Antiparasitic activity of sulfur- and fluorine-containing bisphosphonates against trypanosomatids and apicomplexan parasites

Article abstract of DOI:10.3390/molecules22010082

Based on crystallographic data of the complexes 2-alkyl(amino)ethyl-1,1-bisphosphonates-Trypanosoma cruzi farnesyl diphosphate synthase, some linear 1,1-bisphosphonic acids and other closely related derivatives were designed, synthesized and biologically evaluated against T. cruzi, the responsible agent of Chagas disease and against Toxoplasma gondii, the etiologic agent of toxoplasmosis and also towards the target enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. The isoprenoid-containing 1,1-bisphosphonates exhibited modest antiparasitic activity, whereas the linear α-fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates were unexpectedly devoid of antiparasitic activity. In spite of not presenting efficient antiparasitic activity, these data turned out to be very important to establish a structural activity relationship.

Full text of DOI:10.3390/molecules22010082

molecules
Article
Antiparasitic Activity of Sulfur- and Fluorine-Containing
Bisphosphonates against Trypanosomatids and
Apicomplexan Parasites
Tamila Galaka 1, Mariana Ferrer Casal 1, Melissa Storey 2, Catherine Li 2, María N. Chao 1,
Sergio H. Szajnman 1, Roberto Docampo 2, Silvia N. J. Moreno 2 and Juan B. Rodriguez 1,
*
1
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; galakatamila@qo.fcen.uba.ar (T.G.); mferrer@qo.fcen.uba.ar (M.F.C.);
nchao@qo.fcen.uba.ar (M.N.C.); shs@qo.fcen.uba.ar (S.H.S.)
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology,
University of Georgia, Athens, GA 30602, USA; mtayl04@uga.edu (M.S.); catli@uga.edu (C.L.);
rdocampo@uga.edu (R.D.); smoreno@uga.edu (S.N.J.M.)
2
*
Correspondence: jbr@qo.fcen.uba.ar; Tel.: +54-11-4576-3385; Fax: +54-11-4576-3346
Academic Editor: Artur Silva
Received: 31 October 2016; Accepted: 30 December 2016; Published: 4 January 2017
Abstract: Based on crystallographic data of the complexes 2-alkyl(amino)ethyl-1,1-bisphosphonates–
Trypanosoma cruzi farnesyl diphosphate synthase, some linear 1,1-bisphosphonic acids and other
closely related derivatives were designed, synthesized and biologically evaluated against T. cruzi, the
responsible agent of Chagas disease and against Toxoplasma gondii, the etiologic agent of toxoplasmosis
and also towards the target enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii
(TgFPPS), respectively. The isoprenoid-containing 1,1-bisphosphonates exhibited modest antiparasitic
activity, whereas the linear α-fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates were unexpectedly
devoid of antiparasitic activity. In spite of not presenting efficient antiparasitic activity, these data
turned out to be very important to establish a structural activity relationship.
Keywords: bisphosphonates; farnesyl diphosphate synthase; Trypanosoma cruzi; Toxoplasma gondii;
Chagas disease; toxoplasmosis; antiparasitic agents
1. Introduction
American Trypanosomiasis (Chagas disease) is a chronic parasitosis caused by the kinetoplastid
parasite Trypanosoma cruzi and is the largest parasitic disease burden of the Americas [1,2]. Isoprenoids
are essential compounds of the cellular machinery of all organisms due to their roles in a variety
of biological processes. Several enzymes of this pathway in T. cruzi, involved in the synthesis
of sterols [1,2] and farnesyl diphosphate [3] have been reported to be excellent targets against
pathogenic parasites. Despite their structural and functional variety, all isoprenoids derive from
a common precursor: isopentenyl diphosphate (IPP), and its isomer, dimethylallyl diphosphate
(DMAPP). In T. cruzi, IPP is synthesized only via the so-called mevalonate pathway, which has
the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase as the key regulatory enzyme [
In Apicomplexan parasites such as Toxoplasma gondii, the responsible agent of toxoplasmosis,
IPP is synthesized by the prokaryotic-type 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway [ ],
which localizes to the apicoplast and is essential [ ]. T. gondii possesses a bifunctional farnesyl
diphosphate synthase (FPPS)/geranyl diphosphate synthase (GGPPS) (TgFPPS) that is able to catalyze
the formation of both FPP and GGPP [ ]. It is very relevant to state that it is possible to inhibit the
host mevalonate pathway and the TcFPPS or DOXP pathway of T. gondii synergistically [ ]. Certainly,
4].
5
6
7,8
9
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the use of two approved and widely used drugs, zoledronic acid and atorvastatin exhibited a strong
synergism in the inhibition of T. gondii growth [ ]. In addition, TcFPPS has 50% identity with TgFPPS,
therefore, it would be possible to access very selective inhibitors of the activity of each enzyme [10].
Bisphosphonates ( ) are metabolically stable pyrophosphate analogs ( ) in which a methylene
9
2
1
group replaces the oxygen atom bridge between the two phosphorus atoms of the pyrophosphate
moiety. Substitution at the carbon atom with different side chains has generated a large family of
compounds. 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 [11
the bisphosphonate moiety to the bone mineral [13]. Apart from their ability as inhibitors of bone
resorption, bisphosphonates have turned out to be antibacterial agents [13], anticancer agents [14 15],
selective inhibitors of acid sphingomyelinase [16], and stimulators of γδ T cells [17]. Interestingly,
these compounds behave as antiparasitic agents as well [10 18]. Aminobisphosphonates such as
pamidronate ( ), alendronate ( ), and risedronate ( ) were first found to be effective in the inhibition of
,12]. Selective action on bone is based on the binding of
,
,
3
4
5
T. cruzi in vitro and in vivo without toxicity to the host cells (Figure 1) [19]. In addition, the scope of
these compounds was broadened by finding that some bisphosphonates were also growth inhibitors
of T. gondii, T. brucei rhodesiense, Leishmania donovani and Plasmodium falciparum [20–22]. In vivo testing
in mice has shown that risedronate can have a significantly marked protective effect in mice infected
by T. cruzi [23,24].
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.
Pioneering work of Rogers et al. had shown that apoptosis induced by bisphosphonates in J774
macrophages was associated with the inhibition of post-translational prenylation of proteins such as
Ras and that this effect can be reverted by the addition of components of the mevalonate pathway such
as mevalonic acid, FPP, and GGPP [25]. Many years later, it was established that the primary molecular
target of bisphosphonates was farnesyl diphosphate synthase (FPPS) [26]. Selective inhibition of
parasite growth may be possible due to the incidence of very fast proliferation of parasites together
with the presence of calcium polyphosphate stores in the acidocalcisomes, which mimic the calcium
hydroxyapatite surfaces found in bone, facilitating bisphosphonate drug uptake [27,28].
A relevant achievement of our laboratory was the finding that linear bisphosphonates became
efficient antiparasitic agents [29
usually found in those bisphosphonates currently employed in the treatment of bone disorders,
is absent [26]. Compounds were the first examples of linear bisphosphonates, which turned
out to be antiparasitic agents (trypanosomatids and Apicomplexan parasites) targeting parasitic
FPPS [ 29 32]. For example, is a moderate growth inhibitor of intracellular T. cruzi [29] and also
against tachyzoites of T. gondii [ 32], whereas is effective against P. falciparum [ ]. Compound is
practically devoid of antiparasitic activity [30], whereas shows similar cellular activity against
T. cruzi [31]. In addition, -fluoro-1,1-bisphosphonates 10 and 11 are neither effective against
–31] where, in most of them, the hydroxyl group at the C-1 position,
6–9
7,
–
6
7,
7
7
8
9
α
amastigotes of T. cruzi nor the target enzyme TcFPPS, but they are extremely potent inhibitors of the
enzymatic activity of TgFPPS [33]. Of paramount concern are 2-alkyl(amino)ethyl-1,1-bisphosphonates,
which are potent inhibitors of T. cruzi proliferation acting against the target enzyme (TcFPPS) at the low
nanomolar range [34,35]. Undoubtedly, compounds 12–14 emerge as pertinent members of this type
of bisphosphonates. For example, 12 is considerably more potent than the well-known antiparasitic
agent WC-9 against T. cruzi (amastigotes) [34], under the same assays conditions [36]; while 13 is

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a very effective inhibitor of the enzymatic activities of TcFPPS and of TgFPPS [34]. Moreover, 14
,
the bisphosphonate bearing a long aliphatic chain, is an effective growth inhibitor of T. cruzi [35].
Compound 15 is an interesting example of linear bisphosphonates synthesized by an attempt to
improve structures 12–14. Compound 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 [37]. Compound 15 is devoid of activity against T. cruzi growth and TcFPPS, but exhibited a
potent and selective inhibition of the enzymatic activity towards TgFPPS [37]. Linear sulfur-containing
bisphosphonates are interesting examples of selective anti-Toxoplasma agents as it is the case of 16 and
17 [38]. Certainly, 16 is a potent inhibitor of T. gondii proliferation. This cellular activity was associated
with a potent action against the target enzyme TgFPPS [38], while 17 is an unusually potent inhibitor
towards TgFPPS [38]. The methylsulfonium derivative 18 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 (Figure 2; Table 1) [38].
Figure 2. Chemical structures of representative lineal bisphosphonic acids as putative antiparasitic
agents.
Table 1. Biological activity of bisphosphonates previously prepared in our laboratory against T. cruzi
(amastigotes), T. gondii (tachyzoites), TcFPPS, and TgFPPS.
T. cruzi Growth
ED50 (µM)
TcFPPS
IC50 (µM)
T. gondii Growth
ED50 (µM)
TgFPPS
IC50 (µM)
Compound
10
11
12
13
14
15
16
17
18
>10.0
>10.0
0.84
10.0
0.67
>10.0
>20.0
>20.0
>20.0
>1.0
>1.0
0.49
0.058
0.81
>10.0
0.097
>10.0
0.040
2.67 ± 11.93
>10.0
9.4
0.035
0.060
0.14
0.095
0.093
0.039
0.021
0.009
0.013
>50.0
6.23
2.0
1.8
5.0
7.0
2. Rationale
Prospects in antiparasitic (antileishmanial, antitrypanosomal, and Apicomplexan) drug
research have changed substantially since the availability of the crystal structure of several
complexes of 2-alkylaminoethyl-1,1-bisphosphonates with T. cruzi farnesyl diphosphate synthase [39].
These X-ray crystallographic studies on the interaction of inhibitor 13 with TcFPPS (13–TcFPPS) [39,40]
have indicated that the nitrogen atom does not coordinate the Mg2+ present at the active site of the
target enzyme [40]. The tridentate coordination structure is circumvented to the hydroxyl groups
bonded to the phosphorus atoms either for 2-alkylaminoethyl- or 1-hydroxy-1,1-bisphosphonates [41,42].
X-ray structures and thermodynamic data of the complexes TcFPPS with 12 and 13 indicated that
these compounds bind to the allylic site of TcFPPS having the hydrophobic alkyl chain buried in

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the cavity that binds the isoprenoid chain of the substrate [39]. The structures of 19 and 20 were
envisioned by molecular modeling studies, which suggested that these compounds would increase
affinity substantially (Figure 3) [39].
Figure 3. Chemical structures of the designed target molecules based on crystallographic and molecular
modeling studies.
On the other hand, α-fluoro-2-alkylaminoethyl-1,1-bisphosphonates such as 54–61 are also very
interesting structural variations taking into account the selective and potent action against TgFPPS
exhibited by 1-fluoro-1,1-bisphosphonates [33] and also the selective and efficient biological activity
exhibited by 2-alkylaminoethyl-1,1-bisphosphonates towards both T. cruzi cells and TcFPPS [34,35,39].
3. Results and Discussion
Preparation of the allylamine analog of the lead chemical structures 12
performed according to already published methods [34 35]. Succinctly, the Michael-type acceptor
21 43], which was synthesized from commercially available tetraethyl methylene-1,1-bisphosphonate,
was reacted with 2-methylallylamine in methylene chloride to produce the Michael adduct 22
This compound was hydrolyzed via the method developed by McKenna [44 45] by treatment with
–14 (compound 19) was
,
[
.
,
bromotrimethylsilane in methylene chloride followed by digestion with methanol [38] to yield the
desired compound 19 (Scheme 1). The synthesis of the desired compound 20 was attempted following
a slightly modified method previously described by Mori and Ueda (Scheme 1) [46]. Therefore,
methallyl chloride (23) was coupled with 2-mercaptopyridine (24) to give the corresponding sulfide 25
in 84% yield, which on reaction with n-buthyllithium and isopentenyl bromide at low temperature
◦
gave 27 in 45% yield. This compound was treated with m-chloroperbenzoic acid at
−
20 C to produce
the corresponding sulfoxide 28 as main product and a small amount of the sulfone 29. Basic treatment
of 28 with diethylamine gave rise to the rearranged allyl alcohol 30, which under Mitsunobu-type
conditions by treatment with triphenylphosphine, diisopropyl azodicarboxylate and phtalimide [47
]
yielded amine precursor 31. Cleavage of the phtalimide protecting group by treatment with hidrazine
in refluxing ethanol [47] gave rise to the desired amine 32 as a main product and the hydrated
product 33 as a consequence of the reaction work-up. This compound was reacted with the Michael
acceptor 21 to produce 34 in good yields. Unexpectedly, it was not possible to carry out deprotection
of the tetraethyl ester functionality. The presence of two double bonds indicated that hydrolysis
by treatment with concentrated hydrochloric acid was an unlikely transformation [43]. The most
commonly employed deprotection protocol, that is, reaction with bromotrimethylsilane followed by
digestion with methanol [43], underwent a retro-Michael reaction yielding the corresponding free
amine 32 together with the Michael acceptor 21. Actually, this retro-Michael reaction could take place
during or after silylation; therefore, it is more likely that 21 or its Michael adduct with amine (34
)
upon solvolysis there will probably be no silyl ester, just free acid, product of solvolysis of silyl ester
(after/before Michael addition). Hydrolysis in the presence of 2,4,6-collidine as described for other
closely related compounds [48] was not satisfactory to avoid this behavior. The silyl ester of 21 has
been described as a suitable Michael acceptor for a number of soft nucleophiles [49,50]. However,
in our hands, we were not able to prepare addition product 20 by reaction between the silyl ester
of 21 and the allylic amine 32. Evidently, the presence of the amino group at the C-3 position is not
compatible with the available methodology to perform this transformation and new chemistry has
to be developed to encourage this task. Efforts in these aspects are currently being pursued in our
laboratory. Synthetic efforts to prepare the sulfur-containing analog of the envisioned compound
20, namely, 35 are outlined in Scheme 1 starting form allylic alcohol 30. Thus, 30 was treated under
Mitsunobu-type conditions with triphenylphosphine, diisopropyl azodicarboxylate and thioacetic

Molecules 2017, 22, 82
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acid [51] to yield the thiolacetate 36, which was hydrolyzed by treatment with potassium carbonate [51
]
to produce the corresponding mercaptan 37 in low but reproducible yields. Michael addition of 37 on
21 gave rise to 38, which could not be converted into the envisioned title compound 35.
Scheme 1. Synthetic approach to access the envisioned putative inhibitors of farnesyl pyrophosphate
synthase.
Compounds 39
, 44 and 45 are isoprenoid derivatives, which are structurally related to 19, 20,
and 35 These compounds, envisioned as optimized inhibitors of the target enzyme TcFPPS,
.
are outlined in Scheme 2. Then, following a similar approach, geraniol (40) was converted into 41 by
treatment with triphenylphosphine, diisopropyl azodicarboxylate and phtalimide in 80% yield [51].
Synthesis of the free amine 42 followed by conjugate addition on 21 gave 43 in very good yields.
Neither 43 nor its acetyl derivative 46 was able to produce the desired hypothetical desired compound
39 employing the currently known methods of phosphonates ester hydrolysis. Certainly, phosphonate
esters bearing an amino group at the C-3 position requires a new method for hydrolysis reaction
to occur.
Scheme 2.
Synthetic approach for the preparation of geranyl and farnesyl containing
1,1-bisphosphonates.

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On the other hand, 44 and 45 were straightforwardly prepared starting from geraniol (40) and
farnesol (47), respectively. These alcohols under Mitsunobu type conditions [47] gave thiol acetates 48
and 49 in good yields that on treatment with lithium aluminum hydride produced the corresponding
mercaptans 50 and 51. On reaction with 21, both compounds were converted into precursors 52 and
53, which after final treatment of bromotrimethylsilane and digestion with methanol yielded the title
compounds 44 and 45.
The synthesis of 1-fluoro-2-alkylaminoethyl-1,1-bisphosphonate derivatives 54–61 was
accomplished following a similar approach with other closely related compounds [33] employing
Selectfluor (1-chloromethyl-4-fluoro-1,4-diazobicyclo[2.2.2]octane bis(tetrafluoroborate), F-TEDA BF4)
as fluorinating agent as shown in Scheme 3 [52
Selectfluor; therefore, protection of this group was required for the fluorination reaction to occur. Then,
each tetraethyl 2-alkylaminoethyl-1,1-bisphosphonate 12 14 62 66 was reacted with acetic anhydride
in pyridine to produce the corresponding amides 67 74, which treated with sodium hydride followed
by addition of Selectfluor [54] yielded the respective monofluoro bisphosphonate derivatives 75 82
,53]. The presence of the amino group interfered with
–
,
–
–
–
.
Hydrolysis of these tetraethyl esters by treatment with concentrated hydrochloric acid generated the
title compounds 54–61 (Scheme 3).
Scheme 3. Synthetic approach to access 2-alkylaminoethyl-1-fluoro-1,1-bisphosphonates.
Biological results are shown in Table 2. Most of the envisioned compounds resulted to be devoid
of antiparasitic activity. Compound 19 was inactive against T. cruzi and T. gondii cells but exhibited
a marginal activity against the target enzymes. Sulfur-containing isoprenoid 1,1-bisphosphonates
44 and 45 were also devoid of cellular activity against T. cruzi and T. gondii. Biological evaluation of
α
-fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates 54
–
–
61 was very surprising. Certainly, in spite of
14, which are very effective growth inhibitors
having a small structural variation in comparison to 12
of T. cruzi cells targeting TcFPPS at the low nanomolar range, most of these fluorine derivatives,
with the exception of 59, exhibited vanishing antiparasitic action. Bisphosphonate 59 was a growth
inhibitor of tachyzoites of T. gondii at the low micromolar range (ED50 = 4.0
the work on other bisphosphonates on bone mineral affinity and mevalonate pathway inhibition
evaluations performed by McKenna et al. [55], where there is a drop of activity between -hydroxy
and -fluoro analogs, but desoxy and fluoro analogs behave in a similar way. The derivatives that bear
µM). It is worth mentioning
α
α
long chain length such as 60 and 61 turned out to be toxic compounds against Vero cells; therefore,
they were not further analyzed.
In summary, the modest activity shown by these new families of bisphosphonates bring
about important insights concerning structure–activity relationship. The simple replacement of the
hydrogen atom by a fluorine atom had a dramatic effect on the biological activity, giving rise to
inactive compounds. Effects in optimizing bisphosphonate structures are presently being pursued in
our laboratory.

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Table 2. Biological activity of bisphosphonates against T. cruzi (amastigotes), T. gondii (tachyzoites),
TcFPPS, TgFPPS, human FPPS (HhFPPS) and Vero cells ‡.
T. cruzi Growth
ED50 (µM)
TcFPPS
IC50 (µM)
T. gondii Growth
ED50 (µM)
TgFPPS
IC50 (µM)
HhFPPS
IC50 (µM)
Cytotoxicity
ED50 (µM)
Compound
19
>20
>10.0
4.5 ± 2.8
22.28 ± 11.93
0.183 ± 0.063
6.6 ± 2.4
>200
NT
44
NT
>10.0
0.313 ± 0.027
NT
45
>10.0
NT
NT
>10.0
NT
NT
NT
NT
54
>10.0
NT
NT
NT
55
>10.0
0.097 ± 0.020
>10.0
0.091 ± 0.032
NT
>200
NT
56
>10.0
NT
NT
NT
NT
NT
NT
>10.0
NT
NT
NT
NT
NT
NT
NT
57
>10.0
>10.0
NT
NT
58
>10.0
>10.0
NT
NT
59
>10.0
4.008 ± 1.191
NT
161.7 ± 12.4
60
61
cytotoxic †
cytotoxic †
1.80 ± 0.39
NT
15.0
NT
20.0
benznidazole
‡
Maximum concentration tested. NT = not tested. † visually.
4. 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 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
fully decoupled and are referenced to the middle peak of the solvent CDCl3 at
δ
= 7.26 ppm. Coupling constants are reported in Hz. 13C NMR spectra were
= 77.0 ppm. 31P NMR
δ
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 performed 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. High-resolution mass spectra were obtained using a Bruker micrOTOF-Q II spectrometer,
which is a hybrid quadrupole time of flight mass spectrometer with MS/MS capability. 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
(0.003 M) in concentrated H2SO4 (10%).
·
4H2O (0.04 M), Ce(SO4)2
As judged from the homogeneity of the 1H, 13C, 31P NMR spectra and HPLC analyses of the
title compounds employing a Beckmann Ultrasphere ODS-2 column 5
water–acetonitrile (9:1) at 3.00 mL/min with a refractive index detector indicated a purity >97%.
µ
M, 250
×
10 mm eluting with
Tetraethyl 1-[(2-methylall-1-ylamino)ethyl] 1,1-bisphosphonate (22). A solution of compound 21 (300 mg,
1.0 mmol) in anhydrous methylene chloride (10 mL) was treated with triethylamine (121.2 mg, 168 µL
,
1.2 mmol) and 2-methylallylamine hydrochloride (129 mg, 1.2 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
1
eluent to produce 340 mg (91% yield) of 22 as a colorless oil: H NMR (500.13 MHz, CDCl3)
δ 1.34
(t, J = 7.1 Hz, 12H, H-20), 1.75 (s, 3H, CH3 at C-5), 2.66 (tt, J = 23.5, 5.8 Hz, 1H, H-1), 3.11 (dt, J = 16.9,
5.8 Hz, 2H, H-2), 3.17 (s, 2H, H-4), 4.19 (m, 8H, H-10), 4.83 (br s, 1H, H-6a), 4.89 (br s, 1H, H-6b); 13
NMR (125.77 MHz, CDCl3)
16.4 (dd, J = 6.4, 2.6 Hz, C-20), 20.6 (CH3 at C-5), 37.7 (t, J = 132.5 Hz
C
,
δ
C-1), 44.9 (t, J = 4.3 Hz, C-2), 55.1 (C-4), 62.6 (dd, J = 32.6, 6.5 Hz, C-10), 111.0 (C-6) 143.3 (C-5);
31P NMR (202.46 MHz, CDCl3)
found 372.1712.
δ
22.73. HRMS (ESI) calcd. for (C14H32O6NP2) [M + H]+ 372.1707;
1-[(2-Methylall-1-ylamino)ethyl] 1,1-bisphosphonic Acid (19). To a solution of the tetraethyl ester 22
(320 mg, 0.86 mmol) in anhydrous methylene chloride was added dropwise trimethylsilyl bromide
(1.276 g, 1.10 mL, 8.6 mmol) under an argon atmosphere. The reaction mixture 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 and stirred for 24 h. The solution was

Molecules 2017, 22, 82
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then concentrated under reduced pressure. The residue was dissolved in dry methanol (10 mL) and
subsequently concentrated under reduced pressure twice. The solvent was evaporated and the residue
1
was crystallized from ethanol–water to yield 183 mg (83% yield) of pure 19 as a white solid: H NMR
(500.13 MHz, D2O)
δ 1.74 (s, 3H, CH3 at C-5), 2.44 (m, 1H, H-1), 3.36 m, 2H, H-2), 3.59 (s, 2H, H-4), 5.03
(s, 1H, H-6a), 5.12 (s, 1H, H-6b); 13C NMR (125.77 MHz, D2O)
δ 19.5 (CH3 at C-5), 35.7 (t, J = 128.8 Hz,
C-1), 44.3 (C-2), 52.5 (C-4), 117.4 (C-6) 135.9 (C-5); 31P NMR (202.46 MHz, D2O)
calcd. for (C6H15O6NP2Na) [M + Na]+ 282.0272; found 282.0268.
δ 16.96. HRMS (ESI)
2-((2-Methylallyl)thio)pyridine (25). To a solution of sodium ethoxide (prepared from sodium (620 mg,
27.0 mmol) and anhydrous ethanol (20 mL)) was added 2-mercaptopyridine (24; 3.00 g, 27.0 mmol)
at 0 ◦C. The mixture was stirred for 15 min. Then, methallyl chloride (23; 3.06 mL, 2.81 g, 31.0 mmol)
was added and the mixture was stirred at 0 ◦C for 3 h. The solvent was evaporated and the residue
was partitioned between water (50 mL) and ethyl ether (50 mL). The organic phase was washed with
an aqueous 5% solution of sodium hydroxide (2
was dried (MgSO4) and the solvent was evaporated to produce 3.743 g (84% yield) of 25 as a colorless
×
30 mL) and water (2
×
30 mL). The organic layer
1
oil: H NMR (500.13 MHz, CDCl3)
δ
1.85 (s, 3H, CH3 at C-2), 3.86 (s, 2H, H-1), 4.86 (t, J = 1.4 Hz, 1H,
H-3a), 5.02 (q, J = 0.9 Hz, 1H, H-3b), 6.97 (ddd, J = 7.3, 4.9, 1.0 Hz, 1H, H-50), 7.18 (dt, J = 8.1, 0.9 Hz,
1H, H-30), 7.47 (ddd, J = 7.9, 7.5, 1.8 Hz, 1H, H-40), 8.42 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H, H-60); 13C NMR
(125.77 MHz, CDCl3) δ
21.5 (CH3 at C-2), 37.1 (C-3), 113.7 (C-1), 119.5 (C-50), 122.3 (C-30),135.9 (C-40),
141.3 (C-2), 149.4 (C-60), 159.0 (C-20).
2-((2,6-Dimethylhepta-1,5-dien-3-yl)thio)pyridine (27). A solution of 25 (3.74 g, 22.7 mmol) in anhydrous
tetrahydrofuran (50 mL) cooled at
−
50 ◦C was treated dropwise with an 1.1 M solution of
n-butyllithium in hexane (22 mL). The reaction mixture turned deep-red and was cooled to −78 ◦C.
Then, to the resulting mixture a solution of isopentenyl bromide (26; 2.8 mL, 3.61 g, 24.2 mmol) in
anhydrous tetrahydrofuran (10 mL) was added dropwise. The reaction mixture was stirred at −78 ◦C
for 1 h. Then, the mixture was allowed to reach room temperature gradually. The reaction was
quenched by addition of a saturated solution of ammonium chloride (50 mL). The organic layer was
separated and the aqueous phase was extracted with ethyl ether (50 mL). The combined organic layers
were dried (MgSO4) and the solvent was evaporated to give 2.384 g of 27 (45% yield) as a colorless oil:
1H NMR (500.13 MHz, CDCl3)
δ 1.64 (s, 3H, CH3 at C-6), 1.68 (s, 3H, H-7), 1.82 (s, 3H, CH3 at C-2),
2.43 (m, 1H, H-4a), 2.56 (m, 1H, H-4b), 4.40 (dd, J = 8.6, 6.6 Hz, 1H, H-3), 4.85 (br s, 1H, H-1a), 4.98
(br s, 1H, H-1b), 5.15 (tt, J = 7.0, 1.2 Hz, 1H, H-5), 6.92 (ddt, J = 7.3, 5.1, 1.0 Hz, 1H, H-50), 7.17 (ddd,
J = 8.0, 1.9, 1.0 Hz, 1H, H-30), 7.46 (m, 1H, H-40), 8.42 (ddd, J = 4.0, 1.8, 0.9 Hz, 1H, H-60); 13C NMR
(125.77 MHz, CDCl3)
δ
18.0 (CH3 at C-6), 19.5 (CH3 at C-2), 205.8 (C-7), 32.1 (C-4), 51.2 (C-3), 113.3
0
0
0
0
(C-1), 119.6 (C-5 ), 121.2 (C-3 ), 123.2 (C-5), 133.5 (C-6), 135.9 (C-4 ), 144.2 (C-2), 149.4 (C-6 ), 159.1 (C-2 ).
HRMS (ESI) calcd. for C14H20NS [M + H]+ 234.1316; found 234.1319.
2-((2,6-Dimethylhepta-1,5-dien-3-yl)sulfinyl)pyridine (28) and 2-((2,6-dimethylhepta-1,5-dien-3-yl)sulfon◦yl)
pyridine (29). To a solution of 27 (5.91 mg, 25.3 mmol) in methylene chloride (100 mL) cooled at
−
25 C
under an argon atmosphere was added dropwise a solution of m-chloroperbenzoic acid (77% purity,
5.61 g, 26.6 mmol) in methylene chloride (100 mL). The mixture was stirred at
25 ◦C for 1 h. Then,
the temperature was allowed to reach room temperature. Then, the mixture was extracted with a
saturated solution of potassium carbonate (2 75 mL) and water (2 75 mL). The organic phase was
−
×
×
dried (MgSO4), and the solvent was evaporated. The residue was purified by column chromatography
(silica gel) eluting was hexane–EtOAc (19.1) to give 5.68 g of 28 (91% yield) and 403 mg of 29 (6% yield)
1
as colorless oils: Compound 28 (two diastereomers): H NMR (500.13 MHz, CDCl3)
δ 1.42 (s, 3H, CH3
at C-6), 1.55 (s, 3H, CH3* at C-6), 1.50 (s, 3H, H-7), 1.70 (s, 3H, H-7*), 1.73 (s, 3H, CH3 at C-2), 1.88
(s, 3H, CH3* at C-2), 2.25 (m, 1H, H-4a), 2.60 (m, 1H, H-4a*), 2.47 (m, 1H, H-4b), 2.85 (m, 1H, H-4b*),
3.46 (dd, J = 8.2, 7.9 Hz, 1H, H-3), 3.57 (dd, J = 10.3, 4.9 Hz, 1H, H-3*), 4.48 (br s, 1H, H-1a), 4.77
(p, J = 1.5 Hz, 1H, H-1a*), 4.83 (tp, J = 7.0, 1.4 Hz, 1H, H-3), 5.03 (br s, 1H, H-1b), 5.13 (p, J = 1.5 Hz, 1H,
H-1b*), 5.19 (m, 1H, H-3*), 7.31 (m, 1H, aromatic proton), 7.88–7.97 (m, 2H, aromatic proton), 8.60–8.64
(m, 1H, aromatic proton); compound 29 1H NMR (500.13 MHz, CDCl3)
δ 1.57 (s, 3H, CH3 at C-6), 1.67

Molecules 2017, 22, 82
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(s, 3H, H-7), 1.82 (br s, 3H, CH3 at C-2), 2.48 (m, 1H, H-4a), 2.72 (m, 1H, H-4b), 4.21 (dd, J = 11.2, 4.2 Hz,
1H, H-3), 4.84 (br s, 1H, H-1a), 4.88 (t, J = 7.0 Hz, 01H, H-5), 5.02 (br s, 1H, H-1b), 7.51 (ddd, J = 7.7, 4.7,
0
0
1.1 Hz, 1H, H-5 ), 7.91 (dt, J = 7.7, 1.7 Hz, 1H, H-4 ), 8.02 (d, J = 7.9 Hz, 1H, H-3 ), 8.74 (ddd, J = 4.6, 1.6,
0
0.8 Hz, 1H, H-6 ); 13C NMR (125.77 MHz, CDCl3)
δ 17.9 (CH3 at C-6), 20.2 (CH3 at C-2), 24.6 (C-7), 25.7
(C-4), 67.7 (C-3), 118.4 (C-1), 120.8 (C-50), 123.4 (C-30), 127.1 (C-5), 135.1 (C-6), 136.4 (C-2), 137.7 (C-40),
150.1 (C-60), 156.6 (C-20).
(E)-2,6-Dimethylhepta-2,5-dien-1-ol (30). A solution of 28 (5.65 g, 22.7 mmol) in methanol (100 mL)
was treated with diethylamine (100 mL) and the resulting mixture was stirred at room temperature
overnight. The solvent was evaporated and the residue was partitioned between ethyl ether (100 mL)
and water (100 mL). The aqueous phase was extracted with ethyl ether (2
organic layers were washed with 5% hydrochloric acid (2 30 mL), a saturated solution of sodium
bicarbonate (2 30 mL), brine (3 30 mL). The organic layer was dried (MgSO4) and the solvent
×
50 mL). The combined
×
×
×
was evaporated. The product was purified by column chromatography (silica gel) eluting with a
mixture of hexane EtOAc (19:1) to yield 1.973 g (62% yield) of alcohol 30 as a colorless oil: Rf = 0.51
1
(hexane–EtOAc, 1:1): H NMR (500.13 MHz, CDCl3)
δ
1.64 (s, 3H, C-7), 1.70 (s, 6H, CH3 at C-2, CH3
at C-6), 2.73 (t, J = 7.2 Hz, 2H, H-4), 4.00 (s, 2H, H-1), 5.11 (tp, J = 7.0, 1.3 Hz, 1H, H-3), 5.39 (tq, J = 7.3,
1.3 Hz, 1H, H-5); 13C NMR (125.77 MHz, CDCl3)
δ
13.6 (CH3 at C-2), 17.7 (CH3 at C-6), 25.7 (C-7), 26.7
(C-4), 69.0 (C-1), 122.4 (C-3), 125.1 (C-5), 132.0 (C-6), 134.5 (C-2).
(E)-2-(2,6-Dimethylhepta-2,5-dien-1-yl)isoindoline-1,3-dione (31). To a mixture of alcohol 30 (406 mg,
2.9 mmol), triphenylphosphine (911.3 mg, 3.5 mmol), phtalimide (511.2 mg, 3.5 mmol) in anhydrous
◦
tetrahydrofuran (10 mL) cooled at 0 C was added a solution of diisopropyl azodicarboxylate
(702.6 mg, 3.5 mmol). The reaction mixture was stirred at room temperature overnight. The solvent
was evaporated and the residue was purified by column chromatography (silica gel) eluting with a
1
mixture of hexane–EtOAc (19:1) to give 453.0 mg (58% yield) of pure 31 as a colorless oil: H NMR
(500.13 MHz, CDCl3)
δ 1.60 (s, 3H, C-7), 1.67 (s, 6H, CH3 at C-2, CH3 at C-6), 2.70 (t, J = 7.3 Hz, 2H,
H-4), 4.20 (s, 2H, H-1), 5.05 (tp, J = 7.0, 1.4 Hz, 1H, H-3), 5.36 (tq, J = 7.2, 1.3 Hz, 1H, H-5); 13C NMR
(125.77 MHz, CDCl3)
δ 14.6 (CH3 at C-2), 17.7 (CH3 at C-6), 25.6 (C-7), 26.9 (C-4), 45.0 (C-1), 122.3 (C-3),
0
0
0
123.3 (C-2 ), 126.6 (C-5), 129.0(C-1 ), 132.0 (C-6), 132.1 (C-2), 133.9 (C-3 ), 168.3 (CO). HRMS (ESI) calcd.
for C17H19O2NNa [M + Na]+ 292.1313; found 292.1315.
(E)-2,6-Dimethylhepta-2,5-dien-1-amine (32) and (E)-7-amino-2,6-dimethylhept-5-en-2-ol (33). A solution
of 31 (400 mg, 1.5 mmol) in ethanol (10 mL) was treated with a solution of 60% hidrazine in water
(0.1 mL). The reaction mixture was refluxed for 3 h. The mixture was allowed to cool to room
temperature and was filtered. The solvent was evaporated. The residue was treated with a 20%
aqueous solution of hydrochloric acid (10 mL) and the mixture was filtered. The pH of the solution
was adjusted to 11 by addition of a 1.0 M solution of sodium hydroxide. The aqueous solution
was extracted with methylene chloride (3
The residue was purified by column chromatography (silica gel) employing a mixture of hexane–EtOAc
(4:1) to give 175.4 mg of 32 (84% yield) and 21.2 mg of 33 (9% yield): Compound 32
1H NMR
(500.13 MHz, CDCl3) 1.64 (s, 3H, C-7), 1.66 (CH3 at C-2), 1.70 (CH3 at C-6), 2.72 (t, J = 7.1 Hz, 2H,
H-4), 3.17 (s, 2H, H-1), 5.11 (tq, J = 7.1, 1.2 Hz, 1H, H-3), 5.26 (tt, J = 7.1, 1.0 Hz, 1H, H-5); 13C NMR
×
30 mL), dried (MgSO4) and the solvent was evaporated.
:
δ
(125.77 MHz, CDCl3)
122.9 (C-5), 131.7 (C-6), 136.3 (C-2). Compound 33: H NMR (500.13 MHz, CDCl3)
δ
14.6 (CH3 at C-2), 17.7 (CH3 at C-6), 25.7 (C-7), 26.8 (C-4), 49.9 (C-1), 122.6 (C-3),
1
δ
1.24 (s, 6H, H-7,
CH3 at C-6), 1.36 (s, 2H, NH2), 1.55 (m, 2H, H-5), 1.66 (s, 3H, CH3 at C-2), 2.11 (m, 2H, H-4), 3.17 (s, 2H,
H-1), 5.30 (tq, J = 7.1, 1.2 Hz, 1H, H-3); 13C NMR (125.77 MHz, CDCl3)
δ
14.5 (CH3 at C-2), 22.7 (C-4),
29.2 (C-7, CH3 at C-6), 43.5 (C-5), 49.9 (C-1), 70.8 (C-6), 123.7 (C-3), 136.6 (C-2). HRMS (ESI) calcd. for
C9H19ONNa [M + Na]+ 180.1364; found 180.1353.
Tetraethyl (E)-2,6-dimethylhepta-2,5-dien-1-ylamino ethyl-1,1-bisphosphonate (34). A solution of 21
(327.8 mg, 1.1 mmol) in anhydrous methylene chloride (10 mL) was treated with triethylamine
(133.1 mg, 185
µL, 1.3 mmol) and 32 (152.0 mg, 1.1 mmol) as described for the preparation of 22.
Evaporation of the solvent yielded 483.2 mg (100% yield) of pure 34 as a colorless oil: 1H NMR

Molecules 2017, 22, 82
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(500.13 MHz, CDCl3)
δ
1.34 (t, J = 7.1 Hz, 12H, CH2CH3), 1.63 (s, 3H, C-10), 1.66 (CH3 at C-5), 1.68
(CH3 at C-9), 2.64 (tt, J = 23.7, 5.9 Hz, 1H, H-1), 2.71 (t, J = 6.8 Hz, 2H, H-7), 3.08 (dt, J = 16.8, 5.9 Hz,
2H, H-2), 3.12 (s, 2H, H-4), 4.18 (m, 8H, CH2CH3), 5.09 (tt, J = 7.1, 1.3 Hz, 1H, H-6), 5.29 (t, J = 7.2 Hz
,
1H, H-8); 13C NMR (125.77 MHz, CDCl3)
δ 14.6 (CH3 at C-5), 16.3 (d, J = 3.0 Hz, CH2CH3), 16.4
(d, J = 2.7 Hz, CH2C0H3), 18.7 (CH3 at C-9), 24.7 (C-10), 26.9 (C-7), 37.6 (t, J = 132.4 Hz, C-1), 44.8 (t,
J = 4.3 Hz, C-2), 57.0 (C-4), 62.4 (d, J = 6.6 Hz, CH2CH3), 62.7 (d, J = 6.6 Hz, C0H2CH3), 122.8 (C-6),
125.1 (C-8), 131.5 (C-9), 132.8 (C-5); 31P NMR (202.46 MHz, CDCl3) δ 22.82 ppm.
(E)-S-(2,6-Dimethylhepta-2,5-dien-1-yl) ethanethioate (36). To a mixture of alcohol 30 (471.0 mg,
3.4 mmol), triphenylphosphine (1.32 g, 5.04 mmol), thioacetic acid (255.7 mg, 3.4 mmol) in anhydrous
tetrahydrofuran (10 mL) cooled at 0 ◦C was added a solution of diisopropyl azodicarboxylate (1.02 g,
5.04 mmol) as depicted for the preparation of 31. The product was purified by column chromatography
(silica gel) eluting with a mixture of hexane–EtOAc (49:1) to give 459.0 mg (69% yield) of 36 as a
1
colorless oil: Rf 0.37 (hexane–EtOAc, 19:1); H NMR (500.13 MHz, CDCl3)
δ 1.62 (s, 3H, C-7), 1.65
(m, 3H, CH3 at C-2), 1.68 (d, J = 1.1 Hz, 3H, CH3 at C-6), 2.33 (s, 3H, CH3C(O)), 2.68 (t, J = 6.9 Hz,
2H, H-4), 3.55 (q, J = 0.9 Hz, 2H, H-1), 5.11 (tq, J = 7.1, 1.2 Hz, 1H, H-3), 5.38 (tq, J = 7.3, 1.2 Hz, 1H,
H-5); 13C NMR (125.77 MHz, CDCl3)
δ 15.2 (CH3 at C-2), 17.7 (CH3 at C-6), 25.7 (C-7), 27.2 (C-4), 30.5
(CH3C(O)), 38.2 (C-1), 122.2 (C-3), 127.9 (C-5), 130.0 (C-2), 132.1 (C-6), 195.1 (CH3C(O)).
(E)-2,6-Dimethylhepta-2,5-dien-1-thiol (37). To a solution of 36 (353.0 mg, 1.78 mmol) in 20.0 mL of a
mixture of methanol–water (5:3) was added potassium carbonate (100 mg, 0.72 mmol). The reaction
mixture was stirred at room temperature for 6 h. Then, water (50 mL) was added and the mixture was
extracted with methylene chloride (3
(2 30 mL), dried (MgSO4) and the solvent was evaporated. The residue was purified by column
chromatography (silica gel) eluting with hexane to yield 89 mg (32% yield) of pure 37 as a colorless oil:
Rf 0.57 (hexane–EtOAc, 19:1); 1H NMR (300.18 MHz, CDCl3)
1.40 (t, J = 7.8 Hz, 1H, SH), 1.63 (s, 3H,
×
30 mL). The combined organic layers were washed with brine
×
δ
C-7), 1.69 (s, 3H, CH3 at C-2), 1.75 (q, J = 1.0 Hz, 3H, CH3 at C-6), 2.71 (q, J = 7.9 Hz, 2H, H-4), 3.12
(d, J = 7.8 Hz, 2H, H-1), 5.08 (tp, J = 7.1, 1.4 Hz, 1H, H-3), 5.31 (tq, J = 7.2, 1.0 Hz, 1H, H-5).
Tetraethyl (E)-2,6-dimethylhepta-2,5-dien-1-ylthioethyl-1,1-bisphosphonate (38). A solution of 21 (430.3 mg,
1.43 mmol) in anhydrous methylene chloride (15 mL) was treated with triethylamine (147.1 mg,
205 µL, 1.43 mmol) and 37 (224.1 mg, 1.43 mmol) as described for the preparation of 22. The solvent
was evaporated and the residue was purified by column chromatography (silica gel) employing
1
hexane–EtOAc (4:1) as eluent to give 268 mg (41% yield) of 38 as a colorless oil: H NMR (500.13 MHz,
CDCl3) δ
1.366 (t, J = 7.0 Hz, 6H, CH2CH3), 1.374 (t, J = 7.1 Hz, 6H, CH2CH03), 1.64 (s, 3H, C-10), 1.70
(s, 3H, CH3 at C-5), 1.74 (m, 3H, CH3 at C-9), 2.61 (tt, J = 23.8, 6.2 Hz, 1H, H-1), 2.74 (t, J = 7.6 Hz,
2H, H-7), 2.94 (dt, J = 16.2, 6.2 Hz, 2H, H-2), 3.15 (s, 2H, H-4), 4.21 (m, 8H, CH2CH3), 5.11 (tp, J = 7.0,
1.6 Hz, 1H, H-6), 5.31 (t, J = 7.1 Hz, 1H, H-8); 13C NMR (125.77 MHz, CDCl3)
δ 14.9 (CH3 at C-5),
16.2 (t, J = 3.3 Hz, CH2CH3), 16.4 (dd, J = 6.5, 1.3 Hz, CH2C0H3), 17.7 (CH3 at C-9), 25.6 (C-10), 26.6
(t, J = 5.0 Hz, C-2), 27.2 (C-7), 38.7 (t, J = 131.7 Hz, C-1), 42.4.0 (C-4), 62.7 (d, J = 4.8 Hz, CH2CH3),
62.8 (d, J = 6.7 Hz, C0H2CH3), 122.2 (C-6), 127.9 (C-8), 130.0 (C-4), 132.1 (C-9); 31P NMR (202.47 MHz,
CDCl3) δ 21.77 ppm.
(E)-2-(3,7-Dimethylocta-2,6-dien-1-yl)isoindoline-1,3-dione (41). To a mixture of geraniol 40 (1.00 g,
6.5 mmol), triphenylphosphine (1.70 g, 6.5 mmol), phtalimide (953.3 mg, 6.5 mmol) in anhydrous
tetrahydrofuran (15 mL) cooled at 0 ◦C was added a solution of diisopropyl azodicarboxylate (1.38 g,
6.5 mmol) as described for the preparation of 31. The residue was purified by column chromatography
(silica gel) eluting with hexane–EtOAc (19:1) to give 1.47 g (80 yield) of pure 41 as a colorless oil:
Rf 0.52 (hexane–EtOAc, 4:1); 1H NMR (200 MHz, CDCl3)
δ 1.56 (s, 3H, H-8), 1.63 (s, 3H, CH3 at C-7),
1.82 (s, 3H, CH3 at C-3), 2.00 (m, 2H, H-5), 2.08 (p, J = 7.0 Hz, 2H, H-4), 4.27 (d, J = 7.2 Hz, 2H, H-1),
5.05 (m, 1H, H-6), 5.27 (t, J = 7.4 Hz, 1H, H-2).
(E)-3,7-Dimethylocta-2,6-dien-1-amine (42). A solution of 41 (900 mg, 3.2 mmol) in ethanol (13 mL) was
treated with a solution of 60% hidrazine in water (150 µL, 101 mg, 3.2 mmol). The reaction mixture

Molecules 2017, 22, 82
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was refluxed for 3 h and was treated as described for the preparation of 32 to yield 422 mg (86% yield)
1
as a colorless oil: H NMR (500.13 MHz, CDCl3)
δ
1.60 (s, 3H, H-8), 1.63 (s, 3H, CH3 at C-7), 1.69 (s,
3H, CH3 at C-3), 2.00 (m, 2H, H-5), 2.08 (p, J = 7.0 Hz, 2H, H-4), 3.27 (d, J = 6.8 Hz, 2H, H-1), 5.10
(tp, J = 6.7, 1.3 Hz, 1H, H-6), 5.26 (tp, J = 6.9, 1.2 Hz, 1H, H-2); 13C NMR (125.77 MHz, CDCl3)
δ
16.1
(CH3 at C-3), 17.7 (CH3 at C-7), 25.7 (C-8), 26.5 (C-5), 39.5 (C-4), 39.6 (C-1), 124.1 (C-2), 125.9 (C-6), 131.5
(C-7), 136.3 (C-3).
Tetraethyl 1-[((E)-3,7-dimethylocta-2,6-diene-1-amino)ethyl] 1,1-bisphosphonate (43). A solution of 21
(400.1 mg, 1.33 mmol) in anhydrous methylene chloride (10 mL) was treated with amine 42 (204.0
mg, 1.33 mmol) as described for the preparation of 22. Evaporation of the solvent gave 611.1 mg
1
(100% yield) of pure 43 as a colorless oil: H NMR (500.13 MHz, CDCl3)
δ 1.34 (t, J = 7.1 Hz, 12H,
CH2CH3), 1.63 (s, 3H, H-11), 1.66 (s, 3H, CH3 at C-10), 1.68 (s, 3H, CH3 at C-6), 2.71 (t, J = 6.8 Hz, 4H,
H-7, H-8), 2.65 (tt, J = 23.7, 5.9 Hz, 1H, H-1), 3.08 (dt, J = 16.8, 5.9 Hz, 2H, H-2), 3.13 (br s, 2H, H-4), 5.09
(tt, J = 7.1, 1.3 Hz, 1H, H-9), 5.29 (t, J = 7.2 Hz, 1H, H-5); 13C NMR (125.77 MHz, CDCl3)
δ 14.6 (CH3 at
0
C-6), 16.3 (d, J = 3.0 Hz, CH2CH3), 16.4 (d, J = 2.7 Hz, CH2CH3 ), 17.7 (CH3 at C-10), 25.6 (C-11), 26.9
(C-8), 37.6 (t, J = 132.4 Hz, C-1), 44.8 (t, J = 4.3 Hz, C-2), 57.0 (C-4), 62.4 (d, J = 6.8 Hz, CH2CH3), 62.7
0
(d, J = 6.8 Hz, CH2CH3 ), 122.8 (C-5), 125.1 (C-9), 131.5 (C-10), 132.8 (C-6); 31P NMR (202.46 MHz,
CDCl3) δ 22.82 ppm.
Tetraethyl 1-[((E)-3,7-dimethylocta-2,6-diene-1-acetamido)ethyl] 1,1-bisphosphonate (46). A solution of 43
(227 mg, 0.5 mmol) in anhydrous pyridine (3.0 mL) was treated with acetic anhydride (3.0 mL) and
the mixture was stirred at room temperature overnight. The solvent was evaporated and the residue
was purified by column chromatography (silica gel) eluting with a mixture of CH2Cl2–methanol
1
(19:1) to give 312.2 mg (63% yield) of pure 46 as a colorless o0il: H NMR (500.13 MHz, CDCl3)
δ 1.34
(t, J = 7.0 Hz, 6H, CH2CH3), 1.35 (t, J = 7.1 Hz, 6H, CH2CH3 ), 1.61 (s, 3H, H-11), 1.68 (s, 3H, CH3 at
C-10), 1.69 (s, 3H, CH3 at C-6), 2.00–2.11 (m, 4H, H-7, H-8), 2.07 (s, 3H, C(O)CH3), 3.38 (tt, J = 22.7
,
5.5 Hz, 1H, H-1), 3.71 (ddd, J = 14.7, 11.1, 7.4 Hz, 2H, H-2), 4.04 (d, J = 6.4 Hz, 2H, H-4), 4.18 (m,
8H, CH2CH3), 5.07 (m, 2H, H-5, H-9); 13C NMR (125.77 MHz, CDCl3)
δ 16.3 (CH3 at C-6), 16.34
0
(d, J = 6.7 Hz, CH2CH3), 16.37 (d, J = 6.6 Hz, CH2CH3 ), 17.7 (CH3 at C-10), 21.9 (C(O)CH3), 25.7
(C-11), 26.3 (C-8), 34.4 (t, J = 129.9 Hz, C-1), 39.5 (C-7), 44.4 (t, J = 3.5 Hz, C-2), 48.8 (C-4), 62.4 (d,
0
J = 6.8 Hz, CH2CH3), 62.7 (d, J = 6.8 Hz, CH2CH3 ), 119.7 (C-5), 123.6 (C-9), 131.9 (C-10), 139.4 (C-6),
171.2 (C(O)CH3); 31P NMR (202.46 MHz, CDCl3) δ 21.49 ppm.
(E)-S-(3,7-Dimethylocta-2,6-dien-1-yl) ethanethioate (48). To a mixture of geraniol 40 (1.00 g, 6.5 mmol),
triphenylphosphine (2.55 g, 9.7 mmol),◦thioacetic acid (0.55 mL, 592.2 mg, 7.8 mmol) in anhydrous
tetrahydrofuran (20 mL) cooled at 0 C was added a solution of diisopropyl azodicarboxylate
(1.62 mL, 1.66 g, 9.7 mmol) as depicted for the preparation of 31. The product was purified by
column chromatography (silica gel) eluting with hexane–EtOAc (9:1) to give 828.2 mg (60% yield) of
48 as a colorless oil: Rf 0.39 (hexane–EtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3)
δ 1.59 (s, 3H, H-8),
1.68 (s, 6H, CH3 at C-3, CH3 at C-7), 2.00 (m, 2H, H-4), 2.06 (m, 2H, H-4), 2.32 (s, 3H, SC(O)CH3), 3.54
(d, J = 7.9 Hz, 2H, H-1), 5.06 (tp, J = 6.8, 1.4 Hz, 1H, H-6), 5.21 (tp, J = 7.9, 1.3 Hz, 1H, H-2); 13C NMR
(125.77 MHz, CDCl3)
δ 16.2 (CH3 at C-2), 17.7 (CH3 at C-6), 25.6 (C-1), 26.4 (C-8), 27.3 (C-5), 30.4
(SC(O)CH3), 39.5 (C-4), 118.4 (C-2), 123.8 (C-5), 131.7 (C-7), 140.1 (C-3), 196.1 (SC(O)CH3). HRMS (ESI)
calcd. for C12H20OS [M + Na]+ 235.1133; found 235.1138.
(E)-3,7-Dimethylocta-2,6-diene-1-thiol◦ (50). A solution of 48 (846 mg, 3.98 mmol) in anhydrous
tetrahydrofuran (10 mL) cooled at 0 C was treated with portionwise with lithium aluminum hydride
◦
(181 mg, 4.74 mmol). The mixture was stirred at 0 C for 4 h. The reaction was quenched by addition of
ethyl acetate (2.0 mL). Then, the mixture was partitioned between methylene chloride (50 mL) and an
aqueous saturated solution of potassium tartrate (50 mL). The organic phase was washed with water
(2
chromatography (silica gel) eluting with hexane to give 411 mg (61% yield) of 50 as a colorless oil:
1H NMR (500.13 MHz, CDCl3)
1.40 (t, J = 7.1 Hz, 1H, SH), 1.60 (s, 3H, H-8), 1.66 (d, J = 1.2 Hz, CH3 at
C-7), 1.68 (d, J = 1.2 Hz, CH3 at C-3), 2.00 (m, 2H, H-4), 2.07 (m, 2H, H-4), 3.16 (ddd, J = 7.6, 7.3, 0.4 Hz,
×
30 mL), dried (MgSO4), and the solvent was evaporated. The product was purified by column
δ

Molecules 2017, 22, 82
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2H, H-1), 5.08 (tq, J = 6.9, 1.4 Hz, 1H, H-6), 5.34 (tsxt, J = 7.8, 1.3 Hz, 1H, H-2); 13C NMR (125.77 MHz,
CDCl3) δ 15.8 (CH3 at C-2), 17.7 (CH3 at C-6), 22.1 (C-1), 25.7 (C-8), 26.4 (C-5), 39.4 (C-4), 123.3 (C-2),
123.6 (C-5), 131.7 (C-7), 137.5 (C-3). HRMS (ESI) calcd. for C10H19S [M + H]+ 171.1207; found 171.1194.
Tetraethyl 1-[((E)-3,7-dimethylocta-2,6-diene-1-thio)ethyl] 1,1-bisphosphonate (52). A solution of 21
(1.00 mg, 3.34 mmol) in anhydrous methylene chloride (15 mL) was treated with triethylamine (337 mg,
470 µL, 3.34 mmol) and 50 (580.9 mg, 3.34 mmol) as described for the preparation of 22. The solvent
was evaporated and the residue was purified by column chromatography (silica gel) employing EtOAc
1
as eluent to give 1.163 g (74% yield) of 52 as a colorless oil: H NMR (500.13 MHz, CDCl3)
δ 1.35
(t, J = 7.1 Hz, 12H, CH2CH3), 1.60 (s, 3H, H-11), 1.67 (s, 3H, CH3 at C-10), 1.68 (s, 3H, CH3 at C-6), 2.02
(m, 2H, H-7), 2.07 (m, 2H, H-8), 2.61 (tt, J = 23.8, 6.1 Hz, 1H, H-1), 3.02 (dt, J = 16.2, 6.1 Hz, 2H, H-2),
3.22 (d, J = 7.7 Hz, 2H, H-4), 5.08 (m, 1H, H-9), 5.26 (t, J = 7.7 Hz, 1H, H-5); 13C NMR (125.77 MHz,
CDCl3)
δ 16.2 (CH3 at C-6), 16.4 (d, J = 6.1 Hz, CH2CH3), 17.7 (CH3 at C-10), 25.7 (C-11), 26.5 (C-8),
27.3 (t, J = 5.0 Hz, C-2), 30.7 (C-4), 38.9 (t, J = 131.6 Hz, C-1), 39.6 (C-4), 119.9 (C-5), 123.9 (C-9), 131.7
(C-10), 139.4 (C-6); 31P NMR (202.46 MHz, CDCl3)
[M + Na]+ 493.1919; found 493.1929.
δ 21.72 ppm. HRMS (ESI) calcd. for C20H40O6SP2
1-[((E)-3,7-Dimethylocta-2,6-diene-1-thio)ethyl] 1,1-bisphosphonic Acid (44). A solution of the Michael
adduct 52 (997 mg, 2.1 mmol) and 2,4,6-collidine (2.53 g, 2.76 mL, 21.0 mmol) in anhydrous methylene
chloride (20 mL) was treated with bromotrimethylsilane (3.23 g, 2.73 mL, 21.1 mmol) 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 (10 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. The product was purified by column chromatography
on reverse phase eluting with a mixture of water–acetonitrile (3:2) to give 361.2 mg (48% yield) of 44 as
1
an amorphous solid: H NMR (500.13 MHz, D2O)
δ
1.54 (s, 3H, H-11), 1.61 (s, 3H, CH3 at C-10), 1.62
(s, 3H, CH3 at C-6), 1.94–2.08 (m, 5H, H-1, H-7, H-8), 2.93 (ddd, J = 15.1, 14.0, 7.3 Hz, 2H, H-2), 3.20 (d,
J = 7.7 Hz, 2H, H-4), 5.11 (t, J = 7.0 Hz, 1H, H-9), 5.30 (t, J = 7.5 Hz, 1H, H-5); 13C NMR (125.77 MHz,
D2O)
δ 15.3 (CH3 at C-6), 16.9 (CH3 at C-10), 24.8 (C-11), 25.7 (C-8), 28.7 (t, J = 3.7 Hz, C-2), 29.9 (C-4),
40.1 (t, J = 109.3 Hz, C-1), 38.7 (C-4), 119.8 (C-5), 124.2 (C-9), 133.6 (C-10), 140.2 (C-6); 31P NMR
(202.46 MHz, D2O)
found 425.0298.
δ
15.85 ppm. HRMS (ESI) calcd. for C12H22O6SP2Na3 [M + H]+ 425.0305;
S-((2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trien-1-yl) ethanethioate (49). To a mixture of farnesol 47
(1.00 g, 4.5 mmol), triphenylphosphine (1.77 g, 6.7 mmol), thioacetic acid (0.38 mL, 410.1 mg,
5.4 mmol) in anhydrous tetrahydrofuran (20 mL) cooled at 0 ◦C was added a solution of diisopropyl
azodicarboxylate (1.13 mL, 1.15 g, 6.7 mmol) as depicted for the preparation of 31. The product was
purified by column chromatography (silica gel) eluting with hexane–EtOAc (9:1) to give 840.0 mg
(84% yield) of 49 as a colorless oil: Rf 0.38 (hexane–EtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3)
δ 1.59
(s, 3H, C-12), 1.60 (s, 3H, CH3 at C-7), 1.68 (s, 6H, CH3 at C-3CH3 at C-11), 1.95–2.10 (m, 8H, H-4, H-5,
H-8, H-9), 2.32 (s, 3H, SC(O)CH3), 3.54 (t, J = 7.9 Hz, 2H, H-1), 5.08 (m, 2H, H-6, H-10), 5.21 (tq, J = 7.9,
1.1 Hz, 1H, H-2); 13C NMR (125.77 MHz, CDCl3)
δ 16.0 (CH3 at C-3), 16.2 (CH3 at C-7), 17.7 (CH3 at
C-11), 25.9 (C-12), 26.3 (C-9), 26.7 (C-5), 27.3 (C-1), 30.4 (SC(O)CH3), 39.5 (C-4), 39.7 (C-8), 118.4 (C-2),
123.7 (C-10), 124.3 (C-6), 131.3 (C-11), 135.3 (C-7), 140.2 (C-3), 196.1 (SC(O)CH3). HRMS (ESI) calcd. for
C17H28OS [M + Na]+ 303.1759; found 303.1757.
(2E,6E)-3,7,11-Trimethyldodeca-2,6,10-triene-1-thiol (5◦1). A solution of 49 (840.3 mg, 3.0 mmol) in
anhydrous tetrahydrofuran (10 mL) cooled at 0 C was treated with portionwise with lithium
aluminum hydride (170 mg, 4.5 mmol) as described for the preparation of 50 to obtain 421.8 mg
1
of 51 (59% yield) as a colorless oil: H NMR (500.13 MHz, CDCl3)
δ
1.42 (t, J = 7.1 Hz, 1H, SH), 1.62
(s, 6H, C-12, CH3 at C-3), 1.68 (s, 3H, CH3 at C-7), 1.71 (s, 3H, CH3 at C-11), 1.99–2.14 (m, 8H, H-4, H-5,
H-8, H-9), 3.18 (t, J = 7.2 Hz, 2H, H-1), 5.12 (m, 2H, H-6, H-10), 5.37 (tq, J = 7.8, 1.2 Hz, 1H, H-2); 13
NMR (125.77 MHz, CDCl3) 15.8 (CH3 at C-3), 16.0 (CH3 at C-7), 17.7 (CH3 at C-11), 22.1 (C-1), 25.7
C
δ

Molecules 2017, 22, 82
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(C-12), 26.3 (C-9), 26.7 (C-5), 39.4 (C-4), 39.7 (C-8), 123.3 (C-2), 123.7 (C-10), 124.3 (C-6), 131.3 (C-11),
135.3 (C-7), 137.5 (C-3). HRMS (ESI) calcd. for C15H27S [M + H]+ 239.1833; found 239.1820.
Tetraethyl 1-[((2E,6E)-3,7,11-trimethyldodeca-2,6,10-triene-1-thio)ethyl]-1,1-bisphosphonate (53). A solution
of 21 (303 mg, 1.0 mmol) in anhydrous methylene chloride (10 mL) was treated with triethylamine
(112 mg, 154
µL, 1.1 mmol) and 51 (241 mg, 1.0 mmol) as described for the preparation of 22.
The solvent was evaporated and the residue was purified by column chromatography
(silica gel) employing EtOAc as eluent to yield 412.3 mg (76% yield) of 53 as a colorless oil: Rf
0.50 (EtOAc–methanol, 9:1); 1H NMR (500.13 MHz, CDCl3)
δ 1.35 (t, J = 7.1 Hz, 12H, CH2CH3), 1.36
(t, J = 7.0 Hz, 12H, CH2CH03), 1.60 (s, 6H, C-15, CH3 at C-6), 1.68 (s, 6H, CH3 at C-10, CH3 at C-14),
1.96–2.10 (m, 8H, H-7, H-8, H-11, H-12), 2.61 (tt, J = 23.8, 6.1 Hz, 1H, H-1), 3.02 (dt, J = 18.2, 6.1 Hz, 2H,
H-2), 3.22 (d, J = 7.7 Hz, 2H, H-4), 4.20 (m, 8H, CH2CH3), 5.10 (m, 2H, H-9, H-13), 5.26 (tq, J = 7.6, 1.0
Hz, 1H, H-5); 13C NMR (125.77 MHz, CDCl3)
δ 16.2 (CH3 at C-6), 16.3 (CH3 at C-10), 16.4 (d, J = 6.1 Hz,
CH2CH3), 17.7 (CH3 at C-14), 25.7 (C-15), 27.3 (t, J = 5.0 Hz, C-2), 26.5 (C-12), 26.7 (C-8), 30.7 (C-4), 38.9
0
(t, J = 131.6 Hz, C-1), 39.6 (C-7), 39.7 (C-11), 62.7 (d, J = 6.7 Hz, CH2CH3), 62.8 (d, J = 6.6 Hz, C H2CH3),
119.8 (C-5), 123.8 (C-13), 124.3 (C-9), 131.3 (C-14), 135.3 (C-10), 139.5 (C-6); 31P NMR (202.46 MHz,
CDCl3) δ 21.47. HRMS (ESI) calcd. for C25H48O6SP2 [M + Na]+ 561.2545; found 561.2534.
1-[((2E,6E)-3,7,11-Trimethyldodeca-2,6,10-triene-1-thio)ethyl]-1,1-bisphosphonic Acid (45). A solution of
53 (375 mg, 0.7 mmol), 2,4,6-collidine (843 mg, 920 µL, 7.0 mmol) in anhydrous methylene chloride
(10 mL) was treated with bromotrimethylsilane (1.07 g, 0.9 mL, 7.0 mmol) under an argon atmosphere.
The reaction mixture was stirred at room temperature for 48 h. Then, methanol (1.0 mL) was added
and the reaction mixture was treated as depicted for the preparation of 44. The residue was purified
by reversed-phase column chromatography eluting with a mixture of water–acetonitrile (2:1) to give
1
170 mg (52% yield) of 45 as an amorphous solid: H NMR (500.13 MHz, D2O-CD3OD)
δ
1.61 (s, 6H,
C-15, CH3 at C-6), 1.69 (s, 3H, CH3 at C-10) 1.72 (s, 3H, CH3 at C-14), 1.96–2.17 (m, 9H, H-1, H-7, H-8,
H-11, H-12), 3.06 (dt, J = 14.6, 7.1 Hz, 2H, H-2), 3.31 (d, J = 7.9 Hz, 2H, H-4), 5.14 (m, 2H, H-9, H-13),
5.26 (tq, J = 7.8, 1.0 Hz, 1H, H-5); 13C NMR (125.77 MHz, D2O-CD3OD)
δ 16.1 (CH3 at C-6), 16.3 (CH3
at C-10), 17.9 (CH3 at C-14), 24.3 (C-15), 25.9 (C-12), 30.2 (t, J = 3.7 Hz, C-2), 26.9 (C-12), 27.0 (C-8), 31.4
(C-4), 41.4 (t, J = 109.4 Hz, C-1), 40.09 (C-7), 40.11 (C-11), 120.9 (C-5), 125.23 (C-13), 125.22 (C-9), 133.4
(C-14), 136.7 (C-10), 140.6 (C-6); 31P NMR (202.46 MHz, D2O-CD3OD)
for C17H29O6SP2Na4 [M + H]+ 515.0751; found 515.0737.
δ 18.16 ppm. HRMS (ESI) calcd.
Acetylation Reaction. General Procedure. Tetraethyl 2-(N-n-alkylamino)ethyl bisphosphonates 12
–14,
61 65 (15 mmol) were treated individually with acetic anhydride (3.0 mL) and pyridine (3.0 mL).
–
The mixture was stirred at room temperature overnight. The solvent was evaporated and each residue
was purified by column chromatography (silica gel) eluting with CH2Cl2–methanol (99:1) to produce
the acetylated products 67–74.
Tetraethyl 2-(N-n-propylacetamido)ethyl-1,1-bisphosphonate (67): 66% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ 0.90 (t, J = 7.4 Hz, 3H, H-6), 1.33 (t, J = 7.0 Hz, 6H, CH2CH3), 1.34
(t, J = 7.0 Hz, 6H, CH2CH0 ), 1.55 (p, J = 7.1 Hz, 2H, H-5), 2.06 (s, 3H, C(O)CH3), 3.38 (t, J = 7.7 Hz,
3
1H, H-4), 3.46 (tt, J = 22.6, 7.5 Hz, 1H, H-1), 3.74 (ddd, J = 14.7, 11.2, 7.4 Hz, 2H, H-2), 4.17 (m,
8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)
δ 11.2 (C-6), 16.30 (d, J = 4.1 Hz, CH2CH3), 16.36 (d,
J = 4.1 Hz, CH2CH3), 21.6 ((C(O)CH3), 22.2 (C-5), 34.1 (t, J = 130.1 Hz, C-1), 44.6 (t, J = 3.3 Hz, C-2),
52.7 (C-4), 62.4 (d, J = 6.8 Hz, CH2CH3), 63.0 (d, J = 6.7 Hz, CH2CH3), 171.1 (C(O)CH3); 31P NMR
(202.46 MHz, CDCl3) δ 21.48.
Tetraethyl 2-(N-n-butylacetamido)ethyl-1,1-bisphosphonate (68): 51% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ 0.95 (t, J = 7.3 Hz, 3H, H-7), 1.34 (t, J = 7.0 Hz, 6H, CH2CH3), 1.35
(t, J = 7.1 Hz, 6H, CH2CH0 ), 1.53 (p, J = 7.1 Hz, 2H, H-5), 2.07 (s, 3H, C(O)CH3), 3.41 (t, J = 7.8 Hz
,
3
1H, H-4), 3.44 (tt, J = 22.8, 7.4 Hz, 1H, H-1), 3.74 (ddd, J = 14.7, 11.1, 7.4 Hz, 2H, H-2), 4.19 (m,
8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)
13.8 (C-7), 16.27 (d, J = 4.1 Hz, CH2CH3), 16.33 (d,
J = 4.1 Hz, CH2CH3), 20.0 (C-6), 21.6 ((C(O)CH3), 31.1 (C-5), 34.1 (t, J = 130.1 Hz, C-1), 44.6 (t, J = 3.3 Hz
δ
,

Molecules 2017, 22, 82
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C-2), 50.6 (C-4), 62.4 (d, J = 6.8 Hz, CH2CH3), 62.9 (d, J = 6.8 Hz, CH2CH3), 171.0 (C(O)CH3); 31P NMR
(202.46 MHz, CDCl3) δ 21.47.
Tetraethyl 2-(N-n-penylacetamido)ethyl-1,1-bisphosphonate (69): 47% yield, colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ 0.91 (t, J = 7.2 Hz, 3H, H-8), 1.26 (m, 2H, CH2), 1.34 (t, J = 7.1 Hz, 6H,
CH2CH3), 1.35 (t, J = 7.1 Hz, 6H, CH2CH0 ), 1.53 (p, J = 7.7 Hz, 2H, H-5), 2.07 (s, 3H, C(O)CH3), 3.41
3
(t, J = 7.7 Hz, 1H, H-4), 3.44 (tt, J = 22.8, 7.4 Hz, 1H, H-1), 3.74 (ddd, J = 14.7, 11.1, 7.4 Hz, 2H, H-2),
4.19 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)
δ 13.9 (C-8), 16.3 (d, J = 4.2 Hz, CH2CH3), 16.4
(d, J = 4.2 Hz, CH2CH3), 21.6 ((C(O)CH3), 22.4 (C-7), 28.7 (C-6), 28.9 (C-5), 34.1 (t, J = 130.0 Hz, C-1),
44.6 (t, J = 3.4 Hz, C-2), 50.9 (C-4), 62.4 (d, J = 6.8 Hz, CH2CH3), 62.9 (d, J = 6.7 Hz, CH2CH3), 171.0
(C(O)CH3); 31P NMR (202.46 MHz, CDCl3) δ 21.47.
Tetraethyl 2-(N-n-hexylacetamido)ethyl-1,1-bisphosphonate (70): 51% yield, colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ
0.87 (t, J = 6.90 Hz, 3H, H-9), 1.29 (m, 6H, CH2), 1.34 (t, J = 7.0 Hz, 6H,
CH2CH3), 1.35 (t, J = 7.1 Hz, 6H, CH2CH ), 1.52 (m, 2H, H-5), 2.07 (s, 3H, C(O)CH3), 3.41 (t, J = 7.7 Hz
,
3
1H, H-4), 3.44 (tt, J = 22.8, 7.4 Hz, 1H, H-1), 3.74 (ddd, J = 14.7, 11.1, 7.4 Hz, 2H, H-2), 4.18 (m, 8H,
CH2CH3); 13C NMR (125.77 MHz, CDCl3)
δ
14.0 (C-9), 16.2 (d, J = 4.3 Hz, CH2CH3), 16.4 (d, J = 4.1 Hz,
CH2CH3), 21.6 ((C(O)CH3), 22.6 (C-8), 26.5 (C-6), 29.0 (C-5), 31.5 (C-7), 34.1 (t, J = 130.0 Hz, C-1), 44.6
(t, J = 3.2 Hz, C-2), 50.9 (C-4), 62.4 (d, J = 6.8 Hz, CH2CH3), 62.9 (d, J = 6.7 Hz, CH2CH3), 171.0
(C(O)CH3); 31P NMR (202.46 MHz, CDCl3) δ 21.48.
Tetraethyl 2-(N-n-heptylacetamido)ethyl-1,1-bisphosphonate (71): 66% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ 0.89 (t, J = 7.1 Hz, 3H, H-10), 1.29 (m, 8H, CH2), 1.34 (t, J = 7.1 Hz, 6H,
CH2CH3), 1.35 (t, J = 7.1 Hz, 6H, CH2CH0 ), 1.52 (p, J = 7.5 Hz, 2H, H-5), 2.07 (s, 3H, C(O)CH3), 3.41
3
(t, J = 7.7 Hz, 1H, H-4), 3.44 (tt, J = 23.1, 7.4 Hz, 1H, H-1), 3.74 (ddd, J = 14.7, 11.1, 7.4 Hz, 2H, H-2),
4.18 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)
δ 14.0 (C-10), 16.3 (d, J = 4.4 Hz, CH2CH3), 16.4
(d, J = 4.1 Hz, CH2CH3), 21.6 ((C(O)CH3), 22.6 (C-9), 26.8 (C-6), 29.03 (C-5), 29.04 (C-7), 31.5 (C-8), 34.1
(t, J = 130.0 Hz, C-1), 44.6 (C-2), 50.9 (C-4), 62.4 (d, J = 6.6 Hz, CH2CH3), 62.9 (d, J = 6.8 Hz, CH2CH3),
171.0 (C(O)CH3); 31P NMR (202.46 MHz, CDCl3) δ 21.49.
Tetraethyl 2-(N-n-octylacetamido)ethyl-1,1-bisphosphonate (72): 50% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ 0.89 (t, J = 7.0 Hz, 3H, H-11), 1.29 (m, 10H, CH2), 1.34 (t, J = 7.1 Hz, 6H,
CH2CH3), 1.35 (t, J = 7.1 Hz, 6H, CH2CH0 ), 1.52 (p, J = 7.4 Hz, 2H, H-5), 2.07 (s, 3H, C(O)CH3), 3.41
3
(t, J = 7.8 Hz, 1H, H-4), 3.44 (tt, J = 22.9, 7.4 Hz, 1H, H-1), 3.74 (ddd, J = 14.7, 11.1, 7.4 Hz, 2H, H-2),
4.19 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)
δ 14.0 (C-11), 16.2 (d, J = 3.9 Hz, CH2CH3), 16.3
(d, J = 3.9 Hz, CH2CH3), 21.5 ((C(O)CH3), 22.5 (C-10), 26.7 (C-6), 28.9 (C-8), 29.1 (C-5), 29.2 (C-7), 31.6
(C-9), 34.0 (t, J = 130.0 Hz, C-1), 44.5 (t, J = 3.2 Hz, C-2), 50.8 (C-4), 62.3 (d, J = 6.8 Hz, CH2CH3), 62.8
(d, J = 6.6 Hz, CH2CH3), 170.9 (C(O)CH3); 31P NMR (202.46 MHz, CDCl3) δ 21.44.
Tetraethyl 2-(N-n-nonylacetamido)ethyl-1,1-bisphosphonate (73): 57% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ 0.84 (t, J = 6.9 Hz, 3H, H-12), 1.23 (m, 12H, CH2), 1.30 (t, J = 7.1 Hz, 6H,
CH2CH3), 1.31 (t, J = 7.1 Hz, 6H, CH2CH0 ), 1.49 (p, J = 7.1 Hz, 2H, H-5), 2.03 (s, 3H, C(O)CH3), 3.37
3
(t, J = 7.8 Hz, 1H, H-4), 3.40 (tt, J = 23.0, 7.4 Hz, 1H, H-1), 3.70 (ddd, J = 14.8, 11.2, 7.4 Hz, 2H, H-2),
4.15 (m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)
δ 14.0 (C-12), 16.2 (d, J = 4.0 Hz, CH2CH3), 16.3
(d, J = 3.9 Hz, CH2CH3), 21.5 ((C(O)CH3), 22.5 (C-11), 26.7 (C-6), 29.0 (C-8), 29.1 (C-5), 29.3 (C-7), 29.4
(C-9), 31.8 (C-10), 34.0 (t, J = 130.1 Hz, C-1), 44.5 (t, J = 3.3 Hz, C-2), 50.9 (C-4), 62.3 (d, J = 6.9 Hz,
CH2CH3), 62.9 (d, J = 6.7 Hz, CH2CH3), 170.9 (C(O)CH3); 31P NMR (202.46 MHz, CDCl3) δ 21.45.
Tetraethyl 2-(N-n-decylacetamido)ethyl-1,1-bisphosphonate (74): 73% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ 0.85 (t, J = 6.9 Hz, 3H, H-13), 1.23 (m, 14H, CH2), 1.31 (t, J = 7.0 Hz, 6H,
CH2CH3), 1.32 (t, J = 7.1 Hz, 6H, CH2CH0 ), 1.49 (p, J = 7.1 Hz, 2H, H-5), 2.04 (s, 3H, C(O)CH3), 3.38
3
(t, J = 7.5 Hz, 1H, H-4), 3.44 (tt, J = 23.2, 7.6 Hz, 1H, H-1), 3.71 (ddd, J = 14.6, 11.2, 7.5 Hz, 2H, H-2), 4.15
(m, 8H, CH2CH3); 13C NMR (125.77 MHz, CDCl3)
δ
14.0 (C-13), 16.27 (d, J = 6.8 Hz, CH2CH3), 16.30 (d,
J = 6.6 Hz, CH2CH3), 21.6 ((C(O)CH3), 22.6 (C-12), 26.8 (C-6), 29.0 (C-8), 29.2 (C-5), 29.3 (C-7), 29.4 (C-9),

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29.5 (C-10), 31.8 (C-11), 34.0 (t, J = 130.0 Hz, C-1), 44.5 (t, J = 3.3 Hz, C-2), 50.9 (C-4), 62.4 (d, J = 6.9 Hz
,
CH2CH3), 62.9 (d, J = 6.7 Hz, CH2CH3), 171.0 (C(O)CH3); 31P NMR (202.46 MHz, CDCl3) δ 21.46.
Fluorination Reaction. General Procedure. Sodium hydride (10.5 mmol, 60% in mineral oil) was
washed with anhydrous hexane under an argon atmosphere, and then anhydrous tetrahydrofuran
(10 mL) was added. The mixture, cooled at 0 ◦C, was treated with the corresponding
tetraethyl 2-(N-n-alkylacetamido)ethyl-1,1-bisphosphonate such as 67–74 (10.0 mmol) in anhydrous
tetrahydrofuran (10 mL). The solution was stirred at 0 ◦C for 15 min, then, the mixture was allow◦ed
to reach room temperature and stirred for additional 60 min. The mixture was cooled at 0 C
and Selectfluor (12.5 mmol) was added followed by addition of N,N-dimethylformamide (5.0 mL).
The reaction mixture was allowed to reach room temperature and stirred for 4 h. The mixture
was partitioned between methylene chloride (30 mL) and brine (30 mL). The aqueous phase was
extracted with methylene chloride (2
×
30 mL). The combined organic layers were washed with brine
(5 30 mL), water (2 25 mL), dried (MgSO4), and the solvent was evaporated. The residue was
×
×
purified by column chromatography eluting with a mixture of EtOAc–methanol (49:1) to produce the
corresponding 1-fluoro tetraethyl esters 75–82.
Tetraethyl 2-(N-n-propylacetamido)ethyl-1-fluoro-1,1-bisphosphonate (75): 50% yield; 1H NMR (500.13 MHz,
CDCl3) δ
0.88 (t, J = 7.3 Hz, 3H, H-6), 0.91 (t, J = 7.4 Hz, 3H, H-60), 1.38 (t, J = 7.0 Hz, 12H, CH2CH3),
1.39 (dt, J = 6.9, 3.3 Hz, 12H, CH2CH0 ), 1.53 (sext, J = 7.6 Hz, 2H, H-5), 1.60 (sext, J = 7.5 Hz, 2H,
3
H-50), 2.14 (s, 3H, C(O)CH3), 2.21 (s, 3H, C(O)CH3), 3.45 (t, J = 7.8 Hz, 2H, H-4), 3.47 (t, J = 7.9 Hz, 2H,
H-40), 4.10 (dd, J = 10.1, 8.8 Hz, 2H, H-2), 4.15 (dd, J = 10.2, 8.8 Hz, 2H, H-20), 4.27 (m, 8H, CH2CH3),
two rotamers; 13C NMR (125.77 MHz, CDCl3)
δ
11.1 (C-6), 11.3 (C-60), 16.3 (t, J = 3.3 Hz, CH2CH3),
16.4 (t, J = 2.3 Hz, CH2CH03), 19.8 (C-5), 21.2 (C-50), 21.4 (C(O)CH3), 21.9 (d, J = 3.5 Hz, C(O)CH03),
44.9 (d, J = 17.9 Hz, C-2), 48.4 (d, J = 3.5 Hz, C-4), 48.9 (d, J = 18.3 Hz, C-20), 50.9 (d, J = 4.2 Hz, C-20),
64.2 (m, CH2CH3), 64.7 (t, J = 3.4 Hz, CH2CH3), 171.3 (C(O)CH3), 171.6 (C(O)0CH3), two rotamers;
31P NMR (202.46 MHz, CDCl3) 11.70 (d, J = 70.2 Hz), 11.90 (d, J = 77.4 Hz), two rotamers; 19F NMR
δ
(470.59 MHz, CDCl3) δ −192.55 (t, J = 77.4 Hz), −192.80 (t, J = 71.3 Hz), two rotamers.
Tetraethyl 2-(N-n-butylacetamido)ethyl-1-fluoro-1,1-bisphosphonate (76): 65% yield; colorless oil; 1H NMR
0
(500.13 MHz, CDCl3)
δ
0.91 (t, J = 7.3 Hz, 3H, H-7), 0.94 (t, J = 7.4 Hz, 3H, H-7 ), 1.30 (m, 2H, H-6), 1.37
0
(t, J = 6.8 Hz, 12H, CH2CH3), 1.38 (dt, J = 6.8, 3.3 Hz, 12H, CH2CH ), 1.49 (p, J = 7.7 Hz, 2H, H-5), 1.54
3
0
(p, J = 7.8 Hz, 2H, H-5 ), 2.12 (s, 3H, C(O)CH3), 2.20 (s, 3H, C(O)CH3), 3.46 (t, J = 7.9 Hz, 2H, H-4), 3.49
(t, J = 7.9 Hz, 2H, H-40), 4.10 (dd, J = 10.5, 8.5 Hz, 2H, H-2), 4.14 (dd, J = 10.3, 8.9 Hz, 2H, H-20), 4.27
(m, 8H, CH2CH3), two rotamers; 13C NMR (125.77 MHz, CDCl3)
δ
13.75 (C-7), 13.84 (C-70), 16.39
(t, J = 3.2 Hz, CH2CH3), 16.44 (t, J = 2.6 Hz, CH2CH03), 20.0 (C-6), 20.1 (C-60), 21.5 (C(O)CH3), 22.0
(d, J = 3.6 Hz, C(O)CH03), 28.7 (C-5), 30.1 (C-50), 44.9 (d, J = 18.1 Hz, C-2), 46.5 (d, J = 3.3 Hz, C-4), 49.1
(d, J = 4.2 Hz, C-40), 49.9 (d, J = 18.1 Hz, C-20), 64.2 (m, CH2CH3), 64.7 (m, CH2CH3), 171.2 (C(O)CH3),
171.6 (C(O)0CH3), two rotamers; 31P NMR (202.46 MHz, CDCl3)
δ 11.70 (d, J = 70.2 Hz), 11.90
(d, J = 77.2 Hz), two rotamers; 19F NMR (470.59 MHz, CDCl3) δ −192.46 (t, J = 77.5 Hz),
−
192.81
(t, J = 71.4 Hz), two rotamers. HRMS (ESI) calcd. for (C16H34O7NFP2Na) [M + Na]+ 456.1692;
found 456.1700.
Tetraethyl 2-(N-n-pentylacetamido)ethyl-1-fluoro-1,1-bisphosphonate (77): 75% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ
0.89 (t, J = 7.5 Hz, 3H, H-8), 0.90 (t, J = 7.5 Hz, 3H, H-80), 1.24 (m, 2H, CH2),
0
1.37 (t, J = 6.7 Hz, 12H, CH2CH3), 1.38 (dt, J = 6.5, 2.6 Hz, 12H, CH2CH ), 1.51 (p, J = 7.6 Hz, 2H, H-5),
3
1.55 (p, J = 7.7 Hz, 2H, H-50), 2.12 (s, 3H, C(O)CH3), 2.20 (s, 3H, C(O)CH3), 3.47 (t, J = 7.9 Hz, 2H, H-4),
4.10 (t, J = 9.6 Hz, 2H, H-2), 4.15 (t, J = 9.1 Hz, 2H, H-20), 4.27 (m, 8H, CH2CH3), two rotamers; 13
C
NMR (125.77 MHz, CDCl3)
δ
13.86 (C-8), 13.90 (C-80), 16.36 (m, CH2CH3), 21.4 (C(O)CH3), 21.88 (d.
J = 3.5 Hz, C(O)CH03), 22.3 (C-7), 22.4 (C-70), 26.1 (C-6), 27.6 (C-60), 28.8 (C-5), 29.0 (C-50), 44.8 (d,
J = 18.5 Hz, C-2), 46.7 (d, J = 3.3 Hz, C-4), 49.2 (d, J = 4.2 Hz, C-40), 49.8 (d, J = 18.2 Hz, C-20), 64.1
(m, CH2CH3), 64.6 (m, CH2CH3), 95.8 (dt, J = 153.2, 109.3 Hz, 1H, H-1), 97.3 (dt, J = 153.0, 103.1 Hz,
1H, H-10), 171.1 (C(O)CH3), 171.4 (C(O)0CH3), two rotamers; 31P NMR (202.46 MHz, CDCl3)
δ 11.66

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(d, J = 71.8 Hz), 11.87 (d, J = 78.0 Hz), two rotamers; 19F NMR (470.59 MHz, CDCl3) δ −192.56
(t, J = 77.4 Hz), −192.82 (t, J = 71.2 Hz); two rotamers.
Tetraethyl 2-(N-n-hexylacetamido)ethyl-1-fluoro-1,1-bisphosphonate (78): 36% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ
0.87 (t, J = 6.9 Hz, 3H, H-9), 0.89 (t, J = 6.9 Hz, 3H, H-90), 1.29 (m, 6H, CH2),
0
1.37 (t, J = 6.9 Hz, 12H, CH2CH3), 1.38 (dt, J = 6.9, 2.7 Hz, 12H, CH2CH ), 1.49 (p, J = 7.5 Hz, 2H, H-5),
3
1.54 (p, J = 7.8 Hz, 2H, H-50), 2.12 (s, 3H, C(O)CH3), 2.19 (s, 3H, C(O)CH3), 3.46 (t, J = 7.9 Hz, 2H, H-4),
4.09 (t, J = 9.4 Hz, 2H, H-2), 4.09 (t, J = 9.4 Hz, 2H, H-20), 4.26 (m, 8H, CH2CH3), two rotamers;
13C NMR (125.77 MHz, CDCl3)
δ
13.95 (C-9), 14.00 (C-90), 16.39 (t, J = 3.2 Hz, CH2CH3), 16.44
(t, J = 2.6 Hz, CH2CH03), 21.5 (C(O)CH3), 22.0 (d, J = 3.5 Hz, C(O)CH03), 22.5 (C-8), 22.6 (C-80), 26.56
(C-6), 26.58 (C-60), 28.0 (C-5), 31.5 (C-7), 31.6 (C-70), 44.9 (d, J = 18.2 Hz, C-2), 46.8 (d, J = 3.2 Hz
C-4), 49.3 (d, J = 4.3 Hz, C-40), 49.9 (d, J = 18.4 Hz, C-20), 62.4 (d, J = 6.8 Hz, CH2CH3), 62.9
(d, J = 6.7 Hz, CH2CH3), 171.1 (C(O)CH3), 171.5 (C(O)0CH3), two rotamers; 31P NMR (202.46 MHz,
,
CDCl3)
δ
11.71 (d, J = 69.7 Hz), 11.91 (d, J = 77.1 Hz), two rotamers; 19F NMR (470.59 MHz, CDCl3)
δ −192.50 (t, J = 77.6 Hz),
−
192.78 (t, J = 71.2 Hz); two rotamers. HRMS (ESI) calcd. for (C18H39O7NFP2)
[M + H]+ 462.2188; found 462.2197.
Tetraethyl 2-(N-n-heptylacetamido)ethyl-1-fluoro-1,1-bisphosphonate (79): 53% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3) δ
0.87 (t, J = 6.4 Hz, 3H, H-10), 0.88 (t, J = 6.9 Hz, 3H, H-100), 1.27 (m, 8H, CH2),
1.36 (t, J = 7.0 Hz, 12H, CH2CH3), 1.38 (dt, J = 6.8, 3.4 Hz, 12H, CH2CH0 ), 1.49 (p, J = 7.6 Hz, 2H,
3
H-5), 1.54 (p, J = 7.8 Hz, 2H, H-50), 2.11 (s, 3H, C(O)CH3), 2.19 (s, 3H, C(O)CH3), 3.46 (t, J = 7.9 Hz,
2H, H-4), 4.09 (dd, J = 10.3, 8.6 Hz, 2H, H-2), 4.13 (t, J = 10.2, 8.7 Hz, 2H0, H-20), 4.27 (m, 8H, CH2CH3),
two rotamers; 13C NMR (125.77 MHz, CDCl3)
δ
13.99 (C-10), 14.02 (C-10 ), 16.3 (t, J = 3.2 Hz, CH2CH3),
16.4 (t, J = 2.4 Hz, CH2CH03), 21.5 (C(O)CH3), 21.9 (d, J = 3.5 Hz, C(O)CH03), 22.50 (C-9), 22.53 (C-90),
26.6 (C-6), 26.7 (C-60), 26.9 (C-7), 28.0 (C-5), 31.69 (C-8), 31.74 (C-80), 44.9 (d, J = 17.9 Hz, C-2), 46.8 (d,
J = 3.2 Hz, C-4), 49.3 (d, J = 4.3 Hz, C-40), 49.9 (d, J = 18.3 Hz, C-20), 64.2 (m, CH2CH3), 95.9 (dt,
0
0
J = 153.1, 102.8 Hz, 1H, H-1), 97.4 (dt, J = 153.1, 102.8 Hz, 1H, H-1 ), 171.2 (C(O)CH3), 171.5 (C(O) CH3),
two rotamers; 31P NMR (202.46 MHz, CDCl3)
δ 11.69 (d, J = 72.1 Hz), 11.89 (d, J = 78.4 Hz), two rotamers;
19F NMR (470.59 MHz, CDCl3) δ −192.52 (t, J = 77.5 Hz), −192.79 (t, J = 71.3 Hz); two rotamers.
Tetraethyl 2-(N-n-octylacetamido)ethyl-1-fluoro-1,1-bisphosphonate (80): 52% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ
0.87 (t, J = 7.1 Hz, 3H, H-11), 0.88 (t, J = 6.9 Hz, 3H, H-110), 1.27 (m, 10H, CH2),
1.37 (t, J = 7.0 Hz, 12H, CH2CH3), 1.38 (dt, J = 6.8, 3.4 Hz, 12H, CH2CH0 ), 1.50 (p, J = 7.6 Hz, 2H,
3
H-5), 1.54 (p, J = 7.9 Hz, 2H, H-50), 2.12 (s, 3H, C(O)CH3), 2.19 (s, 3H, C(O)CH3), 3.46 (t, J = 8.2 Hz,
2H, H-4), 4.09 (dd, J = 12.0, 7.2 Hz, 2H, H-2), 4.15 (t, J = 10.2, 8.8 Hz, 2H, H-20), 4.27 (m, 8H, CH2CH3),
two rotamers; 13C NMR (125.77 MHz, CDCl3)
δ
14.01 (C-11), 14.02 (C-110), 16.4 (m, CH2CH3), 21.5
(C(O)CH3), 21.9 (d, J = 3.5 Hz, C(O)CH03), 22.55 (C-10), 22.58 (C-100), 26.6 (C-6), 26.8 (C-60), 26.9 (C-7),
28.0 (C-5), 29.1 (C-8), 29.3 (C-80), 31.74 (C-9), 31.79 (C-90), 44.9 (d, J = 18.0 Hz, C-2), 46.8 (d, J = 3.3 Hz
,
C-4), 49.3 (d, J = 4.2 Hz, C-40), 49.9 (d, J = 18.4 Hz, C-20), 64.2 (m, CH2CH3), 95.9 (dt, J = 153.1,
109.2 Hz, 1H, H-1), 97.4 (dt, J = 153.1, 103.3 Hz, 1H, H-10), 171.2 (C(O)CH3), 171.5 (C(O)0CH3),
two rotamers; 31P NMR (202.46 MHz, CDCl3)
δ 11.69 (d, J = 72.1 Hz), 11.89 (d, J = 79.6 Hz), two rotamers;
19F NMR (470.59 MHz, CDCl3) δ −192.51 (t, J = 77.5 Hz), −192.78 (t, J = 71.3 Hz); two rotamers.
Tetraethyl 2-(N-n-nonylacetamido)ethyl-1-fluoro-1,1-bisphosphonate (81): 59% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ
0.875 (t, J = 7.0 Hz, 3H, H-11), 0.882 (t, J = 6.9 Hz, 3H, H-110), 1.26 (m, 12H,
0
CH2), 1.37 (t, J = 7.0 Hz, 12H, CH2CH3), 1.38 (dt, J = 6.9, 3.3 Hz, 12H, CH2CH ), 1.50 (p, J = 7.6 Hz, 2H,
3
0
H-5), 1.54 (p, J = 7.7 Hz, 2H, H-5 ), 2.12 (s, 3H, C(O)CH3), 2.19 (s, 3H, C(O)CH3), 3.46 (t, J = 7.9 Hz, 2H,
0
0
H-4), 3.48 (t, J = 8.5 Hz, 2H, H-4 ), 4.09 (dd, J = 10.0, 8.9 Hz, 2H, H-2), 4.15 (t, J = 10.2, 8.8 Hz, 2H, H-2 ),
4.27 (m, 8H, CH2CH3), two rotamers; 13C NMR (125.77 MHz, CDCl3)
δ
14.0 (C-12), 014.1 (C-120), 16.4
0
(m, CH2CH3), 21.5 (C(O)CH3), 21.9 (d, J = 3.6 Hz, C(O)CH 3), 22.59 (C-11), 22.61 (C-11 ), 26.6 (C-6), 26.8
(C-60), 26.9 (C-7), 28.0 (C-5), 29.16 (C-8), 29.21 (C-80), 29.28 (C-70) 29.39 (C-50), 29.46 (C-9), 29.50 (C-90),
31.78 (C-10), 31.81 (C-100), 44.9 (d, J = 17.9 Hz, C-2), 46.8 (d, J = 3.4 Hz, C-4), 49.3 (d, J = 4.3 Hz, C-40),
49.9 (d, J = 18.2 Hz, C-20), 64.2 (m, CH2CH3), 64.7 (t, J = 3.2 Hz, CH2CH3), 95.9 (dt, J = 153.0, 109.0 Hz,
1H, H-1), 97.4 (dt, J = 153.0, 103.0 Hz, 1H, H-10), 171.2 (C(O)CH3), 171.5 (C(O)0CH3), two rotamers;

Molecules 2017, 22, 82
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31P NMR (202.46 MHz, CDCl3)
δ
11.70 (d, J = 77.9 Hz), 11.91 (d, J = 77.9 Hz), two rotamers; 19F NMR
(470.59 MHz, CDCl3) δ −192.50 (t, J = 77.4 Hz), −192.77 (t, J = 71.3 Hz); two rotamers.
Tetraethyl 2-(N-n-decylacetamido)ethyl-1-fluoro-1,1-bisphosphonate (82): 54% yield; colorless oil; 1H NMR
(500.13 MHz, CDCl3)
δ
0.877 (t, J = 7.0 Hz, 3H, H-13), 0.881 (t, J = 6.8 Hz, 3H, H-130), 1.26 (m, 14H,
0
CH2), 1.37 (t, J = 7.0 Hz, 12H, CH2CH3), 1.38 (dt, J = 6.9, 3.1 Hz, 12H, CH2CH ), 1.50 (p, J = 7.3 Hz, 2H,
H-5), 1.54 (p, J = 7.6 Hz, 2H, H-5 ), 2.12 (s, 3H, C(O)CH3), 2.19 (s, 3H, C(O)CH3), 3.46 (t, J = 7.9 Hz, 2H,
H-4), 3.48 (t, J = 8.2 Hz, 2H, H-4 ), 4.09 (dd, J = 10.4, 8.6 Hz, 2H, H-2), 4.15 (t, J = 10.1, 8.8 Hz, 2H, H-2 ),
4.27 (m, 8H, CH2CH3), two rotamers; 13C NMR (125.77 MHz, CDCl3)
14.0 (C-13), 16.4 (m, CH2CH3),
21.5 (C(O)CH3), 21.9 (d, J = 3.9 Hz, C(O)CH03), 22.61 (C-12), 22.61 (C-120), 26.6 (C-6), 26.8 (C-60), 26.9
3
0
0
0
δ
0
0
0
0
(C-7), 28.0 (C-5), 29.23 (C-8), 29.25 (C-8 ), 29.27 (C-7 ) 29.39 (C-5 ), 29.45 (C-9), 29.50 (C-10), 29.54 (C-10 ),
31.81 (C-11), 31.83 (C-110), 44.9 (d, J = 18.6 Hz, C-2), 46.8 (d, J = 3.4 Hz, C-4), 49.3 (d, J = 4.5 Hz, C-40),
49.9 (d, J = 18.4 Hz, C-20), 64.2 (m, CH2CH3), 64.7 (t, J = 3.2 Hz, CH2CH3), 95.9 (dt, J = 153.0, 109.0 Hz,
1H, H-1), 97.4 (dt, J = 153.0, 103.1 Hz, 1H, H-10), 171.2 (C(O)CH3), 171.5 (C(O)0CH3), two rotamers;
31P NMR (202.46 MHz, CDCl3)
δ
11.70 (d, J = 71.2 Hz), 11.90 (d, J = 77.7 Hz), two rotamers; 19F NMR
(470.59 MHz, CDCl3) δ −192.50 (t, J = 77.5 Hz), −192.78 (t, J = 71.2 Hz); two rotamers.
Hydrolysys. General Procedure. Esters 75 82 were treated, in independent experiments, with a
–
concentrated aqueous solution of hydrochloric acid (2.0 mL). The resulting mixtures were refluxed for
24 h. The solvent was evaporated and the residues were crystallized from water–ethanol.
◦
1-[(n-Prop-1-ylamino)ethyl]-1-fluoro-1,1-bisphosphonic Acid (54): 54% yield; white solid; m.p. 161 C;
1H NMR (500.13 MHz, D2O)
δ
0.91 (t, J = 7.5 Hz, 3H, H-6), 1.65 (sext, J = 7.4 Hz, 2H, H-5), 3.04
(t, J = 7.3 Hz, 2H, H-4), 3.60 (m, 2H, H-2); 13C NMR (125.77 MHz, D2O)
10.4 (C-6), 19.5 (C-5), 49.6
8.89 (d, J = 65.4 Hz); 19F NMR (470.59
δ
(d, J = 16.1, C-2), 50.2 (C-4); 31P NMR (202.46 MHz, D2O)
δ
MHz, D2O) δ −193.32 (t, J = 65.5 Hz). HRMS (ESI) calcd. for (C5H15O6NFP2) [M + H]+ 266.0359;
found 266.0359.
1-[(n-But-1-ylamino)ethyl]-1-fluoro-1,1-bisphosphonic Acid (55): 70% yield; white solid; m.p. 153–154 ◦C;
1H NMR (500.13 MHz, D2O)
δ
0.85 (t, J = 7.4 Hz, 3H, H-7), 1.32 (sext, J = 7.5 Hz, 2H, H-6), 1.61
(p, J = 7.5 Hz, 2H, H-5), 3.07 (t, J = 7.4 Hz, 2H, H-4), 3.59 (m, 2H, H-2); 13C NMR (125.77 MHz, D2O)
δ
12.7 (C-7), 19.0 (C-6), 27.5 (C-5), 48.1 (C-4), 49.1 (dt, J = 18.1, 2.3 Hz, C-2); 31P NMR (202.46 MHz, D2O)
δ
8.84 (d, J = 65.5 Hz); 19F NMR (470.59 MHz, D2O) δ −193.39 (t, J = 65.6 Hz). HRMS (ESI) calcd. for
(C6H17O6NFP2) [M + H]+ 280.0515; found 280.0514.
1-[(n-Pent-1-ylamino)ethyl]-1-fluoro-1,1-bisphosphonic Acid (56): 65% yield; white solid; m.p. 164–165 ◦C;
1H NMR (500.13 MHz, D2O)
δ
0.81 (t, J = 7.1 Hz, 3H, H-8), 1.28 (m, 4H, CH2), 1.63 (p, J = 7.3 Hz, 2H,
H-5), 3.06 (t, J = 7.4 Hz, 2H, H-4), 3.59 (p, J = 10.1 Hz, 2H, H-2); 13C NMR (125.77 MHz, D2O)
δ
13.0
(C-8), 21.4 (C-7), 25.1 (C-5), 27.7 (C-7), 48.3 (C-4), 49.1 (d, J = 18.3 Hz, C-2); 31P NMR (202.46 MHz, D2O)
δ
8.93 (d, J = 47.3 Hz); 19F NMR (470.59 MHz, D2O) δ −193.32 (t, J = 65.5 Hz). HRMS (ESI) calcd. for
(C7H19O6NFP2) [M + H]+ 294.0672; found 294.0667.
1-[(n-Hex-1-ylamino)ethyl]-1-fluoro-1,1-bisphosphonic Acid (57): 34% yield; white solid; 1H NMR
(500.13 MHz, D2O)
(p, J = 7.5 Hz, 2H, H-5), 3.05 (t, J = 7.5 Hz, 2H, H-4), 3.58 (p, J = 10.6 Hz, 2H, H-2); 13C NMR
(125.77 MHz, D2O) 13.1 (C-9), 21.6 (C-8), 25.2 (C-6), 25.4 (C-5), 30.3 (C-7), 49.1 (dt, J = 18.1, 2.3 Hz,
δ 0.77 (t, J = 7.1 Hz, 3H, H-9), 1.22 (m, 4H, CH2), 1.28 (m, 2H, CH2), 1.61
δ
C-2), 92.2 (dt, J = 184.1, 139.4 Hz, C-1); 31P NMR (202.46 MHz, D2O) 8.91 (d, J = 59.7 Hz); 19F NMR
δ
(470.59 MHz, D2O) δ −193.54 (t, J = 65.9 Hz). HRMS (ESI) calcd. for (C8H21O6NFP2) [M + H]+ 308.0823;
found 308.0822.
1-[(n-Hept-1-ylamino)ethyl]-1-fluoro-1,1-bisphosphonic Acid (58): 56% yield; white solid; m.p. 135–136 ◦C;
1H NMR (500.13 MHz, D2O)
δ
0.76 (t, J = 7.1 Hz, 3H, H-10), 1.20 (m, 4H, CH2), 1.27 (m, 4H, CH2), 1.61
(p, J = 7.4 Hz, 2H, H-5), 3.06 (t, J = 7.7 Hz, 2H, H-4), 3.59 (p, J = 10.7 Hz, 2H, H-2); 13C NMR
(125.77 MHz, D2O) 13.2 (C-10), 21.8 (C-9), 25.4 (C-6), 25.5 (C-7), 27.8 (C-5), 30.7 (C-8), 48.4 (C-4),
49.0 (dt, J = 17.9, 2.3 Hz, C-2), 92.1 (dt, J = 184.2 Hz, 139.6 Hz, C-1); 31P NMR (202.46 MHz, CDCl3)
δ

Molecules 2017, 22, 82
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δ
8.90 (d, J = 60.8 Hz); 19F NMR (470.59 MHz, D2O)
(C9H23O6NFP2) [M + H]+ 322.0985; found 322.0969.
−
193.65 (t, J = 66.0 Hz). HRMS (ESI) calcd. for
1-[(n-Oct-1-ylamino)ethyl]-1-fluoro-1,1-bisphosphonic Acid (59): 31% yield; white solid; m.p. >270 ◦C; 1H
NMR (500.13 MHz, D2O) 0.74 (t, J = 6.9 Hz, 3H, H-11), 1.19 (m, 10H, CH2), 1.59 (p, J = 7.4 Hz, 2H,
H-5), 3.02 (t, J = 7.6 Hz, 2H, H-4), 3.57 (p, J = 10.2 Hz, 2H, H-2); 13C NMR (125.77 MHz, D2O)
13.4
δ
δ
(C-11), 22.0 (C-10), 25.4 (C-6), 25.6 (C-7), 28.1 (C-8), 28.2 (C-5), 31.0 (C-9), 48.2 (C-4), 48.9 (d, J = 18.1 Hz,
C-2), 92.1 (dt, J = 184.6 Hz, 139.8 Hz, C-1); 31P NMR (202.46 MHz, CDCl3) δ 8.87 ppm (d, J = 61.0 Hz);
19F NMR (470.59 MHz, D2O) δ −193.71 (t, J = 66.1 Hz).
◦
1
1-[(n-Non-1-ylamino)ethyl]-1-fluoro-1,1-bisphosphonic Acid (60). White solid; m.p. 147 C; H NMR
(500.13 MHz, D2O) 0.77 (t, J = 7.1 Hz, 3H, H-12), 1.19 (m, 12H, CH2), 1.62 (p, J = 6.67 Hz, 2H, H-5),
3.06 (t, J = 7.1 Hz, 2H, H-4), 3.59 (m, 2H, H-2); 13C NMR (125.77 MHz, D2O)
13.4 (C-12), 22.0 (C-11),
25.4 (C-6), 25.5 (C-7), 28.1 (C-9), 28.3 (C-8), 28.4 (C-5), 31.1 (C-10), 48.4 (C-4), 49.1 (d, J = 18.1 Hz, C-2),
δ
δ
92.3 (dt, J = 184.9 Hz, 139.4 Hz, C-1); 31P NMR (202.46 MHz, CDCl3) 8.83 ppm (d, J = 65.7 Hz); 19
δ F
NMR (470.59 MHz, D2O) δ −193.34 (t, J = 65.6 Hz). HRMS (ESI) calcd. for (C11H27O6NFP2) [M + H]+
350.1298; found 350.1306.
◦
1-[(n-Dec-1-ylamino)ethyl]-1-fluoro-1,1-bisphosphonic Acid (61): 54% yield; White solid; m.p. 151 C;
1H NMR (500.13 MHz, D2O)
δ
0.78 (t, J = 6.7 Hz, 3H, H-13), 1.20 (m, 10H, CH2), 1.27 (m, 4H, CH2), 1.64
(p, J = 7.3 Hz, 2H, H-5), 3.06 (t, J = 7.0 Hz, 2H, H-4), 3.62 (m, 2H, H-2); 13C NMR (125.77 MHz, D2O)
13.4 (C-13), 22.0 (C-12), 25.4 (C-6), 25.5 (C-7), 28.1 (C-10), 28.4 (C-8,C-9), 28.6 (C-5), 31.1 (C-11), 48.4
δ
(C-4), 49.1 (dt, J = 18.1, 2.3 Hz, C-2); 31P NMR (202.46 MHz, CDCl3)
δ F
8.75 ppm (d, J = 65.6 Hz); 19
NMR (470.59 MHz, D2O) δ −199.16 (t, J = 65.8 Hz). HRMS (ESI) calcd. for (C12H23O6NFP2) [M + H]+
364.1454; found 364.1443.
5. Drug Screening
5.1. T. cruzi Amastigote Assays
These experiments were done as reported using tdTomato labeled trypomastigotes with the
modifications described by Recher et al., 2013 [38]. ED50 values were determined by non-linear
regression analysis using SigmaPlot.
5.2. 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 [38]. Plates were read with covered lids, and both excitation (544 nm) and emission (590 nm) were
read from the bottom.
5.3. Cytotoxicity for Vero cells
The cytotoxicity was tested using the Alamar Blue
™ assay as described by Recher et al., 2013 [38].
5.4. 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 [34].
6. Conclusions
In this paper, we have described the synthesis and biological evaluation of isoprenoid and
linear 1,1-bisphosphonates against T. cruzi and T. gondii cells as well as the target enzymes TcFPPS
and TgFPPS. The title compounds exhibited marginal antiparasitic activity. However, the biological
data here presented provide insights concerning structure–activity relationship. Clearly, the simple
replacement of the hydrogen atom by a fluorine atom had a dramatic effect on the biological activity,
giving rise to inactive compounds.

Molecules 2017, 22, 82
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1
Supplementary Materials: Copies of the H NMR, 13C NMR, 31P NMR and 19F NMR spectra of the target
molecules and the corresponding intermediates are included as supporting information. They are available online
at http://www.mdpi.com/1420-3049/22/1/82/s1.
Acknowledgments: This work was supported by grants from the Consejo Nacional de Investigaciones
Científicas y Técnicas (PIP 0797), the Agencia Nacional de Promoción Científica y Tecnológica (PICT 2012 #0457),
and the Universidad de Buenos Aires (20020130100223BA) to J.B.R., and the U.S. National Institutes of Health to
R.D. (AI-107663) and S.N.J.M. (AI-102254).
Author Contributions: Tamila Galaka and Mariana Ferrer Casal performed the synthetic aspects of the experiment
and wrote the draft of paper. Sergio H. Szajnman supervised the synthetic aspects of this work. Melissa Storey,
Catherine Li and María N. Chao performed biological assays. Roberto Docampo and Silvia N. J. Moreno supervised
the biological aspects of this study. Juan B. Rodriguez participated in the writing and revision of the manuscript.
All authors took part in data analysis and discussion. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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