Chemoenzymatic synthesis and biological evaluation of 2- and 3-hydroxypyridine derivatives against Leishmania mexicana

Article abstract of DOI:10.1016/j.bmc.2012.06.028

A series of hydroxyalkyl and acyloxyalkyl derivatives of 2- and 3-hydroxypyridine was synthesized and their biological activity was evaluated as growth inhibitors of protozoan Leishmania mexicana. Thirty novel compounds were obtained through a chemoenzymatic methodology in two reaction steps. The influence of various reaction parameters in the enzymatic step, such as enzyme source, acylating agent/substrate ratio, enzyme/substrate ratio, solvent and temperature, was studied. Some of the evaluated compounds showed a remarkable activity as Leishmania mexicana growth inhibitors, obtaining the best results with the acetylated derivatives. The advantages showed by the enzymatic methodology, such as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to prepare these derivatives of substituted pyridines with application as potential antiparasitic agents.

Full text of DOI:10.1016/j.bmc.2012.06.028

Bioorganic & Medicinal Chemistry 20 (2012) 4614–4624
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemoenzymatic synthesis and biological evaluation of 2- and
3-hydroxypyridine derivatives against Leishmania mexicana
Guadalupe García Liñares a, Gonzalo Parraud a, Carlos Labriola b, Alicia Baldessari a,
⇑
a Laboratorio de Biocatálisis, Departamento de Química Orgánica y UMYMFOR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,
Pabellón 2, piso 3, C1428EGA Buenos Aires, Argentina
b Instituto de Investigaciones Bioquímicas, Av. Patricias Argentinas, 435C1405BWE Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of hydroxyalkyl and acyloxyalkyl derivatives of 2- and 3-hydroxypyridine was synthesized and
their biological activity was evaluated as growth inhibitors of protozoan Leishmania mexicana. Thirty
novel compounds were obtained through a chemoenzymatic methodology in two reaction steps. The
influence of various reaction parameters in the enzymatic step, such as enzyme source, acylating
agent/substrate ratio, enzyme/substrate ratio, solvent and temperature, was studied. Some of the evalu-
ated compounds showed a remarkable activity as Leishmania mexicana growth inhibitors, obtaining the
best results with the acetylated derivatives. The advantages showed by the enzymatic methodology, such
as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to
prepare these derivatives of substituted pyridines with application as potential antiparasitic agents.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 24 April 2012
Revised 8 June 2012
Accepted 15 June 2012
Available online 23 June 2012
Keywords:
Hydroxypyridine derivatives
Lipase-catalyzed
Synthesis
Leishmaniasis
1. Introduction
Second line drugs include pentamidine (3) and amphotericin B (4),
which are mainly employed in resistant cases when the antimoni-
Leishmaniasis is considered by World Health Organization to be
one of the most serious diseases worldwide caused by protozoan
parasites.1 The leishmaniasis are a complex of clinical diseases pro-
duced by at least 17 species and some mutants of the protozoan
Leishmania.2,3 The spectrum of disease comprises three major syn-
dromes: cutaneous, mucocutaneous and visceral leishmaniasis.
Leishmania mexicana is one of the species that produce cutaneous
leishmaniasis.4 Twelve million people suffer from human leish-
maniasis worldwide with an incidence of two million cases per
year and 350 million individuals at risk focused at tropical and sub-
tropical regions.5–7 Moreover, the incidence of leishmania and HIV
coinfection has been increased worldwide due to occurrence of vis-
ceral leishmaniasis in urban areas.8
Control of this disease remains a problem because effective vac-
cines are not available up until now and the chemotherapy is still
deficient, using old drugs associated to long-term treatments, par-
asitic resistance, and different drug sensitivity. The chemotherapy
for leishmaniasis has been based for over 60 years on the use of
pentavalent antimonial drugs: meglumine antimoniate (Glucan-
time)9 (1) or sodium stibogluconate (Pentostam) (2) are still the
first-choice drugs for Leishmaniasis (Fig. 1). However, these drugs
are very unsatisfactory due to their frequent toxic effects and the
growing rates of resistance to them in several parts of the world.10
als fail.11 The disadvantages of these drugs include their high cost,
the need for long-term treatment, the lack of an oral formulation,
the loss of effectiveness due to parasites resistance and serious
toxic effects.10,12,13 Recently, the oral drug miltefosine (5)14 was
approved for the treatment of human visceral Leishmania infec-
tions, but a WHO report indicates that it is teratogenic.15,16 Two
interesting drugs under clinical trials are the antibiotic paromomy-
cin (6)2,17 and sitamaquine (7)18 (Fig. 1).
Extensive studies have been made in the last few years of new
and diverse molecules used as possible antileishmanial drugs. For
example, arylquinuclidine19 derivatives, developed as cholesterol-
lowering agents, were potent in vitro growth inhibitors of
Leishmania
amazonensis
and
1,3,4-thiadiazolium-2-ami-
nide20exhibited significant activity against Leishmania amazonen-
sis. Some tetrahydro-1-benzazepine derivatives were found to
have good activity against Leishmania chagasi promastigotes.21
The activity of hydroxyurea was also tested against Leishmania
mexicana, showing to be a good candidate for drug therapy for
leishmaniasis because it induces parasite death when it is used
at concentrations ranging from 10 to 100 l
g/mL.22 A series of
1,3,5-triazines and pyrimidines derivatives were synthesized and
screened for their in vitro antileishmanial activity against Leish-
mania donovani and some compounds have shown more than
90% inhibition against promastigotes.23 However, the problems of
drug resistance and the side effects of the chemotherapies used
at present, have not been solved. Consequently, the development
⇑
Corresponding author. Tel.: +54 11 4576 3300x262; fax: +54 11 4576 3385.
E-mail address: alib@qo.fcen.uba.ar (A. Baldessari).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.028

G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
4615
COOH
COOH
OH
. 3 Na
CH3NHCH2
HCO
CH2OH
OCH
. 9 H2O
HO
Sb+
O
Sb
O
O
O
Sb
HCO
OCH
CHOH
CHOH
CH2NHCH3
HO
O
O
O
H
H
CHOH
CHOH
CH2OH
OH
OH
2
1
O
P
CH2CH2N+(CH3)3
O
O
CH3(CH2)14CH2
O-
5
O
O
H2N
NH2
NH2
OH
NH
NH
3
O
O
NH2
O
NH2
HO
HO
O
OH
OH
OH
O
O
H2N
6
HO
NH2
OH
OH
OH
O
OH
O
O
HO
O
OH
OH
OH
OH
CH3
N
OH
H3CO
O
O
4
NH(CH2)6N(C2H5)2
HO
OH
NH2
7
Figure 1. Current drugs clinically employed for the treatment of leishmaniasis.
of novel, safe and affordable compounds with potent anti-leish-
manial activity is urgently needed.
are useful in the synthesis of pharmaceuticals and biologically ac-
tive compounds.29–33
On the other hand, it is known that the use of enzymes and
whole cells of microorganisms in synthesis of pharmaceuticals
derivatives is increasing in the last years.24–26 It is recognized that
enzymes are capable of accepting a wide array of substrates, and
catalyze enantio-, chemo- and regioselective reactions. As a result,
biocatalysts allow carrying out different chemical transformations
without the need for tedious protection and deprotection steps,
especially in compounds with several functional groups. Over the
last years, biocatalysis in non-aqueous media has been widely used
for several synthetic reactions such as esterification, transesterifi-
cation, aminolysis, polymerization, etc.27 Enzymes are also well-
known by their high enantioselective behavior and this property
has formed the basis for the widespread use of enzymes for the
synthesis of enantiomerically pure compounds.28
In the present study, we report the synthesis of a series of 2-
and 3-hydroxypyridine derivatives 8a–n and 9a–p (Fig. 2).
The chemoenzymatic strategy allowed us to obtain these novel
compounds which were biologically evaluated against promasti-
gote Leishmania mexicana.
O
O
O
O
R1
OH
n
n
9a-g: 3-OR, n=5-11, R1 = CH3
9h-n: 2-OR, n=5-11, R1 = CH3
9o: 3-OR, n=10, R1 = CH2CH3
9p: 2-OR, n=10, R1 = CH2CH3
N
N
8a-g: 3-OR, n=5-11
8h-n: 2-OR, n=5-11
Studies carried out in our laboratory on the esterification and
transesterification of multiple substrates have shown that lipases
Figure 2. Structures of 2-hydroxy- and 3-hydroxypyridine derivatives.

4616
G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
with alkyl chains containing from six to twelve carbon atoms. The
2. Results and discussion
2.1. Rationale
reaction was performed in a suspension of potassium hydroxide in
dimethyl sulfoxide via a modified Williamson procedure.48 Pyri-
dinyloxyalkanols of general formula 8 were obtained in good to
very good yields. In a second lipase catalyzed step, compounds 8
were treated with esters and, through a transesterification reac-
tion, compounds of general formula 9 were obtained.
Studies of features of the biochemistry and physiology of Leish-
mania spp. have recognized several potential molecular targets for
the design of new drugs. The chemotherapeutic search is based on
metabolic differences between the pathogenic parasite and mam-
mals hosts. Thus, the selective inhibition of key biosynthetic path-
way for parasite survival would not have any toxic effects for the
host. A valid molecular target for antileishmania compounds is
the enzyme dihydrofolatereductase (DHFR).2 DHFR catalyses the
reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is
methylated to form methylene tetrahydrofolate, which is a vital
cofactor to convert deoxyuridine monophosphate into thymidine
monophosphate. DHFR constitutes an interesting target for drug
design, because DHFR inhibition prevents biosynthesis of thymi-
dine, leading to cell death.34 In addition, the leishmanial DHFR
and the corresponding human enzyme show structural differ-
ences.35 A selective inhibition of leishmanial DHFR would lead to
growth impairing of the parasite because these microorganisms
do not have a mechanism of transport of this cofactor from the
host.
Most known DHFR inhibitors contain a heterocyclic aromatic
ring. For example, pyrimidines or triazines with amino substitu-
ents at the 2- and 4-positions of general formula 10–12 inhibited
DHFR activity (Fig. 3).36–38 Some of these compounds were potent
inhibitors of DHFR activity but the maximum activity against par-
asite cell not always correlate with the more potent enzymatic
inhibitors. It was reported that long chain derivatives (8–12 carbon
atoms) were the most potent cell growth inhibitors.37
2.2.2. Enzymatic synthesis of pyridinyloxyakyl esters
2.2.2.1. Optimal conditions.
With the aim of achieving the
optimal conditions for the enzymatic reaction we studied the
behavior of various lipases and some reaction parameters. In
every case 12-(pyridin-3-yloxy)-dodecan-1-ol (8g) was used as
substrate.
To begin, several commercial lipases in different solvents were
evaluated in the acetylation reaction: Candida antarctica lipase B
(CAL B), Candida rugosa lipase (CRL), Lipozyme, lipase from the fun-
gus Rizhomucor miehei (LIP) and Carica papaya lipase (CPL). Reac-
tions were carried out at 30 °C using an enzyme: substrate ratio
of 10 and ethyl acetate as acylating agent and solvent. As it can
be seen from Table 1 (entries 1–5), all the enzymes were active
achieving 9g in total conversion at different reaction time. CAL B
gave the most satisfactory results, affording 9g in 1 h of reaction
at 30 °C. LIP and CRL were also active but showed a lower perfor-
mance. CPL was effective at 55 °C showing total conversion after
96 h.
Then, we performed several experiments changing reaction
parameters such as enzyme/substrate ratio (E/S) and substrate
concentration. Table 1 (entries 6–11 and 12–15) show the results
obtained for a CAL B catalyzed reaction at 30 °C at different E/S ra-
tio and substrate concentration, respectively.
It has been observed that working at E/S: 0.1, total conversion
was obtained at 7 h of reaction. This is a remarkably low value
for CAL B catalyzed reactions compared with previous work in
which it was reported that an E/S: 2 or higher was necessary to
achieve goods results.29
On the other hand, there are several papers reporting biologi-
cally active compounds which exhibit a pyridine ring in their struc-
ture. Different substituted pyridines showed diverse biological
activities, such as topoisomerase I or II inhibition,39,40 anticancer
activity,41–43 antimicrobial activity,44 cyclooxygenase-2 inhibi-
tion,45 kinase inhibition,46 antiviral activity,47 etc. Little has been
reported about new compounds that show to be very effective as
DHFR and parasite growth inhibitors. Considering the above men-
tioned work and taking into account that pyridine derivatives can
be DHFR inhibitors, we have decided to prepare new compounds
containing a pyridine ring in place of aminopyrimidine ring substi-
tuted with alkyloxy groups of different chain length.
As we had obtained good results with ethyl acetate as acylating
agent in previous work,30–32 we tried ethyl esters (ethyl acetate
and ethyl propionate) as acylating agents. In order to get more con-
venient reaction conditions, in the case of acetylation we also
tested activated esters such as vinyl and isopropenyl acetates.
Some enzymatic acetylations need the use of these activated esters
due to low reactivity of the substrate in the presence of the li-
pase.33 But pyridinyloxyalkanols showed to be excellent substrates
for the biocatalytic acetylation with CAL B and ethyl acetate.
Considering that it is more convenient to work with substrate at
the highest possible concentration, we studied the enzymatic
transesterification at substrate concentrations between 0.04 and
0.4 M. It can be observed (Table 1, entry 13) that the optimum con-
centration for the reaction of 8 g was found to be 0.08 M. Total con-
version was achieved at 7 h of reaction. Table 1 (entries 14–15)
shows that a higher substrate concentration significantly increases
reaction time.
2.2. Synthesis
In order to obtain the compounds of interest, a chemoenzymatic
methodology was applied, involving two steps: the first chemical
and the second enzymatic.
The preparation of this new family of hydroxypyridine deriva-
tives is illustrated in Scheme 1.
2.2.1. Synthesis of pyridinyloxyalkanols
Taking into account the studies previously mentioned, we have
chosen as standard conditions for the enzymatic reaction: CAL B as
The first reaction involved a nucleophilic substitution between
the corresponding hydroxypyridine and various lineal haloalkanols
NH2
N
NH2
NH2
OR
OR
OR
N
N
N
H2N
H2N
N
OR
H2N
N
12
11
10
Figure 3. Benzyl-2,4-diaminopyrimidines as inhibitors of leishmanial DHFR activity.

G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
4617
O
OH
O
O
O
R1
OH
b
OH
a
n
n
+
X
n
N
N
N
9a: 3-OR, n=5, R1 = CH3
9b: 3-OR, n=6, R1 = CH3
9c: 3-OR, n=7, R1 = CH3
9d: 3-OR, n=8, R1 = CH3
9e: 3-OR, n=9, R1 = CH3
13a: 3-OH
13b: 2-OH
14a: X = Cl, n=5
14b: X = Br, n=6
14c: X = Br, n=7
14d: X = Br, n=8
14e: X = Br, n=9
14f: X = Br, n=10
14g: X = Br, n=11
8a: 3-OR, n=5
8b: 3-OR, n=6
8c: 3-OR, n=7
8d: 3-OR, n=8
8e: 3-OR, n=9
8f: 3-OR, n=10
8g: 3-OR, n=11
8h: 2-OR, n=5
8i: 2-OR, n=6
8j: 2-OR, n=7
8k: 2-OR, n=8
8l: 2-OR, n=9
8m: 2-OR, n=10
8n: 2-OR, n=11
9f: 3-OR, n=10, R1 = CH3
9g: 3-OR, n=11, R1 = CH3
9h: 2-OR, n=5, R1 = CH3
9i: 2-OR, n=6, R1 = CH3
9j: 2-OR, n=7, R1 = CH3
9k: 2-OR, n=8, R1 = CH3
9l: 2-OR, n=9, R1 = CH3
9m: 2-OR, n=10, R1 = CH3
9n: 2-OR, n=11, R1 = CH3
9o: 3-OR, n=10, R1 = CH2CH3
9p: 2-OR, n=10, R1 = CH2CH3
Scheme 1. Chemoenzymatic synthesis of 2-hydroxy- and 3-hydroxypyridine derivatives.
Table 1
Optimization of reaction parameters for lipase-catalyzed preparation of 12-(pyridin-3-yloxy)-dodecyl acetate (9g)
Entry
Enzyme
Temperature (°C)
E/S
Substrate concentration (M)
t (h) (total conversion)
Lipase
1
2
3
4
5
CAL B
LIP
CRL
CPL
CPL
30
30
30
55
55
10
10
10
10
15
0.04
0.04
0.04
0.04
0.04
1
24
96
144
96
E/S
6
7
8
9
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
30
30
30
30
30
30
2
1
0.5
0.2
0.1
0.02
0.04
0.04
0.04
0.04
0.04
0.04
1
1
2
4
7
10
11
96
Substrate concentration
12
13
14
15
CAL B
30
30
30
30
0.1
0.1
0.1
0.1
0.04
0.08
0.2
7
7
24
72
CAL B
CAL B
CAL B
0.4
Acylating agent and solvent: ethyl acetate.
biocatalyst, temperature: 30 °C, an E/S ratio of 0.1 and substrate
concentration: 0.08 M. These conditions were optimized for the
acetylation reaction of 8g with ethyl acetate.
have tried the same procedure but using tetrahydropyranyl ethers
a protecting groups for the hydroxyl. In this case, the products
were obtained in similar yields but with the disadvantage of add-
ing two additional steps to the synthetic way.
2.2.2.2. Acylation of pyridinyloxyalkanols.
Using the stan-
Moreover, it was observed that reaction yields with 2-hydroxy-
pyridine were lower than with the 3-hydroxy isomer. The keto-
enol tautomerism in 2-hydroxypyridine could explain its lower
reactivity. All compounds 8 were fully characterized by using
NMR and IR spectroscopy and high resolution mass spectrometry.
With regard to the enzyme-catalyzed acylation, acetate deriva-
tives of pyridinyloxyalkanols were obtained in almost quantitative
yield (90–97% of isolated compound), in a slightly higher yield in
the case of the 3-hydroxy derivatives. Yield increased as the alkyl
chain length increased. Two propionate derivatives 9o and 9p were
also prepared using ethyl propionate as acylating agent, which
showed to be not so efficient as ethyl acetate. For the same sub-
strate 8f, the propionyl derivative 9o was obtained only in 60%
yield, 37% lower than the 97% yield in 9f. In the case of the 2-hy-
droxy derivative, the decrease in yield for the propionylation of
8m was even more marked, affording 9p in only 45% yield.
dard conditions described in the previous section, we applied the
enzymatic strategy in the preparation of the acetyl derivatives of
the pyridinyloxyalkanols 9a–n.
Accordingly, the compounds 8a–n, obtained in the first step of
the synthesis, were dissolved in ethyl acetate, that acted both as
acylating agent and solvent. The enzyme was then added and the
suspension was shaken at 30 °C for 1 hour (Scheme 1). The lipase
afforded each acetyl derivative (9a–n) in excellent yield. All prod-
ucts were completely identified by spectroscopic methods.
2.2.3. Synthetic results
Table 2 shows that pyridinyloxyalkanols of general formula 8
were obtained in good to very good yields.
These yields were not influenced by the length of the chain of
haloalkanol. With the purpose of increasing the product yield, we

4618
G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
Table 2
Pyridinyloxyalkanols (8) and their acylated derivatives (9)
n
Pyridinyloxyalkanol (8) % yield
Pyridinyloxyalkanol acetate (9) % yielda
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
2-OH
2-OH
2-OH
2-OH
2-OH
2-OH
2-OH
3-OH
2-OH
5
6
7
8
9
10
11
5
6
7
8
9
10
11
10
10
66
70
62
65
65
68
72
52
55
55
60
58
50
51
—
90
92
90
95
95
97
99
90
90
90
94
95
95
95
60
45
—
Reaction conditions as described in experimental.
a
9o and 9p: propionate.
The enzymatic acylation offered a good alternative to prepare
ester derivatives of the pyridinyloxyalkanols. Although, the syn-
thesis of esters performed by chemical methods is not difficult, it
has the disadvantage of using acetic anhydride and pyridine or
dimethylaminopyridine as reagents.49 The enzymatic approach
showed interesting advantages. The reaction was simple, it was
performed at room temperature and the products were obtained
in excellent yield by simple filtration and solvent evaporation.
Moreover, ethyl esters used as nucleophile and solvent, are eco-
nomic and less toxic than most of acylating agents commonly used
in traditional synthetic procedures. The lipase is biodegradable and
consequently more friendly to the environment than chemical cat-
alysts. In addition, as the enzyme is insoluble in the reaction med-
ium, it is easily removed by filtration and can be re-used. In the
acetylation reaction of pyridinyloxyalkanols, CAL B kept 82% of
activity after six reaction cycles.
the acyl chain, we prepared 9o. This compound showed no inhibi-
tory activity on Leishmania mexicana growth, indicating that this
fine structural difference can influence biological activity.
3. Conclusions
This work describes a chemoenzymatic strategy for the synthe-
sis of thirty novel pyridinyloxyalkanols and acylated derivatives,
which were completely spectroscopically characterized. Lipases
from different sources exhibited different performance as catalysts
in the second step. CAL B lipase gave the best results. The advanta-
ges presented by this methodology are: mild reaction conditions,
economy and low environmental impact. The acylation reaction
with CAL-B was of great interest due to a very low E/S ratio allowed
to obtain the desired products at 7 h of reaction with a 100% con-
version. This result constitutes an additional advantage of this
method.
2.3. Biological evaluation
All of the compounds were biological evaluated against promas-
tigote Leishmania mexicana. Products 8g, 9g and 9e showed to be
very effective as inhibitors of parasite growth with IC50 = 16, 12 y
The effect of different concentrations of the new pyridinylox-
yalkanols (8a–n) and their acylated derivatives (9a–p) on promas-
tigote form of Leishmania mexicana growth was studied during
10 days. Geneticin, a well-known antiparasitic agent, was used as
20 lg/mL. These products can be useful as potential drugs for che-
motherapy of leishmaniasis. In order to establish a more complete
structure-activity relationship, studies of DHFR inhibition are cur-
rently under progress.
a standard inhibitor at a concentration of 100 lg/mL. The evalua-
tion was carried out by reading parasites in a Neubauer chamber
every two day for ten days. A remarkable inhibitory effect on Leish-
mania mexicana growth was observed in the case of 8g, 9g and 9e.
The rest of the tested compounds were inactive even at a concen-
4. Experimental
4.1. General
tration of 50
bition was detected after 6 days of incubation of 8g and 9g. With
20 g/mL, 50% inhibition was observed after 1 day of exposure
lg/mL. For the lowest dosage of 10 lg/mL, 25% inhi-
Chemicals were commercially available and used without fur-
ther purification. Solvents were distilled before use. Dichlorometh-
ane was distilled from phosphorus pentoxide. Lipase from Candida
rugosa (CRL) (905 U/mg solid) was purchased from Sigma Chemical
Co.; Candida antarctica lipase B (CAL B): Novozym 435 (7400 PLU/g)
and Lipozyme RM 1 M (LIP) (7800 U/g) were generous gifts of
Novozymes Spain; Carica papaya lipase (CPL) is the remaining solid
fraction of papaya latex, after wash off of proteases using distilled
water. CPL is a naturally immobilized enzyme and was a generous
gift of Dr. Georgina Sandoval, CIATEJ, México. All enzymes were
used ‘straight from the bottle’. Enzyme/substrate ratio (E/S): en-
zyme amount in mg/substrate amount in mg. Enzymatic reactions
were carried out on Innova 4000 digital incubator shaker, New
Brunswick Scientific Co. at 33 and 55 °C and 200 rpm. To monitor
the reaction progress aliquots were withdrawn and analyzed by
l
and a maximum of 95% and 80%, respectively was reached after
10 days. Figures 4–6 show the growth curve of promastigote Leish-
mania mexicana in the presence of compounds 8g, 9g and 9e.
The three compounds showed to be the most potent against L.
mexicana with IC50 = 16, 12 and 20
the standard inhibitor geneticin (IC50 = 50
l
g/mL, respectively, better than
g/mL). Compound 9d
l
resulted to be moderately effective. The acetylated product 9g,
with 14 carbon atom in the side chain is the most potent, indicat-
ing that the presence of the acetyl group through the alcohol ester-
ification increased the inhibitory activity exhibited by 8g.
Compound 9g has twelve carbon atoms in the alcohol moiety
and two atoms in the acyl chain. With the aim to test the activity
of a compound with the same chain length but containing eleven
carbon atoms in the alcohol moiety and three carbon atoms in

G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
4619
Figure 4. Effect of 8g concentration on Leishmania mexicana growth rate.
Figure 5. Effect of 9g concentration on Leishmania mexicana growth rate.
Figure 6. Effect of 9e concentration on Leishmania mexicana growth rate.
TLC performed on commercial 0.2 mm aluminum-coated silica gel
plates (F254) and visualized by 254 nm UV or immersion in an
aqueous solution of (NH4)6Mo7O24ꢀ4H2O (0.04 M), Ce(SO4)2
(0.003 M) in concentrated H2SO4 (10%). Melting points were mea-
sured in a Fisher Johns apparatus and are uncorrected. 1H NMR and
13C NMR spectra were recorded in CDCl3 as solvent using a Bruker
AC-200 spectrometer operating at 200.13 and 50.32 MHz for 1H
and 13C, respectively, and a Bruker AM-500 NMR instrument oper-
ating at 500.14 and 125.76 MHz for 1H and 13C respectively. The 1H
NMR spectra are referenced with respect to the residual CHCl3 pro-
ton of the solvent CDCl3 at d = 7.26 ppm. Coupling constants are re-
ported in Hz. 13C NMR spectra were fully decoupled and are
referenced to the middle peak of the solvent CDCl3 at
d = 77.0 ppm. Splitting patterns are designated as: s, singlet; d,
doublet; t, triplet; q, quadruplet; qn, quintet; dd, double doublet,
etc. IR spectra were recorded with a Nicolet Magna 550 spectrom-
eter. High Resolution Mass Spectrometry was recorded with Ther-
mo Scientic EM/DSQ II—DIP. The results were within 0.02% of the
theoretical values.
4.2. Synthesis of pyridinyloxyalkanols. General procedure
To a solution of 2-hydroxy or 3-hydroxypyridine (200 mg,
2 mmol) in dimethyl sulfoxide (5.0 mL) was added potassium
hydroxide (450 mg, 8 mmol). The mixture was stirred at room
temperature for 30 min. Then, the corresponding alkylhalide
(2 mmol) was added and the reaction mixture was stirred at room
temperature overnight. The mixture was partitioned between sat-
urated solution ammonium chloride (50 mL) and methylene chlo-
ride (50 mL). The aqueous phase was extracted with methylene

4620
G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
chloride (2 ꢁ 50 mL). The combined organic layers were washed
with saturated solution of sodium chloride (5 ꢁ 50 mL), dried
(MgSO4), and the solvent was evaporated. The residue was purified
by column chromatography (silica gel) employing mixtures of hex-
ane/EtOAc as eluent (7:3–2:3).
[M+Na]+ Calcd C15H25NNaO2 274.1783. Found: C15H25NNaO2
274.1778.
4.2.6. 11-(Pyridin-3-yloxy)-undecan-1-ol (8f)
Yield 68% of white solid mp 49–50 °C. Rf 0.50 (EtOAc); IR (KBr,
cmꢂ1) 2922, 2849, 1638, 1459, 1309, 1215, 1040, 776, 664; 1H
NMR (CDCl3) d 1.29–1.33 (m, 12H, H-3, H-4, H-5, H-6, H-7, H-8),
1.46 (qn, J = 7.3 Hz, 2H, H-9), 1.56 (qn, J = 7.0 Hz, 2H, H-2), 1.79
(qn, J = 7.1 Hz, 2H, H-10), 3.63 (t, J = 6.6 Hz, 2H, H-1), 4.00 (t,
J = 6.5 Hz, 2H, H-11), 7.21 (m, 2 H, H-40, H-50); 8.19 (d, J = 2.9 Hz,
1 H, H-20); 8.30 (s, 1 H, H-60); 13C NMR (CDCl3) d 25.7 (C-3), 25.9
(C-10), 29.1–29.5 (C-4, C-5, C-6, C-7, C-8, C-9), 32.8 (C-2), 63.0
(C-1); 68.4 (C-11); 121.5 (C-40); 124.0 (C-50); 137.6 (C-20); 141.5
(C-60); 155.4 (C-30); HRMS: [M+Na]+ Calcd C16H27NNaO2
288.1939. Found: C16H27NNaO2 288.1934.
4.2.1. 6-(Pyridin-3-yloxy)-hexan-1-ol (8a)
Yield 66% of pure compound as a colorless oil. Rf 0.39 (EtOAc);
IR (KBr, cmꢂ1) 2927, 2852, 1635, 1457, 1312, 1220, 1044, 772,
669; 1H NMR (CDCl3) d 1.44–1.50 (m, 4H, H-3, H-4), 1.60 (qn,
J = 6.8 Hz, 2H, H-2), 1.80 (qn, J = 6.7 Hz, 2H, H-5), 3.65 (t,
J = 6.4 Hz, 2H, H-1), 3.99 (t, J = 6.4 Hz, 2H, H-6), 7.20 (m, 2 H, H-
40, H-50); 8.17 (dd, J1 = 2.2 Hz, J2 = 3.9 Hz, 1 H, H-20); 8.27 (dd,
J1 = 1.1 Hz, J2 = 2.3 Hz, 1 H, H-60); 13C NMR (CDCl3) d 25.5 (C-3),
25.7 (C-4), 29.0 (C-5), 32.6 (C-2), 62.5 (C-1); 68.1 (C-6); 121.1 (C-
40); 123.8 (C-50); 137.8 (C-20); 141.7 (C-60); 155.5 (C-30); HRMS:
[M+Na]+ Calcd C11H17NNaO2 218.1157. Found: C11H17NNaO2
218.1152.
4.2.7. 12-(Pyridin-3-yloxy)-dodecan-1-ol (8g)
Yield 72% of white solid mp 37–38 °C. Rf 0.52 (EtOAc); IR (KBr,
cmꢂ1) 2900, 2850, 1642, 1447, 1310, 1209, 1040, 770, 658; 1H
NMR (CDCl3) d 1.25–1.33 (m, 14H, H-3, H-4, H-5, H-6, H-7, H-8,
H-9), 1.45 (qn, J = 7.4 Hz, 2H, H-10), 1.56 (qn, J = 7.1 Hz, 2H, H-2),
1.80 (qn, J = 7.1 Hz, 2H, H-11), 3.64 (t, J = 6.6 Hz, 2H, H-1), 4.02 (t,
J = 6.5 Hz, 2H, H-12), 7.31 (s, 2 H, H-40, H-50); 8.22 (s, 1 H, H-20);
8.31 (s, 1 H, H-60); 13C NMR (CDCl3) d 25.7 (C-3), 25.8 (C-11),
29.0–29.5 (C-4, C-5, C-6, C-7, C-8, C-9, C-10), 32.8 (C-2), 63.0 (C-
1); 68.7 (C-12); 123.1 (C-40); 124.4 (C-50); 136.0 (C-20); 140.1 (C-
60); 155.8 (C-30); HRMS: [M+Na]+ Calcd C17H29NNaO2 302.2096.
Found: C17H29NNaO2 302.2091.
4.2.2. 7-(Pyridin-3-yloxy)-heptan-1-ol (8b)
Yield 70% of pure compound as a colorless oil. Rf 0.41 (EtOAc);
IR (KBr, cmꢂ1) 2920, 2852, 1629, 1460, 1312, 1225, 1050, 770,
665; 1H NMR (CDCl3) d 1.36–1.40 (m, 6H, H-3, H-4, H-5), 1.57
(m, 2H, H-2), 1.80 (qn, J = 6.7 Hz, 2H, H-6), 3.64 (t, J = 6.4 Hz, 2H,
H-1), 4.00 (t, J = 6.4 Hz, 2H, H-7), 7.23 (s, 2 H, H-40, H-50); 8.20 (s,
1 H, H-20); 8.30 (s, 1 H, H-60); 13C NMR (CDCl3) d 25.5 (C-3), 25.8
(C-4, C-6), 29.1 (C-5), 32.1 (C-2), 62.6 (C-1); 68.2 (C-7); 121.3 (C-
40); 124.0 (C-50); 137.6 (C-20); 141.5 (C-60); 155.3 (C-30); HRMS:
[M+Na]+ Calcd C12H19NNaO2 232.1313. Found: C12H19NNaO2
232.1308.
4.2.8. 6-(Pyridin-2-yloxy)-hexan-1-ol (8h)
Yield 52% of pure compound as a colorless oil. Rf 0.12 (EtOAc);
IR (KBr, cmꢂ1) 3022, 2922, 1615, 1555, 1444, 1282, 1190, 1024,
758, 649; 1H NMR (CDCl3) d 1.31–1.37 (m, 4H, H-3, H-4), 1.51
(qn, J = 6.4 Hz, 2H, H-2), 1.70 (qn, J = 6.8 Hz, 2H, H-5), 3.56 (t,
J = 6.2 Hz, 2H, H-1), 3.87 (t, J = 7.2 Hz, 2H, H-6), 6.13 (dt,
J1 = 1.3 Hz, J2 = 6.7 Hz, 1 H, H-30); 6.50 (d, J = 9.0 Hz, 1 H, H-50);
7.23 (dd, J1 = 1.4 Hz, J2 = 7.0 Hz, 1 H, H-20); 7.28 (m, 1 H, H-60);
13C NMR (CDCl3) d 25.1 (C-3), 26.0 (C-4), 29.1, (C-5), 32.3 (C-2),
49.5 (C-1); 62.2 (C-6); 106.1 (C-30); 120.8 (C-50); 137.4 (C-40);
139.3 (C-60); 162.6 (C-20); HRMS: [M+H]+ Calcd C11H18NO2
196.1338. Found: C11H18NO2 196.1333.
4.2.3. 8-(Pyridin-3-yloxy)-octan-1-ol (8c)
Yield 62% of pure compound as a colorless oil. Rf 0.45 (EtOAc);
IR (KBr, cmꢂ1) 2930, 2850, 1640, 1450, 1309, 1220, 1045, 776,
670; 1H NMR (CDCl3) d 1.36–1.38 (m, 6H, H-3, H-4, H-5), 1.46
(m, 2H, H-6), 1.57 (m, 2H, H-2), 1.80 (m, 2H, H-7), 3.64 (t,
J = 6.6 Hz, 2H, H-1), 4.01 (t, J = 6.5 Hz, 2H, H-8), 7.26 (s, 2 H, H-40,
H-50); 8.21 (s, 1 H, H-20); 8.31 (s, 1 H, H-60); 13C NMR (CDCl3) d
25.6 (C-3), 25.8 (C-7), 29.0 (C-4), 29.2 (C-5), 29.3 (C-6), 32.7 (C-
2), 62.9 (C-1); 68.5 (C-8); 122.3 (C-40); 124.2 (C-50); 136.8 (C-20);
140.8 (C-60); 155.5 (C-30); HRMS: [M+Na]+ Calcd C13H21NNaO2
246.1470. Found: C13H21NNaO2 246.1466
4.2.9. 7-(Pyridin-2-yloxy)-heptan-1-ol (8i)
Yield 55% of pure compound as a colorless oil. Rf 0.15 (EtOAc);
IR (KBr, cmꢂ1) 3025, 2952, 1625, 1550, 1450, 1302, 1220, 1040,
770, 660; 1H NMR (CDCl3) d 1.33 (m, 6H, H-3, H-4, H-5), 1.53 (m,
2H, H-2), 1.74 (m, 2H, H-6), 3.62 (t, J = 6.7 Hz, 2H, H-1), 3.93 (t,
4.2.4. 9-(Pyridin-3-yloxy)-nonan-1-ol (8d)
Yield 65% of pure compound as a colorless oil. Rf 0.46 (EtOAc);
IR (KBr, cmꢂ1) 2900, 2840, 1620, 1465, 1300, 1210, 1040, 765,
659; 1H NMR (CDCl3) d 1.30–1.34 (m, 8H, H-3, H-4, H-5, H-6),
1.46 (m, 2H, H-7), 1.56 (m, 2H, H-2), 1.81 (m, 2H, H-8), 3.64 (t,
J = 6.6 Hz, 2H, H-1), 4.04 (t, J = 6.5 Hz, 2H, H-9), 7. 40 (s, 2 H, H-40,
H-50); 8.25 (s, 1 H, H-20); 8.33 (s, 1 H, H-60); 13C NMR (CDCl3) d
25.7 (C-3), 25.8 (C-8), 28.9 (C-4), 29.1 (C-5), 29.2 (C-6), 29.4 (C-
7), 32.7 (C-2), 63.0 (C-1); 69.0 (C-9); 124.4 (C-40); 124.9 (C-50);
134.7 (C-20); 140.0 (C-60); 156.1 (C-30); HRMS: [M+Na]+ Calcd
J = 7.0 Hz, 2H, H-7), 6.21 (t, J = 6.1 Hz,
1
H, H-30); 6.65 (d,
J = 9.0 Hz, 1 H, H-50); 7.30 (m, 1 H, H-40); 7.37 (m, 1 H, H-60); 13C
NMR (CDCl3) d 25.6 (C-3), 26.5 (C-6), 29.1 (C-4, C-5), 32.6 (C-2),
50.1 (C-1); 62.9 (C-7); 106.5 (C-30); 120.9 (C-50); 137.5 (C-40);
139.6 (C-60); 162.3 (C-20); HRMS: [M+Na]+ Calcd C12H19NNaO2
232.1313. Found: C12H19NNaO2 232.1310.
C14H23NNaO2 260.1626. Found: C14H23NNaO2 260.1621.
4.2.10. 8-(Pyridin-2-yloxy)-octan-1-ol (8j)
Yield 55% of pure compound as a colorless oil. Rf 0.17 (EtOAc);
IR (KBr, cmꢂ1) 3022, 2932, 1615, 1545, 1420, 1300, 1210, 1030,
770, 654; 1H NMR (CDCl3) d 1.28–1.31 (m, 8H, H-3, H-4, H-5, H-
6), 1.53 (m, 2H, H-2), 1.74 (m, 2H, H-7), 3.61 (t, J = 6.6 Hz, 2H, H-
1), 3.92 (t, J = 7.3 Hz, 2H, H-8), 6.21 (t, J = 6.2 Hz, 1 H, H-30); 6.68
(d, J = 8.9 Hz, 1 H, H-50); 7.27 (d, J = 5.7 Hz, 1 H, H-40); 7.35 (t,
J = 7.5 Hz, 1 H, H-60); 13C NMR (CDCl3) d 25.6 (C-3), 26.5 (C-7),
29.0 (C-4), 29.1 (C-5), 29.2 (C-6), 32.6 (C-2), 50.4 (C-1); 62.9 (C-
8); 106.8 (C-30); 120.9 (C-50); 137.6 (C-40); 139.9 (C-60); 162.7 (C-
20); HRMS: [M+Na]+ Calcd C13H21NNaO2 246.1470. Found:
4.2.5. 10-(pyridin-3-yloxy)-decan-1-ol (8e)
Yield 65% of pure compound as a colorless oil. Rf 0.47 (EtOAc);
IR (KBr, cmꢂ1) 2895, 2850, 1625, 1450, 1310, 1230, 10504, 762,
660; 1H NMR (CDCl3) d 1.30–1.33 (m, 10H, H-3, H-4, H-5, H-6, H-
7), 1.44 (m, 2H, H-8), 1.55 (m, 2H, H-2), 1.79 (m, 2H, H-9), 3.63
(t, J = 6.6 Hz, 2H, H-1), 4.00 (t, J = 6.5 Hz, 2H, H-10), 7.27 (t,
J = 2.7 Hz, 2 H, H-40, H-50); 8.21 (t, J = 2.8 Hz, 1 H, H-20); 8.30 (s, 1
H, H-60); 13C NMR (CDCl3) d 25.7 (C-3), 25.9 (C-9), 29.0–29.3 (C-
4, C-5, C-6, C-7, C-8), 32.8 (C-2), 62.9 (C-1); 68.6 (C-10); 122.4
(C-40); 124.2 (C-50); 136.6 (C-20); 140.6 (C-60); 155.6 (C-30); HRMS:
C13H21NNaO2 246.1465.

G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
4621
4.2.11. 9-(Pyridin-2-yloxy)-nonan-1-ol (8k)
1240, 1045, 771; 1H NMR (CDCl3) d 1.44 (m, 2H, H-3), 1.51 (m,
2H, H-4), 1.67 (qn, J = 7.0 Hz, 2H, H-2), 1.82 (qn, J = 6.9 Hz, 2H, H-
5), 2.06 (s, 1H, CH3COO–), 4.02 (t, J = 6.4 Hz, 2H, H-1), 4.08 (t,
J = 6.7 Hz, 2H, H-6), 7.27 (m, 2 H, H-40, H-50); 8.21 (m, 1 H, H-20);
8.32 (m, 1 H, H-60); 13C NMR (CDCl3) d 21.0 (CH3COO–), 25.6 (C-
3, C-4), 28.5 (C-5), 29.0 (C-2), 64.4 (C-1); 68.2 (C-6); 122.4 (C-40);
124.3 (C-50); 136.5 (C-20); 140.7 (C-60); 155.5 (C-30), 171.3
(CH3COO–); HRMS: [M+H]+ Calcd C13H20NO3 238.1443. Found:
Yield 60% of pure compound as a colorless oil. Rf 0.20 (EtOAc);
IR (KBr, cmꢂ1) 3020, 2930, 1620, 1540, 1425, 1310, 1200, 1042,
777, 648; 1H NMR (CDCl3) d 1.28 (m, 10H, H-3, H-4, H-5, H-6, H-
7), 1.50 (m, 2H, H-2), 1.71 (m, 2H, H-8), 3.59 (t, J = 6.5 Hz, 2H, H-
1), 3.89 (t, J = 6.6 Hz, 2H, H-9), 6.15 (t, J = 6.6 Hz, 1 H, H-30); 6.56
(d, J = 9.0 Hz, 1 H, H-50); 7.29 (m, 2 H, H-40, H-60); 13C NMR (CDCl3)
d 25.6 (C-3), 26.5 (C-8), 29.0 (C-4), 29.2 (C-5, C-6), 29.3 (C-7), 32.6
(C-2), 49.9 (C-1); 62.8 (C-9); 106.1 (C-30); 120.9 (C-50); 137.5 (C-40);
139.3 (C-60); 162.6 (C-20); HRMS: [M+H]+ Calcd C14H24NO2
238.1807. Found: C14H24NO2 238.1802.
C13H20NO3 238.1438.
4.3.2. 7-(Pyridin-3-yloxy)-heptyl acetate (9b)
Yield 92% of pure compound as a colorless oil. Rf 0.60 (EtOAc);
IR (KBr, cmꢂ1) 3442, 2930, 2858, 1736, 1652, 1570, 1475, 1360,
1239, 1040, 770; 1H NMR (CDCl3) d 1.36 (m, 6H, H-3, H-4, H-5),
1.62 (m, 2H, H-2), 1.85 (qn, J = 6.7 Hz, 2H, H-6), 2.04 (s, 1H,
CH3COO–), 4.04 (q, J = 6.7 Hz, 4H, H-1, H-7), 7.30 (s, 2 H, H-40, H-
50); 8.23 (s, 1 H, H-20); 8.33 (s, 1 H, H-60); 13C NMR (CDCl3) d 21.0
(CH3COO–), 25.9 (C-3), 28.6 (C-6), 29.1 (C-4, C-5), 29.4 (C-2), 64.6
(C-1); 68.4 (C-7); 121.4 (C-40, C-50); 137.6 (C-20); 141.5 (C-60);
155.6 (C-30), 171.2 (CH3COO–); HRMS: [M+H]+ Calcd C14H22NO3
252.1600. Found: C14H22NO3 252.1596.
4.2.12. 10-(Pyridin-2-yloxy)-decan-1-ol (8l)
Yield 58% of pure compound as a colorless oil. Rf 0.26 (EtOAc);
IR (KBr, cmꢂ1) 3002, 2912, 1635, 1515, 1400, 1320, 1215, 1035,
780, 664; 1H NMR (CDCl3) d 1.27–1.32 (m, 12H, H-3, H-4, H-5, H-
6, H-7, H-8), 1.55 (m, 2H, H-2), 1.74 (m, 2H, H-9), 3.63 (t,
J = 6.9 Hz, 2H, H-1), 3.92 (t, J = 7.0 Hz, 2H, H-10), 6.18 (t,
J = 6.5 Hz, 1 H, H-30); 6.62 (d, J1 = 5.8 Hz,1 H, H-50); 7.27 (d,
J1 = 5.6 Hz, 1 H, H-40); 7.33 (t, J = 6.4 Hz, 1 H, H-60); 13C NMR (CDCl3)
d 25.6 (C-3), 26.5 (C-9), 29.1–29–3 (C-4, C-5, C-6, C-7, C-8), 32.7 (C-
2), 50.1 (C-1); 63.0 (C-10); 106.3 (C-30); 121.0 (C-50); 137.6 (C-40);
139.5 (C-60); 162.7 (C-20); HRMS: [M+H]+ Calcd C15H26NO2
252.1964. Found: C15H26NO2 252.1959.
4.3.3. 8-(Pyridin-3-yloxy)-octyl acetate (9c)
Yield 90% of pure compound as a colorless oil. Rf 0.65 (EtOAc);
IR (KBr, cmꢂ1) 3441, 2932, 2856, 1732, 1650, 1572, 1471, 1352,
1230, 1039, 770; 1H NMR (CDCl3) d 1.38 (m, 6H, H-3, H-4, H-5),
1.49 (m, 2H, H-6), 1.65 (qn, J = 7.6 Hz, 2H, H-2), 1.85 (qn,
J = 7.7 Hz, 2H, H-7), 2.07 (s, 1H, CH3COO–), 4.08 (t, J = 6.7 Hz, 4H,
H-1, H-8), 7.48 (t, J = 7.7 Hz, 2 H, H-40, H-50); 8.30 (s, 1 H, H-20);
8.37 (s, 1 H, H-60); 13C NMR (CDCl3) d 21.0 (CH3COO–), 25.8 (C-3,
C-7), 28.5 (C-4), 28.9 (C-5), 29.0 (C-6), 29.1 (C-2), 64.5 (C-1); 68.7
(C-8); 123.4 (C-40); 124.5 (C-50); 135.7 (C-20); 139.9 (C-60); 155.8
(C-30), 171.2 (CH3COO–); HRMS: [M+H]+ Calcd C15H23NNaO3
288.1576. Found: C15H23NNaO3 288.1571.
4.2.13. 11-(Pyridin-2-yloxy)-undecan-1-ol (8m)
Yield 50% of white solid mp 35–36 °C. Rf 0.39 (EtOAc); IR (KBr,
cmꢂ1) 3012, 2912, 1615, 1540, 1412, 1320, 1205, 1040, 770, 664;
1H NMR (CDCl3) d 1.26–1.31 (m, 14H, H-3, H-4, H-5, H-6, H-7, H-
8, H-9), 1.55 (qn, J = 7.0 Hz, 2H, H-2), 1.73 (qn, J = 6.8 Hz, 2H, H-
10), 3.62 (t, J = 6.6 Hz, 2H, H-1), 3.90 (t, J = 7.5 Hz, 2H, H-11), 6.14
(dt, J1 = 1.3 Hz, J2 = 6.6 Hz, 1 H, H-30); 6.56 (d, J = 9.1 Hz,1 H, H-
50); 7.24 (dd, J1 = 2.0 Hz, J2 = 6.7 Hz,
1
H, H-40); 7.30 (ddd,
J1 = 2.2 Hz, J2 = 6.7 Hz, J3 = 9.0 Hz, 1 H, H-60); 13C NMR (CDCl3) d
25.7 (C-3), 26.6 (C-10), 29.1–29–4 (C-4, C-5, C-6, C-7, C-8, C-9),
32.7 (C-2), 49.9 (C-1); 63.0 (C-11); 105.9 (C-30); 121.1 (C-50);
137.5 (C-40); 139.2 (C-60); 162.6 (C-20); HRMS: [M+Na]+ Calcd
4.3.4. 9-(Pyridin-3-yloxy)-nonyl acetate (9d)
Yield 95% of pure compound as a colorless oil. Rf 0.66 (EtOAc);
IR (KBr, cmꢂ1) 3439, 2933, 2845, 1722, 1650, 1560, 1449, 1342,
1229, 1010, 769; 1H NMR (CDCl3) d 1.32 (m, 8H, H-3, H-4, H-5,
H-6), 1.46 (m, 2H, H-7), 1.61 (m, 2H, H-2), 1.79 (m, 2H, H-8),
2.04 (s, 1H, CH3COO–), 4.00 (t, J = 6.6 Hz, 2H, H-1), 4.04 (t,
J = 6.6 Hz, 2H, H-9), 7. 22 (s, 2 H, H-40, H-50); 8.21 (s, 1 H, H-20);
8.31 (s, 1 H, H-60); 13C NMR (CDCl3) d 21.0 (CH3COO–), 25.8 (C-3,
C-8), 28.5 (C-4), 29.1 (C-5, C-6), 29.4 (C-7), 29.7 (C-2), 64.6 (C-1);
68.4 (C-9); 121.6 (C-40); 124.0 (C-50); 137.4 (C-20); 141.3 (C-60);
155.4 (C-30), 171.2 (CH3COO–); HRMS: [M+H]+ Calcd C16H26NO3
280.1913. Found: C16H26NO3 280.1908.
C16H27NO2 288.1939. Found: C16H27NO2 288.1934.
4.2.14. 12-(Pyridin-2-yloxy)-dodecan-1-ol (8n)
Yield 51% of white solid mp 58–59 °C. Rf 0.56 (EtOAc); IR (KBr,
cmꢂ1) 2992, 2889, 1608, 1515, 1395, 1294, 1200, 1030, 769, 644;
1H NMR (CDCl3) d 1.26–1.33 (m, 16H, H-3, H-4, H-5, H-6, H-7, H-
8, H-9, H-10), 1.57 (m, 2H, H-2), 1.74 (m, 2H, H-11), 3.64 (t,
J = 6.7 Hz, 2H, H-1), 3.92 (t, J = 6.5 Hz, 2H, H-12), 6.16 (dt,
J1 = 1.4 Hz, J2 = 6.7 Hz, 1 H, H-30); 6.57 (dd, J1 = 0.6 Hz, J2 = 9.2 Hz,
1 H, H-50); 7.25 (dd, J1 = 1.9 Hz, J2 = 6.6 Hz, 1 H, H-40); 7.31 (ddd,
J1 = 2.2 Hz, J2 = 6.7 Hz, J2 = 9.0 Hz, 1 H, H-60); 13C NMR (CDCl3) d
25.7 (C-3), 26.6 (C-11), 29.2–29–5 (C-4, C-5, C-6, C-7, C-8, C-9, C-
10), 32.8 (C-2), 49.9 (C-1); 63.0 (C-12); 105.9 (C-30); 121.1 (C-50);
137.5 (C-40); 139.2 (C-60); 162.6 (C-20); HRMS: [M+Na]+ Calcd
4.3.5. 10-(Pyridin-3-yloxy)-decyl acetate (9e)
Yield 95% of pure compound as a colorless oil. Rf 0.70 (EtOAc);
IR (KBr, cmꢂ1) 3445, 2927, 2850, 1732, 1635, 1550, 1479, 1379,
1202, 1012, 768; 1H NMR (CDCl3) d 1.30–1.33 (m, 10H, H-3, H-4,
H-5, H-6, H-7), 1.45 (qn, J = 7.3 Hz, 2H, H-8), 1.61 (qn, J = 7.2 Hz,
2H, H-2), 1.79 (qn, J = 7.1 Hz, 2H, H-9), 2.04 (s, 1H, CH3COO–),
4.00 (t, J = 6.7 Hz, 2H, H-1), 4.04 (t, J = 6.8 Hz, 2H, H-10), 7.24 (s,
2 H, H-40, H-50); 8.21 (s, 1 H, H-20); 8.31 (s, 1 H, H-60); 13C NMR
(CDCl3) d 21.0 (CH3COO–), 25.8 (C-3), 25.9 (C-9), 28.6 (C-4), 29.0
(C-5), 29.1 (C-6), 29.2 (C-7), 29.4 (C-8, C-2), 64.6 (C-1); 68.5 (C-
10); 122.0 (C-40); 124.1 (C-50); 137.0 (C-20); 141.0 (C-60); 155.5
(C-30); 171.2 (CH3COO–); HRMS: [M+Na]+ Calcd C17H27NNaO3
316.1889. Found: C17H27NNaO3 316.1883.
C17H29NNaO2 302.2096. Found: C17H29NNaO2 302.2091.
4.3. Synthesis of pyridinyloxyalkyl esters. General procedure
To a solution of the corresponding pyridinyloxyalkanol (50 mg)
in ethyl acetate or ethyl propionate (2.5 mL) was added CAL-B li-
pase (5 mg). The mixture was shaken at 200 rpm and 30 °C. Once
the reaction was finished, the enzyme was filtered off and the sol-
vent evaporated under reduced pressure. The residue was purified
by column chromatography (silica gel) employing mixtures of hex-
ane/EtOAc as eluent (9:1–3:2).
4.3.6. 11-(Pyridin-3-yloxy)-undecyl acetate (9f)
4.3.1. 6-(Pyridin-3-yloxy)-hexyl acetate (9a)
Yield 97% of pure compound as a colorless oil. Rf 0.75 (EtOAc);
IR (KBr, cmꢂ1) 3430, 2915, 2839, 1726, 1655, 1560, 1456, 1359,
1239, 1040, 772; 1H NMR (CDCl3) d 1.29 (m, 14H, H-3, H-4, H-5,
Yield 90% of pure compound as a colorless oil. Rf 0.59 (EtOAc);
IR (KBr, cmꢂ1) 3440, 2930, 2860, 1732, 1655, 1580, 1465, 1370,

4622
G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
H-6, H-7, H-8, H-9), 1.61 (qn, J = 6.9 Hz, 2H, H-2), 1.79 (qn,
J = 6.9 Hz, 2H, H-10), 2.04 (s, 1H, CH3COO–), 3.99 (t, J = 6.5 Hz,
2H, H-1), 4.04 (t, J = 6.7 Hz, 2H, H-11), 7.19 (m, 2 H, H-40, H-50);
8.19 (dd, J1 = 2.0 Hz, J2 = 6.6 Hz, 1 H, H-20); 8.30 (s, 1 H, H-60); 13C
NMR (CDCl3) d 21.0 (CH3COO–), 25.9 (C-3, C-10), 28.6 (C-4),
29.1–29.5 (C-5, C-6, C-7, C-8, C-9, C-2), 64.6 (C-1); 68.3 (C-11);
121.1 (C-40); 123.7 (C-50); 137.8 (C-20); 141.7 (C-60); 155.5 (C-30),
171.2 (CH3COO–); HRMS: [M+Na]+ Calcd C18H29NNaO3 330.2045.
Found: C18H29NNaO3 330.2040.
4.3.11. 9-(Pyridin-2-yloxy)-nonyl acetate (9k)
Yield 94% of pure compound as a colorless oil. Rf 0.65 (EtOAc);
IR (KBr, cmꢂ1) 3435, 2928, 2852, 1728, 1640, 1560, 1515, 1469,
1389, 1212, 1032, 770; 1H NMR (CDCl3) d 1.30–1.32 (m, 10H, H-
3, H-4, H-5, H-6, H-7), 1.61 (qn, J = 6.9 Hz, 2H, H-2), 1.75 (qn,
J = 6.8 Hz, 2H, H-8), 2.05 (s, 1H, CH3COO–), 3.94 (t, J = 7.0 Hz, 2H,
H-1), 4.05 (t, J = 6.8 Hz, 2H, H-9), 6.21 (t, J = 6.5 Hz, 1 H, H-30);
6.68 (d, J = 9.0 Hz,1 H, H-50); 7.28 (dd, J1 = 0.9 Hz, J2 = 6.7 Hz, 1 H,
H-40); 7.35 (ddd, J1 = 1.0 Hz, J2 = 6.7 Hz, J3 = 8.9 Hz, 1 H, H-60); 13C
NMR (CDCl3) d 21.0 (CH3COO), 25.8 (C-3), 26.6 (C-8), 28.5 (C-4),
29.1 (C-5, C-6), 29.2 (C-7), 29.3 (C-2), 50.2 (C-1); 64.6 (C-9);
106.5 (C-30); 120.9 (C-50); 137.5 (C-40); 139.6 (C-60); 162.6 (C-20);
171.2 (CH3COO); HRMS: [M+Na]+ Calcd C16H25NNaO3 302.1732.
Found: C16H25NNaO3 302.1727.
4.3.7. 12-(Pyridin-3-yloxy)-dodecyl acetate (9g)
Yield 99% of pure compound as a colorless oil. Rf 0.80 (EtOAc);
IR (KBr, cmꢂ1) 3432, 2932, 2860, 1736, 1655, 1571, 1462, 1365,
1240, 1036, 760; 1H NMR (CDCl3) d 1.24–1.32 (m, 14H, H-3, H-4,
H-5, H-6, H-7, H-8, H-9), 1.45 (qn, J = 7.2 Hz, 2H, H-10), 1.61 (qn,
J = 7.0 Hz, 2H, H-2), 1.79 (qn, J = 7.0 Hz, 2H, H-11), 2.03 (s, 1H,
CH3COO–), 4.01 (t, J = 6.6 Hz, 2H, H-1), 4.04 (t, J = 6.7 Hz, 2H, H-
12), 7.26 (s, 2 H, H-40, H-50); 8.21 (s, 1 H, H-20); 8.31 (s, 1 H, H-
60); 13C NMR (CDCl3) d 21.0 (CH3COO–), 25.9 (C-3, C-11), 28.6–
29–5 (C-2, C-4, C-5, C-6, C-7, C-8, C-9, C-10), 64.6 (C-1); 68.6 (C-
12); 122.4 (C-40); 124.2 (C-50); 136.7 (C-20); 140.7 (C-60); 155.6
(C-30), 171.2 (CH3COO–); HRMS: [M+Na]+ Calcd C19H31NNaO3
344.2202. Found: C19H31NNaO3 344.2197.
4.3.12. 10-(Pyridin-2-yloxy)-decyl acetate (9l)
Yield 95% of pure compound as a colorless oil. Rf 0.70 (EtOAc);
IR (KBr, cmꢂ1); 1H NMR (CDCl3) d 1.26–1.31 (m, 12H, H-3, H-4,
H-5, H-6, H-7, H-8), 1.59 (qn, J = 7.1 Hz, 2H, H-2), 1.73 (qn,
J = 7.4 Hz, 2H, H-9), 3.90 (t, J = 7.0 Hz, 2H, H-1), 2.03 (s, 1H,
CH3COO–), 3.90 (t, J = 7.0 Hz, 2H, H-1), 4.03 (t, J = 6.9 Hz, 2H, H-
10), 6.14 (dt, J1 = 1.4, J2 = 6.7 Hz, 1 H, H-30); 6.56 (d, J = 9.1 Hz,1 H,
H-50); 7.24 (dd, J1 = 1.6 Hz, J2 = 6.7 Hz, 1 H, H-40); 7.28 (ddd,
J1 = 2.1 Hz, J2 = 6.7 Hz, J2 = 8.9 Hz, 1 H, H-60); 13C NMR (CDCl3) d
21.0 (CH3COO), 25.8 (C-3), 26.6 (C-9), 28.5 (C-4), 29.1 (C-5, C-6),
29.2 (C-7), 29.3 (C-8, C-2), 49.9 (C-1); 64.6 (C-10); 105.9 (C-30);
121.0 (C-50); 137.5 (C-40); 139.2 (C-60); 162.6 (C-20), 171.2
(CH3COO); HRMS: [M+H]+ Calcd C17H28NO3 294.2069. Found:
4.3.8. 6-(Pyridin-2-yloxy)-hexyl acetate (9h)
Yield 90% of pure compound as a colorless oil. Rf 0.50 (EtOAc);
IR (KBr, cmꢂ1) 3440, 2932, 2858, 1735, 1655, 1580, 1540, 1465,
1367, 1240, 1045, 771; 1H NMR (CDCl3) d 1.37 (m, 4H, H-3, H-4),
1.61 (qn, J = 6.2 Hz, 2H, H-2), 1.75 (qn, J = 6.7 Hz, 2H, H-5), 2.03
(s, 1H, CH3COO–), 3.93 (t, J = 7.4 Hz, 2H, H-1), 4.03 (t, J = 6.6 Hz,
2H, H-6), 6.21 (t, J = 6.5 Hz, 1 H, H-30); 6.64 (d, J = 9.1 Hz, 1 H, H-
50); 7.26 (dd, J1 = 2.0, Hz, J2 = 6.7 Hz, 1 H, H-40); 7.34 (ddd,
J1 = 2.0 Hz, J2 = 6.7 Hz, J3 = 9.0 Hz, 1 H, H-60); 13C NMR (CDCl3) d
20.8 (CH3COO), 25.5 (C-3), 26.2 (C-4), 28.4 (C-5), 29.1 (C-2), 50.0
(C-1); 64.3 (C-6); 106.7 (C-30); 120.9 (C-50); 137.4 (C-40); 139.7
(C-60); 162.9 (C-20); 171.3 (CH3COO); HRMS: [M+H]+ Calcd
C17H28NO3 294.2064.
4.3.13. 11-(Pyridin-2-yloxy)-undecyl acetate (9m)
Yield 95% of pure compound as a colorless oil. Rf 0.71 (EtOAc);
IR (KBr, cmꢂ1) 3440, 2925, 2850, 1736, 1655, 1580, 1530, 1466,
1366, 1241, 1045, 772; 1H NMR (CDCl3) d 1.29–1.31 (m, 14H, H-
3, H-4, H-5, H-6, H-7, H-8, H-9), 1.60 (qn, J = 7.1 Hz, 2H, H-2),
1.73 (qn, J = 7.3 Hz, 2H, H-10), 2.04 (s, 1 H, CH3COO), 3.91 (t,
J = 7.5 Hz, 2H, H-1), 4.04 (t, J = 6.8 Hz, 2H, H-11), 6.15 (dt,
J1 = 1.2 Hz, J2 = 6.7 Hz, 1 H, H-30); 6.56 (d, J = 9.0 Hz, 1 H, H-50);
7.24 (dd, J1 = 2.0 Hz, J2 = 6.7 Hz, 1 H, H-40); 7.30 (ddd, J1 = 2.0 Hz,
C13H20NO3 238.1443. Found: C13H20NO3 238.1440.
J2 = 6.7 Hz, J3 = 9.0 Hz,
1 d 21.0
H, H-60);13C NMR (CDCl3)
4.3.9. 7-(Pyridin-2-yloxy)-heptyl acetate (9i)
Yield 90% of pure compound as a colorless oil. Rf 0.58 (EtOAc);
IR (KBr, cmꢂ1) 3445, 2930, 2860, 1738, 1650, 1570, 1520, 1475,
1369, 1242, 1040, 772; 1H NMR (CDCl3) d 1.29 (s, 6H, H-3, H-4,
H-5), 1.58 (m, 2H, H-2), 1.73 (m, 2H, H-6), 2.04 (s, 1H, CH3COO–),
3.91 (t, J = 7.4 Hz, 2H, H-1), 4.03 (t, J = 6.7 Hz, 2H, H-7), 6.15 (dt,
J1 = 1.0 Hz, J2 = 4.6 Hz, 1 H, H-30); 6.57 (d, J = 9.1 Hz, 1 H, H-50);
7.23 (m, 1 H, H-40); 7.32 (m, 1 H, H-60); 13C NMR (CDCl3) d 21.0
(CH3COO), 25.8 (C-3), 26.5 (C-6), 28.5 (C-4), 29.1 (C-5), 29.2 (C-
2), 50.1 (C-1); 64.6 (C-7); 106.4 (C-30); 120.9 (C-50); 137.5 (C-40);
139.5 (C-60); 162.8 (C-20); 171.2 (CH3COO); HRMS: [M+H]+ Calcd
(CH3COO), 25.9 (C-3), 26.6 (C-10), 28.6–29.2 (C-4, C-5, C-6, C-7,
C-8, C-9), 29.4 (C-2), 49.9 (C-1); 64.6 (C-11); 105.9 (C-30); 121.1
(C-50); 137.5 (C-40); 139.2 (C-60); 162.6 (C-20); 171.3 (CH3COO);
HRMS: [M+Na]+ Calcd C18H29NNaO3 330.2045. Found:
C18H29NNaO3 332.2040.
4.3.14. 12-(Pyridin-2-yloxy)-dodecyl acetate (9n)
Yield 95% of pure compound as a colorless oil: Rf 0.75 (EtOAc);
IR (KBr, cmꢂ1) 3437, 2942, 2858, 1736, 1651, 1570, 1520, 1465,
1369, 1242, 1040, 770; 1H NMR (CDCl3) d 1.24–1.31 (m, 16H, H-
3, H-4, H-5, H-6, H-7, H-8, H-9, H-10), 1.60 (qn, J = 7.0 Hz, 2H, H-
2), 1.73 (qn, J = 7.1 Hz, 2H, H-11), 2.03 (s, 1 H, CH3COO), 3.92 (t,
J = 7.0 Hz, 2H, H-1), 4.03 (t, J = 6.8 Hz, 2H, H-12), 6.18 (t,
J = 6.3 Hz, 1 H, H-30); 6.63 (d, J = 8.9 Hz, 1 H, H-50); 7.31 (dd,
C14H22NO3 252.1600. Found: C14H22NO3 252.1597.
4.3.10. 8-(Pyridin-2-yloxy)-octyl acetate (9j)
Yield 90% of pure compound as a colorless oil. Rf 0.62 (EtOAc);
IR (KBr, cmꢂ1) 3442, 2928, 2862, 1740, 1652, 1572, 1538, 1470,
1360, 1240, 1042, 770; 1H NMR (CDCl3) d 1.32 (s, 8H, H-3, H-4,
H-5, H-6), 1.58 (m, 2H, H-2), 1.74 (m, 2H, H-7), 2.04 (s, 1H,
CH3COO–), 3.92 (t, J = 7.4 Hz, 2H, H-1), 4.03 (t, J = 6.7 Hz, 2H,
H-8), 6.18 (t, J = 6.7 Hz, 1 H, H-30); 6.61 (d, J = 8.9 Hz, 1 H, H-50);
7.25 (m, 1 H, H-40); 7.34 (m, 1 H, H-60); 13C NMR (CDCl3) d 21.0
(CH3COO), 25.8 (C-3), 26.5 (C-7), 28.5 (C-4), 29.0 (C-5,C-6), 29.2
(C-2), 50.1 (C-1); 64.5 (C-8); 106.3 (C-30); 121.0 (C-50); 137.5
(C-40); 139.5 (C-60); 164.4 (C-20); 172.2 (CH3COO); HRMS:
[M+H]+ Calcd C15H23NNaO3 288.1576. Found: C15H23NNaO3
288.1572.
J1 = 1.5 Hz, J2 = 7.3 Hz,
J2 = 6.8 Hz, J3 = 8.8 Hz,
1
1
H, H-40); 7.34 (ddd, J1 = 1.8 Hz,
H, H-60);13C NMR (CDCl3)
d
21.0
(CH3COO), 25.9 (C-3), 26.6 (C-11), 28.6–29.2 (C-4, C-5, C-6, C-7,
C-8, C-9), 29.4 (C-2,, C-11), 50.1 (C-1); 64.6 (C-12); 106.3 (C-30);
120.9 (C-50); 137.6 (C-40); 139.5 (C-60); 162.6 (C-20); 171.2
(CH3COO); HRMS: [M+H]+ Calcd C19H32NO3 322.2382. Found:
C19H32NO3 322.2377.
4.3.15. 11-(Pyridin-3-yloxy)-undecyl propionate (9o)
Yield 60% of pure compound as a colorless oil. Rf 0.74 (EtOAc);
IR (KBr, cmꢂ1) 3429, 2932, 2857, 1735, 1656, 1580, 1462, 1369,

G. García Liñares et al. / Bioorg. Med. Chem. 20 (2012) 4614–4624
4623
1240, 1040, 770; 1H NMR (CDCl3)
d
1.13 (t, J = 7.6 Hz, 3H,
References and notes
CH3CH2COO), 1.29 (m, 14H, H-3, H-4, H-5, H-6, H-7, H-8, H-9),
1.61 (qn, J = 6.6 Hz, 2H, H-2), 1.79 (qn, J = 6.7 Hz, 2H, H-10), 2.31
(q, 2H, CH3CH2COO-), 3.99 (t, J = 6.2 Hz, 2H, H-1), 4.04 (t,
J = 6.6 Hz, 2H, H-11), 7.19 (s, 1 H, H-40), 7.20 (s, 1 H, H-50); 8.20
(m, 1 H, H-20); 8.30 (s, 1 H, H-60); 13C NMR (CDCl3) d 9.2
(CH3CH2COO-), 25.9 (C-3, C-10), 27.7 (CH3CH2COO-), 28.6 (C-4),
29.1–29.3 (C-5, C-6, C-7, C-8), 29.5 (C-2, C-9), 64.5 (C-1); 68.3 (C-
11); 121.2 (C-40); 137.8 (C-20); 141.7 (C-50); 155.5 (C-60), 171.2
(CH3CH2COO-); HRMS: [M+Na]+ Calcd C19H31NNaO2 344.2202.
Found: C19H31NNaO2 344.2197.
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4.3.16. 11-(Pyridin-2-yloxy)-undecyl propionate (9p)
Yield 45% of pure compound as a colorless oil. Rf 0.54 (EtOAc);
IR (KBr, cmꢂ1) 3438, 2930, 2858, 1736, 1657, 1579, 1569, 1466,
1367, 1242, 1043, 773; 1H NMR (CDCl3) d 1.13 (t, J = 7.6 Hz, 3 H,
CH3CH2COO), 1.26–1.31 (m, 14H, H-3, H-4, H-5, H-6, H-7, H-8, H-
9), 1.60 (qn, J = 7.0 Hz, 2H, H-2), 1.73 (qn, J = 7.3 Hz, 2H, H-10),
2.32 (q, J = 7.6 Hz, 2H, CH3CH2COO-), 3.91 (t, J = 7.4 Hz, 2H, H-1),
4.05 (t, J = 6.8 Hz, 2H, H-11), 6.15 (dt, J1 = 1.0 Hz, J2 = 6.7 Hz, 1 H,
H-30); 6.56 (d, J1 = 9.0 Hz,
1
H, H-50); 7.24 (dd, J1 = 2.0 Hz,
J2 = 6.7 Hz,
1
H, H-40); 7.31 (ddd, J1 = 2.0 Hz, J2 = 6.7 Hz,
J3 = 9.0 Hz, 1 H, H-60); 13C NMR (CDCl3) d 9.2 (CH3CH2COO-), 25.9
(C-3), 26.6 (C-10), 27.6 (CH3CH2COO-), 28.6 (C-4), 29.1–29.3 (C-5,
C-6, C-7, C-8), 29.4 (C-2, C-9), 50.0 (C-1); 64.5 (C-11); 105.9 (C-
30); 121.1 (C-50); 137.5 (C-40); 139.3 (C-60); 162.7 (C-20), 174.6
(CH3CH2COO-); HRMS: [M+Na]+ Calcd C19H31NNaO3 344.2202.
Found: C19H31NNaO3 344.2197.
4.4. Drug screening
Leishmania mexicana promastigotes were grown in a culture
medium containing 33 g lꢂ1 brain-heart infusion (Difco); 3 g lꢂ1
tryptose (Difco); 3 g lꢂ1 Na2HPO4; 0.4 g lꢂ1 KCl; 0.3 g lꢂ1 glucose.
pH was about 7.5, without the need for adjustment. After steriliza-
tion (10 min at 121 °C) penicillin (100 IU mLꢂ1), streptomycin
(100 lg/mL), haemin (20 l
g/mL, added as 1 mL of a 2 g mLꢂ1 solu-
tion in 0.1 N NaOH per litre of medium) and 20% v/v heat-inacti-
vated (45 min at 57 °C) fetal calf serum were added. Cultures
were performed at 28 °C in 600 mL cylindrical flasks with screw
caps, containing 100 mL of medium. Shaking was performed man-
ually, twice a day. All cultures were started with inocula taken
from exponential growth parasite in the same medium. The sus-
ceptibilities of the promastigote form of Leishmania to synthetic
compounds were tested by culturing them in cell-free medium at
28 °C. Growth experiments with promastigotes of L. mexicana were
initiated with 15 ꢁ 106 parasites/mL, and the inhibitors were
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Acknowledgments
We thank UBA X010, UBACYT 20020100100304, CONICET PIP
112-200801-00801/09 and ANPCyT PICT 2005-32735 for partial
financial support. A.B., G.G.L. and C.L. are Research Members of
CONICET (Argentina).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.06.028.

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