Synthesis, cytotoxicity and in vitro antileishmanial activity of naphthothiazoles

Article abstract of DOI:10.1111/cbdd.12123

The leishmaniasis is a spectral disease caused by the protozoan Leishmania spp., which threatens millions of people worldwide. Current treatments exhibit high toxicity, and there is no vaccine available. The need for new lead compounds with leishmanicidal activity is urgent. Considering that many lead leishmanicidal compounds contain a quinoidal scaffold and the thiazole heterocyclic ring is found in a number of antimicrobial drugs, we proposed a hybridization approach to generate a diverse set of semi-synthetic heterocycles with antileishmanial activity. We found that almost all synthesized compounds demonstrated potent activity against promastigotes of Leishmania (Viannia) braziliensis and reduced the survival index of Leishmania amastigotes in mammalian macrophages. Furthermore, the compounds were not cytotoxic to macrophages at fivefold higher concentrations than the EC₅₀ for promastigotes. All molecules fulfilled Lipinski's Rule of Five, which predicts efficient orally absorption and permeation through biological membranes, the in silico pharmacokinetic profile confirmed these characteristics. The potent and selective activity of semi-synthetic naphthothiazoles against promastigotes and amastigotes reveals that the 2-amino-naphthothiazole ring may represent a scaffold for the design of compounds with leishmanicidal properties and encourage the development of drug formulation and new compounds for further studies in vivo.

Full text of DOI:10.1111/cbdd.12123

Chem Biol Drug Des 2013; 81: 749–756
Research Article
Synthesis, Cytotoxicity and In Vitro Antileishmanial
Activity of Naphthothiazoles
Juliano S. de Toledo1, Paulo E. S. Junior2,
Key words: fragment embedment, Leishmania braziliensis,
leishmaniasis, naphthothiazoles, neglected tropical disease
Viviane Manfrim2, Camila F. Pinzan1, Alexandre
S. de Araujo3, Angela K. Cruz1 and Flavio S.
Received 26 October 2012, revised 11 December 2012 and
accepted for publication 12 February 2013
Emery2,*
1Departamento de Biologia Celular e Molecular e
^
Bioagentes Patogenicos, Faculdade de Medicina de
Ribeirao Preto, Universidade de Sao Paulo, Av.
Leishmaniasis is caused by protozoan parasites from the
Leishmania genus and comprises two major diseases:
visceral leishmaniasis (VL) and cutaneous leishmaniasis
(CL). While VL is fatal if untreated, CL may heal spontane-
ously or leave disfiguring scars. Leishmaniasis is one of
the most neglected tropical diseases and is associated
with high morbidity levels. Moreover, the mortality rate of
VL is approximately 60 000 per year, which is the second
highest rate among parasitic diseases, only surpassed by
malaria (1). The expansion of the geographical distribution
of leishmaniasis in developing and developed countries in
Europe (e.g. France, Italy, Spain and Greece) and North
America is an alert to health authorities worldwide (2,3).
This scenario makes evident that leishmaniasis is no longer
a poverty-associated disease (2), since climate change (4–
6), human migration (7,8) and additional socio-environmen-
tal factors (9) have become important variables affecting
the incidence of this parasitic infection. Although the dis-
ease is treatable, its control is difficult for the absence of
an effective vaccine, the adaptation of the vector and res-
ervoirs to human environments, and the emergence of
resistant lines. The relative high toxicity of available chemo-
therapy and the lack of more effective, safer and orally
available therapies for all clinical manifestations is another
relevant problem that substantially makes leishmaniasis a
high-risk disease (10). The long-lasting lack of commercial
incentives to support the R&D for drugs against tropical
diseases has been overcome by organized networks
between public–private partnerships (PPPs) (11,12).
~
~
~
Bandeirantes 3900, Ribeirao Preto, SP, 14049-900, Brazil
Departamento de Ciencias Farmaceuticas, Faculdade de
2
^
^
^
^
Ciencias Farmaceuticas de Ribeirao Preto, Universidade
de Sao Paulo, Av. do Cafe s/n-Campus USP, Ribeirao
~
~
ꢀ
~
Preto, SP, 14040-903, Brazil
ꢀ
3
^
Departamento de Fısica, Instituto de Biociencias Letras e
ꢀ
^
~
Ciencias Exatas de Sao Jose do Rio Preto, Universidade
ꢀ ~
ꢀ
Estadual Paulista Julio de Mesquita Filho, Rua Cristovao
~
ꢀ
Colombo, 2265, Jardim Nazareth. Sao Jose do Rio Preto,
SP, 15054-000, Brazil
*Corresponding author: Flavio S. Emery,
flavioemery@fcfrp.usp.br
The leishmaniasis is a spectral disease caused by
the protozoan Leishmania spp., which threatens mil-
lions of people worldwide. Current treatments exhibit
high toxicity, and there is no vaccine available. The
need for new lead compounds with leishmanicidal
activity is urgent. Considering that many lead leish-
manicidal compounds contain
a quinoidal scaffold
and the thiazole heterocyclic ring is found in a number
of antimicrobial drugs, we proposed a hybridization
approach to generate a diverse set of semi-synthetic
heterocycles with antileishmanial activity. We found
that almost all synthesized compounds demonstrated
potent activity against promastigotes of Leishmania
(Viannia) braziliensis and reduced the survival index
of Leishmania amastigotes in mammalian macrophag-
es. Furthermore, the compounds were not cytotoxic
to macrophages at fivefold higher concentrations than
the EC50 for promastigotes. All molecules fulfilled
Lipinski’s Rule of Five, which predicts efficient orally
absorption and permeation through biological mem-
branes, the in silico pharmacokinetic profile con-
firmed these characteristics. The potent and selective
activity of semi-synthetic naphthothiazoles against
promastigotes and amastigotes reveals that the 2-
amino-naphthothiazole ring may represent a scaffold
for the design of compounds with leishmanicidal
properties and encourage the development of drug
formulation and new compounds for further studies
in vivo.
The characterization of physico-chemical factors could
drive the design and innovation of potential leishmanicidal
hits. Specifically, the Rule of Five, which is useful for pre-
diction of good permeability and drug-likeness for com-
pounds, is more likely when H-bond donors (nOHNH) are
ꢀ 5, H-bond acceptors (nON) ꢀ 10, molecular weight
(MW) ꢀ 500 and calculated lipophilicity (xlog P is the
calculated logP using the specific logarithm of the soft-
ware) ꢀ 5 (13,14). In addition, oral bioavailability has also
been correlated with the total polar surface area (TPSA)
parameter, which represents the sum of surfaces of polar
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12123
749

de Toledo et al.
ꢁ2
ꢀ
were obtained from Vetec Quımica Fina Ltda (Duque de
atoms in a molecule ( ꢀ 140 A ) including N, O, P and S
(TPSA_NOPS), the number of rotatable bonds (rotatable
bonds count – rbc ꢀ 10) and water solubility (logS) (15).
To facilitate the identification of effective antileishmanial
drug candidates, Nwaka et al. proposed a set of criteria
based on biological activity, physico-chemical characteris-
tics and pharmacokinetics. According to the authors, a le-
ishmanicidal hit should fit the following criteria: EC50 for
the amastigote (in macrophages) about 1–2 lg/mL, selec-
tivity over parasites >20 (selectivity index, SI), confirmed
and elucidate structure, established synthetic route, good
drug-likeness scores (DLS), no violation of Lipinski’s Rule
of Five and chemically exploitable (16).
~
Caxias, RJ, Brazil) and Synth (Labsynth, Sao Paulo, Brazil).
Iodoxybenzoic acid was freshly prepared as previously
described (24). All reactions were monitored by thin layer
chromatography using Merck Silica gel 60F254 plates,
and synthesized compounds were purified by flash column
chromatography on silica gel 60 (200–430 mesh; Merck
Brasil, Sao Paulo, Brazil) or by recrystallization. High-reso-
lution mass spectra were obtained using an electrospray
ionization time-of-flight mass spectrometer. The NMR data
were recorded with a Bruker DPX-500 instrument using
DMSO-d6, CDCl3 or CD3OD as a solvent and tetrameth-
ylsilane as an internal standard. IR spectra were measured
with a Perkin-Elmer Spectrum RX IFTIR System.
Heteroaromatic rings found in small bioactive molecules
have been successfully exploited for different medicinal
purposes, including bioavailability, due to their shape,
hydrophobicity, hydrogen bond potential, polarizability, and
established diversified reactivity for manipulation of func-
tional groups and structures (17). The thiazole ring system
is a useful structural element in medicinal chemistry. This
structure can be found in numerous biologically active mol-
ecules used for the treatment of allergies, hypertension,
inflammation, fungal and bacterial infections and has found
broad application in drug development (18,19). In particu-
lar, diverse classes of therapeutic drugs, including antimi-
crobials, contain a 2-aminothiazole ring. Several synthetic
and natural naphthoquinones are also known as antipara-
sitic lead compounds, acting by different sort of mecha-
nisms of action against trypanosomatids (20–23). The aim
of this study was to make the fragment embedment of the
thiazole ring into naphthoquinoidal structures to generate a
small library of naphthothiazoles, including a hybrid thiazo-
loquinone, aiming compounds with different pattern of sub-
stitution to search for improved druggability. We adopted
chemical and biological criteria to design and select hit
compounds anti-Leishmania. The compounds containing a
2-aminothiazole core within a naphthalene skeleton were
synthesized, the structure was correlated with drug-like-
ness index and drug-relevant properties, the activity against
promastigote and amastigote forms was tested, and the
cytotoxicity was evaluated.
Drug-relevant properties and DLS were calculated with the
Vortex data analysis and visualization software from Dot-
matics (http://www.dotmatics.co.uk/) and with the web-
based molecular property calculator from OSIRIS Property
Explorer
(http://www.organic-chemistry.org/prog/peo/).
OSIRIS was used to compare the DLS (drug similarity
based on the occurrence of substructure fragments – 80%
of drugs have DLS higher than À4.0) of the naphthothiaz-
oles and antileishmanial drugs on the market. Vortex was
used to compare MW, xlogP, rbc, TPSA, nOHNH and
nON and violations to Lipinski’s Rule of Five. The probable
pharmacokinetic behaviour of the naphthothiazoles was
predicted using the web tool ‘PK/DB Database for Phar-
macokinetics Properties’ (25) which expressed the results
as: human intestinal absorption (HIA); human oral bioavail-
ability (F); plasma protein binding (PPB).
Leishmania culture
The parasites Leishmania (V.) braziliensis, strain H3227
(MHOM/BR/94/H-3227), were maintained in vitro in M199
medium (GIBCO, Grand Island, NY, USA) at 26 °C supple-
mented with 10% heat-inactivated foetal calf serum, 2%
human urine, 20 mM HEPES, 4 mM NaHCO3, 10 U/mL of
penicillin and 100 lg/mL of streptomycin (GIBCO).
Antipromastigote assay
We have provided a new rationale for the development of
novel lead compounds with leishmanicidal activity based
on thiazole ring properties. We have shown a potent and
selective activity of semi-synthetic naphthothiazoles against
promastigotes and amastigotes revealing that the 2-
amino-naphthothiazole ring may represent a scaffold for
the design of molecules with anti-Leishmania activities.
The antipromastigote assay was carried out according to
the method of Dutta et al. (26). The naphthothiazole com-
pounds were serially diluted from 0.02 to 10 lM in Schnei-
der’s medium (supplemented with 10% FBS and 2%
human urine) containing 2 9 105 parasites/mL. A total of
4 9 104 parasites were seeded per well in 96-well micro-
plates incubated at 26 °C until the end of the log phase.
After, 100 lg of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide (MTT) dissolved in 10 lL of PBS was
added per well, and the plates were incubated at 37 °C
for 4 h. Following incubation, the plates were centrifuged
at 3000 9 g for 5 min, the supernatant was removed and
precipitated formazan was dissolved in 100 lL of dimethyl
sulfoxide (DMSO). The absorbance was measured at
492 nm, and the data for two independent assays
Methods and Materials
Chemical synthesis, drug-relevant properties and
pharmacokinetics
All chemicals and reagents were purchased from Sigma-
Aldrich Brasil Ltda (Sao Paulo, Brazil), and the solvents
750
Chem Biol Drug Des 2013; 81: 749–756

In Vitro Antileishmanial Efficacy of Naphthothiazoles
performed in triplicate were analysed using GRAPH PAD PRISM
5.0 software (GraphPad Software Inc., La Jolla, CA, USA).
The EC50 was defined as the effective drug concentration
that inhibits parasite proliferation by 50% compared with a
non-treated culture. Geneticin (G418) was used as a posi-
tive control.
of infected macrophages multiplied by the average number
of amastigotes per macrophage). Analysis of the mean of
two assays performed in duplicate was carried out with
GRAPH PAD PRISM 5.0 software.
Results
Determination of cytotoxicity against mammalian
cells
Synthesis of naphthothiazoles
Despite all the efforts on the R&D to discover new lead
compounds active against Leishmania parasites, the che-
motherapy currently available to treat leishmaniasis is not
suitable (10,27). This work reports a molecular hybridiza-
tion strategy for the design and synthesis of six semi-syn-
thetic naphthothiazoles.
Cytotoxicity was determined using the MTT assay. Briefly,
BALB/c mice were intraperitoneally injected with 1.5 mL
of 5% thioglycolate medium. After 72 h, murine peritoneal
cells were harvested in RPMI 1640 medium (GIBCO Invi-
trogen Corporation, New York, NY, USA), pH 7.6 supple-
mented with 10% heat-inactivated foetal calf serum
(GIBCO). A total of 1 9 105 cells were plated in 96-well
microtiter plates and allowed to adhere for 12 h at 37 °C
in an atmosphere of 5% CO2. The naphthothiazole com-
pounds were serially diluted from 0.024 to 12.5 lM in
RPMI medium, and macrophages were incubated in the
presence of these compounds for 48 h at 37 °C in 5%
CO2. Control cells were incubated in DMSO without
drugs. Analysis of the mean of two assays performed in
triplicate was carried out with GRAPH PAD PRISM 5.0 soft-
ware.
The naphthothiazoles were designed based on a hybridiza-
tion strategy, in which a 2-amino-thiazole ring is embedded
into a naphthoquinoidal skeleton (Figure 1). The naphtho-
thiazoles compounds 4, 5 and 6 were obtained in a single
step by the addition of thiourea to the respective naphtho-
quinones (1, 4-naphthoquinone [1], lawsone [2] and mena-
dione [3]), in an ethanolic/HCl solution, as previously
described (28,29) (Scheme 1). The compound 6 was sub-
jected to double acetylation in presence of acetic anhydride
and triethylamine to produce compound 8, and methylated
in the presence of dimethyl sulphate and sodium carbonate
in ethanol to produce the compound 7 (Scheme 2). The
compound 4 was oxidized in presence of 2-iodoxybenzoic
acid and acetonitrile: water (2:1) to produce the compound
9 (Scheme 3). All reactions gave good-to-average yields of
respective compounds. The crystal and molecular structure
of compound 7 were determined by single crystal X-ray dif-
fraction (Supporting information), and it was deposited at
‘The Cambridge Crystallographic Data Centre’ (www.ccdc.
cam.ac.uk; deposit number: 854691). Details of all synthetic
procedures, structure characterization, spectroscopic and
spectrometric data, and crystallographic results can be
found in the Supporting information.
Anti-amastigote assay
A suspension of 5 9 105 macrophages/mL in RPMI 1640
medium was dispersed into polypropylene tubes and
infected with L. (V.) braziliensis (10 parasites per cell) for
3 h. Non-internalized parasites were removed by centrifu-
gation at 50 9 g, three washing cycles with RPMI med-
ium. The infected macrophages were plated on glass
coverslips (13-mm diameter) in 24-well plates and incu-
bated for 1 h at 33 °C. The naphthothiazole compounds
which show activity against promastigotes were diluted in
RPMI medium, and infected macrophages were incubated
in the presence of these compounds for 48 h at 33 °C in
5% CO2. The coverslips were washed with PBS, stained
ꢀ
ꢀ
with Panotic stain (Panotico Rapido LB kit; Laborclin,
Biological activity
ꢀ
Parana, Brazil), dried, mounted on glass slides with
The anti-Leishmania activity of semi-synthetic naphtho-
thiazoles against Leishmania promastigotes (extracellular
stage) was accessed in biological screening by MTT
reduction, and activity against amastigotes (intracellular
stage) was determined by reduction in infection index.
Tissue-Tekꢀ mounting medium (Sakura Finetek Europe
B.V., Alphen aan den Rijn, Holland) and examined micro-
scopically. Two hundred cells were evaluated per assay.
The results were expressed as infection index (percentage
Figure 1: General strategy for the development of new heterocyclic derivatives from quinones.
Chem Biol Drug Des 2013; 81: 749–756
751

de Toledo et al.
Scheme 1: Schematic representation of the synthesis of aminothiazoles from napthoquinones.
Scheme 2: Methylation and diacetylation of naphthothiazole 6.
Scheme 3: Oxidation of hydroxyl-thiazole 4 to quinone 9.
Table 1: In vitro antipromastigote activity of 2-amino-naphtho-
thiazoles
Compound
Promastigote EC50 [lM (lg/mL)]
Figure 2: Cytotoxicity on macrophages. Naphthothiazoles effect
on peritoneal macrophage from BALB/c mice. Cell viability
determined by MTT method with different concentrations of
1,4-naphthoquinone
Lawsone
Menadione
4
0.11 (0.01)
Inactive
1.78 (0.30)
1.44 (0.31)
1.21 (0.28)
4.6 (1.081)
0.97 (0.304)
1.18 (0.27)
Inactive
compounds 4, 5, 6,
7 and 8. Results expressed in % of
means Æ standard deviations from tree independent experiments.
5
6
excluding the compound 6, which displayed some cyto-
toxicity at 6–12 lM concentration (Figure 2). Based on the
EC50 determined for promastigotes, and given the lack of
cytotoxicity to macrophages, the naphthothiazole com-
pounds (except compounds 6 and 9) were tested at a
concentration of 3 and 6 lM. All derived tested com-
pounds (4, 5, 7 and 8) were able to reduce number of
macrophage′s parasite loads (Figure 3). Compounds 4
and 5 reduced the survival index of intracellular amastigote
Leishmania by 75% at concentrations of 1.29 lg/mL
(6 lM) and 0.69 lg/mL (3 lM), respectively (Table 1). The
hybrid compounds 4, 5, 7 and 8 fit the biological criteria in
terms of efficacy and toxicity (Table 1).
7
8
9
Control (G418)
2.42 (1.2)
The compounds 4, 5 and 6 possess a free basic amine,
and the water-soluble aminothiazole hydrochloride form
was used for biological studies. All compounds tested,
except for the compound 9, showed potent activity
against promastigotes (Table 1). According to EC50 deter-
mined for promastigotes, the cytotoxicity was tested on
mammalian cells. None of the compounds were cytotoxic,
752
Chem Biol Drug Des 2013; 81: 749–756

In Vitro Antileishmanial Efficacy of Naphthothiazoles
drugs, we used the Lipinski’s Rule of Five, rbc, TPSA and
DLS (14). All compounds synthesized adhered to Lipinski’s
Rule of five. The most active compounds (4, 5, 7 and 8) dis-
play values of xlogP (value ranges from 3.75 to 4.88) lower
than miltefosine, which has xlogP-value of 5.59, violating
the Lipinski’s statement (Table 2). All synthesized com-
ꢁ2
pounds show a TPSA lower than 140 A and also lower rbc
(<3), especially when compared to the oral miltefosine
(rbc = 20). The active naphthothiazoles have DLS higher
than À1.9 and comparable to almost all leishmanicidal
drugs, except for miltefosine and pentamidine, which have
very low DLS (Table 2). In addition, according to the phar-
macokinetic prediction, the naphthothiazoles display high
values of HIA (>80% for active compounds) and high values
of oral bioavailability, especially the most active compounds
4 and 7, with F of 66% (Table 2). Furthermore, the com-
pounds are approximately 50% free in plasma. The proba-
ble human HIA, F and PPB of the tested compounds
indicated that naphthothiazoles have a pharmacokinetic
profile which would allow oral administration (Table 3).
Figure 3: In vitro anti-amastigote activity of naphthothiazoles.
Reduction in infection index of peritoneal macrophage infected by
Leishmania and treated with different concentrations of
compounds 4, 5, 7 and 8. Macrophages were infected with
Leishmania (Viannia) braziliensis promastigotes and treated with
naphthothiazoles compounds (3 or 6 lg/mL). The number of
intracellular amastigotes per 200 macrophages was determined
under optical microscope analysis. Values represent the averages
of three independent experiments with the error bars indicating
the range of values; *p < 0.05.
Discussion
Leishmaniasis are among the sixth most threaten infectious
diseases worldwide (http://www.who.int/leishmaniasis/en/
index.html). Nowadays, some PPPs are stimulating R&D of
new therapies for tropical and neglected diseases, including
leishmaniasis (10,30). As a result, some non-antimony-based
oral therapies have become available over the past decade,
including miltefosine and liposomal amphotericin (31). How-
ever, none of these new chemotherapeutics are suitable to
treat all clinical manifestations of leishmaniasis (27).
Drug-like properties and pharmacokinetics
prediction
There is ample evidence that the characterization of physico-
chemical properties could optimize the drug discovery pro-
cess. We have used cheminformatic tools to examine and
compare the drug-like properties, DLS and pharmacokinet-
ics of the naphthothiazoles herein described with the ones
commonly used in chemotherapy against leishmaniasis.
In this study, we employed a molecular hybridization strat-
egy for the design and synthesis of six semi-synthetic
To evaluate and compare the potential oral bioavailability
and drug-likeness of naphthothiazoles and leishmanicidal
Table 2: Predicted molecular properties of synthesized compounds and leishmanicidal drugs
ꢁ2
Compound
MW
xlogP
1.64
HBA
HBD
LIPINSKI
LC
TPSA_NOPS (A )
RBC
DLS
1
158.15
174.15
172.18
217.27
233.27
231.29
244.31
314.36
230.24
924.12
195.21
407.57
615.63
340.42
676.77
2
3
2
3
4
3
3
5
4
17
6
5
19
6
17
0
1
0
2
3
2
1
1
1
11
6
0
13
4
5
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Fail
Pass
Pass
Fail
Pass
Fail
0
0
0
0
0
0
0
0
0
3
1
1
3
0
2
34.1
54.4
34.1
89
109.2
89
76.4
96.5
101.3
299.4
113.2
68.4
347.3
118.2
284.8
0
0
0
0
0
0
1
3
0
3
6
20
9
10
8
À2.44
2
0.89
2.07
4.12
3.75
4.4
4.24
4.88
2
À1.89
À4.08
À1.79
À1.89
À1.90
À1.73
0.28
3
4
5
6
7
8
9
À5.72
À0.14
1.76
Amphotericin B
Meglumine
Miltefosine
Paromomycin
Pentamidine
Stibogluconate
0.21
À3.71
5.59
À10.36
3.01
À6.08
À54.74
2.49
À5.35
1.02
MW, molecular weight; HBA, H-bond acceptor; HBD, H-bond donor; LC, Lipinski count violations; TPSA_NOPS, total polar surface area;
RBC, rotatable bond count; DLS, drug-likeness score; All molecular properties were calculated using Vortex data analysis and visualization
software from Dotmatics, except DLS, which was calculated with the web-based molecular property calculator from OSIRIS Property
Explorer.
Chem Biol Drug Des 2013; 81: 749–756
753

de Toledo et al.
Table 3: Predicted pharmacokinetic behaviour of synthesized
naphthothiazoles
We have applied a preliminary analysis of the physico-
chemical properties and predicted the absorption and
bioavailability for synthesized compounds. We have found
that the semi-synthetic naphthothiazoles (compounds 4–9)
show no violations of Lipinski’s Rule of Five. Among leish-
manicidal drugs available on the market, only miltefosine
does not violate Lipinski’s Rule of Five; all other drugs
have, at least, 1 violation. Moreover, the xlogP and TPSA
value ranges from 2.0 to 4.88 and 76.4 to 109.2, respec-
tively, for naphthothiazoles, suggesting that these com-
pounds are potentially able to cross cell membranes in a
permeation process, which could explain the ability to
reach the amastigote inside of phagolysosome. According
to our theoretical study, the TPSA of most leishmanicidal
Compound
HIA (%)
F (%)
PPB (%)
4
5
6
7
8
9
83.82
56.41
82.59
85.39
83.28
72.50
66.71
55.78
58.66
66.03
43.36
73.89
57.29
51.61
54.28
55.08
59.15
67.31
HIA, human intestinal absorption; F, human oral bioavailability;
PPB, plasma protein binding. The predicted pharmacokinetic
parameters of the compounds were obtained from PK/DB Data-
base for Pharmacokinetics Properties (http://miro.ifsc.usp.br/
pkdb).
ꢁ2
drugs currently on the market is higher than 140 A which
reflects on poor absorption and bioavailability. The good
drug-like properties of the naphthothiazoles are compara-
ble to the ones for miltefosine suggesting a good oral bio-
availability, because only miltefosine has an oral route of
administration, while the other drugs are parenterally
administered. Compared with miltefosine, the compounds
4, 5, 7 and 8 display higher DLS, which indicates that
these compounds are more similar to commercial drugs,
in terms of molecular properties, as those described in this
work, and appear to have potential for oral administration
(Table 2). In addition, the pharmacokinetic behaviour pre-
dicted to naphthothiazoles show a better profile for oral
administration than miltefosine, which is almost 100%
bound to plasma protein (34). There is no available data
for humans about F and HIA, although some experiments
in mice suggest good intestine absorption (35).
naphthothiazoles that significantly reduced the proliferation
of promastigotes and intracellular amastigotes in vitro.
Moreover, none of the tested compounds shown cytotox-
icity to mammalian macrophages up to 12.5 lM.
The potent activity against promastigotes of water-soluble
compounds 4 and 5 (0.31, 0.28 lg/mL), and moderate
activity of 6 (1.08 lg/mL), suggests that the naphthothiaz-
ole ring, oxygenated at C-5, is important for antiparasitic
activity and become a potential fragment for further struc-
ture–activity relationship exploration, as indicated by the
biological data herein presented; free hydroxyl, O-alkylation
or O-acylation did not affect the antiparasitic activity. On
the other hand, the substitution on C-4 is clearly a poten-
tial site for functional group diversification, because, as the
biological activity revealed, comparing to compound 4,
alkylation or hydroxylation of C-4 does not appear to affect
the biological activity of the naphthothiazoles. The com-
pound 5 (the most active against both forms of the para-
site) contains 2 hydroxyl groups at C-4 and C-5, but the
oxidation of this catechol to an o-quinoidal structure (Com-
pound 9) lead to loss of activity. As for the hydroxyl group,
compounds with free amines or N-acyl substitution (e.g.
compounds 4 and 8, respectively) showed activity in both
promastigote and amastigote. It is noticeable that free
amine (compounds 4, 5, 6 and 7) could be an important
structural feature because this functional group is proton-
ated in acidic microenvironment. Considering that the par-
asitophorous vacuoles containing Leishmania parasites
maintain an acidic pH (pH 4.5–6.0) (32,33), this physico-
chemical free amine characteristic could lead to the accu-
mulation of these compounds within the phagolysosomal
compartment increasing the leishmanicidal effect. Molecu-
lar modification of compound 6 by either alkylation (com-
pound 7; EC50 0.97 lg/mL against promastigotes) or
acylation (Compound 8; EC50 1.18 lg/mL against prom-
astigotes) of the hydroxyl group at position C-5 improved
the antiparasitic activity, which could be related to higher
lipophilicity. These preliminary structural requirements
could be explored in a hit-to-lead evolution towards leish-
manicidal compounds.
Thus, as the compounds described in this study demon-
strated leishmanicidal activity, met the biological and
chemical parameters, and potentially overcome the limita-
tions of low oral bioavailability and absorption (36), they
may represent oral drug candidates for hit-to-lead progres-
sion. Moreover, further structural modification of the bioac-
tive compounds should be driven by these parameters to
obtain higher drug score indexes and improved leishmani-
cidal activity.
Conclusions and Future Directions
The identification of the lead compounds or chemical
starting point is essential in drug discovery pipeline. This
study revealed four potential candidates for in vivo stud-
ies that drastically inhibited promastigote proliferation and
the macrophages amastigote loads without affecting host
cell viability, with high selectivity index and very good
probable oral bioavailability. The relatively simple labora-
tory synthesis and the possibility to modify naphthothiaz-
oles chemical structure constitute important advantages
for development of new antileishmanial therapy. The
results of this study provide a new rationale for the
development of novel, semi-synthetic naphthothiazole
lead compounds with leishmanicidal activity, which are
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In Vitro Antileishmanial Efficacy of Naphthothiazoles
derived from natural sources of naphthoquinones. The
potent and selective activity of semi-synthetic naphtho-
thiazoles against amastigotes infection in vitro encourage
the development of pharmaceutical drugs for future stud-
ies in vivo.
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Acknowledgments
We are grateful to Professor Aldina Barral (Centro de Pes-
quisas Goncßalo Moniz, FIOCRUZ-Bahia, Brazil) for providing
the Leishmania strain used in this study and Professor Adri-
ano D. Andricopulo (IFSC – USP) for predicting the pharma-
cokinetic of the compounds. This research was supported
by the INCT_if (CNPq 573663/2008-4), INBEQMed (CNPq
573607/2008-7), and FAPESP (2009/14184-0 and 2009/
51812-0). Juliano S. Toledo is supported by postdoctoral
fellowship from CNPq (PNPD no 151599/2008-4).
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Appendix S1. Complementary data from spectroscopy,
crystallography, synthesis and leishmanicidal activity of
naphthothiazoles.
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