Synthesis, evaluation against Leishmania amazonensis and cytotoxicity assays in macrophages of sixteen new congeners Morita-Baylis-Hillman adducts

Article abstract of DOI:10.1016/j.ejmech.2011.06.036

We report the design, synthesis, in vitro evaluation against Leishmania amazonensis (IC₅₀), cytotoxicity assays in macrophages (CC ₅₀), and selectivity index (SICC₅₀/IC₅₀) of sixteen new congeners aromatic Morita-Baylis-Hillman adducts 1-16. The 1-16 were prepared in good to excellent yields (58%-97%) from the "one pot" Morita-Baylis-Hillman Reaction between the aldehydes 29-36 and the acrylates 27 or 28 under DABCO as promoter. The MBHA 2-[Hydroxy(2-nitrophenyl)propyl] propanoate (1, IC₅₀ = 7.52 μg/mL or 28.38 μM; CC₅₀ = 35.77 μg/mL or 134.98 μM; SI = 4.75) and 2-[Hydroxy(2-nitrophenyl) hydroxyethyl] propanoate (9, IC₅₀ = 5.48 μg/mL or 20.52 μM; CC₅₀ = 29.81 μg/mL or 111.64c μM and, SI = 5.43) were the most effective and safe evaluated compounds.

Full text of DOI:10.1016/j.ejmech.2011.06.036

European Journal of Medicinal Chemistry 46 (2011) 4295e4301
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, evaluation against Leishmania amazonensis and cytotoxicity assays
in macrophages of sixteen new congeners MoritaeBayliseHillman adducts
,
Fábio P.L. Silva a, Priscilla A.C. de Assis b c, Claudio G.L. Junior a, Natália G. de Andrade a,
,
,
*
Saraghina M.D. da Cunha a, Márcia R. Oliveira b c, Mário L.A.A. Vasconcellos a
a Laboratório de Síntese Orgânica Medicinal da Paraíba (LASOM-PB), Departamento de Química, Universidade Federal da Paraíba, Campus I, João Pessoa, PB 58059-900, Brazil
b Departamento de Biologia Molecular, Universidade Federal da Paraíba, Campus I, João Pessoa, PB 58059-900, Brazil
c Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, Campus I, João Pessoa, PB 58059-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design, synthesis, in vitro evaluation against Leishmania amazonensis (IC50), cytotoxicity
assays in macrophages (CC50), and selectivity index (SI]CC50/IC50) of sixteen new congeners aromatic
MoritaeBayliseHillman adducts 1e16. The 1e16 were prepared in good to excellent yields (58%e97%)
from the “one pot” MoritaeBayliseHillman Reaction between the aldehydes 29e36 and the acrylates 27
or 28 under DABCO as promoter. The MBHA 2-[Hydroxy(2-nitrophenyl)propyl] propanoate (1,
Received 22 January 2011
Received in revised form
15 June 2011
Accepted 27 June 2011
Available online 5 July 2011
IC50 ¼ 7.52
nitrophenyl)hydroxyethyl] propanoate (9, IC50 ¼ 5.48
111.64c
m
g/mL or 28.38
m
M; CC50 ¼ 35.77
m
g/mL or 134.98
m
M; SI ¼ 4.75) and 2-[Hydroxy(2-
m
g/mL or 20.52
m
M; CC50 ¼ 29.81 g/mL or
m
Keywords:
Leishmaniasis
Leishmania amazonensis
MoritaeBayliseHillman adducts
Molecular modification strategy
Lipophilicity
m
M and, SI ¼ 5.43) were the most effective and safe evaluated compounds.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Bioisoterism
1. Introduction
number of reported leishmaniasis cases, often in epidemic
proportions [3]. Leishmaniasis infection causes many different
clinical manifestations, such as cutaneous [4], mucocutaneous [5]
and visceral (VL), also called Kala Azar. Human infection with
Leishmania chagasi/Leishmania infantum, the protozoan causing
South American VL, ranges from sub-clinical infection to progres-
sive fatal disease [6] however, the cutaneous form is the most
common. Both the cutaneous and mucocutaneous forms can cause
severe disfigurement to patients, including ulcerative skin lesions
and the destruction of the mucous membranes of the nose, mouth,
and throat, leading to permanent disfigurement (diffuse cutaneous
form of the disease) and frequently social ostracization [7]. The
most common species in the Americas and the most important
causative agent of cutaneous and mucocutaneous leishmaniasis in
Brazil is Leishmania braziliensis, while Leishmania amazonensis is
the primary etiologic agent of the diffuse cutaneous form of the
disease [8]. The treatment options for leishmaniasis are limited and
involve the administration of pentavalent antimonial agents as first
line drugs: sodium stibogluconate (PentostanÒ) and meglumine
antimonite (GlucantimeÒ). The amphotericin B and pentamidine
are second choice drugs [9]. However, the chemotherapy with the
second-line drug pentamidine is also far from satisfactory due to
several side effects including renal and hepatic toxicity,
Neglected tropical diseases (NTDs) have traditionally ranked
low on national and international health agendas [1]. Leishmaniasis
are a complex of NTDs, caused by different species of Leishmania sp.
protozoan parasite, which have significant impact on the world,
especially in developing countries with infections spread over
several hundred million people and a significant cause of morbidity
and mortality [1]. Leishmaniasis affect almost 12 million people in
nearly 90 countries, representing a worldwide public health
problem. More than 350 million people are currently at risk, and 2
million new cases are registered each year. An estimated 51 000
annual deaths for leishmaniasis have been reported [2]. Increased
travel and migration within the tropics, subtropics, Middle East and
Southern Europe as well as global climate and environmental
changes are making leishmaniasis a considerable risk for pop-
ulations in geographic regions previously unaffected by the disease.
As a result, there has been a progressive expansion of leishmaniasis
endemic regions as well as a concomitant increase in the total
* Corresponding author. Tel.: þ55 83 3248 2352; fax: þ55 83 3216 7433.
E-mail address: mlaav@quimica.ufpb.br (M. L.A.A. Vasconcellos).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.036

4296
F.P.L. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4295e4301
etiologic agent of malaria [24,25]. In 2006, we described the
O
OH
NR3 (catalyst)
conditions
EWG
EWG
molluscicidal activities of simple aromatic MBHA against Bio-
mphalaria glabrata (Say) snails, the intermediate host of schisto-
somiasis [26]. In the same year the antiproliferative effects of some
MBHA and a new phthalide derivative on human tumor cell lines
was described. In this article Kohn et al. reinforce what is known,
the higher biological activity of aromatic compounds when
compared to aliphatic compounds [27].
+
N
R
R
N
R= alkyl, aryl, H
EWG= CO2R, CN, COR, others
DABCO
MBHA
Scheme 1. General MoritaeBayliseHillman reactions with e.g. aldehydes.
Our research group described in 2007, that some aromatic
MBHA were very active compounds against the L. amazonensis
(cutaneous and mucocutaneous infections) and some of them are
safe compounds [28]. Following that work, we published the bio-
logical evaluation of aromatic MBHA against L. chagasi (visceral
infections) parasites [29], and in 2010, we have shown that the
MBHA 3-Hydroxy-2-methylene-3-(4-nitrophenyl) propanenitrile,
a high anti-leishmania compound, is a highly active compound
against epimastigote and trypomastigote form of Trypanosoma
cruzi, the parasite that causes Chagas disease [30]. In the same year,
we presented an improved synthesis for sixteen MBHA and their
biological evaluation against L. amazonensis and L. chagasi and we
proposed, at the first time, a StructureeActivity-Relationship (SAR)
analysis for this new promising class of anti-parasitic drugs [31].
In our continuing search for bioactive substances [32e37] and in
connection with our efforts toward the study of reactivity of MBHR
[21,23,38e41], we have been trying to discover a lead compound
against in vitro leishmaniasis, aiming to advance toward the in vivo
studies. In this context we present here the design, synthesis,
inhibitory activities on antipromastigote form of L. amazonensis
(IC50), results of cytotoxicity assays in macrophages (CC50) and
selectivity index (SI) of eight new aromatic MBHA 1e8 and their
eight classic bioisosters 9e16 [42] (Fig. 1).
pancreatitis, hypotension, dysglycemia, and cardiac abnormalities
[10]. Although amphotericin B and its lipid complex, are quite
effective for visceral leishmaniasis, they are expensive and do not
appear to be suitable for treatment of non-visceral diseases [10].
Reproducible evidence of protective efficacy has not emerged from
clinical trials of first generation leishmaniasis vaccines. So far no
vaccine approved for human use is available [11]. Therefore, there is
an increasingly urgent need for the development of new, inex-
pensive, effective and safe drugs for the treatment of leishmaniasis
and then, the discovery of new lead compounds for this disease is
a pressing concern for global health programs.
The MoritaeBayliseHillman reaction (MBHR) is an important
way for CeC bond formation [12,13]. It involves the coupling of
alkenes containing electron-withdrawing groups (EWG) with
aldehydes, ketones or imines, among others. Tertiary amines are
used as nucleophilic catalysts of which 1,4-diazabicyclo[2.2.2]
octane (DABCO) is the most widely used (Scheme 1). The Mor-
itaeBayliseHillman adducts (MBHA) have been extensively used as
starting materials in organic synthesis for a variety of applications,
many of which have biological activity [14e17]. An inconvenience
associated with this reaction is, in several examples, the long
reaction times. There are reports of reactions that, last up to 65 days
[14]. However, due to the synthetic utility of these MBHA adducts,
several protocols have been described to improve the reaction time
and yields, such as the use of microwaves [18], ultrasound [19], high
pressures [20], use of ionic liquids [21], change of reaction
temperature [22] and catalyst [23], and other experimental proto-
cols [17].
2. Results and discussion
2.1. Design of the MBHA 1e16
This design was based on the anti-leishmania activities of 17e24
(Fig. 1) presented by us in previous paper [31]. We modified the
lipophilicity [43] of 17e24 to design the MBHA 1e8; and used the
The first biological evaluation of the MBHA prepared from “one
pot” MBHR was described against Plasmodium falciparum, the
OH
O
OH
O
NO2 OH
O
R
O2N
R
R
O
O
O
O2N
2 R=CH2CH2CH3, LogP=3.16+/- 0.39
10 R=CH2CH2OH, LogP=1.25+/- 0.41
3 R=CH2CH2CH3, LogP=3.16+/- 0.39
11 R=CH2CH2OH, LogP=1.25+/- 0.41
1 R=CH2CH2CH3, LogP=3.16+/- 0.39
9 R=CH2CH2OH, LogP=1.25+/- 0.41
18 R=CH3,
LogP=2.10+/- 0.39
19 R=CH3,
LogP=2.10+/- 0.39
17 R=CH3,
LogP=2.10+/- 0.39
OH
O
OH
O
OH
O
R
R
N
R
O
N
O
O
N
6 R=CH2CH2CH3, LogP=1.94+/- 0.38
14 R=CH2CH2OH, LogP= 0.03+/- 0.40
5 R=CH2CH2CH3, LogP=1.94+/- 0.38
13 R=CH2CH2OH, LogP= 0.03+/- 0.40
4 R=CH2CH2CH3, LogP=1.94+/- 0.38
12 R=CH2CH2OH, LogP=0.03+/- 0.40
22 R=CH3,
LogP= 0.87+/- 0.38
21 R=CH3,
LogP= 0.87+/- 0.38
20 R=CH3,
LogP= 0.87+/- 0.38
OH
O
OH
O
R
R
O
O
Br
8 R=CH2CH2CH3, LogP=4.20+/- 0.43
16 R=CH2CH2OH, LogP=2.29+/- 0.46
7 R=CH2CH2CH3, LogP=4.66+/- 0.38
15 R=CH2CH2OH, LogP=2.75+/- 0.40
23 R=CH3, LogP= 3.60+/- 0.38
24 R=CH3,
LogP=3.14+/- 0.43
Fig. 1. New MBHA 1e16 synthesized and biologically evaluated in this article. The MBHA 17e24, recently described by us, that inspired our design through 1e16 from molecular
modification. In silico LogP calculated from http://pharma-algorithms.com.

F.P.L. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4295e4301
4297
Table 1
Experimental conditions to prepare the MBHA 1e16 (Fig. 2) and its yields. Several
other conditions, changing solvent, temperature, reaction time and catalyst amount
were also investigated.
Entry MBHA Aldehyde Acrylate Solvent
Reac. time Yields(%)a
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
29
30
31
32
33
34
35
36
29
30
31
32
33
34
35
36
27
27
27
27
27
27
27
27
28
28
28
28
28
28
28
28
PrOH
PrOH
PrOH
CH3CN
CH3CN
CH3CN
PrOH
3 days
2 days
3 days
4 days
7 days
8 days
6 days
8 days
24 h
24h
18 h
28 h
29 h
68
73
97
70
80
66
60
64
71
58
62
94
83
80
67
71
Scheme 2. Molecular modifications on the anti-leishmania MBHA 17e24 as strategy to
idealize the new MBHA 1e16.
classic bioisosterism strategy for molecular modification, to
idealize the MBHA 9e16 [42] (Scheme 2 and Fig. 1).
PrOH
The systematic change of the physicochemical properties of
compounds in a series, e.g. lipophilicity, is a way of understanding
the relationship between a drug molecule and drug action. Physi-
cochemical properties help control the processes of drug absorp-
tion, distribution, metabolism, excretion and interaction of the drug
with its molecular target. The distribution coefficient (LogP) is
a measure of a drug’s lipophilicity and an indication of its ability to
cross cell membranes [43]. Drugs having values of P beyond 1 are
classified as lipophilic, whereas those with partition coefficients
smaller than 1 are indicative of hydrophilic drugs. These values can
be in silico calculated or accurately determined by using an HPLC
method [44]. The higher the value, more lipophilic is the drug. In
order to gain further insight into the ability of MBHA 1e24 to cross
cell membranes, LogP values were calculated in silico using the help
of Pharma-algorithms (available from http://pharma-algorithms.
com) and the results of these calculations are described in Fig. 1.
In fact, one of the most reliable methods in medicinal chemistry
to improve in vitro activity is to incorporate properly positioned
lipophilic groups. For example, addition of a single methyl group
that can occupy a receptor ‘pocket’ improves binding by about
0.7 kcal/mol [45]. The Lipinski’s rule of five [46] predicts that poor
absorption or permeation is more likely when there are more than
5 H-bond donors, 10 H-bond acceptors, the molecular weight
(MWT) is greater than 500 and the calculated LogP (CLogP) is
greater than 5 [47].
9
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
10
11
12
13
14
15
16
18h
CH2OHCH2OH 6 days
CH2OHCH2OH 24 h
a
yields obtained from the purified products; no co-product was detected; 100% of
conversion based on the recovery of unreacted aldehyde (from CGMS analysis).
that, Hua et al. described in 2006 a synthesis of 27 with a 61% yield
using ZrOCl2$8H2O at 50 ꢀC for 24 h [49].
2.2.2. Synthesis of MBHA 1e16
The MoritaeBayliseHillman (MBH) reaction is an exquisite
reaction [17]. In the two proposed reaction mechanisms, it is
evident that small modifications on the experimental conditions,
such as change of the aprotic solvent to a protic one, can change the
reaction pathway and lead to the appearance and disappearance of
side co-products. In independent works McQuade [50] and
Aggarwal [51] presented a dualistic nature of the MBHR mecha-
nism, which was supported by Coelho and co-workers through ESI-
MS experiments [52].
In our work, several experimental conditions were investigated,
changing solvents, temperature of reactions, reaction time and
catalyst amount (0.1e1.0 Equiv.). However, we only present in
Table 1 the best results to prepare the MBHA 1e16 (Scheme 4). The
experimental details of the MBHRs between the aldehydes 29e36
(Fig. 2) and the acrylates 27 or 28 (Scheme 3) were presented in
experimental procedures.
2.2. Chemistry
2.2.1. Synthesis of propyl acrylate (27)
We prepared the propyl acrylate (27, Scheme 3) from 1 Equiv. of
acrylic acid and 3 Equiv. of propyl alcohol (no solvent was used)
under catalytic quantity of p-toluenesulfonic acid (PTSA), at 60 ꢀC
for 24h, in high yield, as shown in Scheme 3. No antipolymerization
substance was necessary to be added and we did observe no co-
products in this easy and efficient synthetic protocol. Also, the
2-hydroxyethyl acrylate (28, Scheme 3) is a commercial compound.
The preparation of acrylates from different alcohols is an
important chemical transformation for the synthesis of new
materials, e.g. polymers. Most of these synthetic methodologies are
described in patents. As far as we can tell, propyl acrylate (27) was
prepared for the first time in 2005, using a microbiological method.
This related invention described a method for preparing esters of
acrylic acid and (C2-C8)-aliphatic alcohols involving hydrolysis of
acrylonitrile to acrylic acid ammonium salt by using the strain of
bacterium Alcaligenes denitrificans C-32 VKM 2243 D [48]. After
Finally, the characterization of the new MBHA 1e16 was
established by FTIR, 1H and 13C NMR and HRMS. The purities of all
compounds were also measured by gas chromatography (GC)
(Experimental procedures).
2.3. Biology
Its important to notice that all new 1e16 MBHA synthesized
here have in silico logP between þ4.66 and þ0.03, 1e2 H-bond
MBHA 1-16
O
OH O
1 Equiv. DABCO
O
R
R
O
+
Ar
O
Ar
2mL solvent (Table 1)
1.2 mmol 27 or
1.0 mmol 28
1mmol
58 %-97%
Ar =o-nitrobenzyl
R = CH2CH2CH3
R = CH2CH2OH
Ar =m-nitrobenzyl
Ar = p-nitrobenzyl
Ar = 2-pyridinyl
Ar =2-pyridinyl
Ar = 2-pyridinyl
Ar= p-bromobenzyl
Ar =2-naphytyl
Scheme 3. Synthesis of propyl acrylate (27), and the commercial 2-hydroxyethyl
acrylate (28).
Scheme 4. Synthesis of the 1e16 MoritaeBayliseHillman adducts (results in Table 1).

4298
F.P.L. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4295e4301
O
NO2
O
O
H
O2N
O
N
O
O
N
O2N
29
30
33
31
O
O
O
N
37
N
N
Fig. 3. Seven members IHB (NO2/HO) in compound 37.
32
O
34
O
conformations. This proposal is reasonable, based on our previous
results [54] where it was characterized by QTAIM theory a seven
member Intramolecular Hydrogen Bond (IHB) between the o-nitro
group in the aromatic ring and the hydroxyl group (N]O/HeO) in
MBHA 37 (Fig. 3). It is important to emphasize that this computa-
tional study which characterized the IHBs were performed
considering in silico aqueous environment [55].
Br
36
35
Fig. 2. The corresponding aldehydes 29e36, used to prepare the MBHA 1e16.
Evidently this conformational modification caused by the nitro
group change the interaction between the molecule and its bio-
logical site of action. It is well established that the redox system TR/
Trypanothione is vital for parasite survival within the host cell and
a good target for chemotherapy anti-leishmania [56,57]. Also, this
conformational effect can also modify the reduction potential of
these compounds changing its biological activities [55]. Even if, up
to now, the correct biological mechanism of action of these MBHA is
not known, it is reasonable to assume that both the conformation
induced by the o-nitro group and the redox potentials of these
compounds are key for their activities. The study of the biological
mechanism of action of these MBH adducts will now be
investigated.
donors, 2e5 H-bond acceptors, the molecular weight between 193
and 300 g/mol, (Fig. 1). All of these are consistent with both the
“Lipinski’s rule of five” and the “Reynisson’s rule”, that consider
respectively the drug-like chemical space and the known drug
space (KDS) [53].
We can note in Table 2 that the two more toxic MBHA against
Leishmania amazonenesis and safe in macrophages, are adducts 1
and 9 (Entry 1 and 9 of Table 2) more accurately evaluated due to
their higher selectivity index (SI). It is noteworthy that, both of
them (1 and 9) have the o-nitrophenyl moiety in these structures.
We could not be able to realize in this present study any evident
relationship between the lipophilicity and bioactivity on MBHA
1e16. In fact, both 1 (more lipophilic, LogP ¼ 3.16, IC50 ¼ 28.38
and 9 (less lipophilic, LogP ¼ 1.25, IC50 ¼ 29.81 M) adducts are
more actives against L. amazonensis that the previously described
MBHA 17 (Fig. 1, intermediate lipophilicity, logP 2.10,
M) [31]. However, it is important to highlight that MBHA
mM)
m
3. Conclusion
¼
IC50 ¼ 62
m
In this article we presented the synthesis, the physical charac-
terization and the biological activities (IC50 and CC50) of sixteen
new aromatic MBHA 1e16. All new adducts were very active
against L. amazonensis (Table 2). The similar structures previously
described MBHA 17e24 [31] were, in most studied cases here, less
bioactive against promastigote form of L. amazonensis than the new
1e16 MBHA. Curiously, both the more lipophilic MBHA 1e8
(idealized from change of calculated LogP) and the MBHA 9e16
(idealized from the classic bioisosterism strategy) are, in most cases
presented here, more bioactives than 17e24 [31]. A possible
conclusion for the lack of a direct relationship between lipid solu-
bility and biological activity in the 1e24 MBHA could be the pres-
ence of different numbers of H-bond donor and H-bond acceptor in
9e16. The most of the selectivity indexes (SI) were greater than 1.
The one-step efficient synthesis and the good selectivity indexes
obtained indicates that 1e16 can be a new promising class of anti-
parasitic compounds. Therefore, we assume that the preparations
of each compound were racemates. One stereoisomer could be
much more active than the other, as is often the case with biolog-
ically active molecules. In the future, we intend for determine
in vivo the relative activities of the stereoisomers of the most active
racemate mixtures [58].
9 (the least lipophilic between 1, 9 and 17) also present one more
hydroxyl group, that increase one H-bond donor and H-bond
acceptor into the structure. Of course, this fact surpasses a simple
analysis of bioactivity based only on the logP values.
This fact could also reinforce our proposal that the o-nitrophenyl
group is an important pharmacophore of this new class of anti-
parasitic compounds [31]. We believe that the proximity between
the NO2 and the
b carbonyl OH groups in 1 and 9 may alter its
Table 2
IC50 values (
mg/mL and mM) in cell lines, CC50 values (mg/mL and mM) and selectivity
index (SI).a
MBHA
IC50
g/mL)
7.52
19.14
13.79
42.89
46.59
32.84
15.52
22.26
5.48
17.24
14.16
59.39
72.20
85.11
14.29
17.02
IC50
M)
Cytotoxicity
CC50 g/mL)
Cytotoxicity
CC50 M)
Selectivity
indexa
(m
(
m
(
m
(m
1
2
3
4
5
6
7
8
28.38
72.23
52.04
194.07
210.81
148.60
54.48
74.70
20.52
64.57
53.03
35.77
30.15
32.20
158.10
84.76
60.17
40.35
37.28
29.81
18.42
12.53
166.92
93.25
102.21
32.10
47.26
134.98
113.77
121.50
715.38
383.53
272.26
149.44
125.10
111.64
68.99
4.75
1.57
2.33
3.68
1.81
1.83
2.59
1.67
5.43
1.06
0.88
2.81
1.29
1.20
2.24
2.77
9
10
11
12
13
14
15
16
4. Experimental procedures
46.93
266.32
323.77
381.65
52.54
748.52
418.16
458.34
118.01
157.43
4.1. Chemistry
4.1.1. General methods
Commercially available reagents were purchased from Aldrich
and used without further purification. The compounds Propyl
56.73
a
Selectivity index (SI) defined by the ratio CC50/IC50.

F.P.L. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4295e4301
4299
acrylate 27 synthesized in this work are not new and it was char-
acterized using 1H NMR and 13C NMR by comparison with the
compounds described in literature [44,45]. The new MBHA 1e16
were characterized using 1H NMR and 13C NMR spectra, obtained
by using a Mercury Spectra AC 200 (200 MHz for 1H and 50.3 MHz
for 13C) in CDCl3 or Varian Spectra VNMR S-500 (500 MHz for 1H
and 125 MHz for 13C) using TMS as an internal standard. The Fourier
Transform Infrared Spectroscopy (FTIR) spectra were obtained
using a spectrophotometer IR-Prestige-21 (Shimadzu). The CG were
recorded on an GC:AGILENT-6890/HP-MSD5973 instrument under
electron impact (EI) at 70 e.V., injection temperature ¼ 280 ꢀC,
injection mode ¼ split, total flow ¼ 45 mL/min, column initial
flow ¼ 1.0 mL/min, O.T.P. ¼ rate 20.00 ꢀC/min (40e280 ꢀC). HRMS
spectra were obtained on a GCT Premier (TOF-MS), using the
column: HP 5-MS. TLC was done by using the flexible plates for TLC
silica gel Kieselgel 60 (Whatman) and spots were visualized with
short wavelength UV light 254 nm.
4.1.3.3. 2-[Hydroxy(4-nitrophenyl)propyl]propanoate (3). 97% yield;
colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 0.90 (t, 3H, J ¼ 7.2 Hz,
CH3), 1.64 (sex, 2H, CH2), 2.85 (sl, 1H, OH), 4.08 (t, 2H, J ¼ 6.8 Hz,
CH2), 5.62 (s,1H, CHeOH); 5.86 (s, 1H, CH2]C), 6.39 (s,1H, CH2]C),
7.56 (d, 2H, J ¼ 8.4 Hz, Ar), 8.19 (d, 2H, J ¼ 8.8 Hz, Ar). 13C NMR
(CDCl3, 50 MHz) d: 9.90, 21.38, 66.42, 72.33, 123.162 (2C), 126.61,
126.89 (2C), 140.71, 146.95, 148.30, 165.57; IR (KBr, cmꢂ1): 3481,
1712, 1523, 1350; GC: RT ¼ 15.84 min (100%); HRMS -Mass calcu-
lated: 265.0950, Found: 265.0950; C13H15NO5.
4.1.3.4. 2-[Hydroxy(pyridin-2-yl)propyl]propanoate (4). 70% yield;
colorless oil; 1H NMR (CDCl3, 200 MHz)
d
: 0.84 (t, 3H, J ¼ 7.4 Hz,
CH3), 1.58 (sex, 2H, CH2), 4.03 (t, 2H, J ¼ 6.6 Hz, CH2), 4.18 (sl, 1H,
OH), 5.58 (s, 1H, CHeOH), 5.93 (s, 1H, CH2]C), 6.34 (s, 1H, CH2]C),
7.17 (t, 1H, J ¼ 6.2 Hz, Ar), 7.38 (d, 1H, J ¼ 7.8 Hz, Ar), 7.64 (ddd, 1H,
J ¼ 7.6/7.6/1.4 Hz, Ar), 8.49 (d, 1H, J ¼ 4.8 Hz, Ar); 13C NMR (CDCl3,
50 MHz) d:10.30, 21.76, 66.34, 72.17, 121.17, 122.57, 126.73, 136.95,
141.76, 148.08, 159.49, 166.08; IR (KBr, cmꢂ1): 3151, 1712; GC:
RT ¼ 13.32 min (100%); HRMS - Mass calculated: 221.1052, Found:
221.1052; C12H15NO3.
4.1.2. Synthesis of propyl acrylate 27 [44,45]
The reaction was carried out using 1.0 mmol of acrylic acid 25,
3.0 mmols of propyl alcohol 26 and 0.1 Equiv. p-Toluenesulfonic
acid (PTSA) under heating at 60 ꢀC for 1 day. The reaction was
allowed to reach room temperature, diluted with CH2Cl2 (15 mL)
and washed with a cold solution of 10% NaOH (2 ꢁ 8 mL) and
then with a cold solution of saturated NaCl (1 ꢁ 8 mL). The
organic phase was dried with MgSO4, filtered and distilled at
4.1.3.5. 2-[Hydroxy(pyridin-3-yl)propyl]propanoate (5). 80% yield;
colorless oil; 1H NMR (CDCl3, 200 MHz)
d
: 0.84 (t, 3H, J ¼ 7.4 Hz,
CH3), 1.58 (sex, 2H, CH2), 4.01 (t, 2H, J ¼ 6.8 Hz, CH2), 4.54 (sl, 1H,
OH), 5.55 (s, 1H, CHeOH), 5.93 (s, 1H, CH2]C), 6.36 (s, 1H, CH2]C),
7.22 (dd, 1H, J ¼ 7.8/4.8 Hz, Ar), 7.69 (d, 1H, J ¼ 8.2 Hz, Ar), 8.37 (d,
70 ꢀC, 98% yield; colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 0.96
1H, J ¼ 4.6 Hz, Ar), 8.46 (sl, 1H, Ar); 13C NMR (CDCl3, 50 MHz)
d:
(t, 4H, J ¼ 7.4 Hz); 1.69 (sex, 3H); 4.11 (t, 2H, J ¼ 6.6 Hz); 5.81 (dd,
9.98, 21.45, 66.26, 70.41, 123.11, 125.72, 134.30, 137.22, 141.37,
147.93, 148.21, 165.58; IR (KBr, cmꢂ1): 3191, 1714; GC:
RT ¼ 13.76 min (100%); HRMS - Mass calculated: 221.1052, Found:
221.1052; C12H15NO3.
1H, J ¼ 10,4/1.8 Hz); 6.12 (dd, 1H, J ¼ 17.4/10.4 Hz); 6.40 (dd, 1H,
J ¼ 17.4/1.8 Hz). 13C NMR (CDCl3, 50 MHz)
d: 10.39, 21.96, 66.12,
128.59, 130.48, 166.36.
4.1.3. General synthesis of the MBHA 1e16
4.1.3.6. 2-[Hydroxy(pyridin-4-yl)propyl]propanoate (6). 66% yield;
The reactions were carried out using 2 mL of solvent (Table 1),
1.0 mmol of corresponding aldehydes, 1.2 mmols of acrylate 27 or
1.0 mmols of acrylate 28 (Table 1) at room temperature until all
starting aldehyde was consumed or until the reaction parked, as
indicated by TLC analysis using ethyl acetate/hexane (7:3 by
volume). The mixtures were evaporated and filtered through silica
gel. Purification of the product was done by chromatography
column using 15 g of silica gel (230e400 mesh) with ethyl acetate/
hexane (3:7 by volume) as eluent.
colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 0.84 (t, 3H, J ¼ 7.4 Hz,
CH3), 1.58 (sex, 2H, CH2), 4.02 (t, 2H, J ¼ 6.6 Hz, CH2), 4.73 (sl, 1H,
OH), 5.51 (s, 1H, CHeOH), 5.91 (s, 1H, CH2]C), 6.35 (s, 1H, CH2]C),
7.29 (d, 2H, J ¼ 6 Hz, Ar), 8.40 (d, 2H, J ¼ 5.6 Hz, Ar); 13C NMR (CDCl3,
50 MHz) d: 11.74, 23.22, 68.09, 73.09, 123.12 (2C), 128.07, 142.84,
150.57 (2C), 153.04, 167.31; IR (KBr, cmꢂ1): 3.124, 1708; GC:
RT ¼ 13.81 min (100%); HRMS - Mass calculated: 221.1052, Found:
221.1052; C12H15NO3.
4.1.3.7. 2-[Hydroxy(naphth-2-yl)propyl]propanoate (7). 60% yield;
4.1.3.1. 2-[Hydroxy(2-nitrophenyl)propyl]propanoate (1). 68% yield;
colorless oil; 1H NMR (CDCl3, 500 MHz)
CH3), 1.65 (sex, 2H, CH2), 3.23 (d, 1H, J ¼ 5.5 Hz, OH), 4.09 (t, 2H,
J ¼ 7.0 Hz, CH2), 5.75 (d, 1H, J ¼ 5.5 Hz, CHeOH), 5.88 (s, 1H, CH2]
C), 6.41 (s, 1H, CH2]C), 7.49 (m, 3H, Ar), 7.86 (m, 4H, Ar); 13C NMR
d
: 0.90 (t, 3H, J ¼ 7.5 Hz,
colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 0.85 (t, 3H, J ¼ 7.2 Hz,
CH3), 1.59 (sex, 2H, CH2); 3.15 (sl, 1H, OH); 4.05 (t, 2H, J ¼ 6.6 Hz,
CH2); 5.73 (s, 1H, CHeOH); 6.16 (s, 1H, CH2]C); 6.37 (s, 1H, CH2]
C); 7.45 (ddd, 1H, J ¼ 7.6/7.2/1.6 Hz, Ar); 7.63 (ddd, 1H, J ¼ 7.6/7.2/
1.2 Hz, Ar); 7.73 (dd, 1H, J ¼ 7.8/1.6Hz, Ar); 7.93 (dd, 1H, J ¼ 8.0/
1.2 Hz, Ar). 13C NMR (CDCl3, 50 MHz): 9.84, 21.35, 66.29, 67.23,
124.14, 125.88, 128.25, 128.43, 133.06, 135.78, 140.38, 147.91,
165.55; IR (KBr, cmꢂ1): 3452, 1712, 1527, 1352; GC: RT ¼ 14.97 min
(100%); HRMS - Mass calculated: 265.0950, Found: 265.0950;
C13H15NO5.
(CDCl3, 125 MHz)
d: 10.59, 22.10, 66.81, 73.70, 124.84, 125.75,
126.26, 126.37 (2C), 127.88, 128.33, 128.41, 133.26, 133.48, 138.93,
142.37, 166.69; IR (KBr, cmꢂ1): 3446, 1714; GC: RT ¼ 16.51 min
(100%); HRMS - Mass calculated: 270.1256, Found: 270.1256;
C17H18O3.
4.1.3.8. 2-[Hydroxy(4-bromophenyl)propyl]propanoate (8). 64% yield;
colorless oil; 1H NMR (CDCl3, 500 MHz)
d
: 0.92 (t, 3H, J ¼ 7.5 Hz, CH3),
4.1.3.2. 2-[Hydroxy(3-nitrophenyl)propyl]propanoate (2). 73% yield;
1.66 (sex, 2H, CH2), 3.16 (d, 1H, J ¼ 6 Hz, OH), 4.10 (t, 2H, J ¼ 7.0 Hz,
colorless oil; 1H NMR (CDCl3, 200 MHz)
d
: 0.89 (t, 3H, J ¼ 7.4 Hz,
CH2), 5.52 (d, 1H, J ¼ 5.5 Hz, CHeOH), 5.83 (s,1H, CH2]C), 6.36 (s,1H
CH3), 1.64 (sex, 2H, CH2), 2.89 (sl, 1H, OH), 4.07 (t, 2H, J ¼ 6.8 Hz,
CH2), 5.62 (s, 1H, CHeOH), 5.90 (s, 1H, CH2]C), 6.40 (s, 1H, CH2]C),
7.51 (t, 1H, J ¼ 8.0 Hz, Ar), 7.73 (d, 1H, J ¼ 7.8 Hz, Ar), 8.13 (ddd, 1H,
CH2]C), 7.27 (d, 2H, J ¼ 8.5 Hz, Ar), 7.48 (d, 2H, J ¼ 8.5 Hz, Ar); 13
C
NMR (CDCl3, 125 MHz) d: 10.58, 22.10, 66.89, 73.12, 121.95, 126.43,
128.54 (2C), 131.74 (2C), 140.65, 142.01, 166.48; IR (KBr, cmꢂ1): 3446,
1712; GC: RT ¼ 18.90 min (100%); HRMS - Mass calculated: 298.0204,
Found: 298.0204; C13H15BrO3.
J ¼ 8.2/2.2/1.0 Hz, Ar), 8.24 (sl, 1H, Ar). 13C NMR (CDCl3, 50 MHz)
d:
9.89, 21.37, 66.40, 72.16, 121.12, 122.28, 126.55, 128.90, 132.25,
140.73, 143.30, 147.82, 165.55; IR (KBr, cmꢂ1): 3473, 1710, 1531,
1350; GC: RT ¼ 15.68 min (100%); HRMS - Mass calculated:
265.0950, Found: 265.0950; C13H15NO5.
4.1.3.9. 2-[Hydroxy(2-nitrophenyl)hydroxyethyl]propanoate (9). 71%
yield; colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 3.48 (sl, 2H, OH),

4300
F.P.L. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4295e4301
3.79 (t, 2H, J ¼ 4.6 Hz, CH2), 4.25 (t, 2H, J ¼ 4.6 Hz, CH2), 5.57 (s, 1H,
CHeOH), 6.24 (s, 1H, CH2]C), 6.34 (s, 1H, CH2]C), 7.45 (ddd, 1H,
J ¼ 8.2/7.3/1.4 Hz, Ar), 7.64 (ddd, 1H, J ¼ 7.6/7.4/1.2 Hz, Ar), 7.79 (d,
1H, J ¼ 7.8 Hz, Ar), 7.94 (dd, 1H, J ¼ 8.0/1.2 Hz, Ar); 13C NMR (CDCl3,
1712; GC: RT ¼ 17.68 min (100%); HRMS - Mass calculated: 272.1049,
Found: 272.1049; C16H16O4.
4.1.3.16. 2-[Hydroxy(4-bromophenyl) hydroxyethyl]propanoate (16).
50 MHz)
d
: 60.65, 66.53, 67.11, 124.58, 126.92, 128.64, 128.85,
71% yield; colorless oil; 1H NMR (CDCl3, 500 MHz)
d: 1.79 (sl, 1H,
133.59, 136.49, 141.38, 147.82, 166.12; IR (KBr, cmꢂ1): 3390, 1714,
1525, 1350. GC: RT ¼ 11.76 min (100%); HRMS - Mass calculated:
267.0743, Found: 267.0743; C12H13NO6.
OH), 3.04 (sl, 1H, OH), 3.82 (t, 2H, J ¼ 4.5 Hz, CH2), 4.28 (t, 2H,
J ¼ 4.5 Hz, CH2), 5.55 (s, 1H, CHeOH), 5.87 (s, 1H, CH2]C), 6.42 (s,
1H, CH2]C), 7.28 (d, 2H, J ¼ 8.5 Hz, Ar), 7.48 (d, 2H, J ¼ 8.5 Hz, Ar);
13C NMR (CDCl3, 125 MHz)
d: 61.22, 66.71, 72.96, 122.11, 127.21,
4.1.3.10. 2-[Hydroxy(3-nitrophenyl)hydroxyethyl]propanoate (10). 58%
128.52 (2C),131.85 (2C),140.49,141.77, 166.43; IR (KBr, cmꢂ1): 3390,
1712; GC: RT ¼ 15.91 min (100%); HRMS - Mass calculated:
299.9997, Found: 299.9997; C12H13BrO4.
yield; colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 2.79 (sl,1H, OH), 3.78
(t, 2H, J ¼ 4.8 Hz, CH2), 4.09 (sl, 1H, OH), 4.22 (t, 2H, 3.0 Hz, CH2), 5.65
(s, 1H, CHeOH), 5.87 (s, 1H, CH2]C), 6.42 (s, 1H, CH2]C), 7.50 (t, 1H,
J ¼ 8.0 Hz, Ar), 7.70 (d, 1H, 7.8 Hz, Ar), 8.10 (ddd, 1H, J ¼ 8.2/2.0/1.0 Hz,
Ar), 8.22 (sl, 1H, Ar); 13C NMR (CDCl3, 50 MHz)
d: 60.59, 66.43, 71.94,
4.2. Biological evaluation
121.56, 122.72, 127.73, 129.36, 132.78, 141.30, 143.61, 148.17, 165.97; IR
(KBr, cmꢂ1): 3355, 3326, 1708, 1529,1350; GC: RT ¼ 16.84 min (100%);
HRMS - Mass calculated: 267.0743, Found: 267.0743; C12H13NO6.
4.2.1. Antipromastigotes activities
The promastigotes viability was determined by the ability of
living cells to reduce the yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide, a tetrazole) to purple formazan
[59]. Promastigote forms of L. amazonensis (IFLA/BR/67/PH8) in the
log phase of growth were incubated at 25 ꢀC in 96-well cellular
4.1.3.11. 2-[Hydroxy(4-nitrophenyl)hydroxyethyl]propanoate (11). 62%
yield; colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 2.76 (sl, 2H, OH), 3.81
(t, 2H, J ¼ 4.6 Hz, CH2), 4.26 (t, 2H, J ¼ 4.4 Hz, CH2), 5.66 (s, 1H,
CHeOH), 5.86 (s,1H, CH2]C), 6.44 (s, 1H, CH2]C), 7.57 (d, 2H,
culture plates (TPP, Switzerland) with 1 ꢁ 105 cells/well in 100
mL
J ¼ 8.6 Hz, Ar), 8.19 (d, 2H, J ¼ 8.8 Hz, Ar); 13C NMR (CDCl3, 50 MHz)
d
:
Schneider’s Drosophila medium supplemented with 20% of FBS in
the presence or absence of different concentrations of 1e16 MBHA.
The growth of promastigote forms was evaluated simultaneously in
the presence of GlucantimeÒ, as reference drug. After a 72 h incu-
60.75, 66.51, 72.40, 123.61 (2C), 127.34 (2C), 127.83, 141.09, 147.39,
148.53, 165.99; IR (KBr, cmꢂ1): 3529, 3371, 1699, 1519, 1350; GC:
RT ¼ 17.04 min (100%); HRMS - Mass calculated: 267.0743, Found:
249.0637 [M ꢂ 18]; C12H11NO5 [M ꢂ 18].
bation, 10
of incubation at 25 ꢀC the formed product formazan was dissolved
in 50 L of Sodium Dodecyl Sulfate (SDS) at 10% for 24 h and the
m
L of a 5 mg mLꢂ1 MTT solution was added to it. After 4 h
4.1.3.12. 2-[Hydroxy(pyridin-2-yl)hydroxyethyl]propanoate (12). 94%
m
yield; colorless oil; 1H NMR (CDCl3, 200 MHz)
d
: 3.62 (t, 2H, J ¼ 4.4 Hz,
absorbance was measured by spectrophotometry at a 545 nm
wavelength. The percentage viability was calculated from the ratio
of OD readings in wells with compounds versus wells without
compounds ꢁ 100. The concentration which inhibits 50% of growth
(IC50) was determined by regression analysis using the SPSS 8.0
software for Windows. All experiments were done at least three
times and each experiment was performed in triplicate [31].
CH2), 4.09 (m, 2H, CH2), 5.57 (s,1H, CHeOH), 5.85 (s,1H, CH2]C), 6.29
(s,1H, CH2]C), 7.11 (m,1H, Ar), 7.35 (d,1H, J ¼ 8 HZ, Ar), 7.60 (ddd,1H,
J ¼ 7.8/7.7/1.6 Hz, Ar), 8.34 (d, 1H, J ¼ 5 Hz, Ar); 13C NMR (CDCl3,
50 MHz) d: 59.99, 66.15, 72.08, 121.03, 122.54, 126.86, 137.03, 141.71,
147.71, 159.81, 165.66; IR (KBr, cmꢂ1): 3344, 1716; GC: RT ¼ 14.26 min
(100%); HRMS
- Mass calculated: 223.0845, Found: 205.0739
[M ꢂ 18]; C11H13NO4 [M ꢂ 18].
4.2.2. Cytotoxicity in murine macrophages
4.1.3.13. 2-[Hydroxy(pyridin-3-yl)hydroxyethyl]propanoate (13). 83%
The animals were maintained at constant room temperature
(21 þ 2 ꢀC) and on a 12/12 h lightedark cycle, with free access to
food pellets and water. Initially, the animals were elicited by peri-
toneal injection of 1 mL of sterile 4% thioglycollate solution (Gibco)
and after five days, the mice were euthanized by cervical disloca-
tion and cells were recovered by peritoneal lavage using 10 ml of
cold PBS containing 3% fetal bovine serum (FBS). Cells were
immediately stored on ice. The suspension was centrifuged at
2500 rpm for 10 min and the pellet resuspended in 1 ml of RPMI
yield; colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 3.66 (t, 2H, J ¼ 4.8 Hz,
CH2), 4.10 (t, 2H, J ¼ 4.8 Hz, CH2), 4.37 (sl, 2H, OH), 5.52 (s, 1H,
CHeOH), 5.87 (s, 1H, CH2]C), 6.31 (s, 1H, CH2]C), 7.18 (m, 1H, Ar),
7.65 (m, 1H, Ar), 8.27 (m, 1H, Ar), 8.39 (sl, 1H, Ar); 13C NMR (CDCl3,
50 MHz) d: 59.97, 66.20, 69.80, 123.36, 126.28, 134.78, 137.69, 141.64,
147.75, 147.91, 165.58; IR (KBr, cmꢂ1): 3336, 1714; GC: RT ¼ 18.81 min
(100%); HRMS - Mass calculated: 223.0845, Found: 223.0845;
C11H13NO4.
1640 (Cultilab, Brazil) supplemented with 10% FBS and 50 mg/ml of
4.1.3.14. 2-[Hydroxy(pyridin-4-yl)hydroxyethyl]propanoate (14). 80%
streptomycin and penicillin 1000 U/ml (RPMI complete). Aliquots
of the suspension were used to quantify the number of viable cell
using the trypan blue method at a final concentration of 0.4% in
order to discriminate between live and dead cells. The quantifica-
tion was done in Neubauer hemocytometer chamber, and the
number of cells in suspension was adjusted to 4 ꢁ 106 cells/mL and
yield; colorless oil; 1H NMR (CDCl3, 200 MHz)
d: 1.27 (sl,1H, OH), 3.76
(sl, 1H, OH), 3.85 (t, 2H, J ¼ 5 Hz, CH2), 4.30 (t, 2H, J ¼ 5 Hz, CH2), 5.56
(s, 1H, CHeOH), 5.89 (s, 1H, CH2]C), 6.46 (s, 1H, CH2]C), 7.35 (d, 2H,
J ¼ 6.5 Hz, Ar), 8.61 (d, 2H, J ¼ 6.0 Hz, Ar); 13C NMR (CDCl3, 50 MHz)
d:
59.98, 66.21, 73.09, 123.11 (2C), 128.06, 142.84, 150.56 (2C), 153.05,
167.31; IR (KBr, cmꢂ1): 3309, 1724; GC: RT ¼ 14.73 min (100%); HRMS
- Mass calculated: 223.0845, Found: 224.0921 [M þ 1]; C11H13NO4.
was then incubated in 100 mL of complete RPMI in 96-well plates, in
the absence and presence of different concentrations of the MBHA
1e16. After 24 h of incubation in an incubator at 5% CO2, was
4.1.3.15. 2-[Hydroxy(naphth-2-yl)hydroxyethyl]propanoate (15). 67%
inoculated 10
tetrazolium bromide (MTT) at a concentration of 5 mg/mL and,
after 4 h, was added to 50 l of SDS at a concentration of 10 mM.
After 24 h we became the reading of the plates in a microplate
reader using the absorbance wavelength of 545 nm. We calculated
the concentration that caused a 50% reduction in cell viability
[CC50] using SPSS version 8.0 for Windows.
mL of 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-
yield; colorless oil; 1H NMR (CDCl3, 500 MHz)
d: 1.90 (sl,1H, OH), 3.20
(sl, 1H, OH), 3.82 (t, 2H, J ¼ 4.5 Hz, CH2), 4.26 (t, 2H, J ¼ 4.5 Hz, CH2),
m
5.78 (s, 1H, CHeOH), 5.94 (s, 1H, CH2 ¼ C), 6.47 (s, 1H, CH2]C), 7.51
(m, 3H, Ar), 7.87 (m, 4H, Ar); 13C NMR (CDCl3, 125 MHz)
d: 60.98,
66.46, 73.30, 124.54, 125.56, 126.21, 126.34, 126.98, 127.72, 128.10,
128.34, 133.07, 133.25, 138.63, 141.92, 166.41; IR (KBr, cmꢂ1): 3394,

F.P.L. Silva et al. / European Journal of Medicinal Chemistry 46 (2011) 4295e4301
4301
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Tests of cytotoxicities were performed in murine macrophages
as a model eukaryotic cell of great importance in the cycle of the
disease. The macrophages were collected from swiss female mices
(3 months of age), weighing 28e35 g and this animals were
obtained from the vivarium Thomas George of Laboratory of
Pharmaceutical Technology, Federal University of Paraíba. The
sacrifice of experimental animals followed international recom-
mendations (AVMA, 2007). All the experimental protocols were
previously approved by the Animal Research Ethical Committee
(CEPA) at the Federal University of Paraíba (process number 208/
2007).
Acknowledgements
This work has been supported by CNPq, CAPES, FAPESQ-PB. We
also would like very much to thank Professor Juliana Alves Vale by
to analyze our new MBHA 1e16 by HRMS and GC and, by the
kindness of the Institute of Chemistry/Unicamp (Campinas City, São
Paulo, Brazil) where the analysis could be made.
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