1,2,5-Oxadiazole N-oxide derivatives and related compounds as potential antitrypanosomal drugs: Structure-activity relationships

Article abstract of DOI:10.1021/jm9805790

The syntheses of a new series of derivatives of 1,2,5-oxadiazole N- oxide, benzo[1,2-c]1,2,5-oxadiazole N-oxide, and quinoxaline di-N-oxide are described. In vitro antitrypanosomal activity of these compounds was tested against epimastigote forms of Trypa

Full text of DOI:10.1021/jm9805790

J . Med. Chem. 1999, 42, 1941-1950
1941
1,2,5-Oxa d ia zole N-Oxid e Der iva tives a n d Rela ted Com p ou n d s a s P oten tia l
An titr yp a n osom a l Dr u gs: Str u ctu r e-Activity Rela tion sh ip s
Hugo Cerecetto,*,† Rossanna Di Maio,† Mercedes Gonza´lez,† Mariela Risso,† Patricia Saenz,† Gustavo Seoane,*,†
Ana Denicola,‡ Gonzalo Peluffo,‡, Celia Quijano,‡, and Claudio Olea-Azar§
Department of Organic Chemistry, Faculty of Chemistry, and Department of Biochemistry, Faculty of Medicine,
University of the Republic, CC 1157, 11800 Montevideo, Uruguay, Department of Physical Biochemistry,
Faculty of Sciences, University of the Republic, 11200 Montevideo, Uruguay, and Department of Inorganic and
Analytical Chemistry, Faculty of Pharmacy, University of Chile, Santiago, Chile
Received October 12, 1998
The syntheses of a new series of derivatives of 1,2,5-oxadiazole N-oxide, benzo[1,2-c]1,2,5-
oxadiazole N-oxide, and quinoxaline di-N-oxide are described. In vitro antitrypanosomal activity
of these compounds was tested against epimastigote forms of Trypanosoma cruzi. For the most
effective drugs, derivatives IIIe and IIIf, the 50% inhibitory dose (ID50) was determined as
well as their cytotoxicity against mammalian fibroblasts. Electrochemical studies and ESR
spectroscopy show that the highest activities observed are associated with the facile mono-
electronation of the N-oxide moiety. Lipophilic-hydrophilic balance of the compounds could
also play an important role in their effectiveness as antichagasic drugs.
In tr od u ction
ized using ESR spectroscopy.12,13 The derivatives 1 and
2 showed interesting in vitro trypanocidal activity but
failed to be good trypanocidal agents in vivo because of
their inherent toxicity (principally 2).14 The undesirable
toxicity of 1 and 2 on host is probably due to the nitro
moiety which acts as a nonselective redox-damaging
function.
Trypanosoma cruzi (T. cruzi) is the etiologic agent of
Chagas’ disease.1,2 Trypanosomiasis and leishmaniasis
are major third-world diseases, with several millions of
new infections presenting annually. About 100 000
people in the United States are also infected, probably
due to transfusion of blood products originating from
South America.3 The current chemotherapy against
Chagas’ disease is still inadequate. The main drug in
use is Nifurtimox (Nfx); however, it has undesirable side
effects4,5 and is yet inefficient to treat chronic Chagas’
disease, which can be regarded as a disease with no
cure. A characteristic ESR signal corresponding to the
nitro anion radical (R-NO2•-) appears when Nfx is added
to intact T. cruzi cells,6 suggesting that intracellular
reduction of Nfx followed by redox cycling yielding
superoxide anion may be its major mode of action
against T. cruzi.5-7
Similar to the nitro pharmacophore of antitrypano-
somal drugs, the N-oxide moiety has proved to be
responsible for the biological activity of numerous drugs
(such as antitumoral, antibacterial) through the produc-
tion of free radical species.15,16 These data suggested the
possibility of designing new antitrypanosomal com-
pounds where the N-oxide moiety would be the phar-
macophore that generates the radical toxic species.
Herein we report on the preparation and biological
evaluation of compounds derived from 1,2,5-oxadiazole
N-oxide, benzo[1,2-c]1,2,5-oxadiazole N-oxide, quinoxa-
line 1,4-di-N-oxide, and some reduced analogues. We
have also performed electrochemical measurements,
ESR spectroscopy, and relative lipophilicity to study
structure-activity relationships.
Our general strategy for the design of new structures
was based on the conjunction of N-oxide systems and
the semicarbazide moieties present in 1 and 2 (See
Chart 1). Initially we chose 1,2,5-oxadiazole N-oxide as
the heteroaromatic N-oxide system, due to the struc-
tural similarity with the nitrofuran heterocycle. Then
we extended the studies over benzo[1,2-c]1,2,5-oxadia-
zole N-oxide and quinoxaline di-N-oxide. Subsequently,
“spermidine-mimetic” residues substituted the N4-semi-
carbazide moiety. Previous work17-19 supported the idea
of designing compounds which bear a positive charge
and/or a flexible side chain “spermidine-like”, capable
of inhibiting trypanothione reductase (TR). TR is a
critical enzyme for the parasite, responsible of cata-
lyzing the reduction of trypanothione disulfide to try-
panothione, which participates in free radical- and
oxygen-derived species detoxification.20-22 In addition,
We have previously reported10,11 the synthesis and
biological activity against T. cruzi epimastigote forms
of a series of 5-nitrofurfural and 5-nitrothiophene-2-
carboxaldehyde derivatives. These compounds proved
to generate nitro anion radicals, which were character-
† Department of Organic Chemistry, University of the Republic.
‡ Department of Physical Biochemistry, University of the Republic.
Department of Biochemistry, University of the Republic.
§ University of Chile.
10.1021/jm9805790 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/06/1999

1942 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Cerecetto et al.
Ch a r t 1
the more active derivatives were deoxygenated in order
to corroborate the pharmacological action of the N-oxide
group.
known procedure.11 Thus, 13 and 14 were obtained from
commercially available 3-(dimethylamino)propylamine
and 3-(diethylamino)propylamine by treatment with
phenyl chloroformate to give the corresponding carbam-
ates and further reaction with hydrazine. The amides
IIIe-j were obtained from acid 10, which was succes-
sively reacted with thionyl chloride at reflux and the
corresponding amine.
Ch em istr y
The 1,2,5-oxadiazole derivatives were obtained as
shown in Scheme 1. Crotonaldehyde was treated with
sodium nitrite in acetic acid to afford the basic system
3, which was converted to the corresponding semicar-
bazone derivatives Ia ,b in moderate yields.11,23 Reduc-
tive amination of 3 with “spermidine-mimetic” amines
gave Ic,d .24 These two amines were prepared by treat-
ment of simple amines (morpholine or piperidine) with
acrylonitrile,25 followed by hydrogenation (Pd/Ni/Ac2O)
and hydrolysis.26 Reaction of cinnamyl alcohol with
sodium nitrite in acetic acid afforded the basic system
4.27 Alcohol 4 was converted into the aldehyde 5 by
oxidation with manganese dioxide.27 This compound,
treated with semicarbazides (11, 12, see Scheme 1),
produced the compounds IIa ,b in moderate yields.11
Also, treatment of 4 with thionyl chloride at room
temperature gave the compound 6, which was trans-
formed into compounds IIc,d by reaction with the
corresponding amines in boiling tetrahydrofuran.28
The benzo[1,2-c]1,2,5-oxadiazole system was prepared
using the appropriate nitrophenyl azides 7 and 8.29-31
Cyclocondensation of these azides in boiling toluene
yielded the heterocycles 9 and 10.32 The semicarbazones
IIIa -d were obtained in variable yields (30-70%),
using the corresponding semicarbazide reactants 13 and
14 (Scheme 1), which were prepared according to a
The compounds IIIa -j (and also Va ,b) exist as a
mixture of isomers at room temperature (III and III′):
This phenomenon was observed through the corre-
sponding NMR spectra (proton and carbon), which
showed complex groups of signals in the aromatic zone
(7.20-8.30 and 110-155 ppm, respectively) at room
temperature. The spectra simplified at higher temper-
ature, where one of these isomers predominated. Careful
examination of coupling constants and chemical shifts
indicated that the major isomer at high temperature is
III′, which is the 1,5-disubstituted heterocycle (variable-
temperature NMR data for derivative IIIa is available
as Supporting Information).33-35 Interestingly, the prod-
ucts Ia -d and IIa -d do not exist as isomeric mixtures,
and they were obtained as simple products as confirmed
by 13C NMR and crystallographic studies.28,36

1,2,5-Oxadiazole N-Oxides as Antitrypanosomal Drugs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1943
Sch em e 1a
a
Reaction conditions: (a) NaNO2/AcOH/0-14 °C; (b) N4-butylsemicarbazide (11) or N4-hexylsemicarbazide (12)/p-TsOH/toluene/rt; (c)
amine/NaBH3CN/ZnCl2/MeOH/rt; (d) NaNO2/AcOH/rt; (e) MnO2/CHCl3/rt/1 day; (f) SOCl2/rt/1 day; (g) amine/K2CO3/KI/THF/reflux; (h)
toluene/reflux/3 h; (i) N4-[3-(dimethylamino)propyl]semicarbazide (13) or N4-[3-(diethylamino)propyl]semicarbazide (14)/p-TsOH/MeOH/
reflux; (j) (i) SOCl2/DMF/reflux/3 h, (ii) amine/Et3N/rt/12 h.
Sch em e 2a
a
Reaction conditions: (a) butanone/morpholine/rt; (b) vinyl acetate/Et2NH/DMF/40 °C; (c) malononitrile/Et3N/DMF/0 °C/rt; (d) butanone/
morpholine/rt; (e) SeO2/AcOEt/reflux; (f) 11/p-TsOH/toluene-CH2Cl2/rt.
In another synthetic effort, we expanded the oxadia-
zole system using compound IIIa (Scheme 2), to obtain
the quinoxaline 1,4-dioxide nucleus. When compound
IIIa was reacted with butanone in morpholine as base,
the dimethylquinoxaline IVa was obtained.29 Reaction
of IIIa with vinyl acetate in DMF at 40 °C produced
the compound IVb in low yield (7%).37 Beirut reaction
of IIIa with malononitrile in the presence of triethyl-
amine at low temperature gave the compound IVc in
good yield as a mixture of 6- and 7-substituted iso-
mers.38-41 The compound IVd was obtained from the
aldehyde 1729,42 in moderate yield.
Substituent effects in the benzo system were studied.
The products Va ,b, as the non-electron-withdrawing
analogues of compounds IIIe,a , respectively, were pre-
pared as delineated in Scheme 3. The direct reductive

1944 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Cerecetto et al.
Sch em e 3a
Ta ble 1
percentage growth
inhibition (%)a,b
compd
Nfx
Epc (V) vs SCEc
RM
100
-0.91d
-0.02e
IIIe
IIIf
IIIb
IIIa
IIIj
IIIi
IIIh
IIIg
IIIc
IIId
90
79
55
45
45
19
17
15
11
8
-1.14
-1.19
-1.07
-1.07
-1.12
-1.17
-1.19
-1.29
-1.02
-1.06
0.14
0.52
0.69
0.41
1.44
1.37
1.67
1.47
1.28
1.30
IIb
IId
IIc
IIa
53
38
22
17
-1.49
-1.10
NDf
0.82
0.74
ND
-1.37
0.58
Ib
Ia
Ic
Id
3
0
0
0
-1.59
ND
ND
0.41
ND
ND
ND
a
Reaction conditions: (a) butylamine or 11/NaCNBH3/Et3N/
ND
MeOH/rt; (b) NaCNBH3/Et3N/DMF/reflux; (c) NaBH4/MeOH/0 °C/
rt; (d) SOCl2/reflux; (e) butylamine or 11/K2CO3/KI/THF/reflux;
(f) triphenylphosphine/EtOH/reflux/1.5 h.
IVb
IVc
IVd
IVa
23
17
6
ND
-0.70
ND
-0.83
ND
ND
-0.01
ND
-0.09
0.06
1.24
0
amination of aldehyde 9 produced a very low yield of
Va . Product Vb was not observed in these conditions
or by direct reduction of IIIa with sodium cyanoboro-
hydride. Further, these compounds were obtained by
nucleophilic substitutions of the chloride 19.
Vb
Va
55
21
-0.96
VIc
VIb
VIa
10
0
0
0.49
0.36
0.39
a
Inhibition of epimastigotes growth, dose ) 25 µM. b The results
are the means of three different experiments with SD less than
10% in all cases. c First reduction step of N-oxides or nitro group
The deoxygenated derivatives VIa -c were prepared
by reaction of the corresponding N-oxides (IIIa ,e,f) with
triphenylphosphine in boiling ethanol (Scheme 3).43,44
The reaction was difficult to follow by TLC (SiO2,
petroleum ether:ethyl acetate (30%)), because of the very
close Rf values of reactants and products.
d
of Nfx. Peak potentials (∼(0.01 V) measured at a scan rate of
0.2 V/s. e The mean Rf values obtained were transformed into RM
(as described under Lipophilicity Studies). f Not determined.
malian fibroblasts were grown as described in the
Experimental Section and exposed to 10 µM concentra-
tion of each compound. Cell viability was assessed by
measuring absorbance at 570 nm due to MTT reduction
to formazan after 48 h of culture, which was proved to
be proportional to the number of viable cells present.
Figure 1B shows the percentage of cell survival referred
to the control culture. The percentage of cell survival
in the presence of 10 µM IIIe,f or Nfx was 69, 58, or
75%, respectively.
Electr och em ica l Stu d ies. The voltammetric re-
sponse of N-oxide derivatives was determined in DMSO
at a mercury dropping working electrode.13,14,46 For the
1,2,5-oxadiazole N-oxide, a typical one reduction peak
was observed during forward cathodic scan, which is
linked to an oxidation peak on the reverse anodic scan.
The benzo[1,2-c]1,2,5-oxadiazole N-oxide derivatives
presented one to two reduction peaks, which were linked
to oxidation peaks. For quinoxaline di-N-oxide three to
four reduction peaks were observed during forward
cathodic scan, some of which may be linked to oxidation
peaks on the reverse anodic scan. Only the first reduc-
tion step was studied as a function of voltage sweep rate
and switching potential. Table 1 summarizes Epc (ca-
thodic peak potential) for the first reduction step of
N-oxide derivatives.
Resu lts
An titr yp a n osom a l Activities. All the compounds
were tested in vitro against T. cruzi. Epimastigote forms
of T. cruzi, Tulahuen strain, were grown in axenic media
as described in the Experimental Section. The com-
pounds were incorporated into the media at 25 µM, and
their ability to inhibit growth of the parasite was
evaluated in comparison to the control (no drug added
to the media). Nfx was used as the reference trypano-
cidal drug. Growth of the parasite was followed for 11
days by measuring the increase in absorbance at 600
nm, which was proved to be proportional to the number
of cells present.11,45 The percentage of inhibition, sum-
marized in Table 1, was calculated as follows: percent
(%) ){1 - [(Ap - A0p)/(Ac - A0c)]} × 100, where Ap )
A600 of the culture containing the drug at day 5; A0p
)
A600 of the culture containing the drug right 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.
The ID50 concentration (50% inhibitory dose) was
assessed for compounds presenting higher trypanocidal
activity (IIIe,f, Nfx). Readings were done on day 5 of
growth and were determined as the drug concentration
required to reduce the absorbance to one-half of that of
the control (without drug) (Figure 1A). The ID50 con-
centrations for compounds IIIe,f were 10 and 16 µM,
respectively (compared to 2 µM for Nfx).
Lip op h ilicity Stu d ies. Reverse-phase TLC experi-
ments were performed on precoated TLC-C18 and eluted
with acetone:water (50:50, v/v). The Rf values for the
compounds were converted into RM values using the
relationship: RM ) log[(1/Rf) - 1].47-51 Table 1 shows
Cytotoxicity Assa y. To evaluate the potential cyto-
toxicity of compounds IIIe,f against the host, mam-

1,2,5-Oxadiazole N-Oxides as Antitrypanosomal Drugs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1945
Discu ssion
Some of the N-oxide derivatives investigated showed
antitrypanosomal activity similar to that of Nfx. At 25
µM, the 3-methyloxadiazole N-oxide derivatives (Ia -
d ) were not active; however, the 4-phenyl analogues
(IIa -d ) showed moderate activity. When we changed
the heterocycle (benzo[1,2-c]1,2,5-oxadiazole instead of
1,2,5-oxadiazole), using the same N4-semicarbazide
moiety, compounds were twice as effective (IIIa vs IIa )
or presented comparable activity (IIIb vs IIb). Within
the benzoxadiazole series, when comparing compounds
with identical side chain, the substitution of the linking
residue from amide to semicarbazone was shown to be
critical for antitrypanosomal activity (the amides IIIe,h
were 2-fold more active than the semicarbazones
IIIa ,d ). The change from alkylamino to [(N,N-dialkyl-
amino)alkyl]amino group (“spermidine-mimetic” group)
resulted in a significant decrease in the activity (the
semicarbazones IIIa ,b were 4-fold more active than
IIIc,d , and the amides IIIe,f are 2-fold more active than
IIIg-j). Also, the expansion of the heterocycle produced
a notorious decrease in activity (compound IIIa showed
an average 3-fold increase of activity compared to
compounds IVa -d ). The change of the semicarbazone
moiety from the C-6 (compound IVa ) to the C-2 (com-
pound IVd ) quinoxaline position did not result in a
significant increase of activity. The non-electron-
withdrawing analogues, compounds Va ,b, maintain
antitrypanosomal activity; however, compound Va was
4-fold less active than its electron-withdrawing (IIIe)
counterpart. Interestingly, for the most active benzoxa-
diazoles, the expansion of the conjugation to the side
chain did not result in an increase of activity (compare
Vb with 55% of growth inhibition to IIIa with 45% of
growth inhibition).
F igu r e 1. (A) Determination of ID50 values. Epimastigote
growth was followed in the absence and presence of increasing
concentrations of Nfx ([), IIIe (2), and IIIf (b), as described
in the Experimental Section. (B) Estimation of cytotoxicity.
Mammalian fibroblasts (V79-M8) were grown in the absence
(control) and presence of 10 µM IIIe, IIIf, or Nfx. The number
of viable cells was assesed by the MTT reduction method and
the percentage of survival calculated as described in the
Experimental Section.
Importantly, the absence of the N-oxide moiety pro-
duced a total loss of activity, indicating the direct
participation of this group on the mechanism for try-
panosomal toxicity (compare compounds IIIa ,e,f to
compounds VIa -c, showing 45, 90, and 79% and 0, 0,
and 10% growth inhibition, respectively).
The electrochemical studies show that the potential
for the first reduction step of the N-oxide derivatives is
similar for all compounds and more negative than the
corresponding potential for Nfx. Using heterocycles
capable of increasing radical stability (observed by ESR),
the potential for the first reduction step becomes less
negative, and therefore, the compounds are more readily
reduced and are also more active (compare Ib to IIIb).
The lipophilic-hydrophilic properties, expressed as RM
values, may be related with the antitrypanosomal
activities of the compounds. This can be seen, for
example, in the RM values for the active members of
the benzoxadiazole series (semicarbazones IIIa ,b and
amides IIIe,f), more related to Nifurtimox’s RM than the
less active members (semicarbazones IIIc,d and amides
IIIg-j).
Compounds IIIe,f (with 90 and 79% growth inhibi-
tion, respectively) were the most effective drugs in
inhibiting epimastigote growth (Table 1). The ID50
values obtained for IIIe,f were 10 and 16 µM, respec-
tively, in the same range of that observed for Nfx (2 µM).
In addition, the cytotoxicity of these compounds against
mammalian fibroblasts is comparable to that of the
the RM values obtained for each compound, and Nfx was
used as the reference drug.
ESR Stu d ies. The ability of these N-oxide derivatives
to yield free radicals in solution was investigated using
ESR spectroscopy. One representative compound of each
type of system was chosen: compounds Ib, IIIb,d , and
IVa . The radicals were generated by electrolytical
reduction in situ at room temperature in DMSO, ap-
plying the same cathodic potential obtained from the
cyclic voltammetry experiments.13,14 The half-life of the
generated radicals was not determined, but they were
stable enough to perform the corresponding experiments
(10-30 min). The interpretation of the ESR spectra by
means of a simulation process has led to the determi-
nation of the coupling constants for all magnetic nu-
clei.46 The hyperfine patterns indicated that the un-
paired electron is delocalized exclusively at the N-O
function for compound Ib (oxadiazole system), but for
the other heterocycles (IIIb,d , IVa ) the radicals are able
to delocalize as far as the semicarbazone system (Figure
2).

1946 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Cerecetto et al.
F igu r e 2. ESR spectra (electrochemical generation) of compounds Ib (A) (from ref 46), IIIb (B), IIId (C), and IVa (D).
reference drug. The ratio percentage of mammalian cell
survival (%)/ID50 (µM) could be considered as an index
of drug effectiveness. Although the ratio values obtained
for IIIe,f (7 and 4, respectively) are lower than the one
observed for the reference drug Nfx (36), they are high
enough to be considered as potential antichagasic drugs.
It is worth mentioning that only one cell line was
studied here in order to assess the cytotoxicity of these
new compounds against the host, and the susceptibility
of other cell types could be different.
In summary, our results show that these novel
N-oxide derivatives have potential trypanocidal activity,
and the mechanism of antitrypanosomal action is
through N-oxide radical formation. In vivo studies to
investigate the ability of these drugs to decrease the
parasitemia of infected mice are currently underway.
Exp er im en ta l Section
Ch em istr y. All starting materials were commercially avail-
able research-grade chemicals and used without further
purification. The compounds 3-12, 15-17, Ia ,b, and IIc were
prepared according to the literature.11,23,27-30,52,53 All solvents
were dried and distilled prior to use.54 All the reactions were
carried out in a nitrogen atmosphere. The typical workup
included washing with brine and drying the organic layer with
sodium sulfate. Melting points were determined using a Leitz
microscope heating stage model 350 apparatus and are uncor-
rected. Elemental analyses were obtained from vacuum-dried
samples (over phosphorus pentoxide at 3-4 mmHg, 24 h at
room temperature), were performed on a Fisons EA 1108
CHNS-O analyzer, and were within (0.4% of theoretical
values. Infrared spectra were recorded on a Perkin-Elmer 1310
apparatus, using potassium bromide tablets for solid and oil
products; the frequencies are expressed in cm-1 1H and 13C
.
NMR spectra were recorded on a Varian XL-100 (at 100 MHz)
instrument, Varian Gemini 300 (at 300 and 75.5 MHz)
instrument, and Bruker DPX-400 (at 400 and 100 MHz)
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 at 70 eV. High-resolution mass
spectra were recorded on a VG ULTIMA instrument at 70 eV.
P r ep a r a tion of Am in es Ic,d . Gen er a l P r oced u r e. A
mixture of aldehyde 3 (1 equiv), the corresponding amines (1
equiv), NaCNBH3 (1 equiv), ZnCl2 (0.5 equiv), and MeOH was
stirred at room temperature until the carbonyl compound was
not present (Al2O3, 1% MeOH in CH2Cl2). After the workup
process the residue was purified as indicated.
Con clu sion s
A new series of 1,2,5-oxadiazole N-oxides, benzo[1,2-
c]1,2,5-oxadiazole N-oxides, and quinoxaline di-N-oxides
was synthesized using a facile methodology. The pre-
liminary antitrypanosomal studies of these derivatives
showed that some of them exhibit significant activity
in vitro. The physicochemical properties studied indicate
that the redox potential of the monoelectronation of the
N-oxide moiety and the lipophilic-hydrophilic balance
play an important role in the biological activity. Our
studies indicate that the N-oxide group of these com-
pounds is the pharmacophore and that stabilization of
the free radical species generated by reduction in the
biological medium is related to their antitrypanosomal
activity.
4-[3-(4-Mor ph olin yl)pr opylam in om eth yl]-3-m eth yl-1,2,5-
oxa d ia zole N2-oxid e d ih yd r och lor id e, Ic: purified by
column chromatography (Al2O3, CH2Cl2:MeOH (0-5%)), color-
less oil (44%). The oil was dissolved in MeOH and treated with
MeOH saturated in hydrogen chloride. The dihydrochloride
was crystallized from Et2O:EtOAc:EtOH: white needles (5%);
To our knowledge this is the first time that N-oxide-
containing structures are reported as trypanocidal
compounds.
mp 198.5-200.5 °C; IR νmax 2959, 2463, 1618, 1393, 1260 cm-1
;
1H NMR (MeOH-d4-D2O, 400 MHz) δ 2.25 (s, 3H), 2.33 (quint,
J ) 7.9 Hz, 2H), 3.34 (m, 6H), 3.50 (t, J ) 7.1 Hz, 2H), 4.00

1,2,5-Oxadiazole N-Oxides as Antitrypanosomal Drugs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1947
(m, 4H), 4.52 (s, 2H); 13C NMR (MeOH-d4-D2O, 100 MHz) δ
6.70, 20.67, 41.36, 45.21, 52.36, 54.02, 63.96, 112.43, 151.90;
MS m/z (abundance) 199 (M+. - CH2dCHOCH2, 0.5%), 183
(0.6%). Anal. (C11H20N4O3‚2HCl) C, H, N.
bazide (1 equiv), p-TsOH (catalytic amounts), and MeOH as
solvent was placed in a flask fitted with a Soxhlet extractor
containing Linde 3A molecular sieves, to which was attached
a condenser. The mixture was heated at reflux until 9 was
not present (SiO2, 1% MeOH in CH2Cl2). After the workup
process the residue was purified as indicated.
4-[3-(1-P ip er in yl)p r op yla m in om eth yl]-3-m eth yl-1,2,5-
oxa d ia zole N2-oxid e, Id : purified by column chromatography
(Al2O3, CH2Cl2:MeOH (0-1%)); colorless oil (33%); IR νmax
4-[3-(Dim eth yla m in o)p r op yl]-1-[(ben zo[1,2-c]1,2,5-oxa -
d ia zol-5(6)-yl N1-oxid e)m eth ylid en e]sem ica r ba zid e, IIIc:
purified by column chromatography (Al2O3, CH2Cl2:MeOH (0-
10%)); colorless oil (60%); IR νmax 3350, 3250, 1676, 1537, 1350
3332, 2948, 1611, 1383 cm-1 1H NMR (acetone-d6-DMSO-
;
d6, 100 MHz) δ 1.65 (m, 2H), 1.95 (m, 6H), 2.30 (s, 3H), 2.98
(t, J ) 7.0 Hz, 2H), 3.20 (m, 6H), 4.15 (s, 2H), 4.62 (bs, 1H);
MS m/z (abundance) 255 (M+. + H, 0.2%), 237 (0.6%).
1
cm-1; H NMR (MeOH-d4-D2O, 100 MHz) δ 1.78 (quint, J )
6.0 Hz, 2H), 2.28 (s, 6H), 2.45 (m, 2H), 3.34 (m, 2H), 7.50-
8.20 (m, 4H); MS m/z (abundance) 306 (M+., 1.1%), 290 (1.4%).
4-[3-(Diet h yla m in o)p r op yl]-1-[(b en zo[1,2-c]1,2,5-oxa -
d ia zol-5(6)-yl N1-oxid e)m eth ylid en e]sem ica r ba zid e, IIId :
purified by column chromatography (Al2O3, CH2Cl2:MeOH (0-
10%)); colorless oil (70%); IR νmax 3300, 3220, 1674, 1532, 1375,
1348 cm-1; 1H NMR (MeOH-d4-D2O, 100 MHz) δ 1.04 (t, J )
7.0 Hz, 6H), 1.80 (quint, J ) 7.0 Hz, 2H), 2.60 (m, 6H), 3.38
P r ep a r a tion of Sem ica r ba zon es IIa ,b, IIIa ,b, a n d IVd .
Gen er a l P r oced u r e. A mixture of aldehyde 5, 9, or 17 (1
equiv), the corresponding semicarbazide (1 equiv), p-TsOH
(catalytic amounts), and toluene (toluene-CH2Cl2 for IVd ) as
solvent was stirred at room temperature until the carbonyl
compound was not present (SiO2, 1% MeOH in CH2Cl2). After
the workup process the residue was purified as indicated.
4-Bu t yl-1-[(4-p h en yl-1,2,5-oxa d ia zol-3-yl
N2-oxid e)-
(m, 2H), 7.50-8.30 (m, 4H); MS m/z (abundance) 334 (M+.
,
m eth ylid en e]sem ica r ba zid e, IIa : purified by crystallization
from EtOH; pale yellow needles (49%); mp 137.5-139.5 °C;
0.1%), 318 (0.5%).
IR νmax 3400, 2940, 2860, 1685, 1590, 1355 cm-1 1H NMR
;
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Am in es
IIc,d . A mixture of 6 (1 equiv), the corresponding amine (1
equiv), K2CO3 (1 equiv), KI (0.1 equiv), and THF as solvent
was heated at reflux until 6 was not present (SiO2, 10% EtOAc
in petroleum ether). After the workup process the residue was
purified as indicated.
3-[3-(Dieth ylam in o)pr opylam in om eth yl]-4-ph en yl-1,2,5-
oxa d ia zole N2-Oxid e Dih yd r och lor id e, IIc. The base28
obtained as an oil was dissolved in CH2Cl2 and treated with
Et2O saturated in hydrogen chloride. The hydrochloride was
crystallized from Et2O:EtOAc:MeOH, to give white needles
(15%): mp 135.0-137.0 °C. Anal. (C16H24N4O2‚2HCl‚H2O) C,
H, N.
(CDCl3, 300 MHz) δ 0.91 (t, J ) 7.1 Hz, 3H), 1.24 (sextet, J )
7.9 Hz, 2H), 1.36 (quint, J ) 6.9 Hz, 2H), 3.14 (q, J ) 6.3 Hz,
2H), 5.42 (t, J ) 5.5 Hz, 1H), 7.55 (m, 3H), 7.67 (m, 2H), 7.70
(s, 1H), 10.85 (s, 1H); 13C NMR-DEPT (CDCl3, 75.5 MHz) δ
13.72 (CH3-C), 19.83 (C-CH2-C), 31.93 (C-CH2-C), 39.38
(CH2-NHCO), 112.62 (CdN+-O-), 124.70 (CH-Ar), 126.66
(C-Ar), 128.60 (CH-Ar), 128.87 (CH-Ar), 130.95 (CH-Ar),
155.35 (CdO), 155.84 (CdN); MS m/z (abundance) 303 (M+.,
0.2%), 287 (0.2%). Anal. (C14H17N5O3) C, H, N.
4-H exyl-1-[(4-p h en yl-1,2,5-oxa d ia zol-3-yl N2-oxid e)-
m eth ylid en e]sem ica r ba zid e, IIb: purified by column chro-
matography (SiO2, CH2Cl2:MeOH (0-5%)) and then crystal-
lized from petroleum ether:EtOAc; yellow needles (43%); mp
115.5-118.0 °C; IR νmax 3380, 3320, 2920, 2850, 1675, 1355
3-[3-(4-Mor ph olin yl)pr opylam in om eth yl]-4-ph en yl-1,2,5-
oxa d ia zole N2-oxid e, IId : purified by column chromatogra-
phy (Al2O3, CH2Cl2:MeOH (0-10%)); colorless oil (75%); IR νmax
1
cm-1; H NMR (CDCl3, 100 MHz) δ 0.90 (t, J ) 8.0 Hz, 3H),
3300, 3050, 2945, 2850, 2810, 1595, 1115 cm-1 1H NMR
;
1.30-1.60 (m, 8H), 3.18 (q, J ) 6.0 Hz, 2H), 5.48 (t, J ) 6.0
Hz, 1H), 7.60 (m, 3H), 7.70 (m+s, 3H), 9.72 (bs, 1H); MS m/z
(abundance) 301 (M+. - CH3CH3, 14.3%), 271 (1.8%). Anal.
(C16H21N5O3) C, H, N.
(CDCl3, 100 MHz) δ 1.92 (quint, J ) 6.0 Hz, 2H), 2.68 (m, 6H),
2.93 (t, J ) 6.0 Hz, 2H), 3.95 (m, 4H), 4.14 (s, 2H), 7.20 (bs,
1H), 7.80 (m, 3H), 8.10 (m, 2H); MS m/z (abundance) 319 (M+.
+ H, 0.2%), 301 (0.7%).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Am id es
IIIe-j. A mixture of acid 10 (1 equiv), SOCl2 (10 equiv), and
DMF (catalytic amounts) was heated at reflux for 3 h. The
excess SOCl2 was eliminated by distillation at reduced pres-
sure, and the mixture was cooled to 0 °C. After addition of a
mixture of the corresponding amine (1 equiv), Et3N (2 equiv),
and dry CH2Cl2 as solvent, the reaction was stirred at room
temperature for 12 h. After the workup process the residue
was purified as indicated.
5(6)-(Bu tylca r ba m oyl)ben zo[1,2-c]1,2,5-oxa d ia zole N1-
oxid e, IIIe: purified by column chromatography (Al2O3,
petroleum ether:EtOAc (0-20%)) and then crystallized from
EtOH; yellow needles (30%); mp 164.0-166.0 °C; IR νmax 3300,
3070, 2930, 2850, 1640, 1610, 1310 cm-1; 1H NMR (CDCl3, 100
MHz) δ 0.98 (t, J ) 6.0 Hz, 3H), 1.30-1.80 (m, 4H), 3.50 (q, J
) 5.0 Hz, 2H), 6.30 (bs, 1H), 7.56 (d, J ) 9.0 Hz, 1H), 7.74 (d,
J ) 9.0 Hz, 1H), 7.80 (s, 1H); MS m/z (abundance) 235 (M+.,
34.2%), 219 (9.5%). Anal. (C11H13N3O3) C, H, N.
4-Bu t yl-1-[(b en zo[1,2-c]1,2,5-oxa d ia zol-5(6)-yl N1-ox-
id e)m eth ylid en e]sem ica r ba zid e, IIIa : purified by crystal-
lization from EtOH; yellow needles (52%); mp 227.0-229.0 °C;
IR νmax 3400, 2930, 2860, 1675, 1610, 1350 cm-1 1H NMR
;
(DMSO-d6-D2O, 300 MHz) δ 0.89 (t, J ) 7.1 Hz, 3H), 1.28
(sextet, J ) 7.3 Hz, 2H), 1.46 (quint, J ) 7.1 Hz, 2H), 3.13 (q,
J ) 6.8 Hz, 2H), 7.50-7.90 (m+s, 4H); 13C NMR (DMSO-d6-
D2O, 75.5 MHz) δ 13.78, 19.58, 32.17, 38.78, 110.77, 117.67,
122.14, 131.60, 135.71, 136.55, 136.61, 155.15, 155.22; MS m/z
(abundance) 277 (M+., 4.1%), 261 (0.4%). Anal. (C12H15N5O3)
C, H, N.
4-H exyl-1-[(b en zo[1,2-c]1,2,5-oxa d ia zol-5(6)-yl N1-ox-
id e)m eth ylid en e]sem ica r ba zid e, IIIb: purified by crystal-
lization from petroleum ether:EtOAc; pale orange needles
(30%); mp 140.0-142.0 °C; IR νmax 3400, 3090, 2930, 1680,
1610, 1355 cm-1; 1H NMR (CDCl3, 100 MHz) δ 0.90 (t, J ) 5.5
Hz, 3H), 1.38 (m, 4H), 1.60 (m, 4H), 3.40 (q, J ) 6.0 Hz, 2H),
6.10 (m, 1H), 7.40-8.10 (m, 4H), 9.85 (bs, 1H); HRMS 305.1481
(M+., 7.6%) (MW theoret 305.1488); HPLC (SiO2, acetonitrile,
µ ) 3.3 mL/min, λ ) 254 nm) tR ) 2.5 min. Anal. (C14H19N5O3)
C, H, N.
5(6)-(Hexylca r ba m oyl)ben zo[1,2-c]1,2,5-oxa d ia zole N1-
oxid e, IIIf: purified by column chromatography (Al2O3, pe-
troleum ether:EtOAc (0-20%)) and then crystallized from
petroleum ether:EtOAc; pale yellow needles (19%); mp 115.0-
117.0 °C; IR νmax 3270, 3070, 2920, 2840, 1635, 1610, 1310
4-Bu t yl-1-[(3-m et h ylq u in oxa lin -2-yl
N1,N4-d ioxid e)
m eth ylid en e]sem ica r ba zid e, IVd : purified by column chro-
matography (Al2O3, CH2Cl2:MeOH (0-1%)) and then crystal-
lized from petroleum ether:EtOAc:EtOH; yellow solid (28%);
mp 235.0-237.0 °C; IR νmax 3420, 2930, 2870, 1690, 1600, 1350
1
cm-1; H NMR (CDCl3, 400 MHz) δ 0.93 (t, J ) 6.8 Hz, 3H),
1.38 (m, 6H), 1.66 (quint, J ) 7.2 Hz, 2H), 3.49 (q, J ) 7.1 Hz,
2H), 6.18 (bs, 1H), 7.50-8.10 (m, 3H); MS m/z (abundance)
263 (M+., 16.0%), 247 (15.0%). Anal. (C13H17N3O3) C, H, N.
5(6)-[3-(Dim et h yla m in o)p r op ylca r b a m oyl]b en zo[1,2-
c]1,2,5-oxa d ia zole N1-oxid e, IIIg: purified by column chro-
matography (Al2O3, CH2Cl2); colorless oil (34%); IR νmax 3300,
3090, 2960, 2880, 1660, 1540, 1300 cm-1; 1H NMR (CDCl3, 100
MHz) δ 1.80 (quint, J ) 6.0 Hz, 2H), 2.36 (s, 6H), 2.58 (t, J )
1
cm-1; H NMR (DMSO-d6-D2O, 100 MHz) δ 0.91 (t, J ) 7.0
Hz, 3H), 1.45 (m, 4H), 2.86 (s, 3H), 3.78 (m, 2H), 7.95 (dd, J 1
) 7.0 Hz, J 2 ) 8.0 Hz, 2H), 8.49 (s, 1H), 8.52 (d, J ) 8.0 Hz,
1H), 8.58 (d, J ) 8.0 Hz, 1H); MS m/z (abundance) 317 (M+.,
7.3%), 300 (21.6%). Anal. (C15H19N5O3) C, H, N.
P r ep a r a tion of Sem ica r ba zon es IIIc,d . Gen er a l P r o-
ced u r e. A mixture of 9 (1 equiv), the corresponding semicar-

1948 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Cerecetto et al.
6.0 Hz, 2H), 3.58 (q, J ) 6.0 Hz, 2H), 7.45-8.00 (m, 3H), 9.20
(bs, 1H); MS m/z (abundance) 264 (M+., 1.1%), 248 (0.6%).
MS m/z (abundance) 343 (M+., 1.2%), 327 (6.6%). Anal.
(C15H17N7O3‚H2O) C, H, N.
5(6)-(Hyd r oxym eth yl)ben zo[1,2-c]1,2,5-oxa d ia zole N1-
Oxid e, 18. A solution of 9 (0.4 g, 2.4 mmol) in MeOH (5.0 mL)
was stirred at 0 °C. NaBH4 (90 mg, 2.4 mmol) was then added,
and the resulting solution was stirred for 3 h at room
temperature. The solvent was removed in vacuo, and the
residue was purified by column chromatography (SiO2, petro-
leum ether:EtOAc (10-30%)): colorless oil (76%); IR νmax 3380,
3100, 2920, 2860, 1620, 1590, 1530, 1350, 1045 cm-1; 1H NMR
(CDCl3, 400 MHz) δ 3.25 (bs, 1H), 4.70 + 4.73 (two singlets,
2H), 7.00-7.84 (m, 3H); MS m/z (abundance) 166 (M+.,
100.0%), 150 (4.0%).
5(6)-[3-(Die t h yla m in o)p r op ylca r b a m oyl]b e n zo[1,2-
c]1,2,5-oxa d ia zole N1-oxid e, IIIh : purified by column chro-
matography (Al2O3, CH2Cl2); colorless oil (24%); IR νmax 3300,
3050, 2960, 2880, 1650, 1535, 1290 cm-1; 1H NMR (CDCl3, 100
MHz) δ 1.10 (t, J ) 7.0 Hz, 6H), 1.75 (quint, J ) 6.5 Hz, 2H),
2.60 (q+m, 6H), 3.58 (q, J ) 6.0 Hz, 2H), 7.30-8.30 (m, 3H),
9.50 (bs, 1H); MS m/z (abundance) 292 (M+., 0.3%), 276 (0.8%).
5(6)-[3-(4-Mor p h olin yl)p r op ylca r b a m oyl]b en zo[1,2-
c]1,2,5-oxa d ia zole N1-oxid e, IIIi: purified by column chro-
matography (Al2O3, petroleum ether:CH2Cl2 (50-100%)); col-
orless oil (39%); IR νmax 3310, 3090, 2970, 2870, 1650, 1595,
5(6)-(Ch lor om et h yl)b en zo[1,2-c]1,2,5-oxa d ia zole N1-
Oxid e, 19. A mixture of 18 (0.3 g, 1.8 mmol) and SOCl2 (2.0
mL, 26.4 mmol) was stirred at reflux during 8 h. The mixture
was poured onto ice, and the aqueous mixture was extracted
with EtOAc (3 × 30 mL). The combined extracts were dried
and evaporated in vacuo. The residue was purified by column
chromatography (SiO2, petroleum ether:EtOAc (0-5%)): color-
1
1535, 1310 cm-1; H NMR (CDCl3, 100 MHz) δ 1.83 (quint, J
) 7.0 Hz, 2H), 2.55 (m, 6H), 3.57 (q, J ) 5.5 Hz, 2H), 3.71 (m,
4H), 7.30-8.30 (m, 3H), 8.46 (bs, 1H); MS m/z (abundance)
306 (M+., 0.5%), 290 (0.4%).
5(6)-[3-(1-P iper in yl)pr opylcar bam oyl]ben zo[1,2-c]1,2,5-
oxa d ia zole N1-oxid e, IIIj: purified by column chromatogra-
phy (Al2O3, petroleum ether:CH2Cl2 (50-100%)); colorless oil
1
less oil (52%); IR νmax 3090, 2960, 1620, 1590, 1530 cm-1; H
(26%); IR νmax 3300, 2930, 1645, 1530, 1285 cm-1 1H NMR
;
NMR (CDCl3, 400 MHz) δ 4.58 (s, 2H), 7.20-7.60 (m, 3H); 13
C
(CDCl3, 400 MHz) δ 1.51 (m, 2H), 1.61 (quint, J ) 5.5 Hz, 4H);
1.80 (quint, J ) 5.5 Hz, 2H), 2.49 (m, 4H), 2.58 (t, J ) 5.3 Hz,
2H), 3.57 (t, J ) 5.3 Hz, 2H), 7.35-8.30 (m, 3H), 9.29 (bs, 1H);
13C NMR (CDCl3, 100 MHz) δ 23.80, 24.46, 26.39, 42.15, 55.15,
59.82, 114.65, 115.85, 117.03, 124.24, 128.91, 129.71, 131.07,
132.22, 132.39, 135.18, 146.21, 149.36, 164.16, 164.69; MS m/z
(abundance) 288 (M+. - 16, 1.4%).
NMR (CDCl3, 100 MHz) δ 45.33, 112.25, 114.28, 116.96,
119.48, 130.35, 133.41, 139.26, 142.61, 152.49; MS m/z (abun-
dance) 184 (M+., 85%), 168 (4.0%).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
Va ,b. A mixture of 19 (1 equiv), butylamine or 11 (1 equiv),
K2CO3 (1 equiv), KI (0.1 equiv), and THF as solvent was heated
at reflux until 19 was not present (SiO2, 5% EtOAc in
petroleum ether). After the workup process the residue was
purified as indicated.
5(6)-(Bu t yla m in om et h yl)ben zo[1,2-c]1,2,5-oxa d ia zole
N1-oxid e, Va : purified by column chromatography (SiO2,
petroleum ether:EtOAc (20-70%)); colorless oil (38%); IR νmax
3330, 2960, 2930, 2860, 1530, 1350 cm-1; 1H NMR (CDCl3, 400
MHz) δ 0.91 (t, J ) 7.2 Hz, 3H), 1.37 (sex, J ) 7.7 Hz, 2H),
1.47 (quint, J ) 7.1 Hz, 2H), 1.64 (bs, 1H), 2.61 (q, J ) 7.2 Hz,
2H), 3.86 (s, 2H), 7.44-7.87 (m, 3H); 13C NMR (CDCl3, 100
MHz) δ 14.32, 20.76, 32.55, 49.53, 52.83, 113.25, 116.42,
125.16, 125.37, 131.96, 132.97, 133.64, 141.96, 145.37; MS m/z
(abundance) 221 (M+., 1.5%), 205 (1.2%).
4-Bu tyl-1-[(2,3-d im eth ylqu in oxa lin -6-yl N1,N4-d ioxid e)-
m eth ylid en e]sem ica r ba zid e, IVa . A solution of IIIa (0.1 g,
0.36 mmol) in morpholine (0.9 mL) was stirred for 10 min at
0 °C. Butanone (0.1 mL) was then added, and the resulting
solution was stirred for 18 h at room temperature. After
concentration of the solution under reduced pressure, the crude
oil was precipitated with Et2O: purified by crystallization from
EtOAc:acetone; yellow needles (40%); mp 238.0-240.0 °C; IR
1
νmax 3340, 3230, 2960, 2920, 2860, 1675, 1530, 1315 cm-1; H
NMR (CDCl3, 100 MHz) δ 0.98 (t, J ) 7.0 Hz, 3H), 1.60 (m,
4H), 2.76 (s, 3H), 2.78 (s, 3H), 3.40 (q, J ) 6.0 Hz, 2H), 6.20 (t,
J ) 6.0 Hz, 1H), 8.06 (s, 1H), 8.15 (dd, J 1 ) 2.0 Hz, J 2 ) 9.0
Hz, 1H), 8.62 (d, J ) 9.0 Hz, 1H), 8.71 (d, J ) 2.0 Hz, 1H),
9.80 (bs, 1H); MS m/z (abundance) 331 (M+., 7.2%), 315 (3.0%).
Anal. (C16H21N5O3‚H2O) C, H, N.
4-Bu t yl-1-[(b en zo[1,2-c]1,2,5-oxa d ia zol-5(6)-yl N1-ox-
id e)m eth yl]sem ica r ba zid e, Vb: purified by column chro-
matography (SiO2, CH2Cl2:MeOH (0-5%)) and then crystal-
lized from petroleum ether:EtOAc; yellow needles (25%); mp
149.0-151.0 °C; IR νmax 3390, 3250, 3070, 2960, 2870, 1645,
4-Bu t yl-1-[(q u in oxa lin -6-yl
N1,N4-d ioxid e)m et h yli-
d en e]sem ica r ba zid e, IVb. To a solution of IIIa (0.2 g, 0.72
mmol) and Et2NH (0.07 mL) in DMF (0.5 mL) was added vinyl
acetate (0.13 mL, 1.44 mmol) during 10 min at 0 °C. The
reaction mixture was stirred for 24 h at 40 °C. Vinyl acetate
(1.3 mL, 14.4 mmol) and Et2NH (1.0 mL) were then added,
and the heating was continued at 40 °C during 48 h. The
solvent was removed in vacuo and the oily residue coevapo-
rated with toluene (3 × 10 mL). The crude product was purified
by column chromatography (SiO2, CH2Cl2): colorless oil (7%);
IR νmax 3300, 2950, 2920, 2860, 1675, 1530, 1350 cm-1; 1H NMR
(MeOH-d4-D2O, 100 MHz) δ 0.98 (t, J ) 7.0 Hz, 3H), 1.50
1
1620, 1025 cm-1; H NMR (acetone-d6, 400 MHz) δ 0.88 (t, J
) 7.3 Hz, 3H), 1.26 (sex, J ) 7.6 Hz, 2H), 1.39 (quint, J ) 7.0
Hz, 2H), 3.12 (q, J ) 6.4 Hz, 2H), 4.06 (s, 2H), 4.98 (bs, 1H),
6.22 (bs, 1H), 6.60 (bs, 1H), 7.50-8.00 (m, 3H); 13C NMR
(acetone-d6, 100 MHz) δ 13.55, 20.08, 32.90, 39.15, 55.86,
114.50, 116.12, 126.00, 131.89, 135.00, 143.10, 159.32; MS m/z
(abundance) 279 (M+., 2.2%), 263 (2.6%). Anal. (C12H17N5O3)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion VIa -c. A
mixture of the corresponding N-oxide IIIa ,e,f (1 equiv), Ph3P
(1 equiv), and EtOH as solvent was heated at reflux for 1.5 h.
The EtOH was eliminated at reduced pressure. After the
workup process the residue was purified as indicated.
1-[(Ben zo[1,2-c]1,2,5-oxa d ia zol-5-yl)m eth ylid en e]-4-bu -
tylsem ica r ba zid e, VIa : purified by column chromatography
(SiO2, petroleum ether:EtOAc (0-20%)) and then crystallized
from petroleum ether:EtOAc: yellow needles (50%); mp 202.0-
205.0 °C; IR νmax 3420, 3090, 2950, 2860, 1675, 1550, 1350
(m, 4H), 3.34 (q, J ) 6.0 Hz, 2H), 7.98 (s, 1H), 8.03 (dd, J 1
)
2.0 Hz, J 2 ) 8.0 Hz, 1H), 8.31 (d, J ) 2.0 Hz, 1H), 8.44 (d, J
) 8.0 Hz, 1H), 8.48 (d, J ) 2.0 Hz, 1H), 8.51 (d, J ) 2.0 Hz,
1H); MS m/z (abundance) 301 (M+. - 2, 2.7%), 285 (2.9%).
1-[(2(3)-Am in o-3(2)-cya n oq u in oxa lin -6-yl N1,N4-d iox-
id e)m eth ylid en e]-4-bu tylsem ica r ba zid e, IVc. A mixture
of IIIa (0.2 g, 0.72 mmol) and malononitrile (0.05 mL, 0.76
mmol) was stirred for 10 min at 0 °C. Over the cooled
suspension was added a solution of Et3N (1 drop) in DMF (0.22
mL). The mixture was allowed to stand at room temperature
over 24 h and filtered off. The solid product was washed with
Et2O, boiling MeOH, boiling DMF, and boiling H2O to give a
red solid (67%): mp (the mixture) > 320 °C; IR νmax 3390, 3280,
1
cm-1; H NMR (CDCl3, 100 MHz) δ 1.02 (t, J ) 7.0 Hz, 3H),
1.60 (m, 4H), 3.45 (q, J ) 6.0 Hz, 2H), 6.15 (bt, J ) 5.5 Hz,
1H), 7.75 (dd, J 1 ) 1.0 Hz, J 2 ) 8.5 Hz, 1H), 7.89 (bs, 2H),
8.00 (dd, J 1 ) 1.0 Hz, J 2 ) 9.0 Hz, 1H), 9.80 (bs, 1H); MS m/z
(abundance) 261 (M+., 4.0%), 232 (0.1%). Anal. (C12H15N5O2)
C, H, N.
2930, 2860, 2230, 1685, 1605, 1525, 1340 cm-1 1H NMR
;
5-(Bu tylca r ba m oyl)ben zo[1,2-c]1,2,5-oxa d ia zole, VIb:
purified by column chromatography (Al2O3, petroleum ether:
Et2O (10-30%)) and then crystallized from petroleum ether:
EtOAc: white needles (49%); mp 205.0-207.0 °C; IR νmax 3290,
3090, 2950, 2860, 1630, 1560 cm-1; 1H NMR (CDCl3, 100 MHz)
(DMSO-d6-D2O, 100 MHz) δ 0.97 (t, J ) 7.0 Hz, 3H), 1.45
(m, 4H), 3.27 (m, 2H), 8.11 (d, J ) 8.0 Hz, 0.4H), 8.14 (d, J )
8.0 Hz, 0.6H), 8.29 (s, 0.4H), 8.33 (s, 0.6H), 8.38 (s, 0.6H), 8.42
(s, 0.4H), 8.58 (d, J ) 8.0 Hz, 0.6H), 8.61 (d, J ) 8.0 Hz, 0.4H);

1,2,5-Oxadiazole N-Oxides as Antitrypanosomal Drugs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1949
δ 0.97 (t, J ) 7.0 Hz, 3H), 1.35-1.75 (m, 4H), 3.52 (q, J ) 6.0
Hz, 2H), 6.62 (bs, 1H), 7.83 (dd, J 1 ) 1.0 Hz, J 2 ) 7.0 Hz, 1H),
7.93 (dd, J 1 ) 1.0 Hz, J 2 ) 7.0 Hz, 1H), 8.21 (t, J ) 1.0 Hz,
1H); MS m/z (abundance) 219 (M+., 18.1%), 204 (1.6%). Anal.
(C11H13N3O2) C, H, N.
5-(Hexylca r ba m oyl)ben zo[1,2-c]1,2,5-oxa d ia zole, VIc:
purified by column chromatography (Al2O3, petroleum ether:
Et2O (10-50%)) and then crystallized from petroleum ether:
EtOAc: white needles (65%); mp 93.0-95.0 °C; IR νmax 3280,
3070, 2920, 2850, 1625, 1550, 1270 cm-1; 1H NMR (CDCl3, 100
MHz) δ 0.92 (t, J ) 6.0 Hz, 3H), 1.37 (m, 6H), 1.68 (m, 2H),
3.53 (q, J ) 6.0 Hz, 2H), 6.28 (bs, 1H), 7.84 (dd, J 1 ) 2.0 Hz,
J 2 ) 8.0 Hz, 1H), 7.95 (dd, J 1 ) 2.0 Hz, J 2 ) 8.0 Hz, 1H), 8.18
(t, J ) 2.0 Hz, 1H); MS m/z (abundance) 247 (M+., 15.5%), 218
(9.7%). Anal. (C13H17N3O2) C, H, N.
Electr och em ica l Meth od . Voltammetric responses for the
compounds were obtained by cyclic voltammetry. Experiments
were carried out in DMSO (Aldrich, spectroscopy grade) with
0.1 M tetrabutylammonium perchlorate (Fluka) as the sup-
porting electrolyte and purged with nitrogen at room temper-
ature. Typically 10-12 mg of compound was used in a cell
volume of ∼ 40 mL. A mercury-dropping electrode was used
as the working electrode, a platinum wire as the auxiliary
electrode, and saturated calomel as the reference electrode.
Voltammograms were obtained using a Weenking POS 88
instrument with a Kipp Zenen BD93 recorder. Voltages scan
rates ranged from 0.1 to 0.5 V/s.
Lip op h ilicity Stu d ies. Reverse-phase TLC experiments
were performed on precoated TLC plates SIL RP-18W/UV254
(Macherey-Nagel) and eluted with acetone (Aldrich, HPLC
grade):water (distilled) (50:50, v/v). The plates were developed
in a closed chromatographic tank and dried, and the spots were
located under UV light.
ESR Mea su r em en t. The radicals were generated by elec-
trolytical reduction in situ at room temperature. ESR spectra
were recorder in the X band (9.85 GHz) on a Bruker ECS 106
spectrometer, using a rectangular cavity with a 50-kHz field
modulation, in DMSO (Aldrich, spectroscopy grade). The
hyperfine splitting constants were estimated to be accurate
within 0.05 G.
An titr yp a n osom a l Bioa ssa ys. T. cruzi epimastigotes (Tu-
lahuen 2 strain) were grown at 28 °C in an axenic medium
(BHI-tryptose) as previously described,45 complemented with
10% fetal calf serum. Cells from a 10-day-old culture (station-
ary phase) were inoculated into 50 mL of fresh culture medium
to give an initial concentration of 1 × 106 cells/mL. Cell growth
was followed by measuring everyday the absorbance of the
culture at 600 nm. Before inoculation, the media was supple-
mented 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 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.
To determine ID50 values, 50% inhibitory doses, 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 ID50 value was taken as the concentration of
drug needed to reduce the absorbance ratio to 50%.
Cytotoxicity Assa ys. Chinese hamster lung fibroblasts
(V79-M8) were grown at 37 °C in Dubelcco’s modified Eagle’s
medium (DMEM), pH 7.0, supplemented with 10% fetal calf
serum, 472 U/mL penicillin, and 100 µg/mL streptomycin. The
cells were cultured in humidified air plus 5% CO2. Cells were
plated in 96-well plates (4 × 103 cells/well) and allowed to
proliferate for 16 h (∼2 doubling times). At this time point
the drugs were added and incubated for 24 h. Then, cells were
washed, new media were supplied, and after 4 h MTT (5 mg/
mL) was added. Plates were wrapped in aluminum foil and
further incubated for 4 h. Medium and MTT were removed
from wells, and DMSO in glycine buffer (0.1 M glycine, 0.1 M
NaCl, pH 10.5) was added to dissolve the formazan crystals.
Absorbance was measured at 570 nm immediately. For this
assay, compounds were supplemented from stock solutions in
ethanol. Final concentration of ethanol in culture media never
exceeded 0.14%, and control was run in the presence of 0.14%
ethanol and in the absence of any drug.
Ack n ow led gm en t. Financial support from CONI-
CYT-Uruguay (Grants 347/95 and 1019/95) and SAREC
is acknowledged. The authors thank RELAQ (Red
Latinoamericana de Ciencia Quı´mica) for a scholarship
to M.R. We also thank Dr. Laura Castro for helping with
the cell cultures, Gabriel Sagrera for help with the NMR
experiments, and Dr. Rafael Radi for critical reading of
the manuscript.
Su p p or tin g In for m a tion Ava ila ble: Variable-tempera-
1
ture H NMR data for IIIa . This information is available free
of charge via the Internet at http://pubs.acs.org.
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