Synthesis and leishmanicidal activity of cinnamic acid esters: Structure-activity relationship

Article abstract of DOI:10.1007/s00044-013-0741-y

Several cinnamic acid esters were obtained via Fischer esterification of cinnamic acids derivatives with aliphatic alcohols. Structures of the products were elucidated by spectroscopic analysis. The synthesized compounds were evaluated for antileishmanial activity against L. (V) panamensis amastigotes and cytotoxic activity was evaluated against mammalian U-937 cells. The compounds 11, 15-17, and 23, were active against Leishmania parasite and although toxic for mammalian cells, they still are potential candidates for antileishmanial drug development. A SAR analysis indicates that first, while smaller alkyl chains lead to higher selectivity indices (10, 11 vs. 12-17); second, the degree of oxygenation is essential for activity, primarily in positions 3 and 4 (17 vs. 18-20 and 22); and third, hydroxyl groups increase both activity and cytotoxicity (14 vs. 23). On the other hand, the presence of a double bond in the side chain is crucial for cytotoxicity and leishmanicidal activity (12 vs. 21). However, further studies are required to optimize the structure of the promising molecules and to validate the in vitro activity against Leishmania demonstrated here with in vivo studies.

Full text of DOI:10.1007/s00044-013-0741-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1378–1386
DOI 10.1007/s00044-013-0741-y
ORIGINAL RESEARCH
Synthesis and leishmanicidal activity of cinnamic acid esters:
structure–activity relationship
•
Elver Otero Sara M. Robledo Santiago Dıaz Miguel Carda
•
•
•
´
•
•
•
´
Diana Mun˜oz Julian Pan˜os Ivan D. Velez Wilson Cardona
Received: 8 April 2013 / Accepted: 17 August 2013 / Published online: 1 September 2013
Ó Springer Science+Business Media New York 2013
Abstract Several cinnamic acid esters were obtained via
Fischer esterification of cinnamic acids derivatives with
aliphatic alcohols. Structures of the products were eluci-
dated by spectroscopic analysis. The synthesized com-
pounds were evaluated for antileishmanial activity against
L. (V) panamensis amastigotes and cytotoxic activity was
evaluated against mammalian U-937 cells. The compounds
11, 15–17, and 23, were active against Leishmania parasite
and although toxic for mammalian cells, they still are
potential candidates for antileishmanial drug development.
A SAR analysis indicates that first, while smaller alkyl
chains lead to higher selectivity indices (10, 11 vs. 12–17);
second, the degree of oxygenation is essential for activity,
primarily in positions 3 and 4 (17 vs. 18–20 and 22); and
third, hydroxyl groups increase both activity and cytotox-
icity (14 vs. 23). On the other hand, the presence of a
double bond in the side chain is crucial for cytotoxicity and
leishmanicidal activity (12 vs. 21). However, further
studies are required to optimize the structure of the
promising molecules and to validate the in vitro activity
against Leishmania demonstrated here with in vivo studies.
Keywords Leishmaniasis Á Antiprotozoal Á Caffeic acid Á
Cinnamic acid ester
Introduction
Leishmaniasis is one of the world’s most neglected diseases,
affecting largely the poorest of the poor, mainly in developing
countries; 350 million people are considered at risk of con-
tracting leishmaniasis, and some two million new cases occur
yearly (Murray et al., 2005); the WHO has classified leish-
maniasis as a category 1 disease, i.e., emerging and uncon-
trolled (http://www.who.int/topics/leishmaniasis/en/). This
disease has symptoms from skin lesions to fatal systemic
infection caused by protozoan parasites of the Leishmania
species (Handman, 1999). Recently, a dramaticincreaseinthe
numberofcasesofleishmaniasishasbeenobservedinpatients
with compromised T cell function, such as those infected with
the human immunodeficiency virus (Wolday et al., 1999).
Drugs currently in use, such as the antimony derivative glu-
cantime, the bis-amidines, pentamidine, and stilbamidine or
the glycomacrolide amphotericin B, display high liver and
heart toxicities, develop clinical resistance after a few weeks
of treatment, and moderate and severe side effects (Desjeux,
2004; Ouellette et al., 2004; Barrett and Gilbert, 2002; Croft
and Coombs, 2003; Faraut-Gambarelli et al., 1997; Olliaro
and Bryceson, 1993). For these reasons, it becomes necessary
to discover new, more potent, and selective agents for treating
this increasing parasitosis.
E. Otero Á W. Cardona (&)
´
Quımica de Plantas Colombianas, Instituto de Quımica, Facultad
de Ciencias Exactas y Naturales, Universidad de Antioquia
´
´
(UdeA), Calle 70 No. 52–21, 1226 Medellın, Colombia
e-mail: wcardona@matematicas.udea.edu.co
´
S. M. Robledo Á I. D. Velez
Programa de Estudio y Control de Enfermedades Tropicales
´
(PECET), Sede de Investigacion Universitaria (SIU),
Universidad de Antioquia (UdeA), Calle 70 No. 52–21,
´
1226 Medellın, Colombia
´
S. M. Robledo Á D. Mun˜oz Á I. D. Velez
CIDEPRO-Center for Development of Products against Tropical
´
Diseases, Medellın, Colombia
´
S. Dıaz Á M. Carda Á J. Pan˜os
´
Departamento de Quımica Inorganica y Organica, Universidad
Jaume I, 12071 Castellon, Spain
´
´
´
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Med Chem Res (2014) 23:1378–1386
1379
Caffeic acid (1), 3,4-dihydroxy cinnamic acid, and its
esters derivatives exhibit a broad spectrum of biological
activities, including anti-inflammatory (Jayaprakasam
et al., 2006; Da Cunha et al., 2004), antimicrobial
(Almajano et al., 2007; Noriaki et al., 2005; King
et al.,1999; Valenta et al., 1998; Bowles and Miller, 1994)
antioxidant (Hung et al., 2005; Noriaki et al., 2005; Ki-
kuzaki et al., 2002; Son and Lewis, 2002; Rajan et al.,
2001), and anticarcinogenic effects (De et al., 2011). In
addition, some studies showed that these compounds have
high leishmanicidal activity and high cytotoxicity (Caba-
nillas et al., 2010; Radtke et al., 2003) (Fig. 1).
reaction. Following evaporation and purification by column
chromatography a total of five compounds 10–14 were
obtained (De Campos et al., 2009). Compound 23 was
similarly obtained by starting from caffeic acid. Compound
21 was obtained by catalytic hydrogenation of compound
12 (Cardona et al., 2006) (Scheme 1).
Antileishmanial and cytotoxic activities
The leishmanicidal activity and cytotoxicity of the synthe-
sized compounds were evaluated following the method
previously reported in the literature (Pulido et al., 2012;
Taylor et al., 2011, 2010;Varela et al., 2009; Robledo et al.,
2005, 1999; Weninger et al., 2001). The leishmanicidal
activity and cytotoxicity were reported as effective con-
centration (EC50) and lethal concentration (LC50) values of
compounds respectively, and are shown in Tables 1 and 2.
According to the results shown in Table 1, only com-
pounds 10–13, 15, 17, and 23 showed activity against
axenic amastigotes of L. (V) panamensis with EC50 lower
than 100 lg/ml. The most active compound was 23
(EC50 = 2.3 lg/ml) followed by 11 and 12 exhibiting
EC50 values of 33.0 and 38.1 lg/ml, respectively. Com-
pounds 10, 13, 15, and 17 showed a moderate leishmani-
cidal activity with an EC50 ranging between 55.0 and
60.0 lg/ml. On the other hand, a high toxicity activity was
measured for compounds 11–17 and 23, with LC50
\100 lg/ml. The lower toxic activity was obtained for
compound 10 (EC50 = 162.6 lg/ml). No leishmanicidal
activity or toxicity was detected for compounds 18–22
(EC50 values higher than 100 lg/ml and LC50 higher than
200 lg/ml, respectively). The best selectivity index was
observed for compounds 23, 10, and 11 with values of 4.3,
3.0, and 2.6, respectively. Compound 23 is structurally
similar to compound 4 reported by Cabanillas et al. 2010.
Although the EC50 and LC50 values reported previously for
compound 4 were slightly different, (EC50 = 0.55 lg/mL
and LC50 3.9 lg/mL), both compounds remain highly
active against Leishmania parasites. Differences in the
Leishmania species and cell type used in the assays may
explain the different results obtained for compounds 23 and
4. Thus, compound 4 was tested against axenic amastigotes
of L. (L) amazonensis and murine peritoneal macrophages
(Cabanillas et al., 2010) whereas compound 23 was tested
in this report against axenic amastigotes of L. (V) panam-
ensis and human macrophages.
In the search for active compounds with low toxicity for
the treatment of leishmaniasis and based on the fact that the
biological activity (including leishmaninicidal activity and
cytotoxicity) of molecules can be modified by the meth-
ylation of the aromatic hydroxyl group (Aponte et al.,
2010; Brenzan et al., 2008), several cinnamic acid esters
analogs were synthesized and their cytotoxic and leish-
manicidal activities were determined.
Results and discussion
Chemistry
The design of the compounds were based on both elec-
tronic and steric modifications, which modify the reactivity
of the molecule. Cinnamic acids 5–9 were obtained via
Knoevenagel condensation between malonic acid and dif-
ferent benzaldehydes in water under microwave heating
(Gupta and Purnima, 2007). These compounds were dis-
solved in thionyl chloride, the solution was stirred and
refluxed during 4 h. Then, the mixture was concentrated
under vacuum and the residue, the respective acid chloride,
was added to a solution of cetyl alcohol (dodecyl or este-
aryl alcohol) in dichloromethane, the mixture was stirred
and monitored by thin layer chromatography (Narasimhan
et al., 2004). After evaporation and purification by column
chromatography, a total of six compounds 15–20 were
obtained. Compound 22 was similarly obtained by starting
from cinnamic acid. On the other hand, 3,4-dimethoxy-
cinnamic acid 5 was dissolved in methanol (propanol,
pentanol, hexanol or octanol), acetyl chloride was added
and the solution was stirred under reflux to complete the
The leishmanicidal activity against the intracellular forms
of L. (V) panamensis was also measured for those compounds
that were active against axenic forms. Thus, compounds 14,
and 18–22 were not evaluated against intracellular amastig-
otes. Thecompounds11, 15–17, and23 wereactive(Table 2);
while in compounds 10, 12 and 13, the leishmanicidal activity
was lower than the cytotoxicity and therefore the EC50 values
O
O
HO
HO
HO
HO
O
OH
n
2, n = 1
3, n = 3
4, n = 5
1.
acid
Caffeic
Fig. 1 Caffeic acid and its esters derivatives
123

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Med Chem Res (2014) 23:1378–1386
R1
R4
O
O
R1
R4
O
Malonic acid
TBAB, K2CO3, H2O
R2
R3
5
OH
H3CO
R2
R3
H
n
O
OH
n
AcCl/ reflux
MW
10. n = 0
11. n = 2
12. n = 4
13. n = 5
14. n = 7
H3CO
5 = R1 = R2 = H, R3 = R4 = OCH3
6 = R1 = R2 = OCH3, R3 = R4 = H
7 = R1 = R3 = OCH3, R2 = R4 = H
8 = R1 = R4 = OCH3, R2 = R3 = H
9 = R1 = R2 = R4 = H, R3 = OCH3
12 H2/Pd-C
MeOH
1. SOCl2
2.
OH
n
O
n = 9, 13, 15
H3CO
H3CO
O
2
21
R1
O
R2
R3
O
n
O
n = 9, 13, 15
O
R4
13
22
15. n = 9, R1 = R2 = H, R3 = R4 = OCH3
16. n = 15, R1 = R2 = H, R3 = R4 = OCH3
O
n = 13
HO
HO
O
17. R1 = R2 = H, R3 = R4 = OCH3
18. R1 = R2 = OCH3, R3 = R4 = H
19. R1 = R4 = OCH3, R2 = R3 = H
20. R1 = R2 = R4 = H, R3 = OCH3
5
23
Scheme 1 Synthetic pathway to cinnamic acid ester derivatives
for these compounds (Table 2) were higher than the LC50
values (Table 1). The most active compounds for intracellular
parasites were 23 and 16 with EC50 of 3.2 and 18.3 lg/ml,
respectively (Table 2), followed by compounds 15 and 17
with EC50 values of 25.2 and 26.5 lg/ml. Compound 11 had
the lowest activity with EC50 60.2 lg/ml. The best SI was
observedfor compounds23, 17, and 11 with valuesof3.1, 2.7,
and 2.6, respectively. As expected, meglumine antimoniate
was only active against intracellular amastigotes of L.
(V) panamensis (Table 2) but did not show activity against
axenic amastigotes that is an extracellular formof this parasite
(Table 1). In turn, amphotericine B was active against both
intracellular and axenic amastigotes (Table 1 and 2).
acid esters 10–12, 17, and 23 have also SI values greater
than 1. The in vitro activity of these molecules could be
improved in further studies by modifying their structure.
Further studies are also required to validate the in vitro
results with in vivo studies.
In general, as observed in other studies, no correlation
was found between the activity shown by the compounds
against intracellular and extracellular (axenic) forms of L.
(V) panamensis parasite. This lack of correlation may be
related to the fact that the activity of a compound may be
due to direct action of the compound on the parasite (which
can be detected in the in vitro system using axenic am-
astigotes) or the formation of a derivative product of
metabolism that the compound suffers when it is internal-
ized by the host cell (activity observed in the in vitro
system using intracellular amastigotes).
Overall, compounds 11, 15, 17 and 23 were apparently
the most active, showing activity against both axenic and
intracellular amastigotes of L. (V) panamensis, while
compounds 10, 12, and 13 showed activity only on the
axenic form of this Leishamania species.
Structure–activity relationship
Meglumine Antimoniate and Amphotericin B, drugs
currently used for the treatment of Leishmaniasis had SI
values greater than 1, leading to greater activity against the
parasite than the toxicity against the host cell. Cinnamic
A relationship between the leishmanicidal activity against
axenic amastigotes and compounds structure was observed
(see Table 1). First, while smaller alkyl chains lead to
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Med Chem Res (2014) 23:1378–1386
1381
higher selectivity indices (10, 11 vs. 12–17); second, the
degree of oxygenation is essential for activity, primarily in
positions 3 and 4 (17 vs. 18–20 and 22); and third,
hydroxyl groups increase both activity and cytotoxicity (14
vs. 23), This result is in agreement with the reports by
Aponte et al. 2010 and by Brezan et al. 2008, in which the
leishmanicidal activity and cytotoxicity of chalcones and
coumarins was found to be enhanced by the presence of
polar hydroxyl groups. On the other hand, the presence of a
double bond in the side chain is crucial for cytotoxicity and
leishmanicidal activity (12 vs. 21). The action of these
compounds may be explained by conjugated addition of
nucleophilic amino acid residues present in biomolecules
of the natural receptors, Michael type mechanism. This
mechanism has been reported for other a, b-unsaturated
compounds such as lactones, chalcones and coumarins (De
Fatima et al., 2006; Buck et al., 2003; Cardona and Saez,
2011).
Table 1 In vitro toxicity and leishmanicidal activity against axenic
amastigotes of L. (V) panamensis of Cinnamic acid ester
Compound
Cytotoxicity U937 Leishmanicidal
cells LC50 (lg/ml)a Activity EC50(lg/ml)b
SI
10
11
12
13
14
15
16
17
18
19
20
21
22
23
162.6 49.6
85.3 17.5
49.8 6.3
54.7 7.7
79.9 5.5
49.7 18.1
28.5 1.9
69.1 6.7
[200.0
55.0 14.7
33.0 0.2
38.1 9.7
60.0 4.0
[100.0
3.0
2.6
1.3
0.9
\0.8
0.8
59.0 1.9
108.2 1.5
55.4 2.0
[100.0
0.3
1.2
\2.0
\2.0
\2.0
\2.0
\2.0
4.3
[200.0
[100.0
[200.0
[100.0
[200.0
[100.0
[200.0
[100.0
9.9 1.7
495,9 ? 55,6
2.3 0.5
[200.0
Meglumine
antimoniate
\2.3
Conclusion
Amphotericin B 29.6 4.7
0.05 0.01
592
LC50 Lethal concentration 50
EC50 Effective concentration 50
The design, synthesis, and antileishmanial screening of
fourteen cinnamic acid esters was reported. Several of the
reported compounds have potential as leishmanicidal drugs,
as determined by both leishmanicidal activity and cyto-
toxicity. A structure–activity relationship was observed for
these compounds where the length of the alkyl chains, the
degree of oxygenation or the presence of hydroxyl groups
may modify the leishmanicidal activity but also the cyto-
toxicity. Overall, the compounds 11, 15–17, and 23 are
potential candidates for antileishmanial drug development
based on their activity against Leishmania parasite.
Although toxic for U-937 cells, more studies on toxicity
using other cell types are needed in order to discriminate
whether the toxicity shown by these compounds is against
tumor or non-tumor cells. In addition, further studies are
required to optimize the structure of these promising mol-
ecules and to validate the in vitro activity against Leish-
mania demonstrated here with in vivo studies.
SI selectivity index: LC50/EC50
a
Degree of toxicity: Highly toxic: LC50 \ 10 lg/ml, toxic:
to \ 50 lg/ml; Moderately toxic: LC50 [ 50
LC50 [ 10
to \ 200 lg/ml and potentially non toxic: LC50 [ 200 lg/ml
b
Degree of activity: Highly Active: EC50 \ 10 lg/ml; Active: [ 10
to \ 50 lg/ml; Moderately active: EC50 [ 50 to \ 100 lg/ml; No
Active: EC50 [ 100 lg/ml
Table 2 In vitro activity of cinnamic acid ester against intracellular
amastigotes of L. (V) panamensis
Compound
Leishmanicidal
activity EC50(lg/ml)
SI
10
[162.6 49.6
60.2 1.2
[49.8 6.3
[54.7 7.7
25.2 2.3
18.3 3.3
26.5 2.0
3.2 0.8
\1.0
2.6
11
12
\1.0
\1.0
2.0
13
Experimental procedures
15
16
1.6
Chemistry
17
2.7
23
3.1
IR spectra were recorded on a Perkin-Elmer Spectrum RX I
FT-IR system in a KBr disk. 1H NMR and 13C NMR spectra
were recorded on Varian Unity 500 MHz spectrometer
using CDCl3 as solvent and TMS as an internal standard.
Chemical shifts are expressed in d ppm. High resolution
mass spectra were recorded by the electron impact mode
(EIMS, 70 eV) on VG AutoSpec spectrometer. Silica gel 60
Meglumine antimoniate
Amphotericin B
6.3 ? 0.9
78.7
592
0.06 0.01
EC50 Effective concentration 50
SI selectivity index: LC50/EC50
Active: EC50 \ 50 lg/ml; Moderately active: EC50 \ 100 lg/ml; No
active: EC50 [ 100 lg/ml
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Med Chem Res (2014) 23:1378–1386
Propyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate (11)
(Merck 0.063–0.200 mesh) was used for column chroma-
tography, and precoated silica gel plates (Merck 60 F254
0.2 mm) were used for TLC.
Yield: 7.0 mmol, 1.751 g, 70 %; yellow pale oil; IR (KBr,
cm-1): mmax 2967 (C–H), 1706 (C=O), 1635 (C=C), 1513
(C=CAr), 1260 (C–O–C), 1177 ((C=O)–O), 808 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.97 (3H, t, J = 7.4 Hz),
1.70 (2H, m), 3.88 (6H, s, OCH3), 4.13 (2H, t, J = 6.9 Hz),
6.29 (1H, d, J = 16.0 Hz, –CO–CH=), 6.83 (1H, d,
J = 8.3 Hz, Ar–H), 7.03 (1H, d, J = 1.8 Hz, Ar–H), 7.07
(1H, dd, J = 8.3, 1.6 Hz, Ar–H), 7.60 (1H, d, J = 16.0 Hz,
Ar–CH=C); 13C NMR (CDCl3, 125 MHz): d 10.37 (CH3),
22.02 (CH2), 55.79 (OCH3), 55.86 (OCH3), 65.94 (–
OCH2–), 109.59, 110.98, 115.86 (=C–CO–), 122.44,
127.35, 144.37 (Ar–C=), 149.10, 150.95, 167.15 (C=O).
EIMS: m/z 251.1283 (M?1), Calcd. for C14H18O4: 250,12.
Synthesis of cinnamic acid esters (10–14)
3,4-dimethoxycinnamic acid (10 mmol), acetyl chloride
(0.5 mmol), and 50 mL of alcohol (methanol, propanol,
pentanol, hexanol or octanol), were placed in a 250 ml
3-neck round-bottom flask equipped with a magnetic stir-
ring bar. The mixture was stirred, heated to reflux for a
period of 4 h. The reaction mixture was concentrated on a
rotatory evaporator, and the residue was purified by chro-
matographic column over silica gel eluted with a mixture
hexane–ethyl acetate at different ratios to obtain the cin-
namic acid esters in yield between 75 and 85 %.
Pentyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate (12)
Synthesis of cinnamic acid esters (15–21)
Yield: 8.5 mmol, 2.364 g, 85 %; yellow pale oil; IR (KBr,
cm-1): mmax 2958 (C–H), 1706 (C=O), 1635 (C=C), 1513
(C=CAr), 1259 (C–O–C), 1160 ((C=O)–O), 807 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.92 (3H, t, J = 7.3 Hz),
1.37 (4H, m), 1.70 (2H, m), 3.90 (6H, s, OCH3), 4.19 (2H,
t, J = 6.8 Hz), 6.30 (1H, d, J = 16.0 Hz, –CO–CH =),
6.85 (1H, d, J = 8.4 Hz, Ar–H), 7.05 (1H, d, J = 1.6 Hz,
Ar–H), 7.09 (1H, dd, J = 8.4, 1.6 Hz, Ar–H), 7.61 (1H, d,
J = 16.0 Hz, Ar–CH=C); 13C NMR (CDCl3, 125 MHz): d
13.79 (CH3), 22.28 (CH2), 27.93 (CH2), 28.35 (CH2),
55.80 (OCH3), 55.87 (OCH3), 65.31 (–OCH2–), 109.55,
110.06, 115.78 (=C–CO–), 122.37, 127.29, 144.25 (Ar–
C=), 149.12, 151.01, 167.24 (C=O). EIMS: m/z 279.1596
(M?1), Calcd. for C16H22O4: 278,15.
Cinnamic acid, 5–9 (10 mmol) and thionyl chloride
(10 mL) were placed in a 50 ml 3-neck round-bottom flask
equipped with a magnetic stirring bar. The mixture was
stirred, heated to reflux for a period of 4 h. The reaction
mixture was concentrated on a rotatory evaporator, and the
residue was added to a solution of cetyl alcohol (dodecyl or
stearyl alcohol) in dichloromethane and the mixture was
stirred and monitored by thin layer chromatography. The
reaction was complete after about 4 h. The mixture was
transferred to a separatory funnel and then quenched by
addition of a solution of 20 mL of potassium carbonate; the
organic layer was washed with water, separated, dried on
anhydrous sodium sulfate, and filtered and concentrated
under reduced pressure. The residue was chromatographed
over silica gel (hexane–ethyl acetate, different ratios) to
obtain the cinnamic acid esters in yield between 70 and
85 %.
Hexyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate (13)
Yield: 8.3 mmol, 2.364 g, 83 %; yellow pale oil; IR (KBr,
cm-1): mmax 2957 (C–H), 1704 (C=O), 1600 (C=C), 1512
(C=CAr), 1270 (C–O–C), 1173 ((C=O)–O), 808 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.91 (3H, t, J = 7.0 Hz),
1.33 (4H, m), 1.41 (2H, m), 1.70 (2H, m), 3.91 (6H, s,
OCH3), 4.19 (2H, t, J = 6.8 Hz), 6.31 (1H, d,
J = 15.9 Hz, –CO–CH=), 6.86 (1H, d, J = 8.2 Hz, Ar–H),
7.06 (1H, d, J = 1.8 Hz, Ar–H), 7.10 (1H, dd, J = 8.2,
1.8 Hz, Ar–H), 7.62 (1H, d, J = 15.9 Hz, Ar–CH=C); 13C
NMR (CDCl3, 125 MHz): d 13.85 (CH3), 22.43 (CH2),
22.59 (CH2), 28.74 (CH2), 31.46 (CH2), 55.84 (OCH3),
55.91 (OCH3), 64.76 (–OCH2–), 109.56, 110.92, 115.97
(=C–CO–), 122.44, 127.33, 144.35 (Ar–C=), 149.07,
151.03, 167.07 (C=O). EIMS: m/z 293.1753 (M?1), Calcd.
for C17H24O4: 292,17.
Methyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate (10)
Yield: 7.4 mmol, 1.643 g, 74 %; yellow pale oil; IR (KBr,
cm-1): mmax 2945 (C–H), 1700 (C=O), 1627 (C=C), 1510
(C=CAr), 1270 (C–O–C), 1178 ((C=O)–O), 857 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 3.78 (3H, s, OCH3), 3.90
(6H, s, OCH3), 6.30 (1H, d, J = 15.9 Hz, –CO–CH=), 6.86
(1H, d, J = 8.0 Hz, Ar–H), 7.04 (1H, d, J = 1.6 Hz, Ar–
H), 7.10 (1H, dd, J = 8.0, 1.6 Hz, Ar–H), 7.63 (1H, d,
J = 15.9 Hz, Ar–CH = C); 13C NMR (CDCl3, 125 MHz):
d 51.46 (OCH3), 55.83 (OCH3), 55.91 (OCH3), 109.50,
110.88, 115.51 (=C–CO–),122.57, 127.38, 144.70 (Ar–
C=), 149.18, 151.09, 167.40 (C=O). EIMS: m/z 223.0972
(M?1), Calcd. for C12H14O4: 222,09.
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Med Chem Res (2014) 23:1378–1386
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Octyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate (14)
150.95, 151.03, 167.14 (C=O). EIMS: m/z 433.3318
(M?1), Calcd. for C27H44O4: 432,32.
Yield: 8.0 mmol, 2.425 g, 80 %; yellow pale oil; IR (KBr,
cm-1): mmax 2956 (C–H), 1718 (C=O), 1633 (C=C), 1514
(C=CAr), 1271 (C–O–C), 1176 ((C=O)–O), 802 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.81 (3H, t, J = 6.6 Hz),
1.09–1.38 (10H, m), 1.61 (2H, m), 3.84 (6H, s, OCH3),
4.12 (2H, t, J = 6.7 Hz), 6.24 (1H, d, J = 16.6 Hz, –CO–
CH=), 6.79 (1H, d, J = 8.4 Hz, Ar–H), 6.99 (1H, d,
J = 1.3 Hz, Ar–H), 7.03 (1H, dd, J = 8.4, 1.3 Hz, Ar–H),
7.55 (1H, d, J = 16.6 Hz, Ar–CH=C); 13C NMR (CDCl3,
125 MHz): d 14.52 (CH3), 23.09 (CH2), 26.41 (CH2),
29.15 (CH2), 29.61 (2CH2), 32.20 (CH2), 56.27 (OCH3),
56.36 (OCH3), 65.21 (–OCH2–), 109.88, 111.39, 112.69
(3CH), 116.29 (=C–CO–), 123.01, 144.95 (Ar–C=), 167.68
(C=O). EIMS: m/z 321.2023 (M?1), Calcd. for C19H28O4:
320,20.
Octadecyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate
(17)
Yield: 7.0 mmol, 3.761 g, 70 %; m.p. 154–158 °C; IR
(KBr, cm-1): mmax 2915 (C–H), 1708 (C=O), 1620 (C=C),
1506 (C=CAr), 1274 (C–O–C), 1163 ((C=O)–O), 811 (C–
HAr); 1H NMR (CDCl3, 500 MHz): d 0.87 (3H, t,
J = 7.1 Hz), 1.18–1.39 (30H, m), 1.55 (2H, m), 3.62 (2H,
t, J = 6.6 Hz), 3.92 (6H, s, OCH3), 6.38 (1H, d,
J = 16.0 Hz, –CO–CH=), 6.88 (1H, d, J = 8.3 Hz, Ar–H),
7.07 (1H, d, J = 1.8 Hz, Ar–H), 7.15 (1H, dd, J = 8.3,
1.8 Hz, Ar–H), 7.78 (1H, d, J = 16.0 Hz, Ar–CH=C); 13C
NMR (CDCl3, 125 MHz): d 14.06 (CH3), 22.60 (CH2),
25.70 (CH2), 29.30 (CH2), 29.40–29.80 (11CH2), 31.89
(CH2), 32.77 (CH2), 55.90 (OCH3), 55.98 (OCH3), 63.03
(–OCH2–), 109.96, 111.08, 114.35 (=C–CO–), 123.55,
126.74, 148.47 (Ar–C=), 149.33, 151.95, 162.84 (C=O).
EIMS: m/z 461.3631 (M?1), Calcd. for C29H48O4: 460,36.
Dodecyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate (15)
Yield: 7.5 mmol, 2.822 g, 75 %; m.p. 55–58 °C; IR (KBr,
cm-1): mmax 2939 (C–H), 1697 (C=O), 1625 (C=C), 1512
(C=CAr), 1251 (C–O–C), 1161 ((C=O)–O), 813 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.85 (3H, t, J = 6.9 Hz),
1.15–1.35 (18H, m), 1.53 (2H, m), 3.61(2H, t, J =
6.67 Hz), 3.88 (6H, s, OCH3), 6.28 (1H, d, J = 16.4 Hz,
–CO–CH=), 6.84 (1H, d, J = 8.3 Hz, Ar–H), 7.02 (1H, d,
J = 1.1 Hz, Ar–H), 7.08 (1H, dd, J = 8.3, 1.1 Hz, Ar–H),
7.61 (1H, d, J = 16.4 Hz, Ar–CH=C); 13C NMR (CDCl3,
125 MHz): d 14.65 (CH3), 23.06 (CH2), 26.14 (CH2),
29.75–32.02 (6CH2), 32.28 (CH2), 33.27 (CH2), 56.28
(OCH3), 56.36 (OCH3), 63.45 (–OCH2–), 109.86, 111.39,
113.34, 115.85 (=C–CO–), 122.88, 125.87, 137.42, 145.22
(Ar–C=), 179.05 (C=O). EIMS: m/z 377.2632 (M?1),
Calcd. for C23H36O4: 376,26.
Hexadecyl (2E)-3-(2,3-dimethoxyphenyl)prop-2-enoate
(18)
Yield: 6.0 mmol, 2.594 g, 60 %; m.p. 41–43 °C; IR (KBr,
cm-1): mmax 2920 (C–H), 1719 (C=O), 1635 (C=C), 1515
(C=CAr), 1270 (C–O–C), 1180 ((C=O)–O), 802 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.89 (3H, t, J = 7.1 Hz),
1.13–1.50 (26H, m), 1.72 (2H, m), 3.87 (3H, s, OCH3),
3.89 (3H, s, OCH3), 4.21 (2H, t, J = 6.7 Hz), 6.49 (1H, d,
J = 16.2 Hz, –CO–CH =), 6.94 (1H, d, J = 8.0 Hz, Ar–H),
7.06 (1H, t, J = 8.0 Hz, Ar–H), 7.16 (1H, d, J = 8.0 Hz,
Ar–H), 8.00 (1H, d, J = 16.2 Hz, Ar–CH=C); 13C NMR
(CDCl3, 125 MHz): d 14.04 (CH3), 22.68 (CH2), 25.95
(CH2), 28.74 (CH2), 29.04–29.90 (10CH2), 31.86 (CH2),
55.88 (OCH3), 61.26 (OCH3), 64.66 (–OCH2–), 113.85
(=C–CO–), 119.21, 119.65, 124.11, 128.69, 139.29 (Ar–
C=), 148.50, 153.15, 167.19 (C=O). EIMS: m/z 433.3318
(M?1), Calcd. for C27H44O4: 432,32.
Hexadecyl (2E)-3-(3,4-dimethoxyphenyl)prop-2-enoate
(16)
Yield: 8.7 mmol, 3.761 g, 87 %; m.p. 48–50 °C; IR (KBr,
cm-1): mmax 2918 (C–H), 1719 (C=O), 1635 (C=C), 1500
(C=CAr), 1271 (C–O–C), 1180 ((C=O)–O), 802 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.89 (3H, t, J = 7.1 Hz),
1.15–1.47 (26H, m), 1.70 (2H, m), 3.91 (6H, s, OCH3),
4.20 (2H, t, J = 6.74 Hz), 6.31 (1H, d, J = 16.0 Hz, –CO–
CH=), 6.87 (1H, d, J = 8.3 Hz, Ar–H), 7.06 (1H, d,
J = 1.6 Hz, Ar–H), 7.11 (1H, dd, J = 8.3, 1.6 Hz, Ar–H),
7.63 (1H, d, J = 16.0 Hz, Ar–CH=C); 13C NMR (CDCl3,
125 MHz): d 14.08 (CH3), 22.64 (CH2), 25.70 (CH2),
28.18-29.85 (10CH2), 31.91(CH2), 32.79 (CH2), 55.84
(OCH3), 55.93 (OCH3), 64.59 (–OCH2–), 109.60, 110.90,
116.02 (=C–CO–), 122.39, 127.45, 144.37 (Ar–C=),
Hexadecyl (2E)-3-(2,5-dimethoxyphenyl)prop-2-enoate
(19)
Yield: 8.5 mmol, 3.675 g, 85 %; m.p. 63–65 °C; IR (KBr,
cm-1): mmax 2918 (C–H), 1710 (C=O), 1630 (C=C), 1495
(C=CAr), 1219 (C–O–C), 1177 ((C=O)–O), 802 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.89 (3H, t, J = 7.0 Hz),
1.22–1.44 (26H, m), 1.72 (2H, m), 3.80 (3H, s, OCH3),
3.85 (3H, s, OCH3), 4.20 (2H, t, J = 6.81 Hz), 6.50 (1H, d,
J = 16.2 Hz, –CO–CH=), 6.85 (1H, d, J = 9.0 Hz, Ar–H),
6.92 (1H, dd, J = 9.0, 3.0 Hz), 7.06 (1H, d, J = 3.0 Hz,
Ar–H), 7.97 (1H, d, J = 16.2 Hz, Ar–CH=C); 13C NMR
123

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Med Chem Res (2014) 23:1378–1386
Octyl 3-(3,4-dihydroxyphenyl)prop-2-enoate (23)
(CDCl3, 125 MHz): d 14.08 (CH3), 22.68 (CH2), 25.96
(CH2), 28.77 (CH2), 29.17–29.97 (10CH2), 31.90 (CH2),
55.09 (OCH3), 55.77 (OCH3), 64.56 (-OCH2-), 112.46,
113.24, 116.99, 119.04, 124.07 (=C–CO–), 139.67 (Ar–
C=), 152.77, 153.56, 167.37 (C=O). EIMS: m/z 433.3318
(M?1), Calcd. for C27H44O4: 432,32.
Yield: 7.2 mmol, 2.104 g, 72 %; m.p. 101–104 °C; IR
(KBr, cm-1): mmax 3318 (OH), 2920 (C–H), 1683 (C=O),
1604 (C=C), 1442 (C=CAr), 1282 (C–O–C), 1178 ((C=O)–
1
O), 815 (C–HAr); H NMR (DMSO-D6, 500 MHz): d 0.79
(3H, t, J = 6.5 Hz), 1.03-1.31 (10H, m), 1.53 (2H, m), 4.01
(2H, t, J = 6.0 Hz), 4.43 (2H, m, OH), 6.17 (1H, d,
J = 15.9 Hz, –CO–CH=), 6.75 (1H, d, J = 8.2 Hz, Ar–H),
6.83 (1H, d, J = 8.2 Hz, Ar–H), 7.02 (1H, s, Ar–H), 7.42
(1H, d, J = 15.9 Hz, Ar–CH=C); 13C NMR (DMSO-D6,
125 MHz): d 13.65 (CH3), 22.35 (CH2), 25.73 (CH2),
28.50 (CH2), 28.98 (CH2), 31.41 (CH2), 45.96 (CH2), 63.98
(–OCH2–), 111.65, 121.61 (=C–CO–), 125.93 (2C), 145.21
(2C), 148.20 (Ar–C=C), 167.68, 167.01 (C=O). EIMS: m/
z 293.1732 (M?1), Calcd. for C17H24O4: 294,18.
Hexadecyl (2E)-3-(4-methoxyphenyl)prop-2-enoate (20)
Yield: 7.8 mmol, 3.138 g, 78 %; m.p. 50–55 °C; IR (KBr,
cm-1): mmax 2915 (C–H), 1707 (C=O), 1638 (C=C), 1517
(C=CAr), 1268 (C–O–C), 1182 ((C=O)–O), 826 (C–HAr);
1H NMR (CDCl3, 500 MHz): d 0.89 (3H, t, J = 7.1 Hz),
1.19–1.46 (26H, m), 1.70 (2H, m), 3.84 (3H, s, OCH3),
4.20 (2H, t, J = 6.9 Hz), 6.32 (1H, d, J = 16.0 Hz, –CO–
CH=), 6.92 (2H, d, J = 8.7 Hz, Ar–H), 7.48 (2H, d,
J = 8.7 Hz, Ar–H), 7.64 (1H, d, J = 16.0 Hz, Ar–CH=C);
13C NMR (CDCl3, 125 MHz): d 14.06 (CH3), 22.64 (CH2),
25.97 (CH2), 28.75 (CH2), 29.20-29.82 (10CH2), 31.89
(CH2), 55.32 (OCH3), 64.58 (–OCH2–), 114.24, 115.79
(=C–CO–), 127.24, 129.59, 144.11 (Ar–C=), 161.31, 167.33
(C=O). EIMS: m/z 403.3212 (M?1), Calcd. for C26H42O3:
402,31.
Solubility Compounds 10–22 exhibited low polarity
which makes them soluble in solvents such as dichloro-
methane, ether and ethyl acetate. Compound 23 is soluble
in more polar solvents such as DMF and DMSO, due to the
hydrophilicity provided by hydroxyl groups.
Biological activity assays
Hexadecyl 3-(4-methoxyphenyl)propanoate (21)
The compounds were subjected to in vitro cytotoxic
activity on mammalian cells and leishmanicidal activity on
axenic and intracellular amastigotes of L. panamensis.
Yield: 7.0 mmol, 2.830 g, 70 %; yellow pale oil; IR (KBr,
cm-1): mmax 2929 (C–H), 1735 (C=O), 1515 (C=CAr), 1247
(C–O–C), 1175 ((C=O)–O), 835 (C–HAr); 1H NMR
(CDCl3, 500 MHz): d 0.90 (3H, t, J = 7.1 Hz), 1.23–1.35
(26H, m), 1.60 (2H, m), 2.60 (2H, t, J = 7.8), 2.90, (2H, t,
J = 7.8), 3.79 (3H, s, OCH3), 4.07 (2H, t, J = 7.1 Hz),
6.83 (2H, d, J = 8.3 Hz, Ar–H), 7.13 (2H, d, J = 8.3 Hz,
Ar–H). 13C NMR (CDCl3, 125 MHz): d 14.10 (CH3),
22.60 (CH2), 25.87 (CH2), 28.60 (CH2), 29.01–30.45
(11CH2), 31.94 (CH2), 36.16 (CH2), 55.14 (OCH3), 64.54
(-OCH2-), 113.83, 129.18, 132.32, 158.04, 172.97 (C=O).
EIMS: m/z 405.3369 (M?1), Calcd. for C26H44O3: 404,33.
In vitro cytotoxic activity in mammalian cells
The cytotoxic activity of the compounds was assessed
based on the viability of the human promonocytic cell line
U937 (ATCC CRL-1593.2TM) evaluated by the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bro-
mide) method as described by Taylor et al. 2010, 2011). In
brief, cells were grown in 96-well cell-culture dishes at a
concentration of 100,000 cells/mL in RPMI-1640 supple-
mented with 10 % FBS and the corresponding concentra-
tions of the compounds, starting at 200 lg/mL in duplicate.
The cells were incubated at 37 °C with 5 % CO2 for 72 h
in the presence of the compounds, and then the effect was
determined using MTT assay, incubating at 37 °C for 3 h.
The effect of the compounds was determined by measuring
the activity of the mitochondrial dehydrogenase by adding
10 lL/well of MTT solution (0.5 mg/mL) and incubating
at 37 °C for 3 h. The reaction was stopped by adding a
50 % isopropanol solution with 10 % sodium dodecyl
sulfate for 30 min. Cell viability was determined based on
the quantity of formazan produced, which was measured at
570 nm in a Bio-Rad ELISA. Cultured cells in the absence
of extracts were used as viability controls; meglumine
Hexadecyl (2E)-3-phenylprop-2-enoate (22)
Yield: 9.4 mmol, 3.500 g, 94 %; m.p. 35–36 °C; IR (KBr,
cm-1): mmax 2952 (C–H), 1714 (C=O), 1640 (C=C), 1475
(C=CAr), 1177 ((C=O)–O), 801 (C–HAr); 1H NMR (CDCl3,
500 MHz): d 0.89 (3H, t, J = 7.0 Hz), 1.19–1.48 (26H, m),
1.72 (2H, m), 4.21 (2H, t, J = 6.8 Hz), 6.35 (1H, d,
J = 16.0 Hz, –CO–CH=), 7.39 (3H, m, Ar–H), 7.53 (2H,
dd, J = 7.0, 2.2 Hz, Ar–H), 7.69 (1H, d, J = 16.0 Hz, Ar–
CH=C); 13C NMR (CDCl3, 125 MHz): d 14.06 (CH3), 22.66
(CH2), 26.01 (CH2), 28.72 (CH2), 29.22-29.91 (10CH2),
31.89 (CH2), 64.66 (–OCH2–), 118.32 (=C–CO–), 127.97,
128.82, 130.10, 134.45, 144.47 (Ar–C =), 167 (C=O).
EIMS: m/z 373.3107 (M?1), Calcd. for C25H42O2: 374,32.
123

Med Chem Res (2014) 23:1378–1386
1385
antimoniate and amphotericin B were used as cytotoxicity
controls. Assays were performed twice with three replica-
tions per each concentration tested. The results are
expressed as lethal concentration 50 (LC50), calculated by
the Probit method (Finney, 1971). The degree of toxicity
was established according to the LC50 values, using the
following scale: Highly toxic: LC50 \10 lg/mL, toxic:
LC50 [10 to \50 lg/mL; Moderately toxic: LC50 [50 to
\200 lg/mL and potentially non toxic: LC50[200 lg/mL.
and the leishmanicidal activity using the following for-
mula: IS, IT = CL50/CE50.
Activity against intracellular amastigotes
The effects of cinnamic acid esters against intracellular
amastigotes of L. (V.) panamensis were evaluated by flow
cytometry using the methodology described by Varela
et al. (2009). In brief, U937 cells were dispensed in 24-well
plates at a concentration of 300,000 cells/well, which were
treated with 1 lM of Phorbol Myristate Acetate (PMA) for
48 h at 37 °C, after which they were infected with prom-
astigotes of L. (V.) panamensis in stationary growth phase
(day 5) in modified NNN medium, at ratio of 1:25 cell/
parasite, after 3 h of incubation at 34 °C in 5 % CO2 non-
internalized parasites were washed, and incubated again at
34 °C and 5 % CO2 to allow differentiation to amastigotes
form. After 24 h of incubation, the compounds with the
appropriate dilution, not exceeding the LC50, were added.
Infected and treated cells were maintained at 34 °C and
5 % CO2 for 72 h. The leishmanicidal effect was measured
in a flow cytometer at 488 nm of excitation and 525 nm of
emission, and determined as described by Pulido et al.
(2012), Taylor et al. (2011) and Varela et al. (2009).
Infected cells cultivated in the presence of meglumina
antimoniate and amphotericin B were used as control for
leishmanicidal activity. In addition, infected but untreated
cells were used as control of viability. Assays were per-
formed at least twice with 3 replicates per each concen-
tration tested. The results were also expressed as the EC50
calculated by the Probit method as above (Finney, 1971).
The degree of leishmanicidal activity was established
according to the EC50 values, using the same scale previ-
ously described. SI was calculated as described above.
In vitro leishmanicidal activity on axenic
and intracellular amastigotes
Axenic and intracellular amastigotes of GFP-transfected L.
(V.) panamensis strain (MHOM/CO/87/UA140epirGFP)
were used for the in vitro testing of leishmanicidal activity
of the cinnamic acid esters derivatives.
Activity against axenic amastigotes
The respective ability of the cinnamic acid esters to kill
axenic amastigotes of L. (V.) panamensis was determined
based on the viability of the parasites evaluated by the
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide) method as described previously (Taylor et al.,
2011; 2010). In short, parasites were cultivated in
Schneider’s medium pH 5.4 supplemented with 20 % heat
inactivated FBS for 3 days at 32 °C. Afterward, they were
harvested, washed, and resuspended at 2 9 106 axenic
amastigotes/mL in fresh medium. Each well of a 96-well
plate was seeded with 100 lL of each parasite suspension
(in duplicate), and 100 lL of each concentration of the test
compound was added, starting at 100 lg/mL. Plates were
incubated at 32 °C. After 72 h of incubation, the effect of
drugs was determined by adding 10 lL/well of MTT and
incubating at 32 °C for 3 h. The reaction was stopped, and
the quantity of formazan produced was measured with a
Bio-Rad ELISA reader set at 570 nm. Parasites cultivated
in the absence of the compound but maintained under the
same conditions were used as controls for growth and
viability. Parasites cultivated in the presence of meglumina
antimoniate and amphotericin B were used as controls for
leishmanicidal activity. Assays were performed at least
twice with three replicates per each concentration tested.
The results are expressed as effective concentration 50
(EC50) calculated by the Probit method (Finney, 1971). The
degree of leishmanicidal activity was established according
to the EC50 values, using the following scale: Highly
active: EC50 \10 lg/mL, active: EC50 [10 to\50 lg/mL;
Moderately active: EC50 [50 to \200 lg/mL and poten-
tially non active: EC50 [200 lg/mL.
´
Acknowledgments The authors thank Dr. Javier Garces for his help
in this study. We acknowledge the support by the Universidad de
Antioquia (Estrategia de Sostenibilidad 2013–2014 and CIDE-
PRO) and Colciencias (contract No. 357-2011).
References
´
Almajano MP, Carbo R, Delgado ME, Gordon MH (2007) Effect of
pH on the antimicrobial activity and oxidative stability of oil-in-
water emulsions containing caffeic acid. J Food Sci 72:C258–
C263
Aponte J, Castillo D, Estevez Y, Gonzalez G, Arevalo J, Hammonda
G, Sauvain M (2010) In vitro and in vivo anti-Leishmania
activity of polysubstituted synthetic chalcones. Bioorg Med
Chem Lett 20:100–103
Barrett M, Gilbert I (2002) Perspectives for new drugs against
trypanosomiasisandleishmaniasis.CurrTopMedChem2:471–482
Bowles BL, Miller AJ (1994) Caffeic Acid Activity against
Clostridium botulinum Spores. J Food Sci 59:905–908
Brenzan MA, Vaturu C, Dias B, Ueda T, Young MC, Goncalves A
et al (2008) Structure–activity relationship of (-) mammea A/BB
The Selective Index (SI), also known as Therapeutic
Index (TI) was calculated by dividing the cytotoxic activity
123

1386
Med Chem Res (2014) 23:1378–1386
derivatives against Leishmania amazonensis. Biomed Pharmac-
other 62:651–658
as potent inhibitors of human immunodeficiency virus type 1
integrase and replication. J Med Chem 42:497–509
Buck S, Hardouin C, Ichikawa S, Soenen D, Gauss C, Hwang I,
Swingle M, Bonness K, Honkanen R, Boger D (2003) Funda-
mental role of the fostriecin unsaturated lactone and implications
for selective protein phosphatase inhibition. J Am Chem Soc
125:15694–15695
Cabanillas B, Le Lamer AC, Castillo D, Arevalo J, Rojas R, Odonne
G, Bourdy G, Moukarzel B, Sauvain M, Fabre N (2010) Caffeic
acid esters and lignans from Piper sanguineispicum. J Nat Prod
73:1884–1890
Murray HW, Berman JD, Davies CR, Saravia NG (2005) Advances in
leishmaniasis. Lancet 366:1561–1577
Narasimhan B, Belsare D, Pharande D, Mourya V, Dhake A (2004)
Esters, amides and substituted derivatives of cinnamic acid:
synthesis, antimicrobial activity and QSAR investigations. Eur J
Med Chem 39:827–834
Noriaki K, Yukari K, Kazuya I, Kyo M, Tokio F (2005) In Vitro
Antibacterial, antimutagenic and anti-influenza virus activity of
caffeic acid phenethyl ester. Biocontrol Sci 10:155–161
Olliaro P, Bryceson A (1993) Practical progress and new drugs for
changing patterns of Leishmaniasis. Parasitol Today 9:323–328
Ouellette M, Drummelsmith J, Papadopoulou B (2004) Leishmani-
asis: drugs in the clinic, resistance and new developments. Drug
Resist Updat 7:257–266
Cardona W, Saez J (2011) Antiprotozoal activity of a, b-unsaturated
d-lactones: promising compounds for the development of new
therapeutic alternatives. Trop J Pharm Res 10:671–680
´
Cardona W, Quin˜ones W, Robledo S, Velez I, Murga J et al (2006)
Antiparasite and antimycobacterial activity of passifloricin
analogues. Tetrahedron 62:4086–4092
´
Pulido SA, Mun˜oz DL, Restrepo AM, Mesa CV, Alzate JF, Velez ID,
Croft S, Coombs G (2003) Leishmaniasis-current chemotherapy and
recent advances in the search for novel drugs. Trends Parasitol
19:502–508
Robledo SM (2012) Improvement of the green fluorescent protein
reporter system in Leishmania spp. for the in vitro and in vivo
screening of antileishmanial drugs. Acta Trop 122(1):36–45
Radtke OA, Foo LY, Lu Y, Kiderlen A, Kolodziej H (2003)
Evaluation of sage phenolics for their antileishmanial activity
and modulatory effects on interleukin-6, interferon and tumour
necrosis factor-a-release in RAW 264.7 cells. Z Naturforsch C
58(5–6):395–400
Rajan P, Vedernikova I, Cos P, Berghe DV, Augustynsa K, Haemersa
A (2001) Synthesis and evaluation of caffeic acid amides as
antioxidants. Bioorg Med Chem Lett 11:215–217
Robledo SM, Valencia AZ, Saravia NG (1999) Sensitivity to
Glucantime of Leishmania Viannia isolated from patients prior
to treatment. J Parasitol 85:360–366
Robledo S, Osorio E, Jaramillo L (2005) In vitro and in vivo
cytotoxicities and antileishmanial activities of thymol and hemi-
synthetic derivatives. Antimic Agents Chemoth 49:1652–1655
Son S, Lewis BA (2002) Free radical scavenging and antioxidative
activity of caffeic acid amide and ester analogues: structure–
activity relationship. J Agric Food Chem 50:468–472
Da Cunha FM, Duma D, Assreuy J, Buzzi FC, Niero R, Campos MM,
Calixto JB (2004) Caffeic acid derivatives: in vitro and in vivo
anti-inflammatory properties. Free Radic Res 38:1241–1253
De Campos F, Franzoi C, Antonini G, Fracasso M, Cechinel V, Yunes
R, Niero R (2009) Antinociceptive properties of caffeic acid
derivatives in mice. Eur J Med Chem 44:4596–4602
De Fatima A, Kohn L, Antonio M, De Carvalho J, Pilli R (2006)
Cytotoxic activity of (S)-goniothalamin and analogues against
human cancer cells. Bioorg Med Chem 14:622–631
De P, Baltas M, Bedos-Belval F (2011) Cinnamic acid derivatives as
anticancer agents-a review. Curr Med Chem 18:1672–1703
Desjeux P (2004) Leishmaniasis: current situation and new perspec-
tives. Comp Immunol Microbiol Infect Dis 27:305–318
Faraut-Gambarelli F, Piarroux R, Deniau M, Giusiano B, Marty P,
Michel G, Faugere B, Dumon H (1997) In vitro and in vivo
resistance of leishmania infantum to meglumine antimoniate:
study of 37 strains collected from patients with visceral
leishmaniasis. Antimicrob Agents Chemother 41:827–830
Finney JD (1971) Probit Analysis, 3rd edn. Cambridge University
Press, Cambridge
´
Taylor VM, Mun˜oz DL, Ceden˜o DL, Velez ID, Jones MA, Robledo
SM (2010) Leishmania tarentolae: utility as an in vitro model for
screening of antileishmanial agents. Exp Parasitol 126:471–475
Taylor VM, Ceden˜o DL, Mun˜oz DL, Jones MA, Lash TD, Young
Gupta M, Purnima B (2007) Tetrabutylammoniumbromide mediated
Knoevenagel condensation in water: synthesis of cinnamic acids.
ARKIVOC 1:94–98
Handman E (1999) Cell biology of Leishmania. Adv Parasitol
44:1–39
Hung C, Tsai W, Kuo LY, Kuo Y (2005) Evaluation of caffeic acid
amide analogues as anti-platelet aggregation and anti-oxidative
agents. Bioorg Med Chem 13:1791–1797
´
AM, Constantino MH, Esposito N, Velez ID, Robledo SM
(2011) In vitro and vivo studies of the utility of dimethyl and
diethyl carbaporphyrin ketals in treatment of cutaneous leish-
maniasis. Antimicrob Agents Chemother 55(10):4755–4764
Valenta C, Schwarz E, Bernkop-Schnurch A (1998) Lysozyme-
caffeic acid conjugates: possible novel preservatives for dermal
formulations. Int J Pharm 174:125–132
Jayaprakasam B, Vanisree M, Zhang Y, Dewitt D, Nair M (2006)
Impact of alkyl esters of caffeic and ferulic acids on tumor cell
proliferation, cyclooxygenase enzyme, and lipid peroxidation.
J Agric Food Chem 54:5375–5381
Varela MRE, Mun˜oz DL, Robledo SM, Kolli BK, Dutta S, Chang KP,
Muskus C (2009) Leishmania (Viannia) panamensis: an in vitro
assay using the expression of GFP for screening of antileishma-
nial drug. Exp Parasitol 122:134–139
´
Kikuzaki H, Hisamoto M, Hirose K, Akiyama K, Taniguchi H (2002)
Antioxidant properties of ferulic acid and its related compounds.
J Agric Food Chem 50:2161–2168
KingPJ, MaG, MiaoW, Jia Q, McDougall BR, ReineckeMG, Cornell C,
Kuan J, Kim TR, Robinson WE (1999) Structure–activity relation-
ships: analogues of the dicaffeoylquinic and dicaffeoyltartaric acids
Weninger B, Robledo S, Arango G, Deharo E, Aragon R, Mun˜oz V,
Callapa J, Lobstein A, Anton R (2001) Antiprotozoal activities
of Colombian plants. J Ethnopharmacol 78:193–200
Wolday D, Berhe N, Akuffo H, Britton S (1999) Leishmania-HIV
interation: immunopathogenic mechanism. Parasitol Today
15:182–187
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