1,2,5-Oxadiazole N-oxide derivatives as potential anti-cancer agents: Synthesis and biological evaluation. Part IV

Article abstract of DOI:10.1016/S0223-5234(01)01265-X

Several new 1,2,5-oxadiazole N-oxide derivatives and some deoxygenated analogues were synthesized to be tested as potential selective hypoxic cell cytotoxins. Compounds prepared were designed in order to gain insight into the mechanism of action of this kind of cytotoxin. Compounds were tested in oxia and hypoxia and they proved to be non-selective. 3-Cyano-N₂-oxide-4-phenyl-1,2,5-oxadiazole showed the best cytotoxic activity in oxia. The cytotoxicity observed for these derivatives could be explained in terms of the electronic characteristics of the 1,2,5-oxadiazole substituents. Electrochemical and ESR studies were performed on the more cytotoxic derivative.

Full text of DOI:10.1016/S0223-5234(01)01265-X

Eur. J. Med. Chem. 36 (2001) 771–782
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01265-X/FLA
Original article
1,2,5-Oxadiazole N-oxide derivatives as potential anti-cancer agents: synthesis
and biological evaluation. Part IV
Mariana Boiania, Hugo Cerecettoa,*, Mercedes Gonza´leza,*, Mariela Rissoa, Claudio Olea-Azarb,
Oscar E. Piroc, Eduardo E. Castellanod, Adela Lo´pez de Cera´ine, Olga Ezpeletae,
Antonio Monge-Vegae
aDepartamento de Qu´ımica Orga´nica, Facultad de Qu´ımica y Facultad de Ciencias, Universidad de la Repu´blica,
Montevideo, Uruguay
bDepartamento de Qu´ımica Inorga´nica y Anal´ıtica, Facultad de Ciencias Qu´ımicas y Farmace´uticas, Universidad de Chile,
Santiago, Chile
cUniversidad Nacional de La Plata e Instituto IFLP(CONICET), Departamento de F´ısica, Facultad de Ciencias Exactas,
La Plata, Argentina
dInstituto de F´ısica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sao Carlos, SP, Brazil
eCentro de Investigaciones en Farmacobiolog´ıa Aplicada, Facultad de Farmacia, Universidad de Navarra, Pamplona, Spain
Received 19 March 2001; revised 17 July 2001; accepted 31 July 2001
Abstract – Several new 1,2,5-oxadiazole N-oxide derivatives and some deoxygenated analogues were synthesized to be tested as
potential selective hypoxic cell cytotoxins. Compounds prepared were designed in order to gain insight into the mechanism of action
of this kind of cytotoxin. Compounds were tested in oxia and hypoxia and they proved to be non-selective. 3-Cyano-N2-oxide-4-
phenyl-1,2,5-oxadiazole showed the best cytotoxic activity in oxia. The cytotoxicity observed for these derivatives could be explained
in terms of the electronic characteristics of the 1,2,5-oxadiazole substituents. Electrochemical and ESR studies were performed on
´
the more cytotoxic derivative. © 2001 Editions scientifiques et me´dicales Elsevier SAS
1,2,5-oxadiazole N-oxide / anti-cancer agents / structure–activity relationships
1. Introduction
gen mustard derivatives [9–11] and N-oxide deriva-
tives [12]. Taking the last as pharmacophore
antecedent, our group is undertaking the synthesis
and biological characterisation of new potential N-
oxide-containing prodrugs [13–18]. We have studied
the bioactivity of derivatives of 1,2,5-oxadiazole N-
oxide (e.g. 1–6, figure 1), which could be seen as
misonidazole (a nitro derivative) and tirapazamine
(an N-oxide) related compounds [16–18]. Unfortu-
nately the compounds were totally unselective under
hypoxia conditions, although derivatives 1 and 2
showed appreciable activity in oxia (figure 1), display-
ing excellent V79 cell-growth inhibition at the lowest
concentration assayed (1 mM) [19].
Hypoxic cells present in human tumours give rise to
clinical problems in the chemotherapy of solid tu-
mours because they limit the efficiency of fractionated
radiotherapy and are also resistant to conventional
chemotherapeutic drugs [1–3]. Bioreductive prodrugs,
which are cytotoxic maximally in the absence of oxy-
gen, have attracted considerable attention as selective
cytotoxins towards hypoxic cells [4]. Several types of
compounds that are toxic only under hypoxic condi-
tions are known. These agents belong to the classes of
nitro derivatives [5–7], quinone derivatives [8], nitro-
From the well-known N-oxides mechanism of ac-
tion [21] and the observed activity and the structural
characteristics of derivatives developed, we conclude
* Correspondence and reprints
E-mail addresses: hcerecet@bilbo.edu.uy (H. Cerecetto),
megonzal@bilbo.edu.uy (M. Gonza´lez).

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M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
that the 1,2,5-oxadiazole N-oxides probably act as is
shown in figure 1, where the first step, electronation
process, is not reverted by oxygen. In order to explore
the structure–activity relationships, we have studied
the cytotoxicity of new derivatives of 1,2,5-oxadiazole
N-oxide. In this paper we report the synthesis and the
biological and physicochemical characterisation of 17
1,2,5-oxadiazole N-oxide derivatives.
E-isomer. The differential stereochemistry of these
oximes was, probably, responsible for the yields ob-
tained in the following dehydration process (prepara-
tion of 9 and 10). To change the electrophilic
aldehyde centre, compounds 1 and 6 were trans-
formed into derivatives 11 and 12 using condensation
processes. The Wittig procedure [24, 25] produced the
desired compounds in good yields. Attempts to obtain
the corresponding nitro alkenes, 15 and 16, through
nitroaldol condensation [26], were unsuccessful.
Long-time heating in basic medium (butylamine) of
the reaction mixture, conducted to a total decomposi-
tion of the nitro alcohols intermediate 13 and 14.
These were isolated and biologically evaluated before
decomposition started.
We have synthesized a series of benzylic-like substi-
tuted derivatives of compounds 2 and 5, since sub-
stituents at this position are expected to have an
important electronic effects on the displayed activity
(according to the proposed mechanism). We choose a
series of sulphur derivatives with well-known elec-
tronic effects (seen the corresponding F coefficients of
Swain–Lupton [27]) and different steric parameters.
These derivatives, 17–20, were prepared by the meth-
ods outlined in figure 3. The chloride 2 was trans-
formed into the corresponding thioether derivatives
(17 and 20) using SN conditions [28]. The phenylthio
2. Chemistry
Trying to confirm the proposed mechanism of ac-
tion, we varied the structure of parent compounds in
two ways. On the one hand, we designed compounds
that change the electrophilic characteristic of the ben-
zylic-like position, and on the other hand, we de-
signed deoxygenated analogues of characteristic
products (derivatives 1, 2, 4 and 5).
Firstly, were prepared compounds 7–10 (see figure
2) as electron-withdrawing substituent derivatives of
parent compounds 1 and 6. The oximes 7 and 8 were
obtained by the traditional method [22] and then
converted into the nitriles 9 and 10 by dehydration
with thionyl chloride as reported by Gasco and
coworkers [23]. According to NOE experiments oxime
7 was obtained as the Z-isomer and oxime 8 as the
Figure 1. Parent compounds and mechanism of action proposed.

M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
773
Figure 2. Electrophilic centre modifications.
Figure 3. Benzylic-like centre modifications.

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M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
The reduction was clearly observed through HETCOR
experiments (HMQC and HMBC). The molecular
structure of derivative 23 has been determined by
X-ray diffraction methods. Figure 5 displays the ORTEP
drawing [34], showing the labelling of the non-H atoms
and their displacement ellipsoids at 30% probability
level. When we tried to reduce derivative 2, under the
same conditions, compound 27 was generated as a
reductive dimerisation Zn-prompted process. This de-
sired deoxy analogue, 25, and 26 were prepared from
alcohol 23 through classical procedures [17].
derivative (20) was obtained in a low yield with an
extensive auto-oxidation of thiophenol. Compound 17
was transformed into derivatives 18 and 19 using
an adequate oxidising reagent [28, 29]. Derivative 5
was transformed into the corresponding ester 21 as
shown in figure 3. Attempts to obtain the correspond-
ing tosylate 22 were unsuccessful, in all cases com-
pound 2 was isolated probably as a chloride
tosylate-display in the reaction medium [30].
Finally, in an attempt to demonstrate the impor-
tance of the N-oxide moiety in the mechanism of
action, the deoxy derivatives, 23–26, were prepared
(see figure 4). When triphenylphosphine was used as
deoxygenating agent [31, 32] unfavourable results were
obtained. The use of Zn in NH4Cl solution led to the
deoxy derivatives 23 and 24 in moderate yields [33].
3. Pharmacology
The 1,2,5-oxadiazole N-oxide derivatives were
tested in a cloning assay using V79 cells. Suspension
Figure 4. Deoxygenated derivatives synthesis.
Figure 5. Molecular plot of derivative 23 at 120 K (showing the labelling of the non-H atoms and their displacement ellipsoids at
,
30% probability level) and interatomic bond distances (A) and angles (°).

M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
775
Table I. Physical and biological properties of 1,2,5-oxadiazole N-oxide derivatives.
Compound M.p. (°C) (crystallisation solvent)
wmax (cm−1
)
Biological evaluation
d
Conc. (M)
SFair (%) a b SFhipox (%) b c
R
7
8
9
147.4–149.0 (toluene)
116.0–117.0, CC e
73.0–74.0, CC e
3322, 1599
3312, 1605
2244, 1611
20
20
20
10
5
3591
3396
70912
78923
090
10090
90913
64914
9696
−0.13
–
0.19
090
37920
9398
10
Oil, CC e
2357, 1634
1719, 1590
1707, 1605
3480, 1570
3500, 1575
2870, 1430
1595, 1038
1321, 1148
2860, 1598
1740, 1235
3520, 1595
1592, 1449
1590, 1450
1700, 1595
–
20
20
20
20
20
20
20
20
20
20
20
20
20
20
5
35910
72911
84920
10090 f
87911
79929
6795
–
−0.19
–
11
51.5–51.9, CC e
12
61.8–62.7, CC e
91913
89915
88917
9893
g
13
Oil, CC e
–
14
Oil, CC e
–
–
–
–
–
–
g
17
Oil, CC e
g
18
94.2–95.4 (petroleum ether–EtOAc)
4795
g
19
131.7–133.0 (petroleum ether–EtOAc)
10090 f
1593
10090 f
10090 f
10090 f
9696
g
20
Oil, CC e
Oil, CC e
63.4–64.6, CC e
87.5–90.0, CC e
Oil, CC e
Oil, CC e
–
g
21
10090 f
9695
23
0.00
g
24
10090 f
68916
9498
7792
–
25
68923
9892
0.03
0.13
–
26
Reference h
10090
090
a Survival fraction in air.
b The tests were carried out in duplicate.
c Survival fraction in hypoxia.
d R values for R2 substituents, from R1=Ph derivatives, were taken from Ref. [20].
e Purification by chromatographic column.
f Highest concentration tested: 20 mM.
g Not available.
h Reference: 7-Chloro-3-(3-dimethylaminopropylamino)quinoxaline-2-carbonitrile N1,N4-dioxide, hydrochloride.
cultures were established from exponentially growing
cells and gassed with pure air or nitrogen for 30 min
before dosing with the compounds. Treatment lasted
2 h and gassing was continuous during this time. All
the compounds were tested firstly at 20 mM employing
duplicate flasks in both air and nitrogen and the
survival fraction under both conditions (SFair and
SFhipox) (see Section 6) was determined [17]. When
compounds became active at this concentration, they
were assayed at lower doses. Derivative 9, which was
potent at 20 mM, was also tested at 10 and 5 mM. The
results obtained are summarised in table I.
assayed and their methyl analogues, 9 and 10, were
analysed by cyclic voltammetry in deaerated dimethyl
sulphoxide (DMSO) solution at a mercury dropping
working electrode [35, 36]. Two typical reduction peaks
were observed during forward cathodic scan, which are
linked to the corresponding oxidation peak on the re-
verse anodic scan (figure 6a). Only the first reduction
step, a one-electron process, was studied as a function of
voltage sweep rate and switching potential. Table II
summarises the electrochemical parameters obtained at
a sweep rate of 1.0 V s−1 of derivatives 9 and 10. The
free radical of compound 9 generated at the first reduc-
tion process was investigated using ESR spectroscopy.
This radical was generated by electrolytical reduction in
situ at room temperature in DMSO, applying the same
cathodic potential obtained from the cyclic voltammetry
experiments [16, 37]. The hyperfine patterns indicate
that the unpaired electron is delocalised as far as the
heterocyclic system (figure 6b). A double-triplet reso-
4. Results
4.1. Electrochemical studies
To relate the electrochemical properties to the ob-
served cytotoxicity, the most characteristic derivatives

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M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
Figure 6. (a) Cyclic voltammogram of derivative 9 at 298 K in DMSO; sweep rate 1.0 V s−1. (b) ESR spectrum of derivative 9 at
298 K in DMSO; electrolytical generation.
nance band suggests that two nitrogens (N-heterocyclic
and N-nitrile) are coupling with the free electron.
withdrawing effects of 3-substituents are in accor-
dance with the proposed mechanism shown in figure
1.
The methyl analogue, 10, showed some degree of
cytotoxicity in oxia at 20 mM. The activity of deriva-
tives 9 and 10 is in accordance with the reduction
potential determined by voltammetry. The less nega-
tive potential for compound 9 than compound 10
indicates the greater facility to monoelectronation of
the former. The ESR spectra show that the extended
delocalisation of derivative 9 free radical is in concor-
dance with the reduction potential, as is with the
observed activity.
The benzylic-like substituted derivatives 17, 19, and
21 showed low cytotoxic activity, derivative 18 dis-
played moderate degree of selectivity for hypoxic con-
ditions and compound 20 showed cytotoxic under
aerobic conditions. The activity of these derivatives
could not be related to the electronic effects of the
substituents, considering the F parameters of Swain–
Lupton, though compounds with substituents of simi-
lar F values showed very different biological activities
(compare 2 and 21, FCl=FOAc=0.41; or 18 and 19,
5. Discussion
With the aim to obtain hypoxic-selective cytotoxins
and to analyse the structural requirements in the
cytotoxic activity of 1,2,5-oxadiazole N-oxides, 17
1,2,5-oxadiazole derivatives were prepared and evalu-
ated under hypoxic and aerobic conditions. The com-
pounds proved to be non-selective, compound 9 being
the most potent derivative at 20 mM. But at 5 mM
concentration the reference drug (7-chloro-3-(3-
dimethylaminopropylamino)quinoxaline-2-carbon-
itrile N1,N4-dioxide) shows hypoxic-selectivity, while
compound 9 shows no cytotoxic results.
The lack of hypoxic-selectivity could be explained
for the redox properties of derivatives. All derivatives
previously described [16] and the compounds studied
in this paper showed more negative potential than
that expected for anaerobic selectivity [38–40].
Compound 9 bears a 3-substituent (3-CN) with an
R parameter positive, as the more cytotoxic parent
compounds (derivatives 1 and 2 with 3-formyl and
3-chloromethyl moieties) (see figure 1 and table I). In
the series of phenyl analogues, clearly, the electronic
effect (evaluated as R descriptor) of the substituent in
3-position and the presence/absence of N-oxide moi-
ety showed that they play an important role in the
biological activity displayed. The mesomeric electron
F
SOMe=0.52 and FSO2Me=0.54). The activity could be
Table II. Electrochemical properties of derivatives 9 and 10.
Compound Epc1 (V) vs. SCE pc1−Epa1 (V) pc1/ipa1
E
i
9
10
−0.67
−0.90
0.08
0.07
1.8
2.0

M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
777
related to the capacity of good leaving group as Cl
substituent. This fact and the decrease of activity of
the deoxy analogues, 23–26, are totally in agreement
with the mechanism of action proposed. The chemical
production of compound 27, in a reductive medium,
indicates that the benzylic-like position in compound
2, could be participating in the electron delocalisation
more than the corresponding position in compounds
4 and 5, though they produced chemically the deoxy
derivatives under reductive conditions. This fact is
also in agreement with the proposed mechanism,
compounds 4 and 5 show less cytotoxicity [41].
Compared to quinoxaline N1,N4-dioxide (reference
compounds) [13, 42] these derivatives showed better
solubility in the biological medium. In spite of the
lack of selectivity of compounds 1, 2 and 9, this kind
of physicochemical property transforms them into
good leaders that could be chemically modified in
order to obtain the desired bioactivity.
Elmer 1310 apparatus, using potassium bromide tablets
for solid and oil products. 1H-NMR and 13C-NMR
spectra were recorded on a Bruker DPX-400 (at 400
MHz and 100 MHz) instrument, with tetramethylsilane
as the internal reference and in the indicated solvent; the
chemical shifts are reported in ppm. NOE, COSY and
HETCOR experiments were performed so as to obtain a
correct characterisation of some derivatives. Mass spec-
tra were recorded on a Shimadzu GC–MS QP 1100 EX
instrument at 70 eV.
6.1.2. Synthesis procedures
6.1.2.1. General procedure for preparation of derivatives
7 and 8
A mixture of aldehyde (1 or 6, 1 equiv.), hydroxy-
lamine hydrochloride (1 equiv.) and p-TsOH (catalytic
amounts) in ethanol as solvent was heated at reflux until
the aldehyde was not present (SiO2, petroleum ether–
AcOEt, 8:2). The solvent was evaporated in vacuo and
the residue was dissolved with EtOAc. After the work-
up procedure the residue was purified as indicated in
table I.
Also, the well-known NO-release properties of
derivative 9 and related compounds [43–45] make it a
potential vasodilator at the tumour zone with the
corresponding blood-flux increment. Combining the
solubility, NO-release properties and cytotoxicity
proved in this paper, compound 9 could be a good
target for cancer therapy.
6.1.2.2. 3-Formyl-N2-oxide-4-phenyl-1,2,5-oxadiazole
Z-oxime (7)
1
Biological evaluation in vivo of derivative 9 is cur-
Yield 40%. H-NMR (NOE experiment) (DMSO-d6)
rently in progress.
7.61 (m, 3H, HꢀAr), 7.78 (m, 2H, HꢀAr), 7.93 (s, 1H,
CHꢁN), 12.45 (s, 1H, OH). 13C-NMR (HMQC and
HMBC experiments) (DMSO-d6) 111.51 (CꢁN+O−),
127.00–132.00 (C-Ar), 136.05 (CꢁNOH), 156.42 (CꢁN).
MS (EI, 70 eV); m/z (percentage of relative abundance):
6. Experimental protocols
+
’
6.1. Chemistry
205 (50.5, [M ]), 189 (9.3), 175 (16.5). Anal.
(C9H7N3O3) C, H, N.
6.1.1. General
All starting materials were commercially available
research-grade chemicals and used without further
purification. Compounds 1, 2, 4–6, and 9 were prepared
according to literature [17, 23] methods. All solvents
were dried and distilled prior to use. All the reactions
were carried out in a nitrogen atmosphere. The typical
work-up included washing the organic layer with brine,
drying with sodium sulphate and evaporating in vacuo.
Melting points were determined using a Leitz Micro-
scope Heating Stage Model 350 apparatus and are un-
corrected. Elemental analyses were obtained from
vacuum-dried samples (over phosphorous pentoxide at
room temperature) and performed on a Fisons EA 1108
CHNS-O analyser, and were within 0.4% of theoretical
values. Infrared spectra were recorded on a Perkin–
6.1.2.3. 4-Formyl-3-methyl-N2-oxide-1,2,5-oxadiazole
E-oxime (8)
Column chromatography (SiO2, petroleum ether–
1
EtOAc (0–50%)). Yield 74%. H-NMR (NOE experi-
ment) (DMSO-d6) 2.27 (s, 3H, CH3), 8.27 (s, 1H,
CHꢁN), 12.49 (s, 1H, OH). 13C-NMR (HMQC and
HMBC experiments) (DMSO-d6) 10.03 (CH3), 112.46
(CꢁN+O−), 139.89 (CꢁNOH), 153.61 (CꢁN). MS (EI, 70
eV); m/z (percentage of relative abundance): 143 (23.2,
+
’
[M ]), 113 (28.0), 83 (31.3). Anal. (C4H5N3O3) C, H, N.
6.1.2.4. General procedure for preparation of derivatives
9 and 10
To a mixture of oxime (7 or 8, 1 mmol) and DMF as
solvent (2.0 mL) was added SOCl2 (0.3 mL). The mix-

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M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
+
’
ture was stirred at room temperature until the oxime
was not present (SiO2, petroleum ether–EtOAc 1:2).
The excess of SOCl2 was eliminated with a saturated
aqueous solution of NaHCO3. The nitrile was extracted
three times with EtOAc. After the work-up procedure
the residue was purified by chromatography.
(percentage of relative abundance): 198 (12.8, [M ]),
168 (12.0), 153 (13.9). Anal. (C8H10N2O4) C, H, N.
6.1.2.9. General procedure for preparation of derivatives
13 and 14
A mixture of aldehyde (1 or 6, 1 equiv.), nitromethane
(1 equiv.) and butylamine (catalytic amounts) in ethanol
was heated at reflux until the aldehyde was not present
(SiO2, petroleum ether–EtOAc, 1:1). The solvent was
evaporated in vacuo and the residue was dissolved with
EtOAc. After the work-up procedure the residue was
purified by chromatography (SiO2, petroleum ether–
EtOAc, 4:1).
6.1.2.5. 4-Cyano-3-methyl-N2-oxide-1,2,5-oxadiazole
(10)
Column chromatography (SiO2, petroleum ether–
1
EtOAc (0–20%)). Yield 3%. H-NMR (DMSO-d6) 2.23
(s, 3H, CH3). 13C-NMR (HMQC and HMBC experi-
ments) (DMSO-d6) 8.52 (CH3), 109.07 (CN), 114.43
(CꢁN+O−), 137.74 (CꢁN). MS (EI, 70 eV); m/z (percent-
+
’
age of relative abundance): 125 (100.0, [M ]), 95 (23.5),
6.1.2.10. 3-(1-Hydroxy-2-nitroethyl)-N2-oxide-4-phenyl-
1,2,5-oxadiazole (13)
65 (7.4). Anal. (C4H3N3O2) C, H, N.
Yield 38%. 1H-NMR (COSY experiment) (CDCl3)
4.72 (dd, J1=14.1 Hz, J2=4.2 Hz, 1H, CHNO2), 4.78
(bs, 1H, OH), 4.98 (dd, J1=14.1 Hz, J2=8.7 Hz, 1H,
CHNO2), 5.63 (dd, J1=8.7 Hz, J2=4.1 Hz, 1H,
CHOH), 7.61 (m, 3H, HꢀAr), 7.77 (m, 2H, HꢀAr).
13C-NMR (HMQC and HMBC experiments) (CDCl3)
62.00 (CHOH), 76.00 (CH2NO2), 113.00 (CꢁN+O−),
6.1.2.6. General procedure for preparation of derivatives
11 and 12
To a solution of sodium (1 equiv.) in anhydrous
ethanol with intensive stirring a solution of ethyloxycar-
bonylmethyl triphenyl phosphonium bromide (1 equiv.)
in anhydrous ethanol was added rapidly. Stirring was
continued for 30 min. Then a solution of aldehyde (1 or
6, 1 equiv.) in anhydrous ethanol was added and the
resulting mixture was heated at reflux until the aldehyde
was not present (SiO2, petroleum ether–EtOAc, 1:1).
The mixture was concentrated in vacuo and treated with
EtOAc. After the work-up process the residue was
purified by chromatography (SiO2, petroleum ether–
EtOAc (0–30%)).
125.00-132.00 (C-Ar), 155.00 (CꢁN). MS (EI, 70 eV);
+
’
m/z (percentage of relative abundance): 251 (5.0, [M ]),
233 (4.2), 217 (0.4), 203 (3.1). Anal. (C10H9N3O5) C, H,
N.
6.1.2.11. 4-(1-Hydroxy-2-nitroethyl)-3-methyl-N2-
oxide-1,2,5-oxadiazole (14)
Yield 25%. 1H-NMR (COSY experiment) (CDCl3)
2.29 (s, 3H, CH3), 4.99 (dd, J1=13.6 Hz, J2=9.1 Hz,
1H, CHNO2), 5.15 (dd, J1=13.6 Hz, J2=3.7 Hz, 1H,
CHNO2), 5.72 (dd, J1=8.8 Hz, J2=3.5 Hz, 1H,
CHOH), 5.94 (bs, 1H, OH). 13C-NMR (HMQC and
HMBC experiments) (CDCl3) 7.75 (CH3), 64.00
(CHOH), 78.00 (CH2NO2), 112.68 (CꢁN+O−), 157.81
6.1.2.7. 3-(Ethyloxypropenoyl)-N2-oxide-4-phenyl-1,2,5-
oxadiazole (11)
1
Yield 71%. H-NMR (CDCl3) 1.28 (t, 3H, J=7.0 Hz,
CH3), 4.27 (q, J=7.0 Hz, 2H, CH2O), 7.20 (d, J=14.0
Hz, 1H, ꢁCH), 7.42 (d, J=14.0 Hz, 1H, ꢁCH), 7.58 (m,
5H, HꢀAr). MS (EI, 70 eV); m/z (percentage of relative
(CꢁN). MS (EI, 70 eV); m/z (percentage of relative
+
’
+
’
abundance): 260 (29.9, [M ]), 215 (13.3), 200 (100.0).
abundance): 189 (37.7, [M ]), 171 (1.1), 155 (0.3), 141
Anal. (C13H12N2O4) C, H, N.
(1.6). Anal. (C5H7N3O5) C, H, N.
6.1.2.8. 4-(Ethyloxypropenoyl)-3-methyl-N2-oxide-1,2,5-
oxadiazole (12)
Yield 85%. H-NMR (CDCl3) 1.29 (t, 3H, J=7.2 Hz,
CH3), 2.27 (s, 3H, CH3), 4.24 (q, J=7.1 Hz, 2H,
CH2O), 6.64 (d, J=16.4 Hz, 1H, ꢁCH), 7.40 (d, J=
16.4 Hz, 1H, ꢁCH). 13C-NMR (HMQC and HMBC
experiments) (CDCl3) 8.82 (CH3), 14.46 (CH3), 61.78
(CH2), 111.53 (CꢁN+O−), 127.32 (Cꢁ), 128.67 (Cꢁ),
153.31 (CꢁN), 165.16 (CꢁO). MS (EI, 70 eV); m/z
6.1.2.12. 3-Methylthiomethyl-N2-oxide-4-phenyl-
1,2,5-oxadiazole (17)
1
A mixture of chloride 2 (500 mg, 2.4 mmol), KI (40
mg, 0.24 mmol) and THF (10.0 mL) as solvent was
stirred at 0 °C. Then sodium thiomethoxide (168 mg,
2.4 mmol) was added in three portions and the mixture
was heated at reflux for 6 h. After filtering through celite
and evaporating the solvent in vacuo, the residue was
dissolved with EtOAc. After the work-up procedure the

M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
779
residue was purified by chromatography (SiO2,
petroleum ether–EtOAc (0–5%)). Yield 34%. H-NMR
6.1.2.15. N2-Oxide-4-phenyl-3-phenylthiomethyl-
1,2,5-oxadiazole (20)
1
(CDCl3) 2.19 (s, 3H, CH3), 3.72 (s, 2H, CH2), 7.53 (m,
3H, HꢀAr), 7.79 (m, 2H, HꢀAr). 13C-NMR (HMQC
and HMBC experiments) (CDCl3) 16.44 (CH3), 26.09
(CH2), 114.66 (CꢁN+O−), 126.00–132.00 (C-Ar), 156.90
A mixture of chloride 2 (500 mg, 2.4 mmol), thiophe-
nol (264 mg, 2.4 mmol), K2CO3 (332 mg, 2.4 mmol), KI
(40 mg, 0.24 mmol) and THF (10.0 mL) as solvent was
heated at reflux for 24 h. After filtering through celite
and evaporating the solvent in vacuo, the residue was
dissolved with EtOAc. After the work-up procedure the
residue was purified by chromatography (SiO2,
(CꢁN). MS (EI, 70 eV); m/z (percentage of relative
+
’
abundance): 222 (3.1, [M ]), 205 (2.2), 159 (31.5). Anal.
(C10H10N2O2S) C, H, N.
1
petroleum ether–EtOAc (0–5%)). Yield 12%. H-NMR
(CDCl3) 4.11 (s, 2H, CH2), 7.20-7.76 (m, 10H, HꢀAr).
13C-NMR (HMQC and HMBC experiments) (CDCl3)
28.47 (CH2), 113.72 (CꢁN+O−), 125.00–135.00 (C-Ar),
6.1.2.13. Methyl[(N2-oxide-4-phenyl-1,2,5-
oxadiazole-3-yl)methyl]sulfoxide (18)
A heterogeneous mixture of thioether 17 (100 mg,
0.45 mmol), meta-chloroperbenzoic acid (78 mg, 0.45
mmol), CHCl3 (15.0 mL) and buffer phosphate (pH 8)
(10.0 mL) was stirred at room temperature for 24 h.
After the work-up procedure the residue was purified by
chromatography (SiO2, petroleum ether–EtOAc (0–
100%)) and then the fraction corresponding to the
product was crystallised as indicated in table I. Yield
76%. 1H-NMR (CDCl3) 2.79 (s, 3H, CH3), 4.21 (d,
J=14.2 Hz, 1H, CH), 4.27 (d, J=14.2 Hz, 1H, CH),
7.61 (m, 3H, HꢀAr), 7.97 (m, 2H, HꢀAr). 13C-NMR
(HMQC and HMBC experiments) (CDCl3) 39.69
(CH3), 47.41 (CH2), 110.57 (CꢁN+O−), 126.00–132.00
156.89 (CꢁN). MS (EI, 70 eV); m/z (percentage of
+
’
relative abundance): 284 (6.3, [M ]), 267 (49.8), 223
(16.4). Anal. (C15H12N2O2S) C, H, N.
6.1.2.16. 3-Acetyloxymethyl-N2-oxide-4-phenyl-
1,2,5-oxadiazole (21)
A mixture of alcohol 5 (500 mg, 2.6 mmol), H2SO4
conc. (0.14 mL, 2.6 mmol) and acetic acid (5.0 mL) as
solvent was stirred at room temperature for 24 h. The
acetic acid was evaporated in vacuo and the residue was
dissolved in EtOAc and treated with NaHCO3 saturated
solution three times. After the work-up procedure the
residue was purified by chromatography (SiO2,
petroleum ether–EtOAc (0–10%)). Yield 24%. 1H-
NMR (CDCl3) 2.10 (s, 3H, CH3), 5.14 (s, 2H, CH2),
7.54 (m, 3H, HꢀAr), 7.72 (m, 2H, HꢀAr). 13C-NMR
(HMQC and HMBC experiments) (CDCl3) 20.75
(CH3), 54.47 (CH2), 111.68 (CꢁN+O−), 126.00–132.00
(C-Ar), 158.61 (CꢁN). MS (EI, 70 eV); m/z (percentage
+
’
of relative abundance): 238 (4.0, [M ]), 175 (74.4), 159
(4.1). Anal. (C10H10N2O3S) C, H, N.
6.1.2.14. Methyl[(N2-oxide-4-phenyl-1,2,5-
oxadiazole-3-yl)methyl]sulfone (19)
(C-Ar), 157.11 (CꢁN), 170.31 (CꢁO). MS (EI, 70 eV);
+
’
m/z (percentage of relative abundance): 234 (2.7, [M ]),
A mixture of thioether 17 (100 mg, 0.45 mmol) and
acetic acid (5.0 mL) as solvent was stirred at 0 °C. Then
KMnO4 (142 mg, 0.90 mmol) was added in three por-
tions and the mixture was stirred at room temperature
for 24 h. The acetic acid was evaporated in vacuo and
the residue was dissolved in EtOAc. After the work-up
procedure the residue was purified by chromatography
(SiO2, petroleum ether–EtOAc (0–20%)) and then the
fraction corresponding to the product was crystallised as
218 (0.5), 204 (5.1). Anal. (C11H10N2O4) C, H, N.
6.1.2.17. General procedure for preparation of
derivatives 23 and 24
A mixture of N-oxide (4 or 5, 0.27 mmol), Zn dust
(1.3 mmol), NH4Cl solution (30%) (4.7 mL) and THF
(5.0 mL) was heated at reflux for 3 h. After filtering
through celite the solution was extracted three times
with Et2O (10 mL). After the work-up process the
residue was purified by chromatography.
1
indicated in table I. Yield 55%. H-NMR (CDCl3) 3.13
(s, 3H, CH3), 4.68 (s, 2H, CH2), 7.61 (m, 3H, HꢀAr),
7.93 (m, 2H, HꢀAr). 13C-NMR (HMQC and HMBC
experiments) (CDCl3) 44.73 (CH3), 49.84 (CH2), 108.94
(CꢁN+O−), 125.00–133.00 (C-Ar), 157.91 (CꢁN). MS
6.1.2.18. 3-Hydroxymethyl-4-phenyl-1,2,5-oxadiazole
(23)
Column chromatography (SiO2, petroleum ether-
1
(EI, 70 eV); m/z (percentage of relative abundance): 254
EtOAc (0–50%)). Yield 45% yield. H-NMR (CDCl3)
+
’
(3.7, [M ]), 238 (0.7), 224 (3.1), 175 (4.8). Anal.
2.54 (bs, 1H, OH), 4.99 (s, 2H, CH2O), 7.54 (m, 3H,
(C10H10N2O4S) C, H, N.
HꢀAr), 7.89 (m, 2H, HꢀAr). 13C-NMR (HMQC and

780
M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
HMBC experiments) (CDCl3) 54.69 (CH2OH), 125.00–
132.00 (C-Ar), 152.94 (CꢁN), 154.23 (CꢁN). MS (EI, 70
celite and then the solvent was evaporated in vacuo.
After the work-up process the residue was purified by
chromatography (SiO2, petroleum ether–EtOAc (0–
eV); m/z (percentage of relative abundance): 176 (100.0,
+
’
1
[M ]), 146 (9.9), 131 (20.1). Anal. (C9H8N2O2) C, H, N.
30%)). Yield 30% yield. H-NMR (CDCl3) 7.54 (m, 3H,
HꢀAr), 7.96 (m, 2H, HꢀAr), 10.48 (s, 1H, CHO). 13C-
NMR (HMQC and HMBC experiments) (CDCl3)
124.00–134.00 (C-Ar), 151.58 (CꢁN), 153.79 (CꢁN),
6.1.2.19. 4-Phenyl-3-[4-(pyrimidine-2-yl)piperazine-
1-ylmethyl]-1,2,5-oxadiazole (24)
Column chromatography (SiO2, petroleum ether–
EtOAc (0–30%)). Yield 58%. 1H-NMR (CDCl3) 2.66
(m, 4H), 3.86 (s, 2H), 3.88 (m, 4H), 6.52 (t, J=4.7 Hz,
1H), 7.52 (m, 3H, HꢀAr), 8.03 (m, 2H, HꢀAr), 8.35 (d,
J=4.7 Hz, 2H). 13C-NMR (HMQC and HMBC experi-
ments) (CDCl3) 44.99 (CH2ꢀN), 50.87 (ArꢀCH2ꢀN),
53.11 (CH2N), 110.49 (CPyrimidinyl), 126.00–131.00 (C-
Ar), 150.63 (CꢁN), 155.50 (CꢁN), 158.12 (CPyrimidinyl),
182.32 (CHO). MS (EI, 70 eV); m/z (percentage of
+
’
relative abundance): 174 (100.0, [M ]), 146 (12.0), 129
(25.0). Anal. (C9H6N2O2) C, H, N.
6.2. Pharmacology
6.2.1. Cells
V79 cells (Chinese hamster lung fibroblasts) were
obtained from the European Collection of Animal Cell
Cultures (ECACC) and maintained in logarithmic
growth as subconfluent monolayers by trypsinisation
and subculture to 1–2×104 cells/ cm−2 twice weekly.
The growth medium was Eagle’s minimal essential
medium (EMEM), containing 10% (v/v) foetal bovine
serum (FBS) and penicillin/streptomycin at 100 U per
162.07 (CPyrimidinyl). MS (EI, 70 eV); m/z (percentage of
+
’
relative abundance): 322 (12.7, [M ]), 292 (0.2), 260
(1.1). Anal. (C17H18N6O) C, H, N.
6.1.2.20. 1,2-Bis-(4-phenyl-1,2,5-oxadiazole3-yl)ethane
(27)
Column chromatography (SiO2, petroleum ether–
100 mg mL−1
.
1
EtOAc (0–5%)). M.p. 135.0–135.5 °C; yield 35%. H-
NMR (CDCl3) 2.67 (s, 4H, CH2), 7.50 (m, 6H, HꢀAr),
7.66 (m, 2H, HꢀAr). 13C-NMR (HMQC and HMBC
experiments) (CDCl3) 23.03 (CH2), 127.00–139.00 (C-
6.2.2. Aerobic and hypoxic cytotoxicity: suspension
cultures
Monolayers of V79 cells in exponential growth were
trypsinised, and suspension cultures were set up in 50
mL glass flasks: 2×104 cells mL−1 in 30 mL of EMEM
containing 10% (v/v) FBS and HEPES (10 mM). The
glass flasks were stoppered with rubber caps perforated
with two 19G needles to provide gas inlet and outlet.
The flasks were submerged and stirred in a water bath at
37 °C where they were gassed with humidified air or
pure nitrogen.
Ar), 148.20 (CꢁN), 151.47 (CꢁN). MS (EI, 70 eV); m/z
+
’
(percentage of relative abundance): 316 (1.6, [M −2H]),
314 (4.3), 287 (19.8). Anal. (C18H14N4O2) C, H, N.
6.1.2.21. 3-Chloromethyl-4-phenyl-1,2,5-oxadiazole (25)
A mixture of 23 (500 mg, 2.8 mmol) and thionyl
chloride (1.24 g, 10.4 mmol) was stirred at room temper-
ature for 24 h. The reaction mixture was treated with
ice, NaHCO3 saturated solution (until basic pH), and
extracted three times with EtOAc (20 mL). After the
work-up process the residue was purified by chromatog-
raphy (Al2O3, petroleum ether–EtOAc (0–5%)). Yield
6.2.3. Treatment
Compound solutions were prepared just before dos-
ing. Stock solutions, 150-fold more concentrated, were
prepared in pure DMSO or in sterilised distilled water.
Thirty minutes after the start of gassing, 0.2 mL of the
stock compound solution was added to each flask, two
flasks per dose. In every assay there was one flask with
0.2 mL of DMSO (negative control). 7-Chloro-3-(3-
dimethylaminopropylamino)quinoxaline - 2 - carbonitrile
N1,N4-dioxide, hydrochloride was used as the reference
positive drug.
1
60%. H-NMR (CDCl3) 4.84 (s, 2H, CH2Cl), 7.59 (m,
3H, HꢀAr), 7.83 (m, 2H, HꢀAr). 13C-NMR (HMQC
and HMBC experiments) (CDCl3) 32.99 (CꢀCl),
125.00–132.00 (C-Ar), 150.72 (CꢁN), 153.98 (CꢁN).
MS (EI, 70 eV); m/z (percentage of relative abundance):
+
’
194 (60.0, [M ]), 164 (40.0), 149 (10.0). Anal.
(C9H7ClN2O) C, H, N.
6.1.2.22. 3-Formyl-4-phenyl-1,2,5-oxadiazole (26)
A mixture of 23 (500 mg, 2.8 mmol), MnO2 (1.24 g,
10.4 mmol) and CHCl3 as solvent was stirred at room
temperature for 24 h. The mixture was filtered through
6.2.4. Cloning
After 2 h exposure to the compound the cells were
centrifuged and re-suspended in plating medium

M. Boiani et al. / European Journal of Medicinal Chemistry 36 (2001) 771–782
781
(EMEM plus 15% (v/v) FBS and penicillin/strepto-
mycin). Cell numbers were determined with a haemocy-
tometer and 102–103 cells were plated in 6-well plates to
give a final volume of 2 mL per 30 mm of well. Plates
were incubated at 37 °C in 5% CO2 for 7 days and then
stained with aqueous crystal violet. Colonies with more
than 64 cells were counted. The plating efficiency (PE)
was calculated by dividing the number of colonies by the
number of cells seeded. The percent of control cell
survival for the compound-treated cultures was calcu-
lated as PE (treated)/PE (control)×100.
H-atom was refined isotropically. As expected, the hete-
rocycle shows a symmetric bonding structure, with NꢀO
bond lengths equal to within experimental accuracy
,
(average(dispersion)=1.381(2) A). CꢀN bond distances
are also experimentally equal to each other (=1.305(1)
,
A). The phenyl ring subtends an angle of 19.14(3)° with
the heterocycle. The CH2OH substituent forms a
C2C1C3O2 torsion angle of 74.8(2)°, with their hy-
droxyl H-atom lying nearly on the C1C3O2 plane. The
crystal is stabilised by a relatively strong and linear
intermolecular H-bond involving the hydroxyl group of
a molecule and one of the nitrogen atoms of a neigh-
,
bour, symmetry related, molecule (d(H···N2%)=1.972 A,
6.3. Electrochemical method
Ú(O2ꢀH···N2%)=171°). The X-ray diffraction intensi-
ties were reduced and corrected with DENZO and
SCALEPACK programs [46]. The structure was solved by
direct and Fourier methods and the final molecular
model obtained by anisotropic full-matrix least-squares
refinement of the non-H atoms employing SHELXS [47]
and SHELXL [48] programs.
Cyclic voltammetry experiments were carried out in
DMSO (Aldrich, spectroscopy grade) with 0.1 M tetra-
butylammonium perchlorate (Fluka) as the supporting
electrolyte and purged with nitrogen at room tempera-
ture. 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 us-
ing a Weenking POS 88 instrument with a Kipp Zenen
BD93 recorder. Voltages scan rates ranged from 0.1 to
7. Supplementary material
Listings of atomic anisotropic displacement
parameters, and hydrogen atoms positions and
isotropic displacement parameters (two tables) are
available from the authors on request.
1.0 V s−1
.
6.4. ESR measurement
The radicals were generated by electrolytical reduc-
tion in situ at room temperature. ESR spectra were
recorded 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).
Acknowledgements
This work was financially supported by Comisio´n
Honoraria de Lucha Contra el Ca´ncer (Uruguay),
Proyecto X-2 of CYTED (Programa Iberoamericano de
Ciencia y Tecnolog´ıa para el Desarrollo), CONICET of
Argentina and FAPESP of Brazil. Part of the X-ray
diffraction experiments were carried out at the National
Diffraction Laboratory (LANADI), La Plata,
Argentina.
6.5. Crystallography
Suitable single crystals of 23 were obtained by slow
evaporation from AcOEt. The X-ray diffraction pattern
was collected at 120 K on a Kappa CCD diffractometer
,
employing Mo Ka radiation (u=0.71073 A). Derivative
23 crystallises in the monoclinic P21/n space group, with
References
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1515 reflections with I>2|(I) and refined to an agree-
ment R1-factor of 0.043. The hydrogen atoms were
found in a difference Fourier map. However, all H-
atoms but the hydroxyl one were positioned stereochem-
ically and refined with the riding model. The hydroxyl
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