Microwave-assisted synthesis and antileishmanial activity of 3-methoxycarbonyl-γ-butyrolactone derivatives

Article abstract of DOI:10.5935/0103-5053.20140113

We describe the microwave-assisted synthesis of ten 3-methoxycarbonyl- γ-butyrolactone derivatives and evaluate their in vitro antileishmanial activity against promastigote forms of Leishmania amazonensis. The synthesis furnished most of the compounds in

Full text of DOI:10.5935/0103-5053.20140113

http://dx.doi.org/10.5935/0103-5053.20140113
J. Braz. Chem. Soc., Vol. 25, No. 8, 1331-1337, 2014.
Printed in Brazil - ©2014 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
A
Microwave-Assisted Synthesis and Antileishmanial Activity of 3-methoxycarbonyl-
γ-butyrolactone Derivatives
Marcos F. Pinatto-Botelho,a Antonio E. M. Crotti,a Julia M. de Souza,b
Lizandra G. Magalhãesb and Paulo M. Donate*,a
aDepartamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Avenida Bandeirantes, 3900, 14040-901 Ribeirão Preto-SP, Brazil
bNúcleo de Pesquisa em Ciências Exatas e Tecnológicas, Universidade de Franca,
Avenida Dr. Armando Salles de Oliveira 201 Parque Universitário, 14404-600 Franca-SP, Brazil
Descrevemos a síntese assistida por micro-ondas de dez derivados de 3-metoxicarbonil-γ-
butirolactona e a avaliação in vitro da atividade leishmanicida desses compostos contra formas
promastigotas de Leishmania amazonensis. A síntese forneceu a maioria dos compostos com
80-95% de rendimento e as reações duraram cerca de 10-20 min. A maior parte dos compostos
apresentaram valores de IC50 superiores a 400 uM. Os compostos 5 (trans-3-(p-metoxi)benzil-4-
metil-3-metoxicarbonil-γ-butirolactona) e 10 (trans-3-(p-metoxi)benzil-4-benzil-3-metoxicarbonil-
γ-butirolactona) foram as exceções, pois exibiram valores de IC50 de 170,4 e 179,6 μM,
respectivamente, o que sugere que a atividade leishmanicida das 3-metoxicarbonil-γ-butirolactonas
pode estar relacionada com a natureza e o tamanho do substituinte na posição C-4.
We describe the microwave-assisted synthesis of ten 3-methoxycarbonyl-γ-butyrolactone
derivativesandevaluatetheirinvitroantileishmanialactivityagainstpromastigoteformsofLeishmania
amazonensis. The synthesis furnished most of the compounds in 80-95% yield and reactions lasted
about 10-20 min. Most of the compounds displayed IC50 values higher than 400 μM. Compounds
5 (trans-3-(p-methoxy)benzyl-4-methyl-3-methoxycarbonyl-γ-butyrolactone) and 10 (trans-3-(p-
methoxy)benzyl-4-benzyl-3-methoxycarbonyl-γ-butyrolactone) were the exceptions: they displayed
IC50 values of 170.4 and 179.6 μM, respectively, suggesting that the leishmanicidal activity of
3-methoxycarbonyl-γ-butyrolactones may be related to the nature and size of the substituent at
position C-4.
Keywords: γ-butyrolactone, multicomponent reaction, microwave-assisted synthesis,
Leishmania amazonensis
Introduction
Pentavalent antimonials were first used in the clinical
setting at the beginning of the last century; they remain
the first-choice drugs to treat leishmaniasis. However,
these compounds are toxic and poorly tolerated, require
daily injections for up to 28 days, and are becoming
ineffective due to proliferation of resistant parasites.5
Second-line drugs, like amphotericin B and pentamidine,
are options in combined therapy or in cases of antimony
treatment failure.6,7 Therefore, the development of new
antileishmanial compounds is imperative.1
γ-Butyrolactones are a structural feature of a diversity
of natural products, such as sesquiterpene lactones8-10
and lignan lactones.11,12 The γ-lactone subunit has been
associated with a number of anti-parasitic activities; e.g.,
leishmanicidal,13,14 nematocidal,15 and antiplasmodial
Protozoan parasites of the Leishmania genus cause a
group of diseases known as Leishmaniasis, a parasitosis
that affects more than 12 million people worldwide and
accounts for high mortality rates in tropical and subtropical
countries.1,2 It is estimated that two to three million new
leishmaniasis cases emerge each year, and that some 350
million people are at risk of infection.3 The parasites of the
genus Leishmania can infect humans and several species
of mammals. The infection manifests itself in three main
typical ways: visceral, cutaneous, and mucocutaneous
leishmaniasis.4
*e-mail: pmdonate@usp.br

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Microwave-Assisted Synthesis and Antileishmanial Activity of 3-methoxycarbonyl-g-butyrolactone Derivatives
J. Braz. Chem. Soc.
actions,16 among several other biological activities.17-19 In
this scenario, the interest in developing new methodologies
to synthesize compounds bearing γ-butyrolactones in their
structures has increased, with emphasis on multicomponent
reactions (MCRs).20-24 MCRs are advantageous: they
incorporate most, if not all atoms of the reagents in the
final product in few steps, usually involve a one-pot
reaction, and allow for facile product purification.25 A few
synthetic γ-butyrolactones have been evaluated for their
antileishmanial activity.
Microwave irradiation has gained popularity in the
past decade as a powerful tool to rapidly and efficiently
synthesize a variety of compounds. This process is
advantageous over conventional thermal heating: it
reduces reaction times, improves yields, and suppresses
the generation of side products.26-28
As part of our ongoing project on the synthesis
and biological activities of γ-butyrolactones,29-32 in this
paper we report the use of microwave irradiation as heat
source to promote the fast and efficient synthesis of a
series of 3,3,4-trisubstituted and 3,3,4,4-tetrasubstituted
3-methoxycarbonyl-γ-butyrolactone derivatives obtained
by the multicomponent reaction previously developed by
Le Gall and co-workers.33 Here, we have also evaluated the
antileishmanial activity of these compounds.
Table 1. Yields obtained in the synthesis of compounds 1-10 produced
via Scheme 1a
Conventional heating at
60 oC (oil bath)
Microwave irradiation at
60 oC and 150 W
Compound
Reaction time
Yield / %)b
/ min
Reaction time
Yield / %)b
/ min
1
2
3
4
5
6
7
8
9
10
180
60
35
70
13
20
18
24
13
30
78
83
20
10
85
96
82
80
81
88
86
85
0
60
20
180
120
180
180
60
20
10
10
10
10
180
60
10-30
20
90
aThe reactions were conducted under argon atmosphere with 5 mL of
acetonitrile, zinc powder (0.8 g, 12 mmol), dimethyl itaconate (2 g,
13 mmol), an aldehyde or a ketone (2.5 mmol), and the aryl bromide
(4 mmol). After brief stirring at room temperature, cobalt bromide
(0.13 g, 0.6 mmol), trifluoroacetic acid (0.03 mL), and 1,2-dibromoethane
(0.05 mL) were successively added. The reaction mixture was heated
at 60 °C for 1-3 h in an oil bath, or irradiated for 10-20 min in a CEM
Discovery® focused microwave oven at 60 oC and 150 W; bisolated yield.
as two-dimensional NMR techniques (HMQC, HMBC and
1H-1H COSY). We assigned the relative stereochemistry
of these compounds on the basis of NOE difference
spectroscopy, by irradiating the signal of the hydrogen
at C-4 (for compounds 1, 2, 5, 6, and 10) or the methyl
group at C-4 (for compounds 3, 8, and 9) with respect to
the hydrogen at C-2.
In the biological assays, we tested all the compounds
as mixtures of enantiomers. Table 2 lists the results of
the in vitro evaluation of the leishmanicidal activity
of the 3-methoxycarbonyl-γ-butyrolactones 1-10
against promastigote forms of Leishmania amazonensis
(MHOM/BR/PH8). Most of the tested compounds did not
cause significant lysis at concentrations lower than 50 µM
(data not shown). The most active compounds at 50 μM
Results and Discussion
We obtained the target compounds by MCR (see
Scheme 1), preferably using microwave irradiation as
heat source. Only compound 9 could not be achieved by
microwave irradiation, but was obtained in good yield after
3 h under conventional heating. Table 1 summarizes the
results of these reactions.
Figure 1 shows the chemical structures of the
3-methoxycarbonyl-γ-butyrolactones 1-10. Compounds
1-4 have been synthesized previously,33 but compounds
5-10 are new.
We identified the structures of the synthesized
compounds on the basis of 1H and 13C NMR data, as well
Scheme 1. Preparation of compounds 1-10 by the multicomponent reaction developed by Le Gall and co-workers.33

Vol. 25, No. 8, 2014
Pinatto-Botelho et al.
1333
Figure 1. Chemical structures of the 3-methoxycarboxyl-γ-butyrolactone derivatives 1-10.
Table 2. In vitro leishmanicidal activity of the 3-methoxycarbonyl-γ-
butyrolactone derivatives 1-10 3,34
5 (68.8 4.8%) and 10 (81.3 0.8%) afforded the highest
lysis value at 400 μM; their IC50 (Inhibitory Concentration)
values were 170.4 μM and 179.6 μM, respectively, much
higher than that of amphothericin B (IC50 = 13.7 μM), used
as positive control.
% Lysis SD/concentration / μMa
Compound
IC50
50
100
200
400
1
2
3
4
5
6
7
8
9
10
5.6 5.1 29.6 2.7 40.1 4.9 42.3 5.7
11.4 8.2 28.5 6.1 35.6 7.2 46.0 4.3
14.2 5.8 25.0 7.3 29.1 1.8 30.0 0.7
4.9 1.9 9.6 4.4 43.5 6.4 49.5 2.4
15.8 4.6 38.1 8.9 57.7 4.7 68.8 4.8
412.2
414.9
467.4
401.9
170.4
415.6
1045
405.1
1989
179.6
When we related the IC50 values to the structures
of the γ-butyrolactone derivatives, we noticed that the
antileishmanial activity decreased when the aromatic
ring did not bind to the β-methyl group, as in the case of
compound 9. The methoxyl group at the aromatic moiety
did not seem to increase the leishmanicidal action, as
evidenced by the comparison between the IC50 values of
compounds 1 and 2. In addition, comparison among the IC50
values of compounds 2-8 and 10 clearly indicated that the
leishmanicidal activity of these compounds depended on
the nature of the substituent at C-4. Although these results
demonstrated that the methyl and benzyl groups at position
γ play a key role in the leishmanicidal activity of compounds
5 and 10, further studies are necessary to investigate the
mechanism through which these substituents affect the
leishmanicidal activity of this class of compounds.
0
0
0
0
0
0
22.1 4.5 40.8 7.7
20.61 4.4 21.9 4.1
38.4 2.8 35.0 0.7 44.0 3.1
1.8 3.1 3.0 5.2 8.8 8.6
6.2 2.1 22.2 4.3 54.2 1.5 81.3 0.8
aPercentages of lysis at concentrations of 6.25, 12.5, and 25 µM were not
statistically significant for compounds 1-3. No lyses were observed at
these concentrations for compounds 4-10. Positive control:Amphotericin
B (12 μM): IC50 = 13.7 μM.
were 2 (11.4 8.2% lysis), 3 (14.2 5.8% lysis), and 5
(15.8 4.6% lysis). On the other hand, γ-butyrolactone 8
was found to be the most active among the tested compounds
at 100 μM, causing 38.4 2.8% lysis. These data indicated
that compound 8 did not display a dose-dependent effect at
concentrations lower than 100 µM. The γ-butyrolactones
Conclusion
In summary, our results indicated that the microwave-
assisted synthesis significantly increased the product yields

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Microwave-Assisted Synthesis and Antileishmanial Activity of 3-methoxycarbonyl-g-butyrolactone Derivatives
J. Braz. Chem. Soc.
andreducedreactiontimeascomparedwiththeconventionally
heated systems. Moreover, the antileishmanial activity of the
3-methoxycarbonyl-γ-butyrolactone derivatives was not so
strong as compared with that of amphotericin B and other
antileishmanial drugs currently employed in the clinical
setting. However, compounds 5 and 10, which proved to
be the most active among the evaluated compounds, should
be further investigated, mainly because their synthesis by
microwave-assisted synthesis reduced reaction times and
improved yields.
heating). Next, the reaction mixture was then filtered through
Celite®, which was then washed several times with anhydrous
diethyl ether. The organic fractions were combined and
concentrated under reduced pressure. The crude reaction
product was purified by flash column chromatography
through silica gel (n-hexane/ethyl acetate, 7:3 v/v), to afford
a mixture of stereoisomers of the butyrolactone with yields
ranging from 13 to 83% for conventional thermal heating,
and from 80 to 96% for microwave-assisted heating, with
the exception of compound 9, which was not obtained by
microwave irradiation.
Experimental
trans-3-Benzyl-4-phenyl-3-methoxycarbonyl-γ-
butyrolactone (1)
General
Yields: 35% (conventional thermal heating), 85%
(microwave radiation); IR (KBr) ν/cm-1 3460, 1786, 1736,
1216; 1H NMR (CDCl3, 400 MHz): d 7.45 (m, 2H, 2Ar-H),
7.43 (m, 2H, 2Ar-H), 7.35 (m, 2H, 2Ar-H), 7.23 (m, 2H,
2Ar-H), 6.95 (m, 2H, 2Ar-H), 5.67 (s, 1H, CH), 3.75 (s, 3H,
CH3), 3.12 (d, 1H, J 17.6 Hz, CH2), 2.86 (d, 1H, J 13.9 Hz,
CH2), 2.71 (d, 1H, J 17.6, Hz CH2), 2.17 (d, 1H, J 13.9 Hz,
CH2); 13C NMR (CDCl3, 100 MHz): d 74.5, 173.0, 135.7,
134.3, 129.5, 129.1, 128.5, 127.3, 126.5, 85.7, 56.3, 52.9,
39.0, 35.6; ESI-MS m/z calcd. for C19H18NaO4+: 333.1103,
found: 333.1097 [M+Na]+.
Mass spectra were acquired on an UltroTOF-Q
mass spectrometer (Bruker Daltonics, Billerica, MA,
USA) fitted with an ESI operating in the positive ion
mode. Accurate masses were obtained using TFA-Na+
(sodiated trifluoroacetic acid) as the internal standard.
1H-NMR spectroscopy was performed using a Bruker
DPRX-400 instrument (Bruker, Fällanden, Switzerland)
operatingat400MHzfor 1Handat100MHzfor 13C.TMSwas
used as internal standard. The chemical shifts are reported in
ppm (d); coupling constants (J) values are given in Hertz (Hz).
Signalmultiplicitiesarerepresentedby:s(singlet), d(doublet),
dd (double doublet), dq (double quadruplet), m (multiplet).
Unless noted otherwise, all the solvents and reagents were
commercially available and used without further purification.
trans-3-(p-Methoxy)benzyl-4-phenyl-3-methoxycarbonyl-
γ-butyrolactone (2)
Yields: 70% (conventional thermal heating), 96%
(microwave radiation); 1H NMR (CDCl3, 400 MHz): d 7.43
(m, 2H, 2Ar-H), 7.42 (m, 1H, Ar-H), 7.35 (m, 2H, 2Ar-H),
7.23 (m, 2H, 2Ar-H), 6.85 (d, 2H, J 8.1 Hz, 2Ar-H), 6.74 (d,
2H, J 8.1 Hz, 2Ar-H), 5.73 (s, 1H, CH), 3.76 (s, 3H, OCH3),
3.73 (s, 3H, CH3), 3.12 (d, 1H, J 17.7 Hz, CH2), 2.80 (d, 1H,
J 13.8 Hz, CH2), 2.69 (d, 1H, J 17.7 Hz, CH2), 2.12 (d, 1H,
J 13.8 Hz, CH2); 13C NMR (CDCl3, 100 MHz): d 174.6,
173.1, 158.8, 134.4, 130.6, 129.1, 128.6, 127.6, 126.6, 114.0,
86.0, 56.4, 55.6, 52.8, 38.5, 35.7; ESI-MS m/z calcd. for
C20H20NaO5+: 363.1208, found: 363.1201 [M+Na]+.
General procedure for the synthesis of compounds 1-10
The 3-methoxycarbonyl-γ-butyrolactones 1-10 were
synthesized using the one-pot multicomponent methodology
described by Le Floch and co-workers.33 All the reactions
were performed in duplicate: one employed a conventional
thermal heating with an oil bath; the other used microwave
radiation as heat source. A 25-mL round-bottomed flask
under argon atmosphere was charged with previously
purified34 acetonitrile (5 mL), zinc dust (0.8 g, 12 mmol),
dimethyl itaconate (2 g, 13 mmol), an aldehyde or a ketone
(2.5 mmol), and the aryl bromide (4 mmol). This mixture
was briefly stirred at room temperature. Cobalt bromide
(0.13 g, 0.6 mmol), trifluoroacetic acid (0.03 mL), and
1,2-dibromoethane (0.05 mL) were then added successively
to the previous mixture. The reaction mixture was heated at
60 °C for 1-3 h in an oil bath, or irradiated for 10-20 min in
a CEM Discovery® focused microwave oven at 60 °C and
150 W. The reaction mixture was monitored by thin-layer
chromatography until consumption of the aryl bromide
was complete (between 1 and 3 h for conventional thermal
heating, and between 10 and 20 min for microwave irradiation
trans-3-(p-Methoxy)benzyl-4-methyl-4-phenyl-3-methoxy-
carbonyl-γ-butyrolactone (3)
Yields: 13% (conventional thermal heating), 82%
(microwave radiation); IR (KBr) ν/cm-1 3450, 2980, 1782,
1768, 1734, 1730, 1512, 1250; 1H NMR (CDCl3, 400 MHz):
d 7.34 (m, 2H, 2Ar-H), 7.33 (m, 1H, Ar-H), 7.30 (m, 2H,
2Ar-H), 7.00 (d, 2H, J 8.6 Hz, 2Ar-H), 6.80 (d, 2H, J 8.6 Hz,
2Ar-H), 3.77 (s, 3H, OCH3), 3.65 (d, 1H, J 13.4 Hz, CH2),
3.31 (s, 3H, CH3), 2.91 (d, 1H, J 17.4 Hz, CH2), 2.77 (d,
1H, J 13.4 Hz, CH2), 2.62 (d, 1H, J 17.4 Hz, CH2), 1.95 (s,
3H, CH3); 13C NMR (CDCl3, 100 MHz): d 174.6, 170.9,

Vol. 25, No. 8, 2014
Pinatto-Botelho et al.
1335
158.8, 140.5, 131.0, 128.4, 128.3, 127.6, 125.0, 114.0, 89.2,
60.1, 55.2, 52.0, 37.0, 35.7, 22.9; ESI-MS m/z calcd. for
C21H22NaO5+: 377.1365, found: 377.1367 [M+Na]+.
(microwave radiation); IR (KBr) ν/cm-1 3500, 2950, 1778,
1726, 1510, 1248; 1H NMR (CDCl3, 400 MHz): d 6.92 (2H,
d, J 8.6 Hz, 2Ar-H), 6.74 (2H, d, J 8.6 Hz, 2Ar-H), 3.71 (3H,
s, CH3), 3.65 (3H, s, OCH3), 3.38 (1H, d, J 13.2 Hz, CH2),
2.96 (1H, d, J 18.0 Hz, 2Ar-H), 2.57 (1H, d, J 13.2 Hz, 2Ar-
H), 2.55 (1H, d, J 18.0 Hz, CH2), 1.94 (1H, dq, J 11.9 Hz,
7.3, CH2), 1.92 (1H, dq, J 14.9 Hz, 7.5, CH2), 1.70 (1H, dq,
J 14.9 Hz, 7.5, CH2), 1.55 (1H, dq, J 11.9 Hz, 7.3, CH2),
1.04 (3H, d, J 7.3 Hz, CH3), 0.90 (3H, d, J 7.5 Hz, CH3);
13C NMR (CDCl3, 100 MHz): d 174.2, 172.0, 158.7, 131.0,
127.7, 114.0, 91.2, 58.7, 55.2, 52.2, 37.3, 36.5, 26.3, 24.8,
8.30, 8.06; ESI-MS m/z calcd. for C18H25O5+: 321.1702,
found: 321.1694 [M+H]+.
trans-3-(p-Methoxy)benzyl-4,4-cyclohexyl-3-methoxy-
carbonyl-γ-butyrolactone (4)
Yields: 20% (conventional thermal heating), 80%
(microwave radiation); IR (KBr) ν/cm-1 3440, 1730, 1704,
1
1500, 1250; H NMR (CDCl3, 400 MHz): d 6.88 (d, 2H,
J 8.8 Hz, 2Ar-H), 6.74 (d, 2H, J 8.8 Hz, 2Ar-H), 3.71 (s,
3H, OCH3), 3.67 (s, 3H, CH3), 3.32 (d, 1H, J 13.4 Hz, CH2),
3.00 (d, 1H, J 17.9 Hz, CH2), 2.50 (d, 1H, J 17.9 Hz, CH2),
2.48 (d, 1H, J 13.4 Hz, CH2), 2.00 (m, 2H, CH2), 1.65 (m,
2H, CH2), 1.56 (m, 2H, CH2), 1.25 (m, 2H, CH2), 1.15 (m,
2H, CH2); 13C NMR (CDCl3, 100 MHz): d 173.9, 171.5,
158.4, 130.5, 127.3, 113.8, 87.3, 58.2, 54.9, 52.0, 36.3,
34.9, 33.2, 30.2, 25.0, 22.3, 21.8; ESI-MS m/z calcd. for
C19H24NaO5+: 355.1521, found: 355.1523 [M+Na]+.
3-(p-Methoxy)benzyl-4-methyl-4-isopentenyl-3-methoxy-
carbonyl-γ-butyrolactone (8)
Yields: 30% (conventional thermal heating), 85%
(microwave radiation); IR (KBr) ν/cm-1 3500, 2950, 1782,
1732, 1515, 1252; 1H NMR (CDCl3, 400 MHz): d 6.88 (d,
2H, J 8.8 Hz, 2Ar-H), 6.73 (d, 2H, J 8.8 Hz, 2Ar-H), 5.51
(s, 1H, =CH), 3.70 (s, 3H, OCH3), 3.68 (s, 3H, CH3), 3.39
(d, 1H, J 14.0 Hz, CH2), 2.95 (d, 1H, J 17.9 Hz, CH2), 2.46
(d, 1H, J 17.9 Hz, CH2), 2.39 (d, 1H, J 14.0 Hz, CH2), 1.83
(s, 3H, CH3), 1.77 (s, 3H, CH3), 1.64 (s, 3H, CH3); 13C NMR
(CDCl3, 100 MHz): d 174.1, 172.0, 158.7, 136.6, 130.7,
128.0, 121.0, 114.1, 88.2, 59.0, 55.2, 52.4, 38.1, 34.3, 28.0,
24.6, 19.2; ESI-MS m/z calcd. for C19H25O5+: 333.1702,
found: 333.1695 [M+H]+.
trans-3-(p-Methoxy)benzyl-4-methyl-3-methoxycarbonyl-
γ-butyrolactone (5)
Yields: 18% (conventional thermal heating), 81%
(microwave radiation); 1H NMR (CDCl3, 400 MHz): d 7.02
(d, 2H, J 8.6 Hz, 2Ar-H), 6.84 (d, 2H, J 8.6 Hz, 2Ar-H),
4.54 (q, 1H, J 6.5 Hz, CH), 3.80 (s, 3H, CH3), 3.76 (s, 3H,
OCH3), 3.35 (d, 1H, J 14.0 Hz, CH2), 2.90 (d, 1H, J 17.7 Hz,
CH2), 2.73 (d, 1H, J 14.0 Hz, CH2), 2.55 (d, 1H, J 17.7 Hz,
CH2), 1.35 (d, 3H, J 6.5 Hz, CH3); 13C NMR (CDCl3,
100 MHz): d 173.5, 171.9, 159.0, 130.9, 127.4, 114.2,81.8,
56.0, 55.3, 52.5, 40.0, 35.3, 16.6; ESI-MS m/z calcd. for
C15H18NaO5+: 301.1052, found: 301.1028 [M+Na]+.
3,4-Dimethyl-4-phenyl-3-methoxycarbonyl-γ-butyrolactone
(9)
Yields: 78% (conventional thermal heating), 0%
(microwave radiation); IR (KBr) ν/cm-1 2960, 1788, 1732,
1230; 1H NMR (CDCl3, 400 MHz): d 7.34 (s, 1H, Ar-H),
7.33 (m, 2H, 2Ar-H), 7.32 (m, 2H, 2Ar-H), 3.27 (s, 3H,
CH3), 3.12 (d, 1H, J 17.4 Hz, CH2), 2.55 (d, 1H, J 17.4 Hz,
CH2), 1.78 (s, 3H, CH3), 1.54 (s, 3H, CH3); 13C NMR
(CDCl3, 100 MHz): d 174.3, 172.5, 140.3, 128.2, 128.1,
124.5, 89.2, 54.4, 52.1, 39.9, 23.8, 19.2; ESI-MS m/z calcd.
for C14H16NaO4+: 271.0946, found: 271.0957 [M+Na]+.
trans-3-(p-Methoxy)benzyl-4-isopropyl-3-methoxycarbonyl-
γ-butyrolactone (6)
Yields: 24% (conventional thermal heating), 88%
(microwave radiation); IR (KBr) ν/cm-1 3450, 2940, 1778,
1
1734, 1500, 1250; H NMR (CDCl3, 400 MHz): d 7,00
(d, 2H, J 8.7 Hz, 2Ar-H), 6.81 (d, 2H, J 8.7 Hz, 2Ar-H),
4.13 (d, 1H, J 5.7 Hz, CH), 3.77 (s, 3H, OCH3), 3.74
(s, 3H, CH3), 3.43 (d, 1H, J 14.0 Hz, CH2), 2.80 (d, 1H,
J 17.6 Hz, CH2), 2.73 (d, 1H, J 14.0 Hz, CH2), 2.56 (d, 1H,
J 17.6 Hz, CH2), 1.96 (dq, 1H, J 5.7, 6.7 Hz, CH), 1.12 (d,
3H, J 6.7 Hz, CH3), 0.95 (d, 3H, J 6.7 Hz, CH3); 13C NMR
(CDCl3, 100 MHz): d 174.7, 172.2, 158.9, 131.0, 127.4,
114.0, 90.9, 55.2, 55.1, 52.4, 41.8, 36.5, 30.5, 20.3, 17.7;
trans-3-(p-Methoxy)benzyl-4-benzyl-3-methoxycarbonyl-
γ-butyrolactone (10)
Yields: 83% (conventional thermal heating), 90%
(microwave radiation); IR (KBr) ν/cm-1 3450, 1784, 1722,
1500, 1230; 1H NMR (CDCl3, 400 MHz): d7.32 (m, 2H, 2Ar-
H), 7.30 (m, 2H, 2Ar-H), 7.20 (m, 1H, Ar-H), 7.03 (d, 2H,
J 8.6 Hz, 2Ar-H), 6.86 (d, 2H, J 8.6 Hz, 2Ar-H), 4.55 (dd, 1H,
J 3.1 Hz, CH), 3.77 (s, 3H, OCH3), 3.76 (s, 3H, CH3), 3.33 (d,
1H, J 13.9 Hz, CH2), 2.97 (dd, 1H, J 11.6, 3.1 Hz, CH2), 2.82
(d, 1H, J 17.5 Hz, CH2), 2.81 (d, 1H, J 13.9 Hz, CH2), 2.80
+
ESI-MS m/z calcd. for C17H22NaO5 : 329.1365, found:
329.1341 [M+Na]+.
3-(p-Methoxy)benzyl-4,4-diethyl-3-methoxycarbonyl-γ-
butyrolactone (7)
Yields: 13% (conventional thermal heating), 86%

1336
Microwave-Assisted Synthesis and Antileishmanial Activity of 3-methoxycarbonyl-g-butyrolactone Derivatives
J. Braz. Chem. Soc.
(dd, 1H, J 11.6, 3.1 Hz, CH2), 2.57 (d, 1H, J 17.5 Hz, CH2);
13C NMR (CDCl3, 100 MHz): d 174.2, 171.9, 158.9, 136.4,
131.0, 129.3, 128.0, 127.2, 127.1, 114.1, 85.9, 55.6, 55.3,
52.5, 39.8, 37.2, 36.2; ESI-MS m/z calcd. for C21H22NaO5+:
377.1365, found: 377.1344 [M+Na]+.
2. Ueda-Nakamura, T.; Mendonça Filho, R. R.; Morgado-Dias,
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Antileishmanial assays
The bioassays were performed using Leishmania
amazonensis (MHOM/BR/PH8). Promastigote forms of
L. amazonensis were incubated in M199 medium (Gibco),
supplemented with L-glutamine (2 mM), NaHCO3 (10 mM),
penicillin (100 UI mL-1), streptomycin (100 μg mL-1), and
20% bovine fetal serum (Gibco).After six days of the initial
inoculation, promastigote forms (2 × 106 parasites mL-1) were
incubated in 96-well microtiter plates containing the tested
samples. The γ-butyrolactones derivatives were dissolved in
dimethyl sulfoxide (DMSO) and diluted into the medium,
to give final concentrations of 6.25, 12.5, 25.0, 50.0, 100.0,
200.0, and 400 μM. The plates were incubated at 22 °C
for 24 h, and the lysis percentage was determined by an
MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium
bromide)] (Sigma-Aldrich) colorimetric method.35 The
bioassays were performed in triplicate, using M199 medium
with 0.5% DMSO as negative control and amphotericin B
(Sigma-Aldrich) as positive control group.
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Statistical analysis
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The obtained data are represented as mean S.D.
The data were statistically analyzed by one-way ANOVA
using GraphPad Prism 5.0 software, followed by Tukey’s
multiple comparison test. The IC50 (inhibitory concentration
necessary to cause lysis of 50% of parasites) values were
calculated using sigmoid dose-response curves.
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Supplementary Information
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1H NMR, 13C NMR, IR and mass spectra of compounds,
and the curves used to calculate the IC50 values, are available
free of charge at http://jbcs.sbq.org.br as a PDF file.
18. Vasconcellos, M. C.; Rosa, R. M.; Machado, M. S.;Villela, I. V.;
Crotti, A. E. M.; Lopes, J. L. C.; Pessoa, C.; Moraes, M. O.;
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Acknowledgments
The authors would like to thank FAPESP for financial
support, and CAPES and CNPq for fellowships.
19. Keles, L. C.; Gianasi, F. M.; Souza, R. C.; Brito, B. L.; Schaab,
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Submitted on: March 21, 2014
Published online: May 23, 2014
FAPESP has sponsored the publication of this article.