Novel antiprotozoal products: imidazole and benzimidazole N-oxide derivatives and related compounds.

Article abstract of DOI:10.1002/ardp.200300840

The syntheses and biological evaluation of the first anti-protozoa imidazole N-oxide and benzimidazole N-oxide and their derivatives are reported. They were tested in vitro against two different protozoa, Trypanosoma cruzi and Trichomonas vaginalis. Deriv

Full text of DOI:10.1002/ardp.200300840

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Novel Antiprotozoal Products 259
Gabriela Aguirrea,
Novel Antiprotozoal Products: Imidazole and
Benzimidazole N-Oxide Derivatives and
Related Compounds
Mariana Boiania,
Hugo Cerecettoa,
Alejandra Gerpea,
a
´
Mercedes Gonzalez ,
b
´
Yolanda Fernandez Sainz ,
The syntheses and biological evaluation of the first anti-protozoa imidazole N-
oxide and benzimidazole N-oxide and their derivatives are reported. They were
tested in vitro against two different protozoa, Trypanosoma cruzi and Tricho-
monas vaginalis. Derivative 7c, ethyl-1-(i-butyloxycarbonyloxy)-6-nitrobenzimid-
azole-2-carboxylate, displayed activity on both protozoa. Lipophilicity and redox
potential were experimentally determined in order to study the relationship with
activity of the compounds. These properties are well related with the observed
bioactivity. Imidazole and benzimidazole N-oxide derivatives are becoming lead-
ers for further chemical modifications and advanced biological studies.
Ana Denicolac,
d
´
Carmen Ochoa de Ocariz ,
e
´
Juan Jose Nogal ,
David Monteroe,
e
´
Jose Antonio Escario
a
´
Departamento de Quımica
´
Organica, Facultad de
´
Quımica-Facultad de
Ciencias, Montevideo,
Uruguay
Keywords: Anti-protozoa compounds; Imidazole N-oxide; Structure-activity
relationship
b Facultad de Ciencias,
Received: August 26, 2003; Accepted: January 9, 2004 [FP840]
DOI 10.1002/ardp.200300840
˜
Pz/Misael Banuelos s/n,
Universidad de Burgos,
˜
Burgos, Espana
c
´
Laboratorio de Fisicoquımica
´
Biologica, Facultad de
Ciencias, Montevideo,
Uruguay
d
´
´
Instituto de Quımica Medica,
˜
Madrid, Espana
e Departamento de
´
Parasitologıa, Facultad de
Farmacia, Universidad
Complutense, Madrid,
˜
Espana
plain the need for development of new selective chem-
otherapeutic solutions [3].
Introduction
On the other hand trichomoniasis, an important sex-
Parasitic diseases are among the most widespread of
human diseases. In 1997, of a global total of 52.2
million deaths, 17.3 million were due to infectious and
parasitic diseases, followed by circulatory diseases,
cancer, and respiratory diseases [1]. In South America
these constitute a major health and economic prob-
lem, where Chagas’ disease occupies the third place
in number of deaths per year, after malaria and
schistosomiasis [2]. Commercially, only two drugs
were available for the treatment of this disease: Nifurti-
mox (Nfx, actually discontinued) and Benznidazole
(Bnz) (Figure 1). Unfortunately, they are not consist-
ently effective and have serious side effects that ex-
ually-transmitted protozoa disease, which is frequent
in rich countries, also needs new effective agents. The
treatment of trichomoniasis was revolutionized by me-
tronidazole (Mtz) (Figure 1) [4]. The 5-nitroimidazole
Mtz, a potent trichomononicidal agent under low oxy-
gen concentrations conditions, [5] has shown impor-
tant strain-resistance effects [6].
In previous works [7, 8] we found that some heterocy-
cle N-oxide containing derivatives posses promising
trypanocidal activity. In particular, compound 1 (Figure
1) has an IC50 for Trypanosoma cruzi (T. cruzi) epima-
stigote form (Tulahuen strain) similar to that found for
Nfx. The well known N-oxide bio-reduction and it bio-
logical consequences [9, 10] were used to explain the
trypanocidal activity. These facts induced us to investi-
gate some related heterocycles (Figure 1). So, we
have prepared some selected derivatives of imidazole
N-oxide and benzimidazole N-oxide. We report here
´
Correspondence: Mercedes Gonzalez, Departamento de
´
´
´
Quımica Organica, Facultad de Quımica y Facultad de
´
´
Ciencias, Universidad de la Republica, Igua 4225, 11400
Montevideo, Uruguay. Phone: +598 2 5258618 ext 216, Fax:
+598 2 5250749; e-mail: megonzal@fq.edu.uy
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´
260 Gonzalez et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Figure 1. Designed structures from Nifurtimox (Nfx), Benznidazole (Bnz), Metronidazole (Mtz) and benzofuroxan
(derivative 1).
the synthesis, anti-protozoa (T. cruzi and Trichomona
vaginalis (T. vaginalis)) activity, and the structure-ac-
tivity studies of these series of compounds.
was prepared as it has previously been reported [15].
In order to obtain more apolar derivatives, compound
3a was reacted with methyl iodide and different chloro-
formate derivatives. This procedure yielded deriva-
tives 7aϪd as unique products of reaction (Scheme
1C). The course of reaction, O-alkylation process, was
clearly evidenced by the 7a-d NMR NOE-diff experi-
ments (Figure 2A).
Results and discussion
Chemistry
Compounds 2aϪf were obtained through a simple
chemical transformation as reported [11Ϫ14], or via
cyclization of the corresponding a-substituted amine-
oxime such as depicted in Scheme 1A. The a-substi-
tuted amine-oximes were obtained from the diacetyl
mono-oxime and amines by reductive amination or via
nucleophilic substitution from chloroacetone and p-tol-
uidine (compound II, Scheme 1A). The imidazole sys-
tem was generated by cyclization of the corresponding
orthoester at reflux. Secondary products were iden-
tified as the result of condensation between cyclization
intermediates and the orthoesters.
Benzimidazole N-oxide derivatives 3aϪe, 4a-c, 5, and
6 are present, in solution, as a mixture of tautomers
XI and XI’ [15, 17] (Figure 2B). This fact was clearly
observed in the H NMR spectra and was confirmed
chemically with the exclusive formation of compounds
7aϪd from 3a.
The structures of all derivatives were confirmed by
NMR (1H, 13C, and HETCOR experiments, i.e. HMQC
and HMBC) and MS, and their purity established by
TLC and microanalysis.
1
On the other hand, the basic cyclization of ethyl N-(2-
nitro-4-substitutedphenyl)glycinate afforded the corre-
sponding benzimidazole N-oxide 3aϪc (Scheme 1B)
[15]. Derivatives 3dϪe were obtained by reaction be-
tween the corresponding benzofuroxan and nitroeth-
ane in basic medium [16]. In order to modify the bioac-
tivity of derivative 3a, it was transformed into com-
pounds 4aϪc, 5, 6 (Scheme 1B), and 7aϪd (Scheme
1C). Treatment of 3a with excess of the corresponding
alcohol (i-propanol, n-butanol and n-hexanol), in pres-
ence of p-toluenesulfonic acid (p-TsOH) and molecular
Biological studies
The existence of the epimastigote form as an obligate
mammalian intracellular stage has been revisited [18,
19] and confirmed recently [20]. For this reason, the
compounds were tested in vitro against epimastigote
forms of T. cruzi (Tulahuen strain) at two different con-
centrations, 25 µM and 100 µM, as indicated in Exper-
imental [7]. The percentage of inhibition was calcu-
lated as follows: % = {1-[(Ap-A0p)/(Ac-A0c)]} ϫ 100,
where Ap = A600 of the culture containing the drug at
day 5; A0p = A600 of the culture containing 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;
˚
sieves (4 A), yielded derivatives 4aϪc. Compound 5,
was obtained in the same manner by reacting com-
pound 3a with n-butylamine in toluene. Compound 6
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Novel Antiprotozoal Products 261
Scheme 1. A. Preparation of 1-substituted imidazole N3-oxide derivatives: (a) 1. R2NH2/ZnCl2/MeOH/molecular
3
’
sieves 4 A/rt, 2. NaCNBH3; (b) p-toluidine/K2CO3/KI/acetone/reflux.; (c) R C(OR )3 (RЈ = Me or Et) (solvent)/
˚
p-TsOH/reflux. B. Preparation of the benzimidazole N-oxide derivatives: (d) piperidine/EtOH/reflux for 3a; EtONa/
7
˚
EtOH/rt for 3b and 3c; (e) nitroethane/piperidine/THF/rt; (f) R OH (solvent)/p-TsOH/ molecular sieves 4A/reflux;
˚
(g) butylamine (1 equiv.)/toluene/p-TsOH/molecular sieves 4A/ reflux; (h) HCl (c)/reflux. c) Preparation of derivati-
ves 7a-d: (i) R8X, NaHCO3 (for 7a)/acetone/rt.
A
0c = A600 in the absence of the drug at day 0. Table
Trichomonacidal activity in vitro was developed using
recently isolated strain JH31A no. 4 of T. vaginalis at
1 µg/mL, 10 µg/mL, and 100 µg/mL as indicated in
1 shows the percentage of inhibition for the evalu-
ated derivatives.
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262 Gonzalez et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Figure 2. A. Tautomeric forms of benzimidazole N-oxide derivatives. B. NOE-diff experiments of derivative 7c
confirming the O-acylation process involved in the reaction between derivative 3a and electrophile reactants.
Experimental [21]. Results are expressed as percent-
ages of growth inhibition (cytostatic activity) respective
centages of reduction (% R), because in these cases,
the number of parasites is reduced with respect to in-
itial population, and the activity is cytocidal. Table 1
shows the cytostatic or cytocidal activity in brackets for
the evaluated derivatives. Table 2 shows the percent-
age of reduction of T. vaginalis growth at 48 h for some
selected derivatives.
to controls, as follows:% C.A. = 100-[(G.I.experimental
/
G.I.control) ϫ 100], where G.I. (growth indices) is the
ratio between the number of parasites at 24 (or 48 h)
and the number of parasites at 0 h. When this ratio
(G.I.) is <1, the values so calculated represent per-
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Novel Antiprotozoal Products 263
Table 1.In vitro activity of developed compounds on
T. cruzi and T.vaginalis.
Table 2. Percentage of reduction of T. vaginalis growth
at the indicated doses after 48 h contact with the
listed compounds.
Com-
pound
T. cruzi#
T. vaginalis#
% Inh.†
% C.A.‡
Compound
Dose#
100 µM 25 µM 100 µg/mL 10 µg/mL 1 µg/mL
100 µg/mL
10 µg/mL
1 µg/mL
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
4a
4b
4c
5
26.0
11.0
5.0
19.0
19.0
31.0
5.0
19.0
35.0
5.0
na
na
na
na
na
na
na
na
na
na
na
na
48.2
46.6
34.3
ND
32.4
27.5
11.1
ND
27.5
20.9
0.0
ND
ND
7.1
6.0
19.7
9.1
ND
2a
2b
3a
3c
4c
5
7b
7c
Mtz
16.7
12.2
17.0
53.0
0.0
48.1
0.0
75.3
100.0
4.3
0.0
7.0
33.7
0.0
12.3
0.0
1.5
0.0
2.8
11.0
0.0
5.5
0.0
11.5
92.1
ND
ND
20.2
56.0
40.2
51.5
ND
17.2
13.0
30.8
38.4
ND
16.6
100.0
#
5.0
39.0
ND
ND
ND
Values are means of two experiments.
42.6
57.1
47.5
59.6
23.2
47.9
71.3
(87.5)
43.3
Ϫ
24.1
12.4
17.2
19.2
13.6
32.5
14.4
17.3
22.1
Ϫ
15.9
16.3
7.1
6.7
8.1
19.7
0.0
9.6
15.4
Ϫ
(92.1)
34.0 5.0
61.0 na
38.0 9.0
ND
5.0
34.0 5.0
61.0 9.0
19.0 10.0
process and the subsequent biological damage. In or-
der to explain the biological response observed we de-
termined these physicochemical properties potentially
related with the activity.
6
na
na
7a
7b
7c
7d
Lipophilicity studies [7, 22, 23]
Nfx 100.0 93.0
Mtz
Reversed-phase TLC experiments were performed for
all the derivatives on precoated TLC-C18 and eluted
with acetone:water (50:50, v/v). The Rf values were
converted into RM values via the relationship: RM = log
[(1/Rf)-1]. Nfx, the bio-assays reference compound,
was used as inter-assays reference. Table 3 summar-
izes ∆RM, defined as ∆RM = Nfx RM Ϫ derivative RM,
for each derivative.
Ϫ
Ϫ
(100.0)
(100.0)
# Values are means of two experiments. † % Inh. Ϫ
percentage of inhibition of T. cruzi growth at the
indicated doses at day 5. ‡ % C.A. Ϫ percentage of
inhibition of T. vaginalis growth at the indicated doses
at 24 h. na Ϫ not active, (% Inh. lower than 4.0%).
ND Ϫ not determined. In brackets Ϫ percentage of
reduction (% R).
Electrochemical studies [7, 24؊26]
The redox properties of N-oxide derivatives 2a, 2cϪf
and 3aϪc, 4a, 4c and 5 were studied using the cyclic
voltammetry methodology. The voltammetric re-
sponses of derivatives were comparable in DMF at a
platinum working electrode. Typically one or two well-
defined reduction peaks were observed during forward
cathodic scan. The first wave for all studied com-
pounds corresponds to a reversible one-electron
transfer. The reverse scan showed the anodic counter-
part of the reduction waves. The breadth of the ca-
thodic wave at its half intensity has a relatively con-
stant value of 60 mV. The ratio ipa/ipc has a value
near to one, so the one-electron reduction process is
reversible. Table 3 summarizes Epc for the first re-
duction step of each (benz)imidazole N-oxide deriva-
tive studied.
Structure-activity relationships
The anti-protozoa activity of (benz)imidazole deriva-
tives developed can be related to their lipophilicity, in
general the derivatives including lipophilic moieties in
their structures resulted the more active compounds.
Thus, compound 3a, an ethyl ester, was in general
less active than 4bϪc and 5, compounds bearing a
lipophilic-butyl and -hexyl chains, and 7bϪd deriva-
tives, with a NH function blocked with a NOCO2R moi-
ety, were in general more active than 3a bearing an
NH group. Also, the N-oxide bio-reduction can be re-
lated with the observed activity such as the nitro group
in Nfx or Mtz. Perhaps, in the present case the re-
duction potentials of the imidazole and benzimidazole
N-oxides are not adequate to allow the electronation
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264 Gonzalez et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Table 3. Physicochemical data and bioactivity of imidazole N-oxide derivatives.
†,‡
Compound
∆RM
Epc (V)†,#
Log10 (% Inh.)§
Log10 (% C.A.)††
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
4a
4b
4c
5
Ϫ0.09
Ϫ0.02
Ϫ0.04
Ϫ0.98
Ϫ1.11
Ϫ0.97
0.07
0.07
0.19
0.49
Ϫ0.09
0.13
Ϫ0.13
Ϫ0.49
Ϫ0.31
0.07
Ϫ0.17
Ϫ0.14
Ϫ0.23
Ϫ0.03
Ϫ1.27
Ϫ
1.41
1.04
0.70
1.28
1.28
1.49
0.70
1.28
1.54
0.70
0.70
1.59
1.53
1.79
1.58
ND
1.68
1.67
1.54
ND‡‡
ND
1.31
1.75
1.60
1.71
ND
Ϫ1.47
Ϫ1.45
Ϫ1.31
Ϫ1.50
Ϫ1.40
Ϫ1.49
Ϫ1.25
LS§§
LS
Ϫ1.24
ND
Ϫ1.19
Ϫ1.22
LS
ND
1.63
1.76
1.68
1.78
1.37
1.68
1.85
2.00
1.64
6
7a
7b
7c
7d
Ϫ
Ϫ
Ϫ
Ϫ
0.7
1.53
1.79
1.28
† Values are means of two experiments. ‡ ∆RM Ϫ Nifurtimox’s RMϪderivative’ RM. # Peak potentials ( 0.01 V)
measured at a scan rate of 0.2 V/s. § % Inh. Ϫ percentage of inhibition of T. cruzi growth at 100 µM at day 5.
†† % C.A. Ϫ percentage of inhibition of T. vaginalis growth at 100 µg/mL at 24 h. ‡‡ ND Ϫ not determined. §§ LS
Ϫ The low solubility did not permit to determine the redox potential in non-aqueous medium.
Conclusions
48 h for some derivatives (3c, 5, and 7c, Table 2).
Further, compound 7c showed trichomonacidal activity
at 100 µg/mL and 24 h of contact (Table 1, result in
brackets).
The imidazole N-oxide derivatives (compounds 2) pro-
ved to be the least actives against the protozoa T.
cruzi and T. vaginalis of the developed compounds, in
the assayed conditions.
Lipophilic-hydrophilic properties can be related to the
observed anti-protozoa activity of (benz)imidazole de-
rivatives. Analyzing the correlation between the deter-
mined ∆RM and the activities, we did not obtain a stat-
istically valid correlation. However, we observed a
parabolic or bilinear [27] tendency between the anti-
protozoa activities of derivatives (as expressed in
Table 3) and ∆RM (Figure 3). Derivatives 4c and 7c
showed the best anti-T. cruzi activity, appearing at the
maximum of the curve activity vs ∆RM (Figure 3A). In
the same manner derivatives 7c and 7b presented the
top of the curve showing trichomonacidal activity vs
∆RM (Figure 3B).
Some activity was displayed at the highest tested con-
centration (100 µM), against T. cruzi, however not
comparable to Nfx. The latter were more cytostatic
against T. vaginalis, 2a, 2b showing reduction in the
growth of parasite at the lowest assayed concentration
(1 µg/mL) at 24 h of contact. However, this activity was
not maintained for 48 h (see Table 2).
The benzimidazole derivatives, in the assayed con-
ditions, showed more activity against protozoa than
the imidazole counterparts.
Derivatives 3c, 4aϪ4c, 5, 7b and 7c displayed good
activity against T. cruzi at 100 mM, whereas com-
pound 7c, bearing a lipophilic substituent in the NO-
moiety was one of the best. Against T. vaginalis, benzi-
midazole derivatives showed high activity at 100 µg/
mL and 24 h of contact which was maintained up to
The N-oxide bio-reduction capacity of these deriva-
tives, expressed as Epc, can be related with the ob-
served activity. The reduction potential of the imida-
zole and benzimidazole N-oxide derivatives were
more negative, so the mono-electronation processes
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Novel Antiprotozoal Products 265
Figure 3. Lipophilicity vs anti-protozoa activity of studied compounds. Imidazole N-oxide derivatives (ᮀ), benzimi-
dazole N-oxide derivatives (l, ᭺ derivative 4c), O-substituted benzimidazole N-oxide derivatives (᭡, ᭝ derivative
7c, ᭞ derivative 7b). A. ∆RM vs trypanocidal in vitro activity. B. ∆RM vs trichomonacidal in vitro activity.
Figure 4. Electrochemical properties vs anti-protozoa activity of studied compounds. Imidazole N-oxide derivati-
ves (ᮀ), benzimidazole N-oxide derivatives (l, ᭺ derivative 4c). A. Epc vs trypanocidal in vitro activity. B. Epc vs
trichomonacidal in vitro activity.
are less favourable than for the reference compounds
Nfx and Mtz [24, 26, 28]. Statistic correlation between
Epc and bioactivities were not obtained but a clear
tendency was observed (Figure 4). Compounds bear-
ing more negative first reduction potentials showed
poor activity against both protozoa.
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266 Gonzalez et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Experimental
Chemistry
All starting materials were commercially available research-
grade chemicals and used without further purification. The
compounds I, VI, IX, X, 2a, 3a, 3d, 6, and 4-chloro-3-nitroben-
zoic acid were prepared according to literature procedures
[13, 15, 16, 32Ϫ37]. All solvents were dried and distilled prior
to use. All the reactions were carried out in a nitrogen atmos-
phere. The typical work-up included washing with brine and
drying the organic layer with magnesium sulphate before con-
centration. Melting points were determined using a Leitz
Microscope Heating Stage Model 350 apparatus (Wetzlar,
Germany) and are uncorrected. Elemental analyses were
obtained from vacuum-dried samples (over phosphorous
pentoxide at 3-4 mm Hg, 24 h at room temperature) and per-
formed on a Fisons EA 1108 CHNS-O analyzer (Valencia,
USA). 1H NMR and 13C NMR spectra were recorded on a
Bruker DPX-400 (at 400 MHz and 100 MHz, (Rehinstetten,
Germany)) instrument, with tetramethylsilane as the internal
reference and in the indicated solvent; the chemical shifts are
reported in ppm. Mass spectra were recorded on a Shimadzu
GC-MS QP 1100 EX instrument (Kyoto, Japan) at 70 eV.
Figure 5. Trypanosomatidae glycolysis inhibitor 8 and
derivative 7c.
Statistically significant multiple correlations between
physicochemical properties studied and the anti-proto-
zoa activities were not obtainable.
On the other hand, some of the more active deriva-
tives developed do not have the N-oxide moiety. De-
rivatives 7b and 7c showed high activity toward both
assayed protozoa, thus probably the lipophilic proper-
ties could be related with the bio-response. The in-
crement in lipophilicities of derivatives 7aϪd (compare
their ∆RM with that of derivative 3a, Table 3) allow
them access to the core of protozoa where the carbon-
ate moiety is hydrolyzed and they are transformed to
the corresponding N-oxide. Furthermore, the struc-
tures of derivatives 7aϪ7d have the structure of aden-
osine derivatives developed as trypanosomatidae gly-
colysis inhibitors [29-31] (i.e. 8, Figure 5). This mecha-
nism of action could be the form how compounds
7aϪd display their activities. Further studies of enzy-
matic O-substituted benzimidazole N-oxide deriva-
tives’ inhibition should be performed.
1-(4-Methylphenylamino)propanone oxime II
A mixture of chloroacetone (2.0 g, 21 mmol), p-toluidine (2.3
g, 21 mmol), K2CO3 (2.9 g, 21 mmol), KI (catalytic amounts)
in acetone (50 mL) was heated at reflux during 10 h. The
mixture was concentrated in vacuo and treated with Et2O.
After the work-up process the residue, 1.4 g (40%), was used
in the next step without purification. 1H NMR (CDCl3, 400
MHz) δ (ppm): 2.22 (s, 6H), 3.97 (s, 2H), 4.43 (bs, 1H), 6.52
(d, J = 8.3 Hz, 2H), 6.98 (d, J = 8.3 Hz, 2H). The correspond-
ing α-amine carbonyl derivative (0.16 g, 1 mmol), NH2OH.HCl
(0.07 g, 1 mmol), and anhydrous AcONa (0.13 g, 1 mmol) in
methanol (5 mL) were heated at reflux during 1 h and then
stirred at room temperature for 12 h. The mixture was con-
centrated in vacuo and treated with EtOAc. After the work-up
process the residue was used without further purification
(0.12 g, 70%).
A mixture of I (1.0 equiv.), the corresponding amine (1.5
These compounds are interesting leading substances
for further chemical modifications and biological stud-
ies. In fact, other studies allowing the elucidation of
the reaction mechanisms of these drugs as well as
schedules for toxicity determinations are currently in
progress.
equiv.), ZnCl2 (catalytic amounts), methanol as solvent and
˚
molecular sieve (4 A) was stirred for 24 h at room tempera-
ture. Then NaCNBH3 (1.0 equiv) was added and agitation
was continued for 48 h. The mixture was concentrated in va-
cuo and treated with EtOAc. After the work-up process the
residue was purified by chromatographic column (SiO2,
EtOAc:petroleum ether (1:1)).
General procedure for the synthesis of α-substituted amine-
oxime IIIϪV
Acknowledgments
3-(4-Methoxyphenylamino)-2-butanone oxime III
The present article is dedicated to the memory of our
friend and colleague Prof. Manfred Stud. Financial
support came from Fondo Clemente Estable and
Oil, 0.66 g (32%). 1H NMR (CDCl3, 400 MHz) δ (ppm): 1.38
(d, J = 6.8 Hz, 3H), 1.84 (s, 3H), 3.40 (bs, 1H), 3.75 (s, 3H),
4.07 (q, J = 6.8 Hz, 1H), 6.68 (m, 2H), 6.77 (m, 2H), 9.92
(bs, 1H).
´
´
´
Comision Sectorial de Investigacion Cientıfica (Uru-
guay). Y.F.ϪS. thanks the ICI-AECI (Spain) for a In-
terCampus-fellowship. Collaborative work was per-
formed under the auspices of the Iberoamerican Pro-
gram for Science and Technology (CYTED), SubProg-
ram X, network X-E, RIIDDMED.
3-(2-Phenylethylamino)-2-butanone oxime IV
Oil, 0.98 g (48%). 1H NMR (CDCl3, 400 MHz) δ (ppm): 1.22
(d, J = 6.7 Hz, 3H), 1.78 (s, 3H), 2.80 (m, 4H), 3.45 (q, J =
6.7 Hz, 1H), 7.22 (m, 3H), 7.28 (m, 2H).
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
3-(2-Furylmethylamino)-2-butanone oxime V
Novel Antiprotozoal Products 267
2.11 (s, 3H), 2.13 (s, 3H), 2.39 (s, 3H), 4.87 (s, 2H), 6.10 (d,
J = 3.4 Hz, 1H), 6.29 (d, J = 3.4 Hz, 1H), 7.33 (d, J = 3.4 Hz,
1H). 13C NMR (HMQC, HMBC) (methanol-d4, 100 MHz) δ
(ppm): 9.17, 12.71, 13.52, 41.07, 107.99, 110.78, 122.23,
131.58, 142.96, 143.03, 150.17. MS m/z (rel. int.%): 190
(M+. -16, 18.0), 109 (6.0), 81 (100.0). Anal. Calcd for
C11H14N2O2: C, 64.06; H, 6.84; N, 13.58. Found: C, 63.70;
H, 6.90; N, 13.22.
Oil, 0.86 g (46%). 1H NMR (CDCl3, 400 MHz) δ (ppm): 1.21
(d, J = 6.8 Hz, 3H), 1.84 (s, 3H), 3.43 (q, J = 6.8 Hz, 1H),
3.69 (s, 2H), 5.74 (bs, 2H), 6.17 (m, 1H), 6.29 (m, 1H), 7.34
(m, 1H), 10 (bs, 2H).
General Procedure for the Synthesis of Imidazole N-Oxide
2bϪ2f
Diethyl 3-oxide-1H-benzimidazole-2,5-dicarboxylate 3b
A mixture of oximes II-V, p-TsOH (catalytic amounts) and the
corresponding orthoester as solvent was heated at reflux until
the oxime was not present (SiO2, MeOH:EtOAc (4:6)). The
mixture was concentrated in vacuo and treated with EtOAc.
After the work-up process the residue was purified as indi-
cate.
Ethyl 4-chloro-3-nitrobenzoate: A mixture of 4-chloro-3-nitro-
benzoic acid (4.15 g, 20.6 mmol), H2SO4 (c) (0.3 mL, 6.2
mmol) and EtOH (20 mL) was heated at reflux for 4 h. The
mixture was concentrated in vacuo and the solid residue was
washed with water (4.85 g, 100%).
4-Methyl-1-(4-methylphenyl)imidazole N3-oxide 2b
Ethyl N-(4-ethoxycarbonyl-2-nitrophenyl)glycinate VII
Chromatographic column (SiO2, EtOAc:MeOH (0 to 40%));
beige solid (52%). H NMR (CDCl3, 400 MHz) δ (ppm): 2.32
A mixture of ethyl 4-chloro-3-nitrobenzoate (4.00 g, 17.5
mmol), ethyl glycinate hydrochloride (2.45 g, 17.5 mmol) and
NaHCO3 (2.93 g, 34.9 mmol) in EtOH (20 mL) was heated at
reflux during 6 h. The mixture was concentrated in vacuo and
treated with EtOAc/H2O. The aqueous phase was acidified
and treated with EtOAc. After the work-up process the resi-
due was crystallized from EtOH (0.5 g, 10%).
1
(s, 3H), 2.40 (s, 3H), 6.89 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H),
7.29 (d, J = 8.5 Hz, 2H), 8.17 (s, 1H). 13C RMN (HMQC,
HMBC) (CDCl3, 100 MHz) δ (ppm): 8.50, 21.34, 112.76,
121.04, 124.44, 131.12, 132.26, 135.00, 138.78. MS m/z (rel.
int.%): 188 (M•+, 40.0), 172 (8.0). mp(°C) 202.0 Ϫ 203.0.
Anal. Calcd for C11H12N2O: C, 70.19; H, 6.43; N, 14.88.
Found: C, 70.02; H, 6.27; N, 14.48.
Diethyl 3-oxide-1H-benzimidazole-2,5-dicarboxylate 3b
4,5-Dimethyl-1-(4-methoxyphenyl)imidazole N3-oxide 2c
Sodium (40.4 mg, 1.8 mmol) was dissolved in EtOH (10 mL)
and then VII (200 mg, 0.67 mmol) was added. The mixture
was stirred at room temperature for 12 h. Then it was concen-
trated in vacuo and treated with Et2O/H2O. The aqueous
phase was acidified. The solid was filtered and crystallized
from EtOH (148 mg, 79%). 1H NMR (CDCl3, 400 MHz) d
(ppm) (main tautomer): 1.46 (t, J = 7.1 Hz, 3H), 1.54 (t, J =
7.1 Hz, 3H), 4.46 (q, J = 7.1 Hz, 2H), 4.61 (q, J = 7.1 Hz,
2H), 7.86 (d, J = 8.7 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 8.39
(s, 1H), 11.00 (bs, 1H). 13C RMN (HMQC, HMBC) (CDCl3,
100 MHz) d (ppm) (main tautomer): 14.50, 14.74, 61.75,
64.14, 113.17, 122.04, 125.49, 128.51, 130.00, 134.66,
140.26, 162.55, 166.57. MS m/z (rel. int.%): 278 (M.+, 0.1),
206 (59.0), 178 (25.0), 161 (100.0). mp(°C) 189.9 Ϫ 190.0.
Anal. Calcd for C13H14N2O5: C, 56.11; H, 5.07; N, 10.07.
Found: C, 56.02; H, 5.20; N, 9.87.
Chromatographic column (SiO2, EtOAc:MeOH (0 to 40%));
beige oil (20%). 1H NMR (methanol-d4, 400 MHz) δ (ppm):
2.11 (s, 3H), 2.24 (s, 3H), 3.88 (s, 3H), 7.11 (d, J = 6.8 Hz,
2H), 7.36 (d, J = 6.8 Hz, 2H), 8.32 (s, 1H). 13C NMR (HMQC,
HMBC) (methanol-d4, 100 MHz) δ (ppm): 6.13, 8.04, 55.18,
115.03, 123.70, 126.30, 127.57, 127.74, 129.09, 161.08. MS
m/z (rel. int.%): 218 (M+, 100.0), 202 (49.0), 148 (72.0), 107
(15.0). Anal. Calcd for C12H14N2O2: C, 66.04; H, 6.47; N,
12.84. Found: C, 65.89; H, 6.38; N, 12.50.
4,5-Dimethyl-1-(2-phenylethyl)imidazole N3-oxide 2d
Chromatographic column (SiO2, EtOAc:MeOH (0 to 40%));
brown oil (6%). 1H NMR (methanol-d4, 400 MHz) δ (ppm):
2.04 (s, 3H), 2.18 (s, 3H), 3.00 (t, J = 7.3 Hz, 2H), 4.05 (t,
J = 7.3 Hz, 2H), 7.28 (m, 5H), 7.36 (s, 1H). 13C NMR (HMQC,
HMBC) (methanol-d4, 100 MHz) δ (ppm): 8.56, 12.60, 37.95,
56.14, 122.50, 127.44, 129.04, 129.07, 132.50, 134.81,
137.70. MS m/z (rel. int.%): 200 (M+. -16, 100.0), 138 (28.0),
111 (42.0), 105 (47.0). Anal. Calcd for C13H16N2O: C, 72.19;
H, 7.46; N, 12.95. Found: C, 71.98; H, 7.29; N, 12.72.
Ethyl 5-methyl-3-oxide-1H-benzimidazole-2-carboxylate 3c
Ethyl N-(4-methyl-2-nitrophenyl)glycinate VIII
A mixture of 4-methyl-2-nitroaniline (1.00 g, 6.6 mmol), ethyl
2-bromoacetate (0.73 mL, 6.6 mmol) and NaHCO3 (1.10 g,
13.2 mmol) in acetone (10 mL) was heated at reflux during
6 d. The mixture was concentrated in vacuo and treated with
EtOAc/H2O. After the work-up process the residue was puri-
fied by chromatography (SiO2, petroleum ether:EtOAc (9:1)).
Orange solid (0.48 g, 30%).
2,4,5-Trimethyl-1-(2-phenylethyl)imidazole N3-oxide 2e
Chromatographic column (SiO2, EtOAc:MeOH (0Ϫ40%));
brown oil (54%). 1H NMR (methanol-d4, 400 MHz) δ (ppm):
2.00 (s, 3H), 2.10 (s, 6H), 2.87 (t, J = 8.0 Hz, 2H), 3.91 (t,
J = 8.0 Hz, 2H), 7.03 (d, J = 7.4 Hz, 2H), 7.26 (m, 3H). 13C
NMR (HMQC, HMBC) (methanol-d4, 100 MHz) δ (ppm): 9.10,
12.71, 13.26, 37.33, 45.80, 121.78, 127.30, 129.13, 129.19,
131.50, 138.07, 142.59. MS m/z (rel. int.%): 214 (M+. -16,
98.0), 123 (100.0), 104 (31.0), 82 (21.0). Anal. Calcd for
C14H18N2O: C, 73.01; H, 7.88; N, 12.16. Found: C, 72.66; H,
7.57; N, 11.95.
Ethyl 5-methyl-3-oxide-1H-benzimidazole-2-carboxylate 3c
Sodium (88.1 mg, 3.8 mmol) was dissolved in EtOH (7.3 mL)
and then VIII (176.2 mg, 0.74 mmol) in DMF (0.44 mL) was
added. The mixture was stirred at room temperature during
4 h. Then it was concentrated in vacuo and treated with Et2O/
H2O. The aqueous phase was acidified and extracted with
EtOAc. After the work up the organic phase was purified by
chromatography (SiO2, petroleum ether:EtOAc (8:2)). Red
solid (28.5 mg, 18%). 1H NMR (CDCl3, 400 MHz) δ (ppm)
(main tautomer): 1.49 (t, J=6.9 Hz, 3H), 2.50 (s, 3H), 4.55 (q,
2,4,5-Trimethyl-1-(furylmethyl)imidazole N3-oxide 2f
Chromatographic column (SiO2, EtOAc:MeOH (0 to 40%));
orange oil (42%). 1H NMR (methanol-d4, 400 MHz) δ (ppm):
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´
268 Gonzalez et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
J=6.0 Hz, 2H), 7.19 (d, J=8.0 Hz, 1H), 7.55 (d+s, J=7.6 Hz,
2H), 9.40 (bs, 1H). 13C RMN (HMQC, HMBC) (CDCl3, 100
MHz) δ (ppm) (main tautomer): 14.57, 22.05, 64.20, 113.04,
126.81, 126.97, 128.30, 128.93, 138.36, 147.52, 153.02.
mp(°C) 221.0 Ϫ 222.0. Anal. Calcd for C11H12N2O3: C, 59.99;
H, 5.49; N, 12.72. Found: C, 59.73; H, 5.33; N, 12.83.
N-(n-Butyl) 5-nitro-3-oxide-1H-benzimidazole-2-carboxamide 5
A mixture of 3a (50 mg, 0.20 mmol), n-butylamine (0.02 mL,
0.20 mmol), p-TsOH (catalytic amounts), molecular sieves (4
˚
A) and toluene (5.0 mL) as solvent was heated at reflux for
12 h. After the work-up, the organic phase was purified by
chromatography (SiO2, petroleum ether: EtOAc (9:1)). Brown
solid (10.5 mg, 19%). Ratio of tautomers 5:5Ј in CDCl3 at
30°C was 55:45. Tautomer 5: 1H NMR (CDCl3, 30°C) δ
(ppm): 1.02 (t, J=7.3 Hz, 3H), 1.51 (sext, J = 7.5 Hz, 2H),
1.75 (quint, J=7.0 Hz, 2H), 3.62 (q, J = 7.0 Hz, 2H), 7.68 (d,
J = 9.0 Hz, 1H), 7.74 (bs, 1H), 8.33 (dd, J1 = 9.0 Hz, J2 = 2.0
Hz, 1H), 8.74 (d, J=2.0 Hz, 1H), 12.07 (bs, 1H); 13C NMR
(CDCl3, 30 sC) δ (ppm): 14.06, 20.49, 31.91, 40.12, 112.86,
117.87, 120.76, 138.61, 142.62, 144.94, 148.68, 158.91. Tau-
tomer 5Ј: 1H NMR (CDCl3, 30 sC) δ (ppm): 1.02 (t, J = 7.3
Hz, 3H), 1.51 (sext, J=7.5 Hz, 2H), 1.73 (quint, J = 7.0 Hz,
2H), 3.60 (q, J = 7.0 Hz, 2H), 7.74 (bs, 1H), 7.88 (d, J = 9.0
Hz, 1H), 8.27 (dd, J1 = 9.0 Hz, J2 = 2.2 Hz, 1H), 8.57 (d, J =
2.0 Hz, 1H), 12.31 (bs, 1H); 13C NMR (CDCl3, 30°C) δ (ppm):
14.06, 20.49, 31.91, 40.12, 109.81, 119.24, 121.21, 131.00,
145.53, 147.30, 149.45, 158.91. MS m/z (rel. int.%): 278 (M+,
22.0), 262 (20.2), 261 (72.2), 249 (11.4), 235 (18.5). mp(°C)
206.7 Ϫ 207.6. Anal. Calcd for C12H14N4O4: C, 51.80; H,
5.07; N, 20.13. Found: C, 51.48; H, 5.00; N, 19.98.
1-Hydroxy-2,5-dimethyl-3-oxide-benzimidazole 3e
Compound X (1.0 equiv.) was dissolved in THF (7.3 mL) and
then nitromethane (1.2 equiv.) and piperidine (1.2 equiv.)
were added. The mixture was stirred at room temperature
during 12 h. Then the solid was collected by filtration and
crystallized from MeOH. White solid (34%). It was not pos-
sible to acquire NMR spectra due to the insolubility of 3e in
the assayed solvents (CDCl3, CD3OD, DMSO-d6, and D2O).
MS m/z (rel. int.%): 178 (M.+, 11.0), 177 (32.0), 145 (32.0),
131 (8.0). mp(°C) 224.6 Ϫ 225.6 (d). Anal. Calcd for
C9H10N2O2: C, 60.66; H, 5.66; N, 15.72. Found: C, 61.00; H,
6.02; N, 15.89.
General procedure for the synthesis of derivatives 4aϪ4c
A mixture of 3a, catalytic amounts of p-TsOH, molecular si-
˚
eves (4 A) and the corresponding alcohol (i-PrOH, n-butanol,
and n-hexanol) as solvent was heated at reflux during the
time indicate below. The solvent was distilled in vacuo. After
the work-up the residue was purified by chromatography
(SiO2, petroleum ether:EtOAc).
General Procedure for the synthesis of derivatives 7aϪ7d
A mixture of 3a (50.0 mg, 0.21 mmol), the corresponding
electrophile reactants (methyl iodide, ethyl chloroformate, i-
butyl chloroformate, 2,2,2-trichloroethyl chloroformate, 1.0
equiv.), NaHCO3 (only when CH3I was used as electrophile)
(2.0 equiv.) and acetone (3.0 mL) as solvent, was stirred at
room temperature for the time indicated below. The solvent
was distilled in vacuo. After the work-up the residue was puri-
fied by chromatography (SiO2, petroleum ether:EtOAc).
Isopropyl 5-nitro-3-oxide-1H-benzimidazole-2-carboxylate 4a
Time reflux: 4 h. Brown solid (72%). 1H NMR (DMSO-d6:D2O,
400 MHz) d (ppm) (main tautomer): 1.39 (d, J = 6.2 Hz, 6H),
5.27 (h, J = 6.2 Hz, 1H), 7.96 (d, J = 9.0 Hz, 1H), 8.17 (dd,
J1 = 9.0 Hz, J2 = 2.2 Hz, 1H), 8.40 (d, J = 2.2 Hz, 1H). 13C
NMR (HMQC, HMBC) (CDCl3, 100 MHz) δ (ppm) (main tau-
tomer): 22.35, 71.08, 108.03, 119.16, 122.84, 132.84, 141.49,
143.12, 145.46, 157.81. MS m/z (rel. int.%): 265 (M.+, 20.3),
249 (4.2), 223 (100.0), 205 (80.8). mp(°C) 172.8 Ϫ 173.4.
Anal. Calcd for C11H11N3O5: C, 49.81; H, 4.18; N, 15.84.
Found: C, 49.55; H, 3.99; N, 15.60.
Ethyl 1-methoxy-6-nitrobenzimidazole-2-carboxylate 7a
Time: 7 d. Yellow solid (100%). 1H NMR (CDCl3, 400 MHz)
δ (ppm): 1.53 (t, J=7.0 Hz, 3H), 4.38 (s, 3H), 4.59 (q, J = 7.1
Hz, 2H), 7.97 (d, J = 9.0 Hz, 1H), 8.27 (d, J = 9.0 Hz, 1H),
8.52 (s, 1H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 14.57,
63.47, 68.08,107.10, 119.80, 123.31, 131.00, 134.00, 141.50,
146.00, 158.00. MS m/z (rel. int.%): 265 (M+, 100.0), 248
(3.0), 190 (62.0). mp(°C) 155.0 Ϫ 156.0. Anal. Calcd for
C11H11N3O5: C, 49.81; H, 4.18; N, 15.84. Found: C, 50.02; H,
4.52; N, 15.55.
n-Butyl 5-nitro-3-oxide-1H-benzimidazole-2-carboxylate 4b
Time reflux: 1 h. Yellow solid (68%). 1H NMR (CDCl3, 400
MHz) δ (ppm) (main tautomer): 1.03 (t, J = 7.4 Hz, 3H), 1.55
(m, 2H), 1.91 (m, 2H), 4.61 (t, J = 6.8 Hz, 2H), 7.96 (d, J =
9.1 Hz, 1H), 8.27 (dd, J1 = 9.1 Hz, J2=2.2 Hz, 1H), 8.62 (d,
J = 2.2 Hz, 1H), 9.00 (bs, 1H). 13C NMR (CDCl3, 100 MHz)
δ (ppm) (main tautomer): 13.97, 19.39, 30.77, 68.37, 108.02,
119.65, 123.10, 129.30, 136.00, 140.87, 146.08, 162.50. MS
m/z (rel. int.%): 279 (M.+, 14.0), 262 (59.0), 223 (75.0), 205
(100.0). mp(°C) 189.2 Ϫ 190.0. Anal. Calcd for C12H13N3O5:
C, 51.61; H, 4.69; N, 15.05. Found: C, 51.43; H, 4.35; N,
14.78.
Ethyl 1-ethyloxycarbonyloxy-6-nitrobenzimidazole-2-car-
boxylate 7b
Time: 30 min. Yellow solid (30%). 1H NMR (CDCl3, 400 MHz)
δ (ppm): 1.49 (t, J = 7.1 Hz, 3H), 1.53 (t, J = 7.1 Hz, 3H),
4.56 (q, J=7.1 Hz, 4H), 8.02 (d, J = 9.0 Hz, 1H), 8.31 (dd, J1=
9.0 Hz, J2 = 2.1 Hz, 1H), 8.44 (d, J = 2.0 Hz, 1H). 13C NMR
(CDCl3, 100 MHz)
δ
(ppm): 14.47, 14.50, 63.69,
68.86,106.88, 120.14, 123.50, 131.31, 133.00, 140.85,
146.74, 152.69, 157.24. MS m/z (rel. int.%): 323 (M+, 1.0),
307 (0.2), 279 (5.0), 251 (53.0). mp(°C) 133.3 Ϫ 134.3. Anal.
Calcd for C13H13N3O7: C, 48.30; H, 4.05; N, 13.00. Found:
C, 47.98; H, 3.92; N, 12.78.
n-Hexyl 5-nitro-3-oxide-1H-benzimidazole-2-carboxylate 4c
Time reflux: 3 h. Brown solid (61%). 1H NMR (DMSO-d6:
D2O, 400 MHz) d (ppm) (main tautomer): 0.86 (t, J = 7.0 Hz,
3H), 1.25 (m, 2H), 1.30 (m, 2H), 1.41 (m, 2H), 1.74 (quint,
J = 7.0 Hz, 2H), 4.38 (t, J = 6.6 Hz, 2H), 7.96 (d, J = 9.0 Hz,
1H), 8.16 (dd, J1 = 9.0 Hz, J2 = 2.2 Hz, 1H), 8.40 (d, J = 2.1
Hz, 1H). MS m/z (rel. int.%): 307 (M+, 4.5), 291 (18.6), 290
(100.0), 277 (20.8). mp(°C) 160.2 Ϫ 161.8. Anal. Calcd for
C14H17N3O5: C, 54.72; H, 5.58; N, 13.67. Found: C, 54.31;
H, 5.23; N, 13.44.
Ethyl 1-(i-butyloxycarbonyloxy)-6-nitrobenzimidazole-2-car-
boxylate 7c
Time: 60 min. Yellow solid (74%). 1H NMR (CDCl3, 400 MHz)
δ (ppm): 1.08 (d, J = 6.7 Hz, 3H), 1.50 (t, J = 7.1 Hz, 3H),
2.19 (m, J = 6.7 Hz, 1H), 4.28 (d, J = 6.6 Hz, 2H), 4.56 (q,
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
Novel Antiprotozoal Products 269
J = 7.1 Hz, 2H), 8.03 (d, J = 9.0 Hz, 1H), 8.31 (dd, J1 = 9.0
Hz, J2 = 2.1 Hz, 1H), 8.44 (d, J = 2.0 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ (ppm): 14.54, 19.04, 28.27, 63.69, 78.44,
106.85, 120.13, 123.52, 131.33, 140.91, 146.75, 148.88,
152.85, 157.23. MS m/z (rel. int.%): 351 (M+, 0.1), 336 (9.0),
num wire auxiliary electrode, and a saturated calomel refer-
ence electrode. Voltage scan rates ranged from 0.05-1.0 V/s.
Lipophilicity studies
Reversed-phase TLC experiments were performed on pre-
coated TLC plates SIL RP-18W/UV254 (Macherey-Nagel,
Düren, Germany) and eluted with acetone:water (50:50, v/v).
The plates were developed in a closed chromatographic tank,
dried and the spots were located under UV light. The Rf val-
ues were averaged from two to three determinations, and
converted into RM.
251 (100.0). mp(°C) 112.5
Ϫ 113.0. Anal. Calcd for
C15H17N3O7: C, 51.28; H, 4.88; N, 11.96. Found: C, 50.99;
H, 4.65; N, 11.65.
Ethyl 1-(2,2,2-trichloroethyloxycarbonyloxy)-6-nitrobenzimid-
azole-2-carboxylate 7d
Time: 10 min. Yellow solid (76%). 1H NMR (CDCl3, 400 MHz)
δ (ppm): 1.51 (t, J = 7.2 Hz, 3H), 4.57 (q, J = 7.2 Hz, 2H),
5.05 (s, 2H), 8.06 (d, J = 9.0 Hz, 1H), 8.35 (dd, J1 = 9.0 Hz,
J2 = 2.1 Hz, 1H), 8.45 (d, J = 2.0 Hz, 1H). 13C NMR (CDCl3,
100 MHz) δ (ppm): 14.51, 63.88, 79.34, 93.34, 106.65,
120.43, 123.73, 131.07, 140.54, 140.78, 146.95, 152.20,
References
[1] a) J. W. Tracy, T. Webster, in Goodman Gilman, The
Pharmacological Basis of Therapeutics (Eds.: J. G.
Hardman, L. E. Limbird), McGraw-Hill, New York, 2001,
pp. 1059Ϫ1060. b) http://www.who.int/whr/2001.
157.18. mp(°C) 121.3
Ϫ
121.8. Anal. Calcd for
C13H10Cl3N3O7: C, 36.60; H, 2.36; N, 9.85. Found: C, 36.20;
H, 2.15; N, 9.52.
[2] J. Aldunate, A. Morello, in Free Radicals in Tropical Dis-
eases (Ed.: O. I. Aruoma), Harwood Academic Pub-
lishers, London, 1993, pp. 137Ϫ140.
Biology
Anti-trypanosomal bioassays
´
[3] H. Cerecetto, M. Gonzalez, Current Topic Med. Chem.
2002, 2, 1185Ϫ1211.
T. cruzi epimastigotes (Tulahuen 2 strain) were grown at
28°C in an axenic medium (BHI-Tryptose), complemented
with 10% foetal calf serum. Cells from a 5-day old culture
were inoculated into 50 mL of fresh culture medium to give
an initial concentration of 1·106 cells/mL. Cell growth was fol-
lowed by measuring the absorbance of the culture at 600 nm
daily for 11 d. Before inoculation, the media was sup-
plemented with 25 µM or 100 µM of compounds from a stock
solution in DMSO and their ability to inhibit growth of the para-
site was evaluated in comparison to the control (no drug ad-
ded to the media). The final concentration of DMSO in the
culture media never exceeded 0.4% and the control was run
in the presence of 0.4% DMSO and in the absence of any
drug. No effect on epimastigotes growth was observed by the
presence of up to 1% DMSO in the culture media. Nfx was
used as reference drug.
[4] C. Cosar, P. Ganter, L. Jolou, Presse Med. 1961, 69,
1069.
[5] S. N. J. Moreno, R. P. Masson, R. Docampo, J. Biol.
Chem. 1984, 259, 8252Ϫ8259.
[6] K. M. Land, P. J. Johnson, Exp. Parasitol. 1997, 87, 305.
´
[7] H. Cerecetto, R. Di Maio, M. Gonzalez, M. Risso, P. Sa-
enz, G. Seoane, A. Denicola, G. Peluffo, C. Quijano, C.
Olea-Azar, J. Med. Chem. 1999, 42, 1941Ϫ1950.
´
[8] G. Aguirre, H. Cerecetto, R. Di Maio, M. Gonzalez, G.
Seoane, A. Denicola, M. A. Ortega, I. Aldana, A. Monge,
Arch. Pharm. 2002, 335, 15Ϫ21.
[9] J. H. Elwell, B. G. Siim, J. W. Evans, J. M. Brown, Bio-
chem. Pharmacol. 1997, 54, 249Ϫ257.
Anti-trichomonas bioassays
´
[10] H. Cerecetto, M. Gonzalez, Mini-Review Med. Chem.
Trichomonacidal in vitro activity was developed using recently
isolated strain JH31A no. 4 of T. vaginalis in TYM medium
(Trypticase-Yeast extract-Maltose) supplemented with 10%
of heat-inactivated equine serum at 37°C with 5% CO2. The
assays were carried out using glass tubes containing 100,000
protozoa/mL in a final volume of 2 mL. Compounds were ad-
ded to the cultures 6 h after seeding. For each concentration
assayed, there were three experimental and six growth con-
trols. Viable protozoa were assessed 24 and 48 h after incu-
bation in the presence of the compounds by counting in an
haemocytometer. Mtz (Rhone Poulenc, Courbevoie, France)
was used as reference drug.
2001, 1, 219Ϫ231.
[11] H. Lettau, P. Nuhn, R. Schneider, P. Stenger, Pharmazie
1990, 45, 830.
[12] R. Hossbach, H. Lettau, P. Nuhn, R. Schneider, P.
Stenger, B. Stiebitz, Pharmazie 1991, 46, 412Ϫ415.
´
[13] J. Alcazar, M. Begtrup, A. de la Hoz, J. Chem. Soc. Per-
kin Trans. I 1995, 2467Ϫ2470.
´
[14] J. Alcazar, M. Begtrup, A. de la Hoz, Heterocycles
1996, 1465Ϫ1470.
[15] I. Harvey, M. Mc Farlane, D. Moody, D. Smith, J. Chem.
Soc. Perkin Trans. I 1988, 681Ϫ689.
Electrochemical method
Voltammetric responses for derivatives were obtained by cy-
clic voltammetry. Experiments were carried out using a BAS-
Epsilon EC (Bioanalytical Systems, Inc., West Lafayette,
USA) instrument in a BAS C3 cell, in N,N-dimethylformamide
(Aldrich, Milwaukee, USA, spectroscopy grade) with tetrabu-
tylammonium perchlorate (Fluka, Buchs, Switzerland) (ca.
0.1 mol/mL) as the supporting electrolyte and purged with
nitrogen at room temperature. A three-electrode cell configu-
ration was used, with a platinum working electrode, a plati-
[16] M. Abu El-Hav, J. Org. Chem. 1972, 37, 2519Ϫ2520.
[17] S. Chua, A. Katritzky, M. Cook, J. Chem. Soc. (B)
1971, 2350Ϫ2355.
[18] J. F. Faucher, T. Baltz, K. G. Petry, Parasitol. Res. 1995,
81, 441Ϫ443.
[19] M. Almeida-de-Faria, E. Freymuller, W. Colli, M. J.
Alves, Exp. Parasitol. 1999, 92, 263Ϫ274.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

´
270 Gonzalez et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 259−270
[20] K. M. Tyler, D. M. Engman, Int. J. Parasitol. 2001, 31,
472Ϫ481.
[29] A. M. Aronov, C. L. M. J. Verlinde, W. G. J. Hol, M. H.
Gelb, J. Med. Chem. 1998, 41, 4790Ϫ4799.
´
[21] A. Herrero, C. Ochoa, J. Atienza, J. A. Escario, A. Go-
[30] A. M. Aronov, S. Suresh, F. S. Buckner, W. C. Van
Voorhis, C. L. M. J. Verlinde, F. R. Opperdoes, W. G.
J. Hol, M. H. Gelb, Proc. Natl. Acad. Sci. USA. 1999,
96, 4273Ϫ4278.
´
´
mez-Barrio, A. R. Martınez Fernandez, Arch. Pharm.
1992, 325, 509Ϫ514.
[22] A. Tsantili-Kakoulidou, A. Antoniadou-Vyza, J. Chroma-
togr. 1988, 448, 317Ϫ326.
[31] J. C. Bressi, J. Choe, M. T. Hough, F. S. Buckner, W. C.
Van Voorhis, C. L. M. J. Verlinde, W. G. J. Hol, M. H.
Gelb, J. Med. Chem. 2000, 43, 4135Ϫ4150.
[23] W. A. Denny, J. A. Graham, P. B. Roberts, R. F. Ander-
son, M. Boyd, C. J. L. Lock, W. R. Wilson, J. Med. Chem.
1992, 35, 4832Ϫ4841.
´
[32] H. Cerecetto, M. Gonzalez, S. Onetto, M. Risso, P. Sa-
enz, G. Seoane, A. M. Bruno, J. Alarcon, C. Olea-Azar,
A. Lopez de Cerain, O. Ezpeleta, A. Monge, Med. Chem.
Res. 2001, 10, 328Ϫ337.
[24] H. Cerecetto, B. Mester, S. Onetto, G. Seoane, M. Gon-
´
´
´
zalez, F. Zinola, Il Farmaco 1992, 47, 1207Ϫ1213.
´
´
[25] A. Monge, J. A. Palop, A. Lopez de Cerain, V. Senador,
[33] R. K. Harris, A. R. Katritzky, S. Oksne, A. S. Bailey, W.
´
´
F. J. Martınez-Crespo, Y. Sainz, S. Narro, E. Garcıa, C.
de Miguel, M. Gonzalez, E. Hamilton, A. J. Barker, E.
G. Paterson, J. Chem. Soc. 1963, 197Ϫ199.
´
D. Clarke, D. T. Greenhow, J. Med. Chem. 1995, 38,
[34] A. J. Boulton, A. R. Katritzky, M. J. Sewell, B. Wallis, J.
Chem. Soc. B. 1967, 9, 914Ϫ916.
1786-1792.
´
[26] C. Olea-Azar, C. Rigol, F. Mendizabal, R. Briones, H.
[35] A. J. Boulton, D. Middleton, J. Org. Chem. 1974, 39,
2956Ϫ2962.
´
Cerecetto, R. Di Maio, M. Gonzalez, W. Porcal, M.
Risso, M. Spectrochimica Acta Part A. 2003, 59, 69Ϫ74.
[36] Vogel’s Textbook of Practical Organic Chemistry,4th
edn., Longman Group, London, 1978, pp. 821Ϫ822.
[27] H. Kubinyi, J. Med. Chem. 1977, 20, 625Ϫ629.
´ ˜
´
[28] S. Bollo, L. J. Nunez-Vergara, M. Bonta, G. Chauviere,
´
´
J. Perie, J. A. Squella, J. Electroanal. Chem. 2001,
511, 46Ϫ54.
[37] H. King, W. O. Murch, J. Chem. Soc. 1925, 2632.
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim