Design, synthesis and biological evaluation of WC-9 analogs as antiparasitic agents

Article abstract of DOI:10.1016/j.ejmech.2013.09.009

As a part of our project pointed at the search of new safe chemotherapeutic and chemoprophylactic agents against parasitic diseases, several compounds structurally related to 4-phenoxyphenoxyethyl thiocyanate (WC-9), which were modified at the terminal aromatic ring, were designed, synthesized and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas disease) and Toxoplasma gondii, the etiological agent of toxoplasmosis. Most of the synthetic analogs exhibited similar antiparasitic activity being slightly more potent than the reference compound WC-9. For example, the nitro derivative 13 showed an ED₅₀ value of 5.2 μM. Interestingly, the regioisomer of WC-9, compound 36 showed similar inhibitory action than WC-9 indicating that para-phenyl substitution pattern is not necessarily required for biological activity. The biological evaluation against T. gondii was also very promising. The ED₅₀ values corresponding for 13, 36 and 37 were at the very low micromolar level against tachyzoites of T. gondii.

Full text of DOI:10.1016/j.ejmech.2013.09.009

European Journal of Medicinal Chemistry 69 (2013) 480e489
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of WC-9 analogs
as antiparasitic agents
Pablo D. Elicio a, María N. Chao a, Melina Galizzi b, Catherine Li b, Sergio H. Szajnman a,
,
Roberto Docampo b, Silvia N.J. Moreno b, Juan B. Rodriguez a
*
a Departamento de Química Orgánica and UMYMFOR (CONICETeFCEyN), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2,
Ciudad Universitaria, C1428EHA Buenos Aires, Argentina
b Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of our project pointed at the search of new safe chemotherapeutic and chemoprophylactic
agents against parasitic diseases, several compounds structurally related to 4-phenoxyphenoxyethyl
thiocyanate (WC-9), which were modified at the terminal aromatic ring, were designed, synthesized
and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American
trypanosomiasis (Chagas disease) and Toxoplasma gondii, the etiological agent of toxoplasmosis. Most of
the synthetic analogs exhibited similar antiparasitic activity being slightly more potent than the refer-
Received 31 May 2013
Received in revised form
3 September 2013
Accepted 5 September 2013
Available online 18 September 2013
ence compound WC-9. For example, the nitro derivative 13 showed an ED50 value of 5.2 mM. Interest-
Keywords:
ingly, the regioisomer of WC-9, compound 36 showed similar inhibitory action than WC-9 indicating that
para-phenyl substitution pattern is not necessarily required for biological activity. The biological eval-
uation against T. gondii was also very promising. The ED50 values corresponding for 13, 36 and 37 were at
the very low micromolar level against tachyzoites of T. gondii.
Antiparasitic agents
Trypanosoma cruzi
Toxoplasma gondii
WC-9 analogs
Squalene synthase inhibitors
Chagas disease
Ó 2013 Elsevier Masson SAS. All rights reserved.
Toxoplasmosis
1. Introduction
requirement of having at hand new safe drugs based on the
knowledge of the biochemistry and physiology of the responsible
American trypanosomiasis (Chagas disease) and toxoplasmosis
[1] are major parasitic diseases that affect millions of individuals
according to the World Health Organization [2,3]. The etiologic
agent of American trypanosomiasis is the hemoflagellated proto-
zoan Trypanosoma cruzi, which has a complex life cycle involving
blood-sucking Reduviid insects and mammals [4]. The occurrence
of American trypanosomiasis in countries where this disease is not
endemic has been attributed to transfusion of infected blood or
congenital transmission [5,6]. On the other hand, the opportunistic
pathogen Toxoplasma gondii causes a broad spectrum of disease but
most infections are asymptomatic [7]. This Apicomplexan parasite
has adopted an essential intracellular life style. The parasite actively
penetrates host cells, sets up a privileged compartment in which it
replicates and finally lyses the cell [8,9]. Chemotherapy for these
parasitic diseases is still deficient and mainly based on old or
empirically discovered drugs [10,11]. Therefore, there is an urgent
agents of these diseases.
Isoprenoids are essential compounds of the cellular machinery
of all organisms due to their roles in a variety of biological pro-
cesses. Several enzymes of this pathway in T. cruzi, involved in the
synthesis of sterols [12] and farnesyl diphosphate [13], and in
protein prenylation [14], have been reported to be excellent drug
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 [15,16]. T. gondii lacks the mevalonate pathway and uses
instead
a
prokaryotic-type 1-deoxy-D-xylulose-5-phosphate
(DOXP) pathway to make IPP and DMAPP [7]. The DOXP pathway
localizes to the apicoplast and is essential [17].
Trypanosomatids have a strict requirement for specific endog-
enous sterols for survival and cannot use the abundant supply of
cholesterol present in their mammalian hosts [18e20]. It has been
reported that ergosterol biosynthesis inhibitors with potent in vitro
* Corresponding author. Tel.: þ54 11 4576 3385; fax: þ54 11 4576 3346.
E-mail address: jbr@qo.fcen.uba.ar (J.B. Rodriguez).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.009

P.D. Elicio et al. / European Journal of Medicinal Chemistry 69 (2013) 480e489
481
pharmacophore [21e23,31,32]. However, pharmacokinetic prop-
erties of this representative drug still should be improved.
Although WC-9 is able to impair parasitemia in murine models of
Chagas disease the level of protection is not as efficient as keto-
conazole, used as a positive control [33]. This lack of efficacy may be
attributed to poor pharmacokinetic properties. In this context, we
have demonstrated that structural variations at the B ring have
influenced biological activity. For example, the introduction of a
fluorine atom at the B ring of WC-9 gives rise to compounds 2 and 3,
which have estimated log P values of 4.71 versus log P of 4.51 for
WC-9, indicating a better distribution between water/octanol. In
fact, these two drugs, 2 and 3, are significantly more potent than
WC-9 [32].
The key step for preparing either 2 or 3 is a coupling reaction
between a suitable substituted phenylboronic acid and a conve-
niently functionalized phenol [34,35]. However, this method suf-
fers from some disadvantages such as low reaction yields, the cost
of the phenylboronic acids, and the availability of diverse func-
tionalized products, which are impractical to access from the syn-
thetic point of view. The approach developed by Buchwald results
an interesting alternative to the expensive and commercially not
available phenylboronic acids. Therefore, it is possible to obtain
straightforwardly either O-arylation products of formula 6 or the N-
arylation compounds of formula 7 (Scheme 2) [36].
Fig. 1. Chemical structure of WC-9 (compound 1) and closely related analogs.
activity and special pharmacokinetic properties in mammals can
induce radical parasitological cure in animal models of both acute
and
chronic
experimental
Chagas
disease
[20].
4-
Phenoxyphenoxyethyl thiocyanate (1) known as WC-9 is an inter-
esting compound that presents IC50 values at the low nanomolar
range against the clinically more relevant form of T. cruzi (amasti-
gotes) (Fig. 1) [21e23]. WC-9 induces a dose dependent effect of
growth of the epimastigotes (EP strain) with a minimal inhibitory
concentration of 1
mM. The growth inhibitory effect of WC-9 is
associated with a depletion of the parasite endogenous sterols,
ergosterol and its 24-ethyl analog. An almost complete disappear-
ance of the parasite sterols is observed, with no accumulation of
sterol intermediates or precursors indicating a blockade of the
biosynthetic pathway at a pre-squalene level [24].
Squalene synthase (SQS) catalyzes the first committed step in
sterol biosynthesis, where a reductive dimerization of two mole-
cules of farnesyl pyrophosphate yields squalene. This enzyme is the
target of WC-9. Highly purified glycosomes and mitochondrial
membrane vesicles obtained from T. cruzi epimastigotes have been
used as enzyme source [25]. WC-9 is a potent inhibitor of both
glycosomal and mitochondrial T. cruzi SQS, with IC50 values of
88 nM and 129 nM. The doseeresponse curves for the activity of
WC-9 against TcSQS were consistent with non-competitive inhibi-
tion with Ki ¼ IC50; these Ki values are two to three orders of
magnitude lower that the Km of the substrates [24].
To date crystal structures of TcSQS are not available. However,
the X-ray crystallographic structure of WC-9 bound to dehy-
drosqualene synthase from Staphylococcus aureus has been pub-
lished [37]. This enzyme catalyzes dehydrosqualene formation, a
metabolite that is further transformed into staphyloxanthin. It has
been postulated that WC-9 might bind into the same hydrophobic
S2 pocket in TcSQS as it does in dehydrosqualene synthase keeping
the same polar interactions with the thiocyanate group [37].
3. Results and discussion
As WC-9 targets TcSQS, this fact suggested that WC-9, with its
thiocyanate group bearing an electrophilic carbon atom linked to
the hydrophobic 4-phenoxyphenoxyethyl moiety, could act by
mimicking the carbocationic transition state of the reaction leading
to the formation of the cyclopropylcarbinyl intermediate
presqualene-diphosphate 5 [26,27]. A similar rationale has been
advanced to explain the potent anti-SQS activity of arylquinuclidine
derivatives against both mammalian and T. cruzi SQS [28,29]. Based
on this hypothesis, it should be possible to design new and more
potent SQS inhibitors, using WC-9 as lead drug. The above results
justify the mechanism of action of 4-phenoxyphenoxy derivatives
at a molecular level, suggesting that these drugs represent prom-
ising SQS inhibitors, with potential antiparasitic and cholesterol-
lowering activity in humans (Scheme 1).
For the above reasons, attempts to improve the biological ac-
tivity of WC-9 would be conducted through a classical approach.
The preparation of the title compounds was performed employing
a Buchwald coupling reaction as a key step in all cases [36].
Structural variations at the B ring as well as the relative position of
the B ring to the main chain were contemplated. The nitro deriv-
ative 13 was synthesized starting from the already depicted tetra-
hydropyranyl derivative 8 [23], which on treatment with hydrogen
at 3 atm, in the presence of palladium on charcoal, afforded the free
phenol 9 in 48% yield. 9 was coupled with 1-iodo-3-nitrobenzene in
the presence of 5% cuprous iodide, 10% picolinic acid and potassium
phosphate to give 10 in 52% yield. This compound was deprotected
by treatment with pyridinium p-toluenesulfonate in methanol to
afford free alcohol 11, which was treated with tosyl chloride in
pyridine to afford the expected tosylate 12 in 88% yield. This
compound was further transformed into the thiocyanate derivative
13 by treatment with potassium thiocyanate in N,N-dime-
thylformamide at 100 ꢀC in 61% yield (Scheme 3).
It has been demonstrated that T. gondii does not synthesize
cholesterol and imports it from the host suggesting that inhibitors
of the host SQS could potentially inhibit T. gondii growth [30].
2. Rationale
The preparation of analogs of WC-9 bearing methoxy groups at
the B ring was outlined in Scheme 4 employing 4-iodophenol as
starting material. This compound was transformed into the tetra-
hydropyranyl ether derivative 14 by treatment with 2-bromoethyl
We have established a rigorous SAR studies on WC-9 chemical
structure finding that the phenoxyethyl thiocyanate moiety, which
is colored in red in Fig. 1, can be considered as the structure of the
Scheme 1. First step of the reductive dimerization of FPP.

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P.D. Elicio et al. / European Journal of Medicinal Chemistry 69 (2013) 480e489
Scheme 2. Buchwald coupling reaction to prepare either phenyl amines or phenyl ethers.
tetrahydro-2H-pyran-2-yl ether in a suspension of potassium hy-
droxide in dimethyl sulfoxide, according to a modified Williamson
procedure [38]. Buchwald coupling reaction of iodo derivative 14
either with 4-methoxyphenol or 3-methoxyphenol afforded 15 and
16 in moderately yields, respectively. Following the general strat-
egy each tetrahydropyranyl derivative 15 and 16 was deprotected
by treatment with pyridinium p-toluenesulfonate affording alco-
hols 17 and 18 in good yields, which were tosylated to give 19 and
20. On treatment with potassium thiocyanate, in separate experi-
ments, these compounds were converted into the title compounds
21 and 22, respectively.
An interesting structural variation was the replacement of the
terminal phenyl group by a pyridyl group where the nitrogen atom
occupied the 200 position. The key step in the preparation of this title
compound was the incorporation of the pyridyl unit. Therefore,
Buchwald coupling of 4-benzyloxyphenol with 3-bromopyridine
afforded 23 in a low but reproducible yield. The benzyl group of
23 was cleaved by treatment with hydrogen at 3 atm in the pres-
ence of palladium on charcoal to give the substituted phenol 24.
Similarly to the preparation of 21 and 22, incorporation of the
terminal ethoxy group through a Williamson procedure, followed
by tetrahydropyranyl cleavage and further tosylation and substi-
tution with the thiocyanate ion gave rise to the title compound 28
(Scheme 5).
In order to study whether the relative position of terminal
phenyl group might have influence on biological activity, two WC-9
analogs were envisioned where the phenyl groups were at the C-30
position rather than at C-40 one. The strategy to get these com-
pounds is presented in Scheme 4. Then, 3-iodophenol was reacted
with 2-bromoethyl tetrahydro-2H-pyran-2-yl ether according to
the already depicted Williamson procedure affording the
committed intermediate 29, which was coupled either with phenol
or 4-methoxyphenol gave rise to 30 and 31, respectively. Then,
following the general strategy, each of these compounds experi-
enced tetrahydropyranyl cleavage, tosylation and nucleophilic
displacement of the tosylate group by treatment with potassium
thiocyanate affording the title compounds 36 and 37, respectively
(Scheme 6).
drug WC-9, used as a positive control, under the same assay con-
ditions. The tetrahydropyranyl precursor 10 of 13 was devoid of
biological activity against T. cruzi (amastigotes) proliferation. This
observation was not in agreement with our previous results in
other closely related compounds, where the biological activity of
WC-9 analogs correlated quite well with the activity exhibited by
their synthetic tetrahydropyranyl ether precursors when bonded to
the same aromatic skeleton [22,23,32]. Compound 13 was also a
potent inhibitor of T. gondii (tachyzoites) growth possessing an ED50
values at the very low micromolar level (2.0
and 22 bearing electron donor groups proved to be effective growth
inhibitors of T. cruzi (amastigotes) exhibiting ED50 values of 6.1
and 4.6 M, respectively. 21 and 22 were also potent inhibitors of
T. gondii growth but to a slight lesser extent than 13 showing ED50
values of 3.8 M and 6.7 M, respectively. Pyridyl analog 28 showed
potent antiparasitic action having ED50 values of 7.4 M and 7.5 mM
mM). Compounds 21
mM
m
m
m
m
against T. cruzi and T. gondii, respectively. Its synthetic precursor 25
exhibited vanishing antiparasitic activity. Compounds 36 and 37
are regioisomers of WC-9 and 21, respectively where the terminal
ring is at the C-300 in lieu of C-400. Both of these compounds prac-
tically hold the antiparasitic activity of WC-9 (ED50s of 11.2
mM and
10.1 M against T. cruzi and 4.0 M and 2.9 M against T. gondii,
m
m
m
respectively). These data indicated that the para-aryl substitution
pattern would not be necessarily required for biological activity.
The results are presented in Table 1.
It can be concluded that, from the chemical point of view, the
Buchwald coupling reaction is a powerful tool to access to diverse
WC-9 derivatives not only those modified at the B ring as it is the
case of the present study, but also at the A ring in future works. In
addition, structural variations at the B ring, regardless whether
electron withdrawing groups or electron donor groups was present,
gave rise to potent inhibitors of T. cruzi proliferation exhibiting an
efficacy superior to our lead drug WC-9. These WC-9 analogs also
exhibited excellent prospective as anti-Toxoplasma agents because
they also exhibited potent inhibitory action against T. gondii
growth. The fact that WC-9 analogs have reasonable drug-like
character overcomes the approach to established a rigorous struc-
ture/activity relationship.
Biological activity of these WC-9 analogs was very promising.
The title compound 13 was a potent growth inhibitor of the intra-
cellular form of T. cruzi, which is the clinically more relevant
replicative form of the parasite. Certainly, this compound bearing
an electron withdrawing group at the C-300 position exhibited an
Our results are in agreement with previous reports indicating that
mevalonate pathway inhibitors are active against different Apicom-
plexan parasites such as Babesia divergens [39], Plasmodium falcipa-
rum [39,40], Cryptosporidium parvum [41], and T. gondii [42],
suggesting that these parasites, which lack a mevalonate pathway, are
dependenton host synthesis of precursors of the isoprenoid pathway.
ED50 value of 5.2 mM, being two-fold more potent than our lead
Scheme 3. Synthesis of the 200-nitro derivative of WC-9 at the B ring.

P.D. Elicio et al. / European Journal of Medicinal Chemistry 69 (2013) 480e489
483
Scheme 4. Synthesis of methoxy derivatives of WC-9 at the B ring.
4. Experimental section
The residue was purified by column chromatography (silica gel)
employing hexaneeEtOAc (9:1) as eluant to produce 1.056 g (48%
yield) of pure 9 as a colorless oil: Rf 0.41 (hexaneeEtOAc; 3:2); 1H
The glassware used in air and/or moisture sensitive reactions
was flame-dried and carried out under a dry argon atmosphere.
Unless otherwise noted, chemicals were commercially available
and were used without further purification. Solvents were distilled
before use. Tetrahydrofuran and ethyl ether were distilled from
sodium/benzophenone ketyl. Chloroform and acetonitrile was
distilled from phosphorus pentoxide. Anhydrous N,N-dime-
thylformamide was used as supplied from Aldrich.
NMR (200 MHz, CDCl3) d
1.55e1.74 (m, 6H, H-3000, H-4000, H-5000), 3.54
(m, 1H, H-6000a), 3.80 (m, 1H, H-6000b), 3.90 (m, 1H, H-1a), 4.01 (m, 1H,
H-1b), 4.11 (m, 2H, H-2), 4.72 (t, J ¼ 3.4 Hz, 1H, H-200), 6.77 (mAB, 4H,
aromatic protons); 13C NMR (50 MHz, CDCl3) 19.3 (C-400), 25.4 (C-
d
500), 30.5 (C-300), 62.3 (C-600), 66.0 (C-1), 68.1 (C-2), 99.0 (C-200), 115.9
(C-20), 116.0 (C-30), 149.8 (C-40), 152.7 (C-10). HRMS (ESI) calcd for
C
13H18O4Na [M þ Na]þ 261.1103; found 261.1102.
Nuclear magnetic resonance spectra were recorded using a
Bruker AM-500 MHz spectrometer. Chemical shifts are reported in
4.2. 4-(3-Nitrophenoxy)phenoxyethyl tetrahydro-2H-pyran-2-yl
parts per million (d) relative to tetramethylsilane. Coupling con-
ether (10)
stants are reported in Hertz. 13C NMR spectra were fully decoupled.
Splitting patterns are designated as s, singlet; d, doublet; t, triplet;
q, quartet.
A mixture of compound 9 (960 mg, 4.0 mmol), 1-iodo-3-
nitrobencene (836 mg, 3.4 mmol), copper(I) iodide (31.9 mg,
0.17 mmol), 2-picolinic acid, (41.3 mg, 0.33 mmol), and potassium
phosphate tribasic (1.425 g, 6.7 mmol) under anhydrous conditions
was evacuated and back-filled with argon. This sequence was
repeated twice. Then, dimethyl sulfoxide was added (15.0 mL) and
the reaction mixture was stirred vigorously at 80 ꢀC for 24 h. The
mixture was cooled to room temperature and partitioned between
ethyl acetate (20 mL) and water (20 mL). The aqueous layer was
extracted with ethyl acetate (2 ꢁ 20 mL). The combined organic
phases were washed with brine (5 ꢁ 50 mL), dried (MgSO4), and
the solvent was evaporated. The product was purified by column
chromatography (silica gel) employing hexaneeEtOAc (19:1) as
eluent to afford 635 mg (52% yield) of pure compound 10 as a
colorless oil: Rf 0.39 (hexaneeEtOAc; 7:3); 1H NMR (500.13 MHz,
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.
Melting points were determined using a FishereJohns appa-
ratus and are uncorrected.
Column chromatography was performed with E. Merck silica gel
plates (Kieselgel 60, 230e400 mesh). Analytical thin layer chro-
matography was performed employing 0.2 mm coated commercial
silica gel plates (E. Merck, DC-Aluminum sheets, Kieselgel 60 F254).
As judged from the homogeneity of the 1H, 13C, spectra of the
title compounds and HPLC analyses employing an IMC-Pack ODS-2
column 5
m
M, 250 ꢁ 10 mm eluting with methanolewater (9:1) at
3.00 mL/min with a variable wavelength detector (275 nM) indi-
CDCl3)
d
1.52e1.68 (m, 4H, H-4000, H-5000), 1.73e1.79 (m, 1H, H-3000a),
cated a purity >97%.
1.82e1.87 (m, 1H, H-3000b), 3.55 (m, 1H, H-6000a), 3.84 (ddd, J ¼ 11.0,
6.5, 4.2 Hz,1H, H-6000b), 3.91 (ddd, J ¼ 11.3, 8.2, 3.1 Hz,1H, H-1a), 4.08
(m, 1H, H-1b), 4.18 (m, 2H, H-2), 4.73 (t, J ¼ 3.5 Hz, 1H, H-2000), 6.97
(d, J ¼ 9.4 Hz, 2H, H-20), 7.01 (d, J ¼ 9.4 Hz, 2H, H-30), 7.27 (ddd,
J ¼ 8.3, 2.5, 0.9 Hz, 1H, H-500), 7.46 (t, J ¼ 8.2 Hz, 1H, H-600), 7.71 (t,
J ¼ 2.3 Hz, 1H, H-200), 7.88 (ddd, J ¼ 8.2, 2.2, 0.9 Hz, 1H, H-400); 13C
4.1. 4-Hydroxyphenoxyethyl tetrahydro-2H-pyran-2-yl ether (9)
A solution of 8 (3.032 g, 9.2 mmol) in ethyl acetate (40 mL) in the
presence of 5% palladium on charcoal (40 mg) was treated with
hydrogen at 3 atm. The reaction was stirred at room temperature
for 4 h. The mixture was filtered off and the solvent was evaporated.
NMR (125.77 MHz, CDCl3)
d
19.4 (C-4000), 25.4 (C-5000), 30.5 (C-3000),
Scheme 5. Preparation of nitrogen-containing thiocyanates at the B ring.

484
P.D. Elicio et al. / European Journal of Medicinal Chemistry 69 (2013) 480e489
Scheme 6. Preparation of the regioisomers of WC-9.
62.2 (C-6000), 65.8 (C-1), 67.9 (C-2), 99.0 (C-2000),111.7 (C-200),116.2 (C-
20), 117.0 (C-400), 121.4 (C-30), 123.2 (C-600), 130.2 (C-500), 148.3 (C-300),
150.2 (C-40), 156.2 (C-10), 159.6 (C-100). HRMS (ESI) calcd for
additional hour. The mixture was extracted with methylene chlo-
ride (50 mL) and the organic layer was washed with 5% HCl
(3 ꢁ 50 mL) and H2O (3 ꢁ 50 mL). The organic phase was dried
(MgSO4) and the solvent was evaporated. The product was purified
by column chromatography (silica gel) employing a mixture of
hexaneeEtOAc (19:1) as eluent to afford 171 mg of tosylate 12 (86%
yield) as a colorless oil. Rf 0.32 (hexaneeEtOAc, 7:3); 1H NMR
C
19H21O6NNa [M þ Na]þ 382.1267; found 382.1274.
4.3. 4-(3-Nitrophenoxy)phenoxyethanol (11)
(200 MHz, CDCl3)
d
2.46 (s, 3H, CH3), 4.01 (distorted t, J ¼ 4.0 Hz, 2H,
A solution of compound 10 (420 mg, 1.17 mmol) in methanol
(20 mL) was treated with pyridinium 4-toluensulfonate (30 mg).
The reaction mixture was stirred at room temperature overnight.
Then, water (50 mL) and the mixture was extracted with methylene
chloride (3 ꢁ 50 mL). The combined organic layers were washed
with brine (3 ꢁ 50 mL), dried (MgSO4), and the solvent was evap-
orated. The residue was purified by column chromatography
eluting with hexaneeEtOAc (7:3) to give 257.1 mg (80% yield) of
pure alcohol 11 as a white solid: Rf 0.13; 1H NMR (500.13 MHz,
H-2), 4.10 (distorted t, J ¼ 4.0 Hz, 2 H, H-1), 7.00 (m, 4H, aromatic
protons), 7.30 (m, 1H), 7.46 (t, J ¼ 8.2 Hz, 1H), 7.72 (t, J ¼ 2.3 Hz, 1H,
H-200), 7.82e7.92 (m, 5H, aromatic protons).
4.5. 4-(3-Nitrophenoxy)phenoxyethyl thiocyanate (13)
A solution of tosylate 12 (171 mg, 0.40 mmol) in anhydrous
dimethylformamide (5 mL) was treated with potassium thiocya-
nate (155 mg, 1.6 mmol). The reaction mixture was heated at 100 ꢀC
for 3 h. The mixture was allowed to cool to room temperature and
water (20 mL) was added. The aqueous phase was extracted with
methylene chloride (2 ꢁ 30 mL) and the combined organic layers
were washed with brine (5 ꢁ 30 mL) and water (2 ꢁ 30 mL). The
solvent was dried (MgSO4) and evaporated. The residue was puri-
fied by column chromatography (silica gel) eluting with hexanee
EtOAc (9:1) to give 77 mg (61% yield) of pure compound 13 as a
colorless oil; Rf 0.31 (hexaneeEtOAc, 7:3); 1H NMR (500.13 MHz,
CDCl3)
d
2.03 (t, J ¼ 6.0 Hz, 1H, OH), 4.00 (q, J ¼ 4.9 Hz, 2H, H-1), 4.11
(t, J ¼ 4.8 Hz, 1H, H-2), 6.97 (d, J ¼ 9.1 Hz, 2H, H-20), 7.02 (d,
J ¼ 9.4 Hz, 2H, H-30), 7.27 (ddd, J ¼ 8.3, 2.5, 0.9 Hz, 1H, H-500), 7.45 (t,
J ¼ 8.3 Hz, 1H, H-600), 7.71 (t, J ¼ 2.3 Hz, 1H, H-200), 7.89 (ddd, J ¼ 8.2,
2.1, 0.9 Hz, 1H, H-400); 13C NMR (125.77 MHz, CDCl3)
d 61.5 (C-1),
69.7 (C-2), 111.7 (C-200), 116.0 (C-20), 117.1 (C-400), 121.5 (C-30), 123.3
(C-600), 130.2 (C-500), 148.9 (C-40), 155.8 (C-10), 159.6 (C-100).
4.4. 4-(3-Nitrophenoxy)phenoxyethyl 4-Toluenesulfonate (12)
CDCl3)
d
3.36 (t, J ¼ 5.8 Hz, 2H, H-1), 4.34 (t, J ¼ 5.8 Hz, 2H, H-2), 6.97
(d, J ¼ 9.2 Hz, 2H, H-20), 7.04 (d, J ¼ 9.2 Hz, 2H, H-30), 7.29 (ddd,
J ¼ 8.3, 2.5, 0.9 Hz, 1H, H-500), 7.46 (t, J ¼ 8.2 Hz, 1H, H-600), 7.72 (t,
J ¼ 2.3 Hz, 1H, H-200), 7.90 (ddd, J ¼ 8.2, 2.2, 0.9 Hz, 1H, H-400); 13C
A solution of alcohol 11 (128 mg, 0.47 mmol) in pyridine (5 mL)
was treated with p-toluenesulfonyl chloride (266 mg, 1.4 mmol)
and the mixture was stirred at room temperature for 4 h. Then, 5%
HCl (50 mL) was added and the reaction mixture was stirred for an
NMR (125.77 MHz, CDCl3) d 33.3 (C-1), 66.4 (C-2), 111.7 (SCN), 111.8
(C-200), 116.2 (C-20), 117.2 (C-400), 121.5 (C-30), 123.4 (C-600), 130.3 (C-
500), 149.2 (C-300), 149.4 (C-40), 155.0 (C-10), 159.2 (C-100). HRMS (ESI)
calcd for C15H12O4N2SNa [M þ Na]þ 339.0415; found 339.0419.
Table 1
Biological activity of WC-9 analogs against T. cruzi (amastigotes), T. gondii (tachy-
zoites), and Vero cells.a
Compound
Trypanosoma cruzi Toxoplasma gondii Cytotoxicity on Vero cells
(amastigotes) (tachyzoites) ED50 M)
ED50 M)
(m
4.6. 4-Iodophenoxyethyl tetrahydro-2H-pyran-2-yl ether (14)
ED50
(
m
M)
(m
10
13
21
22
25
28
36
37
>10
5.2
6.1
4.5
>10
3.28 ꢂ 1.36
2.03 ꢂ 0.34
3.81 ꢂ 1.07
6.7 ꢂ 3.1
>50
>50
>100
>50
>50
>50
>50
>50
>50
e
A solution of iodophenol (5.00 g, 22.7 mmol) in dimethyl sulf-
oxide (5 mL) was treated with potassium hydroxide (2.82 g,
50.3 mmol). The suspension was stirred for 30 min at room tem-
perature. Then, bromoethyl tetrahydropyranyl ether (5.70 g,
27.3 mmol) was added; the reaction mixture was stirred at room
temperature overnight. The mixture was partitioned between
methylene chloride (30 mL) and water (30 mL). The aqueous phase
was extracted with methylene chloride (2 ꢁ 70 mL). The combined
organic layers were washed with a saturated solution of sodium
chloride (2 ꢁ 100 mL) and dried (MgSO4), and the solvent was
evaporated. The product was purified by column chromatography
(silica gel) eluting with hexane to afford 4.10 g (50% yield) of pure
3.25% at 10
7.5 ꢂ 0.8
mM
7.4 ꢂ 0.45
11.2
3.95 ꢂ 0.99
2.93 ꢂ 0.93
4.8 ꢂ 0.41
10.1
5.0 ꢂ 1.1
WC-9
Benznidazole 1.75 ꢂ 0.48
Atovaquone
0.032 ꢂ 0.019
e
a
Data are from one experiment in triplicate, or expressed as means ꢂ S.D. of two
(T. cruzi, 28) or three experiments (T. cruzi, benznidazole; T. gondii), each one in
triplicate.

P.D. Elicio et al. / European Journal of Medicinal Chemistry 69 (2013) 480e489
485
compound 14 as a colorless oil: Rf 0.40 (hexaneeEtOAc; 4:1); 1H
NMR (500.13 MHz, CDCl3)
1.51e1.86 (m, 6H, H-300, H-400, H-500),
d
55.7 (OCH3), 61.6 (C-1), 69.7 (C-2), 114.8 (C-300), 115.6 (C-20), 119.5
d
(C-30), 119.7 (C-200).
3.53 (m, 1H, H-6000a), 3.80 (ddd, J ¼ 11.2, 6.4, 4.1 Hz, 1H, H-600b), 3.89
(ddd, J ¼ 11.2, 8.1, 3.2 Hz, 1H, H-1a), 4.04 (m, 1H, H-1b), 4.11 (t,
J ¼ 3.8 Hz, 1H, H-2a), 4.12 (t, J ¼ 4.4 Hz, 1H, H-2b), 4.69 (t, J ¼ 3.7 Hz,
1H, H-200), 6.71 (t, J ¼ 9.2 Hz, 2H, H-20), 7.54 (t, J ¼ 9.2 Hz, 2H, H-30);
4.10. 4-(3-Methoxyphenoxy)phenoxyethanol (18)
To a solution of 16 (430.1 mg, 0.8 mmol) in methanol (20 mL)
was treated with pyridinium 4-toluensulfonate (30 mg). The reac-
tion mixture was stirred at room temperature overnight and was
treated as depicted for the preparation of 11. The product was pu-
rified by column chromatography (silica gel) eluting with hexanee
EtOAc (17:3) to afford 240 mg (72% yield) of pure 18 as a colorless
oil: Rf 0.18 (hexaneeEtOAc, 7:3); 1H NMR (500.13 MHz, CDCl3)
13C NMR (125.77 MHz, CDCl3) 19.3 (C-400), 25.4 (C-500), 30.5 (C-300),
d
62.2 (C-600), 65.7 (C-1), 67.5 (C-2), 82.9 (C-40), 99.0 (C-200), 117.1 (C-
20), 138.1 (C-30), 158.8 (C-10).
4.7. 4-(4-Methoxyphenoxy)phenoxyethyl tetrahydro-2H-pyran-2-
yl ether (15)
d
3.77 (s, 3H, OCH3), 3.97 (dist. t, J ¼ 4.5 Hz, 2H, H-1), 4.08 (dist. t,
J ¼ 4.5 Hz, 2H, H-2), 6.51 (t, J ¼ 2.1 Hz, 1H, H-200), 6.52 (ddd, J ¼ 8.3,
2.3, 0.9 Hz, 1H, H-600), 6.60 (ddd, J ¼ 8.3, 2.3, 0.9 Hz, 1H, H-400), 6.90
(d, J ¼ 9.1 Hz, 2H, H-20), 6.99 (d, J ¼ 9.2 Hz, 2H, H-30), 7.19 (t,
A mixture of p-methoxyphenol (217.3 mg, 1.75 mmol), com-
pound 14 (508.0 mg, 1.46 mmol), copper(I) iodide (13.9 mg,
0.073 mmol), 2-picolinic acid, (18.0 mg, 0.15 mmol), and potassium
phosphate tribasic (619.4 mg, 2.9 mmol) was treated as depicted for
the preparation of 10. Purification of the product by column chro-
matography (silica gel) eluting with hexaneeEtOAc (19:1) afforded
211 mg (42% yield) of pure 15 as a colorless oil: Rf 0.54 (hexanee
J ¼ 8.5 Hz, 1H, H-500); 13C NMR (125.77 MHz, CDCl3)
d 55.3 (OCH3),
61.5 (C-1), 69.7 (C-2), 103.8 (C-200), 108.1 (C-400), 109.8 (C-600), 115.6
(C-20), 121.0 (C-30), 130.0 (C-500), 150.3 (C-40), 155.0 (C-10), 159.6 (C-
100), 160.9 (C-300).
EtOAc); 1H NMR (500.13 MHz, CDCl3)
d
1.51e1.86 (m, 6H, H-3000, H-
4000, H-5000), 3.53 (m, 1H, H-6000a), 3.78 (s, 3H, OCH3), 3.80 (ddd,
J ¼ 11.2, 6.5, 4.4 Hz,1H, H-6000b), 3.90 (ddd, J ¼ 11.4, 8.3, 3.1 Hz,1H, H-
1a), 4.04 (m, 1H, H-1b), 4.12 (m, 2H, H-2), 4.71 (t, J ¼ 3.7 Hz, 1H, H-
200), 6.84 (t, J ¼ 9.2 Hz, 2H, aromatic protons), 6.89 (mAB, 4H, aro-
matic protons), 6.91 (t, J ¼ 9.2 Hz, 2H, aromatic protons); 13C NMR
4.11. 4-(4-Methoxyphenoxy)phenoxyethyl 4-Toluenesulfonate (19)
To a solution of 17 (170 mg, 0.65 mmol) in pyridine (5 mL) was
added p-toluenesulfonyl chloride (373.4 mg, 1.96 mmol) following
the method of the preparation described for 12. The product was
purified by column chromatography (silica gel) eluting with hex-
aneeEtOAc (9:1) to give 104 mg (39% yield) of pure 19 as a
colorless oil: Rf 0.44 (hexaneeEtOAc, 7:3); 1H NMR (200 MHz,
(125.77 MHz, CDCl3)
d
19.3 (C-4000), 25.4 (C-5000), 30.5 (C-3000), 55.6
(OCH3), 62.2 (C-6000), 65.9 (C-1), 68.0 (C-2), 99.0 (C-2000), 114.7 (C-300),
115.7 (C-20), 119.4 (C-30), 119.5 (C-200), 151.5 (C-40), 151.7 (C-100), 154.6
(C-10), 155.3 (C-400).
CDCl3)
d 2.45 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 4.11 (m, 2H, H-1),
4.34 (m, 2H, H-2), 6.73 (d, J ¼ 8.9 Hz, 2H), 6.89 (m, 7H, aromatic
protons), 7.32 (d, J ¼ 8.3 Hz, 2H, H-3000), 7.82 (d, J ¼ 8.3 Hz, 2H, H-
2000); HRMS (ESI) calcd for C22H22O6Na [M þ Na]þ 437.1035; found
437.1014.
4.8. 4-(3-Methoxyphenoxy)phenoxyethyl tetrahydro-2H-pyran-2-
yl ether (16)
A mixture of m-methoxyphenol (287.5 mg, 2.3 mmol), com-
pound 14 (672.0 mg, 1.9 mmol), copper(I) iodide (18.4 mg,
0.010 mmol), 2-picolinic acid, (23.7 mg, 0.19 mmol), and potassium
phosphate tribasic (819.4 mg, 3.9 mmol) was treated as depicted for
the preparation of 10. The crude was purified by column chroma-
tography (silica gel) eluting with hexaneeEtOAc to give 437 mg
(66% yield) of pure compound 16 as a colorless oil: Rf 0.55 (hexanee
4.12. 4-(3-Methoxyphenoxy)phenoxyethyl 4-Toluenesulfonate (20)
A solution of alcohol 18 (240 mg, 0.92 mmol) in pyridine (5 mL)
was treated with p-toluenesulfonyl chloride (527.4 mg, 2.77 mmol)
following a similar method as depicted for the preparation of 12.
Evaporation of the solvent afforded 269 mg (71% yield) of 20 as a
white solid: mp ¼ 77 ꢀC; Rf 0.40 (hexaneeEtOAc, 7:3); 1H NMR
EtOAc, 7:3); 1H NMR (500.13 MHz, CDCl3) 1.51e1.86 (m, 6H, H-3000
d ,
H-4000, H-5000), 3.53 (m, 1H, H-6000a), 3.76 (s, 3H, OCH3), 3.82 (ddd,
J ¼ 11.1, 6.3, 4.2 Hz, 1H, H-6000b), 3.91 (ddd, J ¼ 11.2, 8.2, 3.1 Hz, 1H, H-
1a), 4.06 (m, 1H, H-1b), 4.15 (m, 2H, H-2), 4.72 (t, J ¼ 3.6 Hz, 1H, H-
200), 6.51 (m, 2H, aromatic protons), 6.59 (ddd, J ¼ 8.2, 2.3,1.0 Hz,1H,
aromatic proton), 6.91 (t, J ¼ 9.2 Hz, 2H, aromatic protons), 6.98 (t,
J ¼ 9.2 Hz, 2H, aromatic protons), 7.18 (t, J ¼ 8.5 Hz, 1H, aromatic
(500.13 MHz, CDCl3) d 2.45 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 4.14 (m,
2H, H-1), 4.36 (m, 2H, H-2), 6.50 (m, 2H, aromatic protons), 6.60 (m,
1H, aromatic proton), 6.77 (d, J ¼ 9.1 Hz, 2H, H-30), 6.94 (d,
J ¼ 9.0 Hz, 2H, H-20), 7.18 (t, J ¼ 8.5 Hz, 1H, aromatic proton), 7.85 (d,
J ¼ 8.3 Hz, 2H, H-3000), 7.83 (d, J ¼ 8.3 Hz, 2H, H-2000); 13C NMR
(125.77 MHz, CDCl3) d 21.6 (CH3), 55.3 (OCH3), 66.0 (C-1), 68.1 (C-2),
proton); 13C NMR (125.77 MHz, CDCl3)
d
19.4 (C-4000), 25.4 (C-5000),
103.8 (C-200), 108.2 (C-400), 109.9 (C-600), 115.7 (C-20), 120.9 (C-30),
128.0 (C-2000), 129.9(C-500), 130.0 (C-3000), 132.9 (C-4000), 145.0 (C-1000),
150.6 (C-40), 154.3 (C-10), 159.5 (C-100), 160.9 (C-300).
30.5 (C-3000), 55.3 (OCH3), 62.2 (C-6000), 65.9 (C-1), 68.0 (C-2), 99.0 (C-
2000),103.7 (C-200),108.0 (C-400),109.7 (C-600),115.8 (C-20),120.9 (C-30),
130.0 (C-500), 150.0 (C-40), 155.0 (C-10), 159.8 (C-100), 160.9 (C-300).
4.13. 4-(4-Methoxyphenoxy)phenoxyethyl thiocyanate (21)
4.9. 4-(4-Methoxyphenoxy)phenoxyethanol (17)
A solution of tosylate 19 (50 mg, 0.12 mmol) in anhydrous
dimethylformamide (5 mL) was treated with potassium thiocya-
nate (47 mg, 0.48 mmol). The product was purified by column
chromatography (silica gel) eluting hexaneeEtOAc (9:1) as eluent
to afford 23 mg (64% yield) of pure 21 as a white solid: mp ¼ 60e
A solution of 15 (234.9 mg, 0.68 mmol) in methanol (20 mL) was
treated with pyridinium 4-toluensulfonate (30 mg). The reaction
mixture was stirred at room temperature overnight and was
quenched as described for the preparation of 11. Evaporation of the
solvent afforded 174.0 mg (98% yield) of alcohol 17 as white solid
that was used in the next step without further purification:
mp ¼ 104e105 ꢀC; Rf 0.19 (hexaneeEtOAc); 1H NMR (500.13 MHz,
62 ꢀC; 1H NMR (500.13 MHz, CDCl3)
d
3.32 (t, J ¼ 5.8 Hz, 2H, H-1),
3.79 (s, 3H, OCH3), 4.28 (t, J ¼ 5.8 Hz, 2H, H-1), 6.87 (m, 4H, aromatic
protons), 6.92 87 (m, 4H, aromatic protons); 13C NMR (125.77 MHz,
CDCl3)
d
3.79 (s, 3H, OCH3), 3.96 (m, 2H, H-1), 4.06 (m, 2H, H-2),
CDCl3)
d
33.3 (C-1), 55.6 (OCH3), 66.5 (C-2), 111.7 (SCN), 114.8 (C-300),
6.85e6.93 (m, 8H, aromatic protons); 13C NMR (125.77 MHz, CDCl3)
115.4 (C-20), 115.9 (C-30),119.4 (C-200),151.1 (C-40), 152.7 (C-100),153.4

486
P.D. Elicio et al. / European Journal of Medicinal Chemistry 69 (2013) 480e489
(C-10), 155.5 (C-400). HRMS (ESI) calcd for C16H15O3NSNa [M þ Na]þ
aromatic protons), 8.31 (m, 1H, aromatic proton), 8.36 (m, 1H, ar-
324.0670; found 324.0673.
omatic proton); 13C NMR (50 MHz, CDCl3) 19.4 (C-4000), 25.4 (C-
d
5000), 30.5 (C-3000), 62.2 (C-6000), 65.8 (C-1), 68.0 (C-2), 99.0 (C-2000),
116.1 (C-20), 120.7 (C-30), 124.2 (C-600, C-500), 140.3 (C-200), 143.4 (C-
400), 149.3 (C-40).
4.14. 4-(3-Methoxyphenoxy)phenoxyethyl thiocyanate (22)
To a solution of tosylate 20 (269 mg, 0.65 mmol) in anhydrous
dimethylformamide (5 mL) was added potassium thiocyanate
(252.3 mg, 2.6 mmol) according to the general procedure. The
crude was purified by column chromatography (silica gel)
employing a mixture of hexaneeEtOAc (7:3) as eluent to give
63.1 mg (32% yield) as a colorless oil: 1H NMR (500.13 MHz,
4.18. 4-(Pyridin-3-yloxy)phenoxyethanol (26)
A solution of compound 25 (457 mg, 1.45 mmol) in methanol
(20 mL) was treated with 4-toluensulfonic acid (50 mg). The reac-
tion mixture was stirred at room temperature overnight. After the
usual treatment, the product was purified by column chromatog-
raphy (silica gel) eluting with hexaneeEtOAc (7:3) to afford 160 mg
(47% yield) of pure 26 as a white solid: mp ¼ 73e75 ꢀC; Rf 0.09
CDCl3)
d
3.34 (t, J ¼ 5.8 Hz, 2H, H-1), 3.77 (s, 3H, OCH3), 4.30 (t,
J ¼ 5.7 Hz, 2H, H-2), 6.52 (m, 2H, aromatic protons), 6.61 (m, 1H,
aromatic proton), 6.90 (d, J ¼ 9.1 Hz, 2H, H-30), 7.00 (d, J ¼ 9.2 Hz,
2H, H-20), 7.19 (t, J ¼ 8.5 Hz, 1H, aromatic proton); 13C NMR
(hexaneeEtOAc, 1:1); 1H NMR (500.13 MHz, CDCl3)
d 3.98 (dist. t,
(125.77 MHz, CDCl3)
d
33.3 (C-1), 55.3 (OCH3), 66.4 (C-2), 103.9
J ¼ 4.6 Hz, 2H, H-1), 4.09 (dist. t, J ¼ 4.5 Hz, 2H, H-2), 6.94 (d,
J ¼ 9.2 Hz, 2H, H-30), 7.00 (d, J ¼ 9.2 Hz, 2H, H-20), 7.24 (m, 2H,
aromatic protons), 8.32 (m, 1H, aromatic proton), 8.36 (m, 1H, ar-
(C-200), 108.3 (C-400), 109.9 (C-600), 111.7 (SCN), 115.9 (C-20), 120.9
(C-30), 130.1 (C-500), 150.9 (C-40), 154.1 (C-10), 159.4 (C-100), 160.9
(C-300). HRMS (ESI) calcd for C16H16O3NS [M þ H]þ 302.0845;
found 302.0856.
omatic proton); 13C NMR (125.77 MHz, CDCl3)
d 61.5 (C-1), 69.7 (C-
2), 115.9 (C-20), 120.9 (C-30), 124.1 (C-600), 124.5 (C-500), 140.1 (C-200),
143.4 (C-400), 149.5 (C-40), 155.5 (C-10, C-100).
4.15. 4-(Pyridin-3-yloxy)phenoxyethyl benzyl ether (23)
4.19. 4-(Pyridin-3-yloxy)phenoxyethyl 4-Toluenesulfonate (27)
A mixture of 4-benzyloxyphenol (2.00 g, 10.0 mmol), 3-
bromopyridine (1.58 g, 10.0 mmol), copper(I) iodide (95.2 mg,
0.5 mmol), 2-picolinic acid, (123 mg, 1.0 mmol), and potassium
phosphate tribasic (4.25 g, 20 mmol). The mixture was reacted
similarly as described for the preparation of 10. The product was
purified by column chromatography (silica gel) eluting with hex-
aneeEtOAc (9:1) to afford 912 mg (33% yield) of pure 23 as a
yellowish solid: mp ¼ 64e65 ꢀC; Rf 0.18 (hexaneeEtOAc, 7:3); 1H
A solution of alcohol 26 (154 mg, 0.67 mmol) in pyridine (5 mL)
was treated with p-toluenesulfonyl chloride (380.9 mg, 2.0 mmol)
and the mixture was stirred at room temperature for 6 h according
to the method of preparation depicted for 12. The product was
purified by column chromatography eluting with a mixture of
hexaneeEtOAc (3:1) to give 118 mg (46% yield) of pure 27 as a
colorless oil: Rf 0.25 (hexaneeEtOAc, 2:3); 1H NMR (200 MHz,
NMR (500.13 MHz, CDCl3)
d
5.06 (s, 2H, H-1), 6.99 (mAB, 4H, aro-
CDCl3) d 2.45 (s, 3H, CH3), 4.15 (m, 2H, H-1), 4.37 (m, 2H, H-2), 6.80
matic protons), 7.27 (m, 2H, aromatic protons), 7.32e7.45 (m, 5H,
(d, J ¼ 9.0 Hz, 2H, H-20) 6.96 (d, J ¼ 9.2 Hz, 2H, H-30), 7.23 (m, 2H,
aromatic protons), 7.35 (d, J ¼ 8.0 Hz, 2H, H-3000), 7.83 (d, J ¼ 8.0 Hz,
2H, H-2000), 8.34 (m, 2H, aromatic protons).
aromatic protons), 8.32 (m, 1H, aromatic proton), 8.36 (m, 1H, ar-
omatic proton); 13C NMR (125.77 MHz, CDCl3)
d 70.5 (C-1), 116.2 (C-
20), 120.8 (C-30), 124.2 (C-600), 124.8 (C-500), 127.5 (C-2000), 128.1 (C-
4000), 128.6 (C-3000), 136.8 (C-1000), 139.7 (C-200), 142.9 (C-400), 149.2 (C-
40), 155.2 (C-10), 155.7 (C-100).
4.20. 4-(Pyridin-3-yloxy)phenoxyethyl thiocyanate (28)
To a solution of 27 (118 mg, 0.31 mmol) in N,N-dime-
thylformamide (5 mL) was added potassium thiocyanate (118 mg,
1.2 mmol). The reaction mixture was treated according to the
general procedure. The product was purified by column chro-
matography (silica gel) eluting with hexaneeEtOAc (3:1) to
afford 53.3 mg (63% yield) of 28 as a colorless oil: Rf 0.30 (hex-
4.16. 4-(Pyridin-3-yloxy)phenol (24)
A solution of 23 (907.6 g, 3.3 mmol) in ethyl acetate (30vmL) in
the presence of 5% palladium on charcoal (30 mg) was treated with
hydrogen at 3 atm. The reaction was stirred at room temperature
for 2 h. The mixture was worked-up as described for the prepara-
tion of compound 9 to afford 582 mg (95% yield) of pure 24 as a
white solid. This compound was used as such in the next step:
mp ¼ 134e136 ꢀC; Rf 0.18 (hexaneeEtOAc, 3:2).
aneeEtOAc; 2:3); 1H NMR (500.13 MHz, CDCl3)
d 3.35 (t,
J ¼ 5.8 Hz, 2H, H-1), 4.32 (t, J ¼ 5.8 Hz, 2H, H-2), 6.94 (d,
J ¼ 9.2 Hz, 2H, H-30), 7.01 (d, J ¼ 9.2 Hz, 2H, H-20), 7.23 (m, 2H,
aromatic protons), 8.24 (m, 1H, aromatic proton), 8.37 (m, 1H,
aromatic proton); 13C NMR (125.77 MHz, CDCl3)
d 33.3 (C-1), 66.4
4.17. 4-(Pyridin-3-yloxy)phenoxyethyl tetrahydro-2H-pyran-2-yl
ether (25)
(C-2), 111.7 (SCN), 116.1 (C-20), 120.8 (C-30), 124.0 (C-600), 124.4 (C-
500), 140.5 (C-200), 143.8 (C-400), 150.2 (C- 40), 154.5 (C-10), 154.7 (C-
100). HRMS (ESI) calcd for C14H13O2N2S [M þ H]þ 273.0698; found
273.0702.
A solution of 24 (582 mg, 3.1 mmol) in dimethyl sulfoxide (5 mL)
was treated with potassium hydroxide (698 mg, 12.4 mmol). The
suspension was stirred for 30 min at room temperature. Then,
bromoethyl tetrahydropyranyl ether (975 mg, 4.7 mmol) was
added. The reaction mixture was treated as depicted for the prep-
aration of 14. The product was purified by column chromatography
(silica gel) employing a mixture of hexaneeEtOAc (4:1) as eluent to
afford 457 mg (47% yield) of pure 25 as a colorless oil: Rf 0.33
4.21. 2-(3-Iodophenoxy)ethyl tetrahydro-2H-pyran-2-yl ether (29)
A solution of 3-iodophenol (5 g, 227 mmol) in dimethyl
sulfoxide (5 mL) was treated with potassium hydroxide (2.54 g,
454 mmol). The suspension was stirred for 30 min at room
temperature. Then, bromoethyl tetrahydropyranyl ether (4.75 g,
227 mmol) was added; the reaction mixture was stirred at room
temperature overnight. The mixture was partitioned between
methylene chloride (30 mL) and water (30 mL). The aqueous
phase was extracted with methylene chloride (2 ꢁ 70 mL). The
combined organic layers were washed with a saturated solution
(hexaneeEtOAc, 1:1); 1H NMR (500.13 MHz, CDCl3)
d 1.50e1.88 (m,
6H, H-3000, H-4000, H-5000), 3.54 (m, 1H, H-6000a), 3.82 (ddd, J ¼ 11.2, 6.4,
4.2 Hz,1H, H-6000b), 3.91 (ddd, J ¼ 11.3, 8.4, 3.0 Hz,1H, H-1a), 4.06 (m,
1H, H-1b), 4.15 (m, 2H, H-2), 4.72 (t, J ¼ 3.6 Hz, 1H, H-200), 6.94 (d,
J ¼ 9.5 Hz, 2H, H-30), 6.98 (d, J ¼ 9.2 Hz, 2H, H-20), 7.21 (m, 2H,

P.D. Elicio et al. / European Journal of Medicinal Chemistry 69 (2013) 480e489
487
of sodium chloride (2 ꢁ 100 mL) and dried (MgSO4), and the
4.24. 3-Phenoxyphenoxyethanol (32)
solvent was evaporated. The residue was purified by column
chromatography (silica gel) eluting with hexane to afford 3.12 g
(38% yield) of pure compound 29 as a colorless oil: Rf 0.54
To a solution of compound 30 (130 mg, 0.41 mmol) in meth-
anol (20 mL) was added pyridinium p-toluenesulfonate (10 mg).
The reaction mixture was stirred at room temperature for 14 h.
The mixture was partitioned between water (50 mL) and methy-
lene chloride (50 mL). The aqueous phase was extracted with
methylene chloride (2 ꢁ 30 mL) and the combined organic layers
were washed with brine (2 ꢁ 50 mL), dried (MgSO4), and the
solvent was evaporated to give 92 mg (89% yield) of pure alcohol
32 as a colorless oil that was use as such in the next step without
further purification: Rf 0.18 (hexaneeEtOAc, 4:1); 1H NMR
(hexaneeEtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3)
d 1.54e1.68
(m, 4H, H-400, H-500), 1.76 (m, 1H, H-300a), 1.86 (m, 1H, H-300b), 3.53
(m, 1H, H-600a), 3.80 (ddd, J ¼ 11.1, 6.5, 4.3 Hz, 1H, H-600b), 3.88
(ddd, J ¼ 11.2, 8.2, 3.1 Hz, 1H, H-1a), 4.06 (m, 1H, H-1b), 4.15 (m,
1H, H-2), 4.70 (t, J ¼ 3.6 Hz, 1H, H-200), 6.90 (ddd, J ¼ 8.3, 2.4,
0.7 Hz, 1H, H-600), 6.99 (t, J ¼ 8.0 Hz, 1H, H-500), 7.28 (dt, J ¼ 7.8,
1.0 Hz, 1H, H-400), 7.30 (t, J ¼ 1.9 Hz, 1H, H-200); 13C NMR
(125.77 MHz, CDCl3)
d
19.3 (C-400), 25.4 (C-500), 30.5 (C-300), 62.2
(C-600), 65.7 (C-1), 67.6 (C-2), 94.2 (C-30), 99.0 (C-200), 114.4 (C-60),
123.9 (C-20), 130.0 (C-40), 130.9 (C-50), 159.5 (C-10).
(500 MHz, CDCl3)
d
2.06 (br s, 1H, OH), 3.93 (dist. t, J ¼ 3.9 Hz, 2H,
H-1), 6.58 (t, J ¼ 2.2 Hz, 1H, Ph), 4.04 (t, J ¼ 7.9 Hz, 2H, H-2), 6.58
(t, J ¼ 2.2 Hz, 1H, Ph), 6.61 (dd, J ¼ 8.2, 2.0 Hz, 1H, Ph), 6.66 (dd,
J ¼ 8.3, 2.2 Hz, 1H, Ph), 7.02 (d, J ¼ 8.3 Hz, 2H, Ph), 7.11 (m, 1H, Ph),
7.22 (t, J ¼ 7.2 Hz, 1H, Ph), 7.34 (t, J ¼ 7.9 Hz, 1H, Ph); 13C NMR
4.22. 3-Phenoxyphenoxyethyl tetrahydro-2H-pyran-2-yl ether (30)
To a round bottom flask was added copper(I) iodide (21.9 mg,
0.11 mmol), 2-picolinic acid, (28.3 mg, 0.23 mmol), compound 29
(400 mg, 1.15 mmol), phenol (129 mg, 1.38 mmol) and potassium
phosphate tribasic (487 mg, 2.3 mmol). The flask was evacuated and
back-filled with argon. The evacuation/backfill sequence was
repeatedtwice. Then, dimethyl sulfoxidewas added (2.0mL) and the
reaction mixture was stirred vigorously at 80 ꢀC for 24 h. The reac-
tion mixture was cooled to room temperature and was partitioned
between ethyl acetate (20 mL) and water (20 mL). The aqueous layer
was extracted with ethyl acetate (2 ꢁ 20 mL). The combined organic
layers were washed with brine (5 ꢁ 50 mL), dried (MgSO4), and the
solvent was evaporated. The residue was purified by column chro-
matography (silica gel) eluting with a mixture of hexaneeEtOAc
(97:3) to afford 200 mg (55% yield) of pure compound 30 as a
(125.77 MHz, CDCl3) d
61.4 (C-1), 69.2 (C-2), 105.4 (C-20), 109.2 (C-
60), 111.3 (C-40), 119.2 (C-200), 123.5 (C-400), 129.7 (C-300), 130.2 (C-50),
156.8 (C-100), 158.6 (C-10), 159.9 (C-40). HRMS (ESI) calcd for
C
14H14O3Na [M þ Na]þ 253.0835; found 253.0844.
4.25. 3-(4-Methoxyphenoxy)phenoxyethanol (33)
To a solution of 31 (199 mg, 0.58 mmol) in methanol (30 mL)
was added pyridinium p-toluenesulfonate (10 mg). The reaction
mixture was stirred at room temperature for 14 h. The mixture
was partitioned between water (70 mL) and methylene chloride
(70 mL). The aqueous phase was extracted with methylene
chloride (2 ꢁ 30 mL) and the combined organic layers were
washed with brine (2 ꢁ 50 mL), dried (MgSO4), and the solvent
was evaporated to afford 145 mg (96% yield) of pure alcohol 33 as
a colorless oil that was used as such in the next step: Rf 0.06
colorless oil: 1H NMR (500.13 MHz, CDCl3) 1.49e1.84 (m, 6H, H-3000
d ,
H-4000, H-5000), 3.52 (m, 1H, H-6000a), 3.79 (ddd, J ¼ 11.1, 6.4, 4.2 Hz, 1H,
H-6000b), 3.88 (ddd, J ¼ 11.2, 8.3, 2.9 Hz, 1H, H-1a), 4.03 (m, 1H, H-1b),
4.12 (m, 2H, H-2), 4.69 (t, J ¼ 3.6 Hz,1H, H-2000), 6.60 (m, 2H, Ph), 6.68
(m,1H, Ph), 7.02 (d, J ¼ 8.4 Hz,1H, Ph), 7.10 (t, J ¼ 7.5 Hz,1H, Ph), 7.21
(t, J ¼ 8.5 Hz, 1H, Ph), 7.33 (t, J ¼ 8.0 Hz, 2H, Ph); 13C NMR
(hexaneeEtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3)
d 3.80 (s, 3H,
OCH3), 3.95 (dist. t, J ¼ 4.2 Hz, 2H, H-1) 4.03 (dist. t, J ¼ 4.6 Hz,
2H, H-2), 6.51 (t, J ¼ 2.3 Hz, 1H, H-20), 6.56 (ddd, J ¼ 8.1, 2.2,
0.6 Hz, 1H, H-60), 6.60 (ddd, J ¼ 8.1, 2.2, 0.6 Hz, 1H, H-40), 6.88 (d,
J ¼ 9.1 Hz, 2H, H-300), 6.99 (d, J ¼ 9.1 Hz, 2H, H-200), 7.18 (t,
(125.77 MHz, CDCl3) d
19.3 (C-4000), 25.4 (C-5000), 30.5 (C-3000), 62.2 (C-
6000), 65.7 (C-1), 67.5 (C-2), 99.0 (C-2000),105.6 (C-20),109.5 (C-60),111.2
(C-40), 119.1 (C-200), 123.5 (C-400), 129.7 (C-300), 130.0 (C-50), 157.0 (C-
100), 158.4 (C-10), 160.2 (C-40) 2 HRMS (ESI) calcd for C19H22O4Na
[M þ Na]þ 337.1416; found 337.1421.
J ¼ 8.2 Hz, 1H, H-50); 13C NMR (125.77 MHz, CDCl3)
d 55.6 (OCH3),
61.3 (C-1), 69.2 (C-2), 104.2 (C-20), 108.4 (C-60), 110.1 (C-40), 114.8
(C-300), 121.0 (C-200), 130.1 (C-50), 149.7 (C-100), 156.0 (C-400), 159.83
(C-10), 159.84 (C-30). HRMS (ESI) calcd for C15H16O4Na [M þ Na]þ
283.0946; found 283.0948.
4.23. 3-(4-Methoxyphenoxy)phenoxyethyl tetrahydro-2H-pyran-2-
yl ether (31)
4.26. 3-Phenoxyphenoxyethyl 4-Toluenesulfonate (34)
A mixture of copper(I) iodide (16 mg, 0.09 mmol, 10 mol%), 2-
picolinic acid, 1 (21.2 mg, 0.17 mmol, 20 mol%), compound 29
(300 mg, 0.86 mmol), 3-methoxyphenol (128 mg, 1.03 mmol) and
potassium phosphate tribasic (365 mg, 1.72 mmol) was treated as
depicted for the preparation of compound 30. After the usual
worked-up, the product was purified by column chromatography
(silica gel) eluting with a mixture of hexaneeEtOAc (97:3) to afford
197 mg (67% yield) of pure compound 31 as a colorless oil: 1H NMR
To a solution of alcohol 32 (92.7 mg, 0.40 mmol) in pyridine
(3 mL) cooled at 0 ꢀC was added p-toluenesulfonyl chloride
(229 mg, 1.20 mmol) portion wise, and the mixture was stirred at
room temperature for 3 h. Then, 5% HCl (10 mL) was added and the
reaction mixture was stirred for an additional hour. The mixture
was extracted with methylene chloride (20 mL) and the organic
layer was washed with 5% HCl (3 ꢁ 20 mL) and water (3 ꢁ 20 mL).
The organic phase was dried (MgSO4) and the solvent was evapo-
rated. The residue was purified by column chromatography (silica
gel) eluting with hexane to afford 99.2 mg (64% yield) of pure
compound 34 as a colorless oil: Rf 0.47 (hexaneeEtOAc, 1:1); 1H
(500.13 MHz, CDCl3)
d
1.50e1.84 (m, 6H, H-3000, H-4000, H-5000), 3.52
(m, 1H, H-6000a), 3.80 (m, 1H, H-6000b), 3.88 (m, 1H, H-1a), 4.03 (m, 1H,
H-1b), 4.10 (m, 2H, H-2), 4.69 (t, J ¼ 3.4 Hz, 1H, H-2000), 6.51 (t,
J ¼ 2.4 Hz,1H, H-20), 6.55 (ddd, J ¼ 8.4, 2.5, 0.5 Hz,1H, H-60), 6.61 (m,
1H, H-40), 6.89 (d, J ¼ 9.1 Hz, 2H, H-300), 6.99 (d, J ¼ 9.1 Hz, 2H, H-200),
NMR (500.13 MHz, CDCl3) d 2.46 (s, 3H, CH3), 4.10 (m, 2H, H-2), 4.34
(m, 2H, H-1), 6.41 (t, J ¼ 2.2 Hz, 1H, H-), 6.52 (dd, J ¼ 8.3, 2.2 Hz, 1H,
H-20), 6.62 (dd, J ¼ 8.2, 2.0 Hz, 1H, H-1), 7.00 (d, J ¼ 8.4 Hz, 2H, H-
3000), 7.12 (t, J ¼ 7.4 Hz, 1H), 7.18 (t, J ¼ 8.2 Hz, 1H), 7.32 (m, 2H, ar-
omatic protons), 7.80 (d, J ¼ 8.2 Hz, 2H, H-2000); 13C NMR
7.19 (t, J ¼ 8.2 Hz, 1H, H-50); 13C NMR (125.77 MHz, CDCl3)
d 19.4 (C-
4000), 25.4 (C-5000), 30.5 (C-3000), 55.6 (OCH3), 61.4 (C-1), 62.2 (C-6000),
69.2 (C-2), 99.0 (C-2000), 104.2 (C-20), 108.4 (C-60), 110.2 (C-40), 114.9
(C-300), 121.0 (C-200), 130.1 (C-50), 149.7 (C-100), 156.1 (C-400), 159.7 (C-
10), 159.9 (C-30). HRMS (ESI) calcd for C20H24O5Na [M þ Na]þ
367.1521; found 367.1527.
(125.77 MHz, CDCl3) d
21.6 (CH3), 65.5 (C-2), 68.0 (C-1), 105.5 (C-20),
109.1 (C-60), 111.6 (C-40), 119.1 (C-200), 123.5 (C-400), 128.0 (C-2000),
129.76 (C-300), 129.82 (C-3000), 130.2 (C-50), 132.8 (C-4000), 145.0 (C-

488
P.D. Elicio et al. / European Journal of Medicinal Chemistry 69 (2013) 480e489
1000), 156.8 (C-100), 158.5 (C10), 159.3 (C-40). HRMS (ESI) calcd for
C
Greiner Bio-One) in 100 mL RPMI media (Sigma) with 10% FBS.
21H20O5SNa [M þ Na]þ 407.0924; found 407.0914.
Plates were incubated overnight at 35 ꢀC and 7% CO2. After over-
night incubation, Vero cells were challenged with 3.4 ꢁ 105 try-
4.27. 3-(4-Methoxyphenoxy)phenoxyethyl 4-Toluenenesulfonate
(35)
pomastigotes/well (CL strain overexpressing a tdTomato red
fluorescent protein) in 50
and 7% CO2. After infection, cells were washed once with Hanks
solution (150 L/well) to eliminate any extracellular parasites and
compounds were added in serial dilutions in RPMI media in 150
m
L volume and incubated for 5 h at 35 ꢀC
To a solution of alcohol 33 (128 mg, 0.49 mmol) in pyridine
(3 mL) cooled at 0 ꢀC was added p-toluenesulfonyl chloride
(281 mg, 1.47 mmol) portion wise, and the mixture was stirred at
room temperature for 4 h. The reaction was worked up as depicted
for the preparation of 12. The residue was purified by column
chromatography (silica gel) eluting with hexane to afford 94 mg
(46% yield) of pure compound 35 as a colorless oil: Rf 0.10 (hexanee
m
mL
volumes. Each dilution was tested in quadruplicate. Each plate also
contained controls with host cells and no parasites (for background
check), and controls with parasites and no drugs (positive control).
Drugs were tested on T. cruzi at 5 different concentrations up to a
maximum of 20 or 25 mM. For each set of experiments, benznida-
EtOAc, 4:1); 1H NMR (500.13 Hz, CDCl3)
d
2.44 (s, 3H, CH3), 3.82 (s,
zole was also used as a positive control. After drug addition, plates
were incubated at 35 ꢀC and 7% CO2. At day 3 post-infection, plates
were assayed for fluorescence [43]. IC50 values were determined by
non-linear regression analysis using SigmaPlot.
3H, OCH3), 4.09 (m, 2H, H-1, H-1), 4.33 (m, 2H, H-2), 6.36 (t,
J ¼ 2.3 Hz, 1H, H-20), 6.46 (dd, J ¼ 8.3, 2.3 Hz, 1H, H-40), 6.53 (dd,
J ¼ 8.2, 2.2 Hz, 1H, H-60), 6.89 (d, J ¼ 9.0 Hz, 2H, H-300), 6.97 (d,
J ¼ 9.0 Hz, 2H, H-200), 7.15 (t, J ¼ 8.2 Hz, 1H, H-50), 7.32 (d, J ¼ 8.4 Hz,
2H, H-3000), 7.80 (d, J ¼ 8.2 Hz, 2H, H-2000); 13C NMR (125.77 MHz,
4.30.2. T. gondii tachyzoites assays
CDCl3)
d
21.6 (CH3), 55.6 (OCH3), 65.4 (C-2), 68.0 (C-1), 104.3 (C-20),
Experiments on T. gondii tachyzoites were carried out as
described previously [44] using T. gondii tachyzoites expressing red
fluorescent protein [45]. Cells were routinely maintained in hTERT
cells grown in High Glucose Dulbecco’s modified Eagle’s medium
(DMEM-HG) supplemented with 1% fetal bovine serum, 2 mM
glutamine, 1 mM pyruvate, at 37 ꢀC in a humid 5% CO2 atmosphere.
Confluent monolayers grown in 96-well black plates with optical
bottoms (black, clear bottom plates from Greiner Bio-One) were
used and drugs dissolved in the same medium and serially diluted
in the plates. Freshly isolated tachyzoites were filtered through a
108.3 (C-60), 110.4 (C-40), 114.9 (C-300), 121.0 (C-200), 128.0 (C-2000),
129.8 (C-3000), 130.1 (C-50), 132.8 (C-4000), 145.0 (C-400), 149.7 (C-100),
156.1 (C-400), 159.2 (C-10), 159.8 (C-30). HRMS (ESI) calcd for
C
22H22O6SNa [M þ Na]þ 437.1035; found 437.1010.
4.28. 3-Phenoxyphenoxyethyl thiocyanate (36)
A solution of tosylate 34 (99 mg, 0.24 mmol) in anhydrous N,N-
dimethylformamide (3 mL) was treated with potassium thiocya-
nate (125 mg, 1.29 mmol) according to the general procedure. The
product was purified by column chromatography (silica gel) using a
mixture of hexaneeEtOAc (9:1) as eluent to give 45 mg (64% yield)
of pure thiocyanate 36 as a colorless oil: 1H NMR (500.13 MHz,
3 mm filter and passed through a 25 gauge needle, before use. The
cultures were inoculated with 4 ꢁ 104 achyzoites/well in the same
media. The plates were incubated at 37 ꢀC and read daily in a
Molecular Devices fluorescence plate reader. To preserve sterility
the plates were read with covered lids, and both excitation
(544 nm) and emission (590 nm) were read from the bottom [45].
For the calculation of the EC50, the percent of growth inhibition was
plotted as a function of drug concentration by fitting the values to
the function: I ¼ ImaxC/(EC50 þ C), where I is the percent inhibition,
Imax ¼ 100% inhibition, C is the concentration of the inhibitor, and
EC50 is the concentration for 50% growth inhibition. There was no
evident cytotoxicity on the host cells with any of the drugs tested
(visual assay).
CDCl3)
d
3.32 (t, J ¼ 5.9 Hz, 2H, H-1), 4.28 (t, J ¼ 5.8 Hz, 2H, H-2), 6.58
(t, J ¼ 2.3 Hz, 1H), 6.65 (m, 2H, Ph), 7.03 (dd, J ¼ 8.7, 1.0 Hz, 2H, Ph),
7.13 (tt, J ¼ 7.4, 1.0 Hz, 1H, Ph), 7.23 (d, J ¼ 8.2 Hz, 2H, Ph), 7.35 (dd,
J ¼ 8.6, 7.4 Hz, 2H, Ph); 13C NMR (125.77 MHz, CDCl3)
d 33.2 (C-1),
65.8 (C-2), 105.5 (C-20), 109.2 (C-60), 111.7 (SCN), 111.9 (C-40), 119.2
(C-200), 123.6 (C-400), 129.8 (C-300), 130.4 (C-50), 156.7 (C-100), 158.7 (C-
10), 159.1 (C-40). HRMS (ESI) calcd for C15H13O2NSNa [M þ Na]þ
294.0559; found 294.0553.
4.29. 3-(4-Methoxyphenoxy)phenoxyethyl thiocyanate (37)
4.31. Cytotoxicity for vero cells
To a solution of tosylate 35 (87 mg, 0.22 mmol) in anhydrous
N,N-dimethylformamide (3 mL) was added potassium thiocyanate
(105 mg, 1.08 mmol) according to the general procedure. The res-
idue was purified by column chromatography (silica gel) using a
mixture of hexaneeEtOAc (9:1) as eluent to afford 43 mg (68%
yield) of pure compound 37 as a colorless oil: Rf 0.58 (hexanee
The cytotoxicity was tested using the Alamar BlueÔ assay as
described by Recher et al., 2013 [46].
Acknowledgments
EtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3)
d
3.31 (t, J ¼ 5.8 Hz, 2H, H-
This work was supported by grants from the National Research
Council of Argentina (PIP 1888), ANPCyT (PICT 2008 #1690), and
the Universidad de Buenos Aires (200201001003801) to J.B.R., the
Bunge & Born Foundation to S.H.S, and the U.S. National Institutes of
Health to R.D. (AI-082542) and S.N.J.M. (AI-102254).
1), 3.81 (s, 3H, OCH3), 4.27 (t, J ¼ 5.8 Hz, 2H, H-2), 6.51 (t, J ¼ 2.4 Hz,
1H, H-20), 6.57 (ddd, J ¼ 8.2, 2.3, 0.8 Hz, 1H, H-40), 6.60 (ddd, J ¼ 8.2,
2.3, 0.8 Hz, 1H, H-60), 6.89 (d, J ¼ 9.1 Hz, 2H, H-300), 6.99 (d,
J ¼ 9.2 Hz, 2H, H-200), 7.20 (t, J ¼ 8.2 Hz, 1H, H-50); 13C NMR
(125.77 MHz, CDCl3)
d 33.3 (C-1), 55.6 (OCH3), 65.8 (C-2), 104.3 (C-
20), 108.4 (C-60), 110.4 (C-40), 114.9 (C-300), 121.1 (C-200), 130.3 (C-50),
Appendix A. Supplementary data
149.6 (C-100), 156.2 (C-10) 159.0 (C-40). HRMS (ESI) calcd for
C
16H15O3NSNa [M þ Na]þ 324.0670; found 324.0674.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.009.
4.30. Drug screening
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