 (m,
14H, -CH2-), 1.31 (m, 2H, H-6), 1.49 (p, J = 7.3 Hz, 2H, H-5), 2.10 (tt, J = 22.5, 6.0 Hz,
1H, H-1), 2.84 (t, J =15.5, 6.1 Hz, 2H, H-2); 31P NMR (202.46 MHz, DMSO-d6) δ
18.80 ppm. HRMS (ESI) Calcd. for C13H31O6P2S [M+H]+ 377.1311; found 377.1324.
1-[(n-Dodecylthio)ethyl]-1,1-bisphosphonic acid (57). 80% yield; white solid; mp =
1
110 ºC; H NMR (500.13 MHz, DMSO-d6) δ 0.84 (t, J = 7.0 Hz, 3H, H-15), 1.23 (m,
16H, -CH2-), 1.30 (p, J = 7.6 Hz, 2H, H-6), 1.49 (p, J = 7.3 Hz, 2H, H-5), 2.10 (tt, J =
22.5, 6.0 Hz, 1H, H-1), 2.84 (t, J =15.5, 6.1 Hz, 2H, H-2); 31P NMR (202.46 MHz,
DMSO-d6) δ 18.97 ppm. HRMS (ESI) Calcd. for C14H33O6P2S [M+H]+ 391.1468;
found 391.1453

1-[(n-Undecylsulfinyl)ethyl]-1,1-bisphosphonic acid (55). 81% yield; white solid; mp
= 97 ºC; 1H NMR (500.13 MHz, D2O)  0.87 (t, J = 7.0 Hz, 3H, H-14), 1.20 (m, 12H, -
CH2-), 1.28 (p, J = 5.9 Hz, 2H, H-7), 1.39 (m, 2H, H-6), 1.69 (m, 2H, H-5), 2.39 (m,
1H, H-1), 2.76 (ddd, J = 13.9, 8.5, 6.0 Hz, 1H, H-4a), 2.89 (ddd, J = 13.5, 8.4, 7.5 Hz,
1H, H-4b), 3.15 (m, 2H, H-2); 31P NMR (202.46 MHz, D2O)  16.09 mAB. HRMS (ESI)
Calcd. for C13H30O7P2SNa [M+Na]+ 415.1080; found 415.0797.
1-[(n-Dodecylsulfinyl)ethyl]-1,1-bisphosphonic acid (58). White solid, 67%; mp = 94
ºC; 1H NMR (500.13 MHz, DMSO-d6)  0.84 (t, J = 7.0 Hz, 3H, H-15), 1.23 (m, 14H, -
CH2-), 1.38 (p, J = 6.9 Hz, 2H, H-6), 1.61 (p, J = 7.4 Hz, 2H, H-5), 2.57 (dt, J = 13.7,
31
6.9 Hz, 1H, H-4a), 2.81 (dt, J = 13.1, 8.0 Hz, 1H, H-4b), 3.05 (m, 2H, H-2); P NMR
(202.46 MHz, DMSO-d6)  17.31 mAB.
1-[(n-Undecylsulfonyl)ethyl]-1,1-biphosphonic acid (56). 58% yield; white solid; mp
= 128 ºC; 1H NMR (500.13 MHz, DMSO-d6) δ 0.84 (t, J = 6.9 Hz, 3H, H-14), 1.22 (m,
14H, CH2), 1.33 (p, J = 7.3 Hz, 2H, H-6), 1.65 (p, J = 7.7 Hz, 2H, H-5), 3.26 (m, 2H,
31
H-4), 3.37 (t, J =16.3, 5.2 Hz, 2H, H-2); P NMR (202.46 MHz, DMSO-d6) δ 17.13
ppm. HRMS (ESI) Calcd. for C13H31O8P2S [M+H]+ 409.1209; found 409.1198.
1-[(n-Dodecylsulfonyl)ethyl]-1,1-biphosphonic acid (59). 65% yield; white solid; mp
1
125 ºC; H NMR (500.13 MHz, DMSO-d6) δ 0.84 (t, J = 6.9 Hz, 3H, H-15), 1.23 (m,
14H, -CH2-),1.33 (p, J = 7.0 Hz, 2H, H-6), 1.64 (p, J = 7.6 Hz, 2H, H-5), 3.21 (m, 2H,
13
H-4) 3.38 (dt, J = 16.1, 4.7 Hz, 1H, H-2); C NMR (125.77 MHz, DMSO-d6) δ 14.0
(C-15), 21.5 (C-14), 22.1 (C-5), 27.8 (C-6), 28.6 (C-7), 28.76 (C-8), 28.84 (C-9), 29.0
(C-12), 29.06 (C-10), 29.08 (C-11), 31.3 (C-13), 34.1 (t, J = 122.3 Hz, C-1), 49.7 (t, J =
4.0 Hz, C-2), 52.5 (C-4); 31P NMR (202.46 MHz, DMSO-d6) δ 17.04 ppm. HRMS
(ESI) Calcd. for C14H32O8P2SNa [M+Na]+ 445.1185; found 445.1201.
Drug Screening
T. cruzi amastigote assays
These experiments were done as reported using tdTomato labeled trypomastigotes
with the modifications described by Recher et al., 2013.52 ED50 values were determined

by non-linear regression analysis using SigmaPlot.
T. gondii tachyzoites assays
Experiments on T. gondii tachyzoites were carried out as described previously
using T. gondii tachyzoites expressing red fluorescent protein with the modifications
described by Recher et al., 2013.52 Plates were read with covered lids, and both
excitation (544 nm) and emission (590 nm) were read from the bottom.
Cytotoxicity for Vero cells. The cytotoxicity was tested using the Alamar BlueTM assay
as described by Recher et al., 2013.52
TcFPPS and TgFPPS assays and product analysis. The enzymatic activity of the
target enzymes was performed according to our previous studies as described for
Szajnman et al., 2008.48
1
13
31
Supplementary Data: Copies of the H NMR, C NMR and P NMR spectra of the
target molecules and the corresponding intermediates are included as supporting
information.
Acknowledgments: We thank Melissa Storey for help with T. cruzi in vitro testing.
This work was supported by grants from the Consejo Nacional de Investigaciones
Científicas y Técnicas (PIP 112-201501-00631 CO), Agencia Nacional de Promoción
Científica y Tecnológica (PICT 2015 1349), and the Universidad de Buenos Aires
(20020170100067BA) to J.B.R., and the U.S. National Institutes of Health to R.D. (AI-
107663) and S.N.J.M. (AI-102254).
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