The antileishmanial potential of C-3 functionalized isobenzofuranones against Leishmania (Leishmania) Infantum Chagasi

Article abstract of DOI:10.3390/molecules201219857

Leishmaniases are diseases caused by protozoan parasites of the genus Leishmania. Clinically, leishmaniases range from cutaneous to visceral forms, with estimated global incidences of 1.2 and 0.4 million cases per year, respectively. The treatment of thes

Full text of DOI:10.3390/molecules201219857

Article
The Antileishmanial Potential of C-3 Functionalized
Isobenzofuranones against Leishmania (Leishmania)
Infantum Chagasi
Wagner Luiz Pereira 1,†, Raphael de Souza Vasconcellos 2,3,†, Christiane Mariotini-Moura 2,3
Rodrigo Saar Gomes 4, Rafaela de Cássia Firmino 2,3, Adalberto Manoel da Silva 1,
Abelardo Silva Júnior 5, Gustavo Costa Bressan 6, Márcia Rogéria Almeida 6,
,
Luís Carlos Crocco Afonso 4, Róbson Ricardo Teixeira 1, ,† and Juliana Lopes Rangel Fietto 3,6, ,†
* *
Received: 5 November 2015 ; Accepted: 8 December 2015 ; Published: 14 December 2015
Academic Editor: Derek J. McPhee
1
Departamento de Química, Universidade Federal de Viçosa, Av. P.H. Rolfs, S/N, Viçosa, MG 36.570-900,
Brazil; wagner.pereira@ufv.br (W.L.P.); adalbert31@hotmail.com (A.M.S.)
Departamento de Biologia Geral, Universidade Federal de Viçosa, Av. P.H. Rolfs, S/N, Viçosa,
MG 36.570-900, Brazil; raphael.biomed@gmail.com (R.S.V.); chrismariotini@yahoo.com.br (C.M.-M.);
rafaelacfirmino@gmail.com (R.C.F.)
Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDi),
Instituto de Física de São Carlos, Av. Trabalhador São Carlense, 400, Caixa Postal 369, São Carlos,
SP 13.560-970, Brazil
Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas—ICEB/NUPEB,
Campus do Morro do Cruzeiro, Universidade Federal de Ouro Preto, Ouro Preto, MG 35.400-000, Brazil;
rodrigosaar@nupeb.ufop.br (R.S.G.); afonso@nupeb.ufop.br (L.C.C.A.)
Departamento de Veterinária, Universidade Federal de Viçosa, Av. P.H. Rolfs, S/N, Viçosa, MG 36.570-900,
Brazil; aberlardo.junior@ufv.br
2
3
4
5
6
Departamento de Bioquímica e Biologia Molecular, Av. P.H. Rolfs, S/N, Viçosa, MG 36.570-900, Brazil;
gustavo.bressan@ufv.br (G.C.B.); marcia@ufv.br (M.R.A.)
*
Correspondence: robsonr.teixeira@ufv.br (R.R.T.); jufietto@ufv.br (J.L.R.F.); Tel.: +55-31-3899-3209 (R.R.T.);
+55-31-3899-2374 (J.L.R.F.); Fax: +55-31-3899-2373 (J.L.R.F.)
†
These authors contributed equally to this work.
Abstract: Leishmaniases are diseases caused by protozoan parasites of the genus Leishmania.
Clinically, leishmaniases range from cutaneous to visceral forms, with estimated global incidences
of 1.2 and 0.4 million cases per year, respectively. The treatment of these diseases relies on multiple
parenteral injections with pentavalent antimonials or amphotericin B. However, these pharmaceuticals
are either too toxic or expensive for routine use in developing countries. These facts call for safer,
cheaper, and more effective new antileishmanial drugs. In this investigation, we describe the results of
the assessment of the activities of a series of isobenzofuran-1(3H)-ones (phtalides) against Leishmania
(Leishmania) infantum chagasi, which is the main causative agent of visceral leishmaniasis in the New
World. The compounds were tested at concentrations of 100, 75, 50, 25 and 6.25
72 h. After 48 h of treatment at the 100 M concentration, compounds and
viability to 4% and 6%, respectively. The concentration that gives half-maximal responses (LC50) for
the antileishmanial activities of compounds and against promastigotes after 24 h were 60.48 and
65.93 M, respectively. Additionally, compounds and significantly reduced parasite infection
in macrophages.
µM over 24, 48, and
µ
7
8 decreased parasite
7
8
µ
7
8
Keywords: Leishmania (L.) infantum chagasi; visceral leishmaniasis; isobenzofuranones; phthalides;
in vitro leishmanicidal activity
Molecules 2015, 20, 22435–22444; doi:10.3390/molecules201219857
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Molecules 2015, 20, 22435–22444
1. Introduction
The leishmaniases are parasitic diseases that are caused by protozoa of the genus Leishmania. These
parasites belong to the order Kinetoplastida and family Trypanosomatidae, which includes species of
obligatory intracellular protozoan parasites. Common hosts include rodents, canines and primates,
including humans [1,2]. Leishmania infections can present with different clinical manifestations
depending on the parasite species and the host-parasite relationship. Visceral leishmaniasis (VL)
is a progressive and frequently fatal disease caused by Leishmania (Leishmania) infantum (synonymous
with Leishmania (Leishmania) infantum chagasi) [3]. VL is one of the most neglected diseases in the world
and affects millions of people worldwide [ ]. Approximately 500,000 new cases occur annually, and
4
90% of all VL cases occurring in India, Bangladesh, Sudan, South Sudan, Ethiopia and Brazil [4].
Currently, several investigations are being conducted to search for alternative treatments for
leishmaniases [
5] because of the small number of available drugs and the development of resistance or
decreased sensitivity of parasite strains to existing treatments that are utilized for human therapy [
5].
These studies have sought new methods and targets for diagnosis, new vaccine candidates and new
rationally designed drugs that can be applied not only in humans, but also in dogs because canines
are considered the major reservoirs of several species of Leishmania in the home environment and
outdoors [6,7].
Historically, chemotherapy for leishmaniases has relied on the use of pentavalent antimonial
drugs, such as N-methylglucamine antimoniate, which is one of the most widely used drugs [
However, the use of this drug has limited clinical potential due to the occurrence of serious side effects
and high incidence of disease recurrence [ ]. Pentamidine is another antileishmanial agent, but it is
8].
9
inadequate as a first-line treatment because of its high toxicity [10]. Amphotericin B has been used
as a second-choice drug in the treatment of leishmaniasis since the 1960s, but the rate of resistance
against this drug is high [10]. Two other examples of antileishmanial compounds are miltelfosine
and paromomocyn. The major limitation of miltefosine is teratogenicity and this precludes its use in
women of child-bearing age [11,12]. The most common side effect associated with paromomycin is the
ototoxicity, as well as liver function problems. In patients treated with the ointment formulation skin
rashes, local pruritus and burs have been the most common side effects encountered [13] Additionally,
the drugs that are available for leishmaniasis chemotherapy are, in general, expensive. Another
alternative is combination therapy, which has been used to decrease the duration and price of treatment
and parasite resistance [14]. The aforementioned problems illustrate a pressing need to develop new
antileishmanial drugs.
Figure 1. Example isobenzofuran-1(3H)-ones and their biological activities.
Compounds containing a benzene ring fused to a
γ-lactone ring are termed isobenzofuran-
1(3H)-ones (also known as phthalides). This unit is present in the structure of several natural
products [15
,16]. In particular, isobenzofuran-1(3H)-ones functionalized at the C-3 position stand
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Molecules 2015, 20, 22435–22444
out for their biological activities, which include antiplatelet [17], GABA receptor antagonist [18],
glutamate-induced cytotoxicity inhibitor [19], phytotoxicity [20], cytotoxicity [21,22] and antimicrobial
activity [23]. Examples of isobenzofuranones and their associated bioactivities are shown in Figure 1,
where the isobenzofuran-1(3H)-one unit is highlighted in blue.
Considering their various biological activities and our research interest in isobenzofuran-1(3H)-
ones [22,24], we herein describe the results of our investigation of the antileishmanial activities of C-3
functionalized isobenzofuranones on Leishmania (Leishmania) infantum chagasi.
2. Results and Discussion
It is well known that amphotericin B, which is used for the treatment of visceral leishmaniasis,
is an effective antibiotic that also possesses antifungal activity [10]. Because previous studies have
demonstrated that isobenzofuranones display antifungal activities [25
,26], we hypothesized that
isobenzofuranones 2–11 (Scheme 1) would also exhibit antileishmanial activities.
Scheme 1. Syntheses of isobenzofuranones 2–11.
Thus, we evaluated the antileishmanial activities of isobenzofuranones
against the pathogenic agent of visceral leishmaniasis Leishmania (L.) infantum chagasi (syn. Leishmania
infantum). Because of limited amount of compound available during the progress of the biological
2–5 and 7–11 (Scheme 1)
6
assays, this compound was not evaluated. The first approach involved evaluations of the direct actions
of these compounds on the promastigote form of the parasite.
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Molecules 2015, 20, 22435–22444
We observed that all of the compounds were toxic to the parasite (Figure 2). After 24 h
of treatment, the 100
µM concentrations of compounds 7 and 8 significantly reduced parasite
viability by over 50% (Figure 2A). After 48 and 72 h, all of the isobenzofuranones were effective
in decreasing parasite viability (Figure 2B,C). Additionally, 100% parasite death was observed after
72 h of treatment with compounds
activity values of compounds
7
and
8
(Figure 2C). Table 1 illustrates the LC50 antileishmanial
11 against the promastigote form of L. (L.) infantum
2
–
5
and
7
–
(syn. L. chagasi). The most active compounds, i.e.,
and 65.93–58.46 µM, respectively.
7 and 8, exhibited LC50 values between 60.48–59.75
Thus, the greatest antileishmanial efficacies against the promastigotes were observed for
isobenzofuranones and ; importantly, these compounds share the common feature of an aromatic
ring that is directly attached at the C-3 position of the isobenzofuranone nucleus. Moreover, this
7
8
1
1
aromatic ring is oxygenated at the C-2 /C-6 positions and exhibits an alkyl group (isopropyl) attached
1
to C-4 (see Scheme 1 for numbering). Thus, these structural features seem to be important in terms of
the antileishmanial activities of the investigated compounds. Notably, a previous investigation of the
cytotoxicities of compounds
most active compounds exhibited structural features similar to these aforementioned features. Indeed,
isobenzofuranone was one of the most active compounds against K562 exhibiting an LC50 of 2.79
and was even more potent than the anticancer agent etoposide (VP-16), which was used as a positive
2–11 against the K562 and U937 cancer cell lines [22] revealed that the
7
µM
control. Due to the superior antileishmanial activities of compounds
these compounds were selected for further evaluation.
7
and 8 after 24 h (Figure 2A),
(A)
(B)
(C)
Figure 2. Leishmanicidal activities of isobenzofuranones
viability assays were used to evaluate the promastigotes of L. infantum after 24 (
2
–
5
and
7
–
11 against promastigotes. MTT
); 48 ( ); and 72 h (C)
A
B
of treatment. The results are presented as the averages and the SDs of three independent experiments
that were performed in triplicate. The * indicates significant differences compared with the control at
p < 0.05.
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Molecules 2015, 20, 22435–22444
Table 1. Antileishmanial efficacies of isobenzofuranones against promastigotes.
LC50 (µM) a
Compound
24 h
48 h
72 h
2
3
4
5
7
8
9
10
11
74.54
>100
>100
>100
60.48
65.93
75.01
74.27
>100
65.55
65.73
65.69
61.69
62.72
63.90
54.78
61.84
30.79
58.96
59.08
59.20
58.96
59.75
58.46
60.51
60.53
60.17
a
The LC50 values were calculated at 24, 48 and 72 h of treatment. The data are representative of three
independent experiments.
The second investigation examined the toxicities isobenzofuranones 7 and 8 against macrophages,
which are the main cells that are infected by Leishmania parasites in leishmaniasis diseases. We observed
low levels of toxicity at high isobenzofuranones concentrations (100 M) and selected a lower dose to
perform the infection assays (75 M). Both compounds decreased the levels of infection and reduced
µ
µ
the numbers of intracellular parasites per infected cell. Together, these results suggest that these
compounds may affect both infection capability and intracellular proliferation. In an investigation of
the possible mechanism of action of C-3 functionalized isobenzofuranones against the HL-60 cancer
cell line, Logrado and collaborators found that the active compounds exerted their effect on HL-60 cells
via direct DNA damage or the generation of free radicals [21]. Although the mechanisms of action of
the most active compounds were not investigated here, it is possible that the antileishmanial activities
that were observed for compounds
7 and 8 are also related to these mechanisms. Conceivably, due to
the intense replication of this parasite during acute infection, it could be strongly affected by DNA
damage or free radicals. Indeed, reports in the literature have indicated that anti-cancer drugs that
affect cellular proliferation can be used as anti-microbial agents because both cell types share common
behaviors concerning their high proliferative capabilities [27–29]. Indeed, a recent work evidenced the
antileishmanial actions of other two isobenzofuranone derivatives and indicated that these actions
were due to the induction of reactive oxygen species (ROS)-mediated apoptosis-like cell death and the
inhibition of topoisomerases [30].
To assess whether the compounds tested on the parasites affected cell viability, RAW macrophages
were maintained for 24, 48 and 72 h in the presence of compounds
ranged from 6.25 to 100 M. There were no significant differences in cellular viability after 24 h with
the tested concentrations of compound (Figure 3A). However, the viabilities of the macrophages were
significantly affected after 48 and 72 h of treatment at 100 M (Figure 3B,C). Regarding compound
M concentration affected the viability after 24 h (Figure 3A). However, concentrations of
M and lower did not seem to affect the macrophages (Figure 3). Based on these results, the 75
7 and 8 at concentrations that
µ
7
µ
8
, the 100 µ
75
µ
µM
concentration was selected for the infection assays.
Next, macrophages were infected with L. (L.) infantum chagasi and subsequently treated with
isobenzofuranones and at 75 M for 24 h. Regarding compound , a reduction of the number of
7
8
µ
7
infected cells by approx. 65% was observed (Figure 4A). This effect was even more pronounced for
compound 8, which reduced the number of infected cells by 76% (Figure 4B).
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Molecules 2015, 20, 22435–22444
(A)
(B)
(C)
7 and 8 to RAW macrophages. MTT viability assays were
Figure 3. Toxicities of isobenzofuranones
performed after 24 h (A); 48 h (B) and 72 h (C) of treatment. The results are presented as the averages
and the SDs of three independent experiments that were performed in triplicate. The * indicates
significant differences compared with the control at p < 0.05.
Figure 4. Leishmanicidal activities of compounds
The percentages of infected macrophages ( ); and the numbers of intracellular amastigotes per
100 cells ( ) were assessed. The first column (without DMSO) is the control after 3 h of infection
and before the addition of 1% DMSO. The second column is the sample assay after 24 h of infection
in the presence of DMSO. The last two columns are the samples with isobenzofuranones and at
the concentration of 75 M in 1% DMSO. The data are presented the averages and the SDs of three
7 and 8 in infected RAW macrophages.
A
B
7
8
µ
independent experiments that were performed in triplicate. The * indicates significant differences
compared with the control at p < 0.05.
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Molecules 2015, 20, 22435–22444
Importantly, although compound 7 did not affect the viability of the macrophages as assessed with
the MTT method, we observed visible changes in their morphologies (Figure 5B). This phenotype was
not observed for compound , which did not cause any visible changes in the overall cell morphology
8
(Figure 5C). These findings suggest that the compounds
which should be better investigated in future studies.
7 and 8 differ in their mechanism of action,
Figure 5. Representative micrographs showing RAW macrophages infected with L. (L) infantum chagasi
that were or were not treated compounds or . The infected macrophages were treated for 24 h with
1% DMSO (control) ( ); 75 M of compound ); or 75 M of compound ). The pictures are
7
8
A
µ
7
(
B
µ
8 (C
representative of all of the experiments, which were conducted as independent triplicates. The upper
pictures are magnified at 100x, and the lower panels are zoomed in images of the selected parts of the
upper pictures. The black arrows indicate intracellular amastigotes.
3. Experimental Section
3.1. Syntheses
The syntheses of the isobenzofuranones were performed as previously reported [22]). Compounds
2
–
11 were prepared using condensation, aromatization and acetylation reactions (Scheme 1). Briefly,
DBU-mediated condensation reactions between commercially available phthalaldehydic acid ( ) and
cyclic and acyclic 1,3-diketones produced compounds 10 and 11. The aromatization of
phthalides and produced the aromatic derivatives . The acetylation of compounds and
led to the preparation of substances and . The structural characterizations of these compounds
1
2, 5, 6, 9,
2
6
3
and
7
3
7
4
8
have been described previously [19]. Stock solutions of the compounds were prepared by dissolving
them in 100% DMSO. The solutions were maintained at 4 ˝C until use. Due to the limited amount of
compound 6, this phtalide was not evaluated.
3.2. Obtaining of Promastigotes Parasites and Leishmanicidal Activity Evaluation
L. (L.) infantum chagasi strain M2682 was grown in Grace’s insect medium supplemented with
10% heat-inactivated fetal calf serum, 2 mM L-glutamine and 100 U/mL penicillin G potassium at
˝
pH 6.5 and 26 C. On the third day (exponential phase), the promastigote parasites were washed
with culture medium, plated at 1 M of
106 Leishmania/mL without (control) or with 100
isobenzofuran-1(3H)-ones and
11, and incubated at 26 ˝C for 24, 48 and 72 h. After incubation,
ˆ
µ
2–
5
7–
1 mM of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added and followed
˝
by an incubation for 2 h at 26 C. Subsequently, the parasites were centrifuged at 1400
ˆ
g, the
supernatants were discarded, and 100
µL of dimethylsulfoxide (DMSO) was added to the pellet, which
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Molecules 2015, 20, 22435–22444
was subsequently stirred for 15 min and reading at 595 nm. The calculations of the viable parasites
were performed using the following equation:
absorbance o f the test ´ absorbance o f the plate
ˆ 100
absorbance o f the control ´ absorbance o f the plate
3.3. Cultivation of Macrophages and Cytotoxicity Evaluation
The mouse leukemic macrophage cell line RAW (RAW) were grown in Roswell Park Memorial
Institute Media (RPMI) supplemented with 10% (v/v) heat-inactivated fetal calf serum (LGC Bio®,
Petaluma, CA, USA), 100 U/mL penicillin (Sigma®, St. Louis, MO, USA), and 100
µg/mL streptomycin
˝
(Sigma®) in flasks of 75 or 150 or 300 cm3, which were maintained at 37 C in an atmosphere
with 95% humidity and 5% CO2. Thereafter, the macrophages were transferred to 96-well plates
at a concentration of 1
ˆ
106 macrophages/mL. The compounds were added to the culture medium at
˝
different concentrations and incubated at 37 C (5% CO2) for 24, 48 and 72 h. After incubation, 1 mM
of MTT was added in each well, and the cultures were returned to the incubator for 2 h. Next, the
culture medium was discarded, and 100 µL DMSO was added, and the wells were stirred for 15 min
and subsequently read at 595 nm. The calculations of the percentages of viable cells were performed
as described in the previous section.
3.4. Infection Assays
The macrophages were left to adhere in the 24-well plate (which included a sterile coverslip for
each well) for 90–120 min at a concentration of 1
remove the non-adhered cells. For infection, 3–5
ˆ
106/mL. The cells were washed twice with PBS to
ˆ
106 parasites/mL suspended in RPMI with 10% fetal
calf serum were added to macrophage cells, which were then left to be infected for 3 h (5 Leishmania
per macrophage). Next, the wells were washed twice with PBS to remove the parasites that did not
infect macrophages. Subsequently, RPMI with 10% fetal calf serum containing the compounds at
the different concentrations specified in the figures was added. All samples, including the controls,
contained 1% DMSO (final DMSO concentration in the working solution). The cultures were incubated
for 24 or 48 h in the same conditions described above. After incubation, the cells were fixed on
coverslips with methanol and stained with Fast Panoptic FAST kit (Panreac AppliChem, Darmstadt,
Germany) according to the manufacturer’s recommendations. All assays were performed in triplicate,
and the stained coverslips were analyzed by light microscopy. We evaluated the numbers of infected
macrophages and the numbers of intracellular amastigotes per 100 macrophages.
4. Conclusions
The potential of diverse C-3 substituted isobenzofuran-1(3H)-ones as antileishmanial agents
against Leishmania (L.) infantum chagasi (syn. Leishmania infantum) was evaluated. The efficacies of
the compounds against promastigote viability were dependent on the type of substituent attached
to the C-3 position. Among the compounds evaluated, the isobenzofuranones
7 and 8 were
found to be capable of significantly reducing the percentage of infected macrophages and the
number of intracellular amastigotes per infected macrophage. Based on the results obtained, these
isobenzofuranones may represent a novel scaffold that can be exploited to develop of new drugs for
the treatment of leishmaniasis.
Acknowledgments: This work was supported by the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) fellowship 552459/2011-9 to JLRF and MRA, the MCT/CAPES/FAPEMIG fellowship CNPq
550514/2011-2 and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) CBB-APQ-01354-13;
CEX—BPD-00174-13; CEX APQ 01287/14.
Author Contributions: W.L.P. and A.M.S. synthesized the isobenzofuranones. R.S.V., C.M.M., R.S.G., R.C.F.,
A.S.J., G.C.B., M.R.A., L.C.C.A. designed and conducted the biological assays. R.R.T. and J.L.R.F. wrote the paper.
All of the authors contributed in the revision of manuscript.
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Conflicts of Interest: The authors declare no conflict of interest.
References
1.
WHO. Leishmaniasis; Fact Sheet No. 375; WHO: Geneva, Switzerland, 2015. Available online:
http://www.who.int/mediacentre/factsheets/fs375/en/ (accessed on 30 October 2015).
WHO. Control of leishmaniases. Technical Report Series, No. 949; WHO: Geneva, Switzerland, 2010.
Shawn, J. Further thoughts on the use of the name Leishmania (Leishmania) infantum chagasi for the aetiological
agent of American visceral leishmaniasis. Mem. Inst. Oswaldo Cruz 2006, 101, 577–579. [CrossRef]
Alvonar, J.; Vélez, I.D.; Bern, C.; Herrero, M.; Desjeux, P.; Cano, J.; Jannin, J.; den Boer, M. Leishmaniasis
worldwide and global estimates of its incidence. PLoS ONE 2012, 7, e35671. [CrossRef] [PubMed]
Sundar, S.; Chakravarty, J. Investigational drugs for visceral leishmaniasis. Expert Opin. Investig. Drugs
2015, 24, 43–59. [CrossRef] [PubMed]
De Souza, R.F.; dos Santos, Y.L.; de Souza Vasconcellos, R.; Borges-Pereira, L.; Caldas, I.S.; de Almeida, M.R.;
Bahia, M.T.; Fietto, J.L. Recombinant Leishmania (Leishmania) infantum Ecto-Nucleoside Triphosphate
Diphosphohydrolase NTPDase-2 as a new antigen in canine visceral leishmaniasis diagnosis. Acta Trop.
2013, 125, 60–66. [CrossRef] [PubMed]
2.
3.
4.
5.
6.
7.
8.
9.
Gramiccia, M.; Gradoni, L. The current status of zoonotic leishmaniases and approaches to disease control.
Int. J. Parasitol. 2005, 35, 1169–1180. [CrossRef] [PubMed]
Frezard, F.; Demicheli, C.; Ribeiro, R.R. Pentavalent antimonials: New perspectives for old drugs. Molecules
2009, 14, 2317–2336. [CrossRef] [PubMed]
Garcia Bustos, M.F.; Barrio, A.; Gonzalez Prieto, G.; de Molina Raspi, E.; Cimino, R.; Cardozo, R.M.;
Parada, L.A.; Yeo, M.; Soto, J.; Uncos, D.A.; et al. In Vivo Antileishmanial Efficacy of Miltefosine against
Leishmania (Leishmania) Amazonensis. J. Parasitol. 2014, 100, 840–847. [CrossRef] [PubMed]
10. Croft, S.L.; Sundar, S.; Fairlamb, A.H. Drug resistance in leishmaniasis. Clin. Microbiol. Rev. 2006, 19, 111–126.
[CrossRef] [PubMed]
11. Croft, S.L.; Coombs, G.H. Leishmaniasis—Current chemoteraphy and recent advances in the search for novel
drugs. Trends Parasitol. 2003, 19, 502–508. [CrossRef] [PubMed]
12. Ouellette, M.; Drummelsmith, J.; Papadopoulou, B. Leishmaniasis: Drugs in the clinic, resistance and new
developments. Drug Resist. Update 2004, 7, 257–266. [CrossRef] [PubMed]
13. Monzote, L. Current treatment of leishmaniasis: A review. Open Antimicrob. Agents J. 2009, 1, 9–19.
14. Monge-Maillo, B.; Lopez-Velez, R. Therapeutic options for visceral leishmaniasis. Drugs 2013, 73, 1863–1888.
[CrossRef] [PubMed]
15. Lin, G.; Chan, S.-K.; Chung, H.-S.; Li, S.-L. Chemistry and biological activities of naturally occurring
phthalides. In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier: Amsterdam,
The Nederland, 2005; pp. 611–669.
16. Zhang, W.; Xu, L.; Yang, L.; Huang, Y.; Li, S.; Shen, Y. Phomopsidone A, a novel depsidone metabolite from
the mangrove endophytic fungus Phomopsis sp. A123. Fitoterapia 2014, 96, 146–151. [CrossRef] [PubMed]
17. Ma, F.; Gao, Y.; Qiao, H.; Hu, X.; Chang, J. Antiplatelet activity of 3-butyl-6-bromo-1(3H)-isobenzofuranone
on rat platelet aggregation. J. Thromb. Thrombolysis 2012, 33, 64–73. [CrossRef] [PubMed]
18. Johnston, G.A. Advantages of an antagonist: Bicuculline and other GABA antagonists. Br. J. Pharmacol.
2013, 169, 328–336. [CrossRef] [PubMed]
19. Zhao, N.; Ji, M.-X.; Xu, L.; Ji, B.-S. 3-butyl-6-fluoro-1(3H)-isobenzofuranone (6-F-NBP), a derivative of
dl-n-butylphthalide, inhibits glutamate-induced cytotoxicity in PC12 cells. Drug Dev. Res. 2012, 73, 11–17.
[CrossRef]
20. Arnone, A.; Assante, G.; Nasini, G.; Strada, S.; Vercesi, A. Cryphonectric acid and other minor metabolites
from a hypovirulent strain of Cryphonectria parasitica. J. Nat. Prod. 2002, 65, 48–50. [CrossRef] [PubMed]
21. Logrado, L.P.; Santos, C.O.; Romeiro, L.A.; Costa, A.M.; Ferreira, J.R.; Cavalcanti, B.C.; de Manoel Moraes, O.;
Costa-Lotufo, L.V.; Pessoa, C.; dos Santos, M.L. Synthesis and cytotoxicity screening of substituted
isobenzofuranones designed from anacardic acids. Eur. J. Med. Chem. 2010, 45, 3480–3489. [CrossRef]
[PubMed]
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Molecules 2015, 20, 22435–22444
22. Teixeira, R.R.; Bressan, G.C.; Pereira, W.L.; Ferreira, J.G.; de Oliveira, F.M.; Thomaz, D.C. Synthesis and
antiproliferative activity of C-3 functionalized isobenzofuran-1(3H)-ones. Molecules 2013, 18, 1881–1896.
[CrossRef] [PubMed]
23. Rahman, M.M.; Gray, A.I. A benzoisofuranone derivative and carbazole alkaloids from Murraya koenigii
and their antimicrobial activity. Phytochemistry 2005, 66, 1601–1606. [CrossRef] [PubMed]
24. Teixeira, R.R.; Pereira, J.L.; da Silva, S.F.; Guilardi, S.; Paixão, D.A.; Anconi, C.P.A.; de Almeida, W.B.; Ellena, J.;
Forlani, G. Synthesis, characterization and phytotoxic activity of hydroxylated isobenzofuran-1(3H)-ones.
J. Mol. Struct. 2014, 1061, 61–68. [CrossRef]
25. Kwan, C.N.; Medoff, G.; Kobayashi, G.S.; Schlessinger, D.; Raskas, H.J. Potentiation of the antifungal effects
of antibiotics by amphotericin B. Antimicrob. Agents Chemother. 1972, 2, 61–65. [CrossRef] [PubMed]
26. Strobel, G.; Ford, E.; Worapong, J.; Harper, J.K.; Arif, A.M.; Grant, D.M.; Fung, P.C.W.; Chaud, R.M.W.
Isopestacin, an isobenzofuranone from Pestalotiopsis microspora, possessing antifungal and antioxidant
activities. Phytochemistry 2002, 60, 179–183. [CrossRef]
27. Dorosti, Z.; Yousefi, M.; Sharafi, S.M.; Darani, H.Y. Mutual action of anticancer and antiparasitic drugs: Are
there any shared targets? Future Oncol. 2014, 10, 2529–2539. [CrossRef] [PubMed]
28. Vlahopoulos, S.; Critselis, E.; Voutsas, I.F.; Perez, S.A.; Moschovi, M.; Baxevanis, C.N.; Chrousos, G.P. New
use for old drugs? Prospective targets of chloroquines in cancer therapy. Curr. Drug Targets 2014, 15, 843–885.
[CrossRef] [PubMed]
29. Wang, B.; Yu, W.; Guo, J.; Jiang, X.; Lu, W.; Liu, M.; Pang, X. The antiparasitic drug, potassium antimony
tartrate, inhibits tumor angiogenesis and tumor growth in nonsmall-cell lung cancer. J. Pharmacol. Exp. Ther.
2015, 352, 129–138. [CrossRef] [PubMed]
30. Mishra, A.; Vinayagam, J.; Saha, S.; Chowdhury, S.; Roychowdhury, S.; Jaisankar, P.; Majumder, H.K.
Isobenzofuranone derivatives exhibit antileishmanial effect by inhibiting type II DNA topoisomerase and
inducing host response. Pharmacol. Res. Perspect. 2014, 2, e00070. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 2–11 are available from the authors.
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2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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