Activity of Fluorine-Containing Analogues of WC-9 and Structurally Related Analogues against Two Intracellular Parasites: Trypanosoma cruzi and Toxoplasma gondii

Article abstract of DOI:10.1002/cmdc.201600505

Two obligate intracellular parasites, Trypanosoma cruzi, the agent of Chagas disease, and Toxoplasma gondii, an agent of toxoplasmosis, upregulate the mevalonate pathway of their host cells upon infection, which suggests that this host pathway could be a potential drug target. In this work, a number of compounds structurally related to WC-9 (4-phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, were designed, synthesized, and evaluated for their effect on T. cruzi and T. gondii growth in tissue culture cells. Two fluorine-containing derivatives, the 3-(3-fluorophenoxy)- and 3-(4-fluorophenoxy)phenoxyethyl thiocyanates, exhibited half-maximal effective concentration (EC₅₀) values of 1.6 and 4.9 μm, respectively, against tachyzoites of T. gondii, whereas they showed similar potency to WC-9 against intracellular T. cruzi (EC₅₀values of 5.4 and 5.7 μm, respectively). In addition, 2-[3- (phenoxy)phenoxyethylthio]ethyl-1,1-bisphosphonate, which is a hybrid inhibitor containing 3-phenoxyphenoxy and bisphosphonate groups, has activity against T. gondii proliferation at sub-micromolar levels (EC₅₀=0.7 μm), which suggests a combined inhibitory effect of the two functional groups.

Full text of DOI:10.1002/cmdc.201600505

DOI: 10.1002/cmdc.201600505
Full Papers
Activity of Fluorine-Containing Analogues of WC-9 and
Structurally Related Analogues against Two Intracellular
Parasites: Trypanosoma cruzi and Toxoplasma gondii
Marꢀa N. Chao,[a] Catherine Li,[b] Melissa Storey,[b] Bruno N. Falcone,[a] Sergio H. Szajnman,[a]
Sergio M. Bonesi,[c] Roberto Docampo,[b] Silvia N. J. Moreno,[b] and Juan B. Rodriguez*[a]
Two obligate intracellular parasites, Trypanosoma cruzi, the
agent of Chagas disease, and Toxoplasma gondii, an agent of
toxoplasmosis, upregulate the mevalonate pathway of their
host cells upon infection, which suggests that this host path-
way could be a potential drug target. In this work, a number
of compounds structurally related to WC-9 (4-phenoxyphenox-
yethyl thiocyanate), a known squalene synthase inhibitor, were
designed, synthesized, and evaluated for their effect on T. cruzi
and T. gondii growth in tissue culture cells. Two fluorine-con-
taining derivatives, the 3-(3-fluorophenoxy)- and 3-(4-fluoro-
phenoxy)phenoxyethyl thiocyanates, exhibited half-maximal ef-
fective concentration (EC50) values of 1.6 and 4.9 mm, respec-
tively, against tachyzoites of T. gondii, whereas they showed
similar potency to WC-9 against intracellular T. cruzi (EC50
values of 5.4 and 5.7 mm, respectively). In addition, 2-[3-
(phenoxy)phenoxyethylthio]ethyl-1,1-bisphosphonate, which is
a hybrid inhibitor containing 3-phenoxyphenoxy and bis-
phosphonate groups, has activity against T. gondii proliferation
at sub-micromolar levels (EC50 =0.7 mm), which suggests a com-
bined inhibitory effect of the two functional groups.
Introduction
Obligate intracellular parasites depend on the integrity of their
host cell to survive. They have evolved sophisticated strategies
to manipulate their host and establish with them a close meta-
bolic link in order to complete their development. A case in
point is that of parasites like Trypanosoma cruzi, a trypanosoma-
tid, and Toxoplasma gondii, an Apicomplexan parasite, which
upregulate host genes for the mevalonate pathway upon in-
fection.[1–3] This up-regulation is probably performed to acquire
cholesterol and other isoprenoids needed to accommodate an
increasing intracellular parasite load and/or to provide these
lipids to be scavenged by the intracellular parasites.[3] T. cruzi is
the agent of Chagas disease (American trypanosomiasis), the
largest parasitic disease burden of the Americas.[4] Treatment
of T. cruzi infection is with nifurtimox or benznidazole. Neither
of these compounds are drugs approved by the US Food and
Drug Administration (FDA), and they are only available in the
United States from the Centers for Disease Control and Preven-
tion (CDC) under investigational protocols. On the other hand,
T. gondii is the agent of toxoplasmosis,[5] which affects a wide
range of hosts, particularly humans and other warm-blooded
animals.[6] Toxoplasmosis can be considered as one of the most
prevalent parasitic diseases: it affects almost one billion people
worldwide.[7] This parasite can cause mortality among immune-
compromised individuals, such as AIDS patients and organ-
transplant recipients, as well as in congenitally infected chil-
dren.[8] Toxoplasmosis may also lead to severe ocular disease in
immune-competent patients.[9] The current chemotherapy for
toxoplasmosis is also deficient, because the available drugs
may cause toxic side effects and they are not able to properly
access the central nervous system. Another drawback of the
present chemotherapy is its high cost.[10]
The up-regulation of the mevalonate pathway of the host
by these intracellular parasites provides an additional potential
drug target, because its inhibition could affect the parasite and
the host cell in which the parasite resides. T. gondii does not
synthesize cholesterol and imports it from the host;[11] it is also
able to take up isoprenoids like farnesyl diphosphate (FPP) and
geranylgeranyl diphosphate synthesized by the host. As with
other trypanosomatids, T. cruzi has a strict requirement for spe-
cific endogenous sterols for survival, although it can take up
cholesterol from its mammalian host.[12,13] Appropriate ergo-
sterol biosynthesis inhibitors can induce a parasitological cure
in both acute and chronic experimental models of Chagas dis-
ease.[14] 4-Phenoxyphenoxyethyl thiocyanate (compound 1;
WC-9) is a potent inhibitor of the intracellular amastigote
forms of T. cruzi.[15] WC-9 is a noncompetitive inhibitor of
T. cruzi squalene synthase (TcSQS) that acts at low nanomolar
[a] M. N. Chao, Dr. B. N. Falcone, Dr. S. H. Szajnman, Prof. Dr. J. B. Rodriguez
Departamento de Quꢀmica Orgꢁnica and UMYMFOR (CONICET-FCEyN), Fac-
ultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabel-
lꢂn 2, Ciudad Universitaria, C1428EHA, Buenos Aires (Argentina)
E-mail: jbr@qo.fcen.uba.ar
[b] C. Li, M. Storey, Prof. Dr. R. Docampo, Prof. Dr. S. N. J. Moreno
Center for Tropical and Emerging Global Diseases and Department of Cellu-
lar Biology, University of Georgia, Athens, GA 30602 (USA)
[c] Prof. Dr. S. M. Bonesi
Departamento de Quꢀmica Orgꢁnica and CIHIDECAR (CONICET-FCEyN), Fac-
ultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabel-
lꢂn 2, Ciudad Universitaria, C1428EHA, Buenos Aires (Argentina)
Supporting information (characterization of target compounds and cor-
1
responding intermediates along with H, 13C, and 19F NMR spectra) and
the ORCID identification number(s) for the author(s) of this article can be
found under http://dx.doi.org/10.1002/cmdc.201600505.
ChemMedChem 2016, 11, 1 – 14
1
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
concentration.[16] This enzyme catalyzes the first step in sterol
biosynthesis, which consists of reductive dimerization of two
molecules of farnesyl diphosphate to yield squalene. Additional
synthetic derivatives of WC-9 (2–6) are shown in
Figure 1.[15,17–21]
effect if the mevalonate pathway of the host and the isopre-
noid pathway of the parasite are targeted independently.[27]
For example, zoledronic acid, a bisphosphonate that inhibits
T. gondii farnesyl diphosphate synthase (TgFPPS), and atorvas-
tatin, a statin that inhibits the host 3-hydroxymethyl-glutaryl
coenzyme A (3-HMG-CoA)-reductase, exhibit a marked syner-
gism in the inhibition of T. gondii growth.[27]
Rationale
WC-9 is one of the few examples of a pharmacologically im-
portant lead compound possessing a thiocyanate group cova-
lently bound to its main skeleton.[28] At the present time, there
is no crystal structure available for the complex WC-9–TcSQS.
However, an X-ray crystal structure of WC-9 with human SQS
has been recently reported (Protein Data Bank (PDB) ID:
3WCD).[29] On the other hand, the X-ray crystal structures of
the complexes E5700–TcSQS and ER-119884–TcSQS are avail-
able.[29] The quinuclidine derivatives E5700 (7) and ER-119884
(8) are potent inhibitors of T. cruzi growth that act as TcSQS in-
hibitors (Figure 2).[30,31] Both of these compounds are extremely
potent inhibitors of the enzymatic activity of TcSQS: they ex-
hibit half-maximal inhibitory concentration (IC50) values of 0.84
and 3.5 nm, respectively.[31]
Figure 3 shows a superimposition of the E5700–TcSQS and
ER-119884–TcSQS complexes with the crystal structure of the
WC-9–human SQS complex. A high degree of similarity is ob-
served between the T. cruzi and human protein structures. Fur-
thermore, the quinoclidine inhibitors occupy the same binding
site as WC-9. Given that these inhibitors were found to be
mixed type (7) and noncompetitive (8),[30] they provide evi-
dence that WC-9 may, in fact, occupy the same binding site in
TcSQS.
Figure 1. Structure of WC-9 and other closely related inhibitors of T. cruzi
proliferation.
It is interesting to note that T. gondii lacks the mevalonate
pathway and uses the essential 1-deoxy-d-xylulose-5-phos-
phate pathway to make isopentenyl diphosphate and dimethy-
lallyl diphosphate.[22] As T. gondii does not synthesize cholester-
ol and imports it from the host,[11] it is reasonable to consider
that inhibitors of the host SQS could eventually control
T. gondii growth. Certainly, other mevalonate pathway inhibi-
tors modulate the growth of various intracellular Apicomplex-
an parasites that are devoid of this pathway, such as Babesia
divergens,[23] Plasmodium falciparum,[23,24] Cryptosporidium
parvum,[25] and T. gondii,[26] which indicates that parasites lack-
ing the mevalonate pathway are reliant on host precursors of
isoprenoid biosynthesis. Interestingly, there is a synergistic
Interestingly, TcSQS activity is also inhibited by bisphospho-
nates.[32] Bisphosphonates are the main modulators of FPPS,
a key enzyme of isoprenoid biosynthesis.[33] Moreover, several
X-ray crystal structures of TcSQS are available with a number
of bisphosphonates, such as BPH1344 (9; PDB ID: 3WCG;
Figure 4).[29]
Figure 2. Structures of quinuclidine derivatives E5700 (7) and ER-119884 (8).
Figure 3. a) Superposition of the crystal structures of human SQS with WC-9 and T. cruzi SQS with 7 and 8. A high degree of similarity is observed between
the protein chains. b) Expansion of the structures showing that the quinuclidine derivatives 7 (red) and 8 (blue) occupy the same S2 site (homoallylic site) as
WC-9 (green). The mechanisms of action of these compounds (7 mixed-type and 8 non-competitive) provide further evidence that WC-9 may indeed bind to
the S2 site in T. cruzi SQS.
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
2
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
as target molecules. Compound 10[20] was treated with 2-
fluoro-, 3-fluoro-, and 4-fluorophenol under typical Buchwald
coupling reaction conditions to generate tetrahydropyranyl de-
rivatives 11, 12, and 13 in 53, 33, and 71% yields, respectively.
Each tetrahydropyranyl protecting group present in these com-
pounds was cleaved by treatment with pyridinium p-toluene-
sulfonate to produce the corresponding free alcohols 14, 15,
and 16 in very good yields. The alcohols were tosylated to
give 17, 18, and 19 in 66, 73, and 84% yields, respectively. On
treatment with potassium thiocyanate, in separate experi-
ments, these compounds were converted into the target mole-
cules 20, 21, and 22, respectively, as illustrated in Scheme 1.
The fluoro derivatives of WC-9 at C4’’ and C3’’, namely 2
and 3, were more potent than the lead drug WC-9.[19] It
seemed reasonable to also produce the fluorinated analogue
at C2’’ (compound 27), which had not been prepared before.
The Buchwald coupling reaction of 23[20] with 2-fluorophenol
generated the tetrahydropyranyl derivative 24 in a low but re-
producible yield, which produced the free alcohol 25 in 62%
yield on treatment with pyridinium 4-toluenesulfonate. Tosyla-
tion of this compound to produce 26 followed by treatment
with potassium thiocyanate resulted in the desired compound
27 in 36% yield (Scheme 2).
An interesting structural variation was the replacement of
the terminal phenyl group by a pyridyl group, in which the ni-
trogen atom occupied the 4’’ position (compound 31). We
have previously synthesized and evaluated the corresponding
2-pyridyl[21] and 3-pyridyl[20] derivatives, so the availability of 31
would complete the corresponding SAR analysis. A straightfor-
ward synthesis of 31 was accomplished by starting from 23,[20]
which was treated with 4-hydroxypyridine under typical cou-
pling reaction conditions to generate 28. On treatment with
pyridinium 4-toluenesulfonate, 28 was converted into alcohol
29, which was further transformed into 30 by reaction with N-
bromosuccinimide and triphenylphosphine[40] in 50% yield. On
reaction with potassium thiocyanate, 30 was transformed into
the desired compound 31 in 34% yield (Scheme 2).
Figure 4. Structure of the bisphosphonate compound known as BPH1344
(9).
Figure 5 shows a superposition of the WC-9–human SQS
complex and the BPH1344–TcSQS complex, with the bi-
sphosphonate group deleted, to show the putative binding
site that WC-9 could occupy in TcSQS.[29] As can be observed,
the two structures show a high degree of similarity, except for
the a helix 284–294 in T. cruzi, which acquires a loop organiza-
tion in the corresponding human SQS structure.[29] The X-ray
crystal structure of WC-9 with dehydrosqualene synthase from
Staphylococcus aureus, an enzyme very similar to SQS that cat-
alyzes dehydrosqualene formation, is also available.[34]
At the present time, there is no computer-assisted protocol
to predict binding of WC-9 analogues to TcSQS. However,
there are abundant structure–activity relationship (SAR) data
available on T. cruzi and T. gondii cells that can be used to facil-
itate drug design.[17–21,35] In addition, there is strong evidence
to state that the phenoxyethyl thiocyanate moiety of WC-9
(Figure 1) is the pharmacophore of this family of molecules. A
question that emerges, the answer to which is still pending, is
whether the optimum substitution pattern will be at the C4’ or
C3’ position. The availability of the Buchwald coupling reaction
allowing us to access a variety of WC-9 analogues encouraged
us to go further in searching for better inhibitors against either
T. cruzi or T. gondii cells.[36–39]
Results and Discussion
The introduction of a fluorine atom into the WC-9 structure to
give rise to compounds 2 and 3 was a significant structural
change, which was quite beneficial for the inhibitory action.[19]
Therefore, it seemed of interest to study the biological activity
of the fluorine-containing analogues of the regioisomer of WC-
9, compound 4, and we envisioned compounds 20, 21, and 22
Although a significant number of structural variations have
been made on the structure of WC-9, only a few of them cor-
Figure 5. a) The binding site (amino acids within 4ꢂ of the ligand) of WC-9 in the human SQS structure (amino acids shown in blue licorice representation)
and b) the putative site that WC-9 would occupy in the T. cruzi SQS (amino acids in red licorice representation). A high degree of similarity is observed be-
tween the two binding sites. In terms of sequence, all amino acids in the binding sites are the same, except for Ser 256 in T. cruzi instead of Cys 254 in the
human enzyme.
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
3
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
Scheme 1. Reagents and conditions: a) 5% CuI, 10% picolinic acid, (2-fluoro-, 3-fluoro-, 4-fluorophenol), K3PO4, DMSO, 908C (4 d for 11, 14 d for 12, 4 d for
13); b) PPTs, CH3OH, RT, 16 h; c) ClTs, py, 08C, 6 h; d) KSCN, DMF, 1008C, 6 h. PPTs: pyridinium p-toluenesulfonate; py: pyridine; THP: tetrahydropyranyl; Ts: tol-
uene-4-sulfonyl.
Scheme 2. Reagents and conditions: a) 5% CuI, 10% picolinic acid, 2-fluorophenol, K3PO4, DMSO, 908C, 12 d, 37%; b) PPTs, CH3OH, RT, 24 h, 62%; c) ClTs, py,
RT, 24 h, 92%; d) KSCN, DMF, 808C, 6 h, 36%; e) CuI, 10% picolinic acid, 4-hydroxypyridine, K3PO4, DMSO, 808C, 24 h, 29%; f) PPTs, CH3OH, RT, 24 h, 59%;
g) NBS, Ph3P, 0 8C, 6 h, 50%; h) KSCN, DMF, 808C, 6 h, 34%. NBS: N-bromosuccinimide.
respond to substitutions on the A ring.[17] An interesting
method to incorporate an acetyl group at the C2’ is the photo-
Fries rearrangement reaction.[41] Therefore, 4-phenoxyphenol
(32) was acetylated to give 33 in excellent yield. Compound
33 was then irradiated at 254 nm to produce the correspond-
ing photo-Fries rearranged product 34 in 39% yield. Com-
pound 34 was treated with 2-bromoethyl tetrahydro-2H-
pyran-2-yl ether in a Williamson etherification reaction to give
35 in 62% yield. This compound was deprotected by treat-
ment with pyridinium 4-toluenesulfonate in methanol to gen-
erate free alcohol 36 (92% yield), which was treated with tosyl
chloride in pyridine to give the expected tosylate 37 in 95%
yield. This compound was further transformed into the thio-
cyanate derivative 38 in 71% yield by treatment with potassi-
um thiocyanate in N,N-dimethylformamide at 808C (Scheme 3).
The concept of a hybrid drug, in that one compound pos-
sesses a dual mode of action, is fascinating.[42–44] For this
reason, hybrid molecules, such as 43 and 48, were envisioned
with a phenoxy unit to target SQS and a bisphosphonate
moiety to act against FPPS.[33] The production of 42 was
straightforward by employing the already-described azide
39.[45] This compound was reduced to the corresponding
amine 40 in 77% yield through a Staudinger reaction by treat-
ment with triphenylphosphine in dichloromethane.[46] A Mi-
chael-type addition of the resulting amine 40 with the Michael
acceptor 41[47] yielded 42 in 82% yield. Hydrolysis of 42 by
treatment with hydrochloric acid at reflux produced the de-
sired compound 43. Hybrid compound 48 was straightfor-
wardly prepared by starting from the previously described to-
sylate 44.[15] Upon treatment of 44 with potassium thioacetate
Scheme 3. Reagents and conditions: a) Ac2O, py, RT, 16 h, 97%; b) hn, C6H12, RT, 12 h, 39%; c) KOH, DMSO, BrCH2CH2OTHP, RT, 12 h, 62%; d) PPTs, CH3OH, RT,
16 h, 92%; e) ClTs, py, RT, 6 h, 95%; f) KSCN, DMF, 808C, 6 h, 71%.
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
4
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
in N,N-dimethylformamide at 908C, 45 was produced. When
45 was treated with lithium aluminum hydride in anhydrous
tetrahydrofuran, it yielded the corresponding thiol 46 in good
yield. The Michael addition reaction of this resultant com-
pound with 41 followed by hydrolysis of the phosphonate
esters by treatment with bromotrimethylsilane and digestion
with methanol produced the desired compound 48. By follow-
ing a quite similar approach, 53 was prepared from the al-
ready-described tosylate 49,[20] which was transformed into
azide 50 by treatment with sodium azide in N,N-dimethylfor-
mamide. Under Staudinger-type conditions, 50 was converted
into amine 51, which was then treated with 41 to produce the
Michael adduct 52. Hydrolysis of this compound by treatment
with concentrated hydrochloric acid at reflux produced the de-
sired compound 53. The synthesis of 58 was also straightfor-
ward. The known tosylate 54[20] was treated with potassium
thioacetate in an SN2 reaction in N,N-dimethylformamide at
808C to give rise to 55 in 83% yield. Hydrolysis of the acetyl
group by treatment with potassium carbonate followed by re-
duction with zinc in glacial acetic acid generated the corre-
sponding thiol 56 in 75% yield. A Michael addition with this
thiol followed by hydrolysis by treatment with bromotrimethyl-
silane and methanol digestion produced the desired com-
pound 58 in 65% yield. Similarly, 63 was obtained from the al-
ready-described tosylate 59,[18] which was transformed into 60
by treatment with sodium azide. This compound was trans-
formed into 61, which was then treated with 41 to yield 62.
Treatment with concentrated hydrochloric acid at reflux result-
ed in the desired compound 63 (Scheme 4).
As another structural variation, hydrophobic analogues of
WC-9, such as 68 and 73, were also produced. Both of these
compounds were easily prepared from b-naphthol (64) and a-
naphthol (69), respectively. Therefore, each compound, in sep-
arate experiments, was treated with 2-bromoethyl tetrahydro-
2H-pyran-2-yl ether under Williamson-type conditions to pro-
duce tetrahydropyranyl derivatives 65 and 70, respectively,
which were easily deprotected by treatment with pyridinium
4-toluenesulfonate to yield the corresponding free alcohols 66
and 71, respectively. On treatment with an excess of tosyl chlo-
ride, 66 and 71 were converted into tosylates 67 and 72, re-
Scheme 4. Reagents and conditions: a) PPh3, CH2Cl2, RT, 4 h, 77%; b) 41, CH2Cl2, NEt3, RT, 5 h, 59%; c) HCl (concd), reflux, 7 h, 81%; d) CH3C(O)SK, DMF, 2 h,
908C, 83%; e) LiAlH4, THF, RT, 3 h, 92%; f) 41, CH2Cl2, NEt3, RT, 5 h, 59%; g) 1) BrSi(CH3)3, CH2Cl2, RT, 48 h, 2) CH3OH, RT, 24 h, 69%; h) NaN3, DMF, 808C, 2 h,
90%; i) PPh3, THF, RT, 2 h, 90%; j) 41, CH2Cl2, NEt3, RT, 24 h, 97%; k) HCl (concd), reflux, 7 h, 35%; l) CH3C(O)SK, DMF, 3 h, 808C, 98%; m) 1) K2CO3, CH3OH/H2O,
40 min, RT, 2) Zn/AcOH, 1 h, 1108C, 75%; n) 41, CH2Cl2, NEt3, RT, 16 h, 75%; o) 1) BrSi(CH3)3, CH2Cl2, RT, 48 h, 2) CH3OH, RT, 24 h, 65%; p) NaN3, DMF, 808C, 2 h,
81%; q) PPh3, THF, RT, 2 h, 67%; r) 41, CH2Cl2, NEt3, RT, 16 h, 88%; s) HCl (concd), reflux, 7 h, 11%.
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
5
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
Scheme 5. Reagents and conditions: a) KOH, DMSO, BrCH2CH2OTHP, RT, 5 d, 48%; b) PPTs, CH3OH, RT, 24 h, 85%; c) ClTs, py, RT, 4 h, 83%; d) KSCN, DMF, 808C,
6 h, 55%; f) KOH, DMSO, BrCH2CH2OTHP, RT, 5 d, 65%; g) PPTs, CH3OH, RT, 24 h, 81%; e) ClTs, py, RT, 4 h, 90%; f) KSCN, DMF, 808C, 6 h, 58%.
spectively. On reaction with potassium thiocyanate in N,N-di-
methylformamide, 67 and 72 were converted into the desired
molecules 68 and 73, respectively, as shown in Scheme 5.
Biological evaluation of these new compounds structurally
related to WC-9 gave promising results. All fluorine-containing
derivatives of compound 4, that is, compounds 20, 21, and 22,
were effective inhibitors of tachyzoites of T. gondii and exhibit-
ed half-maximal effective concentration (EC50) values of 3.1,
1.6, and 4.9 mm, respectively, with 20 and 21 being slightly
more potent than 4 (EC50 =4.0 mm).[20] The fluorinated com-
pounds were also effective growth inhibitors of the intracellu-
lar form of T. cruzi (amastigotes), which is the clinically more
relevant replicative form of the parasite. In fact, all of the com-
pounds bearing a fluorine atom bound to the C2’’, C3’’, and
C4’’ positions exhibited relatively similar EC50 values (7.0, 5.4,
and 5.7 mm, respectively) to that of WC-9, used as a positive
control, under the same assay conditions with T. cruzi.[20] The
fluorine analogue of WC-9 at C2’’, compound 27, which is the
regioisomer of 2 and 3, was also a potent inhibitor of T. gondii
tachyzoite growth, with an EC50 value of 4.5 mm. Compound 27
was more potent than 2 (EC50 =15.8 mm)[19] but exhibited quite
similar efficacy to that of 3 (EC50 =3.9 mm).[19] Quite unexpect-
edly, in spite of the regioisomers 2 and 3 being potent growth
inhibitors of amastigotes of T. cruzi,[19] 27 was devoid of anti-
parasitic activity against this parasite. Pyridyl derivative 31 was
free of antiparasitic activity against T. cruzi; however, 31 be-
haved as a growth inhibitor of T. gondii and showed an EC50
value of 7.9 mm. The introduction of an acetyl group at the C2’
position in 38 proved to be quite beneficial for the biological
activity, because this compound showed potent inhibitory
action against T. gondii. In fact, its EC50 value was 2.4 mm. Com-
pound 38 was less effective against T. cruzi with an EC50 value
of 11.3 mm. The hybrid molecules 53 and 58 were both devoid
of activity against T. cruzi, but they showed a strong inhibitory
action against tachyzoites of T. gondii. The sulfur-containing an-
alogue 58 showed a sub-micromolar activity against T. gondii
with an EC50 value of 0.7 mm. In fact, the 3’-phenoxy substitu-
tion pattern found in 53 and 58 was more beneficial for bio-
logical activity against T. gondii than the 4’-phenoxy substitu-
tion present in 43 and 48. Compounds 53 and 58 were also
recognized as potent inhibitors of the target enzyme TgFPPS
and exhibited IC50 values of 0.040 and 0.076 mm, respectively.
The simplified naphthyl derivatives 68 and 73 turned out to be
potent inhibitors of T. gondii with EC50 values of 3.6 and
3.1 mm, respectively, but these compounds exhibited no inhibi-
tory action against T. cruzi. The results are presented in Table 1.
Given the high in vitro activity of hybrid derivatives of WC-9,
we performed molecular-dynamics-based computational stud-
ies to gain further insight into the interactions of this kind of
compound with the active site of FPPS. By taking 58 as a repre-
sentative example and because no crystal structure of TgFPPS
is available, we constructed the structure using homology
modeling based on the crystal structure of Plasmodium vivax
FPPS (PDB ID: 3MAV), the enzyme showing the highest degree
of similarity with TgFPPS. We inserted 58, three Mg2+ ions, and
isopentenyl pyrophosphate as a coligand by alignment with
the TcFPPS alendronate complex structure (PDB ID: 1YHM). We
carried out a 10 ns molecular dynamics simulation in explicit
water, and performed a clustering analysis to obtain the most
populated structure. Based on an implicit solvent energy de-
composition calculation, we identified the key interactions
present with the binding site. Tyr 303 forms a hydrogen bond
with the sulfur atom at position 3 of 58, Gln 204 forms a hydro-
gen bond with the first bridging oxygen atom, and Thr 200
forms a hydrogen bond with the second bridging oxygen
atom. p–p interactions are present between Tyr 303 and ring A
and between Phe 131 and ring B. Hydrophobic interactions
take place between ring B and Leu 307 and Asn 196. The high
degree of specificity of these interactions is an indication of
why meta-substituted 58 shows a much higher potency than
para-substituted 48 (Figure 6). Further investigation of the abil-
ity of different isoprenoids to rescue the growth inhibition
could help to pin down the actual target of the bisphospho-
nate.
We cannot rule out that this inhibitory effect of WC-9 ana-
logues is, in part, due to inhibition of the host squalene syn-
thase and of other host enzymes of the mevalonate pathway.
In this regard, microarray experiments have shown that 3-
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
6
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
Table 1. Biological activity of WC-9 analogues against T. gondii (tachyzoites), T. cruzi (amastigotes), and Vero cells.[a]
Compound
T. gondii growth
EC50 [mm]
TgFPPS
IC50 [mm]
T. cruzi growth
EC50 [mm]
TcFPPS
IC50 [mm]
HhFPPS
IC50 [mm]
Cytotoxicity
EC50 [mm]
20
21
22
27
31
38
43
48
53
58
63
68
3.12Æ0.37
1.63Æ0.36
4.92Æ1.94
4.54Æ0.57
7.86Æ0.78
2.42Æ0.81
5.90Æ0.07
>10
ND
ND
ND
ND
ND
7.01Æ0.51
5.38Æ0.82
5.69Æ0.47
>10
>10
11.3Æ2.5
>10
>20
>10
>10
>10
ND
ND
ND
ND
ND
ND
ND
0.8Æ0.12
ND
ND
ND
ND
ND
>60
>50
>55
>55
>200
75.7Æ7.3
>100
>200
>50
ND
ND
0.094Æ0.020
0.362Æ0.002
0.040Æ0.002
0.076Æ0.019
0.180Æ0.015
ND
>10
>15
>10
>10
>10
ND
ND
ND
ND
2.40Æ0.70
0.67Æ0.23
>10
>10
ND
0.32Æ0.04
ND
ND
ND
>100
ND
3.62Æ0.38
3.13Æ0.58
4.8Æ0.41[20]
ND
>10
>10
ND
7.5
82.6Æ7.3
ND
73
WC-9
benznidazole
ND
ND
ND
5.0Æ1.1[20]
1.52Æ0.10
ND
[a] Values are the meanÆSD from at least three independent experiments, n=3. ND: not determined.
Conclusions
It can be concluded that most of the target compounds exhib-
ited potent action against T. gondii proliferation and, to
a lesser extent, some of them were also growth inhibitors of
T. cruzi. The druglike characters of our lead compound (for in-
stance, it follows all Lipinki rules:[48] no hydrogen-bond donors,
three hydrogen-bond acceptors, molecular weight is 271.3,
and logP is 4.20) and other closely related analogues offer
good chances for optimizing their chemical structures. The
availability of the crystal structure of WC-9 with dehydrosqua-
lene synthase from S. aureus, together with crystallographic
structure of this compound bound to human SQS and proper
docking studies in homology models, will allow the production
of additional more active WC-9 analogues. A more detailed
computational study is under way to further understand the
interactions of the current inhibitors and guide the develop-
ment of new compounds; the results will be published else-
where.
Figure 6. Interaction of 58 with TgFPPS. The structure was formed by homol-
ogy modeling from P. vivax FPPS, and 58 (blue), three Mg ions (pink), and
isopentenyl pyrophosphate (cyan) were inserted by alignment with the
TcFPPS alendronate crystal structure (PDB ID: 1YHM). Residues at a distance
of 4 ꢂ from the ligand are shown in thick line representation, with those
contributing significantly to the binding energy in thin licorice representa-
tion.
Experimental Section
General methods
The glassware used in air- and/or moisture-sensitive reactions was
flame dried, and the reactions were performed under a dry argon
atmosphere. Unless otherwise noted, chemicals were commercially
available and were used without further purification. Anhydrous
N,N-dimethylformamide and anhydrous dimethyl sulfoxide were
used as supplied from Aldrich. Nuclear magnetic resonance spectra
were performed by using a Bruker AM-500 MHz apparatus. Chemi-
cal shifts (d) are reported in parts per million relative to tetrame-
thylsilane. 13C NMR spectra were fully decoupled. High-resolution
mass spectra were carried out by using a Bruker micrOTOF-Q II
spectrometer, which is a hybrid quadrupole time-of-flight mass
spectrometer with MS–MS capability. Melting points were deter-
mined by using a Fisher–Johns apparatus. Column chromatogra-
phy was performed with Merck silica gel plates (Kieselgel 60, 230–
400 mesh). Analytical thin-layer chromatography was performed by
employing 0.2 mm coated commercial silica gel plates (Merck, DC
HMG-CoA reductase, diphosphomevalonate decarboxylase,
and farnesyl diphosphate synthase are induced 24 h after
T. gondii infection of human foreskin fibroblasts (HFF).[1] Squa-
lene epoxidase, the second enzyme in the cholesterol biosyn-
thesis pathway and one of the rate-limiting enzymes, is upre-
gulated fivefold following infection of either HFF[1] or the por-
cine kidney epithelial cell line PK13,[2] which suggests that in-
duction of mevalonate biosynthetic enzymes is necessary to in-
crease cellular levels of squalene that could be scavenged by
the parasite.[1] Similarly, 3-HMG-CoA reductase, diphosphome-
valonate decarboxylase, and farnesyl diphosphate synthase are
induced several-fold 24–72 h post-infection of Macaca mulatta
LLcMK2 kidney cells with T. cruzi.[3]
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
7
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
Aluminum sheets, Kieselgel 60F254). As judged from the homoge-
3-(4-Fluorophenoxy)phenoxyethanol (16):
A
solution of 13
1
neity of the H, 13C, 19F, and 31P NMR spectra and HPLC analyses by
(581 mg, 1.7 mmol) in methanol (10 mL) was treated with pyridini-
um 4-toluenesulfonate (30 mg) as described for the preparation of
14. The crude product was purified by column chromatography
with hexane/EtOAc (41:9) as the eluent to produce 399 mg (92%
yield) of alcohol 16 as a colorless oil.
employing a Beckmann Ultrasphere ODS-2 column (5 mm, 250ꢃ
10 mm, elution with acetonitrile/water (9:1) at
a rate of
3.00 mLminÀ1 with a refractive index detector), the title com-
pounds had purities of >97%.
3-(2-Fluorophenoxy)phenoxyethyl 4-toluenesulfonate (17): A so-
lution of alcohol 14 (253 mg, 0.95 mmol) in pyridine (3 mL) was
treated with 4-toluenesulfonyl chloride (546 mg, 2.9 mmol), and
the mixture was stirred at 08C for 6h. Then, 5% HCl (50 mL) was
added, and the reaction mixture was stirred for an additional hour.
The mixture was partitioned between dichloromethane (50 mL)
and water (50 mL). The organic layer was washed with 5% HCl (3ꢃ
50 mL) and water (3ꢃ50 mL). The organic phase was dried
(MgSO4), and the solvent was evaporated. The product was puri-
fied by column chromatography with hexane/EtOAc (9:1) as the
eluent to produce 226 mg of 17 (66% yield) as a colorless oil.
Syntheses
3-(2-Fluorophenoxy)phenoxyethyl
tetrahydro-2H-pyran-2-yl
ether (11): A mixture of compound 10 (900 mg, 2.6 mmol), 2-fluo-
rophenol (580 mg, 5.2 mmol), copper(I) iodide (49.2 mg,
0.26 mmol), 2-picolinic acid (63.6 mg, 0.52 mmol), and potassium
phosphate tribasic (1.1 g, 5.2 mmol) under anhydrous conditions
was placed in a flask, which was evacuated and back-filled with
argon. This sequence was repeated twice. Dimethyl sulfoxide was
then added (3.0 mL), and the reaction mixture was stirred at 908C
for 4 d. The mixture was cooled to room temperature and parti-
tioned between dichloromethane (20 mL) and water (20 mL). The
aqueous layer was extracted with dichloromethane (2ꢃ20 mL), the
combined organic phases were washed with brine (5ꢃ50 mL) and
dried (MgSO4), and the solvent was evaporated. The product was
purified by column chromatography (silica gel) with hexane/EtOAc
(24:1) as the eluent to produce 453 mg (53% yield) of pure com-
pound 11 as a colorless oil.
3-(3-Fluorophenoxy)phenoxyethyl 4-toluenesulfonate (18): A so-
lution of alcohol 15 (175 mg, 0.70 mmol) in pyridine (3 mL) was
treated with 4-toluenesulfonyl chloride (402 mg, 2.1 mmol), and
the mixture was stirred at 08C for 6h. The reaction was quenched
as described for the preparation of 17. The product was purified
by column chromatography with hexane/EtOAc (91:9) as the
eluent to produce 207 mg of 18 (73% yield) as a colorless oil.
3-(4-Fluorophenoxy)phenoxyethyl 4-toluenesulfonate (19). A so-
lution of alcohol 16 (388 mg, 1.6 mmol) in pyridine (3 mL) was
treated with 4-toluenesulfonyl chloride (894 mg, 4.7 mmol) as de-
scribed for the preparation of 17. The product was purified by
column chromatography with hexane/EtOAc (91:9) as the eluent to
produce 226 mg of 19 (84% yield) as a colorless oil.
3-(3-Fluorophenoxy)phenoxyethyl
tetrahydro-2H-pyran-2-yl
ether (12): Dimethyl sulfoxide (3.0 mL) was added to a mixture of
10 (893 mg, 2.6 mmol), 3-fluorophenol (575 mg, 5.1 mmol), cop-
per(I) iodide (48.9 mg, 0.26 mmol), 2-picolinic acid, (63.2 mg,
0.51 mmol), and potassium phosphate tribasic (1.092 g, 5.1 mmol)
as described for the preparation of 11. The reaction mixture was
stirred at 908C for 14 d. After the usual workup, the product was
purified by column chromatography (silica gel) with hexane/EtOAc
(49:1) as the eluent to produce 280 mg (33% yield) of 12 as a color-
less oil.
3-(2-Fluorophenoxy)phenoxyethyl thiocyanate (20): A solution of
tosylate 17 (381 mg, 0.98 mmol) in anhydrous N,N-dimethylforma-
mide (3.0 mL) was treated with potassium thiocyanate (460 mg,
4.7 mmol). The reaction mixture was heated at 1008C for 6h. The
mixture was allowed to cool to room temperature, and water
(20 mL) was added. The aqueous phase was extracted with di-
chloromethane (2ꢃ30 mL), the combined organic layers were
washed with brine (5ꢃ30 mL) and water (2ꢃ30 mL), then dried
(MgSO4), and the solvent was evaporated. The residue was purified
by column chromatography with hexane/EtOAc (23:2) as the
eluent to produce 226 mg (80% yield) of 20 as a colorless oil.
3-(4-Fluorophenoxy)phenoxyethyl
tetrahydro-2H-pyran-2-yl
ether (13): A mixture of 10 (877 mg, 2.5 mmol), 4-fluorophenol
(565 mg, 5.0 mmol), copper(I) iodide (48.0 mg, 0.25 mmol), 2-pico-
linic acid, (62.0 mg, 0.50 mmol), and potassium phosphate tribasic
(1.072 g, 5.0 mmol) was treated with dimethyl sulfoxide (3.0 mL) as
described for the preparation of 11. The reaction mixture was
stirred at 908C for 4 d. The reaction was quenched as described for
the workup of 11, and the product was purified by column chro-
matography (silica gel) with hexane/EtOAc (24:1) as the eluent to
produce 594 mg (71% yield) of 13 as a colorless oil.
3-(3-Fluorophenoxy)phenoxyethyl thiocyanate (21): A solution of
18 (197 mg, 0.49 mmol) in anhydrous N,N-dimethylformamide
(3.0 mL) was treated with potassium thiocyanate (237 mg,
2.4 mmol). The mixture was treated as described for the prepara-
tion of 20. The residue was purified by HPLC with methanol/H2O
(9:1) as the eluent and by employing a Bechman Ultrasphere 5 mm
column (250ꢃ10 mm) to give rise to 107 mg (68% yield) of 21 as
a colorless oil.
3-(2-Fluorophenoxy)phenoxyethanol (14):
A solution of 11
(426 mg, 1.3 mmol) in methanol (10 mL) was treated with pyridini-
um 4-toluenesulfonate (30 mg). The reaction mixture was stirred at
room temperature overnight. Water (50 mL) was then added, and
the mixture was extracted with dichloromethane (3ꢃ50 mL). The
combined organic layers were washed with brine (3ꢃ50 mL) and
dried (MgSO4), and the solvent was evaporated. The residue was
purified by column chromatography with hexane/EtOAc (85:15) as
the eluent to produce 297 mg (93% yield) of pure alcohol 14 as
a colorless oil.
3-(4-Fluorophenoxy)phenoxyethyl thiocyanate (22): A solution of
19 (514 mg, 1.3 mmol) in anhydrous N,N-dimethylformamide
(3.0 mL) was treated with potassium thiocyanate (647 mg,
6.7 mmol). The reaction mixture was treated as described for the
preparation of 20. The residue was purified by column chromatog-
raphy with hexane/EtOAc (47:3) as the eluent to produce 221 mg
(58% yield) of 22 as a colorless oil.
3-(3-Fluorophenoxy)phenoxyethanol (15):
A solution of 12
(269 mg, 0.81 mmol) in methanol (10 mL) was treated with pyridi-
nium 4-toluenesulfonate (30 mg) as described for the preparation
of 14. The residue was purified by column chromatography with
hexane/EtOAc (83:17) as the eluent to give 182 mg (91% yield) of
alcohol 15 as a colorless oil.
4-(2-Fluorophenoxy)phenoxyethyl
tetrahydro-2H-pyran-2-yl
ether (24): Dimethyl sulfoxide (3.0 mL) was added to a mixture of
23 (858 mg, 2.5 mmol), 2-fluorophenol (549.5 mg, 4.9 mmol), cop-
per(I) iodide (46.0 mg, 0.24 mmol), 2-picolinic acid, (59.4 mg,
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
8
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
0.51 mmol), and potassium phosphate tribasic (1.026 g, 4.9 mmol)
as described for the preparation of 11. The reaction mixture was
stirred at 908C for 12 d. After the usual workup, the product was
purified by column chromatography with hexane/EtOAc (19:1) as
the eluent to produce 295 mg (37% yield) of 24 as a colorless oil.
anhydride (3 mL), and the reaction mixture was stirred at room
temperature for 16 h. Water (10 mL) was then added, and the mix-
ture was stirred for 1 h. The mixture was extracted with dichloro-
methane (2ꢃ30 mL). The combined organic layers were washed
with an aqueous 1n solution of hydrochloric acid (2ꢃ30 mL) and
water (2ꢃ30 mL), then dried (MgSO4), and the solvent was evapo-
rated. The product was purified by column chromatography with
hexane/EtOAc (19:1) as the eluent to give 2.478 g (97% yield) of
33 as a colorless oil.
4-(2-Fluorophenoxy)phenoxyethanol (25):
A solution of 24
(280 mg, 0.84 mmol) in methanol (10 mL) was treated with pyridi-
nium 4-toluenesulfonate (30 mg) as depicted for the preparation
of 14. The residue was purified by column chromatography with
hexane/EtOAc (9:1) as the eluent to give 124 mg (62% yield) of al-
cohol 25 as a colorless oil.
2-Acetoxy-4-phenoxyphenol (34):
A solution of acetate 33
(257.8 mg, 1.13 mmol) in cyclohexane (200 mL), previously de-
gassed with dry nitrogen, in a septum-stopped quartz tube was ir-
radiated with germicide lamps (4ꢃ20 W) centered at 254 nm at
room temperature for 12 h. The solvent was evaporated, and the
residue was purified by column chromatography with hexane/
EtOAc (9:1) to produce 100.5 mg (39% yield) of pure 34 as a white
solid.
4-(2-Fluorophenoxy)phenoxyethyl 4-toluenesulfonate (26). A so-
lution of alcohol 25 (110 mg, 0.44 mmol) in pyridine (3.0 mL) was
treated with 4-toluenesulfonyl chloride (250 mg, 1.3 mmol), and
the mixture was stirred at room temperature for 24 h. The reaction
was quenched as described for the preparation of 17. The product
was purified by column chromatography with hexane/EtOAc (19:1)
as the eluent to produce 163 mg of 26 (92% yield) as a colorless
oil.
2-Acetoxy-4-phenoxyphenoxyethyl
tetrahydro-2H-pyran-2-yl
ether (35): A solution of 34 (457.1 g, 2.0 mmol) in dimethyl sulfox-
ide (5.0 mL) was treated with potassium hydroxide (450 mg,
8.0 mmol). The mixture was stirred at room temperature for 5 min.
Bromoethyl tetrahydropyranyl ether (418 mg, 2.0 mmol) was then
added, and the reaction mixture was stirred at room temperature
overnight. The mixture was partitioned between water (35 mL) and
dichloromethane (35 mL). The aqueous phase was extracted with
dichloromethane (2ꢃ30 mL). The combined organic layers were
washed with a saturated solution of sodium chloride (5ꢃ30 mL),
then dried (MgSO4), and the solvent was evaporated. The product
was purified by column chromatography with hexane/EtOAc (19:1)
as the eluent to yield 442 mg (62% yield) of 35 as a colorless oil.
4-(2-Fluorophenoxy)phenoxyethyl thiocyanate (27). A solution of
26 (151 mg, 0.38 mmol) in anhydrous N,N-dimethylformamide
(3.0 mL) was treated with potassium thiocyanate (215.7 mg,
2.2 mmol). The reaction mixture was heated at 808C for 6 h and
was quenched as described for the preparation of 20. The residue
was purified by column chromatography with hexane/EtOAc (19:1)
as the eluent to produce 39.6 mg (36% yield) of 27 as a colorless
oil.
4-(4-Pyridyl)oxyphenoxyethyl tetrahydro-2H-pyran-2-yl ether
(28): Dimethyl sulfoxide (10.0 mL) was added to a mixture of 23
(2.145 g, 6.25 mmol), 4-hydroxypyridine (1.165 g, 12.3 mmol), cop-
per(I) iodide (115.0 mg, 0.6 mmol), 2-picolinic acid, (149.2 mg,
1.28 mmol), and potassium phosphate tribasic (2.565 g, 12.3 mmol)
as described for the preparation of 11. The reaction mixture was
stirred at 908C for 12 d. After the usual workup, the product was
purified by column chromatography with hexane/EtOAc (1:1) as
the eluent to produce 572 mg (29% yield) of 28 as a colorless oil.
2-Acetoxy-4-phenoxyphenoxyethanol (36):
A solution of 35
(420 mg, 1.2 mmol) in methanol (10 mL) was treated with pyridini-
um 4-toluenesulfonate (30 mg) as described for the preparation of
14. The residue was purified by column chromatography with
hexane/EtOAc (83:17) as the eluent to give 301 mg (92% yield) of
alcohol 36 as a colorless oil.
2-Acetoxy-4-phenoxyphenoxyethyl 4-toluenesulfonate (37):
A
4-(4-Pyridyl)oxyphenoxyethanol (29): A solution of 28 (556 mg,
1.76 mmol) in methanol (10 mL) was treated with pyridinium 4-tol-
uenesulfonate (90 mg) as depicted for the preparation of 14. The
residue was purified by column chromatography with EtOAc/
MeOH (49:1) as the eluent to give 240.1 mg (59% yield) of alcohol
29 as a colorless oil.
solution of alcohol 36 (280.2 mg, 1.03 mmol) in pyridine (3 mL)
was treated with 4-toluenesulfonyl chloride (383 mg, 2.0 mmol),
and the mixture was stirred at room temperature for 6 h. The reac-
tion was quenched as described for the preparation of 17. The
product was purified by column chromatography with hexane/
EtOAc (9:1) as the eluent to produce 417 mg of 37 (95% yield) as
a colorless oil.
4-(4-Pyridyl)oxyphenoxyethyl bromide (30): A mixture of N-bro-
mosuccinimide (187.0 mg, 1.05 mmol) and triphenylphosphine
(275.4 mg, 1.05 mmol) in anhydrous dichloromethane (20 mL)
cooled at 08C was treated with alcohol 29 (220.3 mg, 0.95 mmol),
and the reaction mixture was stirred at 08C for 6 h. The solvent
was evaporated, and the residue was purified by column chroma-
tography with CH2Cl2/MeOH (99:1) as the eluent to give 154.2 mg
(50% yield) of pure 30 as a colorless oil.
2-Acetoxy-4-phenoxyphenoxyethyl thiocyanate (38): A solution
of 37 (348 mg, 0.82 mmol) in anhydrous N,N-dimethylformamide
(3.0 mL) was treated with potassium thiocyanate (321.6 mg,
3.3 mmol). The reaction mixture was heated at 808C for 6 h and
was quenched as described for the preparation of 20. The residue
was purified by column chromatography with hexane/EtOAc (9:1)
as the eluent to produce 186.9 mg (71% yield) of 38 as a colorless
oil.
4-(4-Pyridyl)oxyphenoxyethyl thiocyanate (31): A solution of 30
(130 mg, 0.44 mmol) in anhydrous N,N-dimethylformamide (3.0 mL)
was treated with potassium thiocyanate (172.6 mg, 1.8 mmol). The
reaction mixture was heated at 808C for 6 h and was quenched as
described for the preparation of 20. The residue was purified by
column chromatography with CH2Cl2/MeOH (19:1) as the eluent to
produce 40.8 mg (34% yield) of 31 as a colorless oil.
4-Phenoxyphenoxyethyl amine (40): A solution of azide 39
(1.020 g, 4.0 mmol) in tetrahydrofuran (20 mL) was treated with tri-
phenylphosphine (2.308 g, 8.8 mmol). The reaction mixture was
stirred at room temperature for 4 h. Water (2.0 mL) was then
added, and the mixture was stirred for an additional hour. The sol-
vent was evaporated, and the residue was purified by column
chromatography with CH2Cl2/MeOH (19:1) as the eluent to give
706 mg (77% yield) of 40 as a colorless oil.
4-Phenoxyphenyl acetate (33): A solution of 4-phenoxyphenol
(32; 2.08 g, 11.2 mmol) in pyridine (5 mL) was treated with acetic
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
9
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
Tetraethyl
1-[(4-phenoxyphenoxyethylamino)ethyl]-1,1-bi-
with water/methanol (9:1) as the eluent to produce 159 mg (69%
sphosphonate (42): Amine 40 (345.1 mg, 1.5 mmol) was added to
a solution of 41 (300 mg, 1.5 mmol) in anhydrous dichloromethane
(10 mL) under an argon atmosphere. The reaction mixture was
stirred at room temperature for 4 h. The solvent was evaporated to
produce 651 mg (82% yield) of 42, which was used in next step
without further purification.
yield) of 48 as an amorphous solid.
3-Phenoxyphenoxyethylazide (50): A solution of tosylate 49
(999.6 mg, 2.60 mmol) in anhydrous N,N-dimethylformamide (5 mL)
was treated with sodium azide (845.1 mg, 13.0 mmol). The reaction
mixture was stirred at 808C for 2 h. The mixture was then allowed
to cool to room temperature, and water (50 mL) was added. The
mixture was extracted with dichloromethane (3ꢃ20 mL), and the
combined organic layers were washed with an aqueous saturated
solution of sodium chloride (5ꢃ20 mL) and water (2ꢃ20 mL), then
dried (MgSO4). The solvent was evaporated, and the product was
purified by column chromatography with hexane/EtOAc (97:3) as
the eluent to produce 590.1 mg (90% yield) of 50 as a colorless oil.
1-[(4-Phenoxyphenoxyethylamino)ethyl]-1,1-bisphosphonic acid
(43): Compound 42 (430.1 mg, 0.81 mmol) was treated with a con-
centrated aqueous solution of hydrochloric acid (3.0 mL). The re-
sulting mixture was heated at reflux for 24 h. The solvent was
evaporated, and the residue was crystallized from H2O/ethanol
(1:1) to give 274.1 mg (81% yield) of 52 as a white solid.
S-(2-(4-Phenoxyphenoxy)ethyl) ethanethioate (45): A solution of
tosylate 44 (1.90 g, 4.9 mmol) in anhydrous N,N-dimethylforma-
mide (10 mL) under an argon atmosphere was treated with potas-
sium thioacetate (1.23 g, 10.8 mmol). The reaction mixture was
stirred at 908C for 2 h. The solvent was evaporated, and the resi-
due was partitioned between water (50 mL) and dichloromethane
(50 mL). The aqueous phase was extracted with dichloromethane
(2ꢃ50 mL). The combined organic layers were dried (MgSO4), and
the solvent was evaporated. The product was purified by column
chromatography with hexane/EtOAc (19:1) as the eluent to pro-
duce 1.383 g (98% yield) of pure 45 as a colorless oil.
3-Phenoxyphenoxyethylamine (51): A solution of 50 (545.3 mg,
2.1 mmol) in tetrahydrofuran (10 mL) was treated with triphenyl-
phosphine (616.3 mg, 2.4 mmol). The reaction mixture was stirred
2 h at room temperature. Water (20 mL) was then added, and the
mixture was extracted with dichloromethane (3ꢃ20 mL). The com-
bined organic phases were washed with water (2ꢃ20 mL) and
dried (MgSO4), and the solvent was evaporated. The product was
purified by column chromatography with CH2Cl2/methanol (49:1)
as the eluent to produce 431.1 mg (90% yield) of 51 as a colorless
oil.
Tetraethyl
1-[(3-phenoxyphenoxyethylamino)ethyl]-1,1-bi-
2-(4-Phenoxyphenoxy)ethanethiol (46): A solution of compound
45 (1.35 g, 4.7 mmol) in tetrahydrofuran (8.0 mL) was added to
a mixture of lithium aluminum hydride (238 mg, 63 mmol) in anhy-
drous tetrahydrofuran (20.0 mL) cooled at 08C. The reaction mix-
ture was allowed to reach room temperature and was stirred for
3 h. The reaction was quenched by addition of ethyl acetate
(10 mL). The mixture was partitioned between an aqueous saturat-
ed solution of sodium potassium tartrate (50 mL) and dichlorome-
thane (50 mL). The aqueous layer was extracted with dichlorome-
thane (2ꢃ50 mL). The combined organic layers were dried
(MgSO4), and the solvent was evaporated. The residue was purified
by column chromatography with hexane/EtOAc (99:1) as the
eluent to give 1.064 g (92% yield) of 46 as a colorless oil.
sphosphonate (52): Amine 51 (350.8 mg, 1.5 mmol) was added to
a solution of 41 (451.0 mg, 1.5 mmol) in anhydrous dichlorome-
thane (10 mL) under an argon atmosphere. The reaction mixture
was stirred at room temperature overnight. The solvent was
evaporated to give 770 mg (97% yield) of 52, which was used in
the next step without further purification.
1-[(3-Phenoxyphenoxyethylamino)ethyl]-1,1-bisphosphonic acid
(53): Compound 52 (411.2 mg, 0.78 mmol) was treated with a con-
centrated aqueous solution of hydrochloric acid (2 mL). The result-
ing mixture was heated at reflux for 7 h. The solvent was evaporat-
ed, and the residue was crystallized from H2O/ethanol (1:1) to give
114 mg (35% yield) of 53 as an amorphous solid.
S-[3-Phenoxy)phenoxyethyl] ethanethioate (55): A solution of 54
(660 mg, 1.7 mmol) in anhydrous N,N-dimethylformamide (3.0 mL)
was treated with potassium thioacetate (392 mg, 3.4 mmol). The
reaction mixture was stirred at 808C for 3 h. The mixture was al-
lowed to cool to room temperature, and water (20 mL) was added.
The aqueous phase was extracted with dichloromethane (2ꢃ
30 mL), and the combined organic layers were washed with brine
(5ꢃ30 mL) and water (2ꢃ30 mL), then dried (MgSO4). The solvent
was evaporated. The residue was purified by column chromatogra-
phy with hexane/EtOAc (49:1) as the eluent to give 411 mg (83%
yield) of 55 as a colorless oil.
Tetraethyl
1-[4-phenoxyphenoxyeth-1-ylthio)ethyl]
1,1-bi-
sphosphonate (47): Triethylamine (139 mL, 101 mg, 1.0 mmol) and
thiol 46 (246 mg, 1.0 mmol) were added to a solution of 41
(300 mg, 1 mmol) in anhydrous dichloromethane (10 mL). The reac-
tion mixture was stirred at room temperature for 5 h. Water
(20 mL) was added, and the mixture was extracted with dichloro-
methane (3ꢃ10 mL). The combined organic layers were washed
with brine (20 mL) and dried (MgSO4), and the solvent was evapo-
rated to produce 336 mg (59% yield) of tetraethyl ester 47. The
product was used in the next step without further purification: col-
orless oil.
2-(3-Phenoxyphenoxy)ethanethiol (56): Potassium carbonate (an-
1-[4-Phenoxyphenoxyeth-1-ylthio)ethyl] 1,1-bisphosphonic acid
(48): A solution of the tetraethyl ester 47 (290 mg, 0.53 mmol) in
anhydrous dichloromethane (10 mL) was treated with bromotrime-
thylsilane (10 equiv) under an argon atmosphere. The reaction mix-
ture was stirred at room temperature for 48 h. Methanol (1.0 mL)
was then added, and the solvent was evaporated. The residue was
dissolved in methanol (10.0 mL), and the mixture was stirred at
room temperature for 24 h. The solvent was evaporated, and the
residue was redissolved in methanol and evaporated four times, to
complete the hydrolysis of remaining trimethylsilyl bromide and to
remove the recently formed hydrobromic acid. The residue was
purified by column chromatography (silica gel C18-reversed phase)
hydrous powder) was added to
a solution of 55 (957 mg,
3.3 mmol) in methanol (10 mL) with stirring, then water (3.0 mL)
was added to obtain complete solution. The reaction mixture was
stirred at room temperature for 40 min, and the solvent was
evaporated. The residue was dissolved in glacial acetic acid, and
zinc (1.8 g, 28 mmol) was added. The reaction mixture was heated
at reflux for 1 h at 1108C. The mixture was allowed to cool to
room temperature and was partitioned between water (30 mL) and
dichloromethane (30 mL). The organic phase was washed with
water (2ꢃ30 mL), then dried (MgSO4), and the solvent was evapo-
rated to yield 616 mg (75% yield) of 56 as a colorless oil, which
was used in the next step without further purification.
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
10
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
Tetraethyl 2-[3-(Phenoxy)phenoxyethylthio]ethyl-1,1-bisphosph-
onate (57): Thiol 56 (616 mg, 2.5 mmol) was added to a solution
of 41 (751 mg, 2.5 mmol) in anhydrous dichloromethane (10 mL).
The reaction mixture was stirred at room temperature for 16h.
Water (20 mL) was added, and the mixture was extracted with di-
chloromethane (3ꢃ10 mL). The combined organic layers were
dried (MgSO4), and the solvent was evaporated. The residue was
purified by column chromatography with CH2Cl2/methanol (49:1)
as the eluent to produce 1.1 g (81% yield) of 57 as a colorless oil.
uenesulfonate (30 mg) as described for the preparation of 14. The
residue was purified by column chromatography with hexane/
EtOAc (9:1) as the eluent to give 463.5 mg (85% yield) of alcohol
66 as a colorless oil.
2-(Naphthalen-2-yloxy)ethyl 4-toluenesulfonate (67): A solution
of alcohol 66 (450.3 mg, 2.45 mmol) in pyridine (5 mL) was treated
with 4-toluenesulfonyl chloride (1.422 g, 7.5 mmol), and the mix-
ture was stirred at room temperature for 4 h. The reaction was
worked up as described for the preparation of 17. The product
was purified by column chromatography with hexane/EtOAc (91:9)
as the eluent to produce 696.3 mg of 67 (83% yield) as a colorless
oil.
2-[3-(Phenoxy)phenoxyethylthio]ethyl-1,1-bisphosphonic
acid
(58): A solution of 57 (1.1 g, 2.0 mmol) in anhydrous dichlorome-
thane (10 mL) was treated with bromotrimethylsilane (3.1 g,
20.2 mmol) under an argon atmosphere. The reaction mixture was
stirred at room temperature for 48 h. Methanol (1.0 mL) was then
added, and the solvent was evaporated. The residue was dissolved
in methanol (8 mL), and the mixture was stirred at room tempera-
ture for 24 h. The solvent was evaporated, and the residue was re-
dissolved in methanol and evaporated four times. The residue was
purified by column chromatography (silica gel C18-reversed phase)
with water/methanol (1:1) as the eluent to produce 568 mg (65%)
of 58 as a yellow pale solid after lyophilization.
2-(Naphthalen-2-yloxy)ethyl thiocyanate (68): A solution of 67
(464.1 mg, 1.35 mmol) in anhydrous N,N-dimethylformamide
(3.0 mL) was treated with potassium thiocyanate (531 mg,
5.5 mmol). The reaction mixture was treated as described for the
preparation of 20. The residue was purified by column chromatog-
raphy with hexane/EtOAc (19:1) as the eluent to produce 170.3 mg
(55% yield) of 68 as a colorless oil.
2-(Naphthalen-1-yloxy)ethyl tetrahydro-2H-pyran-2-yl ether (70):
A solution of 69 (537.1 g, 3.82 mmol) in dimethyl sulfoxide (5.0 mL)
was treated with potassium hydroxide (503 mg, 8.9 mmol) and bro-
moethyl tetrahydropyranyl ether (1.1157 g, 5.3 mmol) as described
for the preparation of 35. The product was purified by column
chromatography with hexane/EtOAc (19:1) as the eluent to pro-
duce 675.7 mg (65% yield) as a colorless oil.
Phenoxyethylazide (60): A solution of 59 (1.1054 g, 3.78 mmol) in
anhydrous N,N-dimethylformamide (5 mL) was treated with sodium
azide (1.2290 g, 18.9 mmol). The reaction mixture was stirred at
808C for 2 h. The mixture was then allowed to cool to room tem-
perature, and water (50 mL) was added. The aqueous phase was
extracted with dichloromethane (2ꢃ20 mL), and the combined or-
ganic layers were washed with an aqueous saturated solution of
sodium chloride (5ꢃ20 mL) and water (2ꢃ20 mL), then dried
(MgSO4). The solvent was evaporated, and the residue was purified
by column chromatography with hexane/EtOAc (97:3) as the
eluent to give 498.4 mg (81% yield) of 60 as a colorless oil.
2-(Naphthalen-1-yloxy)ethanol (71): A solution of 70 (562.8 mg,
2.07 mmol) in methanol (10 mL) was treated with pyridinium 4-tol-
uenesulfonate (30 mg) as described for the preparation of 14. The
residue was purified by column chromatography with hexane/
EtOAc (9:1) as the eluent to give 315.6 mg (81% yield) of alcohol
71 as a colorless oil.
Phenoxyethylamine (61):
A solution of azide 60 (463.6 mg,
2.84 mmol) in tetrahydrofuran (10 mL) was treated with triphenyl-
phosphine (819.7 mg, 3.12 mmol) as described for the preparation
of 40. The residue was purified by column chromatography with
hexane/EtOAc (9:1) as the eluent to afford 260.8 mg (67% yield) of
61 as a yellow pale oil.
2-(Naphthalen-1-yloxy)ethyl 4-toluenesulfonate (72): A solution
of alcohol 71 (326.1 mg, 1.77 mmol) in pyridine (5 mL) was treated
with 4-toluenesulfonyl chloride (1.027 g, 5.4 mmol), and the mix-
ture was stirred at room temperature for 4 h. The reaction was
worked up as described for the preparation of 17. The product
was purified by column chromatography with hexane/EtOAc (9:1)
as the eluent to produce 545.5 mg of 72 (90% yield) as a colorless
oil.
Tetraethyl
1-[(Phenoxyethylamino)ethyl]-1,1-bisphosphonate
(62): Amine 61 (194.6 mg, 1.5 mmol) was added to a solution of 41
(452.9 mg, 1.5 mmol) in anhydrous dichloromethane (10 mL) under
an argon atmosphere. The reaction mixture was stirred at room
temperature overnight. The solvent was evaporated to give
574.6 mg (88% yield) of 62, which was used in the next step with-
out further purification.
2-(Naphthalen-1-yloxy)ethyl thiocyanate (73): A solution of 67
(445.4 mg, 1.29 mmol) in anhydrous N,N-dimethylformamide
(3.0 mL) was treated with potassium thiocyanate (550 mg,
5.7 mmol). The reaction mixture was treated as described for the
preparation of 20. The residue was purified by column chromatog-
raphy with hexane/EtOAc (19:1) as the eluent to yield 171.6 mg
(58% yield) of 73 as a colorless oil.
1-[(Phenoxyethylamino)ethyl]-1,1-bisphosphonic acid (63): Com-
pound 62 (547.6 mg, 1.25 mmol) was treated with a concentrated
aqueous solution of hydrochloric acid (4.0 mL). The mixture was
heated at reflux for 7h. The solvent was evaporated, and the resi-
due was crystallized from H2O/ethanol (1:1) to give 44.5 mg (11%
yield) of 63 as a white solid.
Drug screening
2-(Naphthalen-2-yloxy)ethyl tetrahydro-2H-pyran-2-yl ether (65):
A solution of 64 (1.0121 g, 7.02 mmol) in dimethyl sulfoxide
(10.0 mL) was treated with potassium hydroxide (877 mg,
15.6 mmol) and bromoethyl tetrahydropyranyl ether (1.7957 g,
8.6 mmol) as described for the preparation of 35. The product was
purified by column chromatography with hexane/EtOAc (19:1) as
the eluent to produce 917.6 mg (48% yield) of 65 as a colorless oil.
T. cruzi amastigote assays: These experiments were done as report-
ed by using tdTomato-labeled trypomastigotes[49] with the modifi-
cations described by Recher et al.[50] EC50 values were determined
by nonlinear regression analysis with SigmaPlot.
T. gondii tachyzoites assays: Experiments on T. gondii tachyzoites
were carried out as described previously[51] by using T. gondii tachy-
zoites expressing red fluorescent protein[52] with the modifications
described by Recher et al.[50] Plates were read with covered lids,
2-(Naphthalen-2-yloxy)ethanol (66): A solution of 65 (789.0 mg,
2.9 mmol) in methanol (10 mL) was treated with pyridinium 4-tol-
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
11
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
and both excitation (544 nm) and emission (590 nm) were read
from the bottom.
Keywords: antiprotozoal agents · drug discovery · enzymes ·
fluorine · inhibitors
Cytotoxicity for Vero cells: The cytotoxicity was tested by using the
Alamar BlueTM assay as described by Recher et al.[50]
[1] I. J. Blader, I. D. Manger, J. C. Boothroyd, J. Biol. Chem. 2001, 276,
24223–24231.
[2] M. Okomo-Adhiambo, C. R. A. Beattie, Infect. Immun. 2006, 74, 4254–
4265.
Computational methods
[3] Y. Li, S. Shah-Simpson, K. Okrah, A. T. Belew, J. Choi, K. L. Caradonna, P.
Padmanabhan, D. M. Ndegwa, M. R. Temanni, H. Corrada Bravo, N. M. El-
Sayed, B. A. Burleigh, PLoS Pathog. 2016, 12, e1005511.
[4] G. E. G. LiÇares, E. L. Ravaschino, J. B. Rodriguez, Curr. Med. Chem. 2006,
13, 335–360.
[5] C. G. K. Lꢄder, W. Bohne, D. Soldati, Trends Parasitol. 2001, 17, 460–463.
[6] E. A. A. Innes, Zoonoses Public Health 2010, 57, 1–7.
[7] D. E. Hill, S. Chirukandoth, J. P. Dubey, Anim. Health Res. Rev. 2005, 6,
41–61.
[8] S.-Y. Wong, J. S. Remington, Clin. Infect. Dis. 1994, 18, 853–862.
[9] G. N. Holland, Am. J. Ophthalmol. 2004, 137, 1–17.
[10] J. B. Rodriguez, S. H. Szajnman, Expert Opin. Ther. Pat. 2012, 22, 311–
333.
[11] I. Coppens, I. A. P. Sinai, K. A. Joiner, J. Cell Biol. 2000, 149, 167–180.
[12] F. S. Buckner, J. A. Urbina, Int. J. Parasitol. Drugs Drug Resist. 2012, 2,
236–242.
[13] J. A. Urbina, Drugs Future 2010, 35, 409–420.
[14] J. A. Urbina, J. L. Concepcion, S. Rangel, G. Visbal, R. Lira, Mol. Biochem.
Parasitol. 2002, 125, 35–45.
[15] G. M. Cinque, S. H. Szajnman, L. Zhong, R. Docampo, A. J. Schvartzapel,
J. B. Rodriguez, E. G. Gros, J. Med. Chem. 1998, 41, 1540–1554.
[16] J. A. Urbina, J. L. Concepcion, A. Montalvetti, J. B. Rodriguez, R. Docam-
po, Antimicrob. Agents Chemother. 2003, 47, 2047–2050.
[17] S. H. Szajnman, W. Yan, B. N. Bailey, R. Docampo, E. Elhalem, J. B. Rodri-
guez, J. Med. Chem. 2000, 43, 1826–1840.
Model building: The structure of TgFPPS was built with homology
modeling by using SwissModel,[53] based on the structure of
P. vivax FPPS (PDB ID: 3MAV), a protein showing the highest
degree of similarity with TgFPPS. The constructed TgFPPS structure
was aligned with the TcFPPS alendronate complex (PDB ID:
1YHM),[54] the structure of 58 was built on that of the alendronate,
and Mg2+ ions and the coligand isopentenyl pyrophosphate were
added. Charges for 58 and isopentenyl pyrophosphate were ob-
tained by using the RESP method at the Hartree Fock 6-31G* level,
and the compounds were parameterized with the Generalized
Amber force field. The complex was built by using the tleap
module in AmberTools 15.[55] The Amber FF14SB force field was
used to parameterize the protein structure, the charge was neutral-
ized by addition of seven Na+ ions, and the complex was solvated
with a box of TIP3P waters extending 10 ꢂ beyond the complex.
Molecular dynamics simulations: Simulations were run by using
the sander module in AmberTools 15. The Mg and phosphate units
of 58 were restrained during all simulations to their original coordi-
nates by using a 100 KcalmolÀ1 harmonic potential. The complex
was minimized for 1000 steps of steepest descent, followed by
1000 steps of conjugate gradient. It was then heated from 0 to
300 K for 20 ps by using Langevin dynamics, followed by an 80 ps
optimization with the NPT ensemble. The production run consisted
of a 10 ns MD simulation with the NVT ensemble.
[18] E. Elhalem, B. N. Bailey, R. Docampo, I. Ujvꢅry, S. H. Szajnman, J. B. Rodri-
guez, J. Med. Chem. 2002, 45, 3984–3999.
[19] G. G. LiÇares, S. Gismondi, N. O. Codesido, S. N. J. Moreno, R. Docampo,
J. B. Rodriguez, Bioorg. Med. Chem. Lett. 2007, 17, 5068–5071.
[20] P. D. Elicio, M. N. Chao, M. Galizzi, C. Li, S. H. Szajnman, R. Docampo,
S. N. J. Moreno, J. B. Rodriguez, Eur. J. Med. Chem. 2013, 69, 480–489.
[21] M. N. Chao, C. E. Matiuzzi, M. Storey, C. Li, S. H. Szajnman, R. Docampo,
S. N. J. Moreno, J. B. Rodriguez, ChemMedChem 2015, 10, 1094–1108.
[22] S. C. Nair, C. F. Brooks, C. D. Goodman, A. Strurm, G. I. McFadden, S. Sun-
driyal, J. L. Anglin, Y. Song, S. N. J. Moreno, B. Striepen, J. Exp. Med.
2011, 208, 1547–1559.
Energy calculations: Calculation of the binding energy was per-
formed by using the MMPBSA.py module in AmberTools 15. The
Generalized Born Surface Area model was used with igb=5, the
corresponding mbondi2 radii, and a salt concentration of 0.1m.
100 frames at 100 ps intervals were taken from the MD simulation.
Pairwise energy decomposition was carried out with the ligand de-
composed fragment-wise by using an in-house modified version of
MMPBSA.py.
[23] P. Grellier, A. Valentin, V. Millerioux, J. Schrevel, D. Rigomier, Antimicrob.
Agents Chemother. 1994, 38, 1144–1148.
Clustering analysis: Clustering analysis was performed with the
cpptraj module in AmberTools 15 on the MD simulation stripped of
solvent by using the dbscan algorithm, with an epsilon of 0.75 ꢂ
for the residues at 4 ꢂ from the ligand. The MD snapshot corre-
sponding to the most populated cluster was minimized for 1000
steps of steepest descent, followed by 1000 steps of conjugate
gradient, with the explicit waters maintained for the minimization
to assemble Figure 6.
[24] B. Pradines, M. Torrentino-Madamet, A. Fontaine, M. Henry, E. Baret, J.
Mosnier, S. Briolant, T. Fusai, C. Rogier, Antimicrob. Agents Chemother.
2007, 51, 2654–2655.
[25] K. Bessoff, A. Sateriale, K. K. Lee, C. D. Huston, Antimicrob. Agents Che-
mother. 2013, 57, 1804–1814.
[26] E. Cortez, A. C. Stumbo, M. Olieveira, H. S. Barbosa, L. Carvalho, Int. J.
Antimicrob. Agents 2009, 33, 185–186.
[27] Z. H. Li, S. Ramakrishnan, B. Striepen, S. N. J. Moreno, PLoS Pathog.
2013, 9, e1003665.
[28] P. Bazzini, C. G. Wermuth in The Practice of Medicinal Chemistry (Eds.:
C. G. Wermuth, D. Aldous, P. Raboisson, D. Rognan), Academic Press,
London, 2015, pp. 348–349.
[29] N. Shang, Q. Li, T. P. Ko, H. C. Chan, J. Li, Y. Zheng, C. H. Huang, F. Ren,
C. C. Chen, Z. Zhu, M. Galizzi, Z. Li, C. A. Rodrigues-Poveda, D. Gonzalez-
Pacanowska, P. Veiga-Santos, T. M. U. de Carvalho, W. de Souza, J. A.
Urbina, A. H. J. Wang, R. Docampo, K. Li, Y. L. Liu, E. Oldfield, R. T. Guo,
PLoS Pathog. 2014, 10, e1004114.
[30] J. A. Urbina, J. L. Concepcion, A. Caldera, G. Payares, C. Sanoja, T.
Otomo, H. Hiyoshi, Antimicrob. Agents Chemother. 2004, 48, 2379–2387.
[31] M. Sealey-Cardona, S. Cammerer, S. Jones, L. M. Ruiz-Pꢆrez, R. Brun, I. H.
Gilbert, J. A. Urbina, D. Gonzꢅlez-Pacanowska, Antimicrob. Agents Che-
mother. 2007, 51, 2123–2129.
Acknowledgements
This work was supported by grants from the Consejo Nacional de
Investigaciones Cientꢀficas y Tꢃcnicas (Argentina) (PIP 0797), the
Agencia Nacional de Promociꢂn Cientꢀfica y Tecnolꢂgica (Argenti-
na) (PICT 2012 #0457), and the Universidad de Buenos Aires
(20020130100223BA) to J.B.R., and from the US National Insti-
tutes of Health (NIH) (AI-107663 to R.D. and AI-102254 to
S.N.J.M.). The technical assistance of Carolina Exeni is greatly ap-
preciated.
[32] C. A. Rodrꢀgues-Poveda, D. Gonzꢅlez-Pacanowska, S. H. Szajnman, J. B.
Rodrꢀguez, Antimicrob. Agents Chemother. 2012, 56, 4483–4486.
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
12
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
[33] J. B. Rodriguez, B. N. Falcone, S. H. Szajnman, Expert Opin. Drug Discov-
ery 2016, 11, 307–320.
[34] F. Y. Lin, Y. L. Liu, K. Li, R. Cao, W. Zhu, J. Axelson, R. Pang, E. Oldfield, J.
Med. Chem. 2012, 55, 4367–4372.
[35] A. J. Schvartzapel, L. Zhong, R. Docampo, J. B. Rodriguez, E. G. Gros, J.
Med. Chem. 1997, 40, 2314–2322.
[49] A. M. C. Canavaci, J. M. Bustamante, A. M. Padilla, C. M. P. Brandan, L. J.
Simpson, D. Xu, C. L. Boehlke, R. L. Tarleton, PLoS Neglected Trop. Dis.
2010, 4, e740.
[50] M. Recher, A. P. Barboza, Z. H. Li, M. Galizzi, M. Ferrer-Casal, S. H. Szajn-
man, R. Docampo, S. N. J. Moreno, J. B. Rodriguez, Eur. J. Med. Chem.
2013, 60, 431–440.
[36] D. Maiti, S. L. Buchwald, J. Am. Chem. Soc. 2009, 131, 17423–17429.
[37] N. C. Bruno, S. L. Buchwald, Org. Lett. 2013, 15, 2876–2879.
[38] B. Bhayana, B. P. Fors, S. L. Buchwald, Org. Lett. 2009, 11, 3954–3957.
[39] B. P. Fors, D. A. Watson, M. R. Biscoe, S. L. Buchwald, J. Am. Chem. Soc.
2008, 130, 13552–13554.
[40] R. Meier, S. Strych, D. Trauner, Org. Lett. 2014, 16, 2634–2637.
[41] M. A. Miranda, CRC Handbook of Organic Photochemistry and Photobiolo-
gy (Eds.: W. M. Horspool, P. S. Song), CRC Press, Boca Raton, FL, 2004,
pp. 1–11.
[42] B. Meunier, Acc. Chem. Res. 2008, 41, 69–77.
[43] J. Lee, W. Huang, J. M. Broering, A. E. Barron, J. Seo, Bioorg. Med. Chem.
Lett. 2015, 25, 2849–2852.
[44] C. M. Dehnhardt, A. M. Venkatesan, Z. Chen, E. Delos-Santos, S. Ayral-Ka-
loustian, N. Brooijmans, K. Yu, I. Hollander, L. Feldberg, J. Lucas, R.
Mallon, Bioorg. Med. Chem. Lett. 2011, 21, 4773–4778.
[45] M. Ferrer-Casal, A. P. Barboza, S. H. Szajnman, J. B. Rodriguez, Synthesis
2013, 45, 2397–2404.
[51] M. Gubbels, C. Li, B. Striepen, Antimicrob. Agents Chemother. 2003, 47,
309–316.
[52] S. Agrawal, G. G. van Dooren, W. L. Beatty, B. Striepen, J. Biol. Chem.
2009, 284, 33683–33691.
[53] L. Bordoli, F. Kiefer, K. Arnold, P. Benkert, J. Battey, T. Schwede, Nat.
Protoc. 2008, 4, 1–13.
[54] S. B. Gabelli, J. S. McLellan, A. Montalvetti, E. Oldfield, R. Docampo, L. M.
Amzel, Proteins Struct. Funct. Bioinf. 2006, 62, 80–88.
[55] D. A. Case, J. T. Berryman, R. M. Betz, D. S. Cerutti, T. E. Cheatham III, T. A.
Darden, R. E. Duke, T. J. Giese, H. Gohlke, A. W. Goetz, N. Homeyer, S.
Izadi, P. Janowski, J. Kaus, A. Kovalenko, T. S. Lee, S. LeGrand, P. Li, T.
Luchko, R. Luo, B. Madej, K. M. Merz, G. Monard, P. Needham, H.
Nguyen, H. T. Nguyen, I. Omelyan, A. Onufriev, D. R. Roe, A. Roitberg, R.
Salomon-Ferrer, C. L. Simmerling, W. Smith, J. Swails, R. C. Walker, J.
Wang, R. M. Wolf, X. Wu, D. M. York, P. A. Kollman, AMBER 2015, Univer-
sity of California, San Francisco, 2015.
[46] H. Saneyoshi, T. Ochikubo, T. Mashimo, K. Hatano, Y. Ito, H. Abe, Org.
Lett. 2014, 16, 30–33.
[47] J. B. Rodriguez, Synthesis 2014, 46, 1129–1142.
[48] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug Delivery
Rev. 2001, 46, 3–26.
Manuscript received: October 4, 2016
Revised: October 20, 2016
Final Article published: && &&, 0000
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
13
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
M. N. Chao, C. Li, M. Storey, B. N. Falcone,
S. H. Szajnman, S. M. Bonesi, R. Docampo,
S. N. J. Moreno, J. B. Rodriguez*
&& – &&
Activity of Fluorine-Containing
Analogues of WC-9 and Structurally
Related Analogues against Two
Intracellular Parasites: Trypanosoma
cruzi and Toxoplasma gondii
Potent against parasites: Derivatives of
the squalene synthase inhibitor WC-9
were designed, synthesized, and evalu-
ated against the parasites Trypanosoma
cruzi and Toxoplasma gondii. Derivative
21 exhibited good potency against T.
gondii and similar potency to WC-9
against T. cruzi. Compound 58, a hybrid
inhibitor, has sub-micromolar activity
against T. gondii, which suggests a com-
bined inhibitory effect of the two func-
tional groups. Compound 58 was mod-
elled in the active site of T. gondii farne-
syl diphosphate synthase.
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
14
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!