Design, Antileishmanial Activity, and QSAR Studies of a Series of Piplartine Analogues

Article abstract of DOI:10.1155/2019/4785756

Piplartine is an alkamide found in different Piper species and possesses several biological activities, including antiparasitic properties. Thus, the aim of the present study was to evaluate a series of 32 synthetic piplartine analogues against the Leishmania amazonensis promastigote forms and establish the structure-activity relationship and 3D-QSAR of these compounds. The antileishmanial effect of the compounds was determined using the MTT method. Most compounds were found to be active against L. amazonensis. Among 32 assayed derivatives, compound (E)-(-)-bornyl 3-(3,4,5-trimethoxyphenyl)-acrylate exhibited the most potent antileishmanial activity (IC₅₀ = 0.007 ± 0.008 μM, SI > 10), followed by benzyl 3,4,5-trimethoxybenzoate (IC₅₀ = 0.025 ± 0.009 μM, SI > 3.205) and (E)-furfuryl 3-(3,4,5-trimethoxyphenyl)-acrylate (IC₅₀ = 0.029 ± 0.007 μM, SI > 2.688). It was found that the rigid substituents contribute to increasing antiparasitic activity against L. amazonensis promastigotes. The presence of the unsaturated heterocyclic substituent in the phenylpropanoid chemical structure (furfuryl group) resulted in a bioactive derivative. Molecular simplification of benzyl 3,4,5-trimethoxybenzoate by omitting the spacer group contributed to the bioactivity of this compound. Furthermore, bornyl radical appears to be important for antileishmanial activity, since (E)-(-)-bornyl 3-(3,4,5-trimethoxyphenyl)-acrylate exhibited the most potent antileishmanial activity. These results show that some derivatives studied would be useful as prototype molecules for the planning of new derivatives with profile of antileishmanial drugs.

Full text of DOI:10.1155/2019/4785756

Hindawi
Journal of Chemistry
Volume 2019, Article ID 4785756, 12 pages
https://doi.org/10.1155/2019/4785756
Research Article
Design, Antileishmanial Activity, and QSAR Studies of a
Series of Piplartine Analogues
1
2
1
3
´
´
Flavio R. Nobrega, Larisse V. Silva, Carlos da Silva M. Bezerra Filho, Tamires C. Lima,
Yunierkis P. Castillo,4 Daniel P. Bezerra ,5 Tatjana K. Souza Lima,2
and Damião P. de Sousa
1
1
´
Laboratory of Pharmaceutical Chemistry, Universidade Federal da Paraıba, 58051-900 João Pessoa, PB, Brazil
2
´
Department of Cellular and Molecular Biology, Universidade Federal da Paraıba, 58051-900 João Pessoa, PB, Brazil
3Department of Pharmacy, Federal University of Sergipe, 49100-000 São Cristo´vão, SE, Brazil
4
´
´
´
Escuela de Ciencias Fısicas y Matematicas, Universidad de Las Americas, Quito, Ecuador
5Gonçalo Moniz Institute, Oswaldo Cruz Foundation (IGM-FIOCRUZ/BA), Salvador, Bahia 40296-710, Brazil
Correspondence should be addressed to Damião P. de Sousa; damiao_desousa@yahoo.com.br
Received 14 October 2018; Accepted 17 December 2018; Published 8 January 2019
Academic Editor: Andrea Trabocchi
´
´
Copyright © 2019 Flavio R. Nobrega et al. .is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Piplartine is an alkamide found in different Piper species and possesses several biological activities, including antiparasitic properties.
.us, the aim of the present study was to evaluate a series of 32 synthetic piplartine analogues against the Leishmania amazonensis
promastigote forms and establish the structure-activity relationship and 3D-QSAR of these compounds. .e antileishmanial effect of
the compounds was determined using the MTTmethod. Most compounds were found to be active against L. amazonensis. Among 32
assayed derivatives, compound (E)-(−)-bornyl 3-(3,4,5-trimethoxyphenyl)-acrylate exhibited the most potent antileishmanial activity
(IC50 � 0.007 0.008 μM, SI > 10), followed by benzyl 3,4,5-trimethoxybenzoate (IC50 � 0.025 0.009 μM, SI > 3.205) and (E)-furfuryl
3-(3,4,5-trimethoxyphenyl)-acrylate (IC50 � 0.029 0.007 μM, SI > 2.688). It was found that the rigid substituents contribute to
increasing antiparasitic activity against L. amazonensis promastigotes. .e presence of the unsaturated heterocyclic substituent in the
phenylpropanoid chemical structure (furfuryl group) resulted in a bioactive derivative. Molecular simplification of benzyl 3,4,5-
trimethoxybenzoate by omitting the spacer group contributed to the bioactivity of this compound. Furthermore, bornyl radical
appears to be important for antileishmanial activity, since (E)-(−)-bornyl 3-(3,4,5-trimethoxyphenyl)-acrylate exhibited the most
potent antileishmanial activity. .ese results show that some derivatives studied would be useful as prototype molecules for the
planning of new derivatives with profile of antileishmanial drugs.
weight loss, and progressive anemia, and if left untreated, it
can be fatal in 95% of cases [1–3].
1. Introduction
Leishmaniasis is a tropical disease prevalent in 98 countries
and has high impact on South America, Africa, and Asia
(especially India). .is illness is caused by protozoan par-
asites of the Leishmania genus and is transmitted to humans
by the bite of infected female phlebotomine sandflies. .ere
are at least twenty species of Leishmania that cause clinical
manifestations in humans, and they can occur in three
different forms: (i) visceral leishmaniasis, (ii) cutaneous
leishmaniasis, and (iii) mucocutaneous leishmaniasis. .e
most severe form is visceral leishmaniasis, characterized by
prolonged fever, hepatomegaly, splenomegaly, substantial
.e drugs currently available for treatment of leish-
maniasis are pentavalent antimonials glucantime, bis-
amidines (pentamidine and stilbamidine), miltefosine, and
amphotericin B. Unfortunately, all these drugs have several
limitations, such as low efficacy, toxicity to the liver and
heart, elevated cost, and parasite resistance. For these rea-
sons, new and more efficient therapeutic approaches are
needed for prevention and treatment of leishmaniasis [1].
Natural products provide a virtually unlimited source of
inspiration for new, powerful and selective drug leads. It is
estimated that 20,000 plant species have antiparasitic

2
Journal of Chemistry
activities. A wide variety of secondary metabolites have
antileishmanial activity, namely, alkaloids, triterpenes, ter-
penoids, chalcones, saponins, glycosides, acetogenins, and
ꢀavonoids [4–6].
O
O
3
O
O
N
2
Piperaceae family, also known as pepper family, com-
prises over 1,000 species distributed pantropically. e Piper
genus is the most diverse of the Piperaceae family and
produces a number of structural classes (lignans, phenyl-
propanoids in the form of amides, and other derivatives of
phenylpropanoids) with several biological activities, including
antileishmanial activity [7–9]. In a study [7], antileishmanial
activity of n-hexane, ethyl acetate, acetone, and methanol
extracts from Piper cubeta fruits and Piper retrofractum stem
bark was investigated against Leishmania donovani pro-
mastigotes. Among these substances, piplartine demonstrated
the highest leishmanicidal activity, with an IC50 value of
7.5 μM. Furthermore, this molecule was 3 times more potent
than positive control pentamidine (IC50 ꢀ 25 μM) [7, 8].
Piplartine (1) (Figure 1), also known as piperlongumine,
is an alkamide found in large quantities in long pepper (Piper
longum L.). In addition to antileishmanial and trypanocidal
activities, this alkamide has been also reported as having
other pharmacological activities, including antitumor, cy-
totoxic, antinociceptive, antiplatelet aggregation, and anti-
metastatic activities [10]. us, the goals of the present study
were to synthesize a series of 32 piplartine analogues and
evaluate the structure-activity relationship among these
derivatives against Leishmania amazonensis promastigotes.
O
Figure 1: Chemical structure of piplartine (1). Modi’cations in the
green and blue substructures of the molecule were investigated in
the present study.
L. amazonensis was evaluated using the MTT method.
e experiments were performed against promastigote
forms, that is the proliferative form found in the invertebrate
host, and the tests compounds were assayed at following
concentrations: 3.125, 6.25, 12.5, 25, 50, 100, 200, and
400 µg/ml. e antileishmanial activity was assessed as IC50
and expressed in µM. IC50 values of the piplartine analogues
are summarized in Table 1.
Most compounds tested against the promastigote forms
of L. amazonensis were bioactive (6–13, 15, 17, 18, 20, 21, 23,
25, 27, 28, and 36). Among the 32 assayed derivatives,
compound 20 (IC50 ꢀ 0.007 0.008 μM) exhibited the best
antileishmanial activity, followed by 36 (IC50 ꢀ 0.025
0.009 μM), 18 (IC50 ꢀ 0.029 0.007 μM), and 21 (IC50
ꢀ
0.042 0.011 μM). For compounds 31, 33, and 34, their IC50
cannot be determined. Amphotericin B, standard drug,
exhibited an IC50 value of 0.0015 μM.
2. Results and Discussion
2.1. Chemistry and Antileishmanial Activity of Compounds
5–36. For this study, a series of 32 analogues of 1 (Scheme 1)
were synthesized, preserving the (E)-3-(3,4,5-trimethox-
yphenyl)-acryloyl moiety on cinnamic esters and amides
and changing this moiety on benzoic esters by removing
the ethylene group between carbons 2 and 3. Side-chain
modi’cations were also evaluated changing the radical R to
methyl (5), ethyl (6), propyl (7), iPr (8), butyl (9), pentyl
(10), decyl (11), 2-methoxyethyl (12), 4-methoxybenzyl (13),
phenylethyl (14), 4-methylphenylethyl (15), carvacryl (16),
CHPh2 (17), furfuryl (18), eugenyl (19), (−)-bornyl (20)
and piperonyl (21) on cinnamic esters, and butyl (22),
N,N-diethyl (23), octyl (24), cyclohexyl (25) benzyl (26),
pyrrolidyl (27), 4-methylbenzyl (28), 4-methoxybenzyl (29),
4-ꢀuorobenzyl (30), 4-chlorobenzyl (31), 4-bromobenzyl
(32) 2,4-dimethoxybenzyl (33), and 3,4-dimethoxybenzyl
(34) on amides. e benzoic esters synthetized were 2-
methoxyethyl-3,4,5-trimethoxybenzoate (35) and benzyl
3,4,5-trimethoxybenzoate (36) [8–12].
e statistical parameters of the obtained 3D-QSAR
model as well as superposition of the 3D structures from
which it was derived are summarized in Figure 2. In this
’gure are also presented the general scažold of the com-
pounds under study, the plot of the predicted vs. observed
pIC50 values for the training set, and the results of LOO
cross-validation procedures. e obtained statistical pa-
rameters show that the developed model is accurate and
robust. is is supported by the high values of q2 obtained
during the LOO and LMO cross-validation experiments.
Regarding the inꢀuence of the steric and electrostatic factors
on bioactivity, it was found that both contribute almost
equally to explain the observed bioactivity.
In general, compounds containing O in the X position
(see Figure 2) were more active than those containing NH at
the same position. is ežect can be clearly observed for
compounds 17 and 23 whose only dižerence is the presence
of NH (17) vs. O (23) at position X, compound 23 being 6.5-
fold more active than 17. is can be due to electrostatic
’elds surrounding this position, as shown in Figure 3(a).
ese ’elds indicate that negatively charged substituents are
favored at position X, which is the case of the compounds
with O at this position. is observation can be generalized
to all the compounds in the dataset. A special case is
compound 29 that lacks the double bond spacer. In terms of
electrostatic potential, the O atom at the X position remains
close to the electronegative ’eld while the lack of the spacer
To synthesize the analogues, the following starting ma-
terials were used: 3,4,5-trimethoxycinnamic acid (2); 3,4,5-
trimethoxybenzoic acid (3); 3′,4′,5′-trimethoxyacetophenone
(4); and ROH to esters and primary or secondary amines to
amides. Most products were obtained in high yields, and the
reactions (a, b, c, d, e, and f) were performed in one step
(Scheme 1).
e antileishmanial potential of all piplartine ana-
logues (5–36) against the strains IFLA/BR/1967/PH8

Journal of Chemistry
3
O
O
Cinnamic esters (R)
3
O
N
2
(5)a
(16)c
(11)b
O
(1)
Cinnamic esters
O
O
(12)a
O
O
(6)a
(7)a
(8)a
(20)d
O
OH
(2)
a, b, c, d
3
e
O
O
O
O
O
R
(17)b
(13)b
O
2
O
O
O
(21)d
e
e
(14)c
O
O
(9)a
Amides
(18)d
(19)d
O
NR
H
O
(15)b
(10)b
O
O
N
O
O
O
O
(23)
O
O
O
N
Amides (R)
R1
R2
(27)
O
(22)
Benzoic esters
O
R3
R1
R2
R3
O
O
O
H
H
H
H
H
H
H
CH3
OCH3
F
(28)
(29)
(30)
(31)
(32)
3 (33)
3 (34)
O
a
(24)
O
OH
H
O
(35)
(3)
O
H
O
O
O
H
Cl
(25)
O
H
Br
O
O
O
O
O
f
OCH3
H
OCH
O
OCH3 OCH
(4)
(26)
(36)
O
Scheme 1: Synthesis method: (a) ROH, H2SO4, reflux; (b) Et3N, RBr or RCl, acetone, reflux; (c) SOCl2, reflux (d) DCC, DMAP, CH2Cl2, r.t.;
(e) RNH2, (CH3CH2)2NH or pyrrolidine, BOP, Et3N, CH2Cl2, DMF; (f) ROH, CuBr, pyridine, chlorobenzene, BF3·O(Et)2.
Table 1: Antileishmanial activity of compounds 5–36 against the strains IFLA/BR/1967/PH8 of Leishmania (L.) amazonensis.
Cinnamic esters
Amides
Benzoic esters
IC50
(μM)
IC50
(μM)
IC50
(μM)
SI
SI
SI
5
0.641 0.052
0.257 0.041
0.09 0.032
0.194 0.04
0.149 0.025
0.133 0.014
0.076 0.009
0.076 0.022
0.101 0.031
0.975 0.241
0.125 0.035
0.513 0.065
0.083 0.016
0.029 0.007
>0.154
22
23
24
25
26
27
28
29
30
31
32
0.577 0.080
0.091 0.015
0.564 0.061
0.117 0.027
0.734 0.252
0.353 0.080
0.109 0.010
0.662 0.188
1.137 0.252
>0.148
35
36
>0.656 0.05
>0.141
6
>0.364
>0.126
>0.02 0.0
>3.205∗
7
>0.992
>0.126
8
>0.459
>0.678
9
>0.570
>0.104
10
11
12
13
14
15
16
17
18
19
>0.608
>0.243
>0.868
>0.063
>1.106
>0.105
>0.688
>0.062
>0.069
>1.108
>0.062
>0.561
0.486 0.043
>0.127
>0.142
33
34
>1.033
>0.062
>0.062
>0.748
>1.033
>2.688∗
>1.041
>0.062
20
21
0.007 0.008
0.042 0.011
>10∗
Positive control: amphotericin B
0.0015
>1.582
-
∗Cytotoxic effects on MRC5 (Medical Research Council cell strain 5—fibroblast derived from lung tissue); positive control: doxorubicin (IC50 0.00287
(0.0017–0.0050) μM). SI: selectivity index.
places the trimethoxyphenyl group in a region where an
electronegative MIF is present. .us, compounds without
the spacer will have higher bioactivity compared to those
containing the double bond spacer. Finally, the electro-
positive potential observed around position R correlates with
the favorable influence of aromatic substitutions at this
position.
We also analyzed the influence of the steric molecular
interaction fields. .ese fields are summarized in
Figure 3(b), and it can be seen that the substituents attached

4
Journal of Chemistry
O
CH3
O
R
5.5
5
X
H3C
O
O
4.5
4
H3C
3.5
3
2.5
2
Parameter
Value
8
0.98
0.81
0.72
Num. PCs
R2
2
3
4
5
6
LOO q2
Observed pIC50
LMO q2
Training
LOO cross validation
Steric
51%
contriburion
Electrostatic
contriburion
49%
Figure 2: Summary of the QSAR modeling procedure.
to the R position are surrounded by a steric favorable region
beyond which a steric unfavorable potential is located. In
consequence, bulky substituents at R are favorable for
bioactivity. Para-substituted phenyl rings are tolerated,
whereas meta- and ortho-substituted rings decrease bio-
activity. .e steric effect for different groups at R is depicted
in Figure 3(c) for compounds 27, 10, and 21.
From Figure 3, it can be seen that compound 27 lies
entirely within the steric favorable region, while compounds
10 and 21 bear substituents which protrude into the un-
favorable steric region. In addition to 27, the other two most
active compounds (29 and 25) are also substituted at R with
groups completely falling within the steric favorable region.
On the other hand, large substituents such as those present
in compounds 9 and 26 reach the steric unfavorable region,
which reduce their bioactivity. To avoid falling within the
steric unfavorable region, if rings are used at R, no more than
one methyl group should link them to X. Furthermore,
nonbulky groups at R such as aliphatic substitutions lead to
nonoptimal bioactivity because of their inability to com-
pletely occupy the steric favored region. .e structure-
activity relationship (SAR) derived from these analyses is
summarized in Figure 3(c).
paramount importance to improve disease treatment and
control. Several medicinal plant secondary metabolites have
shown interesting antileishmanial activities, including al-
kaloids, steroids, terpenoids, phenolic acids, and phenyl-
propanoids [13–15].
.e chloroform extract of Valeriana wallichii rhizomes
was investigated to identify the structures responsible for
its antileishmanial activity. A bioassay-guided fraction-
ation was undertaken and resulted in (−)-bornyl caffeate,
α-kessyl alcohol, two cinnamic acid derivatives, and four
valtrates. All these compounds exhibited antileishmanial
activity against L. major promastigotes, with IC50 values
varying between 0.8 and 48.8 µM. Furthermore, some
compounds were more potent than the positive control
miltefosine (IC50 � 36.2 µM) [16].
A phytochemical study guided by the antileishmanial
activity of the crude extract from the stem bark of Tecoma
mollis yielded seven phenylpropanoid glycosides. .ese
secondary metabolites were tested against the promastigote
forms of L. donovani, and pentamidine and amphotericin B
were used as positive controls. .e compounds acteoside,
luteoside A, and luteoside B shared the highest IC50 values:
30.08, 15.07, and 6.71 µg/ml, respectively. .e IC50 values
of the positive controls were 0.34 µg/ml to amphotericin B
and 1.31 µg/ml to pentamidine. .e obtained results also
revealed that ortho-dihydroxy phenyl group (catechol
moiety) was important to the antileishmanial activity [17].
Piplartine (1), a plant metabolite, is classified as an
alkamide and contains in its structure a 3,4,5-trimethox-
ycinnamic acid (2) moiety and a δ-lactam ring. Both 1 and 2,
used as starting materials to synthesize some piplartine
analogues, were tested against IFLA/BR/1967/PH8 L.
amazonensis. .e IC50 values found were 179 0.05 μg/ml
2.2. Anti-Leishmania Activity of Compounds 5–36.
Pentavalent antimonials, in use for more than six decades,
are still the first-line of treatment for visceral leishmaniasis,
also known as kala-azar black fever. However, this thera-
peutic option presents several limitations, including side
effects, the need for daily injections, and drug resistance, and
requires a long treatment regimen. .us, developing and
testing of new compounds with leishmanicidal activity is of

Journal of Chemistry
5
17
10
23
29
21
27
(a)
(b)
No linker
preferred
over linked
Bulky substitutions are preferred
Rings unsubstituted or para-substituted
with small groups are preferred
Ortho- and meta-substitutions on rings
decrease bioactivity
Rings substitutions are preferred over
aliphatic groups
CH3
O
O
R
X
O preferred
over NH
H3C
O
O
H3C
(c)
Figure 3: Summary of the 3D-QSAR model. (a) Influence of the electrostatic properties of the compounds on bioactivity. (b) Influence of
the steric factor on bioactivity. (c) Summary of the SAR derived from the 3D-QSAR analyses.
(0.564 μM) for 1 and 145 μg/ml (0.608 μM) for 2 [18, 19]. In
addition, piplartine (1) was tested against promastigote
forms of L. infantum and L. amazonensis and reached IC50
values of 7.9 and 3.3 μM, respectively (positive control:
amphotericin B, IC50 � 0.2 μM). .ese results demonstrate
the potential antileishmanial activity of piplartine and

6
Journal of Chemistry
3,4,5-trimethoxycinnamic acid, justifying the choice of these
compounds as starting materials for the planning of new
derivatives with higher antileishmanial activities [20].
important for the antileishmanial activity. .e most active
compounds (IC50 values varying between 15.6 and 50.2 μM)
were all esters of borneol, namely caffeic acid (−)-bornyl
ester, caffeic acid isobornyl ester, cinnamic acid (−)-bornyl
ester, and phenyl propanoic acid (−)-bornyl ester [1].
Esters 14 (IC50 � 0.975 0.241 μM), 16 (IC50 � 0.513
0.065 μM), and 19 (IC50 > 1.041 μM), containing aromatic
groups, decreased the antileishmanial activity when compared
with some amides (28, IC50 � 0.109 0.010 µM). However,
the esters 17 (IC50 � 0.083 0.016 µM) and 20 (IC50 � 0.007
0.008 µM) gave betters results, demonstrating that the bi-
ological effect of these changes is not easily predictable [9, 12].
.e introduction of methyl groups contributes to increase
lipophilicity. It was observed that the methyl group of the
ester 15 (IC50 � 0.125 0.035 μM) in the para-position on the
phenylethyl moiety was the determinant change to improve
the antileishmanial activity when compared with ester 14
(IC50 � 0.975 0.24 μM) lacking this group [9, 12].
2.3. Structure-Activity Relationship (SAR). .e assayed
compounds in this study were divided into three groups:
analogues 5–21 (cinnamic esters), analogues 22–34 (am-
ides), and analogues 35–36 (benzoic esters). Previous studies
have investigated different compounds containing the 3,4,5-
trimethoxyphenyl ring in order to understand its contri-
bution to the biological activity [21]. For example, in a study
performed by Dong-Jun et al., the authors showed that 3,4,5-
trimethoxyphenyl moiety at the N-1 position of the β-lac-
tam-azide derivatives was crucial for the antiproliferative
activity against MGC-803, MCF-7, and A549 cancer cell
lines [22]. Furthermore, anticancer compounds such as
colchicine, steganacin, phenstatin, podophyllotoxin, com-
bretastatin, and other synthetic analogues of these com-
pounds share a 3,4,5-trimethoxyphenyl moiety as a common
structural feature [23]. .us, herein we preserved the (E)-3-
(3,4,5-trimethoxyphenyl)-acryloyl moiety on cinnamic es-
ters and amides and changed this moiety on benzoic esters
(without the spacer group in the chemical structure).
Analysis on the structure-activity relationship revealed
that, in general, an acrylate moiety on cinnamic esters ap-
pears be more important for improving antileishmanial
activity when compared to the acrylamide moiety on amides.
.is effect can be clearly observed comparing compounds 13
(IC50 � 0.101 0.031 μM) and 29 (IC50 � 0.662 0.188 μM),
whose only difference is the replacement of the OH group 13
by NH 29 at this position, being compound 13 6.6-fold more
active than 29.
.e chemical structure of ester 36 (IC50 � 0.025
0.009 µM) differs from other compounds by a molecular
simplification of the (E)-3-(3,4,5-trimethoxyphenyl)-acryloyl
moiety with removal of the ethylene group (carbons 2 and 3)
and another simplification by changing from phenylethyl
radical to benzyl. .is structural modification contributed to
increase the antileishmanial activity, since compound 36
(IC50 � 0.02 0.01 µM) was more active than the ester 14
(IC50 � 0.975 0.241). In addition, the comparison can also
be made with amide 26 (IC50 � 0.734 0.252 µM), a struc-
tural analogue containing the same benzyl radical attached
to the (E)-3-(3,4,5-trimethoxyphenyl)-acrylamide moiety
[12, 19, 24, 25]. Similarly, ester 36 showed more potent
antileishmanial activity than amide 26.
.e introduction of methylene groups in general in-
creases the lipophilicity, providing lower IC50 values. Table 1
shows that the antileishmanial activity of esters 5 (IC50
0.641 0.052 µM), 6 (IC50 � 0.257 0.041 µM), and 7 (IC50
�
�
0.09 0.032 µM) was inversely proportional to the length of
the carbon chain attached to (E)-3-(3,4,5-trimethox-
yphenyl)-acrylate. For esters 9 (IC50 � 0.149 0.025 µM) and
10 (IC50 � 0.133 0.014 µM), the IC50 values have increased
compared to 7, demonstrating that from four carbons the
antileishmanial activity begins to decrease, with exception
of ester 11 (IC50 � 0.076 0.009 µM) that even with decyl
radical provided IC50 near the ester 7 (IC50 � 0.09
0.032 µM) [12, 26].
Branches on side-chains R of esters 8 (IC50 � 0.194
0.04 µM) and 17 (IC50 � 0.083 0.016 µM) did not provide
better results compared to esters of saturated chains.
However, the aromatic rings of ester 17 were determinant
to improve the antileishmanial activity when compared to
ester 8. .e exchange of side-chain R, from butyl in ester 9
(IC50 � 0.149 0.025 µM) to methoxyethyl in ester 12
(IC50 � 0.076 0.022 µM), reduced the IC50 value. On ester
18 (IC50 � 0.029 0.007 µM), the furfuryl radical reduced the
molecular flexibility and, compared to ester 12, improved
the antileishmanial activity. A molecular simplification
approach was tried on ester 35 (IC50 � 0.656 0.049 µM)
compared to ester 12, but did not provide better results.
However, the presence of oxygen on the methoxyethyl
group on side-chain R of ester 36 (IC50 � 0.025 0.009 µM)
contributed to increase the antileishmanial activity
[12, 24, 25].
.e ester 20 (IC50 � 0.007 0.008 µM) containing the
monoterpenic substructure bornyl attached on the (E)-3-
(3,4,5-trimethoxyphenyl)-acryloyl moiety yielded the best
IC50 value, near the positive control amphotericin B
(IC50 � 0.0015 µM). .is result demonstrates the importance
of this radical on antileishmanial activity and can be a
prototype for the planning of new derivatives with higher
antileishmanial activities. In an earlier study, a series of
compounds based on caffeic acid bornyl ester was synthe-
sized and tested in vitro against L. major, L. donovani
promastigotes, and L. major amastigotes to investigate the
SAR. .e results showed that the bornyl moiety was
.e increase of stiffness on side-chain R of ester 21
(IC50 � 0.042 0.011 µM), containing two atoms of oxygen
of the dioxolane group fused on benzyl, increased the
antileishmanial activity when compared to ester 13 (IC50
0.101 0.031 µM) with 4-methoxylbenzyl radical. Compar-
ing the saturated side-chain R on amides 22 (IC50
�
�
0.577 0.080 µM) and 24 (IC50 � 0.564 0.061 µM) with the
six-membered ring on amide 25 (IC50 � 0.117 0.027 µM),
it was observed that the replacement of saturated side-
chains R by rigid groups increased the antileishmanial
activity also on amides [12, 24, 25]. Compound 23

Journal of Chemistry
7
(IC50 � 0.091 0.015 µg/ml) with a tertiary amide provides
better result compared to 27 (IC50 � 0.353 0.080 µM).
Replacing the methyl groups attached to the aromatic
ring with the methoxy group dramatically decreased the
antileishmanial activity in series of amide analogues, and this
reduction was dependent on the number of substituted
groups. For example, compound 28 (R3 � CH3) exhibited an
IC50 value of 0.109 0.010 μM, whereas compounds 29
(R3 � OCH3), 33 (R1 and R3 � OCH3), and 34 (R1 and
R3 � OCH3) revealed IC50 values of 0.662 0.188, >1.033
and > 1.033, respectively. Similarly, the addition of halogens
atoms (Br, F or Cl) to the aromatic ring of amide series
also reduced the antileishmanial activity, since compounds
3.89 (s, 9H), 3.77–3.66 (m, 2H), 3.40 (s, 3H); 13C NMR
(50 MHz, CDCl3): δC 166.0, 152.7, 142.1, 124.9, 106.8, 70.4,
−1
64.0, 60.7, 58.8, 56.0; IR νmax (KBr, cm ): 2941, 2839, 1715,
1589, 1504, 864; microOTOF: m/z [M + Na]+ 293.3107
(calcd. for C13H18O6: 293.1103).
3.4. General Procedure for Preparation of Compound 13.
In a 50 ml round-bottom flask, 0.42 mmol (0.1 g) of 3,4,5-
trimethoxycinnamic acid were dissolved in 5.0 ml of ace-
tone. .en, 1.68 mmol (0.22 ml) of Et3N and 0.43 mmol of
alkyl halide were added in the same solution, and the re-
action mixture was refluxed for 48 h. .e acetone was re-
moved under reduced pressure, and the content of the
reactions was diluted in 10 ml of ethyl acetate and 10 ml of
water. .e products were extracted thrice with 10 ml of ethyl
acetate. Afterwards, organic phases were successively
washed with 5% (w/v) NaHCO3 and water, dried over an-
hydrous Na2SO4, and filtered. .e product was purified via a
silica gel 60 column chromatography (mobile phase: hexane-
ethyl acetate (1 :1)) [30].
30 (R3 � Br, IC50 �1.137 0.252 μM), 31 (R3 � Br, IC50
>
1.108 μM), and 32 (R3 � Br, IC50 � 0.486 0.043 μM) showed
weak or no activity.
3. Experimental
1
3.1. Chemical Characterization and Reagents. .e H and
13C NMR and IR spectral data were recorded, and their
signals were compared with published data. For compounds
18, 19, 21, 32, 33, 34, and 35, the spectrometric methods LS-
MALDI TOF/TOF or ESI-TOF were used to confirm the
chemical structures. .e preparation and structural char-
acterization data of compounds 5–9, 10–12, 14–17, 22, 23,
26, 28, 29, and 31 were published in a previous article [21].
All used reagents were of reagent grade and purchased
from Sigma Aldrich. .e following equipments were used:
Irprestige-21 Shimadzu Fourier Transform spectropho-
tometer (Shimadzu, Kyoto, Japan) for IR, and Varian
Mercury spectrometer (Palo Alto, CA, USA) 200 MHz for
1H NMR and 50 MHz for 13C NMR. .e standard for
chemical shifts was the CDCl3 or TMS solvent peak. For HR-
MS, the LS-MALDI TOF/TOF apparatus was used equipped
with a high-performance laser (λ � 355 nm) and a reflector
operated by the software FlexControl 2.4 (Bruker Daltonics
G, bsH, Bremen, Germany), and microOTOF operated by
software Bruker Compass DataAnalysis 4.0 (Bruker Dal-
tonics G, bsH, Bremen, Germany). Melting points were
measured on an equipment Tecnal PFM-II, 220 V. To
measure the optical activity of 20 and of starting material
(−)-borneol a digital polarimeter P-2000 (Jasco) was used
after standardization with 98% anhydrous methanol.
3.4.1. (E)-4-Methoxybenzyl 3-(3,4,5-trimethoxyphenyl)-
acrylate (13). Yield 77.4%; white solid; MM: 358.14 g/mol;
1
m.p.: 110–111°C; H NMR (200 MHz, CDCl3): δH 7.61 (d,
J � 16 Hz, 1H), 7.34 (d, J � 2.0 Hz, 2H), 6.90 (d, J � 2.0 Hz,
2H), 6.73 (s, 2H), 6.37 (d, J � 16 Hz, 1H), 5.17 (s, 2H), 3.85 (s,
9H), 3.80 (s, 3H); 13C NMR (50 MHz, CDCl3): δC 166.8;
159.7; 153.5; 145.0; 140.2; 130.2; 129.9; 128.2; 117.3; 114.0;
−1
105.3; 66.3; 61.0; 56.2; 55.3; IR νmax (KBr, cm ): 2999, 2939,
2837, 1709, 1636, 1034, 1005, 1584, 1506, 825, 609; LS-
MALDI TOF/TOF m/z [M]+ 358.1353 (calcd. for
C20H22O6: 358.1416).
3.5. General Procedure for Preparation of Compounds 18–21.
To a solution of 0.1 g (0.42 mmol) of 3,4,5-trimethoxycin-
namic acid dissolved in 6.3 ml of CH2Cl2, 0.54 mmol of ROH
and 0.015 g of DMAP were added. After 5 min of stirring,
0.1 g (0.54 mmol) of DCC was also added. Stirring was
continued overnight at room temperature. .e solvent was
removed under vacuum, and the remaining content was
diluted in 10 ml of ethyl acetate and 10 ml of water. .e
product was extracted with 10 ml ethyl acetate (three times),
and the organic phases were successively washed with 5%
(w/v) NaHCO3 and water, dried over anhydrous Na2SO4,
and filtered. After removing ethyl acetate under vacuum, the
products were purified on a silica gel 60 column chroma-
tography (mobile phase: hexane-ethyl acetate).
3.2. General Procedure for Preparation of Compound 35.
To a solution of 0.25 g of carboxylic acid in 250 ml of ROH
under stirring, 0.5 ml of 96% (v/v) H2SO4 were added and
solution was refluxed for 3 h. After removing of half ROH
under reduced pressure, the solution was diluted in 10 ml of
water, and product was extracted with ethyl acetate. .en,
the product was successively washed with 5% (w/v) NaHCO3
and water and dried with anhydrous Na2SO4. Finally, the
solvent was evaporated to yield the pure compound [27–29].
3.5.1. (E)-Furfuryl 3-(3,4,5-trimethoxyphenyl)-acrylate (18).
Yield 79%; brown oil; MM: 318.11 g/mol; 1H NMR
(200 MHz, CDCl3): δH 7.58 (d, J � 16.0 Hz, 1H), 7.43–7.37
(m, 1H), 6.69 (s, 2H), 6.45–6.16 (m, 3H), 5.15 (s, 2H), 3.81 (s,
6H), 3.83 (s, 3H); 13C NMR (50 MHz, CDCl3): δC 166.4,
153.3, 149.5, 145.3, 143.3, 140.0, 129.7, 116.7, 110.7, 110.6,
−1
3.3. 2-Methoxyethyl 3,4,5-trimethoxybenzoate (35). Yield
105.5, 60.8, 58.0, 56.0; IR νmax (KBr, cm ): 2997, 2939,
1
91%; brown solid; MM: 270.11 g/mol; m.p.: 32–35°C; H
2839, 1713, 1634, 1003, 1582, 1504, 827; LS-MALDI TOF/
NMR (200 MHz, CDCl3): δH 7.31 (s, 2H), 4.51–4.40 (m, 2H),
TOF m/z [M]+ 318.1103 (calcd. for C17H18O6: 318.1103).

8
Journal of Chemistry
3.5.2. (E)-Eugenyl 3-(3,4,5-trimethoxyphenyl)-acrylate (19).
Yield 86%; white solid; MM: 384.16 g/mol; m.p.: 170–172°C;
1H NMR (200 MHz, CDCl3): δH 7.77 (d, J � 16.0 Hz, 1H),
7.00 (d, 1H, J � 6.0 Hz), 6.83–6.73 (m, 4H), 6.58 (d,
J � 16.0 Hz, 1H), 6.08–5.83 (m, 1H), 5.18–5.02 (m, 2H), 3.88
(s, 1H), 3.83 (s, 9H), 3.34 (q, J � 6.0 Hz, 2H), 1.62–1.44 (m,
2H), 1.31–1.22 (m, 10H), 0.84 (t, J � 6.0 Hz, 3H); 13C NMR
(50 MHz, CDCl3): δC 166.0, 153.4, 147.6, 140.7, 139.5, 130.6,
120.3, 104.7, 61.0, 56.1, 39.9, 31.9, 29.7, 29.4, 27.1, 22.7, 14.2;
−1
IR νmax (KBr, cm ): 3290, 3003, 2955, 2924, 2853, 1655,
(s, 3H), 3.86 (s, 6H), 3.80 (s, 3H), 3.37 (d, J � 6.0 Hz, 2H); 13
C
1622, 1580, 1504, 827 [32].
NMR (50 MHz, CDCl3): δC 165.0, 153.4, 151.0, 146.4, 140.2,
139.0, 137.9, 137.0, 129.7, 122.6, 120.6, 116.3, 116.1, 112.7,
3.6.2.
(E)-N-Cyclohexyl-3-(3,4,5-trimethoxyphenyl)-acryl-
−1
105.3, 60.9, 56.0, 55.8, 40.1; IR νmax (KBr, cm ): 3067, 2968,
amide (25). Yield 59%; white solid; MM: 319.18 g/mol; m.p.:
320–321°C; 1H NMR (200 MHz, CDCl3): δH 7.49 (d,
J � 16.0 Hz, 1H), 6.68 (s, 2H), 6.34 (d, J � 16.0 Hz, 1H), 5.90
(s, 1H), 3.83 (s, 3H), 3.81 (s, 6H), 2.01–1.05 (m, 11H); 13C
NMR (50 MHz, CDCl3): δC 165.0, 153.4, 140.5, 139.3, 130.6,
120.7, 104.8, 61.0, 56.1, 48.4, 33.3, 25.6, 24.9; IR νmax (KBr,
2939, 2839, 1722, 1630, 1034, 1009, 1582, 1508, 824; LS-
MALDI TOF/TOF m/z [M]+ 384.1575 (calcd. for C22H24O6:
384.1573).
3.5.3. (E)-(−)-Bornyl 3-(3,4,5-trimethoxyphenyl)-acrylate
(20). Yield 87%; white solid; MM: 374.21 g/mol; m.p.:
100–102°C; 1H NMR (200 MHz, CDCl3): δH 7.54 (d,
J � 16.0 Hz, 1H), 6.73 (s, 2H), 6.34 (d, J � 16.0 Hz, 1H), 4.98
(d, J � 8.0 Hz, 1H), 3.85 (s, 9H), 2.47–2.28 (m, 1H), 2.05–1.96
(m, 1H), 1.67 (d, J � 4.0 Hz, 1H), 1.37–1.15 (m, 3H), 1.07–
0.95 (m, 1H), 0.90 (s, 3H), 0.84 (s, 6H); 13C NMR (50 MHz,
CDCl3): δC 167.2, 153.4, 144.2, 139.9, 130.0, 118.0, 105.1,
79.9, 60.9, 56.1, 48.9, 47.9, 44.9, 36.9, 28.1, 27.3, 19.7, 18.9,
−1
cm ): 3287, 3003, 2928, 2853, 1663, 1612, 1582, 1508, 822
[33].
3.6.3. (E)-1-(Pyrrolidine-1-yl)-3-(3,4,5-trimethoxyphenyl)-
prop-2-en-1-one (27). Yield 72%; white solid; MM:
291.15 g/mol; m.p.: 233–235°C; 1H NMR (200 MHz, CDCl3):
δH 7.59 (d, J � 16.0 Hz, 1H), 6.72 (s, 2H), 6.59 (d, J � 16,0 Hz,
1H), 3.86 (s, 6H), 3.84 (s, 3H), 3.68–3.51 (m, 4H), 2.07–1.79
(m, 4H), 13C NMR (50 MHz, CDCl3): δC 164.8, 153.4, 142.0,
139.5, 131.0, 118.0, 105.1, 61.0, 56.2, 46.8, 46.2, 26.2, 24.4; IR
−1
13.6; IR νmax (KBr, cm ): 2953, 2839, 1697, 1634, 1026,
25°C
1007, 1582, 1504, 831; [ ∝ ]D � −5.5° (c 0.001, MeOH),
RSD: 4.3% [31].
−1
νmax (KBr, cm ): 3049, 2963, 2939, 2839, 1645, 1605, 1580,
1504, 820 [29].
3.5.4.
(E)-Piperonyl-3-(3,4,5-trimethoxyphenyl)-acrylate
(21). Yield 30%; white solid; MM: 372.12 g/mol; m.p.:
123–126°C; 1H NMR (200 MHz, CDCl3): δH 7.61 (d,
J � 16.0 Hz, 1H), 6.93–6.76 (m, 3H), 6.74 (s, 2H), 6.36 (d,
3.6.4. (E)-N-(4-Fluorobenzyl)-3-(3,4,5-trimethoxyphenyl)-
acrylamide (30). Yield 87%; white solid; MM: 345.14 g/
mol; m.p.: 195–196°C; 1H NMR (200 MHz, CDCl3): δH
7.57 (d, J � 16.0 Hz, 1H), 7.30–7.19 (m, 2H), 6.99 (t,
J � 8.0 Hz, 2H), 6.69 (s, 2H), 6.39 (d, J � 14.0 Hz, 1H), 4.50 (d,
J � 6.0 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 6H); 13C NMR (50 MHz,
CDCl3): δC 166.0, 164.7, 159.8, 153.4, 141.5, 139.6, 134.1,
130.4, 129.6, 129.5, 119.8, 115.8, 115.4, 105.0, 61.0, 56.1, 43.2;
J � 16.0 Hz, 1H), 5,95 (s, 2H), 5,12 (s, 2H), 3,86 (s, 9H); 13
C
NMR (50 MHz, CDCl3): δC 166.8, 153.4, 147.9, 147.7, 145.1,
140.1, 129.9, 129.8, 122.4, 117.2, 109.1, 108.3, 105.2, 101.2,
−1
66.4, 61.0, 56,2; IR νmax (KBr, cm ): 3067, 2926, 2849, 1703,
1630, 1038, 1005, 1582, 1502, 854; LS-MALDI TOF/TOF m/z
[M + H]+ 373.1288 (calcd. for C20H20O7 + H: 373.1268).
−1
IR νmax (KBr, cm ): 3291, 3012, 2959, 2932, 2839, 1651,
1616, 1580, 1508, 829 [34].
3.6. General Procedure for Preparation of Amides. To a so-
lution of 0.1 g of 3,4,5-trimethoxycinnamic in 0.84 ml of
DMF in a round-bottom flask, 0.06 ml (0.42 mmol) of tri-
methylamine was added. .e solution was cooled in an ice
water bath and 0.42 mmol of amine were added, followed by
a solution of 0.42 mmol of BOP (0.84 ml) in CH2Cl2. After
stirring for 30 min, the reaction continued at room tem-
perature for 3 h. CH2Cl2 was removed under vacuum, and
the remaining solution was diluted in 10 ml of ethyl acetate
and 10 ml of water. .e product was extracted with 10 ml of
ethyl acetate (three times), and the organic phases were
washed successively with 1 N HCl, water, 1 M NaHCO3, and
water, dried over anhydrous Na2SO4, filtered, and evapo-
rated. .e product was purified on a silica gel 60 column
chromatography (mobile phase: hexane-ethyl acetate (1 :1)).
3.6.5. (E)-N-(4-Bromobenzyl)-3-(3,4,5-trimethoxyphenyl)-
acrylamide (32). Yield 99.8%; white solid; MM: 405.6 g/
mol; m.p.: 360–363°C; 1H NMR (200 MHz, CDCl3): δH
7.57 (d, J � 16.0 Hz, 1H), 7.43 (d, J � 8.00 Hz, 2H), 7.17 (d,
J � 8.00 Hz, 2H), 6.70 (s, 2H), 6.45–6.15 (m, 2H), 4.49 (d,
J � 6.0 Hz, 2H), 3.86 (s, 3H), 3.84 (s, 6H); 13C NMR (50 MHz,
CDCl3): δC 166.0, 153.4, 141.6, 139.7, 137.4, 131.8, 13.,4,
129.5, 121.4, 119.7, 105.0, 61.0, 56.2, 43.2; IR νmax (KBr,
−1
cm ): 3292, 3044, 2957, 2932, 2835, 1651, 1614, 1582, 1506,
824; LS-MALDI TOF/TOF m/z [M]+ 405.0558 (calcd. for
C19H20BrNO4: 405.0576).
3.6.6.
(E)-N-(2,4-Dimethoxybenzyl)-3-(3,4,5-trimethox-
yphenyl)-acrylamide (33). Yield 23%; white solid; MM:
3.6.1.
(E)-N-Octyl-3-(3,4,5-trimethoxyphenyl)-acrylamide
387.17 g/mol; m.p.: 235–238°C; 1H NMR (200 MHz, CDCl3):
(24). Yield 62.4%; white solid; MM: 349.23 g/mol; m.p.:
133–134°C; 1H NMR (200 MHz, CDCl3): δH 7.50 (d,
J � 16.0 Hz, 1H), 6.69 (s, 2H), 6.33 (d, J � 16.0 Hz, 1H), 6.00
δ
H 7.50 (d, J � 16,0 Hz, 1H), 7.20 (d, J � 8,0 Hz, 1H), 6.67 (s,
2H), 6.42 (s, 1H), 6.41–6.23 (m, 3H), 4.46 (d, J � 6.0 Hz, 2H),
3.83 (s, 3H), 3.82 (s, 6H), 3.80 (s, 3H), 3,76 (s, 3H); 13C NMR

Journal of Chemistry
9
(50 MHz, CDCl3): δC 165.6, 160.6, 158.6, 153.4, 140.8, 139.4,
(1 × 106 cells) and treated with different concentrations of
piplartine derivatives (5–36) (400, 200, 100, 50, 25, 12.5,
6.25, and 3.125 μg/ml) diluted in Schneider’s medium
(Sigma) supplemented with 10% FBS. .e plates were in-
cubated for 72 hours at 34°C. After the incubation period,
10 μl of a 5 mg/ml MTT solution in PBS was added to each
well. After treatment, the plate was incubated for additional
4 hours, and at the end 50 μl of a solution of 10% sodium
dodecyl sulphate (Sigma Chemical-st. Louis, MO, USA) was
added and left overnight to allow dissolution of the crystals
of formazan [37]. Finally, the absorbance reading was
performed using a spectrophotometer (Biotek model
Elx800) at 540 nm. .e Schneider medium was used as
negative control, and amphotericin B, a reference drug for
treatment of visceral leishmaniasis, was used as positive
control [37]. .e IC50 values were defined as the concen-
tration of each compound that reduced the absorbance of
treated cells by 50% when compared with the cell control. All
assays were performed in triplicate.
130.6, 130.6, 120.4, 118.8, 104.9, 104.0, 98.6, 61.0, 56.1, 55.4,
−1
39.1; IR νmax (KBr, cm ): 3283, 3040, 3001, 2932, 2837,
1651, 1605, 1585, 1506, 820; LS-MALDI TOF/TOF m/z [M]+
387.1674 (calcd. for C21H25NO6: 387.1682).
3.6.7.
(E)-N-(3,4-Dimethoxybenzyl)-3-(3,4,5-trimethox-
yphenyl)-acrylamide (34). Yield 75%; white solid; MM:
387.17 g/mol; m.p.: 170–173°C; 1H NMR (200 MHz, CDCl3):
δH 7.48 (d, J � 16.0 Hz, 1H); 6.80–6.64 (m, 3H); 6.61 (s, 2H),
6.41 (d, J � 16.0 Hz, 1H), 4.38 (d, J � 6.0 Hz, 2H), 3.76 (s, 3H),
3.73 (s, 3H), 3.71 (s, 9H); 13C NMR (50 MHz, CDCl3): δC
165.9, 153.2, 148.9, 148.2, 140.8, 139.3, 130.7, 130.3, 120.1,
−1
120.0, 111.0, 104.8, 60.8, 55.9, 43.5; IR νmax (KBr, cm ):
3370, 3061, 2961, 2926, 2853, 1670, 1585, 1502, 827; LS-
MALDI TOF/TOF m/z [M + Na]+ 410.1579 (calcd. for
C21H25NO6 + Na: 410.1580).
3.7. General Procedure for Preparation of Compound 36.
To a 5 ml round-bottom flask with 0.1 g (0.47 mmol) of
3′,4′,5′-trimethoxyacetophenone in 0.1 ml (0.94 mmol) of
benzylic alcohol, the solution of 0.007 g of CuBr
(0.047 mmol) in 0.07 ml (0.94 mmol) of pyridine was added,
and then the solution of 0.025 mmol BF3·Et2O in 0.5 ml of
chlorobenzene was added dropwise. .e mixture was then
stirred at 130°C open air for 10 h. After cooling at room
temperature, chlorobenzene was removed under reduced
pressure. .e remaining content was diluted in 10 ml of ethyl
acetate and 10 ml of water. .e product was extracted with
10 ml of ethyl acetate (three times), and the organic phase
was successively washed with 5% (w/v) NaHCO3 and water,
dried over anhydrous Na2SO4, and filtered. After the re-
moval of ethyl acetate under vacuum, the product was
purified on a silica gel 60-column chromatography (mobile
phase: hexane-ethyl acetate) [35].
3.10. Alamar Blue Assay. To assess the cytotoxicity against
normal cells, the cell line MRC-5 (human lung fibroblast)
was obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA) and was cultured as recom-
mended by the ATCC. .e cell culture was tested for my-
coplasma using Mycoplasma Stain Kit (Sigma-Aldrich) to
validate the use of cells free from contamination. .e cell
viability was measured colorimetrically using the alamarBlue
assay [38]. Shortly, compounds 18, 20, and 36 (dissolved in
DMSO and diluted in the RPMI-1640 medium at a range of
eight different concentrations from 0.19 to 25 μg/ml) were
added to each well and incubated for 72 h. Doxorubicin
(Sigma-Aldrich) was used as the positive control. Four hours
before the end of incubation, 20 μl of a stock solution
(0.312 mg/ml) of resazurin (Sigma-Aldrich) was added to
each well. Absorbance was measured at 570 nm and 600 nm
using the SpectraMax 190 Microplate Reader (Molecular
Devices, Sunnyvale, CA, USA). .e half maximal inhibitory
concentration (IC50) values with 95% confidence intervals
were obtained by nonlinear regression using GraphPad
Prism (Intuitive Software for Science; San Diego, CA, USA).
3.7.1. Benzyl 3,4,5-trimethoxybenzoate (36). Yield 56%;
yellow oil; MM: 307.12 g/mol; 1H NMR (200 MHz, CDCl3):
δH 7.37-7.27 (m, 5H), 7.25 (s, 2H), 5.28 (s, 2H), 3.81 (m, 6H),
3.80 (s, 3H); 13C NMR (50 MHz, CDCl3): δC 166.1, 153.0,
142.3, 136.1, 128.7, 128.3, 128.2, 125.2, 107.0, 66.9, 61.0, 56.3;
−1
IR νmax (KBr, cm ): 2999, 2939, 2837, 1713, 1589, 1502, 864
4. Methods
[36].
.e first step for modeling was obtaining the initial 3D
structure of each compound using the OpenEye’s Omega
software [39]. Fragments for 3D structures generation were
obtained with MAKEFRAGLIB, which is a part of the
OMEGA suite. .e Merck Molecular Force Field (MMFF94s)
excluding the Coulomb interactions (mmff94s_NoEstat) was
employed for fragment generation. Fragments with 4.0 kcal
above the global minimum as well as those with a RMS
distance lower than 0.1 from any conformer in the database
were discarded. Default parameters were used for generating
one 3D conformation per compound with OMEGA. Con-
formational exploration and alignment of the compounds
were performed with the Open3DALIGN software [40]. A
conformational library of each compound was obtained
3.8. Leishmania Culture Conditions. Leishmania ama-
zonensis promastigote forms (IFLA/BR/67/PH8) were
cultivated at 26°C in Schneider’s medium (Sigma) supple-
mented with 10% fetal bovine serum (FBS) (Gibco BRL,
Gaithersburg, MD, USA) and the antibiotic gentamicin
(40 µg/ml) (Schering–Plough, Rio de Janeiro, Brazil).
3.9. MTT Assay. .e antileishmanial activity on promasti-
gote forms of L. amazonensis was evaluated using the
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) method (Amaresco, Ohio, USA). .e promasti-
gotes of L. amazonensis were distributed in a 96-well plate

10
Journal of Chemistry
through quenched molecular dynamics (QMD) conforma-
tional searches that employed TINKER with an implicit
solvent model [41]. Two hundred molecular dynamic sim-
ulations of length 100 ps were performed for each compound.
.e remaining parameters for the QMD in Open3DALIGN
were kept to their default values. Structural alignment took
place was performed using a method that combines atom-
and pharmacophore-based alignments as implemented in
Open3DALIGN.
contains a furfuryl portion attached to the (E)-3-(3,4,5-
trimethoxyphenyl)-acryloyl moiety. .is modification sig-
nificantly improved the bioactivity. Molecular simplification
of 36 in relation to other derivatives formed an analogue
with better biological activity against Leishmania. Further-
more, bornyl radical appears to be important for the bio-
activity, given that compound 20 revealed the strongest
antileishmanial activity. Together, these results show that
compounds 18, 20, and 36 are promising lead compounds in
the development of new options of pharmacotherapeutic
drugs for leishmaniasis.
3D-QSAR models were developed with the Open3-
DQSAR suite [42], which performs partial least squares
(PLS) regression models from molecular interaction fields
(MIF). Unless otherwise noted, default parameters were
employed for Open3DQSAR. .e input to Open3DQSAR is
a set of aligned conformers of the dataset with associated
bioactivities. .e best alignment produced by Open3-
DALIGN in the previous step was used for 3D-QSAR models
generation. In this study, compounds 19, 26, 31, and 32
were removed from the modeling process because of their
undetermined IC50. .e bioactivity of the remaining com-
pounds was transformed to pIC50 values (pIC50 � −log10
IC50), provided that IC50 values are expressed in molar units.
A grid was constructed around the aligned molecules in
Data Availability
.e data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
.e authors declare that they have no conflicts of interest.
Acknowledgments
˚
such a way that its box exceeded in 5 A the largest molecule
.is work was supported by the Brazilian agencies Conselho
˚
and grid spacing was set to 0.5 A. Steric and electrostatic
´
´
Nacional de Desenvolvimento Cientıfico e Tecnologico
molecular mechanics of MIFs were computed using the
Merck force field (MMFF94). For the computation of both
MIFs, an alkyl carbon (charge +1) was used as probe. Before
model construction, variables were filtered to discard those
with van der Waals energies above 104 kcal/mol. Corre-
sponding points on the electrostatic MIF were also removed.
For both MIFs, energies greater than 30 kcal/mol or lower
than −30 kcal/mol were set to 30 kcal/mol and −30 kcal/mol,
respectively. Also values of energy between −0.05 and
0.05 kcal/mol were set to 0 in the two MIFs, and all variables
with standard deviation lower than 0.1 were discarded. All
variables spanning up to four levels were removed from both
fields. As a final filter, variables were scaled employing the
AUTO option of Open3DQSAR.
Initial PLS models considered 10 principal components.
.e optimal number of principal components (PCs) was
selected based on the value of the Leave One Out cross-
validation R2 (q2). Variables were then grouped using the
Smart Region definition procedure implemented in
Open3DQSAR taking into account the previously identified
optimal number of PCs. .e grouped variables were subject
to a selection procedure according to the Fractional Factorial
Design using Leave Many Out (LMO) cross validation with
20 runs and 5 groups. Only selected variables in previous
step were kept on the dataset. After these variable selection
procedures, the PLS model was recomputed considering the
optimal number of PCs and validated using the LOO and
LMO strategies.
(CNPq) and Coordenação de Aperfeiçoamento de Pessoal de
´
Nıvel Superior (CAPES).
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