Antileishmanial activity and structure-activity relationship of triazolic compounds derived from the neolignans grandisin, veraguensin, and machilin G

Article abstract of DOI:10.3390/molecules21060802

Sixteen 1,4-diaryl-1,2,3-triazole compounds 4-19 derived from the tetrahydrofuran neolignans veraguensin 1, grandisin 2, and machilin G 3 were tested against Leishmania (Leishmania) amazonensis intracellular amastigotes. Triazole compounds 4-19 were synthetized via Click Chemistry strategy by 1,3-dipolar cycloaddition between terminal acetylenes and aryl azides containing methoxy and methylenedioxy groups as substituents. Our results suggest that most derivatives were active against intracellular amastigotes, with IC₅₀ values ranging from 4.4 to 32.7 μM. The index of molecular hydrophobicity (ClogP) ranged from 2.8 to 3.4, reflecting a lipophilicity/hydrosolubility rate suitable for transport across membranes, which may have resulted in the potent antileishmanial activity observed. Regarding structure-activity relationship (SAR), compounds 14 and 19, containing a trimethoxy group, were the most active (IC₅₀ values of 5.6 and 4.4 μM, respectively), with low cytotoxicity on mammalian cells (SI = 14.1 and 10.6). These compounds induced nitric oxide production by the host macrophage cells, which could be suggested as the mechanism involved in the intracellular killing of parasites. These results would be useful for the planning of new derivatives with higher antileishmanial activities.

Full text of DOI:10.3390/molecules21060802

molecules
Article
Antileishmanial Activity and Structure-Activity
Relationship of Triazolic Compounds Derived
from the Neolignans Grandisin, Veraguensin,
and Machilin G
Eduarda C. Costa 1, Tatiana B. Cassamale 2, Diego B. Carvalho 2, Lauriane S. S. Bosquiroli 1,
Mariáh Ojeda 3, Thalita V. Ximenes 4, Maria F. C. Matos 3, Mônica C. T. Kadri 4,
Adriano C. M. Baroni 2 and Carla C. P. Arruda 1,
*
1
Laboratório de Parasitologia Humana, Centro de Ciências Biológicas e da Saúde,
Universidade Federal de Mato Grosso do Sul, 79090-900 Campo Grande—MS, Brazil;
eduarda_c.costa@hotmail.com (E.C.C.); lauri.bosqui@gmail.com (L.S.S.B.)
Laboratório de Síntese e Química Medicinal-LASQUIM, Centro de Ciências Biológicas e da Saúde,
Universidade Federal de Mato Grosso do Sul, 79090-900 Campo Grande—MS, Brazil;
tatibortolo@gmail.com (T.B.C.); diegob.carvalho@hotmail.com (D.B.C.);
adrianobaroni@hotmail.com (A.C.M.B.)
2
3
4
Laboratório de Biologia Molecular e Culturas Celulares, Centro de Ciências Biológicas e da Saúde,
Universidade Federal de Mato Grosso do Sul, 79090-900 Campo Grande—MS, Brazil;
mariahojeda@live.com (M.O.); matosmfc@gmail.com (M.F.C.M.)
Laboratório de Biofisiofarmacologia, Centro de Ciências Biológicas e da Saúde,
Universidade Federal de Mato Grosso do Sul, 79090-900 Campo Grande—MS, Brazil;
thalitario@hotmail.com (T.V.X.); monica.kadri@ufms.br (M.C.T.K.)
*
Correspondence: carla.arruda@ufms.br; Tel.: +55-67-3345-7369
Academic Editor: Marcello Iriti
Received: 26 May 2016; Accepted: 16 June 2016; Published: 20 June 2016
Abstract: Sixteen 1,4-diaryl-1,2,3-triazole compounds
veraguensin , grandisin , and machilin G were tested against Leishmania (Leishmania) amazonensis
intracellular amastigotes. Triazole compounds 19 were synthetized via Click Chemistry strategy
4–19 derived from the tetrahydrofuran neolignans
1
2
3
4–
by 1,3-dipolar cycloaddition between terminal acetylenes and aryl azides containing methoxy and
methylenedioxy groups as substituents. Our results suggest that most derivatives were active against
intracellular amastigotes, with IC50 values ranging from 4.4 to 32.7 µM. The index of molecular
hydrophobicity (ClogP) ranged from 2.8 to 3.4, reflecting a lipophilicity/hydrosolubility rate suitable
for transport across membranes, which may have resulted in the potent antileishmanial activity
observed. Regarding structure-activity relationship (SAR), compounds 14 and 19, containing a
trimethoxy group, were the most active (IC50 values of 5.6 and 4.4 µM, respectively), with low
cytotoxicity on mammalian cells (SI = 14.1 and 10.6). These compounds induced nitric oxide
production by the host macrophage cells, which could be suggested as the mechanism involved in the
intracellular killing of parasites. These results would be useful for the planning of new derivatives
with higher antileishmanial activities.
Keywords: neglected diseases; biological activity; cytotoxicity; synthetic compounds;
neolignan derivatives
1. Introduction
Cutaneous leishmaniasis (CL) is a parasitic infectious disease that affects the skin, cartilage and
mucosa of the upper respiratory tract. The resulting ulcers can develop into destructive and disabling
Molecules 2016, 21, 802; doi:10.3390/molecules21060802
www.mdpi.com/journal/molecules

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injuries, making the illness a serious public health problem [
1]. There have been between 0.7 and
1.3 million new annual cases throughout the world, and 95% occur in the Americas, Mediterranean
basin, Middle East and Central Asia [ ]. Leishmania (Leishmania) amazonensis Lainson and Shaw 1972
2
is one of the etiological agents of CL. It produces the typical localized lesions or the diffuse form of
the disease, in which the parasite spreads due to impaired cell-mediated immune response. Thus,
the lesions tend not to heal spontaneously and to be more resistant to treatment [3,4].
The first choice drugs for CL are the pentavalent antimonials, which are associated to hepato,
cardio and nephrotoxicity [
as amphotericin B, pentamidine or paramomycin are indicated, despite their degree of toxicity [
Resistance and high cost are other aspects that lead to the urgent need for new therapeutic options
arising from natural products [ 10]. However, these products may have undesirable properties such
5]. When these drugs are ineffective or cannot be prescribed, drugs such
6].
7
–
as high toxicity, low solubility and bioavailability, which can be minimized by the development of
synthetic derivatives [11].
Cassamale et al. [12] have synthetized a series of triazole derivatives from the tetrahydrofuran
neolignans veraguensin
important scaffolds in molecular modification studies due to their antileishmanial and antichagasic
activities [13 16]. Cassamale et al. [12] demonstrated the antileishmanial activity of these triazole
1, grandisin 2, and machilin G 3. These compounds have been considered
–
derivatives on promastigote forms; now the present work shows their activity on L. (L.) amazonensis
intracellular amastigotes, searching for structure-activity relationship information to support the
development of new drug candidates for CL.
2. Results and Discussion
Synthetic derivatives of tetrahydrofuran neolignans
moderately active (20–50 M), and potentially inactive (>50
Most compounds were considered active against L. (L.) amazonensis intracellular amastigotes (
10 11 12 14, and 19). Compounds 15 16 17, and 18 have been moderately active and 13 did not
show potential activity on the parasites (Table 1).
1–
3
were classified as active (<20
µM),
µ
µM) according to Upegui et al. [17].
4, 6, 8, 9,
,
,
,
5,
7
,
,
,
The ability to inhibit the growth of parasites apparently depends on the presence and ratio
of lipophilic/hydrophilic substituents on aromatic rings [18]. The transport of a compound across
membranes may be influenced by the molecular hydrophobicity described by the octanol/water
partition coefficient (ClogP) [19]. According to Lipinski’s Rule of Five [20–22], compounds with
logP < 5 have better absorption and permeation in vivo. Daunes and D’Silva [23] verified that among a
series of molecules, the ones with higher logP (>2.7) were the most active against trypanosomatids
once entering the host cell more easily. In other words, the molecular hydrophobic character improves
the antileishmanial activity, which may indicate that the active compound must interact with a target
system such as an enzyme or receptor, where the binding site is generally hydrophobic.
In our study, synthetic derivatives showed lipophilicity 2.8 < ClogP < 3.4 (Table 1), reflecting an
adequate lipophilicity for the transport across membranes and resulting in potent antileishmanial activity.
We observed that ring B containing the trimethoxy substituent has influenced the antileishmanial
activity independently of the substituent on the ring A (compounds 6, 10, 14 and 19) (Table 1).
Grandisin derivative 6 was active on intracellular amastigotes (IC50 value of 9.4 µM), with the
highest selectivity index. This compound was 66 times more toxic to amastigotes than to mammalian
cells (Table 1). The substitution of tetrahydrofuran ring by triazolic ring may have resulted in
increased activity, once it was described an IC50 value of 98.05
on L. (L.) amazonensis promastigotes [24]. Furthermore, its coefficient of hydrophobicity is lower than of
its precursor (2.8 and 3.7, respectively), which may have resulted in a better solubility and consequent
µM (42.4
µ
g
mL´1) for grandisin
¨
2
in vitro activity. Indeed, more soluble grandisin derivatives have been synthetized in order to reduce
its lipophilicity, which may limit in vivo studies [25]. It is important to note that the intracellular
amastigote form is the target for drug candidates in the mammalian host.

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Table 1. Molecular hydrophobicity, in vitro antileishmanial activity and cytotoxicity of 1,4-diaryl-1,2,3-triazole derivatives 4–19 of the neolignans 1–3.
Intracellular Amastigotes
J774.A1 Cells
Compounds
R1
R2
R3
R4
R5
R6
ClogP 1
SI 4
IC50 (µM) 2
IC50 (µM) 3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
-H
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-H
-H
-OCH3
-H
-H
-H
-H
-OCH3
-H
-H
-H
-OCH3
-H
-H
-OCH3
-H
-H
-OCH3
-OCH3
-OCH2O-
-OCH3
-H
-OCH3
-H
-OCH2O-
-H
-OCH3
-OCH3
-OCH3
-H
-H
-OCH3
-H
-H
-H
-OCH3
-H
-H
-H
-OCH3
-H
3.4
3.1
2.8
2.9
3.3
3.3
3.1
3.1
3.2
3.2
2.9
2.9
3.0
3.0
2.9
2.9
13.1
21.3
9.4
877.2
76.4
>622.8
86.2
79.2
22.6
65.4
67.5
96.5
134.6
79.1
76.9
>768.5
9.2
66.9
3.6
>66.2
4.2
4.9
1.2
3.9
4.1
6.5
2.6
14.1
2.6
>23.5
0.3
2.2
10.6
-OCH2O-
20.4
16.0
18.3
16.8
16.5
14.8
50.7
5.6
29.2
32.7
29.9
29.8
4.4
-H
-OCH3
-H
-OCH3
-H
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH2O-
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH2O-
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH2O-
-H
-H
-H
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH2O-
66.9
46.9
0.5
19
Doxorubicin 5
Amphotericin B 5
1
-OCH2O-
-OCH3
-
0.7
-
3.1
2.2
ClogP, octanol/water partition coefficient; 2 IC50, half maximum inhibitory concentration on intracellular amastigotes; 3 IC50, half maximum inhibitory concentration on J774.A1 cells;
SI (selectivity index), IC50 on mammalian cells/IC50 on intracellular amastigotes; 5 Positive controls, amphotericin B for L. (L.) amazonensis and doxorubicin for J774.A1 cells. The data
4
are representative of three independent experiments.

Molecules 2016, 21, 802
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Treatment with compound
of 6.25
has induced an increased production of nitric oxide (NO) compared to untreated infected cells
(control) (Figure 2A). Thus, NO production may be suggested as the mechanism of leishmanicidal
action, especially because does not seem to have a direct action on L. (L.) amazonensis once it was
6
had significantly decreased the infection index at the concentration
µg
¨
mL´1, reaching 98.6% reduction (p < 0.0001) at the concentration of 50
µg
mL´1 (Figure 1A).
¨
6
6
proven inactive on promastigote forms [12].
Figure 1. Effect of 1,4-diaryl-1,2,3-triazole derivatives (4–19) of the neolignans 1–3 on intracellular
amastigotes. Peritoneal macrophages were infected with L. amazonensis and treated with different
concentrations of the compounds. Infection index was calculated 24 h after treatment. Bars represent
the mean
untreated cells (control) (Student’s t-test). (
series; (D) methylenedioxy series (substitution pattern of ring A).
˘
SD of six replicates. * p
ď
0.05, ** p
ď
0.01, *** p
) trimethoxy series; (
ď
0.001 for the different concentrations versus
A
B
) dimethoxy series; ( ) methoxy
C
Figure 2. Nitric oxide release by L. amazonensis infected macrophages 24 h after treatment with
1,4-diaryl-1,2,3-triazole derivatives ( 19) of the neolignans . Bars represent the mean SD of six
replicates. * p 0.05, ** p 0.01, for the different concentrations compared to untreated cells (control)
(Student’s t-test). ( ) trimethoxy series; ( ) dimethoxy series; ( ) methoxy series; ( ) methylenedioxy
series (substitution pattern of ring A).
4–
1–
3
˘
ď
ď
A
B
C
D

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The other compounds from trimethoxy series (with substituents on ring A) were active (11) and
moderately active (15 and 18), but less selective, with important cytotoxicity on mammalian cells
(Table 1). All of them were able to significantly decrease infection index, mainly 18, reaching 92.1%
reduction (p < 0.0001) (Figure 1A). This compound, however, did not induce a significant production
of NO. 11 acted variably on NO release, with a production lower than untreated cells at the lowest
concentrations, but higher than control at the highest concentrations tested (Figure 2A).
Compound
derivative) was moderately active on intracellular amastigotes (IC50 value of 21.3
selectivity (SI = 3.6). Silva Filho et al. [16] have demonstrated the activity of veraguensin on L. donovani
promastigote forms (48.3 M; 18
mL´1). Once again we may educe that the insertion of triazole
ring may have improved the antileishmanial activity due to reduction of octanol/water partition
coefficient (3.1 ( ) versus 4.2 ( )). Compound has significantly reduced the infection index from the
concentration of 12.5
mL´1 (Figure 1B). Furthermore, the mechanism of leishmanicidal action
5
with a dimethoxy group as substitution pattern on rings A and B (veraguensin
1
µM) with relative
µ
µg
¨
5
1
5
µg
¨
seems also to be independent of NO, once NO production was lower than control at the concentration
´1
of 25 µg¨ mL (Figure 2B). These data corroborate those obtained by Konishi et al. [26], who verified
the inhibition of NO production from LPS-activated murine macrophages by three veraguensin’s
position isomers.
The other compounds from dimethoxy series were active (
9 and 14), excepting 16, which was
moderately active but selective (IC50 = 32.7 M; SI > 23.5). Despite its moderate activity, 16 was able to
µ
significantly induce NO production (Figure 2B).
Compound 14 was active and selective (IC50 = 5.6 µM; SI = 14.1) (Table 1), and reduced the
infection index at all concentrations tested (86.4% at the highest concentration, p = 0.0003) (Figure 1B).
It’s important to note that 14 was a hybrid from veraguensin and grandisin , and it had the best
1
2
activity of the whole dimethoxy series. Furthermore, it significantly induced NO production at the
lowest concentrations tested, and this may be suggested as a possible mechanism of leishmanicidal
action (Figure 2B).
Compounds from methoxy series showed IC50 values from 13.1 to 16.8
µM (
4,
8
,
10, and 12
,
Table 1). Compound was the most active of this series, and highly selective (IC50 = 13.1
4
µM;
SI = 66.9). As well as 12, this compound was able to induce an increase of NO production at the highest
concentration tested (Figure 2C).
The most active compound on intracellular amastigotes (19) came from methylenedioxy series
(IC50 = 4.4 µM). Compound 19 is a hybrid from machilin G 3 and grandisin 2 and was also quite
selective (SI = 10.6) (Table 1). 19 has significantly reduced the infection index and in addition it
induced NO production twice as high than control at the highest concentrations tested (Figure 2D).
Cassamale et al. [12] have demonstrated this compound as highly active against L. (L.) amazonensis
promastigote forms (IC50 = 7.2
µM), and this suggests its direct action on the parasite. On the other
hand, position isomer 18 (IC50 = 29.8
µM, Table 1) was less active than 19, indicating the role of minor
structural differences on the antileishmanial activity of these compounds.
Compound 13 did not show potential activity and selectivity (Table 1). Despite this, 13 was able
to induce an increase in NO production (Figure 2D), as well as its position isomer 12 (Figure 2C).
We should point out that all compounds from methylenedioxy series have induced NO production
in greater or lesser degree (Figure 2D). This implies that this group may be associated to cellular
activation and/or cytotoxicity. For example, compound 17 (machilin G
3 analog) was toxic for the
macrophages (SI = 0.3) with moderate antileishmanial activity (Table 1).
3. Materials and Methods
3.1. Triazole Derivatives of Neolignans
Sixteen 1,4-diaryl-1,2,3-triazole derivatives with substitution patterns found in the neolignans
veraguensin 1, grandisin 2, and machilin G 3 (Figure 3) were tested. They were designed based on the

Molecules 2016, 21, 802
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concept of the bioisosterism of rings, where the tetrahydrofuran core was substituted by a 1,2,3-triazole
ring (Figure 4) [12].
Figure 3. Structure of the neolignans veraguensin 1, grandisin 2, and machilin G 3.
Figure 4. Structural design of 1,4-diaryl-1,2,3-triazole derivatives of the neolignans 1–3.
Triazole derivatives
cycloaddition reactions between terminal acetylenes 25a
and methylenedioxy substitution patterns [12]. The synthesis of starting materials began by preparing
aryl bromides 21a via a bromination reaction of 20a in the presence of NBS, p-TsOH, CH2Cl2 and
SiO2 [12 27]. Subsequently, a cross-coupling Sonogashira reaction between bromobenzenes 21a and
4–
19 were obtained via Click Chemistry strategy from 1,3-dipolar
–d
and aromatic azides 27a with methoxy
–d
–c
–c
,
–c
2-methyl-3-butyn-2-ol in the presence of PdCl2(PPh3)2/CuI, Et3N provided the acetylene alcohols
22a–c with 81%–86% yields, after 24 h reaction time [12,28].
Retro-Favorski reaction of 22a
acetylenes 25a with 75% to 79% yield [12
synthesized by the Corey-Fuchs method (Scheme 1) [12,31].
–
c
with KOH under reflux in toluene generated the terminal
–
c
,28–30]. Ethynyl-1,2,3-trimethoxybenzene 25d was
Scheme 1. Synthesis of aryl acetylenes 25a
(75 mmol), TsOH (10 mmol), SiO2 (37 g), CH2Cl2 (210 mL), room temperature, 3 h, 21a = 79%, 21b = 82%,
21c = 85%; (b) 21a (50 mmol), 2-methyl-3-butyn-2-ol (183 mmol), PdCl2(Ph3)2 (2.5 mmol), CuI
(5 mmol), Et3N (250 mL), reflux, 24 h, 22a = 81%, 22b = 82%, 22c = 86%; (c) 22a (30 mmol), KOH
–d [12]. Reagents and reaction: (a) 20a–c (75 mmol), NBS
–
c
–
c
(90 mmol), toluene (225 mL), reflux, 24 h, 25a = 75%, 25b = 78%, 25c = 79%; (d) CCBr4 (100 mmol),
PPh3 (200 mmol), CH2Cl2 (100 mL), 0 ˝C, then 23 (50 mmol), 0 ˝C-room temperature, 5 h, 24 = 87%;
(
e
) 24 (10 mmol), THF (50 mL), n-BuLi 2.5 M in hexanes (22 mmol),
´
25 ˝C—room temperature, 1 h,
25d = 83%.
Next, aromatic azides 27a–d were prepared by the reaction of aromatic amines 26a–d with
t-BuONO/TMSN3 using the protocol reported by Moses et al. (Scheme 2) [12,32].

Molecules 2016, 21, 802
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Scheme 2. Synthesis of aryl azides 27a–d [12]. Reagents and reaction: (a) 26a–d (20 mmol), t-BuONO
(43 mmol), CH3CN, 15 min, 0 ˝C, then TMSN3 (32.6 mmol), rt, 5–12 h; 27a = 79%, 27b = 86%, 27c = 89%,
27d = 90%.
The 1,3-dipolar cycloaddition occurred when terminal acetylenes 25a
using CuSO4 H2O, sodium ascorbate and CH2Cl2/H2O 1:1 as solvents, yielding the compounds
4–19 in 78% to 92% yield (Scheme 3) [12,33].
–d reacted with aryl azides
27a
–
d
¨
Scheme 3. General method for the obtaining of 1,4-diaryl-1,2,3-triazole derivatives
(a) 25a (2 mmol), 27a (2 mmol), CuSO4 5H2O (0.128 mmol), sodium ascorbate (0.352 mmol),
CH2Cl2/H2O (4 mL), room temperature, 24 h.
4–19 [12,31]:
–
d
–
d
¨
3.2. General Procedure for the Synthesis of Triazoles 4–19
To a solution of terminal acetylenes 25a (2 mmol, 1.0 equiv) and azides 27a
in dichloromethane (2 mL) and water (2 mL), were added CuSO4 5H2O (0.128 mmol, 0.064 equiv) and
–
d
–d (2 mmol, 1.0 equiv)
¨
sodium ascorbate (0.352 mmol, 0.176 equiv). The reaction mixture was stirred for 24 h. Then it was
added a saturated solution of NH4Cl (30 mL) and the product was extracted with dichloromethane
(3
ˆ
20 mL). The organic phase was dried over anhydrous MgSO4, and the solvent was evaporated
under reduced pressure. The products were purified by recrystallization from ethyl acetate.
3.3. ClogP
In order to estimate the molecular hydrophobicity of triazole derivatives 4–19, theoretical values
of logP (ClogP) were calculated with ChemAxon software. The program estimated octanol/water
partition coefficient based on a modified version of the method of Viswanadhan et al. [34].

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3.4. Antileishmanial Activity
3.4.1. Parasites and Peritoneal Macrophages
In vitro antileishmanial activity was evaluated on peritoneal macrophages infected with.
L. (L.) amazonensis intracellular amastigotes. Parasites (IFLA/BR/1967/PH8 strain) were routinely
isolated from BALB/c mice and maintained as promastigotes at 25 ˝C in Schneider’s Insect Medium
(Sigma-Aldrich®, St. Louis, MO´, 1USA) supplemented with 20% fetal calf serum (FCS-Cultilab®
Campinas, Brazil) and 140
µg
mL gentamicin (Sigma-Aldrich®). Macrophages were obtained from
¨
peritoneal wash of BALB/c mice after euthanasia. 10 mL of RPMI 1640 (Sigma-Aldrich®) supplemented
´1
´1
with 2% L-glutamine, 2.8% bicarbonate buffer, 100 U
¨
mL penicillin and 100 mg mL streptomycin
¨
were injected into the peritoneal cavity. After massage area, liquid was aspirated and transferred to
tubes on ice. Peritoneal cells were quantified in Neubauer chamber after cellular exclusion with Trypan
Blue staining (Sigma-Aldrich®).
3.4.2. In Vitro Antileishmanial Activity on Intracellular Amastigotes
Peritoneal cells (1
ˆ
105 cells/well) were added to 24-well plates containing circular coverslips.
˝
Plates were incubated for one hour at 37 C/5% CO2 to allow cell adhesion and then 1
L. (L.) amazonensis promastigotes were added to each well. Plates were incubated at 35 C/5% CO2 for
four hours and then cells were treated for 24 h with synthetic compounds 19 (6.25–50
mL´1).
Amphotericin B (Sigma-Aldrich®) was used as the reference drug (0.25 to 2 µg¨ mL´1) and untreated
cells were used as negative control. Coverslips were processed as described by Rizk et al. [35].
The overall number of amastigotes was determined by counting 100 cells in six replicates. The half
maximal inhibitory concentration (IC50) was calculated using a nonlinear regression curve. Infection
index was obtained as described by Paladi et al. [36].
ˆ
106
˝
4–
µg
¨
3.5. Nitric Oxide Production
To evaluate the nitric oxide production (NO) by infected and treated peritoneal cells, 50 µL of
culture supernatant from the antileishmanial assay were collected and incubated with equal volume
of Griess reagent (1% sulfanilamide /0.1% naphthalene diamine in 5% phosphoric acid) for 10 min
at room temperat´ure. According to Ding et al. [37], the absorbance was determined at 540 nm and
converted to NO2
(µM) by comparing to a standard curve of known concentrations of sodium nitrite
(1–10 µM) in RPMI medium (Sigma-Aldrich®).
3.6. Cytotoxicity Assay
Murine macrophages (J774.A1, Rio de Janeiro Cell Bank, Brazil) were seeded in 96-well plates
˝
(1
0.25–250
drug (0.025–25
ˆ
105 mL´1) and incubated with compounds at 37 C/5% CO2 for 48 h at concentrations of
µg
¨
mL´1 to estimate IC50. Amphotericin B (Sigma-Aldrich®) was used as the reference
µ
g
mL´1). Cell growth was evaluated according to Skehan et al. [38] using the
¨
sulforhodamine B assay. Dimethyl sulfoxide (DMSO, Vetec®, Rio de Janeiro, Brazil) was used as
negative control at the concentration used to solubilize the highest concentration of compounds.
IC50 was calculated by nonlinear regression curve. Selectivity index was calculated according to
Medeiros et al. [39].
3.7. Ethical Aspects
This study received approval from the local Animal Experimentation Ethical Committee
(CEUA/UFMS) under protocol 503/2013.

Molecules 2016, 21, 802
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4. Conclusions
Among 16 synthetic derivatives of tetrahydrofuran neolignans veraguensin
1, grandisin 2
and machilin G , 15 showed high or moderate antileishmanial activity. Compounds 14 and 19
3
,
containing a trimethoxy substituent on ring B, were the most active against intracellular amastigotes,
with low cytotoxicity on mammalian cells. These compounds induced nitric oxide production by
the host macrophage cells, which could be suggested as the mechanism involved in the intracellular
killing of parasites. These results would be useful for the planning of new derivatives with higher
antileishmanial activities.
Acknowledgments: We gratefully acknowledge financial support received from the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq, Protocol 563977/2010-8), Fundação de Apoio ao Desenvolvimento
do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT, Protocol 23/200.137/2010), and the
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Author Contributions: C.C.P.A., A.C.M.B., M.F.C.M., and M.C.T.K. conceived and designed the experiments;
E.C.C., L.S.S.B., M.O., and T.V.X. performed the experiments; E.C.C. analyzed the data; T.B.C. and D.B.C.
contributed analysis tools; E.C.C., C.C.P.A, and A.C.M.B wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
Abbreviations
The following abbreviations are used in this manuscript:
C.L.
FCS
Cutaneous Leishmaniasis
Fetal Calf Serum
RPMI
IC50
DMSO
S.I.
Roswell Park Memorial Institute
Half maximal inhibitory concentration
Dimethyl sulfoxide
Selectivity Index
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Sample Availability: Samples of the compounds 4–19 are available from the authors.
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