Selenium-containing analogues of WC-9 are extremely potent inhibitors of Trypanosoma cruzi proliferation

Article abstract of DOI:10.1016/j.bmc.2017.10.016

The obligate intracellular parasite, Trypanosoma cruzi is the etiologic agent of Chagas disease or American trypanosomiasis, which is the most prevalent parasitic disease in the Americas. The present chemotherapy to control this illness is still deficient

Full text of DOI:10.1016/j.bmc.2017.10.016

Accepted Manuscript
Selenium-containing analogues of WC-9 are extremely potent inhibitors of
Trypanosoma cruzi proliferation
María N. Chao, Melissa Storey, Catherine Li, Maricel G. Rodríguez, Florencia
Di Salvo, Sergio H. Szajnman, Silvia N.J. Moreno, Roberto Docampo, Juan B.
Rodriguez
PII:
S0968-0896(17)31731-5
https://doi.org/10.1016/j.bmc.2017.10.016
BMC 14021
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 August 2017
6 October 2017
15 October 2017
Please cite this article as: Chao, M.N., Storey, M., Li, C., Rodríguez, M.G., Di Salvo, F., Szajnman, S.H., Moreno,
S.N.J., Docampo, R., Rodriguez, J.B., Selenium-containing analogues of WC-9 are extremely potent inhibitors of
Trypanosoma cruzi proliferation, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.
2017.10.016
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Selenium-containing analogues of WC-9 are extremely potent inhibitors of Trypanosoma
cruzi proliferation
María N. Chao,a Melissa Storey,b Catherine Li,b Maricel G. Rodríguez,c Florencia Di Salvo,c
Sergio H. Szajnman,a,* Silvia N. J. Moreno,b Roberto Docampo,b and Juan B. Rodrigueza,*
aDepartamento de Química Orgánica and UMYMFOR (CONICET–FCEyN), Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria,
C1428EHA, Buenos Aires, Argentina, bCenter for Tropical and Emerging Global Diseases and
Department of Cellular Biology, University of Georgia, Athens, Georgia, 30602, USA,
cDepartamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Buenos
Aires, C1428EGA, Argentina
Abstract− The obligate intracellular parasite, Trypanosoma cruzi is the etiologic agent of
Chagas disease or American trypanosomiasis, which is the most prevalent parasitic disease in the
Americas. The present chemotherapy to control this illness is still deficient particularly in the
chronic stage of the disease. The ergosterol biosynthesis pathway has received much attention as
a molecular target for the development of new drugs for Chagas disease. Especially, inhibitors of
the enzymatic activity of squalene synthase were shown to be effective compounds on T. cruzi
proliferation in in vitro assays. In the present study we designed, synthesized and evaluated the
effect of a number of isosteric analogues of WC-9 (4-phenoxyphenoxyethyl thiocyanate), a
known squalene synthase inhibitor, on T. cruzi growth in tissue culture cells. The selenium-
containing derivatives turned out to be extremely potent inhibitors of T. cruzi growth. Certainly,
3-phenoxyphenoxyethyl, 4-phenoxyphenoxyethyl, 4-(3-fluorophenoxy)phenoxyethyl, 3-(3-
fluorophenoxy)phenoxyethyl selenocyanates and ( )-5-phenoxy-2-(selenocyanatomethyl)-2,3-
dihydrobenzofuran arose as relevant members of this family of compounds, which exhibited
effective ED50 values of 0.084 µM, 0.11 µM, 0.083, µM, 0.085, and 0.075 µM, respectively. The
results indicate that compounds bearing the selenocyanate moiety are at least two orders of
magnitude more potent than the corresponding skeleton counterpart bearing the thiocyanate
1

group. Surprisingly, these compounds exhibited excellent selectively index values ranging from
900 to 1,800 making these molecules promising candidates as antiparasitic agents.
2

Introduction
Trypanosoma cruzi is the etiologic agent of a chronic zoonotic disease namely American
trypanosomiasis or Chagas disease, which is one of the important parasitic diseases worldwide.
As a result of public policies for the control of vectors the number of infected people with T.
cruzi was reduced from 18 million in 1991 to 6 million in 2010.1–3 However, even now, it is the
most prevalent parasitic disease in the Americas.4 Like other trypanosomatids, T. cruzi has a
complex life cycle that involves Reduviid bugs with blood-sucking activity and mammalian
hosts.5 The parasite multiplies in the insect gut as an epimastigote form and is released as a non-
dividing metacyclic trypomastigote in the insect excrements and invades the host through the
intact mucosa or wounds produced by the blood-sucking activity of the vector. In the mammalian
host, T. cruzi proliferates intracellularly as an amastigote form, which is further released into the
blood stream as a non-dividing highly infective trypomastigote.5 Infection of T. cruzi can also
take place via the placenta or through blood transfusion or organ transplantation, which is the
way of transmission in areas where of Chagas disease is not endemic due to increasing migration
of infected people.6
The present chemotherapy is still deficient and based on two empirically-discovered drugs:
nifurtimox (Lampit®, Bayer - El Salvador, 1) and benznidazole (Abarax®, Elea - Argentina, 2),
which are not FDA-approved in the United States where they are available only from CDC under
investigational protocols (Figure 1).1 These compounds are able to cure at least 50% of recent
infections, but they are not effective against the chronic stage of the disease.7,8 Certainly, therapy
with benzonidazole of chronic patients is not satisfactory at all, but a beneficial effect of
treatment in such individuals has been well demonstrated.9,10 Their major drawback is long-term-
treatment associated to severe side effects such as vomiting, anorexia, peripheral neuropathy and
allergic dermopathy.7 So far, there are no vaccines available to prevent infection of T. cruzi.8
Figure 1. Chemical structures of the antiparasitic agents nifurtimox and benznidazole.
Squalene synthase (SQS) is essential in ergosterol biosynthesis. This enzyme catalyzes the first
committed step consisting in a two-step reductive dimerization reaction between two molecules
3

of farnesyl diphosphate (FPP) to give rise to one molecule of squalene,11 which is an obligated
metabolite for the biosynthesis of the required endogenous sterols. There is strong evidence to
indicate that the reaction occurs in two well-defined biosynthetic steps.12,13 As shown in Scheme
1 one molecule of farnesyl diphosphate losses its diphosphate moiety to produce a carbocation
species that attacks another molecule of farnesyl diphosphate via an 1′-2,3-condensation to give
rise to the corresponding intermediate cyclopropylpresqualene diphosphate, which experiences,
after leaving the residual diphosphate, a rearrangement reaction of the resulting
cyclopropylcarbocation. This carbocation experiences a cyclopropyl ring-opening followed by
hydride attack employing NADPH as hydride source.12,13 Human SQS is a highly conserved
membrane-bound protein,14 and it was crystallized as truncated and active form.15 A soluble
recombinant truncated enzyme from T. cruzi (TcSQS), expressed in Escherichia coli was also
crystallyzed.16 TcSQS has a dual subcellular localization being equally distributed between
glycosomes and mitochondria.17 SQS has no homologous pyridine dinucleotide-binding motifs
suggesting that this enzyme would adapt a distinctive NADPH binding mode from the Rossmann
fold in the active site.18 In this sense, SQS might have one or two overlapping catalytic sites to
perform both consecutive reactions.18 Structural determination of a membrane protein is not a
trivial task. 19
Scheme 1. Reductive dimerization of two molecules of farnesyl diphosphate to produce one molecule of squalene.
There are a number of ergosterol biosynthesis inhibitors that can induce parasitological cure
in both acute and chronic experimental models of Chagas disease.17 It worth mentioning that
quinuclidine derivatives E5700 and ER-119884, two potent orally active inhibitors of the
enzymatic activity of SQS, arise as relevant members of inhibitors targeting SQS. The former
4

one, E5700, has shown to have both in vitro and in vivo anti-T. cruzi activity.20 4-
Phenoxyphenoxyethyl thiocyanate (compound 1; WC-9) is a potent inhibitor of the intracellular
amastigote forms of T. cruzi21 WC-9 acting as a non-competitive inhibitor of TcSQS at a low
nanomolar range.22 Additional synthetic derivatives of WC-9 such as 2−8 are shown in Figure
2.21,23–28
Figure 2. Chemical structure of WC-9 and other closely related inhibitors of T. cruzi proliferation.
Rationale
A thiocyanate moiety covalently bonded to the main frame in the molecule of WC-9
constitutes one of the few cases where this group behaves as a key functional group in a
pharmacologically important lead structure.29 In order to get further insights about the role of the
sulfur atom present in the thiocyanate group, isosteric analogues of WC-9 were envisioned by
replacing this sulfur atom by a selenium atom or an oxygen atom leading to selenocyanate or
cyanate derivatives respectively. In addition, to study the influence of the special orientation of
the thiocyanate group, conformationally rigid analogues of WC-9 were considered based on the
concept that it should exist an optimal conformation for molecular recognition.30,31 A variety of
32
biological properties of the selenocyanate group have been described
and very recently
molecules bearing this functionality have been reported as moderate inhibitors of Leishmania
infantum and L. braziliensis33,34
Although the target of WC-9 has been identified as TcSQS, little is known about the precise
mode of action of this allosteric inhibitor. The crystal structure of the complex WC-9−TcSQS is
not yet available, but an X-ray crystal structure of WC-9 with human SQS has been recently
reported.35 A high degree of similarity is observed between the T. cruzi and human protein
structures. However, these crystallographic data did not provide enough information concerning
several points: (a) there was no inhibition data, that is, IC50 values were not available; (b) it was
not clear which was the role of the sulfur atom present in the thiocyanate moiety; (c) WC-9 in
the crystal WC-9−hSQS was present in an unusual unfavorable conformation, which was more
5

than 210 kJ/mol to that found in the crystal structure of WC-9 alone. Coordinates of WC-9 in the
crystal WC-9−hSQS were taken from Protein Data Bank (PDB) (3WCD) while the coordinates
of WC-9 are provided in the present study either by single crystal X-ray diffraction
determination or by electronic structure calculations by DFT (Density Functional theory). Single
point energy calculations for comparison were done employing Gaussian 16 program at
DFT/B3LYP using 6-311 ++G(d,p) basic set.36 Moreover, the crystalline structure of WC-9 with
dehydrosqualene synthase from Staphylococcus aureus, an enzyme that catalyzes
dehydrosqualene formation, did not provide further insight in the precise mode of action of this
compound.37
Results and Discussion
In our experience, small structural variations on the WC-9 chemical structure had a deep
effect on the biological activity. The development of the Buchwald coupling reaction has proven
to be very useful to access a variety of WC-9 analogues, which might be putative growth
inhibitors of either T. cruzi or T. gondii cells.38–42 Since our finding that insect juvenile hormone
analogues are cell growth inhibitors, particularly, for T. cruzi proliferation,43 we were able to
establish a rigorous structure activity relationship (SAR) on WC-9 chemical structure.21,23–28,44
The development of WC-9 as a lead structure has been reviewed.1,45,46
A sulfur atom as a linker between the two aromatic rings was the first isosteric modification
of WC-9 and related analogues conceived. The introduction of this sulfur atom to form the
corresponding asymmetric diarylthioether skeletons was accomplished via a Buchwald coupling
reaction employing potassium orthophosphate as a base, thiophenol, and ethylenglycol as a co-
solvent and as a ligand.47 Therefore, the already described synthetic intermediates 1127 and 1527
under these particular Buchwald coupling reaction conditions gave the respective
tetrahydropyranyl derivatives 12 and 16 in low but reproducible yields. Each tetrahydropyranyl
protecting group present in these compounds was removed by treatment with pyridinium 4-
toluenesulfonate producing the corresponding alcohols 13 and 17 in excellent yields, which were
tosylated to give 14 and 18 in 72% and 82% yields, respectively. On treatment with potassium
thiocyanate, in separate experiments, these compounds were converted into the target molecules
9 and 10, respectively, as illustrated in Scheme 2.
6

Scheme 2. Synthetic approach for the preparation of thiocyanate derivatives bearing a diarylthioether as a non polar
skeleton.
Conformationally constrained analogues of WC-9 were straightforwardly prepared starting
from 4-phenoxyphenol (21). Then, this compound was reacted with allyl bromide via a
Williamson etherification reaction to give the allyl ether 22 in 93% yield. Claisen rearrangement
on 22 by treatment with N,N-dimethylaniline at 210 ºC produced the respective rearranged
product 22,48,49 which was epoxidized by treatment with m-chloroperoxybenzoic acid in
chloroform to produce the corresponding racemic epoxyphenol 24. Nucleophilic ring opening of
the epoxy ring under acidic conditions (diluted hydrochloric acid) gave the expected fused five-
membered ring 25 according to the literature.48,50,51 Certainly, nucleophilic attack took place at
the most substituted carbon atom as the reaction via a carbocationic transition state.48,50–52 Once
the conformationally constrained 25 was at hand, tosylation of this compound followed by SN2
nucleophilic substitution with potassium thiocyanate produced the title compound 19.
Aromatization of the ring on 19 was successfully carried out by treatment with 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ) in refluxing 1,4-dioxane53 to yield the target molecule 20 in
24% yield. The synthesis of these conformationally restricted analogues is shown in Scheme 3.
7

Scheme 3. Synthetic strategy to access conformationally constrained analogues of WC-9.
The isosteric analogues of WC-9 were easily synthesized. The selenocyate group was
introduced by a SN2 reaction between a suitable tosylate and the commercially available
potassium selenocyanate in refluxing tetrahydrofuran in the presence of 18-crown-6.54 Then, the
known tosylates 3127 and 33,21 prepared from 3-phenoxyphenoxyphenol (30) and 4-
phenoxyphenoxyphenol (32) were transformed, in independent experiments, into the
selenocyanate derivatives 27 and 28, by reaction with potassium selenocyanate in 64% and 71%
yields, respectively. The cyanate 29 was synthesized starting from already depicted 4-
phenoxyphenoxyethanol (32),55,56 which on treatment with trichloroacetyl isocyanate at −15 ºC
followed by digestion with potassium carbonate in methanol-water gave carbamate 34. This
compound was converted into the title compound 29 by treatment with trichloroacetyl isocyanate
at −15 ºC in 98% yield55,56 as shown in Scheme 4.
Scheme 4. Straightforward preparation of the first selenium-containing analogues of WC-9 and the cyanate
derivative 29.
As preliminary results indicated that selenocyanates derivatives 27 and 28 behaved as very
8

effective growth inhibitors of T. cruzi growth, it was decided to incorporate this group into the
relevant skeletons of the thiocyanate series.21,23–28 Scheme 5 illustrates the preparation of
conformationally constrained selenium-containing analogues of WC-9. Therefore, compound 35
was straightforwardly prepared from tosylate 26 by treatment with potassium selenocyanate in
refluxing tetrahydrofuran in the presence of 18-crown-6 in 86% yield.54 The title compounds 36
and 37 were synthesized starting from 3-phenoxyphenol (38), which on reaction with N,N-
dimethylaniline at 210 ºC for 10 hours48,49 was converted into a non-separable mixture of the
Claisen rearranged regioisomers 40 and 41. Epoxidation of the mixture 40/41 by reaction with
m-chloroperoxybenzoic acid in chloroform led to the respective epoxides, which experienced a
spontaneous ring-closing to form alcohols 42 and 43.50–52 Each alcohol was treated with tosyl
chloride, in independent experiments, and the resulting tosylates 44 and 45 yielded after
treatment with potassium selenocyanate,54 under the usual conditions, the target molecules 36
and 37 in 36% and 75% yields, respectively.
Scheme 5. Synthetic approach to access conformationally constrained selenocyanate derivatives.
The rest of the designed selenium-containing compounds were directly prepared from the
appropriate tosylate, which had previously been used for the preparation of the thiocyanate
counterpart. Therefore, the title compounds 46−55 were available by treatment of the respective
already depicted tosylated (56,26 57,26 58,25 59,24 60,24 61,28 62,28 63,28 64,27 and 6527) with
potassium selenocyanate as illustrated in Scheme 6.
9

Scheme 6. Preparation of selected selenocyanate derivatives based on privileged skeletons previously evaluated
with the thiocyanate moiety.
Biological evaluation of these new isosteric analogues of WC-9 was very encouraging.
Title compounds 9 and 10 exhibited quite similar biological activity against intracellular T. cruzi
and tachyzoites of Toxoplasma gondii than their isosteric counterparts, that is, WC-9 and
compound 4, indicating that the replacement of the oxygen bridge between both phenyl groups
by a sulfur atom had a weak effect on the activity against these parasites.
Conformationally constrained analogues of WC-9 such as 19 and 20 were potent
inhibitors of T. cruzi growth showing similar activity to our lead drug WC-9 with ED50 values of
6.0 µM and 5.2 µM, respectively, whereas WC-9 had an ED50 value of 5.0 µM.27 Evidently, this
fixed conformation was not still optimal for a better recognition. In fact, the potency of these
compounds is maintained.
Biological data of isosteric derivatives of WC-9 where the sulfur atom at the thiocyanate
unit was replaced either by a selenium atom or an oxygen atom to give rise to 28 and 29 were
very relevant. Definitely, the selenium-containing derivative 28 was an extremely potent
inhibitor of T. cruzi proliferation showing an ED50 value of 0.11 µM, that is, almost 50 times
more potent than our lead drug WC-9 (ED50 = 5.0 µM).27 In addition, this compound exhibited
very low in vitro toxicity against Vero cells (ED50 > 100 µM) presenting a Selectivity Index (SI)
close to 1,000. Interestingly, 28 exhibited practically the same degree of activity against T.
gondii (ED50 = 3.7 µM) compared to WC-9 (ED50 = 4.8 µM).27 Cyanate derivative 29 was
virtually devoid of antiparasitic activity against T. cruzi indicating that the atom bonded to the
cyanide moiety had a strong influence on biological activity. In this case, the inhibitory action
10

certainly increases as the number in the periodic table increases: OCN < SCN < SeCN. The
obliged synthetic precursor 34 was inactive against T. cruzi. The effect of the selenium
replacement was more noticeable in compound 27, the isosteric analogue of 4. 27 was a very
potent growth inhibitor of T. cruzi (amastigotes) possessing an ED50 value as low as 0.085 µM
being 130-fold more potent than the parent molecule 4 with a SI value of 1,765. 27 behaved
similarly to 28 against T. gondii showing similar potency than its thiocyanate counterpart
(compound 4). Therefore, 28 exhibited an ED50 value of 2.9 µM against T. gondii versus 4.0 µM
by 4.26
Bearing in mind the above results, our efforts were focused on T. cruzi cells. Of the
conformationally rigid selenium-containing analogues 35, 36 and 37, the first one emerged as the
most potent molecule having EC50 values of 0.083 µM, 0.12 µM and 0.41 µM, respectively.
These results offered further insights on the spatial distribution either of the rigid non-polar
skeleton as well as the orientation of the selenocyanate group. In fact, superimposition of the
chemical structures indicated that there was a marked difference among the disposition of the
terminal phenyl ring that matched the differences of the inhibitory action observed as illustrated
in Figure 3a. Moreover, because all of these regioisomers are racemic mixtures, it seems
reasonable to assume, particularly for 35, that only one enantiomer should have the proper
orientation of the selenocyanate unit for better molecular recognition. Figure 3b shows clearly
that it is reasonable to assume that one enantiomer should be accountable for biological action.
(a)
(b)
Figure 3. (a) Superimposition of regioisoimers 35−37 alienated on the selenocyanate group 35 blue, 36 green and
37 magenta; (b) different spatial orientations of both enantiomers of 35: the R enantiomer blue and the S enantiomer
green.
11

The rest of the designed compounds also exhibited promising inhibitory action against
amastigotes of T. cruzi. With the exception of the iodine-containing derivatives 46 and 47, the
rest of the target molecules consisting of a privileged skeleton bonded to an ethyl selenocyanate
moiety exhibited a very efficient action as inhibitors of T. cruzi growth. Fluorine-containing
molecules 49, 52 and 53 turned out to be extremely potent growth inhibitors of T. cruzi
proliferation. All of them were at least 50 times more potent than the thiocyanate counterpart
being 52 the most potent member of this selenium family of compounds with an ED50 value as
low as 0.075 µM. 49 and 53 showed ED50 values of 0.085 µM and 0.099 µM, respectively.
These three compounds had low in vitro toxicity having SI values of more than 1,000. The
chlorine-containing compound 54 was very potent as well with an ED50 value of 0.11 µM and a
SI value greater than 1,000 (Table 1). At the present time, the precise mode of action is still
unknown but it could be assumed that the selenium atom of the inhibitors might form a new
selenium-sulfur bond with a cysteine residue at the binding site.32
The difference in the observed inhibitory action against T. cruzi and T. gondii may be
attributed by the fact that T. gondii does not synthesize cholesterol and imports it from the host57
suggesting that inhibitors of the host SQS could possibly slow T. gondii multiplication. As a
matter of fact, mevalonate pathway inhibitors modulate proliferation of different Apicomplexan
parasites including Plasmodium falciparum,58–60 indicating that microorganisms where the
mevalonate pathway is absent are reliant on host metabolites of isoprenoid biosynthesis. T.
gondii lacks the mevalonate pathway and utilizes a prokaryotic-type 1-deoxy-D-xylulose-5-
phosphate (DOXP) pathway instead to produce isopentenyl diphosphaste and dimethylallyl
diphosphate. The DOXP pathway localizes to the apicoplast and is essential.61,62
X-Ray single crystal structure determination of WC-9 and 28
Molecular structure description. The molecular structures for WC-9 (CCDC number: 1561895)
and 28 (CCDC number: 1561896) have been unequivocally established by X-ray crystallography
(Figure 4) and are in agreement with the spectroscopic data.21 Crystal and data collection details
are shown in Table 2 and Supplementary Data. Whereas WC-9 crystallizes in the P21/n space
group, 28 does it in P-1. Both molecular structures show the diphenyl ether moiety with
comparable bond distances and angles (Supplementary Data) but the C6O1C7 angle and
12

C5C6O1C7 torsion angle show some differences: 117.60(14)º and 118.5(3)º for C6O1C7 and
4.28(1)º and –11.43(6)º for C5C6O1C7, respectively, but all of the values according to expected
values (see CSD search analysis). The C13 and C14 next to the XCN group exhibit similar
structural parameters and as expected, the identity of the X atom, S or Se, impacts in the XCN
structural features (Table 2). The electronic effect exerted by the identity of the chalcogen in the
XCN group could be influencing the structural differences observed in the diphenyl ether moiety.
Intramolecular parameters studies using Cambridge Structural Database (CSD). Using the
Mogul program,63 a knowledge base of molecular geometry derived from the CSD, the
intramolecular parameters of WC-9, 28 and WC-9 crystallized in SQS35 (WC-9−hSQS) were
compared with the corresponding ones from similar molecules deposited at the CSD. Bond
lengths and angle of WC-9 and 28 are in agreement with the CSD information but, WC-9−hSQS
present some unusual structural values. The C10O2C13 (Figure 4) angle bond to the SCN and
torsion angles C13C14S1(Se1)C15 and N1C15S1C14 related to the C–SCN lie far away from
the Gaussian exhibited by the database information (Supplementary Data). These results would
demonstrate unusual structural features observed for WC-9−hSQS through a comparison and
statistical analysis based on experimental data of related compounds deposited at the CSD.
Figure 4. Molecular structure of WC-9 and 28 with the atomic numbering scheme. H atoms labelling is omitted for
clarity, ellipsoids are drown with 50% probability.
Supramolecular behaviour. The supramolecular packing of WC-9 and 28 shows some similar
features, but there is a distinct aspect related with the presence of the chalcogen. The
selenocyanate derivative shows a weak H-bond interaction between the N and the C12–H which
13

is not present in the thiocyanate analogue (Supplementary Data). The electronic effect exerted by
the Se results in an increase of the electronic density in the N atom acting as H-bond acceptor
which thus, gives place to the mentioned interaction. This result could be considered as an
experimental demonstration of the supramolecular interactions that each derivative is able to
experiment in different environments such as solvent and/or molecular targets. Electron density
maps of both structures were created using the program Tonto implemented in CrystalExplorer64
using DFT at the B3LYP/STO-3G65 level to confirm the observed interactions in general and the
different role of the chalcogen being the SeCN involved in stronger intermolecular interactions
(Supplementary Data).
It can be concluded that most target molecules, that is, the selenium-containing analogues
of WC-9 designed and synthesized in the present work, exhibited potent inhibitory action against
T. cruzi proliferation. These isoteric analogues of WC-9 showed increased potency. In fact, each
skeleton increased its efficacy when a thiocyanate group was replaced by a selenocyanate moiety
by a factor ranging from 46 to 140. Figure 5 shows the increment in efficacy of the selenium-
containing compounds versus the sulfur ones. Table 1, columns # 4 and # 5, indicates the precise
increment values. This isosteric structural variation was not beneficial against T. gondii
maintaining the same level of inhibitory action as it was the case for compounds 27 and 28.
Moreover, all of these title compounds can be considered as having drug-like characteristics66
offering great potential for optimization of their chemical structure (For details parameters
calculated by DataWarrior,67 see Table S1 in Supplementary Data). Efforts to further optimize
the structure of selenium-containing analogues of WC-9 analogues are currently being pursued
in our laboratory.
14

12
11
10
9
Thiocyanate family
Selenocyanate family
8
7
6
5
4
3
2
i
z
u
r
0,1
c
.
T
Figure 5. T. cruzi amastigote replication ratio between ED50 values (µM) of thiocyanate/selenocyanate derivatives.
15

Table 1. Biological activity of isosteric analogues of WC-9 against T. cruzi (amastigotes), T.
gondii (tachyzoites) and Vero cells.
Compd T. cruzi growth T. gondii growth
T. cruzi growth
SCN counterpart
ED50 (µM)
Ratio T. cruzi
growth
Cytotoxicity
ED50 (µM)
Selectivity
Index b
ED50 (µM)a
ED50 (µM)a
SCN/SeCN
5.73 0.41
> 10.0
4.583 1.512
3.581 0.944
> 10.0
-
-
> 62.5
> 50.0
> 62.5
> 10.9
5
9
10
-
-
5.99 0.55
5.19 0.32
0.085 0.004
0.109 0.011
> 10.0
-
-
> 10.4
19
-
-
131.8
45.9
-
20
2.867 0.924
3.704 0.463
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
4.8 0.4127
11.226
5.0 1.126
> 150
> 100
> 1,765
> 917
27
28
-
29
> 10.0
-
-
34
0.083 0.019
0.120 0.016
0.407 0.051
> 10.0
5.99 0.55
72.2
-
> 125
> 125
> 1,500
> 1,042
> 307
5
35
-
36
-
-
> 125
37
-
-
> 50
46
5.44 0.23
1.38 0.29
0.085 0.020
0.271 0.010
0.126 0.04
0.075 0.005
0.099 0.020
0.114 0.020
2.71 0.31
5.0 1.127
-
-
> 62.5
> 11.5
> 45.3
1,796
1,227
1,114
>> 833
> 1,263
> 1,096
17.6
47
>10025
4.324
3.724
-
> 62.5
48
50.6
13.6
55.6
71.3
57.5
55.0
2.8
-
152.7 63.8
121.5 20.4
140.4 35.3
>> 62.5
> 125
49
50
7.01 0.5128
5.38 0.8228
5.69 0.4728
6.27 0.7527
7.49 1.3927
5.0 1.127
51
52
53
> 125
54
47.8 27.5
82.6 7.327
55
16.5
WC-9
benznid
azol
1.50 0.08
b
aData are the mean_SD of one experiment carried out in triplicate. Selectivity index:
(ED50cytotoxicity)/(ED50parasite).
16

Table 2. Crystal data and structure refinement details for WC-9 and 28.
Compound WC-9 28
CCDC Number
1561895
1561896
C15H13NO2Se
318.22
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a/Å
C15H13NO2S
271.32
298.15
298.15
monoclinic
P21/n
triclinic
P-1
8.5141(5)
18.0328(6)
9.7129(5)
90
8.7845(6)
8.8130(6)
10.0457(9)
111.320(8)
92.726(7)
100.516(6)
706.79(10)
2
b/Å
c/Å
α/°
β/°
111.742(6)
90
γ/°
Volume/Å3
1385.16(12)
4
Z
ρcalcg/cm3
µ/mm-1
1.301
1.495
0.230
2.653
F(000)
568.0
320.0
Crystal size/mm3
0.5 × 0.2 × 0.1
MoKα (λ = 0.71073)
0.5 × 0.3 × 0.1
Radiation
MoKα (λ = 0.71073)
7.482 to 57.82
2Θ range for data collection/° 8.014 to 58.388
-11 ≤ h ≤ 11, -24 ≤ k ≤ 24, -11 ≤ h ≤ 11, -11 ≤ k ≤ 11, -13
Index ranges
-13 ≤ l ≤ 13
≤ l ≤ 13
Reflections collected
Independent reflections
17783
16553
3430 [Rint = 0.0487, Rsigma 3406 [Rint = 0.0705, Rsigma
= 0.0312] 0.0532]
=
17

Data/restraints/parameters
Goodness-of-fit on F2
3430/0/172
3406/0/172
1.045
1.023
R1 = 0.0474, wR2 =
0.1116
Final R indexes [I>=2σ (I)]
R1 = 0.0434, wR2 = 0.0933
R1 = 0.0815, wR2 =
0.1314
Final R indexes [all data]
R1 = 0.0759, wR2 = 0.1137
0.35/-0.39
Largest diff. peak/hole / e Å-3 0.19/-0.22
Supplementary Data. The Supplementary Data is available at . Physical data and spectral
information of the target molecules and the corresponding intermediates as well as copies of the
1H NMR, 13C NMR, and 19F NMR spectra are included as Supplementary Data. Moreover,
additional Figures indicating intramolecular interaction, electronic density surface, CSD
structural studies, X-ray diffraction studies tables and atomic coordinates for compounds WC-9
and 28.
Experimental Section
The glassware used in air- and/or moisture-sensitive reactions was flame-dried and reactions
were carried out under argon. Unless otherwise noted, chemicals were commercially available
and used without further purification. Solvents were distilled before use. Tetrahydrofuran was
distilled from benzophenone ketyl. Dichloromethane was distilled from phosphorus pentoxide.
Nuclear magnetic resonance spectra were recorded with a Bruker Avance II 500 MHz or a
1
Bruker Fourier 300 spectrometers. The H NMR spectra are referenced with respect to the
residual CHCl3 proton of the solvent CDCl3 at δ = 7.26 ppm. Coupling constants are reported in
13
Hz. C NMR spectra were fully decoupled and are referenced to the middle peak of the solvent
CDCl3 at δ = 77.0 ppm. Splitting patterns are designated as s, singlet; d, doublet; t, triplet; q,
quadruplet; dd, double doublet, etc. Melting points were determined with a Fisher–Johns
apparatus and are uncorrected. IR spectra were recorded with a Nicolet Magna 550 spectrometer.
Elemental analyses were performed with an Exeter CE-440 Elemental Analyzer. Analytical TLC
was performed on commercial 0.2 mm aluminum-coated silica gel plates (F254) and visualized by
18

254 nm UV or immersion in an aqueous solution of (NH4)6Mo7O24•4H2O (0.04 M), Ce(SO4)2
(0.003 M) in concentrated H2SO4 (10%).
Synthesis of selenocyanates. General procedure
A solution of the corresponding tosylate (10 mmol), potassium selenocyanate (11 mmol), and
18-crown-6 (0.1 mmol) in anhydrous tetrahydrofuran (30 mL) was refluxed for 10 h. The
solution was cooled to room temperature and the mixture was partitioned between brine (50 mL)
and methylene chloride (30 mL). The aqueous phase was extracted with methylene chloride (3 ×
25 mL). The combined organic layers were dried (MgSO4) and the solvent was evaporated. The
product was purified by column chromatography (silica gel) employing mixtures of hexane-
EtOAc as eluent or by HPLC eluting with mixtures of MeOH−H2O or MeCN−H2O.
4-(Phenylthio)phenoxyethyl Tetrahydro-2H-pyran-2-yl Ether (12). A mixture of compound
11 (1.549 g, 4.5 mmol), thiophenol (490.2 mg, 4.5 mmol), copper(I) iodide (42.3 mg, 0.22
mmol), ethylene glycol (0.57 mL; 641.3 mg, 10.4 mmol), and potassium phosphate tribasic (1.1
g, 5.2 mmol) under anhydrous conditions was evacuated and back-filled with argon. This
sequence was repeated twice. Then, 2-propanol (5.0 mL) was added and the reaction mixture
was stirred at 80 ºC for 4 days. The mixture was cooled to room temperature and was partitioned
between methylene chloride (20 mL) and water (20 mL). The aqueous layer was extracted with
methane chloride (2 × 20 mL) and 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 hexane−EtOAc (19:1) as eluent to produce 312.3 mg
(21% yield) of pure 12 as a colorless oil: Rf 0.51 (hexane−EtOAc (4:1); 1H NMR (500.13 MHz,
CDCl3) δ 1.51−1.77 (m, 6H, H-3′′′, H-4′′′, H-5′′′), 3.53 (m, 1H, H-6′′′a), 3.82 (ddd, J = 11.3, 6.3,
4.2 Hz, 1H, H-6′′′b), 3.91 (ddd, J = 11.3, 8.3, 3.1 Hz, 1H, H-1a), 4.06 (m, 1H, H-1b), 4.17 (m, 2H,
H-2), 4.71 (dd, J = 3.9, 3.3 Hz, 1H, H-2′′′), 6.93 (d, J = 8.9 Hz, 2H, H-3′), 7.11-7.25 (m, 5H,
13
aromatic protons), 7.40 (d, J = 8.9 Hz, 2H, H-2′); C NMR (50 MHz, CDCl3) δ 19.3 (C-4′′′),
25.4 (C-5′′′), 30.5 (C-3′′′), 62.2 (C-6′′′), 65.7 (C-1), 67.5 (C-2), 99.0 (C-2′′′), 115.7 (C-2′), 124.4
(C-4′′), 125.8 (C-4′), 128.2 (C-3′) 128.9 (C-3′′), 135.3 (C-2′′), 138.6 (C-1′′), 159.1 (C-1′).
19

4-(Phenylthio)phenoxyethanol (13). A solution of 12 (301.1 mg, 0.9 mmol) in methanol (10
mL) was treated with pyridinium 4-toluenesulfonate (30 mg). The reaction mixture was stirred at
room temperature overnight. Then, water (50 mL) was added 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 evaporated. The product was purified by column
chromatography (silica gel) eluting with hexane−EtOAc (17:3) to give 221.3 mg (100% yield) of
13 as a colorless oil: Rf 0.15 (hexane−EtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3) δ 4.00 (t, J =
4.6 Hz, 2H, H-1), 4.12 (t, J = 4.6 Hz, 2H, H-2), 6.94 (d, J = 8.8 Hz, 2H, H-3′), 7.17-7.30 (m, 5H,
aromatic protons), 7.43 (d, J = 8.8 Hz, 2H, H-2′); 13C NMR (125.77 MHz, CDCl3) δ 61.3 (C-1),
69.3 (C-2), 115.5 (C-2′), 116.9 (C-4′′), 125.0 (C-4′), 125.9 (C-4′), 128.4 (C-3′) 128.9 (C-3′′),
135.2 (C-2′′), 138.3 (C-1′′), 158.8 (C-1′).
4-(Phenylthio)phenoxyethyl 4-Toluenesulfonate (14). A solution of 13 (192 mg, 0.78 mmol) in
pyridine (3 mL) was treated with p-toluenesulfonyl chloride (650 mg, 3.4 mmol) and the mixture
was stirred at 0 ºC for 2 h. Then, 5% HCl (50 mL) was added and the reaction mixture was
stirred for an additional hour. The mixture was extracted with methylene chloride (50 mL) and
the organic layer was washed with 5% HCl (3 × 50 mL) and water (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 hexane−EtOAc (91:9) as eluent to give
1
243.5 mg of 14 (72% yield) as a colorless oil: Rf 0.35 (hexane−EtOAc, 4:1); H NMR (500.13
MHz, CDCl3) δ 2.45 (s, 3H, CH3), 4.15 (m, 2 H, H-1), 4.37 (m, 2H, H-2), 6.77 (d, J = 8.8 Hz,
2H, H-3′), 7.13-7.24 (m, 5H, aromatic protons), 7.34 (d, J = 7.9 Hz, 2H, H-3′′′), 7.36 (d, J = 8.8
13
Hz, 2H, H-2′), 7.82 (d, J = 8.4 Hz, 2H, H-2′′′); C NMR (125.77 MHz, CDCl3) δ 21.7 (CH3),
65.5 (C-1), 67.9 (C-2), 115.5 (C-2′), 126.0 (C-4′′), 127.2 (C-4′), 128.0 (C-2′′′), 128.6 (C-3′) 129.0
(C-3′′), 129.9 (C-3′′′), 132.8 (C-4′′′), 135.0 (C-2′′), 138.3 (C-1′′), 145.0 (C-1′′′), 158.1 (C-1′).
4-(Phenylthio)phenoxyethyl Thiocyanate (9). A solution of 14 (198.3 mg, 0.50 mmol) in
anhydrous N,N-dimethylformamide (3 mL) was treated with potassium thiocyanate (235 mg,
2.42 mmol). The reaction mixture was heated at 90 ºC for 5 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
20

mL) and water (2 × 30 mL). The organic phase was dried (MgSO4), and the solvent was
evaporated. The residue was purified by column chromatography (silica gel) eluting with
hexane−EtOAc (93:7) to give 89.1 mg (62% yield) of 9 as a colorless oil:: Rf 0.32
(hexane−EtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3) δ 3.69 (t, J = 5.8 Hz, 2 H, H-1), 4.35 (t, J
= 5.8 Hz, 2H, H-2), 6.92 (d, J = 8.9 Hz, 2H, H-3′), 7.18-7.30 (m, 5H, aromatic protons), 7.44 (d,
13
J = 8.9 Hz, 2H, H-2′); C NMR (125.77 MHz, CDCl3) δ 33.2 (C-1), 65.9 (C-2), 111.6 (SCN),
115.6 (C-2′), 126.1 (C-4′′), 126.3 (C-4′), 128.8 (C-3′) 129.0 (C-3′′), 134.9 (C-2′′), 137.8 (C-1′′),
157.8 (C-1′). HRMS (ESI) calcd. for C15H14O2NS2 [M+H]+ 288.0517; found 288.0523.
3-(Phenylthio)phenoxyethyl Tetrahydro-2H-pyran-2-yl Ether (16). A mixture of compound
15 (1.00 g, 2.9 mmol), thiophenol (316 mg, 2.9 mmol), copper(I) iodide (27.3 mg, 0.14 mmol),
ethylene glycol, (0.37 mL; 413.7 mg, 6.7 mmol), and potassium carbonate (793 mg, 5.7 mmol)
was evacuated and back-filled with argon. This sequence was repeated twice. Then, 2-propanol
(3.0 mL) was added and the reaction mixture was stirred vigorously at 80 ºC for 7 days. The
mixture was cooled to room temperature and was partitioned between methylene chloride (20
mL) and water (20 mL). The aqueous layer was extracted with dichloromethane (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 hexane−EtOAc (97:3) as eluent to afford 419 mg (44% yield) of 16 as a colorless oil:
1
Rf 0.48 (hexane−EtOAc, 4:1); H NMR (500.13 MHz, CDCl3) δ 1.50−1.64 (m, 4H, H-4′′′, H-
5′′′), 1.70−1.75 (m, 1H, H-3′′′a), 1.79−1.86 (m, 1H, H-3′′′b), 3.51 (m, 1H, H-6′′′a), 3.79 (ddd, J =
11.0, 6.5, 4.3 Hz, 1H, H-6′′′b), 3.88 (ddd, J = 11.3, 8.3, 3.0 Hz, 1H, H-1a), 4.01 (m, 1H, H-1b),
4.10 (m, 2H, H-2), 4.68 (t, J = 3.8 Hz, 1H, H-2′′′), 6.81 (ddd, J = 8.3, 2.4, 1.0 Hz, 1H, H-6′), 6.92
(m, 2H, aromatic protons), 7.25 (m, 1H, aromatic proton), 7.30 (m, 2H, aromatic protons), 7.35
(m, 2H, aromatic protons); 13C NMR (50 MHz, CDCl3) δ 19.3 (C-4′′′), 25.4 (C-5′′′), 30.5 (C-3′′′),
62.2 (C-6′′′), 65.7 (C-1), 67.5 (C-2), 99.0 (C-2′′′), 113.6 (C-6′), 116.9 (C-2′), 123.2 (C-4′), 127.2
(C-4′′), 129.2 (C-3′′) 129.9 (C-5′), 131.3 (C-2′′), 135.4 (C-1′′), 137.0 (C-3′), 159.4 (C-1′).
3-(Phenylthio)phenoxyethanol (17). A solution of 16 (399 mg, 1.2 mmol) in methanol (10 mL)
was treated with pyridinium 4-toluenesulfonate (30 mg). The reaction mixture was stirred at
room temperature overnight. Then, water (50 mL) was added and the mixture was extracted with
21

methylene chloride (3 × 50 mL). The combined organic layers were washed with brine (3 × 50
mL), dried (MgSO4), and the solvent was evaporated. The residue was purified by column
chromatography (silica gel) eluting with hexane−EtOAc (17:3) to give 290 mg (97% yield) of
pure alcohol 17 as a colorless oil: Rf 0.13 (hexane−EtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3)
δ 2.01 (t, J = 6.2 Hz, 1H, OH), 3.93 (m, 2H, H-1), 4.02 (t, J = 6.2 Hz, 2H, H-2), 6.79 (ddd, J =
8.3, 2.5, 0.9 Hz, 1H, H-6′), 6.87 (dd, J = 2.4, 1.8 Hz, 1H, H-2′), 6.93 (ddd, J = 7.7, 1.7, 0.9 Hz,
1H, H-4′), 7.21 (t, J = 8.0 Hz, 1H, H-5′), 7.27 (m, 1H, H-4′′), 7.32 (m, 2H, H-3′′), 7.38 (m, 2H,
13
H-2′′); C NMR (50 MHz, CDCl3) δ 61.4 (C-1), 69.2 (C-2), 113.2 (C-6′), 116.4 (C-2′), 123.2
(C-4′), 127.4 (C-4′′), 129.2 (C-3′′) 130.0 (C-5′), 131.6 (C-2′′), 135.0 (C-1′′), 137.5 (C-3′), 159.1
(C-1′).
3-(Phenylthio)phenoxyethyl 4-Toluenesulfonate (18). A solution of 17 (280 mg, 1.1 mmol) in
pyridine (3 mL) was treated with 4-toluenesulfonyl chloride (650 mg, 3.4 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 additional hour. The mixture was extracted with methylene chloride
(50 mL) and the organic layer was washed with 5% HCl (3 × 50 mL) and water (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 hexane−EtOAc (91:9) as eluent
1
to give 376 mg of 18 (82% yield) as a colorless oil: Rf 0.37 (hexane−EtOAc, 4:1), H NMR
(500.13 MHz, CDCl3) δ 2.44 (s, 3H, PhCH3), 4.08 (m, 2H, H-1), 4.33 (t, J = 5.8 Hz, 2H, H-2),
6.65 (ddd, J = 8.3, 2.6, 0.5 Hz, 1H, H-6′), 6.70 (t, J = 2.5 Hz, 1H, H-2′), 6.90 (ddd, J = 7.7, 1.4,
0.8 Hz, 1H, H-4′), 7.16 (t, J = 8.0 Hz, 1H, H-5′), 7.27 (m, 1H, H-4′′), 7.31 (d, J = 8.6 Hz, 2H, H-
13
3′′′), 7.32 (m, 2H, H-3′′), 7.35 (m, 2H, H-2′′), 7.79 (d, J = 8.4 Hz, 2H, H-2′′′); C NMR (50
MHz, CDCl3) δ 21.6 (PhCH3), 65.4 (C-1), 68.0 (C-2), 113.2 (C-6′), 116.5 (C-2′), 123.5 (C-4′),
127.4 (C-4′′), 128.0 (C-2′′′), 129.3 (C-3′′), 129.8 (C-3′′′), 130.0 (C-5′), 131.5 (C-2′′), 132.8 (C-
4′′′), 135.0 (C-1′′), 137.5 (C-3′), 145.0 (C-1′′′), 158.4 (C-1′).
3-(Phenylthio)phenoxyethyl Thiocyanate (10). A solution of 18 (369 mg, 0.92 mmol) in
anhydrous N,N-dimethylformamide (3 mL) was treated with potassium thiocyanate (447 mg, 4.6
mmol). The reaction mixture was heated at 80 ºC for 3 h. The mixture was allowed to cool to
room temperature and water (20 mL) was added. The aqueous phase was extracted with
22

methylene chloride (2 × 30 mL) and the combined organic layers were washed with brine (5 × 30
mL) and water (2 × 30 mL). The organic phase was dried (MgSO4), and the solvent was
evaporated. The residue was purified by column chromatography (silica gel) eluting with
hexane−EtOAc (93:7) to give 175 mg (66% yield) of 10 as a colorless oil: Rf 0.37
(hexane−EtOAc, 4:1); 1H NMR (500.13 MHz, CDCl3) δ 3.30 (t, J = 5.8 Hz, 2H, H-1), 4.25 (t, J
= 5.8 Hz, 2H, H-2), 6.79 (ddd, J = 8.3, 2.5, 0.8 Hz, 1H, H-6′), 6.86 (dd, J = 2.4, 1.8 Hz, 1H, H-
2′), 6.94 (ddd, J = 7.7, 1.7, 0.9 Hz, 1H, H-4′), 7.22 (t, J = 8.0 Hz, 1H, H-5′), 7.28 (m, 1H, H-4′′),
13
7.33 (m, 2H, H-3′′), 7.38 (m, 2H, H-2′′); C NMR (50 MHz, CDCl3) δ 33.2 (C-1), 65.8 (C-2),
111.6 (SCN), 113.2 (C-6′), 116.3 (C-2′), 123.7 (C-4′), 127.5 (C-4′′), 129.3 (C-3′′) 130.1 (C-5′),
131.7 (C-2′′), 134.8 (C-1′′), 138.0 (C-3′), 158.2 (C-1′). HRMS (ESI) calcd. for C15H13ONS2Na
[M+Na]+ 310.0336; found: 310.0336.
4-Phenoxyphenyl Prop-2-en-1-yl Ether (22). A solution of 4-phenoxyphenol (21; 1.00 g, 5.4
mmol) in dimethyl sulfoxide (5.0 mL) was treated with potassium hydroxide (60.3 mg, 10.7
mmol) and the mixture was stirred for 5 min. Then, allyl chloride (41.1 mg, 0.44 ml, 5.4 mmol)
was added dropwise. The reaction mixture was stirred at room temperature for 30 min. The
reaction mixture was extracted with methylene chloride (2 × 25 mL) and the combined organic
layers were washed with brine (5 × 50 mL), dried (MgSO4), and the solvent was evaporated. The
product was purified by column chromatography (silica gel) eluting with a mixture of
hexane−AcOEt 9:l to give 1.13 g (93% yield) of 22 as a yellow pale oil: Rf = 0.79
1
(hexane−EtOAc, 4:1); IR (film, cm−1) 3050,2950, 1580, 1470, 1200, 1100, 900, 800; H-NMR
(CDCl3, 300.18 MHz) δ 4.52 (dt, J = 5.3, 1.6 Hz, 2H, H-1), 5.30 (dq, J = 10.5, 1.4 Hz, 1H, H-3cis
to H-2); 5.42 (dq, J = 17.2, 1.6 Hz, 1H, H-3trans to H-2), 6.06 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H, H-2),
13
6.88-7.07 (m, 7H, aromatic protons), 7.27-7.32 (m, 2H, aromatic protons); C NMR (CDCl3,
75.48 MHz) δ 69.3 (C-1), 115.8 (C-2′′); 117.65 (C-3), 117.68 (C-2′), 120.7 (C-3′), 122.5 (C-4),
129.6 (C-3′′), 133.3 (C-2), 150.3 (C-4′), 154.9 (C-1′), 158.4 (C-1′′).
2-(Prop-2-en-1-yl)-4-phenoxyphenol (23). A solution of 22 (1.2 g, 5.3 mmol) in N,N-
dimethylaniline (1.0 mL) was stirred at 210 ºC for 10 h. The mixture was allowed to cool to
room temperature. Then, methylene chloride (20 mL) was added and the mixture was extracted
with a 10% aqueous solution of sodium hydroxide (2 × 15 mL). The aqueous phase was acidified
23

with 10% hydrochloric acid, and then was extracted with methylene chloride (2 × 25 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
hexane−EtOAc (97:3) as eluent to afford 220 mg (18% yield) of 23 as a colorless oil: Rf = 0.39
(hexane-EtOAc, 4:1); 1H-NMR (CDCl3, 300.18 MHz) δ 3.38 (dt, J = 6.4, 1.4 Hz, 2H, H-1), 5.14
(m, 1H, H-3cis to H-2); 5.18 (m, H-3trans to H-2, 5.99 (ddt, J = 17.5, 9.7, 6.4 Hz, H-2), 6.79-6.84 (m,
3H, aromatic protons), 6.91-6.96 (m, 2H, aromatic protons), 7.03 (tt, J = 7.4, 1.0, 1H, H-4′′), 7.29
(dd, J = 8.6, 7.4 Hz, 2H, H-3′′); 13C NMR (75.48 MHz, CDCl3) δ 35.1 (C-1), 116.7 (C-5′), 116.8
(C-3), 117.5 (C-2′′), 119.0 (C-6′), 121.7 (C-3′), 122.3 (C-4′′), 124.5 (C-6′), 126.8 (C-2′), 129.6
(C-3′′), 135.9 (C-2), 150.0 (C-1′), 150.3 (C-4′), 158.5 (C-1′′).
2-(Oxiran-2-ylmethyl)-4-phenoxyphenol (24). A solution of 23 (202 mg, 0.89 mmol) in
methylene chloride (20 mL) was added m-chlorobenzoic acid (308 mg, 1.8 mmol). The solution
was stirred at room temperature for 5 days. Then, the mixture was washed with a saturated
solution of sodium bicarbonate (3 × 30 mL), and water (2 × 20 ml). The organic phase was dried
(MgSO4), and the solvent was evaporated to give 205 mg (95% yield) of 24 as a yellowish oil,
which was used in the next step without further purification: Rf = 0.32 (hexane−EtOAc, 4:1); 1H
NMR (CDCl3, 300.18 MHz) δ 2.68 (dd, J = 15.1, 7.4 Hz, 1H, H-1a), 2.74 (dd, J = 4.3, 2.9 Hz,
1H, H-3a), 2.94 (t, J = 4.1 Hz, 1H, H-3b), 3.19 (dd, J = 15.1, 2.4 Hz, 1H, H-1b), 3.30 (ddt, J = 5.0,
3.9, 2.5 Hz, 1H, H-2), 6.81-6.96 (m, 5H, aromatic protons), 7.01-7.07 (m, 1H, aromatic proton),
13
7.27-7.32 (m, 2H, aromatic protons); C NMR (75.48 MHz, CDCl3) δ 34.8 (C-1), 48.1 (C-3),
53.5 (C-2), 117.6 (C-6′), 118.2 (C-2′′), 120.0 (C-3′), 122.1 (C-5′), 122.4 (C-4′′), 124.5 (C-6′),
129.6 (C-3′′), 151.8 (C-4′),149.8 (C-1′), 158.5 (C-1′′).
(5-Phenoxy-2,3-dihydrobenzofuran-2-yl)methanol (25). Compound 24 (205 mg, 0.85 mmol)
was dissolved in acidified CHCl3 (prepared by shaking 180 ml of CHCl3 with 7 drops of
concentrated HCl). After 20 min the mixtures was extracted with 1% aqueous NaHCO3. The
organic phase was dried (Na2SO4) and the solvent was evaporated. The product was purified by
column chromatography (silica gel) employing hexane−EtOAc (39:11) as eluent to afford 110
1
mg (54% yield) of 25 as a colorless oil: Rf = 0.36 (hexane−EtOAc, 3:2); H-NMR (CDCl3,
300.18 MHz) δ 3.04 (dd, J = 16.0, 7.6 Hz, 1H, H-3a), 3.25 (dd, J = 15.8, 9.3 Hz, 1H, H-3b), 3.77
24

(dd, J = 12.0, 6.1 Hz, 1H, CHaHOH), 3.89 (dd, J = 12.1, 3.2 Hz, 1H, CHbHOH), 4.96 (dddd, J =
9.4, 7.6, 6.1, 3.3 Hz, 1H, H-2), 6.76 (d, J = 8.5 Hz, 1H, H-7), 6.81-6.97 (m, 4H, aromatic
protons), 7.02-7.08 (m, 1H, aromatic proton), 7.31 (dd, J = 8.6, 7.4 Hz, 2H, aromatic protons);
13C NMR (75.48 MHz, CDCl3) δ 31.5 (C-3), 64.8 (CH2OH), 83.5 (C-2), 109.7 (C-7), 117.1 (C-
6), 117.5 (C-2′), 119.5 (C-4), 122.3 (C-4′), 128.0 (C-4a), 129.6 (C-3′), 150.4 (C-5), 155.4 (C-7a),
158.7 (C-1′).
(5-Phenoxy-2,3-dihydrobenzofuran-2-yl)methyl 4-Toluenenesulfonate (26). A solution of
alcohol 25 (98.7 mg, 0.41 mmol) in pyridine (3 mL) was treated with 4-toluenesulfonyl chloride
(233.0 mg, 1.2 mmol) and the mixture was stirred at room temperature for 4 h. The mixture was
extracted with methylene chloride (50 mL) and the organic layer was washed water (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 hexane-EtOAc (91:9) as eluent to
1
afford 140 mg of 26 (87% yield) as a colorless oil: Rf = 0.18 (hexane-EtOAc; 8:2); H-NMR
(CDCl3, 300.18 MHz) δ 3.04 (dd, J = 16.0, 7.0 Hz, 1H, H-3a), 3.27 (dd, J = 16.0, 9.6 Hz, 1H, H-
3b), 4.19 (d, J = 5.1 Hz, 2H, CH2OH), 4.97 (ddt, J = 9.7, 7.1, 5.0 Hz, 1H, H-2), 6.64 (d, J = 8.5
Hz, 1H, H-7), 6.76-6.93 (m, 4H, aromatic protons), 7.01-7.07 (m, 1H, aromatic proton), 7.28 (d,
J = 7.4 Hz, 1H, aromatic proton), 7.31 (d, J = 7.5 Hz, 1H, aromatic proton), 7.35 (d, J = 8.0 Hz,
2H, H-3′′), 7.80 (d, J = 8.4 Hz, 2H, H-2′′).
( )-5-Phenoxy-2-(thiocyanatomethyl)-2,3-dihydrobenzofuran (19). A solution of 26 (163 mg,
0.34 mmol) in anhydrous N,N-dimethylformamide (3.0 mL) was treated with potassium
thiocyanate (167 mg, 1.7 mmol). The reaction mixture was stirred at 100 ºC 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 combined organic phases were dried
(MgSO4) and the solvent was evaporated. The residue was purified by column chromatography
(silica gel) eluting with hexane−EtOAc (9:1) to give 80.1 mg (82% yield) of 19 as a colorless oil:
Rf =0.33 (hexane−EtOAc, 4:1); 1H-NMR (CDCl3, 500.13 MHz) δ 3.06 (dd, J = 16.1, 6.4 Hz, 1H,
H-3a), 3.25 (dd, J = 13.6, 6.4 Hz, 1H, CHaHSCN), 3.28 (dd, J = 13, 3.2 Hz, 1H, CHbHOH), 3.46
(dd, J = 16.1, 9.2 Hz, 1H, H-3b), 5.10 (ddt, J = 9.2, 6.4, 5.5 Hz, 1H, H-2), 6.77 (d, J = 8.5 Hz,
25

1H, H-7), 6.84 (ddt, J = 8.6, 2.4, 0.7 Hz, 1H, aromatic proton), 6.89 (m, 1H, aromatic proton),
6.94 (m, 2H, aromatic protons), 7.05 (tt, J = 7.4, 1.0 Hz, 1H, aromatic proton), 7.29 (d, J = 7.4
13
Hz, 1H, aromatic proton), 7.31 (d, J = 7.4 Hz, 1H, aromatic proton); C NMR (125.76 MHz,
CDCl3) δ 34.8 (C-3), 38.3 (CH2SCN), 80.6 (C-2), 110.2 (C-7), 111.7 (SCN), 116.9 (C-6), 117.7
(C-2′), 119.9 (C-4), 122.6 (C-4′), 126.5 (C-4a), 129.5 (C-3′), 151.0 (C-5), 154.7 (C-7a), 158.4 (C-
1′).
5-Phenoxy-2-(thiocyanatomethyl)benzofuran (20). To a solution of 19 (21.9 mg, 7.73 × 10-2
mmol) in anhydrous dioxane (1.5 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) dissolved in 1.5 ml of the same solvent. The solution was refluxed for 24 h and then was
cooled. The solid was filtered off and the filtrate was evaporated under reduced pressure. The
residue was purified by column chromatography (silica gel) eluting with hexane-EtOAc (49:1) to
1
give 5.3 mg (24% yield) of 20 as a colorless oil: Rf = 0.30 (hexane−EtOAc, 4:1); H-NMR
(CDCl3, 500.13 MHz) δ 4.29 (s, 2H, CHaHSCN), 6.76 (s, 1H, H-3), 6.98 (m, 2H, aromatic
protons), 7.07 (m, 2H, aromatic protons), 7.18 (d, J = 2.5 Hz, 1H, aromatic proton), 7.32 (d, J =
7.4 Hz, 1H, aromatic proton), 7.33 (d, J = 7.4 Hz, 1H, aromatic proton), 7.44 (d, J = 8.9 Hz, 1H,
aromatic proton); 13C NMR (125.76 MHz, CDCl3) δ 31.3 (CH2SCN), 107.4 (C-7), 111.2 (SCN),
111.4 (C-3), 112.2 (C-4), 118.0 (C-6), 118.1 (C-2′), 122.9 (C-4′), 128.7 (C-4a), 129.7 (C-3′),
150.9 (C-7a), 151.9 (C-5), 153.0 (C-2), 158.2 (C-1′).
3-Phenoxyphenoxyethyl 4-Toluenesulfonate (31). A solution of 3026 (288.4 mg, 1.2 mmol) in
pyridine (3.0 mL) cooled at 0 °C was treated 4-toluenesulfonyl chloride (710.3 mg, 3.7 mmol)
portion wise, and the mixture was stirred at 0 ºC for 3 h. Then, a 5% aqueous solution of
hydrochloric acid (10 mL) was added and the reaction mixture was stirred for an additional hour.
The mixture was extracted with methylene chloride (30 mL) and the organic layer was washed
with 5% hydrochloric acid (3 × 25 mL) and water (3 × 30 mL). The organic phase was dried
(MgSO4) and the solvent was evaporated. The product was purified by column chromatography
(silica gel) eluting with hexane−EtOAc (19:1) to give 312 mg (68 % yield) of 31 as a colorless
1
oil: Rf = 0.47 (hexane−EtOAc, 1:1); H NMR (500.13 MHz, CDCl3) δ 2.46 (s, 3H, CH3), 4.10
(m, 2H, H-2), 4.34 (m, 2H, H-1), 6.41 (t, J = 2.2 Hz, 1H, H-2′), 6.52 (dd, J = 8.3, 2.2 Hz, 1H, H-
6′), 6.62 (dd, J = 8.2, 2.0 Hz, 1H, H-4′), 7.00 (d, J = 8.4 Hz, 2H, H-2′′), 7.12 (t, J = 7.4 Hz, 1H,
26

H-5′), 7.18 (t, J = 8.2 Hz, 1H, H-4′′), 7.32 (d, J = 8.3 Hz, 2H, H-3′′′), 7.34 (t, J = 8.0 Hz, 2H, H-
13
3′′), 7.80 (d, J = 8.2 Hz, 2H, H-2′′′); C NMR (125.77 MHz, CDCl3) δ 21.6 (CH3), 65.5 (C-2),
68.0 (C-1), 105.5 (C-2′), 109.1 (C-6′), 111.6 (C-4′), 119.1 (C-2′′), 123.5 (C-4′′), 128.0 (C-2′′′),
129.76 (C-3′′), 129.82 (C-3′′′), 130.2 (C-5′), 132.8 (C-4′′′), 145.0 (C-1′′′), 156.8 (C-1′′), 158.5
(C1′), 159.3 (C-4′).
(4-Phenoxyphenoxyethyl) 4-Toluenesulfonate (33). Alcohol 3221 (436.4 mg, 1.9 mmol) in
pyridine (3.0 mL) was treated with 4-toluenesulfonyl chloride (1,049 g, 5.1 mmol) as depiected
for the preparation of 31. The residue was purified by column chromatography (silica gel)
employing hexane-EtOAc (9:1) as eluent to afford 499.3 mg of 33 (72% yield) as a white solid:
1
mp 65 ºC; H NMR (500.13 MHz, CDCl3) δ 2.45 (s, 3 H, CH3), 4.14 (m, 2 H, H-2), 4.37 (m, 2
H, H-1), 6.77 (d, J = 9.1 Hz, 2H, H-2′), 6.92 (m, 2H, H-2′′), 6.93 (d, J = 9.1 Hz, 2H, H-3′), 7.05
(tt, J = 7.4, 1.0 Hz, 1H, H-4′′), 7.30 (dd, J = 8.4, 7.4, 2H, H-3′′), 7.35 (d, J = 8.0 Hz, 2H, H-3′′′),
13
7.85 (d, J = 8.3 Hz, 2H, H-2′′′); C NMR (125.76 MHz, CDCl3) δ 21.6 (CH3), 66.0 (C-1), 68.1
(C-2), 115.8 (C-2′′), 117.8 (C-2′), 120.7 (C-3′), 122.6 (C-4′′), 128.0 (C-2′′′), 129.6 (C-3′′), 129.8
(C-3′′′), 132.9 (C-4′′′), 145.0 (C-1′′′), 150.8 (C-4′), 154.2 (C-1′), 158.2 (C-1′′).
3-Phenoxyphenoxyethyl Selenocyanate (27). A solution of 31 (99 mg, 0.24 mmol) in
anhydrous N,N-dimethylformamide (3.0 mL) was treated with potassium selenocyanate (125 mg,
1.29 mmol) according to the general procedure. The product was purified by column
chromatography (silica gel) using a mixture of hexane-EtOAc (9:1) as eluent to give 45 mg (64%
yield) of 27 as a colorless oil: Rf = 0.32 (hexane−EtOAc; 4:1); 1H NMR (500.13 MHz, CDCl3) δ
3.41 (t, J = 6.0 Hz, 2H, H-1), 4.35 (t, J = 6.0 Hz, 2H, H-2), 6.57 (t, J = 2.3 Hz, 1H, aromatic
proton), 6.64 (m, 2H, aromatic protons), 7.03 (dd, J = 8.7, 1.0 Hz, 2H, aromatic protons), 7.13 (t,
J = 7.4 Hz, 1H, aromatic proton), 7.23 (t, J = 8.3 Hz, 1H, aromatic proton), 7.35 (dd, J = 8.3, 8.0
Hz, 2H, aromatic protons); 13C NMR (125.77 MHz, CDCl3) δ 28.0 (C-1), 66.5 (C-2), 101.6
(SeCN), 105.5 (C-2′), 109.2 (C-6′), 111.9 (C-4′), 119.2 (C-2′′), 123.6 (C-4′′), 129.8 (C-3′′), 130.4
(C-5′), 156.7 (C-1′′), 158.7 (C-1′), 159.1 (C-4′). HRMS (ESI) calcd. for C15H13O2NSeNa
[M+Na]+ 342.0009; found 342.0005.
4-Phenoxyphenoxyethyl Selenocyanate (28). Tosylate 33 (350.1 mg, 1.0 mmol) was treated
27

according to the general procedure. The product was purified by column chromatography (silica
gel) employing a mixture of hexane−EtOAc (9:1) as eluent to obtain 225.9 mg (71% yield) of 28
as a white solid: mp 54 ºC; Rf = 0.59 (hexane−EtOAc, 7:3); IR (film, cm−1) 3041, 2868, 2152,
1588, 1501, 1486, 1212, 1008, 839, 755, 690; 1H NMR (500.13 MHz, CDCl3) δ 3.44 (t, J = 5.9
Hz, 2 H, H-1), 4.37 (t, J = 5.9 Hz, 2H, H-2), 6.90 (d, J = 9.2 Hz, 2H, H-2′), 6.95 (m, 2H, H-2′′),
6.99 (d, J = 9.2 Hz, 2H, H-3′), 7.06 (tt, J = 7.4, 1.0 Hz, 1H, H-4′′), 7.31 (dd, J = 8.7, 7.4, 2H, H-
3′′); 13C NMR (125.77 MHz, CDCl3) δ 28.1 (C-1), 67.1 (C-2), 101.1 (SeCN), 115.9 (C-2′′),
117.9 (C-2′), 120.8 (C-3′), 122.7 (C-4′′), 129.7 (C-3′′), 151.2 (C-4′), 153.9 (C-1′), 158.1 (C-1′′).
HRMS (ESI) calcd. for C15H13O2NSeNa [M+Na]+ 342.0009; found 341.9996. Anal. Calculated
for (C15H13O2NSe) C 56.61, H 4.12, N 4.40; found C 56.50, H 4.27, N 3.92.
4-Phenoxyphenoxyethyl carbamate (34). To a solution of 4-phenoxyphenyethanol (32; 500
mg, 2.2 mmol) in anhydrous methylene chloride (5.0 mL), cooled at −15 ºC, was added
trichloroacetyl isocyanate (451 mg, 0.28 mL, 2.4 mmol) dropwise under an argon atmosphere.
The reaction mixture was stirred at this temperature for 10 min. Then, methanol (1.0 mL) was
added to get rid of the excess of trichloroacetyl isocyanate. Then, the mixture was concentrated
under reduced pressure. The residue was dissolved in methanol (15 mL) and an aqueous 2.0 M
solution of potassium carbonate (7.5 mL) was added. The resulting mixture was stirred at room
temperature for additional 5 h. The reaction mixture was extracted with methylene chloride (2 ×
25 mL) and the combined organic layers were washed with brine (5 × 50 mL), dried (MgSO4),
and the solvent was evaporated to give 584.0 mg (98% yield) of 34 as a white solid, which was
used as such in the next step: mp 126 ºC; Rf = 0.27 (hexane−EtOAc, 3:2); IR (KBr, cm-1) 1684,
1504, 1491, 1233, 1065, 839,754, 689; 1H NMR (300.18 MHz, CDCl3) δ 4.16 (m, 2H, H-1), 4.43
(m, 2H, H-2), 4.72 (br s, 2H, C(O)CH2), 6.89 (d, J = 9.2 Hz, 2H, H-2′), 6.94 (m, 2H, H-2′′), 6.98
(d, J = 9.2 Hz, 2H, H-3′), 7.05 (tt, J = 7.4, 1.0 Hz, 1H, H-4′′), 7.30 (dd, J = 8.6, 7.4, 2H, H-3′′);
13C NMR (75.48 MHz, CDCl3) δ 63.6 (C-1), 66.8 (C-2), 115.7 (C-2′′), 117.7 (C-2′), 120.8 (C-3′),
122.5 (C-4′′), 129.6 (C-3′′), 1506.2 (C-4′), 156.7 (C-1′), 156.5 (OC(O)NH2), 158.3 (C-1′′).
4-Phenoxyphenoxyethyl Cyanate (29). To a solution of carbamate 34 (50 mg, 0.18 mmol) in
anhydrous methylene chloride (16 mL), cooled at −78 ºC, was added anhydrous
diisopropylethylamine (14.2 mg, 0.19 mL, 1.1 mmol) followed by addition of
28

trifluoromethanesulfonic anhydride (77.0 mg, 45.8 µL, 0.27 mmol) under an argon atmosphere.
The solution was stirred at −78 ºC for 1.5 h. Then, the reaction was quenched by addition of a
5% aqueous solution of sodium bicarbonate. The solution was allowed to warm to room
temperature and the organic layer was separated, washed with a 5% aqueous solution of sodium
bicarbonate, dried (MgSO4), and the solvent was evaporated. The product was purified by
column chromatography (silica gel) employing hexane−EtOAc (83:17) as eluent to afford 11.1
mg (24% yield) of 29 as a colorless oil: Rf = 0.62 (hexane−EtOAc, 3:2); IR (film, cm-1) 2250,
1506, 1484, 1220, 871, 843, 688; 1H NMR (300.18 MHz, CDCl3) δ 4.33 (t, J = 5.9 Hz, 2 H, H-
1), 4.74 (t, J = 5.9 Hz, 2H, H-2), 6.93 (d, J = 9.2 Hz, 2H, H-2′), 6.97 (m, 2H, H-2′′), 7.02 (d, J =
13
9.2 Hz, 2H, H-3′), 7.08 (tt, J = 7.4, 0.9 Hz, 1H, H-4′′), 7.33 (dd, J = 8.5, 7.4, 2H, H-3′′); C
NMR (75.48 MHz, CDCl3) δ 78.0 (C-1), 65.8 (C-2), 116.0 (C-2′′), 117.9 (C-2′), 120.7 (C-3′),
122.8 (C-4′′), 129.7 (C-3′′), 151.4 (C-4′), 153.8 (C-1′), 158.1 (C-1′′).
3-Phenoxyphenyl Prop-2-en-1-yl Ether (39). A solution of 3-phenoxyphenol (38; 1.5 g, 8.0
mmol) in DMSO (5.0 ml) was added KOH (904 mg, 16.1 mmol) and was stirred 5 min. Then
was added allyl chloride (617 mg, 0.66 mL, 8.6 mmol) slowly. After stirred 10 min, the reaction
mixture was extracted with methylene chloride (2 × 25 ml). The combined organic phases were
washed with brine (5 × 50 mL), dried (MgSO4), and the solvent was evaporated affording 1.61 g
(90% yield) of 39 as colorless oil which was used as such in the next step: Rf = 0.71
(hexane−EtOAc, 4:1); 1H-RMN (300.18 MHz, CDCl3) δ 4.50 (dt, J = 5.3, 1.5 Hz, 2H, H-1), 5.28
(dq, J = 10.4, 1.4 Hz, 1H, H-3cis to H-2); 5.39 (dq, J = 17.3, 1.6 Hz, 1H, H-3trans to H-2), 6.03 (ddt, J
= 17.3, 10.6, 5.3 Hz, 1H, H-2), 6.59 (m, 2H, aromatic protons), 6.66 (m, 1H, aromatic proton),
7.02 (m, 2H, aromatic protons), 7.11 (tt, J = 7.4, 1.1 Hz, 1H, H-4′′), 7.21 (m, 1H, aromatic
proton), 7.35 (dd, J = 8.5, 7.5 Hz, 2H, H-3′′); 13C NMR (75.48 MHz, CDCl3) δ 68.9 (C-1), 105.5
(C-2′), 109.5 (C-6′), 111.1 (C-4′), 117.9 (C-3), 119.1 (C-2′′), 123.4 (C-4′′), 129.7 (C-3′′), 130.1
(C-5′), 133.0 (C-2), 156.9 (C-1′′), 158.4 (C-1′), 159.9 (C-4′).
2-Allyl-5-phenoxyphenol (40) and 2-Allyl-3-phenoxyphenol (41). A solution of 39 (1.80 g, 7.9
mmol) in N,N-dimethylaniline (6.0 mL) was stirred at 210 ºC for 10 h. The reaction mixture was
allowed to cool to room temperature; then methylene chloride (20 mL) was added. The mixture
was extracted with 10% aqueous solution of sodium hydroxide (2 × 15 mL). The aqueous phase
29