Indazole N-oxide derivatives as antiprotozoal agents: Synthesis, biological evaluation and mechanism of action studies

Article abstract of DOI:10.1016/j.bmc.2006.01.007

A series of indazole N-oxide derivatives have been synthesized and their antichagasic and leishmanocidal properties studied. 3-Cyano-2-(4-iodophenyl)-2H-indazole N¹-oxide exhibited interesting antichagasic activity on the two parasitic strains

Full text of DOI:10.1016/j.bmc.2006.01.007

Bioorganic & Medicinal Chemistry 14 (2006) 3467–3480
Indazole N-oxide derivatives as antiprotozoal agents:
Synthesis, biological evaluation and mechanism of action studies
a
a
a
a,
*
´
Alejandra Gerpe, Gabriela Aguirre, Lucıa Boiani, Hugo Cerecetto,
a,
b
b
c
*
´
Mercedes Gonzalez, Claudio Olea-Azar, Carolina Rigol, Juan D. Maya,
c
d
e
f
´
Antonio Morello, Oscar E. Piro, Vicente J. Aran, Amaia Azqueta,
Adela Lopez de Cerain, Antonio Monge, Marıa Antonieta Rojas and Gloria Yaluff
f
f
g
g
´
´
´
aDepartamento de Quı´mica Orga´nica, Facultad de Quı´mica-Facultad de Ciencias, Universidad de la Repu´blica,
Igua´ 4225, Montevideo 11400, Uruguay
bDepartamento de Quı´mica Inorga´nica y Analı´tica, Facultad de Ciencias Quı´micas y Farmace´uticas,
Universidad de Chile, Olivos 1007, 233 Santiago, Chile
cDepartamento de Farmacologı´a Clı´nica Molecular, Facultad de Medicina, Universidad de Chile, PO Box 70000, 7 Santiago, Chile
dDepartamento de Fı´sica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata-Instituto IFLP(CONICET),
C.C. 67, 1900 La Plata, Argentina
eInstituto de Quı´mica Me´dica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
fCentro de Investigacio´n en Farmacobiologı´a Aplicada, Universidad de Navarra, Irunlarrea s/n,
31080 Pamplona, Navarra, Espan˜a, Spain
gInstituto de Investigaciones en Ciencias de la Salud, Dpto. de Medicina Tropical, Universidad Nacional de Asuncio´n,
Rı´o de la Plata y Lagerenza, Asuncio´n, Paraguay
Received 5 December 2005; revised 30 December 2005; accepted 3 January 2006
Available online 17 February 2006
Abstract—A series of indazole N-oxide derivatives have been synthesized and their antichagasic and leishmanocidal properties stud-
ied. 3-Cyano-2-(4-iodophenyl)-2H-indazole N1-oxide exhibited interesting antichagasic activity on the two parasitic strains and the
two parasitic stages evaluated. Furthermore, besides its trypanocidal activity, 3-cyano-2-(4-nitrophenyl)-2H-indazole N1-oxide
showed leishmanocidal activity in the three parasitic strains evaluated. To gain insight into the mechanism of action, electrochemical
behaviour, ESR experiment, inhibition of parasitic respiration and QSAR were performed.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
currently discontinued) and Benznidazole (Bnz), being
these chemotherapeutic agents still inadequate due to
their undesired side effects.3
Parasitic diseases are the foremost worldwide health
problem today, particularly in the underdeveloped
countries. In South America, Chagas’ disease occupies
the third place in number of deaths per year, after
malaria and schistosomiasis.1–4 Trypanosoma cruzi (T.
cruzi) the aetiological agent of this illness, possesses a
complex cycle of life that does not permit to obtain an
efficient drug. Only two drugs are commercially avail-
able for the treatment of this disease, Nifurtimox (Nfx,
On the other hand, the leishmaniases are a series of diseas-
es caused by Leishmania species. There are approximately
1.5 million cases of leishmaniases each year from Central
and South America, West Asia and Europe.5 The drugs,
which have been most frequently used to treat the leish-
maniases, are the pentavalent antimonials, Pentostam
and Glucantime.6 However, these drugs are quite toxic
and in some areas resistance can be as high as 40%.7 Cur-
rently, WHO/TDR is developing a research programme
with Miltefosine, a very promising leishmanocidal drug,
but new therapeutic alternatives should be found in order
to increase the pharmaceutical arsenal.
Keywords: Antichagasic activity; Leishmanocidal activity; Indazole
N-oxide.
*
Corresponding authors. Tel.: +598 2 5258618x216; fax: +598 2 525 07
49; e-mail addresses: hcerecet@fq.edu.uy; megonzal@fq.edu.uy
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.01.007

3468
A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
OR1
N
and Leishmania braziliensis). Also, we present the elec-
R4
O2N
N
N
trochemical and ESR studies that allowed to explain
their biological behaviour. Moreover, we study the mod-
ification in the uptake of oxygen promoted by indazole.
Finally, we developed a QSAR study.
O
N
O
Z
NR2R3
2. Methods and results
2.1. Synthesis
R5
R7
N
The indazole N1-oxide derivatives were prepared follow-
ing the pathways showed in Scheme 1. The synthetic
route described for the preparation of this system in-
volves the formation of the Schiff base between 2-nitro-
benzaldehyde and the corresponding amines. Treatment
with sodium cyanide converts the Schiff base to its a-
aminonitrile derivatives, which in turn undergo basic
cyclization.11 The cyclization occurs under extremely
mild condition and indazole N1-oxide yield depends on
the reaction solvent, being acetic acid the best condi-
tion.12 We applied successfully this methodology to
derivatives 1–8, using Et3N as base and to derivative 9
using NaHCO3. For derivatives 10–13, acidic conditions
yielded the indazole N1-oxide spontaneously. When we
used 4-aminomorpholine as nucleophile, the corre-
sponding a-aminonitrile derivative was not generated
because the hydrazone moiety was not susceptible to
the cyanide nucleophilic attack, yielding 4-(2-nitroben-
zylideneamino)morpholine (14a. Scheme 2). Attempts
to reduce this hydrazone moiety were performed in dif-
ferent conditions. In mild conditions, namely NaBH4,
the system did not react. However, when we used a
strong reductant, LiAlH4, a very complex mixture of
reaction products was obtained. As a marginal product
N
N
O
R8
N
R6
O
Chart 1. Design concept of indazole N-oxide as antiprotozoal agents.
Recently, we have studied in vitro antichagasic and anti-
trichomona activity of 5-nitroindazoles, showing
remarkable antiprotozoal properties in both parasites.8
We found that in this family of compounds the nitro
moiety did not play a crucial role in the trypanocidal
activity.8b On the other hand, we have found that cer-
tain N-oxide containing heterocycles displayed excellent
trypanocidal activity.9 The well-known N-oxide bio-re-
duction process and its biological consequences10 were
found as crucial steps in the trypanocidal activity dis-
played by this group of compounds. This turns the N-
oxide moiety as an excellent pharmacophore. These
facts prompted us to propose a new potential antiproto-
zoal entity, which combines indazole system and N-ox-
ide moiety (Chart 1). Biological behaviour of these
heterocyclic systems has not been reported yet. There-
fore, we studied the indazole N-oxide derivatives as
new antiprotozoal agents.
1
the indazole N1-oxide 14 was identified (by H NMR
spectroscopy). No further attempts were performed to
improve the preparation of 14. Indazole N2-oxide, deriv-
ative 15, was synthesized following the procedure de-
scribed by Zenchoff et al. (Scheme 2).13
Herein, we report the synthesis of indazole N1- and N2-
oxide derivatives and their antiprotozoa activity (against
T. cruzi, Leishmania amazonensis, Leishmania infantum
1
2
3
4
5
6
7
8
R= Ph
CN
N
R= p-OHPh
R= p-CH3Ph
R= p-ClPh
R= p-IPh
R
N
R= p-NO2Ph
R= m-NO2Ph
R= CH2furyl
O
ii
CN
CN
CHO
iii
i
NR
NO2
NHR
N
(CH2)3CH3
9
N
NO2
NO2
O
1a-13a R= Ph
p-OHPh
p-CH3Ph
p-ClPh
CN
N
10 R= CH2CH2Cl
11 R= CH2CH2Ph
12 R= 2-naphthyl
13 R= 1-naphthyl
p-IPh
R
p-NO2Ph
m-NO2Ph
CH2furyl
(CH2)3CH3
(CH2)2Cl
(CH2)2Ph
2-naphthyl
1-naphthyl
N
O
Scheme 1. Reagents and conditions: (i) H2NR, KCN, CH3CO2H, rt. (ii) Et3N, rt. (iii) NaHCO3, rt.

A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
3469
O
N
ii
N
H
O
CHO
NO2
NO2
N
N
i
iii
NO2
14
N
N
O
14a
N
O
Complex mixture of reaction
Cl
Cl
Cl
15
O
OH
NHCH3
iv
v
N
O
N
NHCH3
CH3
Scheme 2. Reagents and conditions: (i) 4-aminomorpholine, KCN, CH3CO2H, rt. (ii) NaBH4, MeOH, reflux. (iii) LiAlH4, THF, rt. (iv) NaBH4,
MeOH, rt. (v) 1—NaNO2, HCl (concd); 2—H2SO4 (concd), rt.
In order to investigate the relevance of the N-oxide
moiety in the biological behaviour, we synthesized
three deoxygenated derivatives (16–18) (Scheme 3).14
These derivatives were obtained with moderate yield
treating the corresponding indazole N1-oxide with
PPh3.
2.2. Biological characterization
2.2.1. Antitrypanosomal activities. All the compounds
were tested in vitro against two parasitic strains (Brener
and Tulahuen 2) and two parasitic stages (epimastigote
and trypomastigote forms) of T. cruzi at different con-
centrations. Table 1 shows the percentage of growth
All new compounds were characterized by NMR (1H-,
13C-, HMQC and HMBC), MS and IR analysis and
their purity established by TLC and microanalysis. Sin-
gle crystals of derivative 13 adequate for structural
X-ray diffraction studies15 were obtained by slow evap-
oration from an EtOH solution. Figure 1 shows the
ORTEP molecular drawing of derivative 13.
Table 1. In vitro antichagasic activity of Indazole N-oxide derivatives
Compound
Epimastigote growth
inhibition (% GI)a,b
Trypomastigote
reductionb,c
Brener
strain
Tulahuen
strain
Brener strain
1
2
13
5
25
31
1
16
67
3
5
53
NDd (53)e
0 (0)
61
4
39
46
CN
N
CN
N
5
51
70 (16.7)f
52
6
79 (16.2)f
0
R
R
7
47
12
ND (39)e
34
N
N
8
49
13
O
16 R=p-OHPh
17 R=p-IPh
18 R=p-NO2Ph
9
0
ND (59)e
ND
10
11
12
13
15
16
17
18
Nfx
GVg
0
31
0
23
7
26
0 (12)e
ND
6 (40)e
ND (59)e
ND (29)e
—
Scheme 3. Reagents and condition: PPh3, EtOH, reflux.
16
52
25
44
0
21
33
4
3
12
99 (8.5)f
17
34
100 (7.7)f
—
—
100
a Values expressed as the percentage of inhibition of control growth
(% GI). Compounds tested at 25 lM concentration.
b Results are means of three different experiments with SD less than
10% in all cases.
c Values correspond to the percentage of reduction of control try-
pomastigotes. Compounds tested at 100 lg/mL.
d Not determined.
e Values in parentheses correspond to the inhibition using 250 lg/mL
of each compound.
f Values in parentheses correspond to the IC50 (lM).
g GV, gentian violet.
Figure 1. Molecular plot of derivative 13 showing the labelling of the
non-H atoms and their displacement ellipsoids at the 30% probability
level.

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A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
Table 3. Cytotoxicity to MCF-7, TK-10 and HT-29 cell lines at
100 lM
inhibition (% GI) of the indazole derivatives at 25 lM
for the epimastigote form and at 100 or 250 lg/mL for
the bloodstream form, trypomastigote. From concentra-
tion–activity curves the most active derivative against
both epimastigote strains was 6 with IC50 values in the
same order as that of the reference drug (Nfx).
Compound
% SPa
MCF-7
TK-10
HT-29
1
3
4
5
86
83
79
87
88
89
84
83
95
100
86
Compounds 4, 5, 7 and 13 showed medium activity at
the assayed concentrations against the two studied
strains, while compound 8 showed moderated activities
against both forms, epimastigote of Tulahuen and tryp-
omastigote of Brener. Derivatives 2 and 5 were the most
active derivatives against trypomastigote form. Deriva-
tive 5 also showed remarkable antiprotozoal properties
against both epimastigote and trypomastigote forms.
In general, the antiepimastigote activities of the studied
derivatives were slightly higher for Tulahuen than for
Brener strain, results which are similar to those previ-
ously reported for nitroimidazoles.16 In general, the
anti-T. cruzi activities (on epimastigote and trypomasti-
gote forms) of the deoxygenated derivatives 16–18 de-
creased compared with those of their N-oxide
analogues, indicating that the presence of the N-oxide
group is important for the antichagasic activity.
100
a % SP, cell survival percentage.
showed antineoplastic activity since the survival of the
three cell lines was very high.
2.3. Study of anti-T. cruzi mechanism of action
In order to confirm an oxidative stress-mediated mecha-
nism of action, we performed some experiments related
to this fact. We studied the N-oxides’ electrochemical
behaviour, the production of free radicals induced by
the studied compounds into the parasite by ESR exper-
iments and the compounds effect on the parasitic oxygen
consumption.
2.3.1. Electrochemical behaviour. We performed voltam-
metric studies in DMSO solutions, analysing the first
reduction peak for the N-oxide derivatives. Table 4 lists
the values of the first voltammetric cathodic peaks for
all compounds and Nfx.17 The N-oxide reduction oc-
curred between À1.3 and À1.6 V with the absence of
anodic peak, thus indicating an electron irreversible
transfer process. In the case of derivatives 6 and 7 which
possess in their structures a nitro group, two extra
reduction peaks appeared. The first, near À0.9 V, corre-
sponds to one-electron reversible transfer process that is
attributed to the generation of the nitro anion radical.
The second peak corresponds to the reduction of the ni-
tro anion radical to the hydroxylamine by a three-elec-
tron process.18 The cathodic peaks of derivatives 1–13
corresponding to N-oxide reduction are irreversible in
2.2.2. Leishmanocidal activities. According to their
behaviour against epimastigote and trypomastigote
forms of T. cruzi, we selected three different indazole
N-oxides for evaluation as leishmanocidal agents, the
two most actives against bloodstream form (2 and 5)
and the most active against epimastigote form (6).
Derivatives 2, 5 and 6 were tested in vitro against pro-
mastigote forms of L. amazonensis, L. infantum and L.
braziliensis strains at 100 lg/mL (Table 2). Derivative
6 was the most active compound against these strains,
showing an immobility or decrease of the growth of par-
asites that corresponds to 70–90% of parasite lysis.
2.2.3. Cytotoxic activity. According to their behaviour
against the bloodstream form, we selected four different
indazole N-oxides, the most active (5), one mid-active
(1), and two inactive derivatives (3 and 4), for evaluation
as mammal cytotoxic agents. Then, these compounds
were tested against three tumour cell lines, human colon
adenocarcinoma (HT-29), human mammary adenocar-
cinoma (MCF-7) and human kidney carcinoma (TK-
10) at 100 lM. Cell survival percentages (% SP) are
gathered in Table 3. None of the tested compounds
the whole range of sweep rates used (0–2000 mVsÀ1
)
and involved one electron.19 We attributed this feature
Table 4. Cyclic voltammetric parameters in DMSO versus saturated
calomel electrode (sweep rate 2 VsÀ1
)
a
Epc (V)
Compound
1
À1.33
À1.36
À1.36
À1.28
À1.26
À0.92
À0.98
À1.47
À1.61
À1.55
À1.27
À1.35
À0.91b
2
3
Table 2. Leishmanocidal activity
4
Compound
L. amazonensisa L. infantuma L. braziliensisa
5
6
2
5
6
+
+
+
7
+
++
+
+
8
++
+++
0
++
+++
0
9
Pentamidine +++
DMSO
11
12
13
Nfx
0
+++, total lysis of parasites (100% of lysis); ++, immobility or decrease
of the growth of parasites (between 70% and 90% of lysis of parasites);
+, similar growth of parasites to the control but with little mobility; 0,
lysis of parasites is not observed. Initial inocula, 2 · 106 parasites/mL.
a At 100 lg/mL.
a Epc, potential of the first cathodic peak.
b Ref. 17.

A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
3471
800
a
CN
N R
N
O
CN
.
OH
# (*)
# (*)
# (*) # (*)
#
#
+
-
600
400
200
0
+
N R
+
H
+
1e
N
-12,0
b
a
-10,0
-8,0
-6,0
-4,0
-2,0
0,0
b
c
-200
-400
-600
-800
3440
3460
3480
3500
3520
Figure 3. ESR spectrum obtained with T. cruzi extracts incubated with
derivative 5. The ESR signals were observed 10 min after incubation at
37 ꢁC with T. cruzi extract (4 mg protein/mL), NADPH (1 mM),
EDTA (1 mM), in phosphate buffer (20 mM), pH 7.4, DMPO
(100 mM) and 5 (2 mM in acetonitrile). Spectrometer conditions:
microwave frequency 9.68 GHz microwave power 20 mW, modulation
amplitude 0.4 G, scan rate 0.83 G/s, time constant 0.25 s and number
of scan: 10.
scan
-1,8
-1,0
-1,2
-1,4
-1,6
-2,0
E/V vs. saturated calomel
Figure 2. (a) Reduction mechanism proposed to N-oxide derivatives.
(b) Cyclic voltammograms in DMSO: (a) derivative 1, (b) GSH:
derivative 1 (1:1) and (c) GSH:derivative 1 (2:1).
in vivo, we incubated derivative 5 with T. cruzi homog-
enates in the presence of NADPH, EDTA and the spin
trapping DMPO.22 A well-resolved ESR spectrum ap-
peared when DMPO was added to the T. cruzi–5 system.
In this ESR spectrum, it was possible to observe the cor-
responding trapped hydroxyl radical (DMPO-OH spin
adduct aN = a = 15.09 G, Figure 3 marked ( )).22b Fur-
thermore, other six lines appeared that were consistent
with the trapped N-oxide radical according to the hyper-
fine pattern and the hyperfine constants (aN = 15.84 G,
aH = 25.90 G, Figure 3 marked #).22a These results are
in agreement with the electrochemical behaviour of
indazole N-oxide derivatives, when the system is
mono-electronated the hydroxyl radical is released.
to the production of the deoxygenated derivative with
hydroxyl radical release (Fig. 2a).
The electrochemical properties of these compounds in
the presence of a biologically relevant thiol, glutathione
(GSH), have been studied. According to a previous re-
port, this thiol could biologically act as a radical scav-
enger, an oxidizing or a reducing agent, or it is able to
display all these three functions depending on the con-
ditions.20,21 We studied the reactivity of GSH with the
radical of indazole N1-oxide by cyclic voltammetry,
adding increasing amounts of aqueous GSH solution
(0.1 M in phosphate buffer, pH 7.4) to the medium.
Figure 2b shows the typical cyclic voltammograms of
derivatives in DMSO solution in the absence and in
the presence of increasing amounts of GSH. Curve
(a) illustrates the cyclic voltammogram that involves
the reduction process of derivative 1. Afterwards, when
GSH was added to the medium, it produces a signifi-
cant increase in the first cathodic current with a con-
comitant displacement to low potential and
decreasing of the second cathodic peak (curve (b)).
After the addition of GSH in ratio GSH: derivative
(2:1), only the first cathodic peak appeared (curve
(c)). The GSH signals, at the studied concentrations,
did not interfere with the signals of the corresponding
N-oxide.
*
H
2.3.3. Effect on the parasitic respiration. Derivatives 1, 3,
5 and 6 were selected to be tested for their capacity to
inhibit parasitic respiration. These derivatives were
chosen taking into account their anti-T. cruzi activi-
ties, 1 and 3 being inactive compounds and 5 and 6
active. These experiments afforded to investigate the
products’ ability to produce oxidative stress. Respira-
tion measurements were carried out polarographically
treating T. cruzi (epimastigote form, Tulahuen 2
strain) with different concentrations of the studied
compounds (Table 5).17c These derivatives inhibited
oxygen uptake in a concentration-dependent manner
(for derivative 6 it was not possible to perform con-
centration–response studies because 6 precipitates in
the experimental conditions). Only derivative 5 inhibit-
ed respiration close to 30% at IC50 equivalent concen-
tration (see Section 5).
These electrochemical behaviours suggest that a new
electro-active entity was formed by reaction of the inda-
zole N-oxide and GSH. This could show that the radical
of the N-oxide species chemically reacts with GSH.
The studied compounds were unable to produce redox
cycling. These results confirm that the hydroxyl radical
observed in the ESR experiments could be the result
of OHÅ-releasing capacity of compounds after a bio-re-
duction process.
2.3.2. ESR spectroscopic studies. In order to analyse the
capacity of these compounds to generate free radicals

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A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
Table 5. Effect of indazole N-oxide derivatives on respiration of
T. cruzi
Table 6. Crystallographic and PM3/6.31G* optimized structure of
compound 13
a
Compound
IC50 (lM)
Inhibition of respirationb
Crystallographic structure
PM3/6.31G* optimised structure
1
3
5
6
>100
>100
25.1
0.0 5.5
16.7 4.8
27.2 4.8
5.5 4.9
C4
C3a
C7a
C5
C6
C3
N2
16.7
a IC50 values for Tulahuen strain.
N1
O
b Values are expressed as the percentage with respect to control and
correspond to means standard deviation of three independent
experiments. Compounds added at a final concentration equal to
IC50 equivalent concentrations (see Section 5). Control respiration
was 31.5 nanoatoms of oxygen per minute and per milligram of
protein.
C7
13
Crystallographic PM3/6.31G* Relative
Distances
and
anglesa,b
data
optimized
geometry
error
|(dPM3/6.31G*
À
dcrist)/dcrist
|
2.4. Structure–activity relationships
O–N1
N1–N2
N2–C3
1.270
1.257
1.380
1.389
0.01
0.02
0.02
0.25
0.01
0.01
0.03
0.00
0.04
0.02
0.02
0.02
0.00
0.01
0.01
0.01
0.00
0.02
1.408
1.368
1.467
1.437
1.409
1.418
1.378
1.466
1.398
120.95
108.25
107.54
Molecular modelling studies were performed on the
developed indazole N1-oxide derivatives by calculating
the stereoelectronic properties in order to understand
the mechanism of action. These properties were deter-
mined using DFT/B3LYP calculations.23,24 A detailed
conformational search for each of the molecules was
performed, using MM methods, to find the minimum
energy and highest abundance conformer. The geometry
of this conformer was fully optimized by applying PM3/
6-31G* in the gas phase that allows us to obtain acute
results with low time of computational calculi. Then,
single point B3LYP/6-31G* density functional calcula-
tion was performed. The crystallographic molecular
structure of derivative 13 was used as template in order
to assess the validity of the theoretical level used. As
shown in Table 6, PM3/6.31G* geometry describes ade-
quately bond distances and angles and also dihedral an-
gles. The properties determined and examined in this
study were total energy, solvation (water) energy, mag-
nitude of dipolar moment, volume, HOMO’s and LU-
MO’s energies, gap (E_LUMO–E_HOMO) and the
logarithm of the partition coefficient of the non-ionized
molecules (logP). Theoretical logP (clogP) was calcu-
lated using the Villar method, implemented in PC SPAR-
TAN 04 package24 at the PM3 semiempirical level.
C3–C3a
C3a–C4
C3a–C7a
C4–C5
1.404
1.417
1.420
1.377
C6–C7
1.376
1.408
1.370
C7–C7a
C7a–N1
O–N1–N2
N1–N2–C3
N2–C3–C3a
122.92
110.03
107.03
106.52
118.06
121.35
123.26
109.11
C3–C3a–C7a 107.38
C3a–C4–C5
C5–C6–C7
119.63
119.73
C7–C7a–C3a 123.35
N1–C7a–C3a 106.69
a
˚
Distances in A and angles in ꢁ.
b Numbers according to figure.
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
-0,2
In the equations and models, n represents the number of
data points, r2 is the correlation coefficient, s is the stan-
dard deviation of the regression equation, the F value is
related to the F-statistic analysis (Fischer test) and r2adj
defines the cross-validated correlation coefficient. Activ-
ity used in the structure–activity relationship studies was
the inhibitory effect (compounds 1–13) on the growth of
T. cruzi (Brener and Tulahuen strains) expressed as the
percentage of growth inhibition at day 5 at 25 lM con-
centration, % GI. We used log10 (GI) values as the
dependent variables in the linearization procedure.
First, one-variable and multivariable regressions be-
tween both activities and the physicochemical properties
(calculated descriptors and redox potential) were stud-
ied. Non-statistical significant correlations were ob-
tained when Epc was included as independent variable,
but it was observed a clear tendency between activities
and redox potential, namely the most active compounds
possess the least negative Epc (Fig. 4). This could indi-
log10GITULAHUEN
log10GIBRENER
-1,7
-1,6
-1,5
-1,4
-1,3
-1,2
-1,1
-1,0
-0,9
Epc (V)
Figure 4. Potential of the first cathodic peak (Epc) versus activities.
cate that other the properties participate in the displayed
activities.
Only structure–activity models having a value of r2adj
above 0.5 were considered. In the case of trypanocidal
activity against Brener strain, the best equation was
obtained when we analysed the correlation between
activity and the independent clogP and the magnitude

A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
3473
of dipolar moment, l (Eq. 1, Table 7). Dipolar mo-
ments of derivatives are orientated with similar direc-
tions (Fig. 5a). For the same strain another statistical
accepted correlation was obtained between the activi-
ties and clogP and E_HOMO (Eq. 2). E_HOMO
and l magnitude are not orthogonal,25 indicating that
equations 1 and 2 present the same kind of results.
Figure 5b shows the distribution of HOMO. In the
case of trypanocidal activity against Tulahuen strain,
the correlations were statistically less indicative than
in the case of the Brener strain. However, like in the
Brener strain the two best results were obtained with
l (Eq. 3, Table 7) and E_HOMO (Eq. 4) but in these
cases with volume instead of clogP. clogP and volume
are not orthogonal,25 indicating that equations 3 and 4
present the same kind of results. Figure 6 shows the
plot of experimental activity versus calculated ones
for Eq. 1–4. Besides, the correlation matrix for the
used physicochemical descriptors was performed and
cross-correlations between the descriptors used in each
equation were not obtained.25 These parameters are
therefore orthogonal, a fact that affords their use in
the multilinear regression procedure.26
zole N-oxide derivatives has been reported for the first
time. Some of them showed good anti-T. cruzi activity
with low cytotoxicity. In particular, three of the studied
derivatives (4–6) showed interesting properties. Com-
pound 6 exhibited remarkable antiepimastigote activity,
but did not show antitrypomastigote activity. Com-
pounds 4 and 5 displayed antiepimastigote activity,
and derivative 5 maintained trypanocidal activity in
bloodstream form (trypomastigote). As it has been pre-
viously observed,9a,9d,9e,27 the absence of the N-oxide
moiety produces the drop of antiepimastigote activity,
confirming that this group plays a key role in the mech-
anism of indazole N-oxides’ antitrypanosomal activity
(compare the activity of derivative 6 with the activity
of deoxy-derivative 18, Table 1). Derivative 6 displayed
good activity against the three studied Leishmania
strains, producing between 70% and 90% of parasite ly-
sis. The antiprotozoal activity is not due to cytotoxicity
since at the assayed concentration the compounds did
not show a cytotoxic activity against mammary cells
(Table 3).
Some important pieces of information were achieved
from the experiments performed in order to clarify the
mechanism of anti-T. cruzi activity. From the electro-
chemical studies it is possible to conclude that the N-ox-
ide reduction is an irreversible process, indicating that
the production of OH radical (Fig. 2a) could take place
in the biological medium. However, a statistical correla-
tion between activity and values of Epc was not obtained
3. Discussion
New indazole N1-oxide derivatives were synthesized by a
simple methodology and with good yields. Antiproto-
zoal activity, anti-T. cruzi and antileishmania, of inda-
Table 7. Summary of QSAR results
Equation
Statistic parameters
Expressions
r2adj
r2
s
p
F value
n
1
2
3
0.780
0.734
0.573
0.820
0.782
0.679
0.294
0.323
0.356
0.0004
0.0010
0.0130
20.49
16.18
6.36
12
12
13
log10 (GIBRENER) = À3.63( 0.84) + 0.33( 0.06) l 0.73( 0.14) clogP
log10 (GIBRENER) = À19( 4) À3.04( 0.67) EHOMO + 0.71( 0.15) clogP
log10 (GITULAHUEN) = À19( 10) + 0.14( 0.08) l 0.15( 0.08) vol
À 0.0003( 0.0001) vol2
4
0.533
0.650
0.372
0.0190
5.56
13
log10 (GITULAHUEN) = À27( 10) À1.09( 0.76) EHOMO + 0.17( 0.08) vol
À 0.0003( 0.0001) vol2
a
b
9
6
2
5
Figure 5. (a) Two different views of the dipoles of the studied indazole N1-oxide derivatives. (b) Electron density (solid white, isovalue = 0.02) and
HOMO (solid, isovalue = 0.032) isosurfaces for active (down) and inactive (up) derivatives.

3474
A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
2,4
2,2
2,0
1,8
1,6
1,4
1,2
1,0
2,0
1,5
1,0
0,5
0,0
r = 0.9055 s = 0.253 n = 12 p < 0.0001
r = 0.8846 s = 0.272 n = 12 p = 0.0001
0,8
0,6
0,0
0,5
1,0
1,5
2,0
0,0
0,5
1,0
1,5
2,0
Experimental activity
Experimental activity
2,6
2,4
2,2
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
2,5
2,0
1,5
1,0
0,5
r = 0.8045 s = 0.291 n = 13 p = 0.0009
r = 0.8239 s = 0.2808 n = 13 p = 0.0005
0,0
0,5
1,0
1,5
2,0
0,0
0,5
1,0
1,5
2,0
Experimental activity
Experimental activity
Figure 6. Plot of log10 of percentage of trypanosome growth inhibition, Brener (up) and Tulahuen (down) strains, at 25 lM (log10% GI)
experimental versus calculated values from Eqs. 1 to 4.
but only a soft tendency was observed (Fig. 4). On the
other hand, the electrochemical study with GSH showed
that the compounds could react with an electroactive
compound trapping it. The presence of the new entity
was supported by the increase of the cathodic peak cur-
rent corresponding to the anion radical generated by the
GSH addition. The ESR experiment was in agreement
with this fact demonstrating that one of the most active
derivatives (5, Fig. 3) is able to generate free radicals
within the parasitic cells, where OHÅ was detected. In
the studies of the derivatives’ effect on the oxygen con-
sumption of T. cruzi, we observed that compounds pres-
ent a little oxygen uptake inhibition in a concentration-
dependent manner without oxygen recycling.
ies, the trypanocidal activity of indazole N-oxides also
depends on lipophilicity and molecular volume.
4. Conclusions
The results presented above indicate that indazole N-ox-
ide system constitutes a starting point for further chem-
ical modifications in order to improve the antiprotozoal,
T. cruzi and Leishmania, activity. The results provide
supporting evidence to stimulate further in vivo studies
of these compounds in appropriate animal models of
Chagas’ disease.
QSAR studies demonstrated the relevance of the dipolar
moment module, or E_HOMO, and lipophilic proper-
ties for the trypanocidal activity on Brener strain and
dipolar moment module, or E_HOMO, and volume
for the trypanocidal activity on Tulahuen strain. The
relationships between activity and dipolar moment mod-
ule could indicate the electronic requirements for the
derivatives to have optimal interactions with a target
biomolecule. Observing the directions and ways of dipo-
lar moments (Fig. 5a) the system N-oxide and CN deter-
mined these orientations. In this sense, it was possible to
note that the most active compounds against both
strains, derivatives 5 or 6 (Fig. 5b), showed the highest
HOMO’s contribution on indazole oxygen, nitrogen
and carbon 3-, 4- and 6-. These results indicate that
the compounds could act as nucleophiles through these
atoms reacting with electrophile biomolecules and pro-
ducing damages into parasite cell.28 As it is well known,
drug lipophilicity and volume play significant roles in
biological responses such as the transport of the drugs
through the cell membrane.29 According to QSAR stud-
5. Experimental
5.1. Chemistry
Compound 15 was prepared according to literature pro-
cedure.13 All starting materials were commercially avail-
able research-grade chemicals and used without further
purification. All solvents were dried and distilled prior
to use. All the reactions were carried out in a nitrogen
atmosphere. Melting points were determined on a Leitz
Microscope Heating Stage Model 350 capillary appara-
1
tus and are uncorrected. H NMR (400 MHz) and 13C
NMR (100 MHz) spectra were recorded on a Bruker
DPX-400 spectrometer. The chemical shifts are reported
in ppm from TMS (d scale). J values are given in Hz.
The assignments have been performed by means of dif-
ferent standard homonuclear and heteronuclear correla-
tion experiments (HMQC and HMBC). Electron impact
(EI) mass spectra were obtained at 70 eV on a Shimadzu
GC–MS QP 1100 EX instrument. IR spectra were
recorded on a Perkin-Elmer 1310 spectrometer, using

A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
3475
potassium bromide tablets; the frequencies are expressed
in cmÀ1. DC-Alufolien silica gel 60 PF254 (Merck, layer
thickness 0.2 mm) and silica gel 60 (Merck, particle size
0.040–0.063 mm) were used for TLC and flash column
chromatography, respectively. Elemental analyses were
obtained from vacuum-dried samples (over phosphorus
pentoxide at 3–4 mm Hg, 24 h at room temperature) and
performed on a Fisons EA 1108 CHNS-O analyser.
1277, 1232, 1033, 821, 747. Anal. Calcd for
C14H9N3O2 (251.07): C, 66.93; H, 3.61; N, 16.72.
Found: C, 66.61; H, 4.00; N, 16.41.
5.1.1.3. 3-Cyano-2-(4-tolyl)-2H-indazole N1-oxide (3).
2-(2-Nitrophenyl)-2-(4-tolylamino)acetonitrile (3a). Yel-
low solid 0.75 g (60%). H NMR (400 MHz, DMSO-
1
d6) d ppm: 2.19 (3H, s), 6.38 (1H, d, J = 10.0 Hz), 6.59
(1H, d, J = 10.0 Hz), 6.71 (2H, d, J = 8.2 Hz), 7.00
(2H, d, J = 8.2 Hz), 7.72 (1H, m), 7.86 (1H, m), 7.93
(1H, d, J = 7.3 Hz), 8.09 (1H, d, J = 7.8 Hz).
5.1.1. Synthesis of indazole N1-oxide derivatives (1–8 and
10–13). General method A (1–8). A mixture of o-nitro-
benzaldehyde (0.50 g, 3.3 mmol), KCN (0.40 g,
6.6 mmol) and the corresponding amine (3.3 mmol) in
glacial acetic acid (30 mL) was stirred at room tempera-
ture for 24 h. After addition of 20 mL water, the precip-
itated product was collected by filtration and air-dried.
The solid (a-aminonitrile derivative) was treated with
Et3N (10 mL) and was stirred at room temperature for
24–48 h. After addition of 20 mL water the precipitated
product was collected by filtration and air-dried, yielding
the corresponding indazole N1-oxide. The product was
purified by crystallization from the indicated solvent.
3-Cyano-2-(4-tolyl)-2H-indazole N1-oxide (3). Yellow
solid 0.25 g (74%) (method A); mp 203.0–205.0 ꢁC (eth-
anol). 1H NMR (400 MHz, DMSO-d6) d ppm: 2.46 (3H,
s), 7.52 (4H, m), 7.80 (2H, d, J = 8.2 Hz), 7.84 (1H, d,
J = 6.6 Hz), 7.86 (1H, d, J = 8.4 Hz). 13C NMR
(100 MHz, DMSO-d6) d ppm: 21.82, 92.42, 111.80,
115.03, 119.77, 122.92, 128.39, 128.56, 129.07, 129.52,
129.59, 130.92, 142.61. MS (EI, 70 eV) m/z (%): 249
(M+, 100); 233 (M+-16, 22); 219 (M+-30, 22). IR (KBr,
cmÀ1): 2201, 1516, 1488, 1438, 1348, 1242, 1035, 809,
745. Anal. Calcd for C15H11N3O (249.09): C, 72.28; H,
4.45; N, 16.86. Found: C, 72.08; H, 4.62; N, 16.80.
General method B (10–13). A mixture of o-nitrobenzalde-
hyde (0.50 g, 3.3 mmol), KCN (0.40 g, 6.6 mmol) and the
corresponding amine (3.3 mmol) in glacial acetic acid
(30 mL) was stirred at room temperature for 36–48 h.
After addition of 20 mL water, the precipitated product
was collected by filtration and air-dried, yielding the cor-
responding indazole N1-oxide. The product was purified
by crystallization from the indicated solvent.
5.1.1.4. 2-(4-Chlorophenyl)-3-cyano-2H-indazole N1-
oxide (4). 2-(4-Chlorophenylamino)-2-(2-nitrophenyl)ace-
tonitrile (4a). Brown solid 0.20 g (21%). 1H NMR
(400 MHz, DMSO-d6) d ppm: 6.45 (1H, d, J = 9.5 Hz),
6.82 (2H, d, J = 8.6 Hz), 6.99 (1H, d, J = 9.5 Hz), 7.23
(2H, d, J = 8.6 Hz), 7.67 (3H, m), 8.11 (1H, d,
J = 8.0 Hz).
5.1.1.1. 3-Cyano-2-phenyl-2H-indazole N1-oxide (1).
2-(2-Nitrophenyl)-2-phenylaminoacetonitrile (1a). Yel-
low solid 0.51 g (61%). H NMR (400 MHz, DMSO-
2-(4-Chlorophenyl)-3-cyano-2H- indazole N1-oxide (4).
Brown solid 0.29 g (62%) (method A); mp 202.5–
1
1
d6) d ppm: 6.44 (1H, d, J = 9.8 Hz), 6.78 (4H, m), 7.19
(2H, t, J = 8.1 Hz), 7.74 (1H, m), 7.90 (2H, m), 8.1
(1H, d, J = 8.1 Hz).
203.2 ꢁC (ethanol). H NMR (400 MHz, DMSO-d6) d
ppm: 7.52 (2H, m), 7.84 (6H, m). 13C NMR
(100 MHz, DMSO-d6) d ppm: 92.57, 111.70, 115.05,
119.82, 123.08, 128.59, 129.11, 129.75, 130.63, 130.76,
130.82, 137.27. MS (EI, 70 eV) m/z (%): 269 (M+, 46);
253 (M+-16, 7); 204 (29); 111 (57); 75 (100). IR (KBr,
cmÀ1): 2211, 1504, 1440, 1351, 1240, 1087, 1014, 821,
748. Anal. Calcd for C14H8ClN3O (269.04): C, 62.35;
H, 2.99; N, 15.58. Found: C, 62.15; H, 3.01; N, 15.44.
3-Cyano-2-phenyl-2H-indazole N1-oxide (1). Yellow sol-
id 0.29 g (62%) (method A); mp 193.6–194.3 ꢁC (etha-
nol). 1H NMR (400 MHz, DMSO-d6) d ppm: 7.50
(2H, m), 7.73 (3H, m), 7.85 (4H, m). 13C NMR
(100 MHz, DMSO-d6) d ppm: 92.42, 111.77, 115.06,
119.80, 123.04, 128.47, 128.81, 129.11, 129.66, 130.48,
132.07, 132.41. MS (EI, 70 eV) m/z (%): 235 (M+, 75),
219 (M+-16, 31), 206 (M+-29, 26), 204 (8), 77 (100). IR
(KBr, cmÀ1): 2206, 1507, 1474, 1344, 1287, 1237, 1035,
865, 742. Anal. Calcd for C14H9N3O (235.07): C 71.48;
H 3.86; N 17.86. Found: C, 71.32; H, 3.95; N, 17.89.
5.1.1.5. 3-Cyano-2-(4-iodophenyl)-2H-indazole N1-ox-
ide (5). 2-(4-Iodophenylamino)-2-(2-nitrophenyl)acetoni-
trile (5a). Yellow solid 0.71 g (57%). 1H NMR
(400 MHz, DMSO-d6) d ppm: 6.44 (1H, d, J = 9.5 Hz),
6.66 (2H, d, J = 8.7 Hz), 7.01 (1H, d, J = 9.5 Hz), 7.49
(2H, d, J = 8.7 Hz), 7.74 (1H, m), 7.87 (2H, m), 8.11
(1H, d, J = 8.3 Hz).
5.1.1.2.
3-Cyano-2-(4-hydroxyphenyl)-2H-indazole
N1-oxide (2). White solid 0.25 g (54%) (method A); mp
230.0–231.9 ꢁC (methanol). 1H NMR (400 MHz,
DMSO-d6) d ppm: 7.03 (2H, d, J = 8.8 Hz), 7.48 (1H,
dd, J = 6.8, 8.4 Hz), 7.52 (1H, dd, J = 6.8, 8.3 Hz),
7.57 (2H, d, J = 8.8 Hz), 7.81 (1H, d, J = 8.4 Hz), 7.85
(1H, d, J = 8.3 Hz), 10.34 (1H, br s). 13C NMR
(100 MHz, DMSO-d6) d ppm: 92.00, 112.00, 115.00,
116.84, 119.72, 122.69, 123.00, 128.24, 128.92, 129.45,
130.38, 160.89. MS (EI, 70 eV) m/z (%): 251 (M+,
100); 235 (M+-16, 24); 220 (M+-31, 41); 93 (14). IR
(KBr, cmÀ1): 3500–3200, 2224, 1596, 1511, 1445, 1351,
3-Cyano-2-(4-iodophenyl)-2H-indazole N1-oxide (5).
Yellow solid 0.30 g (64%) (method A); mp 212.8–
213.3 ꢁC (ethanol). H NMR (400 MHz, DMSO-d6) d
ppm: 7.49 (1H, dd, J = 6.8, 8.6 Hz), 7.54 (1H, dd,
J = 6.8, 8.2 Hz), 7.62 (2H, d, J = 8.4 Hz), 7.83 (1H, d,
J = 8.6 Hz), 7.87 (1H, d, J = 8.2 Hz), 8.11 (2H, d,
J = 8.4 Hz). 13C NMR (100 MHz, DMSO-d6) d ppm:
92.00, 99.82, 111.71, 115.05, 119.82, 123.11, 128.58,
129.00, 129.74, 130.67, 131.69, 139.43. MS (EI, 70 eV)
m/z (%): 361 (M+, 100); 345 (M+-16, 7); 76 (9). IR
1

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A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
(KBr, cmÀ1): 2210, 2198, 1503, 1439, 1351, 1243, 1035,
1010, 813, 744. Anal. Calcd for C14H8IN3O (360.97):
C, 46.56; H, 2.23; N, 11.64. Found: C, 47.06; H, 2.57;
N, 11.52.
with water (20 mL) and aqueous solution of NaHCO3
(5%) until a basic pH was reached, and was stirred at
room temperature for 48 h, precipitating a brown solid
that was filtered, dried and purified by column chroma-
tography (SiO2, CH2Cl2). Brown solid 0.11 g (15%); mp
5.1.1.6. 3-Cyano-2-(4-nitrophenyl)-2H-indazole N1-ox-
ide (6). Yellow solid 0.30 g (63%) (method A); mp 223.1–
64.0–66.2 ꢁC. H NMR (400 MHz, DMSO-d6) d ppm:
1
0.91 (3 H, t, J = 7.3 Hz), 1.32 (2H, m), 1.84 (2H, m),
4.53 (2H, t, J = 7.0 Hz), 7.43 (1H, dd, J = 6.9, 7.8 Hz),
7.49 (1H, dd, J = 6.9, 7.5 Hz), 7.79 (1H, d, J = 7.8 Hz),
7.81 (1H, d, J = 7.5 Hz). 13C NMR (100 MHz,
DMSO-d6) d ppm: 14.13, 19.95, 30.39, 46.57, 91.30,
111.64, 114.69, 119.54, 122.34, 127.94, 129.19 (two car-
bons). MS (EI, 70 eV) m/z (%): 215 (M+, 23); 198
(M+-17, 26); 159 (100); 57 (14). IR (KBr, cmÀ1): 2206,
1615, 1490, 1445, 1347, 1209, 1034, 745. Anal. Calcd
for C12H13N3O (215.11): C, 66.96; H, 6.09; N, 19.52.
Found: C, 67.00; H, 6.23; N, 19.40.
1
224.3 ꢁC (ethanol). H NMR (400 MHz, DMSO-d6) d
ppm: 7.52 (1H, dd, J = 6.8, 8.4 Hz), 7.57 (1H, dd,
J = 6.8, 8.1 Hz), 7.86 (1H, d, J = 8.4 Hz), 7.90 (1H, d,
J = 8.1 Hz), 8.17 (2H, d, J = 8.9 Hz), 8.56 (2H, d,
J = 8.9 Hz). 13C NMR (100 MHz, DMSO-d6) d ppm:
92.60, 111.59, 115.09, 119.90, 123.48, 125.69, 128.94,
129.33, 130.05, 130.24, 136.78, 149.70. MS (EI, 70 eV)
m/z (%): 280 (M+, 88); 264 (M+-16, 7); 204 (71); 76
(100). IR (KBr, cmÀ1): 2205, 1612, 1536, 1438, 1353,
1299, 1239, 1035, 845, 749. Anal. Calcd for
C14H8N4O3 (280.06): C, 60.00; H, 2.88; N, 19.99.
Found: C, 59.95; H, 2.74; N, 19.69.
5.1.1.10. 2-Chloroethyl-3-cyano-2H-indazole N1-oxide
(10). White solid 0.45 g (20%) (method B); mp 150.9–
151.5 ꢁC. 1H NMR (400 MHz, CDCl3) d ppm: 4.09
(2H, t, J = 5.7 Hz), 4.90 (2H, t, J = 5.7 Hz), 7.43 (1H,
dd, J = 5.3, 7.1 Hz), 7.46 (1H, dd, J = 5.3, 7.4 Hz),
7.72 (1H, d, J = 7.1 Hz), 7.84 (1H, d, J = 7.4 Hz). 13C
NMR (100 MHz, CDCl3) d ppm: 39.95, 48.40, 94.00,
110.50, 114.52, 119.28, 122.00, 128.00, 128.93, 129.00.
MS (EI, 70 eV) m/z (%): 205 (M+-16, 28); 143 (100);
88 (28); 77 (30); 63 (38). IR (KBr, cmÀ1): 2361, 1684,
1601, 1489, 1424, 1300, 1266, 932, 758. Anal. Calcd
for C10H8ClN3O (221.04): C, 54.19; H, 3.64; N, 18.86.
Found: C, 53.93; H, 3.33; N, 18.59.
5.1.1.7. 3-Cyano-2-(3-nitrophenyl)-2H-indazole N1-ox-
ide (7). 2-(2-Nitrophenyl)-2-(3-nitrophenylamino)aceto-
nitrile (7a). Brown solid 0.65 g (66%). 1H NMR
(400 MHz, DMSO-d6) d ppm: 6.64 (1H, d, J = 9.1 Hz),
7.24 (1H, dd, J = 1.9 Hz, J = 8.0 Hz), 7.91 (8H, m).
3-Cyano-2-(3-nitrophenyl)-2H-indazole N1-oxide (7).
Orange solid 0.14 g (30%) (method A); mp 207.1–
1
208.0 ꢁC (ethanol). H NMR (400 MHz, DMSO-d6) d
ppm: 7.48 (1H, dd, J = 6.6, 8.5 Hz), 7.52 (1H, dd,
J = 6.6, 8.0 Hz), 7.77 (1H, d, J = 8.0 Hz), 7.89 (1H, d,
J = 8.5 Hz), 7.91 (1H, dd, J = 7.9, 8.2 Hz), 8.17 (1H, d,
J = 7.9 Hz), 8.54 (1H, d, J = 8.2 Hz), 8.68 (1H, s). 13C
NMR (100 MHz, DMSO-d6) d ppm: 92.00, 111.00,
115.08, 119.36, 123.15, 123.64, 126.37, 128.71, 129.00,
129.87, 131.29, 132.29, 133.07, 149.00. MS (EI, 70 eV)
m/z (%): 280 (M+, 100); 264 (M+-16, 8); 250 (M+-30,
4); 204 (8); 76 (18); 43 (87). IR (KBr, cmÀ1):2207,
1536, 1503, 1440, 1353, 1276, 1237, 1167, 1114, 742.
Anal. Calcd for C14H8N4O3 (280.06): C, 60.00; H,
2.88; N, 19.99. Found: C, 59.96; H, 2.78; N, 19.68.
5.1.1.11. 3-Cyano-2-phenethyl-2H-indazole N1-oxide
(11). White solid 0.17 g (20%) (method B); mp 120.6–
121.7 ꢁC (ethanol). 1H NMR (400 MHz, CDCl3) d ppm:
3.30 (2H, t, J = 7.2 Hz), 4.80 (2H, t, J = 7.2 Hz), 7.20
(2H, d, J = 8.0 Hz), 7.31 (3H, m), 7.42 (2H, m), 7.63
(1H, d, J = 8.4 Hz), 7.87 (1H, d, J = 8.8 Hz). 13C NMR
(100 MHz, CDCl3) d ppm: 34.49, 48.32, 92.00, 110.52,
114.57, 119.13, 122.20, 127.62, 127.89, 128.63, 129.41,
129.43, 129.60, 136.19. MS (EI, 70 eV) m/z (%): 263
(M+, 30); 247 (M+-16, 2); 115 (9); 105 (100); 91 (39). IR
(KBr, cmÀ1): 2207, 1489, 1435, 1339, 1248, 1032, 822,
741. Anal. Calcd for C16H13N3O (263.11): C, 72.99; H,
4.98; N, 15.96. Found: C, 73.04; H, 5.05; N, 16.10.
5.1.1.8. 3-Cyano-2-(2-furylmethyl)-2H-indazole N1-
oxide (8). Yellow solid 0.19 g (24%) (method A); mp
1
143.2–144.8 ꢁC (ethanol). H NMR (400 MHz, CDCl3)
d ppm: 5.78 (2H, s), 6.40 (1H, dd, J = 1.8, 3.2 Hz),
6.70 (1H, d, J = 3.2 Hz), 7.38 (1H, dd, J = 6.7, 7.4 Hz),
7.41 (1H, dd, J = 6.7, 7.6 Hz), 7.45 (1H, d, J = 1.8 Hz),
7.68 (1H, d, J = 7.4 Hz), 7.84 (1H, d, J = 7.6 Hz). 13C
NMR (100 MHz, CDCl3) d ppm: 42.29, 92.00, 111.42,
112.30, 112.44, 114.80, 119.18, 122.81, 127.68, 128.81,
129.00, 144.40, 145.50. MS (EI, 70 eV) m/z (%): 239
(M+, 41); 222 (M+-17, 7); 179 (45); 144 (14); 81 (100);
53 (100). IR (KBr, cmÀ1): 2209, 1489, 1362, 1291,
1040, 777, 749. Anal. Calcd for C13H9N3O2 (239.07):
C, 65.27; H, 3.79; N, 17.56. Found: C, 65.08; H, 4.04;
N, 17.76.
5.1.1.12. 3-Cyano-2-(2-naphthyl)-2H-indazole N1-ox-
ide (12). Yellow solid 0.71 g (75%) (method B); mp
218.3–220.0 ꢁC (ethanol). H NMR (400 MHz, CDCl3)
1
d ppm: 7.46 (1H, dd, J = 6.7, 7.9 Hz), 7.51 (1H, dd,
J = 6.7, 8.3 Hz), 7.68 (2H, m), 7.75 (1H, dd, J = 2.0,
8.8 Hz), 7.79 (1H, d, J = 7.9 Hz), 7.92 (1H, d,
J = 8.3 Hz), 8.00 (1H, d, J = 6.3 Hz), 8.02 (1H, d,
J = 6.8 Hz), 8.13 (1H, d, J = 8.8 Hz), 8.21 (1H, d,
J = 2.0 Hz). 13C NMR (100 MHz, CDCl3) d ppm:
92.60, 111.04, 115.08, 119.33, 123.5, 123.67, 127.48,
127.97, 128.03, 128.45, 128.90, 128.95, 129.18, 129.23,
129.60, 130.43, 133.29, 134.51. MS (EI, 70 eV) m/z
(%): 285 (M+, 14); 268 (M+-17, 8); 179 (67); 144 (59);
127 (100); 115 (35). IR (KBr, cmÀ1): 2207, 1489, 1435,
1339, 1248, 132, 855, 741. Anal. Calcd for C18H11N3O
(285.09): C, 75.78; H, 3.89; N, 14.73. Found: C, 75.46;
H, 3.77; N, 14.59.
5.1.1.9. 2-Butyl-3-cyano-2H-indazole N1-oxide (9). A
mixture of o-nitrobenzaldehyde (0.50 g, 3.3 mmol),
KCN (0.40 g, 6.6 mmol) and butylamine (0.24 g,
3.3 mmol) in glacial acetic acid (30 mL) was stirred at
room temperature for 24 h. The solution was treated

A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
3477
5.1.1.13. 3-Cyano-2-(1-naphthyl)-2H-indazole N1-ox-
ide (13). Black solid 0.46 g (49%) (method B); mp 184.6–
185.8 ꢁC (ethanol). 1H NMR (400 MHz, CDCl3) d ppm:
7.25 (1H, d, J = 8.0 Hz), 7.48 (1H, dd, J = 6.8, 8.0 Hz),
7.54 (1H, dd, J = 6.8, 8.0 Hz), 7.60 (1H, dd, J = 6.8,
8.0 Hz), 7.66 (1H, dd, J = 6.8, 8.4 Hz), 7.72 (1H, d,
J = 4.2 Hz), 7.74 (1H, d, J = 5.4 Hz), 7.81 (1H, d,
J = 8.0 Hz), 7.95 (1H, d, J = 8.0 Hz), 8.06 (1H, d,
J = 8.4 Hz), 8.22 (1H, dd, J = 4.2, 5.4 Hz). 13C NMR
(100 MHz, CDCl3) 8 ppm: 92.00, 110.69, 115.25,
119.42, 122.32, 123.30, 125.59, 127.93, 128.04, 128.13,
128.18, 129.05, 129.21, 129.26, 129.50, 130.01, 133.41,
134.75. MS (EI, 70 eV) m/z (%): 285 (M+, 100); 268
(M+-17, 9); 256 (39); 179 (47); 144 (17); 127 (62); 91
(25). IR (KBr, cmÀ1): 2209, 1487, 1458, 1381, 1262,
1059, 795, 774. Anal. Calcd for C18H11N3O (285.09):
C, 75.78; H, 3.89; N, 14.73. Found: C, 75.88; H, 3.68;
N, 14.65.
119.95, 126.07, 126.18, 126.32, 127.76, 127.93, 129.76,
143.76, 149.35. MS (EI, 70 eV) m/z (%): 264 (M+, 35);
217 (9); 164 (21); 140 (22); 122 (34); 105 (100). IR (KBr,
cmÀ1): 2220, 1597, 1528, 1354, 857, 750. Anal. Calcd for
C14H8N4O2 (264.06): C, 63.64; H, 3.05; N, 21.20. Found:
C, 63.29; H, 2.94; N, 21.01.
5.2. Biology
5.2.1. Trypanocidal (epimastigotes) in vitro test. Trypan-
osoma cruzi epimastigotes (Brener or Tulahuen 2
strains) were grown at 28 ꢁC in an axenic medium
(BHI-Tryptose) as previously described,9 complemented
with 5% foetal bovine serum (FBS). Cells from a 10-day-
old culture (stationary phase) were inoculated into
50 mL of fresh culture medium to give an initial concen-
tration of 1 · 106 cells/mL. Cell growth was followed by
measuring every day the absorbance of the culture at
600 nm. Before inoculation, the media were supplement-
ed with the indicated amount of the drug from a stock
solution in DMSO. The final concentration of DMSO
in the culture media never exceeded 0.4% and the con-
trol was run in the presence of 0.4% DMSO and in the
absence of any drug. No effect on epimastigote growth
was observed by the presence of up to 1% DMSO
in the culture media. The percentage of inhibition was
5.1.2. Synthesis of indazole derivatives (16–18). General
method. A mixture of the corresponding indazole N1-ox-
ide (0.50 g, 1 equiv) (2, 5 and 6) and PPh3 (1 equiv) in
ethanol (30 mL) was heated at reflux for 5–6 h. Then
the mixture was evaporated to dryness and the residue
was purified by column chromatography and then crys-
tallized from the indicated solvent.
calculated
as
follows:
% = {1 À [(Ap À A0p)/
5.1.2.1.
3-Cyano-2-(4-hydroxyphenyl)-2H-indazole
(Ac À A0c)]} · 100, where Ap = A600 of the culture con-
taining the drug at day 5; A0p = A600 of the culture con-
taining the drug just after addition of the inocula (day
0); Ac = A600 of the culture in the absence of any drug
(control) at day 5; A0c = A600 in the absence of the drug
at day 0. To determine IC50 values, 50% inhibitory con-
centrations, parasite growth was followed in the absence
(control) and presence of increasing concentrations of
the corresponding drug. At day 5, the absorbance of
the culture was measured and related to the control.
The IC50 value was taken as the concentration of drug
needed to reduce the absorbance ratio to 50%.
(16). Yellow solid 0.22 g (46%); mp 177.9–181.0 ꢁC
(chloroform). H NMR (400 MHz, DMSO-d6) d ppm:
1
7.03 (2H, d, J = 8.8 Hz), 7.43 (1H, dd, J = 6.8, 8.4 Hz),
7.53 (1H, dd, J = 6.8, 8.8 Hz), 7.72 (2H, d, J = 8.8 Hz),
7.90 (1H, d, J = 8.4 Hz), 7.97 (1H, d, J = 8.8 Hz),
10.21 (1H, br s). 13C NMR (100 MHz, DMSO-d6) d
ppm: 107.18, 112.27, 116.88, 119.32, 119.71, 126.76,
126.97, 127.02, 128.67, 131.14, 148.59, 159.91. MS (EI,
70 eV) m/z (%): 235 (M+, 100); 206 (M+-30, 43); 193
(9); 179 (9); 65 (12). IR (KBr, cmÀ1): 3450–3230, 2220,
1590, 1430, 1345, 1035, 830, 750. Anal. Calcd for
C14H9N3O (235.07): C, 71.48; H, 3.86; N, 17.86. Found:
C, 71.67; H, 3.55; N, 17.49.
5.2.2. Trypanocidal (trypomastigotes) in vitro test30.
Products were evaluated in vitro on the trypomastigote
form of T. cruzi (CL Brener). BALB/c mice infected with
T. cruzi were used 7 days after infection. Blood was ob-
tained by cardiac puncture using 3.8% sodium citrate as
anticoagulant in a 7:3 blood/anticoagulant ratio. The
parasitaemia in infected mice ranged from 1 · 105 to
5 · 105 parasites/mL. The products were dissolved in
minimal quantity of DMSO and added to PBS to give
a final concentration of 100 and 250 lg/mL. Aliquots
(10 lL) of each extract in triplicate were mixed in micro-
titre plates with 100 lL infected blood containing para-
sites at a concentration near to 106 parasites/mL.
Infected blood and infected blood containing GV at
100 and 250 lg/mL concentration were used as control.
The plates were shaken for 10 min at room temperature
and kept at 4 ꢁC for 24 h. Each solution was examined
microscopically (OLYMPUS BH2) for parasite count-
ing. The activity (% of parasites reduction) was com-
pared with the standard drug GV.
5.1.2.2. 3-Cyano-2-(4-iodophenyl)-2H-indazole (17).
Yellow solid 0.28 g (58%); mp 157.6–159.8 ꢁC (ethanol).
1H NMR (400 MHz, CDCl3) d ppm: 7.42 (1H, dd,
J = 7.6, 8.4 Hz), 7.50 (1H, dd, J = 7.6, 8.4 Hz), 7.69 (2H,
d, J = 8.8 Hz), 7.84 (1H, d, J = 8.4 Hz), 7.93 (1H, d,
J = 8.4 Hz), 7.97 (2H, d, J = 8.8 Hz). 13C NMR
(100 MHz, CDCl3) d ppm: 95.91, 111.69, 119.07,
119.67, 125.67, 125.70, 126.98, 128.00, 128.67, 139.00,
139.34, 149.26. MS (EI, 70 eV) m/z (%): 239 (M+-I, 16);
218 (21); 179 (37); 81 (100); 53 (48). IR (KBr, cmÀ1):
2218, 1489, 1368, 1007, 824, 745. Anal. Calcd for
C14H8IN3 (344.98): C, 48.72; H, 2.34; N, 12.17. Found:
C, 49.01; H, 2.56; N, 11.99.
5.1.2.3. 3-Cyano-2-(4-nitrophenyl)-2H-indazole (18).
Yellow solid 0.17 g (37%); mp 180.7–182.5 ꢁC (ethanol).
1H NMR (400 MHz, DMSO-d6) d ppm: 7.52 (1H, dd,
J = 6.8, 8.4 Hz), 7.60 (1H, dd, J = 6.8, 8.7 Hz), 7.97 (1H,
d, J = 8.4 Hz), 8.03 (1H, d, J = 8.7 Hz), 8.27 (2H, d,
J = 9.0 Hz), 8.56 (2H, d, J = 9.0 Hz). 13C NMR
(100 MHz, DMSO-d6) d ppm: 107.93, 111.45, 119.63,
5.2.3. Leishmanocidal in vitro test. The isolation, cultiva-
tion and maintenance of Leishmania promastigotes and

3478
A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
the technique used have been previously described.31
Briefly, promastigote inhibition studies were performed
on L. amazonensis (IFLA/BR/67/PH8), L. infantum
trode, a platinum wire as auxiliary electrode, and a sat-
urated calomel as reference electrode were employed.
(MHOM/ET/1967/L82,LV9)
and
L.
braziliensis
5.4. ESR experiments
(MHOM/BR/75/M2903) grown at 25 ꢁC in Schneider’s
drosophila medium containing 20% FBS. Compounds
were dissolved in 40 lL DMSO (final concentration of
this solvent 0.4%) or Tween 80, then diluted in the medi-
um and placed in microtitre plates in triplicate, with a
final compound concentration of 100 or 250 lg/mL.
The microtitre plates of 96 bowls utilized contain
2 · 106 parasites/mL and the same proportion v/v with
the product in each bowl. The activity of compounds
was evaluated after 72 h by optical observation of a
drop of each culture using an inverted microscope
OLYMPUS IMT2 and comparison with control cell cul-
tures and with those treated with the reference drug
pentamidine. The evaluation is qualitative observing
the lysis of parasites.
NADPH, EDTA, DMPO and acetonitrile were obtained
from Sigma. Incubations consisted of homogenized epi-
mastigotes (Tulahuen 2 strain) of T. cruzi (4 mg protein/
mL), NADPH (1 mM), EDTA (1 mM), DMPO
(100 mM) in phosphate buffer, pH 7.4 (20 mM), and
N-oxide derivative (2 mM) dissolved in acetonitrile. In
all cases, NADPH was added immediately before the
ESR spectrum was recorded. ESR spectra were recorded
at room temperature in the X band (9.7 GHz) using a
Bruker ECS 106 spectrometer with a rectangular cavity
and 50 kHz field modulation. The hyperfine splitting
constants were estimated to be accurate within 0.05.
5.5. Respiration experiments
5.2.4. Antineoplastic assay. The compounds were tested
at 10À4 M in the following tumour cell lines: MCF-7
(human mammary adenocarcinoma) (ATCC HTB-38),
TK-10 (human kidney carcinoma) (NCI) and HT-29
(human colon adenocarcinoma) (ATCC HTB-38). The
cytotoxicity test was carried out according to previously
described procedures32 with some modifications. Cells.
An adequate cell suspension was prepared for each cell
line (MCF-7, TK-10 or HT-29) in RPMI medium, sup-
plemented with L-glutamine (1%), penicillin/streptomy-
cin (1%), non-essential amino acids (1%) and 10% (v/v)
FBS. Then, 225 lL was added into 96-well plates. The
cultures were maintained at 37 ꢁC and 5% CO2 for
48 h. Treatment. Compound solutions were prepared
just before dosing. Stock solutions, 1 mM, were pre-
pared in 10% DMSO and 25 lL (final concentration
10À4 M) was added to each well. The cells were exposed
for 24 h at 37 ꢁC in 5% CO2 atmosphere. Measurement.
After exposure to the compound, the medium was elim-
inated and the cells were washed with PBS. The cells
were fixed with 50 lL of trichloroacetic acid (50%) and
200 lL of culture medium (without FBS) for 1 h at
4 ꢁC. Then, the cells were washed with purified water
and treated with sulforhodamine B (0.4% wt/vol in 1%
acetic acid) for 10 min at room temperature. The plates
were washed with 1% acetic acid and dried overnight.
Finally, 100 lL Tris buffer (pH 10.0) was added and
absorbance at 540 nm was determined. Calculations.
The cell survival percentage (SP) was calculated for all
of the compounds as [(absorbance of cells post-treat-
ment with product À absorbance of blank)/(absorbance
of cells post-treatment with solvent À absorbance of
blank)] · 100.
Tulahuen strain T. cruzi epimastigotes were harvested
by 500g centrifugation, followed by washing and re-sus-
pension in 0.05 M sodium phosphate buffer, pH 7.4, and
containing 0.107 M sodium chloride. Respiration mea-
surements were carried out polarographically with a
Clark No. 5331 electrode (Yellow Springs Instruments)
in a 53 YSI model (Simpson Electric Co). The chamber
volume was 2 mL and the temperature was 28 ꢁC. The
amount of parasite used was equivalent to 2 mg protein.
The IC50 equivalent concentration corresponds to the fi-
nal concentration used in the respiration experiments.
This concentration was calculated considering that the
IC50 (trypanocidal in vitro experiments) was determined
using 3 · 106 parasites/mL, equivalent to 0.0375 mg pro-
tein/mL as initial parasite mass. Whereas 80 · 106 para-
sites/mL, equivalent to 1 mg protein/mL, was used in the
respiration experiments. In order to maintain the para-
site mass-drug ratio constant in these two types of exper-
iments, the IC50 was corrected by this 26-fold parasite
mass increase in the respiration experiment. Values are
expressed as means SD for three independent experi-
ments. No effect of DMSO alone was observed.
5.6. Crystallography study
The molecular structure of derivative 13, C18H11N3O,
was determined by X-ray diffraction methods. The sub-
stance crystallizes in the monoclinic P21/c space group,
with
˚
c = 8.692(1) A,
a = 8.024(1),
b = 20.186(3),
b = 95.17(1)ꢁ and Z = 4. The structure was solved from
1840 reflections with I > 2r(I) and refined to agreement
R1-factors of 0.050. The hydrogen atoms were posi-
tioned stereochemically and refined with the riding
model. Intramolecular bond distances and angles, along
with other crystallographic data, are given as Support-
ing information. Crystallographic data (excluding struc-
ture factors) for the structure in this paper have been
deposited at the Cambridge Crystallographic Data
Centre as supplementary publication numbers CCDC-
284059. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK [fax: 144-(0)1223-336033 or
e-mail: deposit@ccdc.cam.ac.uk].
5.3. Cyclic voltammetry
Cyclic voltammetry was carried out using a Metrohm
693 VA instrument with a 694 VA Stand convertor
and a 693 VA Processor, in DMSO (ca 1.0 mM), under
a nitrogen atmosphere at room temperature, with tetra-
butylammonium perchlorate (TBAP, ca 0.1 mM) as
supporting electrolyte. Three-electrode cell was used
where a mercury-dropping electrode as working elec-

A. Gerpe et al. / Bioorg. Med. Chem. 14 (2006) 3467–3480
3479
´
Cerain, A.; Piro, O. E.; Castellano, E. E. Bioorg. Med.
Chem. 2005, 13, 3197; (b) Olea-Azar, C.; Cerecetto, H.;
5.7. QSAR studies
´
Gerpe, A.; Gonzalez, M.; Aran, V. J.; Rigol, C.; Opazo, L.
Spectrochim. Acta Part A 2005, 63, 36–42.
´
The compounds were built with standard bond lengths
and angles using the Spartan’04, 1.0.1 version,24 suite of
programs and the geometry of each molecule was fully
optimized by applying semiempirical PM3 method in
gas phase from the most stable conformer obtained using
molecular mechanics (MMFF) methods. Angles and
bond lengths of compound 13 were compared with the
correspondingcrystallographic data tovalidate the geom-
etry optimization. Then, a single point calculation using
density functional methodology (Becke’s exchange func-
tional (B) and Becke’s three-parameter adiabatic connec-
tion (B3) hybrid exchange functional were used in
combination with the Lee–Yang–Parr correlation func-
tional).33 The standard 6-31G* basis set of DZP quality
was used for orbital expansion to solve the Kohn–Sham
equations for first- and second-row elements. For heavier
atoms a pseudopotential base (LACVP*) was used. Lipo-
philic properties of the compounds were included into the
analyses, as clogP (logP calculated by the Villar method).
´
9. (a) Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Risso, M.;
Saenz, P.; Seoane, G.; Denicola, A.; Peluffo, G.; Quijano,
C.; Olea-Azar, C. J. Med. Chem. 1999, 42, 1941; (b)
´
Aguirre, G.; Cerecetto, H.; Di Maio, R.; Gonzalez, M.;
Seoane, G.; Denicola, A.; Ortega, M. A.; Aldana, I.;
Monge, A. Arch. Pharm. 2002, 335, 15; (c) Aguirre, G.;
´
Boiani, M.; Cerecetto, H.; Gerpe, A.; Gonzalez, M.;
Fernandez, Y.; Denicola, A.; Ochoa, C.; Nogal, J. J.;
´
Montero, D.; Escario, J. A. Arch. Pharm. Med. Chem.
2004, 337, 259; (d) Aguirre, G.; Boiani, L.; Cerecetto, H.;
´
Di Maio, R.; Gonzalez, M.; Porcal, W.; Thomson, L.;
Tortora, V.; Denicola, A.; Moler, M. Bioorg. Med. Chem.
´
2005, 13, 6324; (e) Aguirre, G.; Boiani, L.; Boiani, M.;
´
Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Porcal, W.;
Denicola, A.; Piro, O. E.; Castellano, E. E.; Sant’Anna,
M.; Barreiro, E. J. Bioorg. Med. Chem. 2005, 13, 6336.
10. (a) Elwell, J. H.; Siim, B. G.; Evans, J. W.; Brown, J. M.
Biochem. Pharmacol. 1997, 54, 249; (b) Cerecetto, H.;
´
Gonzalez, M. Mini-Rev. Med. Chem. 2001, 1, 219.
11. (a) Behr, L. C. J. Org. Chem. 1954, 76, 3672; (b) Behr, L.
C.; Alley, E. G.; Levand, O. J. Org. Chem. 1962, 27, 65.
12. Johnston, D.; Smith, D. M.; Shepherd, T.; Thompson, D.
J. Chem. Soc., Perkin Trans. I 1987, 495.
Acknowledgements
13. Zenchoff, G. S.; Walser, A.; Fryer, R. I. J. Heterocyclic
Chem. 1976, 13, 33.
Financial support from Collaborative Project Consejo
´
Superior Investigaciones Cientıficas (Spain) – UdelaR
14. (a) Howard, E.; Olszewski, W. F. J. Am. Chem. Soc. 1959,
81, 1483; (b) Boyer, J. H.; Ellzey, S. E. J. Org. Chem. 1961,
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(Uruguay) (#2004UY0009), CSIC (Uruguay), CONI-
CYT (Uruguay) and PEDECIBA (Uruguay) is acknowl-
edged. The authors thank AMSUD-PASTEUR network
for a scholarship to LB. COA thanks FONDECYT
(Chile) (#1030949, #7040037, AT-4040020) and PBCT
15. (a) In derivative 13, the indazole ring is planar. The 3-
cyano-2H-indazole N1-oxide plane is nearly perpendicular
to the naphthalene substituent. The N–O bonding to the
indazole heterocycle ring produces a slight lengthening of
the adjacent N–C and N–N bonds (atomic coordinates,
bond distances and angles, crystal data, data collection
procedure, structure determination method and refinement
results are given as Supplementary information).(b) Enraf-
Nonius (1997–2000). COLLECT. Nonius BV, Delft, The
Netherlands. K. Harms and S. Wocadlo, XCAD4 – CAD4
Data Reduction., University of Marburg, Marburg, Ger-
many, 1995.; (c) PLATON, A Multipurpose Crystallo-
graphic Tool, Utrecht University, Utrecht, The
Netherlands, A. L. Spek, 1998; (d) Sheldrick, G. M.
SHELXS-97. Program for Crystal Structure Resolution;
University of Go¨ttingen: Go¨ttingen, Germany, 1997; (e)
Sheldrick, G. M. SHELXL-97. Program for Crystal Struc-
tures Analysis; University of Go¨ttingen: Go¨ttingen, Ger-
many, 1997; (f) Johnson, C. K. ORTEP-II. A Fortran
Thermal-Ellipsoid Plot Program. Report ORNL-5138; Oak
Ridge National Laboratory: Tennessee, USA, 1976.
16. Enanga, B.; Keita, M.; Chauviere, G.; Dumas, M.;
Bouteille, B. Trop. Med. Int. Health 1998, 3, 736.
´
(Chile) Anillo en Ciencia y Tecnologıa. OEP thanks
CONICET (Argentina).
Supporting information
Crystallographic data for derivative 13 (five tables).
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2006.
01.007.
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