Hybrid furoxanyl N-acylhydrazone derivatives as hits for the development of neglected diseases drug candidates

Article abstract of DOI:10.1016/j.ejmech.2012.10.047

Neglected diseases represent a major health problem. It is estimated that one third of the world population is infected with tuberculosis and additionally Leishmaniosis and Chagas disease affect approximately 30 million people. N-Acylhydrazone moiety is a

Full text of DOI:10.1016/j.ejmech.2012.10.047

European Journal of Medicinal Chemistry 59 (2013) 64e74
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Hybrid furoxanyl N-acylhydrazone derivatives as hits for the development
of neglected diseases drug candidates
,
Paola Hernández a, Rosario Rojas b, Robert H. Gilman b, Michel Sauvain c d, Lidia M. Lima e,
,
a
,
a,
*
Eliezer J. Barreiro e , Mercedes González , Hugo Cerecetto
**
*
a Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Igua 4225, 11400 Montevideo, Uruguay
b Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru
c Université de Toulouse, UPS, UMR 152 PHARMA DEV, Toulouse, France
d Mission IRD, Lima, Peru
e LASSBio-Laboratório de Avaliação e Síntese de Substâncias Bioativas, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, PO Box 68023, ZIP 21941902 Rio de Janeiro,
RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Neglected diseases represent a major health problem. It is estimated that one third of the world pop-
ulation is infected with tuberculosis and additionally Leishmaniosis and Chagas disease affect approxi-
mately 30 million people. N-Acylhydrazone moiety is a repeated functional group present in several
prototypes and drug candidates for these neglected diseases. On the other hand, furoxan system has been
studied as pharmacophore for Leishmaniosis and Chagas diseases. Here we report on the design and
preparation of forty hybrid furoxanyl N-acylhydrazones and on their activity on Mycobacterium tuber-
culosis, H37Rv and MDR strains, Trypanosoma cruzi, and Leishmania amazonensis. Among them,
four derivatives displayed excellent to good selectivity indexes against the three different microorgan-
isms. Hybrid compound N0-(4-phenyl-3-furoxanylmethylidene)isoniazide 9 showed the best antibacte-
rial profile with MIC value 4.5 lesser than the value for the reference isoniazid against MDR strain.
Furoxanyl N-acylhydrazone (E)-2-methyl-N0-(4-phenyl-3-furoxanylmethylidene)-4H-imidazo[1,2-a]
pyridine-3-carbohydrazide 15 was ten-fold more potent against T. cruzi Amastigotes than the standard
drug nifurtimox. On the other hand, derivatives (E)-N0-(5-benzofuroxanylmethylidene)benzo[d][1,3]
dioxole-5-carbohydrazide 25 and (E)-N0-(4-hydroxy-3-methoxyphenylmethylidene)-3-methylfuroxan-4-
carbohydrazide 37 emerged as leads for the development of new leishmanicidal agents. The adequate
stability, in simulated biological system and plasma, and the lack of mutagenicity of these derivatives
allow us to propose them as candidates for further pre-clinical studies.
Received 22 August 2012
Received in revised form
24 October 2012
Accepted 26 October 2012
Available online 7 November 2012
Keywords:
N-Acylhydrazone
Furoxane
Anti-M. tuberculosis
Anti-Leishmania
Anti-T. cruzi
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
of patients. Of particular concern is the development of multi-drug-
resistant forms of the disease (MDR-TB), defined as forms resistant
Mycobacterium tuberculosis (M. tuberculosis), and to a lesser
extent Mycobacterium bovis and Mycobacterium africanum, can
cause a chronic and fatal condition in humans known as tubercu-
losis (TB). With the discovery of several active anti-TB agents the
disease was considered to be curable, however, in only a few years,
became evident that the use of drugs as single agents led to rapid
drug resistance and treatment failures among a substantial number
to two or more of the frontline anti-TB agents. It is estimated that
one third of the world’s population is infected with TB, with about
eight million new cases and dying 3.1 million annually [1,2]. The
current frontline therapy for TB consists of administering three
different drugs (the antibiotic rifampicin, and the azaheterocycles
isoniazid and pyrazinamide, Isnz and Pyzd, Fig. 1) over an extended
period of time as well as the problems that arise due to MDR-TB.
Consequently, it is necessary to develop new, potent, fast-acting
anti-tuberculosis drugs with low-toxicity profiles, actives against
MDR-TB and able to be given in conjunction with drugs used to
treat HIV infections [3,4]. Another pharmacological strategy is the
use of thioacetazone (Tzn, Fig. 1) in the cases of resistances by
rifampicin and Isnz.
* Corresponding authors. Tel.: þ598 2 5258618x216; fax: þ598 2 5250749.
** Corresponding author. Tel./fax: þ55 21 25626644.
E-mail addresses: ejbarreiro@ccsdecania.ufrj.br (E.J. Barreiro), megonzal@
fq.edu.uy (M. González), hcerecet@fq.edu.uy (H. Cerecetto).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.047

P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
65
S
F
CN
CN
H
N
O
NHNH2
O
N
NH2
N
N
O
O
N
H
NH2
O
N
N
N
N
O
O
O
N
Me
F
HN
F
2
1
O
Isnz
Pyzd
Tzn
O
Me
O
Ph
O
N
N
N
N
N
O2N
N
N
H
O
N
N
H
N
N
H
SO2
NO2
N
OH
N
OH
Nfx
Bnz
3
4
-O O
P
N+
O
O
H
H
N
Mts
N
N
Ph
Fig. 1. Some of the therapeutic tools for tuberculosis (Isnz, Pyzd, and Tzn), Chagas
disease (Nfx and Bnz) and leishmaniosis (Mts).
S
S
O
Besides TB, the parasitic diseases represent a major health
problem in Third World countries. More specifically, Chagas
disease, or American trypanosomiasis, caused by the protozoan
Trypanosoma cruzi (T. cruzi), and leishmaniosis, caused by
a protozoan belong to the genus Leishmania, are the largest
parasitic diseases burden in the American continents. Chagas
disease affects approximately 20 million people from the
southern United States to southern Chile. Even though the
enforcement of public health programs towards vector elimina-
tion in some Latin American countries has decreased the inci-
dence of new infections, the disease is still endemic in large areas
[5]. Currently, there are only two clinically used drugs, nifurti-
mox (Nfx, Fig. 1) and benznidazole (Bnz, Fig. 1). These drugs are
very deficient and there is an urgent need for the development of
safe and effective drugs [6]. On the other hand, leishmaniosis is
widespread around the world except in South-east Asia.
Currently, infections are found in 16 countries in Europe. The
current anti-leishmaniosis therapy involves the use of pentava-
lent antimonials (meglumine antimoniate and sodium stibo-
gluconate), with manifested resistant in the treatment of visceral
and mucocutaneous forms [7], the use of Amphotericin B (Amph)
and miltefosine (Mts, Fig. 1). Mts is active against visceral and
cutaneous leishmaniosis.
Since beginnings of the nineties we have investigated and
developed new hits for neglected disease drugs [8e15]. In this
sense, we have identified quinoxaline dioxides 1 (Fig. 2), with
excellent selectivity for M. tuberculosis H37Rv strain, and the lead 2
(Fig. 2), that combined anti-M. tuberculosis and anti-T. cruzi activi-
ties. Moreover, N-acylhydrazones (3 and 4) and thio-
semicarbazones (5, Fig. 2) have emerged as potential T. cruzi-
cysteine protease inhibitors where quinoxaline 4 and benzofuroxan
5 displayed good selectivity index for the parasites over mamma-
lian cells. Further, the furoxans 6 and 7 displayed excellent anti-
T. cruzi and anti-Leishmania activities. These compounds (1e7)
possess some common structural features, i.e. the presence of
amide or N-acylhydrazone moieties and N-oxide containing
heterocycles (Fig. 2). On the other hand, the amide moiety together
EtO
SO2 Ph
N
N
N
N
N
N
O
O
O
O
O
O
5
6
7
Fig. 2. Previous identified hits with anti-M. tuberculosis, anti-T. cruzi and anti-Leish-
mania activities. Common moieties are marked (dotted: N-oxide; line: amide).
aromatic cycles also constitute the backbone of current drugs for
tuberculosis and Chagas diseases (Fig. 1).
This information encourage us to design and develop hybrid
agents (Scheme 1), which combine different heterocyclic (hetm)
and furoxanyl moieties (fxm) connected by a N-acylhydrazone
(NAH) linker. As hetm we used the standards Isnz, Pyzd, Nfx, Bnz,
and the prototypes 3, and 4 as templates. Consequently, aza-
heterocycle (pyridine-3-, and 4-yl), diaza-heterocyle (1H-imid-
azole-4-yl, 4H-imidazo[1,2-a]pyridine-3-yl, and 1-phenyl-1H-pyr-
azole-4-yl), and other heterocyles (furoxan-4-yl) as the
combination of furan and imidazole heterocycles, and benzo[d][1,3]
dioxole-5-yl- were proposed as substituent of the acyl-position of
the NAH framework (Scheme 1a). The studied fxm, linked to the
imine group of the NAH unit, were 3-methyl-4-furoxanyl, 4-
phenyl-3-furoxanyl and 5-benzofuroxanyl (Scheme 1b). Addition-
ally, when the acyl-position of the NAH was furoxan-4-yl group, the
imine position was substituted by different aromatic groups
(Scheme 1c) in order to cover different stereoeelectronic
parameters.
Forty hybrid furoxanyl N-acylhydrazones (fx-NAH), belonging to
seven different families of heterocycles (AeG), have been synthe-
sized and their in vitro abilities to decrease the growth of
M. tuberculosis, T. cruzi, and Leishmania have been determined. The
selectivity indexes were evaluated analysing their in vitro cyto-
toxicity against J774 murine macrophages. Besides, stability, in
simulated biological system and plasma, and mutagenicity of some

66
P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
O
N
fxm(Ar)
NAH
hetm
N
H
templates
Me
N
N
N
N
N
N
O
N
a)
hetm
Me
N
c)
Ar
N
N
N
X
X= CH, N
O
O
N
R= variable
R
R
Me
X= O, S
R= variable
N
O
N
N
N
X
N
O
b)
fxm
Me
Ph
N
N
N
N
N
N
O
O
O
O
O
O
Scheme 1. Design of hybrid furoxanyl N-acylhydrazone (fx-NAH) as neglected disease drugs.
of selected derivatives were studied in order to confirm their
potentials as novel antiparasitic drug candidates.
were also prepared [17]. All the new compounds, 8e10, and 42e49,
were characterized by 1H NMR, 13C NMR, COSY, HMQC, and HMBC
experiments, IR and MS. As previously it was observed for deriva-
tives 11 and 20 [16] in accordance to the NMR experiments (1H
NMR and NOE-diff) [16e20] and characteristic fragmentations in
MS experiments, the Isnz-derivatives (8e10 and 48), were obtained
as mixture of E- and Z-isomers around the ylidenic moiety, with
ratio of E:Z near to 91:9. The purity of the synthesized compounds
was established by TLC and microanalysis. Only compounds with
analytical results for C, H and N, within ꢀ0.4 of the theoretical
values were considered pure enough.
2. Methods and results
2.1. Chemistry
The first designed family of fx-NAH was the Isnz derivatives
(family A 8e10, Scheme 2) prepared by the condensation of Isnz
with the corresponding furoxanyl aldehydes (I, II or III). The same
procedure was used to prepare fx-NAH family B (nicotinic acid
derivatives, 11e13, Scheme 2), family C (imidazo[1,2-a]pyridine
derivatives, 14e16, Scheme 2), family D (imidazole derivatives, 17e
19, Scheme 2), family E (pyrazol derivatives, 20e22, Scheme 2),
family F (benzo[d][1,3]dioxole derivatives, 23e25, Scheme 2) and
family G (furoxan derivatives, 26e47, Scheme 2) [16]. In order to
probe the relevance of the N-oxide moiety in the biological activity
the deoxygenated analogues of 10 and 13 (48 and 49, Scheme 2)
2.2. Biological characterization
2.2.1. In vitro anti-M. tuberculosis activity
All the hybrid fx-NAHs (8e47) and the reduced analogues (48
and 49) were tested in vitro for their anti-mycobacterial activities
against sensitive M. tuberculosis H37Rv strain and multidrug-

P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
67
hetm-
Me
Me
HN
O
O
N
N
N
N
N
N
N
Ph
family A family B
family C
family D family E family F
O
Me
Ph
N
O
O
O
8
9
11
12
14
17
18
20
21
23
24
N
N
N
hetm
hetm
N
H
N
O
(I), (II), (III), or (IV)
15
O
N
hetm
NHNH2
N
H
EtOH anh. or toluene anh.
AcOH (cat)
O
(V)-(X)
N
N
O
O
O
n=1 10
13
49
16
19
22
25
N
hetm
N
H
n
n=0 48
Ar
O
N
O
O
N
NH
Me
Me
Me
OMe
NHNH2
NH2NH2 (0.9 eq)-0 O
C
Ar-CHO
family G
N
O
N
N
N
N
toluene anh. / AcOH (cat)
O
O
O
O
O
Ar-
(XI)
(XII)
O
O
Me
Ph
N
N
HO
Me
Ph
O
N
N
N
N
N
N
N
N
O
O
O
O
NO2
O
O
X
O
O
O
O
X
O
N
N
H
H
O
H
26
27
28
X=S, 29
X=S, 31
X=O, 30 X=O, 32
33
n
(I)
(II)
n=1, (III)
R
OR'
OR
n=0, (IV)
X
R'
Y
Me
CONHNH2
Me
HN
CONHNH2
CONHNH2
R"
R=R'=Me, 34 R=Me, R'=H, 36 Y=CO2H, 38
R-R'=CH2, 35 R=H, R'=Me, 37 Y=CF3, 39
N
N
N
N
Y=Br,
Y=(NMe)2, 41
40 X=CH, R=R'=H, R"=OH, 45
4-yl, (V)
3-yl, (VI)
R=R'=R"=OMe, 46
(VII)
(VIII)
Y=NHAc, 42 X=N, R=R'=R"=H,
47
Y=NO2,
Y=H,
43
CONHNH2
CONHNH2
O
O
44
N
N
Ph
(IX)
(X)
Scheme 2. Synthetic procedures used to prepare the studied fx-NAH.
resistant (MDR) clinical isolate strain (Table 1). A modified micro-
plate Alamar blue assay [21,22], where Alamar was substituted by
tetrazolium bromide, was used to detect viable M. tuberculosis
[23,24]. This tetrazolium microplate assay allowed us to determine
the MIC values in 6e7 days. Isnz (V) was included as reference drug.
Furthermore, unspecific cytotoxicity of the studied hybrid
compounds was studied using J774.1 murine macrophages
(Table 1).
Results revealed that fx-NAH 9 was better anti-mycobacterial
agent against MDR strain than the reference drug, with a MIC
value 4.5 lower than the value for Isnz and a selectivity index (SI,
Table 1) higher than the value for Isnz. The behaviour of 9 against
H37Rv strain and macrophages is comparable to Isnz. The other two
members of the family A, fx-NAHs 8 and 10, also possess very good
biological profiles against both Mycobacterium strains. The posi-
tional isomers 11e13, belonging to family B, displayed similar
biological behaviours as fx-NAHs 8e10 being phenylfuroxan 12 the
most selective derivative with more than two-fold active against
MDR strain than Isnz. The most active derivative against MDR strain
was phenylfuroxan 15, belonging to family C, however, its high
unspecific cytotoxicity produces lower SIs than compounds 9, 10 or
12. Similarly, fx-NAH 19, member of family D, had lower SIs with
good behaviour against MDR strain. Derivatives 31 and 32 were the
exclusive active members of family G. The presence of nitro-
heterocyclic substituents in the Ar position of these fx-NAHs
(Scheme 2) could be the responsible of these activities being the
nitrophenyl analogue 43 inactive. In this family of fx-NAH deriva-
tive 42 with an acetamidophenyl scaffold like the drug Tzn (Fig. 1)
was also inactive. The rest members of this family and members of
families E and F displayed irrelevant biological behaviours in these
assays. The absence of N-oxide moiety, derivatives 48 and 49,
confirm the relevance of this function in the anti-Mycobacterium
activity and macrophages toxicity. These benzofurazan derivatives
were less actives and cytotoxic than the N-oxide analogues.
2.2.2. In vitro anti-T. cruzi activity
The hybrid fx-NAHs 8e33, 35, 38e45, 47, and the reduced
analogues (48 and 49) were tested in vitro for their

68
P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
Table 1
Anti-mycobacterial activity and unspecific cytotoxicity of hybrid fx-NAH derivatives.
Family Compd. MIC H37Rv (
mM) MIC MDR (
m
M) IC50 J774.1 (
m
M) SIH37Rva (SIMDR
)
Family Compd. MIC H37Rv (
m
M) MIC MDR (
m
M) IC50 J774.1 (
m
M) SI
A
B
8
9
1.6
0.6
0.35
>93.6
>101.2
40.5
>25.4
10.1
11.0
>93.6
>101.2
20.2
22.1
46.8
>83.3
8.6
38.6
>100.0
>80.1
21.9
>80.1
>66.8
>17.9
>86.2
>71.0
>76.7
>400.0
>400.0
134.9
>400.0
>400.0
194.9
31.0
208.9
>400.0
56.2
141.2
>400.0
>400.0
33.9
295.0
41.7
>250
>667 (>40)
385 (12)
e
G
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
>93.6
>75.8
82.2
>99.2
>106.0
42.1
>93.6
>75.8
82.2
>99.2
>106.0
42.1
>400.0
>400.0
>400.0
>400.0
>400.0
>400.0
68.0
e
e
10
48
11
12
13
49
14
15
16
17
18
19
20
21
22
23
24
25
>5
e
e
e
5 (10)
0.7 (1.4)
<2 (5)
e
>10
1.5
<3
e
44.2
44.5
44.5
>93.6
>83.3
17.3
>95.4
>81.6
>86.2
>85.6
>85.6
>86.2
>79.6
>74.0
>86.5
>82.5
>85.9
>101.6
>95.4
>74.4
>101.2
>95.4
>81.6
>86.2
>85.6
>85.6
>86.2
>79.6
>74.0
>86.5
>82.5
>85.9
>101.6
>95.4
>74.4
>101.2
246.0
C
D
E
F
>400.0
>400.0
>400.0
>400.0
>300.0
>400.0
162.0
3.2 (6.5)
1.8 (3.7)
e
e
77.2
e
>100.0
>80.1
43.7
>80.1
>66.8
>17.9
>86.2
>71.0
>76.7
e
e
e
0.8 (1.5)
<4
<0.6
<2
<1.5
<2
<2
e
<2
<1.5
<3
e
129.0
235.0
37.1
131.8
162.0
173.9
>300.0
>400.0
>400.0
>300.0
>400.0
e
e
e
Isnz
0.4
45.6
>400.0
>1000 >9
e
a
SI ¼ IC50,macrophages/IC50,mycobacteria
.
trypanosomicidal properties using two different forms of T. cruzi.
Axenic epimastigotes of Tulahuen 2 strain and amastigotes Tula-
huen C4 strain growing in VERO (normal African green monkey
epithelial) cells were used in the in vitro models [25,26]. The IC50
and the SI are showed in Table 2. Nfx was included as reference
drug. The furoxan intermediate (XI) and the hydrazide reactants,
VeVII, IX, X and XII, were also included to analyse their anti-T. cruzi
activities.
In general, members of the families AeF displayed some activity
against the epimastigote form of the parasite being, except for
derivative 15, lower than the Nfx activity. Again, the structural
motifs 5-nitrothienyl and 5-nitrofuryl conferred activity to
members of family G, i.e. 31 and 32. This nitroheterocyclic
substituent together o-hydroxyphenyl, in fx-NAH 33, are
framework-templates belonging to the parents compounds Nfx, 3
and 4 (Figs. 1 and 2).
Phenylfuroxan derivatives 9, 15, 24, and 27 were the best anti-
epimastigote member in each family showing that this structural
scaffold could play a role in the studied biological response.
Regarding to the anti-amastigote activity, the most relevant form
thinking in therapy, a clear relationship between anti-epimastigote
and anti-amastigote activities was observed. The most relevant fx-
NAHs were the pyrimidin-4-yl derivatives 9 and 10 with similar
activities and selectivities to that of Nfx. Compound 9 has excellent
SI (>63), when murine macrophages were taken into account as
mammal system, and good SI (2.5) when VERO cells were consid-
ered. Similarly occurred with family A member compound 10.
The best anti-amastigote derivative was fx-NAH 15, which was
Table 2
Trypanosomicidal activity of hybrid fx-NAHs.
Family
Compd.
IC50,epi
(
mM)
IC50,ama
(m
M)
SIa,b
Family
Compd.
IC50,epi
(m
M)
IC50,ama
(m
M)
SIa,b
A
8
9
>300.0
11.0
33.4
>150.0
>150.0
58.9
>40.5
6.4
5.6
e
G
26
27
28
29
30
31
32
33
35
38
39
40
41
42
43
44
45
47
81.3
25.1
>150.0
>150.0
>150.0
50.0
>37.5
>30.3
>32.9
>39.7
>42.4
>33.7
>35.6
>38.7
>34.5
>34.5
>31.9
>29.6
>34.6
>33.0
>34.4
>40.7
>38.2
>40.5
e
>63 (2.5)
24 (2.5)
e
e
10
48
11
12
13
49
14
15
16
17
18
19
20
21
22
23
24
25
e
>37.5
>40.5
>32.4
>35.3
>37.5
>33.3
0.91
>30.9
>40.0
>32.1
>35.0
>32.1
>26.7
>30.9
>34.5
<30.7
>28.4
e
B
e
e
<6
<0.9
<5.5
e
e
35.5
50.1
39.8
<2
<6
e
>150.0
>150.0
5.0
>150.0
>150.0
>150.0
171.5
>150.0
>150.0
29.5
C
D
E
F
>150.0
>150.0
>150.0
>150.0
>150.0
>150.0
>150.0
151.4
62 (12)
<4
e
e
e
<5
<3
e
e
<1
e
e
e
e
e
>150.0
>150.0
e
>150.0
50.0
50.0
<1.5
>5
<5
e
VII
IX
X
XI
XII
>150.0
ns
>150.0
>50.0
>50.0
>52.6
>49.5
>55.6
>62.3
>62.3
e
e
e
e
e
Nfx
V
VI
7.7
>150.0
>150.0
10.4
nsc
>73.0
27 (7.7)
e
e
a
SI ¼ IC50,macrophages (Table 1)/IC50,amastigotes
.
b
c
Values in parenthesis are SIs according to IC50,VERO/IC50,amastigotes
ns: not studied.
.

P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
69
eleven-fold more active than Nfx with excellent SIs, near to two-
fold more selective in both systems than Nfx. The corresponding
hydrazide, VII, was inactive against both forms of the parasite. The
rest of the studied intermediates, V, VI, IXeXII, did not display
relevant activities. Once again, the absence of N-oxide moiety,
derivatives 48 and 49, produced compounds with lower activities.
antiparasitic agents to treat neglected diseases. The compounds,
designed as hybrid entities combining different aromatic residues
and furoxan systems connected by N-acylhydrazone moiety, were
evaluated against M. tuberculosis, T. cruzi, and L. amazonensis.
Hybrid furoxan 9 (Fig. 3a), belonging to family A, was the best
in vitro anti-M. tuberculosis derivative. It displayed good activity
against both studied strains of bacteria with excellent
selectivity indexes, macrophage/bacteria, especially considering
MDR-strain. Additionally, derivative 9 was not mutagenic, against
S. typhimurium TA98, and the stability at different pH and on
plasma showed that it could not act as Isnz pro-drug.
On the other hand, one derivative from family C, the hybrid
compound 15 (Fig. 3a), exhibited excellent profile as anti-T. cruzi
agent comparing to the reference drug Nfx (1.5- and 11.4-fold
higher active than Nfx against epimastigote and amastigote form,
respectively). In addition, it had higher selectivity than Nfx and it
was not mutagenic in absence and presence of S9. Only, in basic
aqueous milieu, pH 8.3, compound 15 showed some degree of
instability after 24 h of incubation at 37 ꢁC.
Regarding, in vitro anti-Leishmania activity, we identified
excellent molecular hits with higher selectivity than the reference
drug Amph. The best hybrid compound was the member of family
G derivative 37 (Fig. 3a). This compound was 50-fold more selec-
tive than the reference drug. Other product with excellent bio-
logical behaviour was derivative 25 (Fig. 3a) that possessed
a selectivity index 28.5-fold higher than the selectivity for Amph.
Furthermore, these hybrid derivatives, 25, and 37, were not
mutagenic.
Lipinski’s rule of five [30] serves as a guide to determine if
compounds will be orally bioavailable. Analysis of compounds 9, 15,
25, and 37 suggested that it would be orally administered (Fig. 3b).
On the other hand, the lipophilicity of these compounds resulted
very different, see molecular lipophilicity potential (MLP, Fig. 3c),
virtual LogP [31,32], as their biological activities.
2.2.3. In vitro anti-Leishmania activity
Some selected hybrid fx-NAHs were evaluated against L. ama-
zonensis axenic amastigotes. For that, MTT colorimetric assay using
MHOM/BR/76/LTB-012 strain was employed [26]. The IC50 and the
SI are showed in Table 3. Amph was included as reference drug.
All the studied compounds displayed remarkable anti-Leish-
mania activities being the best the fx-NAHs 22, 25, and 37. However,
these compounds displayed lower leishmanicidal activities than
Amph. Nonetheless, according to the cytotoxicity against murine
macrophages (Table 1), they were five- to fifty-fold more selective
than the reference drug. Besides the most selective hybrid
compounds, derivatives 25 and 37, derivatives 14, member of family
A, 23, member of family F, and 36, 43, and 45, members of family G,
also displayed very good SIs.
2.3. Hybrid fx-NAHs as drug candidates
Considering the great potential therapeutic use of this class of
compounds, it is indispensable for pharmaceutical development to
assess their bio-safety. Consequently, we evaluated the in vitro
mutagenic potential of the selected compounds 8e10, 15, 25, and
37 using Ames test with Salmonella typhimurium TA98 strain [28] in
absence and presence of S9 in order to simulate the metabolic
process (Table 4).
The negative results of these mutagenicity studies, except for
benzofuroxanyl fx-NAHs 10 and 19 in absence of S9, showed the
safety of this class of compounds to be further developed.
Moreover, the potential capability of these hybrid compounds to
act as pro-drug, where the hydrolysis of acylhydrazone moiety
could produce the active drug, was studied. For that, we analysed
the stability, of selected derivatives 8e10, 13, 15, and 24, on aqueous
solution at different pHs and in the presence of rat plasma (Table 5).
All the studied derivatives, and specially 8e10, the potential pro-
dugs of Isnz, were stable on plasma for 3 h at 37 ꢁC. Additionally, all
the compounds were stable during 24 h on aqueous solution of pH
2.0, 5.0, 7.4, and 8.3. On aqueous solution at pH 8.3 derivative 15
showed some degree of instability, lower than 10% of decomposition
(no attempts to isolate the decomposition products were done).
In line with these findings, hybrid compounds 9, 15, 25, and 37
are excellent leads for further studies. Currently we are performing
mechanism of action and in vivo studies.
4. Experimental
Reagents were purchased from Aldrich and used without
further purification. Melting points were performed using an
Electrothermal Engineering Ltd melting point apparatus, and the
results were uncorrected. Infrared spectra were recorded on
a Bomem FTLA2000 spectrophotometer instrument as films on
KBr discs. 1H and 13C NMR spectra were recorded in the indi-
cated solvent on Bruker DX 400 MHz spectrometer. Chemical
shifts are quoted in parts per million downfield from TMS and
the coupling constants are in Hertz and, in case of E/Z mixture,
for the main isomer. Mass spectra were recorded on a Hewlett
Packard MSD 5973 (electronic impact, EI) or on a Hewlett
Packard LC/MS Series 1100 (electrospray ionization, ESI) instru-
ments. All solvents were dried and distilled prior to use. All the
reactions were carried out in a nitrogen atmosphere. Reactions
were monitored by TLC using commercially available precoated
plates (Merck Kieselgel 60 F254 silica) and developed plates
were examined under UV light (254 nm) or as iodine vapour
stains. Column chromatography was performed using 200 mesh
silica gel. To determine the purity of the compounds, micro-
analyses were done on a Fisons EA 1108 CHNSeO instrument
from vacuum-dried samples and were within ꢀ0.4 of the values
obtained by calculated compositions. Compounds 11e41, IeIV,
and VIeXII were prepared following synthetic procedures
previously reported [16,17].
3. Discussion and conclusions
We reported the synthesis and biological evaluation of new
hybrid furoxanyl N-acylhydrazones as antimicrobial and
Table 3
Leishmanicidal activity of hybrid fx-NAHs.
Family
Compd.
IC50
(
m
M)
SIa
A
C
E
F
8
14
22
23
25
36
37
43
25.3
12.6
1.4
3.3
1.3
8.3
1.7
7.9
4.5
>16
>32
27
40
134
>48
>235
>38
>89
4.7b
>13
G
45
Amph
VII
0.11
29.8
a
SI ¼ IC50,macrophages (Table 1)/IC50,amastigotes
.
b
Using as IC50,macrophages ¼ 0.52
mM (from reference [27]).

70
P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
Table 4
Number of revertants of studied derivatives on TA98 S. typhimurium strain.
Type of furoxanyl moiety
3-Methyl-4-furoxanyl
4-Phenyl-3-furoxanyl
9
5-Benzofuroxanyl
10
Family
S9
8
Controls
D
Da
NRb,c
Md
D
NR
M
D
NR
M
NR
M
(A)
(ꢂ)
500.0
166.7
55.6
18.5
6.2
500.0
166.7
55.6
18.5
6.2
12.5 ꢀ 0.7
9.0 ꢀ 5.7
7.0 ꢀ 1.4
8.5 ꢀ 0.7
12.0 ꢀ 2.8
12.0 ꢀ 1.4
11.5 ꢀ 7.8
17.0 ꢀ 5.7
14.5 ꢀ 6.4
11.5 ꢀ 2.1
Mꢂ
400.0
133.3
44.4
14.8
4.9
400.0
133.3
44.4
14.8
4.9
14.0 ꢀ 4.2
9.5 ꢀ 2.1
12.0 ꢀ 5.7
17.0 ꢀ 1.4
8.5 ꢀ 0.7
9.0 ꢀ 5.7
8.5 ꢀ 0.7
9.5 ꢀ 3.5
8.0 ꢀ 5.7
6.5 ꢀ 3.5
M-
150.0
50.0
16.7
5.6
76.0 ꢀ 7.1
60.5 ꢀ 19.1
37.5 ꢀ 3.5
19.0 ꢀ 2.8
15.5 ꢀ 0.7
82.5 ꢀ 6.4
21.0 ꢀ 0.5
19.0 ꢀ 5.7
12.0 ꢀ 2.8
12.0 ꢀ 2.8
Mþ
wte
e
11.7 ꢀ 4.4
e
NPDf
20.0
1460 ꢀ 580
Mþ
1.9
g
(þ)
(ꢂ)
Mꢂ
M-
150.0
50.0
16.7
5.6
Mþ/ꢂ
wt
e
11.5 ꢀ 5.1
900 ꢀ 290
e
2-AFh
10.0
Mþ
1.9
(C)
(D)
(F)
(G)
15
250.0
83.3
27.8
9.3
6.0 ꢀ 2.8
4.5 ꢀ 2.1
9.5 ꢀ 2.1
7.5 ꢀ 0.7
7.5 ꢀ 2.1
7.0 ꢀ 4.2
9.0 ꢀ 1.4
8.0 ꢀ 2.8
8.0 ꢀ 1.4
6.5 ꢀ 0.7
M-
M-
wt
NPD
e
11.7 ꢀ 4.4
e
20.0
1460 ꢀ 580
Mþ
3.1
(þ)
(ꢂ)
250.0
83.3
27.8
9.3
wt
2-AF
e
11.5 ꢀ 5.1
900 ꢀ 290
e
10.0
Mþ
3.1
19i
500.0
166.7
55.6
18.5
6.2
500.0
166.7
55.6
18.5
12.0 ꢀ 7.1
25
500.0
166.7
55.6
18.5
6.2
500.0
166.7
55.6
18.5
6.2
47.0 ꢀ 8.5
39.5 ꢀ 6.4
30.5 ꢀ 4.9
23.5 ꢀ 2.1
10.5 ꢀ 2.1
27.0 ꢀ 14.1
17.5 ꢀ 0.7
13.0 ꢀ 1.4
12.0 ꢀ 5.7
Mþ
wt
NPD
e
10.0 ꢀ 3.0
e
20.0
1900 ꢀ 200
Mþ
(þ)
(ꢂ)
Mþ/ꢂ
wt
2-AF
e
11.0 ꢀ 1.0
844 ꢀ 80
e
10.0
Mþ
24i
250.0
83.3
27.8
9.3
11.0 ꢀ 1.4
7.0 ꢀ 1.4
10.0 ꢀ 3.0
9.0 ꢀ 4.0
8.5 ꢀ 0.7
13.0 ꢀ 3.0
12.0 ꢀ 3.0
3.5 ꢀ 3.5
4.0 ꢀ 1.3
3.0 ꢀ 1.4
M-
M-
25.0 ꢀ 1.4
9.0 ꢀ 2.8
13.0 ꢀ 4.2
7.5 ꢀ 3.5
8.0 ꢀ 0.5
9.0 ꢀ 3.5
9.0 ꢀ 1.4
4.5 ꢀ 2.1
11.0 ꢀ 2.8
7.5 ꢀ 2.1
Mꢂ
Mꢂ
wt
NPD
e
10.0 ꢀ 2.0j
e
20.0
1900 ꢀ 200j
Mþ
3.1
(þ)
(ꢂ)
250.0
83.3
27.8
9.3
wt
2-AF
e
8.0 ꢀ 4.0j
844 ꢀ 80j
e
10.0
Mþ
3.1
37
500.0
166.7
55.6
18.5
6.2
500.0
166.7
55.6
18.5
6.2
10.5 ꢀ 0.7
7.5 ꢀ 6.4
Mꢂ
Mꢂ
12.5 ꢀ 2.1
11.0 ꢀ 4.2
5.5 ꢀ 0.7
wt
NPD
e
11.7 ꢀ 4.4
e
20.0
1460 ꢀ 580
Mþ
(þ)
22.0 ꢀ 1.4
13.5 ꢀ 7.8
15.0 ꢀ 7.1
11.0 ꢀ 1.4
11.5 ꢀ 0.7
wt
2-AF
e
11.5 ꢀ 5.1
900 ꢀ 290
e
10.0
Mþ
a
D: doses in
m
g/plate.
b
c
d
e
f
NR: number of revertants.
The results are the means of two independent experiments ꢀSD.
M: mutagenicity, according to reference [29] (see Experimental Section for details).
wt: without treatment.
NPD: 4-nitro-o-phenylendiamine.
g
h
i
Mþ/ꢂ: Only the highest studied dose was mutagenic. Higher doses did not study due to solubility problems.
AF: 2-aminofluorene.
From reference [16].
j
Values for derivative 24 [16]. Derivative 25 controls were the same that the other herein studied hybrid compounds.

P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
71
Table 5
Stability of selected hybrid fx-NAHs in physiological conditions. As example the UV-spectra of selected derivative 13 in the different conditions were showed.
Family
Compd.
Stability in aqueous solution
Stability in plasma
13
pH 2.0
pH 5.0
pH 7.4
pH 8.3
Stable24a
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
Stable24
p.d.p.c
Stable3b
Stable3
Stable3
Stable3
Stable3
Stable3
1.0
0.8
0.6
0.4
0.2
0.0
A
8
9
10
13
15
24
pH 2.0
B
C
F
Stable24
250
300
350
(nm)
400
450
λ
1.0
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
2.0
1.5
1.0
0.5
pH 8.3
pH 5.0
pH 7.4
0.8
0.6
0.4
0.2
0.0
0.0
250
300
350
400
450
250
300
350
400
450
250
300
350
400
450
λ
λ
(nm)
(nm)
λ
(nm)
a
b
c
Stable 24 h, @ 37 ꢁC.
Stable 3 h, @ 37 ꢁC.
p.d.p.: presence of decomposition products was observed at the end of the assay.
4.1. General procedure for the preparation of hybrid derivatives 8e
10, 48, and 49
142.7, 146.6, 150.1, 150.9, 162.5. IR (KBr)
y
(cmꢂ1): 3176, 1690, 1617,
1596,1548,1267,1010, 846, 803. MS (ESI) m/z (%): 284 (Mþ þ H,100),
ꢃ
268 (Mþ þ H-16, 5), 224 (40). Calculated analysis for C13H9N5O3: C:
ꢃ
To a solution of Isnz (1 equiv.) or VI in dry EtOH (2.5 mL) was
added to the corresponding aldehyde (I, II, III, or IV, 0.9 equiv.) and
a drop of glacial AcOH. The mixture was stirred until the aldehyde
was consumed (checked by TLC). The solid precipitated was filtered
and washed with cold EtOH.
55.1; H: 3.2; N: 24.7. Found: C: 54.9; H: 2.8; N: 24.8.
4.1.4. N0-(5-Benzofurazanylmethylidene)isoniazide (48)
(E:Z proportion ¼ 97:3). White solid (75%). 1H NMR (DMSO-d6)
d
ppm: 7.85 (2H, d, J ¼ 4.0), 8.14 (2H, bs), 8.36 (1H, s), 8.61 (1H, s),
8.81 (2H, d, J ¼ 4.0), 12.45 (1H, s). 13C NMR (DMSO-d6)
d ppm: 117.2,
4.1.1. N0-(2-Methyl-3-furoxanylmethylidene) isoniazide (8)
117.6, 122.1, 129.8, 138.8, 140.5, 147.1, 149.6, 149.7, 150.9, 162.5.
Calculated analysis for C13H9N5O2: C: 58.4; H: 3.4; N: 26.2. Found:
C: 58.1; H: 3.5; N: 25.9.
(E:Z proportion ¼ 91:9). White solid (40%). E-isomer: 1H NMR
(DMSO-d6)
d
ppm: 2.40 (3H, s), 7.84 (2H, d, J ¼ 4.0), 8.50 (1H, s), 8.84
(2H, d, J ¼ 4.0), 12.61 (1H, s). 13C NMR (DMSO-d6)
d ppm: 9.6, 112.2,
121.9, 137.9, 140.1, 151.0, 154.4, 164.6. IR (KBr)
y
(cmꢂ1): 3200, 1679,
4.1.5. (E)-N0-(5-Benzofurazanylmethylidene) nicotinohydrazide (49)
1611, 1552, 1289, 1029, 855, 797. MS (ESI) m/z (%): 248 (Mþ þ H,
100). Calculated analysis for C10H9N5O3: C: 48.6; H: 3.7; N: 28.3.
Found: C: 48.4; H: 3.6; N: 28.1.
White solid (25%); mp: 181.4e182.9 ꢁC 1H NMR (DMSO-d6)
d ppm:
ꢃ
7.60 (1H, dd, J1 ¼ 7.2, J2 ¼ 8.0), 8.08 (2H, s), 8.29 (1H, dd, J1 ¼ 8.4,
J2 ¼ 6.4), 8.37 (1H, s), 8.59(1H, s), 8.80(1H, d, J¼ 4.0), 9.10(1H, s),12.45
(1H, s). 13C NMR (DMSO-d6)
d ppm: 117.0, 117.2, 124.2, 129.9, 130.0,
4.1.2. N0-(4-Phenyl-3-furoxanylmethylidene) isoniazide (9)
130.1, 146.5, 149.2, 149.5, 153.1, 162.6. Calculated analysis for
C13H9N5O2: C: 58.4; H: 3.4; N: 26.2. Found: C: 58.5; H: 3.1; N: 26.4.
(E:Z proportion ¼ 98:2). White solid (42%). E-isomer: 1H NMR
(DMSO-d6)
d
ppm: 7.62 (3H, m), 7.85 (2H, d, J ¼ 4.0), 7.97 (2H, d,
J ¼ 8.0), 8.40 (1H, s), 8.81 (2H, d, J ¼ 4.0), 12.45 (1H, s). 13C NMR
4.1.6. General procedure for the synthesis of compounds 42e47
To a solution of (XII) (1 equiv.) in dry toluene (5 mL/mmol of
hydrazide) was added the corresponding aldehyde (0.9 equiv.) and
a drop of glacial AcOH. The mixture was stirred until the aldehyde
was consumed (checked by TLC). The solid precipitated was filtered
and washed with cold toluene.
(DMSO-d6) d ppm: 113.2,121.5,129.2,129.5,131.0,131.5,135.0,140.1,
151.0, 156.5, 162.0. IR (KBr)
y
(cmꢂ1): 3239, 1669, 1584, 1548, 1297,
1277, 1046, 854, 784. MS (ESI) m/z (%): 310 (Mþ þ H, 100). Calcu-
lated analysis for C15H11N5O3: C: 58.2; H: 3.6; N: 22.6. Found: C:
58.1; H: 3.4; N: 22.5.
ꢃ
4.1.3. N0-(5-Benzofuroxanylmethylidene)isoniazide (10)
4.1.7. (E)-N0-(4-Acetamidophenylmethylidene)-3-methylfuroxan-4-
carbohydrazide (42)
(E:Z proportion ¼ 91:1). White solid (83%). 1H NMR (DMSO-d6)
d
ppm: 7.30e8.20 (3H, bm), 7.85 (2H, d, J ¼ 4.0), 8.52 (1H, s), 8.81 (2H,
Beige solid (21%); mp: 205.0e207.4 ꢁC 1H NMR (DMSO-d6)
d, J ¼ 4.0),12.45 (1H, s).13C NMR (DMSO-d6)
d
ppm: 112.1,123.4,130.6,
d ppm: 2.05 (3H, s), 2.31 (3H, s), 7.70 (4H, m), 8.46 (1H, s), 10.14 (1H,

72
P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
Fig. 3. a) New identified hits with anti-M. tuberculosis, anti-T. cruzi and anti-Leishmania activities from the herein studied hybrid furoxanyl N-acylhydrazones. b) Lipinski’s phys-
icochemical properties for 9, 15, 25, and 37. c) Molecular lipophilicity potential (MLP), virtual Log P [22,23], for 9, 15, 25, and 37. Up: Molecules are represented by CPK and MLP as
solid colours are red/orange/yellow for hydrophilic regions; blue/violet for lipophilic regions; green for intermediate regions. Down: Molecules are represented by tubes and MLP as
dotted colours. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
s), 12.46 (1H, s). 13C NMR (DMSO-d6)
128.0, 129.0, 142.0, 151.0, 152.0, 154.0, 168.0. IR (KBr)
d
ppm: 9.0, 25.0, 115.0, 119.0,
(cmꢂ1): 3189,
112.0, 127.9, 129.2, 131.0, 134.6, 150.7, 152.5, 154.0. IR (KBr) y
(cmꢂ1):
y
3100, 1682, 1651, 1559, 1539, 1259, 1059, 835, 758. MS (EI) m/z (%):
246 (Mþꢃ, 32), 147 (55), 143 (26), 119 (14), 103 (26), 90 (45).
Calculated analysis for C11H10N4O3: C: 53.7; H: 4.1; N: 22.8. Found:
C: 53.4; H: 3.9; N: 22.9.
1676, 1610, 1583, 1520, 1258, 1036, 899, 831. MS (ESI) m/z (%): 304
(Mþ þ H, 33). Calculated analysis for C13H13N5O4: C: 51.5; H: 4.3; N:
ꢃ
23.1. Found: C: 51.2; H: 4.1; N: 23.2.
4.1.8. (E)-N0-(4-Nitrophenylmethylidene)-3-methylfuroxan-4-
4.1.10. (E)-N0-(3-Hydroxyphenylmethylidene)-3-methylfuroxan-4-
carbohydrazide (45)
carbohydrazide (43)
Yellowsolid(52%);mp:189.8e192.1 ꢁC1HNMR(DMSO-d6)
d
ppm:
2.31 (3H, s), 8.01(2H, d,J ¼ 8.0), 8.28(2H, d,J¼ 12.0), 8.63(1H, s),12.50
(1H, s).13CNMR (DMSO-d6)
ppm:8.9,113.7,124.6,128.9,140.4,148.5,
148.7,151.9,154.4. IR(KBr)
Yellow solid (77%); mp: 190.7e193.2 ꢁC 1H NMR (DMSO-d6)
d
ppm: 2.40 (3H, s), 4.54 (1H, s), 6.90 (1H, m), 7.26 (1H, s), 7.28 (1H,
m), 7.31 (1H, m), 8.34 (1H, s), 9.91 (1H, s). 13C NMR (DMSO-d6)
d ppm: 7.0, 112.0, 114.0, 118.0, 119.6, 129.5, 134.9, 151.0, 151.5, 155.0,
d
y
(cmꢂ1): 3216, 1702, 1606, 1553,1516,1346,
1306,1240,1039, 854, 827. MS (EI) m/z (%): 291 (Mþꢃ,14),192 (50),149
(15), 148 (1), 143 (18), 67 (100). Calculated analysis for C11H9N5O5: C:
45.4; H: 3.1; N: 24.1. Found: C: 45.1; H: 2.9; N: 23.9.
157.7. IR (KBr) y
(cmꢂ1): 3372, 3234, 1694, 1614, 1567, 1558, 1252,
1038, 884, 847. MS (EI) m/z (%): 262 (Mþꢃ, 100), 163 (66), 143 (10),
135 (20), 119 (89). Calculated analysis for C11H10N4O4: C: 50.4; H:
3.8; N: 21.4. Found: C: 50.2; H: 3.9; N: 21.5.
4.1.9. (E)-3-Methyl-N0-(4-phenylmethylidene) furoxan-4-
carbohydrazide (44)
4.1.11. (E)-N0-(3,4,5-Trimethoxyphenylmethylidene)-3-
methylfuroxan-4-carbohydrazide (46)
White solid (42%); mp: 180.4e184.7 ꢁC 1H NMR (DMSO-d6)
d
ppm: 2.39 (3H, s), 7.49 (1H, d, J ¼ 3.0), 7.50 (2H, d, J ¼ 3.0), 7.82
White solid (70%); mp: 179.2e181.9 ꢁC 1H NMR (DMSO-d6)
(2H, m), 8.61 (1H, s), 11.37 (1H, s). 13C NMR (DMSO-d6)
d
ppm: 8.1,
d ppm: 2.25 (3H, s), 3.74 (3H, s), 3.77 (6H, s), 7.04 (2H, s), 8.44 (1H,

P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
73
s), 12.61 (1H, s). 13C NMR (DMSO-d6)
113.7, 129.4, 140.1, 150.9, 152.1, 153.7, 154.0. IR (KBr)
1673, 1610, 1579, 1564, 1241, 1008, 848, 740. MS (EI) m/z (%): 336
(Mþꢃ, 100), 237 (16), 193 (47). Calculated analysis for C14H16N4O6: C:
50.0; H: 4.8; N: 16.7. Found: C: 49.8; H: 4.9; N: 16.4.
d
ppm: 8.8, 56.5, 60.6, 105.1,
(cmꢂ1): 3239,
was calculated as follows {1 ꢂ [(Ap ꢂ A0p)/(Ac ꢂ A0c)]} ꢄ 100,
where Ap ¼ A590 of the culture containing the compound to be
analysed at day 5; A0p ¼ A590 of the culture containing the
compound to be analysed just after addition of the inocula (day 0);
Ac ¼ A590 of the culture in the absence of any compound (control) at
day 5; A0c ¼ A590 in the absence of the compound at day 0. To
determine ID50 values, parasite growth was followed in the absence
(control) and presence of increasing concentrations of the corre-
sponding compound. The ID50 values were determined as the drug
concentrations required to reduce the absorbance by half that
measured for untreated controls.
y
4.1.12. (E)-N0-(2-Pyridinylmethylidene)-3-methyl furoxan-4-
carbohydrazide (47)
White solid (53%); mp: 174.8 ꢁC (d). 1H NMR (DMSO-d6)
d ppm:
2.41 (3H, s), 7.45 (1H, dd, J1 ¼ 5.0, J2 ¼ 2.0), 7.91 (1H, dd, J1 ¼ 7.8,
J2 ¼ 2.0), 8.13 (1H, d, J ¼ 8.0), 8.62 (1H, s), 8.65 (1H, d, J ¼ 5.0), 11.63
(1H, s). 13C NMR (DMSO-d6)
150.1, 151.0, 151.6, 153.7, 154.3. IR (KBr)
d
ppm: 8.2, 113.0, 120.7, 125.1, 136.9,
The anti-trypanosome activity in amastigote Tulahuen C4 strain
y
(cmꢂ1): 3169, 1693, 1620,
transfected with
the colorimetric method based on the reduction of the substrate
chlorophenol red -galactopyranoside (CPRG) by -galactoside
b-D-galactosidase (Lac Z) gene, was evaluated by
1590, 1562, 1242, 1040, 858, 835. MS (EI) m/z (%): 247 (Mþꢃ, 5), 120
(100), 92 (83). Calculated analysis for C10H9N5O3: C: 48.6; H: 3.7; N:
28.3. Found: C: 48.5; H: 3.8; N: 28.0.
b-
D
b
resulting from the expression of the gene. A monolayer of VERO
cells was seeded in a 96 well plate in complete RPMI 1640 medium
without red phenol complemented with 10% of foetal calf serum.
The cells were inoculated with 5 ꢄ 104 tripomastigotes (Tulahuen
C4) per well and then, incubated for 24 h at 37 ꢁC, 5% CO2. After that,
4.2. Biology
4.2.1. Anti-M. tuberculosis assay
A suspension of MTB was made by mixing growth from slants
(20e30 days old) with 100 mL of Tween 80 into 0.2% bovine serum
10
37 ꢁC, 5% CO2 during 5 days. The anti-T. cruzi activity was deter-
mined by adding 25 L of 900 M CPRG substrate (Roche) solution.
mg/mL of each NAHs were added and the plate was incubated at
albumin. Turbidity of the suspension was then adjusted to
m
m
a McFarland standard No. 1 (3 ꢄ 107 CFU/mL) by adding Tween 80
The plate was incubated at 37 ꢁC during 4e5 h until the colour
developed. The compounds that showed anti-parasitic activity
(>50% growing inhibition) were re-evaluated to determine the
inhibitory concentration for 50% growth of the parasites (IC50). The
compounds were evaluated at concentrations 10.0, 2.0, 0.40, 0.80
and bovine serum albumin. The bacterial suspension (300 mL) was
further transferred to 7.2 mL of 7H9GC broth (4.7 g of Middlebrook
7H9 broth base, 20 mL of 10% glycerol, 1 g of Bacto Casitone, 880 mL
of distilled water, 100 mL of OADC (oleic acid, albumin, dextrose,
catalase)). For the bioassay, the compounds were resuspended in
DMSO at a concentration of 4 mg/mL (stock solution). These stock
solutions were further diluted with appropriate volumes of 7H9GC
and 0.16
mg/mL by duplicate. The colour intensity resulting from the
cleavage of CPRG by
b-galactosidase was measured at 570 nm using
a microplate reader. The IC50 of compounds were calculated by
logarithmic regression of their OD values compared to the
untreated control. All active compounds also went through an
evaluation of their cytotoxicity in VERO cells.
broth to yield final concentration of 0.4e25
concentrations ranges of standard antibiotics used as positive
controls were 0.125e32 lg/mL for isoniazid and 0.063e16 g/mL for
rifampin. The drug (100 L) was mixed in the wells with 100 L of
mg/mL. Final drug
m
m
m
bacterial inoculums, resulting in a final bacterial concentration of
approximately 1.2 ꢄ 106 CFU/mL. The wells in column 11 served as
inoculums only controls. Solvent (DMSO) was included in every
experiment as a negative control. Plants were sealed in plastic bags
4.2.3. Anti-Leishmania in vitro test
Experiments were carried out on axenic amastigotes of L. ama-
zonensis (strain MHOM/BR/76/LTB-012) maintained by weekly
passages in MAA/20 medium at 32 ꢁC with 5% CO2 in 25 cm2 tissue
culture flasks. Cultures were initiated with 5 ꢄ 105 amastigotes
and then incubated at 37 ꢁC for 5 days. On day 5, 50
mL of
tetrazolium-Tween 80 mixture {1.5 mL of tetrazolium[3-(4,5-
dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] at a dilu-
tion of 1 mg/mL, in absolute ethanol and 1.5 mL of 10% Tween 80}
was added to the wells, and the plate was incubated at 37 ꢁC for
24 h. After this period, the growth of the microorganism was
visualized by the change in colour of the dye from yellow to purple.
The tests were carried out in triplicate. MIC is defined as the lowest
drug concentration that prevents the aforementioned change in
colour.
with 5 mL of medium. To determine the activity of the NAHs, 100 mL
of axenically grown amastigotes were seeded in 96 well plate. The
compounds, dissolved in DMSO, were added at final concentrations
ranging from 100 to 1
mg/mL. The reference compound, Ampho-
tericin B, was evaluated at 0.1
m
g/mL. After 72 h of incubation, 10 mL
of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT, Sigma) (10 mg/mL) was added to each well and plates were
further incubated for 4 h. The enzymatic reaction was then stopped
with 100 mL of 50% isopropanol-10% sodium dodecyl sulphate and
incubated for an additional 30 min under agitation at room
temperature. Finally, absorbance was measured at 570 nm with
a microplate reader. All experiments were performed in triplícate.
Percent growth inhibition of the parasite was calculated by the
following formula: % of inhibition ¼ ((OD control ꢂ OD drugs)/OD
control) ꢄ 100. The concentration inhibiting 50% of the parasite
growth (IC50) was calculated after evaluating percent growth
inhibition at different concentrations.
4.2.2. Anti-T. cruzi in vitro test
T. cruzi epimastigotes (Tulahuen 2 strain) were grown axenically
at 28 ꢁC in BHI-Tryptose complemented with 5% foetal calf serum.
Cells were harvested in late log phase, suspended in fresh medium,
counted in a Neubauer chamber and placed in 24-well plates
(3 ꢄ 106/mL). Cell growth was measured as the absorbance of the
culture at 590 nm, which was found to be proportional to the
number of cells. Before inoculation, the medium was supplemented
with the indicated amount of the compound to be analysed from
a stock solution in DMSO. The final concentration of DMSO in the
culture media never exceeded 1% and a negative control was run
with 1% DMSO and the absence of any compound, and a positive
control with Nfx was included in each experiment. No effect on
epimastigote growth was observed in the presence of up to 1%
DMSO in the culture media. The percentage of growth inhibition
4.2.4. Unspecific mammalian in vitro cytotoxicity
J774.1 murine macrophage cells (ATCC, USA) were grown in
DMEM culture medium containing 4 mM L-glutamine and sup-
plemented with 10% heat-inactivated foetal calf serum. The cells
were seeded in a 96 well plate (5 ꢄ 104 cells in 200
mL culture
medium) and allowed to attach for 48 h in a humidified 5% CO2/95%
air atmosphere at 37 ꢁC. Afterwards, cells were exposed for 48 h to

74
P. Hernández et al. / European Journal of Medicinal Chemistry 59 (2013) 64e74
the compounds (12.5e400.0
mM) or vehicle for control and addi-
a 37 ꢁC shaker (gentle) for 3 h. The reaction was quenched with cold
tional controls (cells in medium) were used in each test. Cell
viability was then assessed by measuring the mitochondrial-
dependent reduction of MTT to formazan. For this propose, MTT
in sterile PBS (0.2% glucose) pH 7.4 was added to cells to a final
concentration of 0.1 mg/mL and cells were incubated at 37 ꢁC for
3 h. After removing the media, formazan crystals were dissolved in
acetonitrile followed immediately by mixing and centrifugation at
10,000 rpm, 4 min, 10 ꢁC. The supernatant was used for the
determination of stability comparing with a solution of the
compound in acetonitrile prepared at the moment of the read-out.
Acknowledgements
180 mL of DMSO and 20 mL MTT buffer (0.1 M glycine, 0.1 M NaCl,
0.5 mM EDTA, 10.5 pH) and the absorbance at 560 nm was read
using a microplate spectrophotometer. The IC50 is determined as
the concentration which reduces absorbance by 50% compared to
control treated with the solvent of the compounds, and was
determined by linear regression analysis.
We thank PEDECIBA-ANII for scholarship to P.H., and CNPq-
PROSUL network for fellowships to P.H. E.J.B., and L.M.L. thanks
CNPq (BR) for fellowships. We thank INCT-INOFAR (CNPq CNPq
573.564/2008-6 and FAPERJ E-26/170.020/2008) and RID-
IMEDCHAG-CYTED for financial support.
The cytotoxicity against VERO cells (normal African green
monkey kidney epithelial cells) at a maximum concentration of
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