Synthesis and in vitro evaluation of leishmanicidal activity of 7-hydroxy-4-phenylcoumarin derivatives

Article abstract of DOI:10.1007/s00044-016-1729-1

Eight coumarin derivatives (2–8) were synthesized from 7-hydroxy-4-phenylcoumarin 1 and were evaluated for their in vitro leishmanicidal activity against promastigote and amastigote forms of Leishmania amazonensis, as well their toxicity in murine macroph

Full text of DOI:10.1007/s00044-016-1729-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1729-1
ORIGINAL RESEARCH
Synthesis and in vitro evaluation of leishmanicidal activity
of 7-hydroxy-4-phenylcoumarin derivatives
Isael A. Rosa1 Letícia de Almeida2 Karina F. Alves2 Marcos J. Marques2
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Antônio M. Fregnan3 Claudinei A. Silva1,4 Juliana O. S. Giacoppo5
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Teodorico C. Ramalho5 Diogo T. Carvalho3 Marcelo H. dos Santos6
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Received: 29 March 2016 / Accepted: 30 September 2016
© Springer Science+Business Media New York 2016
Abstract Eight coumarin derivatives (2–8) were synthe-
sized from 7-hydroxy-4-phenylcoumarin 1 and were eval-
uated for their in vitro leishmanicidal activity against
promastigote and amastigote forms of Leishmania amazo-
nensis, as well their toxicity in murine macrophages.
Compounds 4 and 7 showed the most significant results
against promastigote forms of L. amazonensis. They were at
least three-fold more active than 1 and Compound 4 was as
effective as Amphotericin B. Compound 4, a 7-O-pre-
nylated derivative, and 7, a tetra-O-acetyl-β-D-glucopyr-
anosyl derivative, presented IC50 values of 21.35 and 10.03
µM against promastigote and IC50 values of 58.10 and
34.93 µM, respectively against amastigote forms. Further-
more, they do not cause toxicity in mammalian or Leish-
mania cells in vitro. This study shows that these coumarin
derivatives are potential prototypes for the development of
novel drugs with leishmanicidal activity.
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Keywords Coumarin derivatives Leishmaniasis
Leishmania amazonensis
Introduction
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1729-1) contains supplementary material,
which is available to authorized users.
Leishmaniasis is a neglected tropical disease (NTD) that
represents the most common illnesses of the world’s poorest
people (Hotez et al. 2012). This disease has been reported in
88 countries on five continents, Africa, Asia, Europe, North
America, and South America (van Assche et al. 2011),
among which the majority are represented by developing
countries (Bhargava and Singh 2012). The outcome of the
disease is divided into several clinical presentations: cuta-
neous leishmaniasis (CL), muco-cutaneous atleishmaniasis
(MCL), visceral leishmaniasis (VL) also known as Kala-
azar and post-Kala-azar dermal leishmaniasis (Beattie and
Kaye 2011). It is a parasitic and vector-borne disease caused
by several different species of protozoan parasites of the
genus Leishmania, which maintain their life cycle through
transmission between an insect (phlebotomine sandfly) and
a mammalian host (Kaye and Scott 2011). The parasite
exists in two distinct forms: a promastigote form found in
the vector and an amastigote form, which develops intra-
cellularly in the susceptible mammalian host (van Griens-
ven and Diro 2012).
* Claudinei A. Silva
clalvess@ufg.br
* Marcelo H. dos Santos
marcelo_hs@yahoo.com.br
1
Laboratório de Fitoquímica e Química Medicinal – LFQM,
Universidade Federal de Alfenas, Alfenas-MG 37130-000, Brazil
2
Laboratório de Parasitologia/Biologia Molecular de
Microrganismos, Universidade Federal de Alfenas, Alfenas-MG
37130-000, Brazil
3
Laboratório de Pesquisa em Química Farmacêutica – LQFar,
Faculdade de Ciências Farmacêuticas, Universidade Federal de
Alfenas, Alfenas-MG 37130-000, Brazil
4
CIEXA-JAT, Universidade Federal de Goiás, Regional Jataí, Jataí-
GO 75801-615, Brazil
5
Departamento de Química, Universidade Federal de Lavras,
Lavras-MG 37200-000, Brazil
6
Departamento de Química, Universidade Federal de Viçosa,
Viçosa-MG 36570-000, Brazil

Med Chem Res
Current therapeutics for leishmaniasis include pentava-
lent antimony (sodium stibogluconate or meglumine anti-
moniate), the polyene amphotericin B (as the deoxycholate
compound in mammalian cells (murine peritoneal
macrophages).
salt or
a
liposomal formulation), the alkyl
phosphocholine miltefosine and the aminoglycoside par-
omomycin (Croft and Olliaro 2011; Seifert et al. 2011).
However, those drugs are far from satisfying the current
demands in individuals, who live in a region where Leish-
mania infections are endemic, due to their cost (Fidalgo
and Gille 2011) variation in the sensitivity of Leishmania
species to drugs, drug-induced toxicity, and variation in
host immune response-drug interaction (Croft et al. 2006)
The search for new bioactive molecules in natural pro-
ducts has proven to be a powerful approach for discovery
and development of new drugs for the treatment of
neglected human diseases (Cragg 1997; Ndjonka et al.
2013). Thus, the use of natural compounds for the treatment
of various diseases caused by parasites, such as the causa-
tive agents of trypanosomiasis, leishmaniasis, schistoso-
miasis, lymphatic filariasis and onchocerciasis, have
stimulated the research for new agents. Among the bioac-
tive molecules some alkaloids, chalcones, lactones, tetra-
lones, saponins and coumarins have been explored.
(Akendengue et al. 1999; Cragg 1997; Ndjonka et al. 2013;
Vieira et al. 2001). Since coumarins have been prospected
for their in vitro and in vivo leishmanicidal properties,
this compound class can be exploited to discover new
effective, low toxic drugs for the treatment of leishmaniasis
(Brenzan et al. 2007; Napolitano et al. 2004; Tiuman
et al. 2012).
Materials and methods
Chemistry
The reactions were monitored by thin-layer chromatography
(TLC) on silica gel 60 F254 (Merck®). Melting points were
determined by using a Mettler melting point apparatus
(Mettler-Toledo, Leicester, UK) and were not corrected.
NMR spectra were recorded on BRUKER Avance DPX
200 or BRUKER Avance DRX 400 spectrometers. Che-
mical shifts (δ) were reported in parts per million (ppm)
with reference to tetramethylsilane (TMS). The coupling
constants (J) were reported in hertz (Hz) and signal multi-
plicities were reported as singlet (s), doublet (d), doublet of
doublet (dd), multiplet (m) and sextet (sext).
Synthesis of 7-Hydroxy-4-phenylcoumarin (1)
Equimolar amounts of resorcinol and ethyl benzoyl acetate
were stirred in perchloric acid at room temperature for 1.5 h.
The reaction mixture was then poured in crushed ice under
constant stirring; the solid product 1 thus formed was iso-
lated by suction filtration and was purified by recrystalli-
zation from hot ethanol.
Synthesis of O-alkyl (2, 3 and 4) and O-acyl (5 and 6)
A compound belonging to the class of coumarins, 7-
geranyloxycoumarin, showed significant growth inhibition
against the tropical parasite Leishmania major (Napolitano
et al. 2004). Also, prenylated coumarins auraptene, umbel-
liprenin, and galbanic acid, isolated from Ferula szowitsiana
(Apiaceae), showed in vitro antileishmanial activity against
promastigotes of L. major (Singh et al. 2014). Isopropenyl
coumarins isolated from the extracts of the leaves of G.
panamensis T. S. Elias (Rutaceae) demonstrated significant
inhibition against axenic amastigote forms of Leishmania
panamensis (Arango et al. 2010). Another coumarin-type,
(-) mammea A/BB, purified from the crude extract of
Calophyllum brasiliense leaves has shown significant
activity against both forms of Leishmania amazonensis, and
it was recently used to assess the treatment of lesions in
animal models (Brenzan et al. 2007, 2008). These studies
encouraged us to investigate the leishmanicidal activity of
new synthetic coumarin derivatives to warrant higher
potency against these human pathogens.
coumarin derivatives
Potassium carbonate (2 eq.) and the alkyl or acyl halide (1
eq.) were added to a stirred solution of 1 (1 eq.) in acetone
(Scheme 1). The reaction mixture was kept under stirring at
room temperature and was monitored by TLC (hexane/ethyl
acetate, 8:2 v/v). The reaction mixture was then extracted
with ethyl acetate (3 × 20 mL). The organic layers were
combined, dried over anhydrous sodium sulfate, filtered and
the solvent evaporated under reduced pressure. Products
were obtained in good yields and were used further without
needing purification.
Synthesis of coumarin glucosides (7 and 8)
Equimolar amounts of potassium hydroxide and 1 were
stirred in an acetone-water mixture (1:1, v/v) under room
temperature until total solubilization. Then 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranosyl bromide (0.3 eq.) was added in
acetone solution. This mixture was left under the same
reaction conditions and was monitored by TLC (dichlor-
omethane/hexane, 9:1, v/v). Acetone was then eliminated
under reduced pressure and the raw product was extracted
The aim of this work was to obtain and evaluate
the leishmanicidal activity against both forms, promasti-
gotes and amastigotes,of L. amazonensis of eight coumarin
derivatives. We also evaluate the cytoxicity of this

Med Chem Res
Scheme 1 Rote for the synthesis
of 7-Hydroxy-4-phenylcoumarin
(1), O-alkyl (2, 3 and 4),
O-acyl (5 and 6) coumarin
derivatives and coumarin
glucosides (7 and 8)
with dichloromethane (3 × 20 mL). The organic phase was
washed with NaOH (10 % w/v/ 3 × 20 mL) and then dried
with anhydrous sodium sulfate. The solvent was eliminated
under reduced pressure and the pure product 5 was obtained
after recrystallization from water/ethanol (6.5:3.5, v/v).
Following, Compound 5 was dissolved in a 1 mol L−1
solution of MeONa in dry MeOH (2 mL/100 mg of com-
pound). The solution was stirred at room temperature and
the reaction was followed by TLC (ethyl acetate/ hexane,
8:2 v/v). The solution was then made neutral by using
Amberlite® IRA-120 ion exchange resin, and the filtrate
cooled to 0 °C when the pure and solid product 6 was
obtained after filtration and cold methanol washings (Con-
chie et al. 1957; Zweckmair et al. 2014).
7.56–7.61 (m, 5H, H Ph); 13C NMR (50 MHz, DMSO-d6): δ
162.47 (C-2), 161.27 (C-7), 156.62 (C-4), 141.82(C-9),
136.22 (C-1′), 130.66 (C-5), 129.90 (C-3′ and C5′), 129.45
(C-2′ and C-6′), 129.17(C-4′), 114.29(C-3), 111.72 (C-10),
111.39 (C-6), 103.76 C-8). HRMS m/z 261.0611 C15H10O3
Na+ (calcd. 261.0528).
7-O-Methyl-4-phenylcoumarin (2) (Rizzi et al. 2006)
White solid (yield 56 %); m.p. 111–112 °C; IR (KBr) υmax
(cm−1) 3062 (υ C–H Ph); 1743.36 (υ C=O); 1207.44 (υ
C–O–C). 1H NMR (CDCl3, 400 MHz):δ 7.43–7.52 (m, 5H,
H Ph), 7.38–7.40 (d, 1H, J = 8.0 Hz H5) 6.89–6.90 (d, 1H,
J = 2.4 Hz H6) 6.78–6.81 (dd, 1H, J = 2.8, 4.0, 2.8 Hz, H8),
6.22 (s, 1H, H3), 3.89 (s, 3H, H7′ OCH3) 13C NMR
(CDCl3, 100 MHz):δ 163.17 (C-2), 161.56 (C-7), 156.39
(C-4), 156.17 (C-9), 135.95 (C-1′), 129.92 (C-4′), 129.17
(C-3′ and C5′), 128.72(C-2′ and C-6′), 128.33, (C-5) 112.90
(C-10), 112.68 (C-3), 112.24 (C-6), 101.46 (C-8), 56.14(C-
7′). HRMS m/z 275.0049 C16H12O3Na+ (calcd. 275.0684).
7-Hydroxy-4-phenylcoumarin (1) (Kuarm et al. 2012)
White crystalline solid (yield 89 %); m.p. 254 °C; IR (KBr)
υmax (cm−1) 3097 (υ O–H); 1711.22 (υ C=O); 1238.17 (υ
1
C–O–C). H NMR (DMSO-d6, 400 MHz): δ 6.19 (s,1H,
H3), 6.81–6.82 (d, 1H, J = 4.0 Hz H8), 6.84–6.85 (dd, 1H,
J = 2.0, 4.0, 2.0 Hz, H6) 7.31–7.33 (d, 1H, J = 8.0 Hz, H6),
7-O-allyl-4-phenylcoumarin (3) (El-Ansary et al. 2012)
White solid (yield 75 %); m.p. 183–184 °C; IR (KBr) υmax

Med Chem Res
(cm−1) 3079.41 (υ C–H2); 3020.58 (υ C–H Ph) 1729.21 (υ
C=O); 1124.52 (υ C–O–C) 748.39 and 623.02 (δ C–H Ph).
4), 155.19 (C-7), 153.69 (C-9), 135.46 (C-1′), 130.14 (C-4′),
129.29 (C3′ and C5′), 128.72 (C2′ and C6′), 128.17(C-5),
118.45 (C-6), 117.15(C-10), 114.82 (C-8), 111.03 (C-3), 36.50
(C-8′), 18.66 (C-9′), 13.92 (C-10′). HRMS m/z 331.1186
C19H16O4 Na+ (calcd. 331.0947).
1H NMR (CDCl3, 200 MHz):
δ 7.41–7.51(m, 6H),
6.89–6.90 (d, 1H, J = 4.0 Hz H8) 6.79–6.87 (dd, 1H,
J = 2.0; 10.0; 2.0 Hz H6), 6.22 (s, 1H), 5.95–6.14 (ddd, 1H
J = 6.0, 12.0, 18.0 Hz H8′) 5.32–5.49, (m, 2H) 4.60–4.62
(d, 2H, J = 4.0 Hz, H7). 13C NMR (CDCl3, 50 MHz) δ
162.04 (C-2), 161.60 (C-7), 156.24 (C-4), 156.16 (C-9),
135.87 (C-1′), 132.44 (C-8′), 129.93 (C-4′), 129.16 (C-3′
and C5′), 128.71 (C-2′ and C-6′), 128.32 (C-5), 118.94 (C-
9′), 113.18 (C-3), 112.95 (C-10), 112.25 (C-6), 102.29 (C-
8) , 69.60 (C-7′). HRMS m/z 301.0515 C18H14O3Na+
(calcd. 301.0841).
7-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-4-phenylcou-
marin (7) (Garazd et al. 2005) Light yellow solid (yield 61
%); m.p. 180 °C; IR (KBr) υmax (cm−1) 3471 (υ C–H2);
3078.33, 2970.38 and 2881.85 (υ C–H3); 1752.22 and
1
1739.79 (υ C=O); 1157.29 (υ C–O–C). H NMR (CDCl3,
400 MHz): δ 7.52–7.39 (m, 5H, H Ph), 7.02 (d,1H, J =
6.4Hz, H-5, 6.87), 6.87–6.84 (dd,1H, J = 2.4, 6.4, 2.4 Hz,
H-6), 6.27 (s, 1H, H3), 5.35–5.28 (m, 1H, H6″), 5.17–5.19
(m, 1H, H-4″), 4.27–4.30 (m, 1H, H-2″), 4.17–4.20 (m, 1H,
H-1″), 3.90–3.94 (m, 1H, H-5″), 2.12 (s, 3H, H-8″), 2.06 (s,
6H, H-14′; H10′), 2.04 (s, 3H, H-11″) 13C NMR (CDCl3,
100 MHz): δ170.93 (C-13″), 170.49 (C-7″), 169.73 (C-9″),
169.56 (C-12″), 161.02(C-2), 159.66 (C-9), 155.92 (C-7),
155.81 (C-4), 135.58 (C-10), 130.10 (C-3), 129.27(C-6′ and
C-2′), 128.67 (C-5′ C-3′), 128.56(C-4′), 114.87 (C-1′),
114.30 (C-8), 113.50 (C-6), 104.54 (C-5), 98.71 (C-1″),
72.93 (C-2″), 72.83 (C-3″), 71.31 (C-4″), 68.45(C-5″), 62.17
(C-6″), 21.03 (C-14″), 20.98 (C-8″), 20.91 C-10″C-11″).
HRMS m/z 591.1538 C29H28O12 Na+ (calcd. 591.1479).
7-O-Prenyl-4-phenylcoumarin (4) (Subramanyam Raju and
Subba Rao 1974) White crystalline solid (yield 57 %); m.p.
202–205 °C; IR (KBr) υmax (cm−1) 3032.10 (υ C–H2);
2862.36 and 2966.52 (υ C–H3); 1708.93 (υ C=O); 1149.57
1
(υ C–O–C). H NMR (CDCl3, 400 MHz):δ 7.43–7.52 (m,
5H, H Ph) 7.38 (d, 1H, J = 8.8 Hz H5) 6.89–6.90 (d, 1H, J =
2.4 Hz H6), 6.78–6.81 (dd, 1H, J = 4.0, 6.0, 4.0 Hz, H8),
6.21 (s, 1H, H3), 5.47–5.49 (t, 1H J = 4.0, 4.0 Hz H8′,
OCH2CHC(CH3)2) 4.58–4.60 (d, 2H J = 8.0 Hz, H7′,
OCH2CHC(CH3)2), 1.81 (s, 3H, H11′, OCH2CHC(CH3)2)
1.77 (s, 3H, H10′,OCH2CHC(CH3)2), 13C NMR (CDCl3,
100 MHz): δ 162.54 (C-2), 162.41(C-7), 156.28 (C-4),
156.24 (C-9), 139.71 (C-1), 135.93(C-9′), 129.90(C-4′),
129.15(C-3′C5′), 128.72(C-2′C6′), 128.26 (C-5), 118.93 (C-
8′), 113.32 (C-3), 112.72 (C-10), 112.05 (C-6), 102.10 (C-8),
65.75 (C-7′), 26.17 (C-10′), 18.64 (C-11′). HRMS m/z
329.0928 C20H18O3 Na+ (calcd. 329.1154).
7-O-(β-D-glucopyranosyl)-4-phenylcoumarin (8) (Garazd
et al. 2005) White solid (yield 69 %); m.p. 233–234 °C; IR
(KBr) υmax (cm–1), 3456.44 (υ O–H); 2885.51 (υ C–H2);
1
1701.22 (υ C=O); 1161.15 (υ C–O–C). H NMR (DMSO-
d6, 400 MHz): δ 7.50–7.56 (m, 5H, H Ph), 7.33–7.35 (d,
1H, J = 8.8 Hz H5) 7.13, (s, 1H, H5″) 6.97–6.99 (d, 1H J =
8.8 Hz) 6.25 (s, 1H, H3), 5.46–5.59 (m, 4H, H7″H8″H9″
H10″), 5.02–5.03 (d, 1H, J = 4,0 Hz H5″) 3.70–3.14 (m, 4H,
H6″H4″H3″ H2″), 13C NMR (DMSO-d6, 100 MHz) δ
161.25 (C-2), 160.90 (C-3), 155.98 C-9), 135.85 (C-1″),
130.64 (C-8), 129.81 (C-6′, and C-2′), 129.39 (C-5′ and C-
3′), 128.71 (C-4′), 114.70 (C-1′), 113.68 (C-7), 112.83 (C-
6), 104.60 (C-5), 100.96 (C-10), 78.13 (C-5″), 77.43 (C-2″),
74.06 (C-4), 70.56 (C-3″), 61.58 (C-6″). HRMS m/z
423.1375 C21H20O8 Na+ (calcd. 423.1056).
7-O-Acetyl-4-phenylcoumarin (5) (Subramanyam Raju and
Subba Rao 1974) White solid (yield 68 %); m.p. 211–213 °C;
IR (KBr) υmax (cm−1) 3077.48 (υ C–H3); 1762.15 and 1733.07
(υ C=O); 1142.84 (υ C–O–C). 1H NMR (CDCl3, 400 MHz): δ
7.44–7.54 (m, 5H, H Ph), 7.50–7.48 (d, 1H, J = 2.4 Hz H8),
7.19–7.20 (d, 1H, J = 2.4 Hz H5), 6.99–7.02 (dd, 1H, J = 2.4,
6.4, 2.4 Hz, H-6), 6.35 (s, 1H, H3), 2.34 (s, 1H, H-7′).13C NMR
(CDCl3, 100 MHz): δ 168.47 (C-2), 160.21 (C-7′), 155.03 (C-
4), 154.60 (C-7), 152.97 (C-9), 130.85 (C-1′), 129.58 (C-3′ and
C-5′), 128.73 (C-2′ and C-6′), 128.14 (C-4′), 127.65 (C-5),
117.84 (C-6), 114.31 (C-8), 110.47 (C-3), 20.87 (C-8′). HRMS
m/z 303.0515 C17H12O4 Na+ (calcd. 303.0634).
Biological evaluation
Leishmanicidal in vitro activity against promastigotes
7-O-butanoyl-4-phenylcoumarin (6) White solid (yield 68 %);
m.p. 198–199 °C; IR (KBr) υmax (cm–1) 3030.10 (υ C–H2);
2951.09 (υ C–H3); 1757.15 and 1739.79 (υ C=O); 1165.00 (υ
C–O–C). 1H NMR (CDCl3, 400 MHz): δ 7.43–7.53 (m, 5H, H
Ph), 7.18–7.19 (d, 1H, J = 2.0 Hz H5), 6.98–7.00 (dd,1H, J =
2.4, 8.8, 2.4 Hz, H6), 6.34 (s, 1H, H3), 2.60–2.56 (t, 2H, J =
4.0, 4.0 Hz H7′), 1.60–1.85, (sext, 2H, J = 8.0, 8.0, 8.0, 8.0, 8.0
Hz, H8′) 1.04–1.07 (t, 3H, J = 8.0, 8.0 Hz H9′) 13C NMR
(CDCl3, 100 MHz): δ 171.77 (C-6′), 160.81 (C-2), 155.62 (C-
Promastigote forms of L. amazonensis (MHOM/BR/71973/
M2269) were cultivated in Schneider’s Drosophila medium
(Sigma, USA) supplemented with 10.0 % (v/v) heat-
inactivated fetal bovine serum and 1.0 % penicillin
(10,000 UI/ mL)/streptomycin (10.0 mg/mL) (Sigma, USA).
Cells were counted in Neubauer’s chamber and adjusted to a
concentration of 1 × 106 cells/mL. Leishmania promastigotes
were re-suspended in fresh medium and harvested on a

Med Chem Res
24-well plate. Coumarin derivatives, solubilized in dime-
thylsulfoxide (DMSO), were added to promastigote culture
plates and incubated for 72 h at 25 °C. After that, the sur-
viving parasites were counted in a Neubauer’s chamber and
compared with controls for the determination of 50.0 %
inhibitory growth concentration (IC50). All tests were per-
formed in triplicate and Amphotericin B (Eurofarma) was
used as the reference drug (Maciel-Rezende et al. 2013).
1 mL of DMSO was added to each well and homogenized
for 15 min. Next, the absorbance of each individual well,
minus the control value, was calculated at 570 nm according
to the following formula.
ꢀ
ꢁ
ODcontrol ꢀ ODdrugs
% inhibition ¼
´ 100
ODcontrol
Each experiment was performed in triplicate, and the
percentage of viable cells was calculated in relation to
control cultures in the medium with only DMSO at the
concentration of 0.6 % v/v.
Leishmanicidal in vitro activity against intracellular
amastigotes
Murine peritoneal macrophages were maintained in RPMI-
1640 medium, supplemented with 10.0 % heat-inactivated
fetal bovine serum and 1.0 % penicillin (10,000 UI/mL)/
streptomycin (10 mg/mL). Cells, at 8 × 105 cells/mL con-
centration, were cultivated in 24-well plates on 13 mm glass
slides (Nunc, USA). After 30 min for adhesion, the cells
were infected with L. amazonensis promastigotes at a
multiplicity of infection of 10:1 (parasite/macrophage). The
plates were incubated in a 5.0 % CO2 air mixture at 37 °C
for 24 h. The nonphagocytosed promastigotes were then
removed while washing, and the compounds were added.
After 72 h, chamber slides were fixed in absolute methanol,
stained with Giemsa and examined under an oil immersion
objective lens of a light microscope. At least 200 macro-
phages were counted per well for calculating the percent
inhibition for the determination of IC50 value. All tests were
performed in triplicate on three different occasions and
Amphotericin B (Eurofarma) was used as the reference drug
(Pereira et al. 2010).
Statistical analysis
The leishmanicidal activity of the compounds was expres-
sed as the concentration that inhibits the growth of 50.0 %
of protozoan forms. Statistical analysis was performed using
nonlinear regression to obtain the IC50 and CC50 (cytotoxic
concentration for 50.0 % of macrophages) values, followed
by variance analyses and Tukey’s test. Differences were
significant when the p value was lower than 0.05.
Evaluation of lipophilicity by LogP (octanol/water)
and ab initio calculations
Lipophilicity values were estimated through theoretical
determination of LogP (o/w) by using the XLOGP3 pro-
gram (Urzúa et al. 2008). The QikProp program calculates
the LogP (o/w) values from regression equations using
experimental data and molecule physical descriptors
(hydrogen bond counts, atom types and charges, rotor
counts, etc.) through Monte Carlo statistical mechanics
simulations (Jorgensen and Duffy 2000) calculated lipo-
philicity expressed by LogP (o/w) of the studied compounds
are shown in Table 1. Actually, partition coefficient data are
quite useful to estimate the solubility of a solute in a specific
solvent (Yalkowsky 1999) (Fig. 1).
Evaluation of cytotoxicity
For the cytotoxicity assay a suspension of 8 × 105 cells/mL
of murine peritoneal macrophages, in RPMI 1640 medium,
supplemented with 10.0 % heat-inactivated fetal bovine
serum and 1.0 % penicillin (10,000 UI/mL)/streptomycin
(10 mg/mL) were added to each well of 24-well plates, on
13 mm glass slides. The plates were incubated in a 5.0 %
CO2 air mixture at 37 °C for cell adhesion. After 24 h, the
non-adherent cells were removed by washing with the
RPMI 1640 medium. Thus, several concentrations of
compounds (in the range of 0.05–160.0 μg/mL) were added
to the wells containing the cells. All target compounds were
solubilized in DMSO at a final concentration of 0.6 % v/v
and the plates were incubated for another 72 h. The non-
adherent cells were then removed by washing with the
RPMI 1640 medium and 50.0 μL of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to
each well at a concentration of 5.0 mg/mL, followed by
incubation for another 4 h, as described by Mossman
(Mosmann 1983). After this, the medium was removed and
The previous predictions of LogP (o/w) values were
confirmed by ab initio theoretical calculations of Lipophi-
licity values by using Gibbs free energies of solvation
of the chemical species. In this methodology, we initially
carried out
a conformational analysis using Monte
Carlo (MC)-simulated annealing (from T = 5000 K to
T = 300 K) (da Cunha et al. 2009; Hohenberg and
Kohn 1964). The selected conformations were subse-
quently refined at the DFT level. Thus, each selected
conformer was then fully optimized by DFT with the
B3LYP functional and basis set 6-311+g (d, p). The solvent
effect was taken into account using PCM calculations
(Miertus et al. 1981).

Med Chem Res
Table 1 Chemical structure and biological activity of coumarin derivatives against both forms of L. amazonensis, compared to amphotericin
Compound
Chemical structure
Promastigote
IC50 (µM)
Amastigote
IC50 (µM)
Amastigote
cytotoxicity
LogPa//Pb
1
70.10 5.56
58.19 1.71
37.37 0.45*
21.35 0.83*
93.60 3.32
83.05 2.56
91.81 1.09
58.10 1.58
>671.59
>634.27
>574.92
>522.26
3.51//3.2
HO
O
O
2
3
4
3.63//3.75
4.40//4.60
5.33//6.20
O
O
O
O
O
O
O
O
O
5
6
7
45.33 0.88*
38.63 0.89*
10.03 1.21*
112.11
2.01
>570.88
>518.92
>281.43
3.06//3.12
4.12//5.70
3.69//3.71
O
O
O
O
89.38 3.1
O
O
O
O
34.93 1.86
OAc
O
O
O
AcO
AcO
OAc
8
91.78 1.69
n.d.
>399.62
1.55//1.58
OH
O
O
O
HO
HO
OH
Amphotericin B
5.09
6.15
27.05
–
n.d. not determined
* Values marked with one asterisk means that the derivative differs statistically from their starting compound when p < 0.05 by Tukey’s test
a
Calculated lipophilicity expressed by Log P (oct/wat) using XLOGP3 program version 3.0.1
b
Calculated lipophilicity expressed by Log P (oct/wat) using Gibbs free energy of solvation from Gaussian program

Med Chem Res
Structure-Activity Relationship
8
not selected to evaluate its activity against intracellular form
of the parasite. All results are shown in Table 1.
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
Compound 4, which presented an O-prenyl group at
position C-7, as well as 7, with a penta-O-acetyl-gluco-
pyranosyl group in this same position, were the most active
coumarins against promastigote forms of L. amazonensis
(IC50 values = 21.35 and 10.03 µM, respectively). Both
compounds were also the most effective against amastigote
forms (IC50 values = 58.10 and 34.93 µM, respectively).
Moreover they presented lower cytotoxicity than the refer-
ence drug, Amphotericin B (cytotoxicity value = 27.05
µM). Compounds 1, 2, 3, 5 and 6, which present free
phenol, O-methyl, O-allyl, O-acetyl and O-butanoyl groups,
respectively, were the least active against both promastigote
and amastigote forms. The presence of free-hydroxyls β-D-
glucopyranosyl group at position C-7 does not seem to be a
promising alternative considering the leishmanicidal activ-
ity, since Compound 8 was not active against promastigotes
of L. amazonensis. On the other hand, it was observed that
per-O-acetyl glucopyranosyl moiety, present in Compound
7, had an important effect on the desired biological action.
Comparing the chemical structures of Compounds 7 and 8,
one may suggest that lipophilicity is an important issue for
the leishmanicidal activity of the Compound 7 (Fig. 1).
Pereira and co-workers showed that benzophenones with
higher lipophilicity present higher activities against Leish-
mania species, which could in fact be related to the ease of
entering the macrophage and reaching the parasite (Pereira
et al. 2010). Maciel-Rezende and co-workers recently evi-
denced that an increase in the size of side chain substituents
led to a proportional increase in lipophilicity of benzophe-
none derivatives and leishmanicidal potency (Maciel-
Rezende et al. 2013). Indeed, these results indicate that
changing the substituents in the coumarin nucleus to
influence on the polarity of the molecule also changes its
biological activity. The results of this study suggest that
some chemical features, as lipophilicity, in the synthesized
coumarins are relevant for the leishmanicidal activity, but
further investigations must be performed to confirm this
hypothesis.
Compound
1
2
5
6
3
4
7
0,0
0
1
2
3
4
5
6
7
Log P (o/w)
Fig. 1 Structure–activity relationship by IC50 values to promastigote
forms and lipophilicity expressed by Log P (o/w) by using Gibbs free
energy of solvation from Gaussian program
Results and discussion
In order to prepare a variety of coumarin derivatives,
Coumarin 1 was prepared as the starting compound. As
depicted in Scheme 1, Compound 1 was synthesized via
Pechmann reaction (Pechmann 1884), which is the most
widely used method to obtain coumarins. This reaction
involves the use of simple starting materials, such as phe-
nols and β-ketoesters in acidic medium and generally works
well at room temperature. The advantages of this procedure
are short reaction times, operational simplicity and high
product yields. Many coumarins are readily purified by
crystallization, such as those synthesized in this work,
which were recrystallized from a mixture of ethanol/water
(7:3 v/v) furnishing high purity products without using
column chromatography purification. All derivatives were
obtained in good yields. The structural assignments of all
1
synthesized compounds were based on H and 13C NMR,
IR and mass spectra analyses.
A series of synthetic coumarins is shown here that were
evaluated in vitro against both forms of L. amazonensis and
the results support the leishmanicidal potential of this class
of compounds. The studies reported by Kappagoda and co-
workers with antiparasitic coumarin derivatives were
focused on coumarins bearing a 5,7-dihydroxy skeleton, a
phenyl group attached to C-4 and prenyl or acyl substituents
attached to C-6 or C-8 (Kappagoda et al. 2011). In this
study, the phenyl group attached to the C-4 position was
maintained and the nature of substituents at the C-7 position
was varied. The most promising derivatives were 4 and 7,
which exhibited good activity against both promastigote
and amastigote forms of Leishmania. Concentrations up to
281.43 µM showed no cytotoxicity and the same profile was
observed in tests beyond these concentrations. Derivative 8
showed high IC50 value against promastigote forms but was
In order to shed some light on the experimental results, a
computational study was developed to evaluate the physico-
chemical properties of the synthesized compounds to check
the influence of the nature of the environment on the sta-
bility of the molecules and to obtain data that could allow
proposing the biological action mechanism of these drugs.
In this regard, we carried out partition coefficient calcula-
tions. Currently, the CCl4/water and n-octanol/water
biphasic systems are the mimic models most widely used
for the water-membrane system (Liu et al. 2011). It should
be kept in mind that the partition coefficient is a very
important issue of the Lipinski’s rule-of-five. In 1997,
Lipinski and colleagues postulated some structural and

Med Chem Res
electronic features that are associated with orally active
drugs in humans, which is denominated as the “rule-of-five”
(Lipinski et al. 2012).
to better understand the action mechanism of these mole-
cules and their effectiveness against amastigote forms of
Leishmania.
In fact, from the theoretical side, a consolidate metho-
dology for the LogP calculation is to use information of the
free energy of solvation in water and in octanol or CCl4 to
estimate the partition coefficient. From Gibbs free energies
of solvation in two different phases at temperature T, one
can calculate the corresponding partition coefficient,
according to the following expression: where ΔhydG is the
hydration free energy and ΔsolvG is the Gibbs free energy of
solvation in 1-octanol.
Acknowledgments The authors are grateful to Rede Mineira de Quí-
mica (RQ-MG) (CEX-RED-00010-14), Fundação de Amparo à Pesquisa
do Estado de Minas Gerais (FAPEMIG), Financiadora de Estudos e
Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de
Ensino Superior (CAPES) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) for the financial support.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Δ
hydG ꢀ ΔsolvG
2:303 RT
LogP ¼
References
In accordance with Table 1, Compounds (4) and (7)
reveal the high hydrophobicity for both theoretical meth-
odologies used. Interestingly, those compounds were the
most active coumarins against promastigote and amastigote
forms of L. amazonensis. Therefore, this pattern clearly
points out and reinforces the proposition regarding the
biological action of phenylcoumarins. In fact, the interac-
tion with the membrane likely plays an important role in the
biological action mechanism of these drugs.
The evaluation of the coumarin derivatives in relation to
toxicity in mammalian cells showed no significant toxicity.
These results indicate that Compounds 4 and 7 showed the
most significant results against promastigote and amasti-
gotes forms of L. amazonensis in vitro and no toxicity.
Coumarin derivatives may be potential new drugs for the
treatment of leishmaniasis that could become available for
low-income populations. This study is part of a continued
search for new drugs with high activity and few side effects
that can be used to treat diseases associated with protozoan
parasites, such as leishmaniasis.
Akendengue B, Ngou-Milama E, Laurens A, Hocquemiller R (1999)
Recent advances in the fight against Leishmaniasis with natural
products. Parasite 6:3–8
Arango V, Robledo S, Séon-Méniel B, Figadère B, Cardona W, Sáez
J, Otálvaro F (2010) Coumarins from Galipea panamensis and
their activity against Leishmania panamensis.
J Nat Prod
73:1012–1014
Beattie L, Kaye PM (2011) Leishmania–host interactions: What has
imaging taught us?. Cell Microbiol 13:1659–1667
Bhargava P, Singh R (2012) Developments in diagnosis and antil-
eishmanial drugs. Interdiscip Perspect Infect Dis 2012:626838
Brenzan MA, Nakamura CV, Dias Filho BP, Ueda-Nakamura T,
Young MCM, Cortez DAGC (2007) Antileishmanial activity of
crude extract and coumarin from calophyllum brasiliense leaves
against Leishmania amazonensis. Parasitol Res 101:715–722
Brenzan MA, Nakamura CV, Dias Filho BP, Ueda-Nakamura T,
Young MCM, Corrêa AG, Alvim Júnior J, dos Santos AO,
Cortez DAGC (2008) Structure–activity relationship of (-)
Mammea A/BB derivatives against leishmania amazonensis.
Biomed Pharmacother 62:651–658
Conchie J, Levvy GA, Marsh CA (1957) Methyl and phenyl glyco-
sides of the common sugars. Adv Carbohydr Chem 12:157–187
Cragg GM (1997) Natural products in drug discovery and develop-
ment. J Nat Prod 60:52–60
Croft SL, Olliaro P (2011) Leishmaniasis chemotherapy-challenges
and opportunities. Clin Microbiol Infect 17:1478–1483
Croft SL, Sundar S, Fairlamb AH (2006) Drug resistance in Leish-
maniasis. Clin Microbiol Rev 19:111–126
Conclusions
da Cunha EFF, Sippl W, Ramalho TC, Antunes OAC, de Alencastro
RB, Albuquerque MG (2009) 3D-QSAR CoMFA/CoMSIA
models based on theoretical active conformers of HOE/BAY-793
analogs derived from HIV-1 protease inhibitor complexes. Eur J
Med Chem 44:4344–4352
El-Ansary SL, Hussein MM, Rahman DEA, Hamed MIAL (2012)
Synthesis and docking studies of furobenzopyrones of potential
antimicrobial and photochemotherapeutic activities. Life Sci J
9:1114–1125
Fidalgo LM, Gille L (2011) Mitochondria and trypanosomatids:
Targets and drugs. Pharmaceut Res 28:2758–2770
Garazd YL, Garazd MM, Khilya VP (2005) Modified coumarins. 19.
Synthesis of Neoflavone D-Glycopyranosides. Chem Nat Compd
41:663–668
Eight coumarin derivatives (Compounds 1–8) were pre-
pared in good chemical yields and their biological activity
against L. amazonensis was tested. The most distinguished
results seem to be related to Compounds 4 and 7 against
both promastigote and amastigote forms of L. amazonensis.
These results indicated that probability that the variation of
the substituents on the coumarin scaffold seems to influence
on the polarity of the molecule and its biological activity.
The potency could in fact be related to the facility to
penetrate the macrophage and reach the parasite. Our the-
oretical findings from LogP (o/w) calculations support this
proposal. Thus, these series of derivatives could be explored
as candidates for development of new drug prototypes
against leishmaniasis. Further studies should be conducted
Hohenberg P, Kohn W (1964) Inhomogeneous electron gas. Phys Rev
136:B864–871
Hotez PJ, Savioli L, Fenwick A (2012) Neglected tropical diseases of
the middle East and North Africa: review of their prevalence,

Med Chem Res
distribution, and opportunities for control ed. Serap Aksoy. PLoS
Rizzi E, Dallavalle S, Merlini L, Pratesi G, Zunino F (2006) Short
synthesis of cytotoxic 4‐arylcoumarins. Synth Commun
36:1117–1122
Seifert K, Munday J, Syeda T, Croft SL (2011) In vitro interactions
between sitamaquine and Amphotericin B, sodium stibogluco-
nate, miltefosine, paromomycin and pentamidine against Leish-
mania Donovani. J Antimicrob Chemother 66:850–854
Singh N, Mishra BB, Bajpai S, Singh RK, Tiwari VK (2014) Natural
product based leads to fight against Leishmaniasis. Bioorg Med
Chem 22:18–45
Subramanyam Raju M, Subba Rao NV (1974) Search for physiolo-
gically active compounds. Part XXIV. synthesis of 7, 8-Furano,
Pyrono and 3-Methyl-4-Phenylcoumarins. Proc Indian Acad Sci
Sect A 79:223–229
Tiuman TS, Brenzan MA, Ueda-Nakamura T, Dias Filho BP,
Cortez DGC, Nakamura CV (2012) Intramuscular and topical
treatment of cutaneous Leishmaniasis lesions in mice infected
with Leishmania amazonensis using Coumarin (-) Mammea A/
BB. Phytomedicine 19:1196–1199
Urzúa A, Echeverría J, Rezende MC, Wilkens M (2008) Antibacterial
properties of 3 H-spiro[1-benzofuran-2,1′-cyclohexane] deriva-
tives from Heliotropium Filifolium. Molecules 13:2385–2393
v. Pechmann H (1884) Neue Bildungsweise Der Cumarine. Synthese
Des Daphnetins I. Ber Deut Chem Ges 17:929–936
van Assche T, Deschacht M, da Luz RAI, Maes L, Cos P (2011)
Leishmania-macrophage interactions: Insights into the redox
biology. Free Radic Bio Med 51:337–351
Negl Trop Dis 6:e1475
Jorgensen WL, Duffy EM (2000) Prediction of drug solubility from
Monte Carlo simulations. Bioorg Med Chem Lett 10:1155–1158
Kappagoda S, Singh U, Blackburn BG (2011) Antiparasitic therapy.
Mayo Clin Proc 86:561–583
Kaye P, Scott P (2011) Leishmaniasis: complexity at the host-
pathogen interface. Nat Rev Microbiol 9:604–615
Kuarm BS, Madhav JV, Rajitha B (2012) Xanthan sulfuric acid: an
efficient and recyclable solid acid catalyst for Pechmann con-
densation. Synth Commun 42:1770–1777
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2012) Experi-
mental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv
Drug Deliv Rev 64(SUPPL):4–17
Liu X, Testa B, Fahr A (2011) Lipophilicity and Its relationship with
passive drug permeation. Pharmaceut Res 28:962–977
Maciel-Rezende CM, de Almeida L, Costa ED’M, Pires FR, Alves KF,
Viegas Junior C, Dias DF, Doriguetto AC, Marques MJ, dos
Santos MH (2013) Synthesis and biological evaluation against
Leishmania amazonensis of a series of Alkyl-substituted benzo-
phenones. Bioorg Med Chem 21:3114–3119
Miertus SE, Scrocco E, Tomasi J (1981) Electrostatic interaction of a
solute with a continuum. A direct utilization of AB initio mole-
cular potentials for the prevision of solvent effects. Chem Phys
55:117–129
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63
van Griensven J, Diro E (2012) Visceral leishmaniasis. Infect Dis Clin
N Am 26:309–322
Napolitano HB, Silva M, Ellena J, Rodrigues BDG, Almeida ALC,
Vieira PC, Oliva G, Thiemann OH (2004) Aurapten, a Coumarin
with growth inhibition against Leishmania major promastigotes.
Braz J Med Biol Res 37:1847–1852
Vieira PC, Mafezoli J, Pupo MT, Fernandes JB, da Silva MFGF, de
Albuquerque S, Oliva G, Pavão F (2001) Strategies for the iso-
lation and identification of trypanocidal compounds from the
rutales. Pure Appl Chem 73:617–622
Ndjonka D, Rapado LN, Silber AM, Liebau E, Wrenger C (2013)
Natural products as a source for treating neglected parasitic dis-
eases. Int J Mol Sci 14:3395–3439
Pereira IO, Marques MJ, Pavan AL, Codonho BS, Barbiéri CL, Beijo
LA, Doriguetto AC, D’Martin EC, dos Santos MH (2010)
Leishmanicidal activity of benzophenones and extracts from
Garcinia Brasiliensis Mart. fruits. Phytomedicine 17:339–345
Yalkowsky SH (1999) Solubility and solubilization in aqueous media,
1st ed. American Chemical Society, Washington, DC
Zweckmair T, Becker M, Ahn K, Hettegger H, Kosma P, Rosenaua T,
Potthast A (2014) A novel method to analyze the degree of
acetylation in biopolymers. J Chromatogr A 1372:212–220