Tyrosol and hydroxytyrosol derivatives as antitrypanosomal and antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2016.04.047

Trypanosomiasis and leishmaniasis keep being a real challenge for health and development of African countries. Existing treatments have considerable side effects and increase resistance of the parasites. We have measured antitrypanosomal and antileishmanial activity of natural phenols, tyrosol (TYR) and hydroxytyrosol (HT) and several of their esters and metabolites. We found significant IC₅₀ values against Trypanosoma brucei for HT decanoate ester and HT dodecanoate ester (0.6 and 0.36 μM, respectively). This represents a large increase in activity with respect to HT (79 and 132 fold, respectively). Moreover, both compounds displayed a high selectivity index against MRC-5, a non-tumoral human cell line (118 and 106, respectively). Then, we synthesized a focused library of compounds to explore structure-activity. We found the ether and thiourea analogs of HT decanoate ester and HT dodecanoate ester also showed IC₅₀ values against T. brucei in the low micromolar range. In conclusion, the di-ortho phenolic ring and medium size alkyl chain are essential for activity whereas the nature of the chemical bond among them seems less important.

Full text of DOI:10.1016/j.ejmech.2016.04.047

European Journal of Medicinal Chemistry 119 (2016) 132e140
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Tyrosol and hydroxytyrosol derivatives as antitrypanosomal and
antileishmanial agents
a
a
a, b
~
Efres Belmonte-Reche , Marta Martínez-García , Pablo Penalver
,
Veronica Gomez-Perez , Ricardo Lucas b, Francisco Gamarro a,
a
a,
ꢀ
ꢀ
ꢀ
Jose María Perez-Victoria **, Juan Carlos Morales a
a
,
, b, *
ꢀ
ꢀ
a
ꢀ
Department of Biochemistry and Molecular Pharmacology, Institute of Parasitology and Biomedicine Lopez-Neyra, CSIC, PTS Granada, Avda. del
Conocimiento, 17, 18016, Armilla, Granada, Spain
b Department of Bioorganic Chemistry, Institute of Chemical Research, CSIC e University of Seville, Avda. Americo Vespucio, 49, 41092, Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Trypanosomiasis and leishmaniasis keep being a real challenge for health and development of African
countries. Existing treatments have considerable side effects and increase resistance of the parasites. We
have measured antitrypanosomal and antileishmanial activity of natural phenols, tyrosol (TYR) and
hydroxytyrosol (HT) and several of their esters and metabolites. We found significant IC50 values against
Received 3 March 2016
Received in revised form
28 March 2016
Accepted 17 April 2016
Available online 26 April 2016
Trypanosoma brucei for HT decanoate ester and HT dodecanoate ester (0.6 and 0.36 mM, respectively).
This represents a large increase in activity with respect to HT (79 and 132 fold, respectively). Moreover,
both compounds displayed a high selectivity index against MRC-5, a non-tumoral human cell line (118
and 106, respectively). Then, we synthesized a focused library of compounds to explore structure-
activity. We found the ether and thiourea analogs of HT decanoate ester and HT dodecanoate ester
also showed IC50 values against T. brucei in the low micromolar range. In conclusion, the di-ortho
phenolic ring and medium size alkyl chain are essential for activity whereas the nature of the chemi-
cal bond among them seems less important.
Keywords:
Antitrypanosomal
Antileishmanial
Tyrosol
Hydroxytyrosol
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
both diseases present important drawbacks such as high toxicity,
increased resistance and variability on their efficacy depending on
Infectious diseases caused by protozoan parasites affect millions
of people around the world, especially in tropical and subtropical
areas. Among them, leishmaniasis (caused by different species of
Leishmania) is a major global health problem with around 12
million people infected and causing 1e2 million new cases every
year. Trypanosoma brucei, transmitted by the tsetse fly, is respon-
sible for sleeping sickness in humans and preclude the develop-
ment of productive livestock and agricultural activity based on
domesticated animals where it causes Nagana [1,2]. For these rea-
sons, this parasite is considered to be one of the major root causes
of hunger and poverty in sub-Sahara Africa. Current treatments for
the different strain of the parasite. Thus, the development of new
antiparasitic therapies continues to be necessary.
Natural products have been tested for decades to find new
antitrypanosomal and antileishmanial agents. Among them,
several phenolics and polyphenols have shown relevant activity.
For example, flavonoids such as 7,8-dihydroxyflavone, 3-
hydroxyflavone and rhamnetin showed low micromolar IC50
values for T. brucei rhodesiense and others such as luteolin and
quercetin also displayed low micromolar IC50s for Leishmania
donovani [3]. In fact, quercetin glycosides present in the aqueous
extract of Kalanchoe pinnata were active by oral administration in
experimental cutaneous and visceral leishmaniasis infections pro-
duced in vivo [4,5].
Tyrosol (TYR, 1) and hydroxytyrosol (HT, 2) (Fig. 1) are natural
phenolic antioxidants present in olives and olive oil. They have
shown a diverse biological activity such as antibacterial [6,7],
antiviral [8], anti-inflammatory [9e12], neuroprotective [13] and
anticancer activity [14], inhibition of human LDL oxidation [15] and
* Corresponding author. Department of Biochemistry and Molecular Pharma-
ꢀ
cology, Institute of Parasitology and Biomedicine Lopez-Neyra, CSIC, PTS Granada,
Avda. del Conocimiento, 17, 18016, Armilla, Granada, Spain.
** Corresponding author.
ꢀ
E-mail addresses: josepv@ipb.csic.es (J.M. Perez-Victoria), jcmorales@ipb.csic.es
(J.C. Morales).
http://dx.doi.org/10.1016/j.ejmech.2016.04.047
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

E. Belmonte-Reche et al. / European Journal of Medicinal Chemistry 119 (2016) 132e140
133
sulfating reagent, NEt3 as base, and acetonitrile as solvent at 100 ꢁC
under microwave radiation. The reaction afforded the sulfated TYR
derivative in good yield. Final acyl deprotection and reverse phase
purification gave TYR sulfate 9.
We identified hydroxytyrosol decanoate ester 13 and hydrox-
ytyrosol dodecanoate ester 14 as relevant hit compounds against
T. brucei (see below, Table 2) after carrying out the first in vitro
screening on T. brucei and L. donovani. We decided then to perform
structure-activity studies on 13 and 14 by changing the phenolic
hydroxyl groups, the type of chemical bond between the phenolic
ring and the alkyl chain, and the length of the alkyl chain (Fig. 2) in
order to improve its biological activity.
Fig. 1. Chemical structures of tyrosol (TYR, 1) and hydroxytyrosol (HT, 2).
prevention of platelet aggregation [16]. Recently, Moradi-Afrapoli
We synthesized a hydroxyl protected version of compound 14
by formation of the acetal derivative 22 and also a shorter version
with a decanoate alkyl chain, compound 21 (Scheme 2). The cyclic
acetal intermediate 20 was obtained by reaction of HT with CH2Cl2
in basic conditions and then acylated using the enzymatic condi-
tions used previously for HT yielding the desired compounds. The
ether analog of 14, compound 29, was prepared as reported pre-
viously [33] by benzyl protection of the phenolic groups, alkylation
with 1-iodododecane under basic conditions and final hydroge-
nation. The HT ether decanoate derivative 28 was prepared
following the same procedure. We also prepared the p-methox-
ybenzyl (PMB) protected HT derivative 24 trying to improve the
yields of the route. However, deprotection with trifluoroacetic acid
of the ether intermediate 27 was unsuccessful. The synthesis of the
thioether analog of 14 was attempted next. Tosylation of derivatives
23 and 24 was followed by nucleophilic displacement with 1-
dodecanethiol producing the protected thioether derivatives 32
and 33 with moderate yields. Neither hydrogenation nor reaction
under strong acidic conditions yielded the final thioether depro-
tected products.
Dopamine hydrochloride was the starting material for the syn-
thesis of the amide and thiourea analogs of 13 and 14 (compounds
37e40) (Scheme 3). Dopamine was also synthesized to compare its
activity with HT. The thiourea derivatives were synthesized by re-
action of the isothiocyanate intermediate 36 with the corre-
sponding primary alkylamine to obtain the products in moderate
yields. The amide analogs of 13 and 14 were prepared by reaction of
dopamine hydrochloride with the corresponding fatty acids using
HATU as the coupling reagent.
et al. reported medium activity of TYR against T. brucei rhode-
siense (IC50 > 15 mM) [17]. To the best of our knowledge, HT has not
been examined for activity against T. brucei. Though, moderate
antileishmanial activity was reported for HT against both, pro-
mastigotes of Leishmania infantum, L. donovani, and Leishmania
major, and against L. donovani amastigotes that parasitize J774A.1
macrophages [18].
The preparation of chemically-modified natural products to
improve their antiparasitic activity is still a very active source of
potential new drugs. This is the case of chalcone [19] or caracasine
acid derivatives [20], or the preparation of hybrids such as Cinchona
alkaloids with bile acids [21] or caffeine-based chalcones [22].
Tyrosol and hydroxytyrosol derivatives have also been synthesized
in order to improve the antioxidant and biological properties of the
parent compounds. Hydroxytyrosol fatty acid esters increased the
protection of proteins and lipids against oxidation caused by per-
oxyl radicals in a brain homogenate as an ex vivo model [23].
Similarly, hydroxytyrosol acetate was able to reduce the metabolic
imbalance induced by a high-cholesterol diet in rats to a higher
extent than HT [24]. Finally, HT alkyl ether derivatives, more stable
under biological conditions than HT, have been reported to exert
antiproliferative [25], neuroprotective [26,27], antiplatelet and
anti-inflammatory effects [28] that are greater than those of HT.
Due to the improved biological activity found for several HT
derivatives with respect to HT itself, we decided to investigate the
antitrypanosomal and antileishmanial activity of a series of TYR
and HT derivatives. We prepared a series of tyrosol and hydrox-
ytyrosol fatty acid esters together with three metabolites of HT and
TYR, tyrosol sulfate, tyrosol glucuronate and hydroxytyrosol glu-
curonate for a first in vitro screening. Later, we synthesized a
focused library of compounds to explore structure-activity rele-
vance varying the number of phenolic OH groups, the type of
chemical bond between the phenolic ring and the alkyl chain, and
the length of the alkyl chain.
2.2. Biological evaluation
The in vitro antiparasitic activities of TYR, HT and their de-
rivatives 3e18 were evaluated against T. brucei brucei and against
axenic amastigotes of L. donovani. Those compounds exhibiting
over 40% inhibition of axenic amastigotes at 20
mM were also
2. Results and discussion
evaluated on intracellular amastigotes of L. donovani. The cytotox-
icity of these compounds was also evaluated against a human non-
tumoral lung cell line (MRC-5). Suramin and amphotericin B were
used as positive drug controls for T. brucei and L. donovani,
respectively. Selectivity indices (SI) were calculated according to
the formula: IC50 (MRC-5)/IC50 parasite. The activity gain (AG) of
each compound with respect to the reference compound (TYR or
HT) was calculated according to the formulas: IC50 (TYR)/IC50
compound and IC50 (HT)/IC50 compound.
2.1. Chemistry
Tyrosol and hydroxytyrosol fatty acid esters (3e7 and 11e16,
respectively) were synthesized by enzymatic acylation using
Novozym 435 and the corresponding vinyl acyl donor in t-butyl
methyl ether as reported previously [29e31] (Scheme 1). Tyrosol
and hydroxytyrosol glucuronates 8 and 17, 18 were prepared by
glycosylation of the acetyl protected TYR or HT derivatives (3 and
10, respectively). We used the acetyl protected trichloroacetimidate
glucuronosyl derivative 19 as glycosyl donor and boron trifluoride
etherate as the catalyst in the glycosylation step. Final acetyl
Table 1 presents the antiparasitic and cytotoxic data for TYR and
its derivatives 3e9. IC50 values against T. brucei ranged from 10 to
62.7 mM and selectivity indices from 1.5 to 5. None of the TYR de-
rivatives improved the activity of TYR itself as can be observed from
deprotection yielded glucuronate derivatives
8
and 17,18 as
the AG values. In the case of L. donovani, compounds 4 and 5
described previously [32]. TYR sulfate 9 was prepared following a
similar strategy to the one used for the glucuronate derivative.
Sulfation of tyrosol acetate 3 was carried out with SO3ꢀNMe3 as
showed inhibition over 40% of axenic amastigotes at 20
centration. These two derivatives were evaluated on intracellular
amastigotes and showed IC50 values > 10 M. Aissa et al. [34]
mM con-
m

134
E. Belmonte-Reche et al. / European Journal of Medicinal Chemistry 119 (2016) 132e140
Scheme 1. Reagents and condition: (a) vinyl alkyl ester, Novozym 435, tBuOMe, 60 ꢁC; (c) 19, BF3$OEt2, CH2Cl2; (d) Na2CO3, MeOH, H2O; (e)SO3$NMe3, TEA, CH3CN, 100 ꢁC, 20 min,
MW.
Table 1
Cytotoxic, trypanocidal and leishmanicidal activities of tyrosol derivatives.
Cytotoxicity MRC-5
T. brucei
L. donovani
Compounds
R1
R2
IC50
(m
M)
IC50
(m
M)
SI
AG
Axenic amastigotes
Intracellular amastigotes
% Inhib. (20
m
M)
IC50
(m
M)
SI
AG
TYR (1)
3
4
5
6
7
8
9
H
H
H
H
H
H
H
>500
>10
>5
1.4
e
50
28
44
46
34
32
36
36
>10
n.d.
>10
>10
n.d.
n.d.
n.d.
n.d.
e
5
e
e
e
1
COCH3
113.6 11.3
166.4 0.6
133.0 9.8
171.5 15.7
67.2 0.2
110.2 14.4
106.4 17.2
e
79.4 23.1
59.1 2.3
56.8 8.3
81.4 19.3
16.2 3.8
61.5 23.8
41.6 12.6
0.038 0.003
0.026 0.002
e
0.13
0.16
0.22
0.16
0.52
0.17
0.16
CO(CH2)4CH3
CO(CH2)6CH3
CO(CH2)8CH3
CO(CH2)10CH3
H
2.8
2.3
2.1
4.1
1.8
2.6
16.6
13.3
e
1
e
e
e
e
e
GlcAc
e
H
SO-3Kþ
e
Suramin
Eflornitin
Amphotericin B
e
e
e
0.10 0.01
Experiments on L. donovani were carried out in axenic amastigotes and intracellular amastigotes.
SI ¼ Selectivity Index (IC50 MRC-5 / IC50 parasite); AG ¼ Activity gain (IC50 tyrosol / IC50 compound).
Table 2
Cytotoxic, trypanocidal and leishmanicidal activities of hydroxytyrosol derivatives.
Cytotoxicity MRC-5
T. brucei
IC50 M)
L. donovani
IC50
(
m
M)
(
m
SI
AG
Axenic amastigotes
Intracellular amastigotes
Compounds
R1
R2, R3
% Inhib. (20
m
M)
IC50
(
mM)
SI
AG
HT (2)
10
11
12
13
14
15
16
17e18
H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
>50
>50
47.5 16.9
43.4 14.0
9.4 1.4
>1
>1
8
e
0
48
48
45
54
56
53
52
31
n.d.
>10
>10
>10
8.44 1.6
>10
>10
>10
n.d.
e
e
e
e
e
e
e
e
e
e
e
COCH3
1
5
5
CO(CH2)2CH3
CO(CH2)6CH3
CO(CH2)8CH3
CO(CH2)10CH3
CO(CH2)12CH3
CO(CH2)14CH3
H
71.5 15.5
103.5 15.2
71.1 6.9
38.1 2.4
>50
7.1
9.6 0.8
11
10.3
8.4
e
0.6 0.24
0.36 0.01
2.29 0.48
2.43 0.33
62.3 16.8
118
106
>22
>21
2
79
132
21
e
>50
118.8 21.5
20
e
GlcAc, H
0.8
e
Experiments on L. donovani were carried out in axenic amastigotes and intracellular amastigotes.
SI ¼ Selectivity Index (IC50 MRC-5/IC50 parasite); AG ¼ Activity gain (IC50 hydroxytyrosol/IC50 compound).
derivatives 5e7 (e.g. tyrosol decanoate 6: 65 and 132 mM, against
L. major and L. infantum, respectively). The authors proposed this
effect could be due to the surfactant activity reported for these
medium chain tyrosyl fatty acid esters [35].
When HT and its derivatives 10e18 were evaluated against
T. brucei (Table 2), compounds 13e16 showed IC50s in the low
micromolar range (0.36e2.43
mM). Remarkably, hydroxytyrosol
Fig. 2. Parts of compound 14 to focus structure-activity studies.
decanoate 13 and hydroxytyrosol dodecanoate 14 displayed a high
selectivity index (118 and 101, respectively) and a large increase in
activity with respect to HT (AG values of 79 and 132, respectively). A
length of the alkyl chain around 10 to 12 carbons seems to be the
optimum on this series. When these compounds were examined
measured antileishmanial activity against L. major and L. infantum
parasite species for tyrosol fatty acid esters from C2 to C18 chain
length. They found the best IC50 values for medium chain tyrosyl

E. Belmonte-Reche et al. / European Journal of Medicinal Chemistry 119 (2016) 132e140
135
Scheme 2. Reagents and conditions: (a) CH2Cl2, K2CO3, DMF, Reflux; (b) vinyl alkyl ester, Novozym 435, tBuOMe; (c) BrBn or PMBCl, K2CO3, Acetone, Reflux; (d) I(CH2)nCH3, KOH,
DMSO, 50 ꢁC; (e)H2, Pd, 4 atm, THF; (f) TsCl, TEM, DCM, 0 ꢁC; (g) HS(CH2)11CH3, K2CO3, THF.
Scheme 3. Reagents and conditions: (a) KOH, MeOH; (b) CS2, TEA, MEOH, THF; (c) H2N(CH2)nCH3, TEA, Pyridine; (d) CH3(CH2)nCOOH, HATU, TEA, THF.
against axenic amastigotes of L. donovani, three of them (com-
pounds 11e13) exhibited over 40% inhibition at 20 M concentra-
tion. When they were evaluated on intracellular amastigotes, the
less important for their activity. When both phenolic hydroxyl
groups and the medium size alkyl chain still remain as part of the
structure of the compounds, the functional group connecting them
seems to be less relevant. Actually, the hydroxytyrosol ether ana-
logs 28 and 29, and the thiourea analogs 37 and 38 showed similar
m
best IC50 value was displayed by hydroxytyrosol decanoate 13
(8.44
m
M). Kyriazis et al. [18] had previously reported IC50 data for
M against L. donovani and in the same range
hydroxytyrosol of 393
m
IC50 values (0.63e1.29
14. In contrast, the amide derivatives 39 and 40 presented slightly
worse antitrypanosomal activity (>10 and 8.74 M, respectively)
mM) to those of the ester derivatives 13 and
for L. infantum and L. major. We have observed that the addition of a
fatty acid ester group to HT improves its activity with respect to HT
against L. donovani although only to IC50s in the low micromolar
range.
m
than the ester derivatives. However, none of the new compounds
displayed better selectivity index than compounds 13 and 14 and
their activity gain with respect to HT was lower too. Finally, the
length of the alkyl chain seems optimum at 10e12 carbon atoms
independently of the bond connecting it to the HT scaffold. Only a
two-fold difference is observed for the ester and ether derivatives
with decyl and dodecyl chains.
Hydroxytyrosol decanoate 13 and hydroxytyrosol dodecanoate
14 possess relevant surfactant properties, even better than medium
chain tyrosyl fatty acid esters [35]. So, it could be argued that 13 and
14 are acting as nonspecific detergents disrupting the parasite's
membrane. Another possibility is they could be inhibiting fatty acid
biosynthesis, through inhibition of FabG, FabI or FabZ enzymes. The
structural similarity of 13 and 14 with trans-2-hexadecenoyl-(N-
acetylcysteamine)-thioester, a FabI inhibitor [36,37], or with 2-, 5-,
6-, and 9-hexadecynoic acids (HDAs), inhibitors of FabG, FabI and
After performing this first in vitro screening on T. brucei and
L. donovani for TYR, HT and their derivatives 3e18, we decided to
focus our efforts only on new compounds against T. brucei. We
carried out a structural-activity study of analogs of hydroxytyrosol
fatty acid esters 13 and 14 (Table 3) in order to probe the relevance
of the phenolic hydroxyl groups, the type of chemical bond be-
tween the phenolic ring and the length of the alkyl chain.
We found that the phenolic hydroxyl groups seem to be
essential for activity against T. brucei since changing them to a
methylene acetal (compounds 21 and 22) or protecting them with
benzyl or p-methoxybenzyl groups (compounds 25, 27, 32 and 33)
increased their IC50 values (>10 mM) with respect to those of 13 and
14. The type of bond linking the alkyl chain to the HT scaffold on
these compounds (ester, ether or thioether) appears to be much

136
E. Belmonte-Reche et al. / European Journal of Medicinal Chemistry 119 (2016) 132e140
Table 3
Cytotoxic and trypanocidal activities of hydroxytyrosol derivatives 19e40.
Cytotoxicity MRC-5
T. brucei
Compounds
R1
R2, R3
IC50
(
m
M)
IC50
(
m
M)
SI
>5
AG
21
22
25
27
28
29
32
33
35
36
37
38
39
40
OCO(CH2)8CH3
OCO(CH2)10CH3
O(CH2)9CH3
O(CH2)9CH3
O(CH2)9CH3
O(CH2)11CH3
S(CH2)11CH3
S(CH2)11CH3
NH2
eCH2e
eCH2e
Bn, Bn
PMB, PMB
H, H
>50
>50
>50
>50
>50
13.19 2.90
>50
>50
68.9 15.11
21.13 8.78
24.8
2.96 1.20
>50
>10 (n ¼ 2)
>10 (n ¼ 3)
>10 (n ¼ 3)
>10 (n ¼ 2)
1.29 0.21
0.63 0.07
>10 (n ¼ 2)
>10 (n ¼ 2)
>10 (n ¼ 2)
9.87 1.02
0.74 0.36
0.77 0.28
>10 (n ¼ 4)
8.74 1,15
>5
>5
>5
>5
37
75
5
5
5
5
>5
>5
>5
>38
21
>5
>5
7
2
33
4
>5
>5
H, H
Bn, Bn
PMB, PMB
H, H
H, H
H, H
H, H
H, H
H, H
NCS
NHSNH(CH2)8CH3
NHSNH(CH2)10CH3
NHCO(CH2)8CH3
NHCO(CH2)10CH3
64
62
5
>50
5
SI ¼ Selectivity Index (IC50 MRC-5/IC50 T. brucei); AG ¼ Activity gain (IC50 hydroxytyrosol/IC50 compound).
FabZ of P. falciparum [38], could be pointing to these potential
therapeutic targets. In both cases, the inhibitors possess long alkyl
chains (13e16 atoms in length) together with one or two groups at
the end of the compound capable of accepting or donating
hydrogen bonds resembling the scaffolds of compounds 13 and 14.
4.2. Synthesis
4.2.1. Preparation of 2-(benzo[d][1,3]dioxol-5-yl)ethyl decanoate
(21)
To a solution of 20 [39] (45 mg, 0.271 mmol) in tert-butyl methyl
ether (3 ml), vinyl decanoate (0.061 ml, 0.271 mmol) and Candida
antarctica lipase (Novozym 435, 200 mg) were added. The reaction
was stirred by an orbital shaker for 24 h at 55 ꢁC and then the
enzyme was separated and washed. The solvent was concentrated
and purified by silica-gel flash column chromatography using a
mixture of hexane/ethyl acetate, 9:1 (v/v). Evaporation of the sol-
vents yielded 18.6 mg of the pure product as a white solid
(Yield ¼ 22%; Rf ¼ 0.67 (Hexane/ethyl acetate, 4:1)). 1H NMR
3. Conclusions
We have examined the trypanocidal and leishmanicidal activ-
ities of several tyrosol and hydroxytyrosol fatty acid esters together
with three of TYR and HT metabolites. We found notable IC50 values
against T. brucei for HT decanoate ester 13 and HT dodecanoate
(300 MHz, CDCl3)
d
¼ 6.77e6.47 (m, 3H, CHarom), 5.86 (s, 2H,
ester 14 (0.6 and 0.36
mM, respectively) and against L. donovani for
OCH2O), 4.16 (t, J ¼ 7.0 Hz, 2H, CH2O), 2.77 (t, J ¼ 6.9 Hz, 2H,
CH2CH2O), 2.21 (t, J ¼ 7.5 Hz, 2H, CH2COO), 1.51 (d, J ¼ 6.2 Hz, 2H,
CH2CH2COO), 1.19 (s, 12H, CH2), 0.81 (t, J ¼ 5.4 Hz, 3H, CH3); 13C
14 (8.44 M). We synthesized several analogs of 13 and 14 to
m
investigate structureeactivity relationship in T. brucei. We observed
that the di-ortho phenolic ring and the medium size alkyl chain are
essential for activity but the nature of the chemical bond among
them is not so relevant.
NMR (75 MHz, CDCl3)
d
¼ 174.0 (COO), 147.9 (C3ipsoO), 146.4
(C4ipsoO), 131.9 (C1ipso), 122.0 (C6arom), 109.6 (C2arom), 108.5 (C5arom),
101.1 (OCH2O), 65.1 (C1), 35.1 (C2), 34.6 (CH2COO), 32.1 (CH2), 29.7
(CH2), 29.5 (CH2), 29.4 (CH2), 25.2 (CH2), 22.9 (CH2), 14.4 (CH3); ESI-
HRMS [M þ Na] calcd for C19H28O4 343.1885, found 343.1875.
4. Experimental section
4.2.2. Preparation of 2-(benzo[d][1,3]dioxol-5-yl)ethyl dodecanoate
(22)
4.1. General
Following the same procedure used to prepare compound 21,
with vinyl dodecanoate (0.075 ml, 0.289 mmol) as the acylating
agent, afforded 71.5 mg as a white solid (Yield ¼ 71%; Rf ¼ 0.65
(Hexane/ethyl acetate, 4:1)). 1H NMR (300 MHz, CDCl3)
All chemicals were used without further purification, unless
otherwise noted. All reactions were monitored by TLC on precoated
Silica-Gel 60 plates F254, and detected by heating with 5% sulfuric
acid in ethanol or Mostain (500 ml of 10% H2SO4, 25 g of
(NH4)6Mo7O24ꢀ4H2O, 1 g Ce(SO4)2ꢀ4H2O). Products were purified
by flash chromatography with Silica gel 60 (200e400 mesh). Low
resolution mass spectra were obtained on an ESI/ion trap mass
spectrometer. High resolution mass spectra were obtained on an
ESI/quadrupole mass spectrometer. NMR spectra were recorded on
a 300, 400 or 500 MHz [300 or 400 MHz (1H), 75 or 100 (13C)] NMR
spectrometers, at room temperature for solutions in CDCl3, D2O or
CD3OD. Chemical shifts are referred to the solvent signal. 2D ex-
periments (COSY, TOCSY, ROESY, and HMQC) were done when
necessary to assign the new compounds. Chemical shifts are in
ppm. Data were processed using manufacturer software, raw data
were multiplied by shifted exponential window function prior to
Fourier transform, and the baseline was corrected using polynomial
fitting.
d
¼ 6.72e6.53 (m, 3H, CHarom), 5.86 (s, 2H, OCH2O), 4.16 (t,
J ¼ 7.0 Hz, 2H, CH2O), 2.77 (t, J ¼ 7.0 Hz, 2H, CH2CH2O), 2.21 (t,
J ¼ 7.5 Hz, 2H, CH2COO), 1.58e1.47 (m, 2H, CH2CH2COO), 1.19 (s,
16H, CH2), 0.81 (t, J ¼ 6.6 Hz, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d
¼ 174.0 (COO), 147.9 (C3ipsoO), 146.4 (C4ipsoO), 131.9 (C1ipso), 122.0
(C6arom), 109.6 (C2arom), 108.5 (C5arom), 101.1 (OCH2O), 65.1 (C1), 35.1
(C2), 34.6 (CH2COO), 32.2 (CH2), 29.9 (CH2), 29.7 (CH2), 29.6 (CH2),
29.5 (CH2), 29.4 (CH2), 25.2 (CH2), 22.9 (CH2), 14.4 (CH3); ESI-HRMS
[M þ Na] calcd for C21H32O4 371.2198, found 371.2204.
4.2.3. Preparation of 2-(3,4-bis(benzyloxy)phenyl)ethan-1-ol (23)
Benzyl bromide (1.62 ml, 13.62 mmol) was added to a me-
chanically stirred degassed suspension of hydroxytyrosol (1 g,
6.49 mmol) and potassium carbonate (3.59 g, 25.9 mmol) in dry

E. Belmonte-Reche et al. / European Journal of Medicinal Chemistry 119 (2016) 132e140
137
acetone (20 ml) and the resulting mixture was refluxed for 12 h.
The reaction was then filtered through a celite © pad and puri-
fied by silica-gel flash chromatography column (hexane/ethyl
acetate, 3:1 (v/v) as solvents) obtaining 1.68 g of the desired
product as a white solid (Yield ¼ 77%; Rf ¼ 0.16 (hexane: ethyl
acetate, 3:1)). The spectroscopic data coincide with the previous
report [33].
and the organic phases were combined, concentrated and purified
by silica gel column eluting with hexane/ethyl acetate, 3:1 (v/v) to
give a yellow oil (Yield ¼ 98%). 1H NMR (300 MHz, CDCl3)
d
¼ 7.71
(d, J ¼ 8.2 Hz, 2H, HTs), 7.48 (d, J ¼ 7.0 Hz, 4H, HBn), 7.39 (dd, J ¼ 16.2,
8.5 Hz, 6H, HBn), 7.30 (d, J ¼ 8.1 Hz, 2H, HTs), 6.87 (d, J ¼ 8.1 Hz, 1H,
HHT), 6.75 (s, 1H, H02), 6.67 (d, J ¼ 8.1 Hz, 1H, HHT), 5.14 (d,
J ¼ 15.3 Hz, 4H, CH2OBn), 4.19 (t, J ¼ 7.0 Hz, 2H, CH2O), 2.89 (t,
J ¼ 7.9 Hz, 2H, CH2CH2O), 2.44 (s, 3H, CH3); 13C NMR (75 MHz,
4.2.4. Preparation of 2-(3,4-bis(4-methoxybenzyloxy)phenyl)
ethan-1-ol (24)
CDCl3)
d
¼ 149.2 (Cipso), 148.2 (Cipso), 144.9 (Cipso), 137.6 (Cipso), 137.4
(Cipso), 133.3 (Cipso), 130.0 (Cipso), 129.8 (Carom), 128.7 (Carom), 128.1
(Carom), 127.6 (Carom), 127.5 (Carom), 122.1 (Carom), 116.1 (Carom), 115.5
(Carom), 71.6 (CH2O), 71.6 (CH2O), 71.0 (CH2O), 35.1 (CH2CH2O), 21.9
(CH3); ESI-HRMS [M þ Na] calcd for C29H28O5S 511.1555, found
511.1561.
Following the same procedure used to prepare compound 23,
with hydroxytyrosol (175 mg, 1.135 mmol), potassium carbonate
(377 mg, 2.72 mmol) and 4-methoxybenzyl chloride (0.339 ml,
2.497 mmol) in DMF (2 ml), afforded 249.5 mg of the pure product
as a white solid (yield ¼ 56%; Rf ¼ 0.35 (Hexane/ethyl acetate, 2:1)).
Solvent purification mixture: Hexane/ethyl acetate, 4:1 (v/v). 1H
NMR (500 MHz, CDCl3)
d
¼ 7.23 (dd, J ¼ 8.7, 2.0 Hz, 4H, HPMB), 6.76
4.2.7. Preparation of 3,4-bis((4-methoxybenzyl)oxy)phenethyl 4-
methylbenzenesulfonate (31)
(dd, J ¼ 8.5, 1.8 Hz 5H, HPMB and H02), 6.70 (d, J ¼ 1.9 Hz, 1H, H05),
6.61 (dd, J ¼ 8.1, 2.0 Hz, 2H, H06), 4.94 (s, 2H, CHP2MBO), 4.93 (s, 2H
CHP2MBO), 3.69 (s, 3H, CH3O), 3.69 (s, 3H, CH3O), 3.66 (t, J ¼ 6.4 Hz,
2H, CH2OH), 2.64 (t, J ¼ 6.5 Hz, 2H, CH2CH2OH); 13C NMR (75 MHz,
Following the same procedure used to prepare compound 30,
with compound 24 (200 mg, 0.507 mmol) as starting material, tosyl
chloride (145 mg, 0.761 mmol) and triethylamine (0.212 ml,
1.521 mmol) in 3 ml of CH2Cl2, afforded 184 mg of pure product as a
yellow oil (Yield ¼ 66%, Rf ¼ 0.46 (hexane/ethyl acetate/Acetone,
CDCl3)
d
¼ 159.3 (Cipso), 149.1 (Cipso), 147.9 (Cipso), 131.8 (Cipso), 129.5
(Cipso), 129.4 (Cipso), 129.1 (CPMBarom), 129.0 (CPMBarom), 121.9
(C6arom), 116.5 (C2arom), 115.8 (C5arom), 113.8 (CPMBarom), 71.4
(CPMBH2O), 71.2 (CPMBH2O), 63.7 (CH2OH), 55.3 (CH3O), 38.7
(CH2CH2OH); ESI-HRMS [M þ Na] calcd for C24H26O5 417.1678,
found 417.1658.
4:2:1)). 1H NMR (300 MHz, CDCl3)
d
¼ 7.52 (d, J ¼ 8.2 Hz, 2H, HTs),
7.20 (d, J ¼ 7.3 Hz, 4H, HPMB), 7.10 (d, J ¼ 8.0 Hz, 2H, HTs), 6.74 (dd,
J ¼ 8.6, 2.9 Hz, 4H, HPMB), 6.68 (d, J ¼ 8.2 Hz, 1H, H05), 6.57 (s, 1H,
H02), 6.48 (d, J ¼ 8.1 Hz, 1H, H06), 4.88 (s, 2H, CH2OPMB), 4.84 (s, 2H,
CH2OPMB), 4.01 (t, J ¼ 6.9 Hz, 2H, CH2O), 3.64 (s, 3H, OCH3), 3.63 (s,
4.2.5. Preparation of 1,2-bis((4, 40-methoxybenzyl)oxy)-4-(2-
(decyloxy)ethyl)benzene (27)
3H, OCH3), 2.69 (t, J ¼ 6.9 Hz, 2H, CH2CH2O), 2.24 (s, 3H, CH3); 13
C
NMR (75 MHz, CDCl3)
d
¼ 159.6 (Cipso), 159.6 (Cipso), 149.4 (Cipso),
Compound 24 (50 mg, 0.127 mmol) and potassium hydroxide
(49.8 mg, 0.887 mmol) were added to DMSO (2 ml) in a round-
bottomed flask. 1-Iododecane (0.081 ml, 0.380 mmol) was then
added and the reaction was heated to 50 ꢁC and stirred overnight.
The reaction mixture was diluted with hydrochloric acid (5%, 25 ml)
and extracted with CH2Cl2 (3 ꢂ 25 ml). The combined organic layers
were then dried with MgSO4, filtered and concentrated. The crude
was purified by silica-gel flash column chromatography eluting
with hexane/ethyl acetate, 3:1 (v/v) obtaining 40.5 mg of the pure
product as a white solid (Yield ¼ 60%; Rf ¼ 0.8 (hexane/ethyl ace-
148.3 (Cipso), 145.0 (Cipso), 133.2 (Cipso), 130.1 (Cipso), 129.8 (Cipso),
129.7 (Cipso), 129.6 (Carom), 129.4 (Carom), 129.3 (Carom), 128.1 (Carom),
122.1 (Carom), 116.4 (Carom), 115.8 (Carom), 114.1 (Carom), 71.5 (CH2O),
71.4 (CH2O), 71.1 (CH2O), 55.5 (CH3O), 35.1 (CH3O), 21.9 (CH3); ESI-
HRMS [M þ Na] calcd for C31H32O7S 571.1766, found 571.1771.
4.2.8. Preparation of (3,4-bis(benzyloxy)phenethyl)(dodecyl)
sulfane (32)
tate, 2:1)). 1H NMR (300 MHz, CDCl3)
d
¼ 7.27 (dd, J ¼ 8.5, 4.7 Hz,
To a mechanically stirred two socket flask under argon atmo-
sphere was potassium carbonate (141 mg, 1.025 mmol) suspended
in acetonitrile (5 ml) and heated to reflux. In a second and third
flask under argon atmosphere was dodecane-1-thiol (0.054 ml,
0.225 mmol) dissolved in acetonitrile (1 ml) and compound 30
(100 mg, 0.205 mmol) dissolved in acetonitrile (2 ml), respectively.
The second and third flask solutions were simultaneously added to
the refluxing two socket flask and left to react for 48 h. The reaction
product was then filtered, concentrated and purified by silica-gel
flash chromatography column using a mixture of hexane/ethyl ac-
etate, 9:1 (v/v) as solvents to obtain 50 mg of the desired product as
a yellow oil (Yield ¼ 47%, Rf ¼ 0.83 (Hexane: ethyl acetate,4:1)). 1H
4H, HPMB), 6.84e6.73 (m, 6H, HPMB, H02 and H05), 6.68e6.61 (m, 1H,
H06), 4.97 (s, 2H, CHP2MBO), 4.95 (s, 2H, CH2PMBO), 3.73 (s, 3H, CH3O),
3.72 (s, 3H, CH3O), 3.48 (t, J ¼ 7.3 Hz, 2H, CH2O), 3.33 (t, J ¼ 6.7 Hz,
2H, CH2O), 2.70 (t, J ¼ 7.2 Hz, 2H, CH2CH2O), 1.49 (dd, J ¼ 13.5,
6.8 Hz, 2H, CH2Cipso), 1.18 (s, 16H, CH2), 0.80 (t, J ¼ 6.6 Hz, 3H,
CH3CH2); 13C NMR (126 MHz, CDCl3)
d
¼ 159.3 (Cipso), 159.2 (Cipso),
149.0 (Cipso), 147.6 (Cipso), 132.6 (Cipso), 129.6 (Cipso), 129.5 (Cipso),
129.1 (CPMBarom), 129.0 (CPMBarom), 121.7 (C6arom), 116.4 (C2arom),
115.7 (C5arom), 113.8 (CPMBarom), 113.8 (CPMBarom), 71.9 (CH2O), 71.4
(CH2O), 71.2 (CH2O), 71.1 (CH2O), 55.3 (CH3O), 35.9 (CH2), 31.9
(CH2), 29.8 (CH2), 29.6 (CH2), 29.6 (CH2), 29.5 (CH2), 29.3 (CH2), 26.2
(CH2), 22.7 (CH2), 14.1 (CH3CH2); ESI-HRMS [M þ Na] calcd for
NMR (300 MHz, CDCl3)
d
7.37 (dd, J ¼ 8.0, 2.4 Hz, 4H, HBn), 7.26 (dtd,
C
34H46O5 557.3243, found 557.3237.
J ¼ 8.6, 6.7, 1.8 Hz, 6H, HBn), 6.79 (d, J ¼ 8.2 Hz, 1H, H05), 6.73 (d,
J ¼ 1.9 Hz, 1H, H02), 6.64 (dd, J ¼ 8.1, 1.9 Hz, 1H, H06), 5.07 (s, 2H,
CH2O), 5.05 (s, 2H, CH2O), 2.74e2.64 (m, 2H, CH2S), 2.64e2.55 (m,
2H, CH2S), 2.46e2.34 (m, 2H, CipsoCH2CH2), 1.52e1.45 (m, 2H,
CH2CH2S), 1.18 (s, 18H, CH2), 0.80 (t, J ¼ 6.6 Hz, 3H, CH3); 13C NMR
4.2.6. Preparation of 3,4-bis(benzyloxy)phenethyl 4-
methylbenzenesulfonate (30)
Tosyl chloride (0.855 g, 4.49 mmol) was dissolved in CH2Cl2
(1.5 ml) in a 10 ml flask under argon atmosphere. Compound 23
(1 g, 2.99 mmol) and triethylamine (1.250 ml, 8.97 mmol) were
dissolved in CH2Cl2 (1.5 ml) in a second 10 ml flask under argon
atmosphere and then cooled to 0 ꢁC. The tosyl chloride solution was
added at a rate of 0.75 ml/h (time ¼ 2 h) whilst stirring and the
reaction was then left to react for another 21 h at room tempera-
ture. The reaction mixture was then washed with water (3 ꢂ 20 ml)
(75 MHz, CDCl3)
d
¼ 149.3 (Cipso), 147.8 (Cipso), 137.7 (Cipso), 137.6
(Cipso), 134.5 (Cipso), 128.7 (CBn), 128.0 (CBn), 128.0 (CBn), 127.6 (CBn),
127.6 (CBn), 121.6 (C6arom), 116.0 (C2arom), 115.5 (C5arom), 71.7 (OCH2),
71.7 (OCH2), 36.2 (SCH2), 34.0 (SCH2), 32.6 (CH2), 32.2 (CH2), 29.9
(CH2), 29.9 (CH2), 29.8 (CH2), 29.6 (CH2), 29.5 (CH2), 29.2 (CH2), 23.0
(CH2), 14.4 (CH3). ESI-HRMS [M þ H] calcd for C34H46O2S 519.3297,
found 519.3295.

138
E. Belmonte-Reche et al. / European Journal of Medicinal Chemistry 119 (2016) 132e140
4.2.9. Preparation of (3,4-bis((4-methoxybenzyl)oxy)
phenethyl)(dodecyl)sulfane (33)
ethyl acetate,1:1)). 1H NMR (300 MHz, CDCl3)
d
¼ 6.68 (d, J ¼ 8.0 Hz,
2H, C2arom and C5arom), 6.53 (dd, J ¼ 8.0, 2.0 Hz, 1H, C6arom), 3.61 (s,
2H, CH2N), 3.30 (dt, J ¼ 3.2, 1.6 Hz, 2H, CH2N), 2.71 (t, J ¼ 7.2 Hz, 2H,
CH2Cipso), 1.56e1.46 (m, 2H, CH2CH2N), 1.29 (s, 18H, CH2), 0.89 (t,
Following the same procedure used to prepare compound 32,
with compound 31 (100 mg, 0.182 mmol), potassium carbonate
(126 mg, 0.911 mmol) and dodecane-1-thiol (0.048 ml, 0.2 mmol),
afforded 97 mg of pure product as a yellow oil (Yield ¼ 92%, Rf ¼ 0.3
J ¼ 6.2 Hz, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d
¼ 196.0 (NHCNH),
146.3 (C4arom), 144.5 (C3arom), 131.9 (C1arom), 121.1 (C6arom), 116.9
(Hexane: ethyl acetate, 6:1)). 1H NMR (300 MHz, CDCl3)
d
7.26 (dd,
(C2
or C5arom), 116.4 (C2
or C5arom), 35.7 (CH2), 35.7 (CH2),
arom
arom
J ¼ 8.4, 4.6 Hz, 4H, HPMB), 6.86e6.75 (m, 5H, HPMB and H05), 6.72 (d,
J ¼ 1.5 Hz, 1H, H02), 6.63 (d, J ¼ 8.1 Hz, 1H, H06), 4.97 (s, 2H, CH2O),
4.95 (s, 2H, CH2O), 3.72 (s, 3H, CH3O), 3.72 (s, 3H, CH3O), 2.75e2.65
(m, 2H, CH2S), 2.65e2.56 (m, 2H, CH2S), 2.42 (t, J ¼ 7.4 Hz, 2H,
33.1 (CH2), 30.7 (CH2), 30.4 (CH2), 28.0 (CH2), 23.7 (CH2), 14.4 (CH3);
ESI-HRMS [M
381.2563.
þ
H] calcd for C21H36N2O2S 381.2576, found
C
ipsoCH2CH2), 1.57e1.42 (m, 2H, CH2CH2S), 1.18 (s, 18H, CH2), 0.80 (t,
4.3. Biological assays
J ¼ 6.5 Hz, 3H, CH3); 13C NMR (75 MHz, CDCl3)
d
¼ 159.6 (Cipso),
159.5 (Cipso), 149.3 (Cipso), 147.9 (Cipso), 134.5 (Cipso), 129.8 (Cipso),
129.7 (Cipso), 129.4 (Carom), 129.3 (Carom), 121.6 (Carom), 116.3 (Carom),
115.9 (Carom), 114.1 (Carom), 71.6 (CH2O), 71.6 (CH2O), 55.5 (CH3O),
36.2 (SCH2), 34.1 (SCH2), 32.6 (CH2), 32.2 (CH2), 30.0 (CH2), 29.9
(CH2), 29.9 (CH2), 29.8 (CH2), 29.6 (CH2), 29.6 (CH2), 29.2 (CH2), 23.0
(CH2), 14.4 (CH3); ESI-HRMS [M þ H] calcd for C36H50O4S 579.3508,
found 579.3486.
4.3.1. In vitro antitrypanosomal activity
Bloodstream forms (BSF) of T. brucei brucei ‘single marker’ S427
(S16) were grown at 37 ꢁC, 5% CO2 in HMI-9 medium supplemented
with 10% (heat-inactivated fetal bovine serum, hiFBS). Drug sus-
ceptibility assay was performed as described in Ref. [42]. Briefly,
parasites (1 ꢂ 104 BSF per ml) were incubated in 96-well plates
with increasing concentration of drugs/compounds for 72 h at
37 ꢁC, 5% CO2 in culture medium. Cell proliferation was determined
using the alamarBlue® assay [43]. Fluorescence was recorded with
an Infinite® F200 microplate reader (Tecan Austria GmbH, Austria)
equipped with 550 and 590 nm filters for excitation and emission
wavelengths, respectively.
4.2.10. Preparation of 4-(2-isothiocyanatoethyl)benzene-1,2-diol
(36)
Triethylamine (1.617 ml, 11.60 mmol) was added to a stirring
suspension of dopamine hydrochloride 34 (2 g, 10.55 mmol) in THF
(26 ml). Methanol (20.80 ml) was slowly added to dissolve the
dopamine hydrochloride, forming a clear solution. Carbon disulfide
(3.17 ml, 52.7 mmol) was added to the mixture which was stirred
for 1 h at 28 ꢁC under argon atmosphere. The yellowish reaction
mixture was cooled to 0 ꢁC and hydrogen peroxide (0.942 ml, 30% in
water) was added drop wise (30%). The solution was immediately
acidified with concentrated hydrochloric acid and then concen-
trated in vacuo. The resulting mixture was filtered and rinsed with
water. The filtrate was extracted with ethyl acetate and the com-
bined organic layers were dried over MgSO4, filtered, and concen-
trated affording 740 mg of crude product as an oil (Yield ¼ 36%). The
spectroscopic data coincide with the previous report [40,41].
4.3.2. In vitro antileishmanial activity
The experiments of drug susceptibility on L. donovani were
carried out in axenic amastigotes and intracellular amastigotes.
Axenic L. donovani MHOM/ET/67/HU3 amastigote parasites were
grown in Schneider medium supplemented with 20% hiFBS, pH 5.4
at 37 ꢁC and 5% CO2. 106 axenic amastigotes/ml in a 96-well plate
were incubated with increasing concentrations of compounds for
72 h at 37 ꢁC, followed by a resazurin-based assay, as described [44].
Additionally, we use intracellular amastigotes of a L. donovani
(MHOM/ET/67/HU3) line with luciferase gene integrated into the
parasite genome [45]. The susceptibility of intracellular L. donovani
amastigotes to synthesized compounds was determined using the
Luciferase Assay System Kit (Promega, Madison, Wis.) as previously
described [44]. Briefly, macrophage-differentiated-THP-1 cells
were plated at a density of 3 ꢂ 104 macrophages/well in 96-well
white polystyrene microplates and were infected at a macro-
phage/parasite ratio of 1:10 with late-stage promastigotes. Infected
cell cultures were incubated at different compound concentrations
in RPMI 1640 medium plus 10% hiFBS at 37 ꢁC with 5% CO2 for 72 h
and then lysed. Luminescence intensity was measured as indicative
of the intracellular parasite growth, according to the instructions of
the supplier.
4.2.11. Preparation of 1-(3,4-dihydroxyphenethyl)-3-decylthiourea
(37)
Compound 36 (355.7 mg, 1.822 mmol) and decan-1-amine
(0.364 ml, 1.820 mmol) were dissolved in pyridine (10 ml) to give
a yellow solution. The reaction was stirred for 3 h and triethylamine
(0.254 ml,1.822 mmol) was then added. The reaction was stirred for
1 h and then concentrated, diluted with 20 ml of hydrochloric acid
(5%) and extracted with ethyl acetate (3 ꢂ 20 ml). The combined
organic phases were concentrated and purified via silica-gel flash
column chromatography while eluting with hexane/ethyl acetate,
2:1 (v/v) to give 338 mg of the pure product as a yellow solid
(Yield ¼ 53% Rf ¼ 0.2 (hexane/ethyl acetate, 2:1)). 1H NMR
4.3.3. Cytotoxicity assay
Human myelomonocytic cell line THP-1 were grown at 37 ꢁC
and 5% CO2 in RPMI-1640 supplemented with 10% hiFBS, 2 mM
(300 MHz, CDCl3)
d
¼ 6.72 (d, J ¼ 8.1 Hz, 2H, C2arom and C5arom), 6.56
(d, J ¼ 7.9 Hz, 1H, C6arom), 3.64 (t, J ¼ 7.1 Hz, 2H, CH2N), 3.39 (m, 2H,
CH2N), 2.74 (t, J ¼ 7.1 Hz, 2H, CH2Cipso),1.55 (t, 2H, CH2CH2N),1.31 (s,
16H, CH2), 0.91 (t, J ¼ 6.7 Hz, 3H, CH3); 13C NMR (75 MHz, CDCl3)
glutamate, 100 U/ml penicillin and 100
mg/ml streptomycin.
3 ꢂ 104 cells/well in 96-well plates were differentiated to mac-
rophages with 20 ng/ml of PMA treatment for 48 h followed by
24 h of culture in fresh medium [44]. MRC-5 cells, a SV-40
transformed human fetal lung fibroblast cell line, were main-
tained at 37 ꢁC and 5% CO2 in DMEM supplemented with 10%
d
¼ 202.0 (NCN), 145.1 (C4arom), 143.6 (C3arom), 130.8 (C1arom), 120.0
(C6arom), 115.8 (C2arom or C5arom), 115.3 (C2arom or C5arom), 34.6 (CH2),
32.0 (CH2), 29.6 (CH2), 29.3 (CH2), 26.9 (CH2), 22.6 (CH2), 13.4 (CH3);
ESI-HRMS [M
353.2263.
þ
H] calcd for C19H32N2O2S 353.2252, found
hiFBS, 100 U/ml penicillin and 100
mg/ml streptomycin. Cells
were harvested by treatment with 0.05% (w/v) trypsin plus
0.48 mM EDTA for 5 min, diluted to 4 ꢂ 104 cell/ml in 96-well
plates and incubated at 37 ꢁC and 5% CO2 before toxicity assay
[44]. Cellular toxicity of all compounds was determined using the
colorimetric MTT-based assay after incubation at 37 ꢁC for 72 h in
the presence of increasing concentrations of compounds [44].
4.2.12. Preparation of 1-(3,4-dihydroxyphenethyl)-3-
dodecylthiourea (38)
Following the same procedure used to prepare compound 37,
from dodecan-1-amine (0.42 ml, 1.822 mmol), afforded 392.7 mg of
the pure product as a yellow solid (Yield ¼ 57% Rf ¼ 0.5 (hexane/

E. Belmonte-Reche et al. / European Journal of Medicinal Chemistry 119 (2016) 132e140
139
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Acknowledgments
We gratefully acknowledge to Junta de Andalucía (Grant No.
FQM-7316 (JCM), BIO-1786 (JMPV), CTS-7282 (FG)) and Spanish
Ministry of Economy and Competitiveness (Grant No. SAF2011-
28215 (JMPV) and SAF2012-34267 (FG)) for financial support.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.047.
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