Heteroarylnitrones as drugs for neurodegenerative diseases: Synthesis, neuroprotective properties, and free radical scavenger properties

Article abstract of DOI:10.1021/jm8006432

New 1,2,4-thiadiazolylnitrones and furoxanylnitrones were developed and evaluated as neuroprotective agents on a human neuroblastoma (SH-SY5Y) cells model. They inhibited at low micromolar concentrations the oxidative damage and the death induced by exposure to hydrogen peroxide. These heteroarylnitrones showed excellent peroxyl free radical absorbance capacities, analyzed by oxygen radical absorbance capacity (ORAC) assay with fluorescein as the fluorescent probe, ranging from 1.5- to 16.5-fold the value of the reference nitrone, α-phenyl-N-tert-butylnitrone (PBN). The electron spin resonance spectroscopy (ESR) demonstrated the ability of these derivatives to directly trap and stabilize oxygen, carbon, and sulfur-centered free radicals. These results demonstrated the potential use of these heteroarylnitrones as neuroprotective agents in preventing the death of cells exposed to enhanced oxidative stress and damage.

Full text of DOI:10.1021/jm8006432

6150
J. Med. Chem. 2008, 51, 6150–6159
Heteroarylnitrones as Drugs for Neurodegenerative Diseases: Synthesis, Neuroprotective
Properties, and Free Radical Scavenger Properties
Williams Porcal,‡ Paola Herna´ndez,‡ Mercedes Gonza´lez,‡ Ana Ferreira,§ Claudio Olea-Azar,# Hugo Cerecetto,*,‡ and
Ana Castro*,|,†
Departamento de Qu´ımica Orga´nica, Facultad de Ciencias-Facultad de Qu´ımica, and Catedra de Inmunolog´ıa, UniVersidad de la Repu´blica,
MonteVideo, Uruguay, Departamento de Qu´ımica Inorga´nica y Anal´ıtica, Facultad de Ciencias Qu´ımicas y Farmace´uticas, UniVersidad de
Chile, Santiago, Chile, and Instituto de Qu´ımica Me´dica (CSIC), Madrid, Spain
ReceiVed May 28, 2008
New 1,2,4-thiadiazolylnitrones and furoxanylnitrones were developed and evaluated as neuroprotective agents
on a human neuroblastoma (SH-SY5Y) cells model. They inhibited at low micromolar concentrations the
oxidative damage and the death induced by exposure to hydrogen peroxide. These heteroarylnitrones showed
excellent peroxyl free radical absorbance capacities, analyzed by oxygen radical absorbance capacity (ORAC)
assay with fluorescein as the fluorescent probe, ranging from 1.5- to 16.5-fold the value of the reference
nitrone, R-phenyl-N-tert-butylnitrone (PBN). The electron spin resonance spectroscopy (ESR) demonstrated
the ability of these derivatives to directly trap and stabilize oxygen, carbon, and sulfur-centered free radicals.
These results demonstrated the potential use of these heteroarylnitrones as neuroprotective agents in preventing
the death of cells exposed to enhanced oxidative stress and damage.
Introduction
Reactive oxygen species (ROS) and reactive nitrogen species
(RNS) produced by oxidative metabolism are capable of
damaging cellular components through molecular modifications
to a polyunsaturated membrane’s lipids, proteins, and nucleic
acids.1,2 Much evidence suggests that biological oxidation in
the human body generates highly pathogenic ROS and RNS
such as hydroxyl free radical (•OH), superoxide anion (O2•-),
peroxynitrite (ONOO-), and lipid peroxide free radicals (ROO•),
causing cellular injury.3-5 These pathological events have
important roles in many degenerative disorders, for example,
atherosclerosis, rheumatoid arthritis, and several neurodegen-
erative diseases such as ischemic conditions, stroke, Parkinson’s
disease, and Alzheimer’s disease.6-8 The central nervous system
(CNS) is especially sensitive to oxidative damage for reasons
such as (a) high use of oxygen during the metabolic process
and therefore greater production of ROS, (b) fewer antioxidants
defenses than other organs, for example, liver and heart, (c)
Figure 1. Antioxidant and neuroprotective mechanisms of action
postulated for PBN.
enrichment in the more easily peroxidable fatty acids, (d) areas
with high levels of the Fe3+/ascorbate pro-oxidant system that
•
reduce H2O2 to form OH, a potent oxidant.9 Recently, it has
neurodegenerative diseases is an important area of neurochemi-
cal research.
been reported that ꢀ-amyloid solutions liberate hydrogen
•
Recently, it has been proved that nitrone-free radical trap,
i.e., R-phenyl-N-tert-butylnitrone (PBN,a Figure 1), could be
used in the treatment of neurodegenerative diseases as well as
in the prolongation of life span.11 Initially, the neuroprotective
activity was attributed to the nitrone group because of its ability
to act as free radical scavenger (FRS, Figure 1). Subsequently,
observations that PBN has a good neuroprotective activity at
lower doses than those necessary to act as an effective FRS
demonstrated the ability of PBN to inhibit signal transduction
processes such as suppression of proinflammatory cytokines,
genes, and mediators associated with enhanced neuroinflam-
matory processes (Figure 1).12 The neuroprotective activity of
the nitrone pharmacophore depends in great part on the
connectivity and the nature of substituents on the nitrone group.
In this sense, the chemical and pharmacological aspects of
different heteroarylnitrones have been reviewed,13 describing
peroxide and, subsequently, OH converted via Fenton’s reac-
tion.10 Consequently, the search of effective treatments that
prevent oxidative stress associated with premature aging and
* To whom correspondence should be addressed. Phone: +34 918061130.
Fax: +34 918034660. E-mail: acastro@neuropharma.es.
‡
Departamento de Qu´ımica Orga´nica, Universidad de la Repu´blica.
Catedra de Inmunolog´ıa, Universidad de la Repu´blica.
Universidad de Chile.
Instituto de Qu´ımica Me´dica (CSIC).
§
#
|
†
Present address: NeuroPharma, S. A.; Avda. de la Industria 52, 28760
Tres Cantos, Madrid, Spain.
a Abbreviations: BBB, blood-brain barrier; DMPO, 5,5-dimethyl-1-
pyrroline N-oxide; FL, fluorescein; FRS, free radical scavenger; HFS,
hyperfine splitting; LDH, lactate dehydrogenase; NAC, N-acetyl cysteine;
ORAC, oxygen radical absorbance capacity; PAMPA, parallel artificial
membrane permeation assay; PBL, porcine brain lipid; PBN, R-phenyl-N-
tert-butylnitrone; RP, reduction percentage.
10.1021/jm8006432 CCC: $40.75
2008 American Chemical Society
Published on Web 09/13/2008

Heteroarylnitrones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6151
Figure 2. Heteroarylnitrones with neuroprotective and spin trapping properties.
Scheme 1. Heteroarylnitrones Designed as Potential Drugs for Neurodegenerative Diseases
some patented nitrone containing furans (I-III, Figure 2) with
good activity against the neuronal cell damage induced by
ꢀ-amyloid.14 On the other hand, imidazolylnitrones (IV and V,
Figure 2) has been developed and biologically evaluated,
showing in vivo neuroprotective properties.15 Additionally
several nitrones has been widely used as spin trap for the specific
thiadiazole, 1,2,4-thiadiazole, 1,2,5-oxadiazole N-oxide, and
benzo[1,2-c]1,2,5-oxadiazole N-oxide derivatives.17,18 The ni-
trones syntheses were planned through the condensation between
the corresponding heteroaromatic aldehyde and N-substituted
hydroxylamine,19 our first goal being the preparation of the
corresponding aldehyde derivatives. For the synthesis of 3- and
5-formyl-1,2,4-thiadiazole aldehydes, 6 and 9, respectively, the
synthetic routes shown in Scheme 2a were employed. First, the
reaction of benzothioamide with N,N-dimethylalkanamide di-
methyl acetals produced the corresponding N′-(thioaroyl)-N,N-
dimethylamidines 1 and 2 in excellent yield.20 These interme-
diates were converted by amination-cyclization into the 1,2,4-
thiadiazole 3 and 4. The 3-formyl-1,2,4-thiadiazole 6 was
obtained through bromination-oxidation of thiadiazole 4.
Attempts to obtain aldehyde 6 using formylation reaction
between 5-phenyl-1,2,4-thiadiazole 3 and n-BuLi in DMF were
unsuccessful. In these conditions, the ring-opening product, 5,
was mainly generated. Oxidation of the methyl substituent in
compound 4 using selenium dioxide was also explored in the
preparation of aldehyde 6; however, no reaction was observed.
Second, nitrile sulfide, obtained in situ through thermolysis of
the 1,3,4-oxathiazol-2-one 7 (Scheme 2a), was treated with ethyl
cyanoformate, yielding ester 8 via a 1,3-dipolar cycloaddition
process.21 Subsequently, the desired aldehyde 9 was prepared
by reduction of 8 with DIBAL-H and subsequent oxidation with
MnO2. In order to know the 1,2,4-thiadiazole-substituents
stereoelectronic/antioxidant effects (Scheme 1), different 3-aryl
substitutions were studied (Scheme 2b). Consequently, alde-
hydes 18, 20, and 21 were prepared following the same synthetic
•
detection of transient radicals (e.g., OH, O2•-, ROO•) or
relatively stable radicals (e.g., NO) that are undetectable under
normal conditions,16 in the electron spin resonance (ESR)
spectroscopy. With this aim two nitrones have been commonly
used in the spin trapping technique: the linear nitrone PBN
(Figure 1) and the cyclic nitrone DMPO (5,5-dimethyl-1-
pyrroline N-oxide VI, Figure 2).
On the basis of that, herein we describe the design and
synthesis of new heteroarylnitrones combining, in their struc-
tures, fragments able to show neuroprotection properties, such
as nitrone moiety, antioxidant fragments, and heterocyclic
groups able to stabilize the generated free radical (Scheme 1).
In order to assess the protective effects of heteroarylnitrones,
the effects of H2O2-injured in neuronal cells (SH-SY5Y) and
in vitro unspecific cytotoxicity against J774-mouse macrophages
were evaluated. Additionally, the antioxidant activities and the
spin trapping properties have been investigated by evaluating
the direct reaction with oxygen-, carbon-, and sulfur-centered
free radicals by antioxidant assays and ESR spectroscopy.
Results and Discussion
Chemistry. Four different families of heteroarylnitrones were
selected to study the influence on the biological activity: 1,2,3-

6152 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Scheme 2. Synthesis of Formyl-Heterocycle Derivativesa
Porcal et al.
a Reagents and conditions: (a) room temp; (b) HSA, Py, EtOH, room temp; (c) n-BuLi, THF, -78 °C, then DMF, 0 °C; (d) SeO2, dioxane or ClCH2CH2Cl,
reflux; (e) NBS, PDBO, CCl4, reflux, then DSMO, 105 °C; (f) Cl(CdO)SCl, toluene, reflux; (g) decaline, 140-160 °C; (h) EtO2C-CN; (i) (1) DIBAL-H,
THF, 0 °C; (2) MnO2, CHCl3, reflux; (j) (1) H2NNHCO2Met, p-TsOH, toluene, reflux; (2) SOCl2, 60 °C; (k) NaBH4, EtOH, room temp; (l) MnO2, CHCl3,
reflux; (m) NBS, PDBO, CCl4, reflux, then DSMO, 120 °C; (n) NaNO2, AcOH, 0 °C; (o) MnO2, CHCl3, room temp; (p) (1) NaN3, DMSO, 60-70 °C; (2)
DMSO, 90-100 °C.
route for aldehyde 9 preparation. Attempts to obtain the desired
4-NO2 analogue 19 were unsuccessful because of the low
stability of ester 15 in the reduction conditions (i) (Scheme 2b).
The 1,2,3-thiadiazole carbonylic reagents were prepared from
ethyl acetoacetate following a condensation with methyl car-
bazate, forming the corresponding acylhydrazone, and subse-
quent cyclization via Hurd-Mori reaction in the presence of
thionyl chloride (Scheme 2c).22 Then thiadiazole 22 was
transformed into the key aldehydes 24 and 25 by reduction/
oxidation for the first case and by benzylic-like bromination/
oxidation for the second. Finally, the synthetic procedures for
furoxan and benzofuroxan systems included, for the first
heterocycle, addition to alkenes followed by intramolecular
cyclization and thermointramolecular cyclization for the second
one (Scheme 2d). Consequently, the reaction of cinnamyl
alcohol or crotonaldehyde with NaNO2 in AcOH produced the
furoxan derivatives 26 and 28, respectively.23 Alcohol 26 was
converted into the aldehyde 27 by oxidation with MnO2.23 The
formylbenzofuroxan 29 was obtained from 4-chloro-3-nitroben-
zaldehyde in a one-pot process via nucleophilic substitution with
the azide anion and subsequent cyclization by pyrolysis of the
corresponding o-nitrophenylazide intermediate.24 The desired
nitrones, 30-41, were obtained in good yield for reaction
between the corresponding aldehydes 6, 9, 18, 20, 21, 24, 25,
27-29 and N-substituted hydroxylamine hydrochloride in the
presence of NaHCO3 (Scheme 3). Taking into account that the
nitrone stability, as well as the stability of the corresponding
spin-adduct (nitroxyl free radical), is highly influenced by the
N-alkyl substituent,13 we developed derivatives 31-33 by

Heteroarylnitrones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6153
Scheme 3. Synthesis and Structure of New Heteroarylnitronesa
a Asterisk (/) indicates at room temperature.
varying R1 (Scheme 3) in order to evaluate the effect of this
modulation in the bioactivity studies.
measuring the activity of LDH released and expressed as the
difference between nitrones-treated and untreated cells. The
basal percentage of viable neurons after treatment with 100 µM
H2O2 was 45%, and N-acetylcysteine (NAC) was used as
positive test at 5 mM (Figure 3). The values summarized in
Table 1 show that the 1,2,4-thiadiazolylnitrones 30-36 and the
furoxanylnitrone 40 protect similarly to or slightly higher against
the cellular damage than the parent nitrone PBN, with values
between 33% and 46%. Interestingly, the treatment of SH-SY5Y
cells with the 1,2,4-thiadiazolylnitrone 36 at 1 µM significantly
protects against H2O2 induced neurotoxicity, with a value of
20% (Figure 3). Furthermore, the benzofuroxanylnitrone 41
showed lower capacity to protect neuroblastoma human cells
from death induced by oxidative stress than PBN, while the
1,2,3-thiadiazolylnitrones (37 and 38) and furoxanylnitrone 39
did not show neuroprotective effects. These results revealed the
consequence of the connectivity and the nature of the nitrone-
pharmacophore substituents in the neuroprotective activity. The
1,2,4-thiadiazolylnitrones 31, 32, and 33, substituted in the
nitrogen atom of the nitrone by a tert-butyl, cyclohexyl, and
benzyl group, respectively, exhibited differential activity, indi-
cating some N-alkyl moieties dependence.
All the structures of the desired nitrones, 30-41, were
established by NMR (1H, 13C, HMQC, and HMBC experi-
ments), IR, and MS. The purity was established by TLC and
microanalysis. The stereochemistry around the olefinic carbon-
nitrogen bond of the nitrone function was established using
NOE-diff experiments, and they are Z-isomers in all the cases.
Biology. Neuroprotective Activity. The neuroprotective
activity of the new developed heteroarylnitrones, 30-41, was
studied using a human neuronal-like cellular system (SH-SY5Y
cells) exposed to H2O2 as oxidative damaging agent.25 First,
SH-SY5Y cytotoxicity of the developed heteroarylnitrones and
PBN was evaluated in vitro in the range 0.05-10 µM for 24 h.
The cell viability was determined by measuring the activity of
lactate dehydrogenase (LDH) released into the culture medium
and expressed as the difference between nitrones-treated and
untreated cells. All the nitrones displayed no cytotoxicity for
neuronal cells, 100% of survival, at assayed neuroprotective
doses (10 µM). Second, each one of the heteroarylnitrones, at
10 µM doses, was administered to the cells 1 h before the
incubation with H2O2. The cell viability was determined by

6154 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Porcal et al.
Figure 4. Prediction of the brain penetration using PAMPA-BBB assay
by 1,2,4-thiadiazolylnitrones 35 and 36 (CNS +: compounds able to
cross the BBB).
Table 2. Free-Radical Scavenger/Antioxidant Activity of
Heteroarylnitrone Derivatives Using ORACFL Method and DPPH
Captured
ref
ORAC (PBN equiv)a
RP of DPPH (%)b
PBN
30
31
32
33
34
35
36
37
38
39
40
41
1.0
0 (20c)
41 ( 3
0
11 ( 3
0
0
10 ( 4
40 ( 3
0
0
0
8 ( 1
17 ( 5
2.0 ( 0.1
5.5 ( 0.1
4.6 ( 0.2
3.5 ( 0.3
1.5 ( 0.2
7.0 ( 0.4
6.0 ( 0.2
2.5 ( 0.1
3.0 ( 0.1
6.0 ( 0.2
2.5 ( 0.1
16.5 ( 0.4
Figure 3. Cellular viability in different experimental conditions: H2O2
(100 µM), nitrone 36 (at 10 and 1 µM), and NAC (5 mM) used as
positive test. Viability was determined through LDH activity after 24 h
of incubation (mean ( SD values from three independent experiments).
Table 1. Effect of Heteroarylnitrones on Cell viability in SH-SY5Y
Cells Preincubated with H2O2 (100 µM) and Heteroarylnitrones
Cytotoxicities (J774 Mouse Macrophages)
difference in cell
c,d
ref
viabilitya-c
% neuroprotection
J774 IC50 (µM)e
a Data are expressed as µmol of PBN equiv/µmol of tested heteroarylni-
trones and are the mean (n ) 3) ( SD. b All heteroarylnitrones were assayed
at 0.1 mM. c Evaluated at 10 mM.
PBN
30
31
32
33
34
35
36
37
38
39
40
41
20.6 ( 0.2
20.0 ( 1.9
20.7 ( 9.4
23.6 ( 10.7
18.1 ( 3.0
19.1 ( 2.1
25.0 ( 5.2
25.2 ( 5.6
0
37
36
38
43
33
34
46
46
>400
>400
>400
100
240
200
>400
>400
>400
>400
100
profile and the lowest mammalian cytotoxicity (Table 1), the
capacity to cross the blood-brain barrier (BBB) by means of a
parallel artificial membrane permeation assay for BBB (PAMPA-
BBB) using a lipid extract of porcine brain was determined.
The assay validation was made by comparing experimental
permeabilities of 20 commercial drugs to reported values.27
From this experiment and taking into account the limit
established by Di et al. for BBB permeation, compounds with
permeability (Pe) above 4.4 × 10-6 cm s-1 could cross the BBB
(Figure 4).28
Free-Radical Scavenger Properties Antioxidant Activity. The
antioxidant activity of the newly developed heteroarylnitrones
was determined using two in vitro assays, the oxygen radical
absorbance capacity (ORAC) and the measurement of reduction
percentage (RP) of the stable diphenylpicrylhydrazyl free radical
(DPPH). ORAC assay29 measures peroxyl free radical scavenger
compounds capability and using fluorescein (FL) as the fluo-
rescent probe. The peroxidation of FL yields nonfluorescent
products at 520 nm, so the remaining fluorescence of FL in the
presence of the free radical scavenger compound is a measure
of the antioxidant activity of the test compound. In this assay
the results were expressed in reference to PBN, as PBN equiv
(µmoles of PBN equivalents per µmoles of tested compound)
(Table 2). On the other hand, the RP of DPPH is based on the
reaction between the deep-violet stable free radical (character-
ized by an absorption band in ethanol solution centered at 520
nm) and the studied free radical scavenger measuring the
absorbance of nitrone-treated and untreated DPPH solutions
(Table 2).30 The peroxyl free radical scavenger property of our
nitrones, according to ORAC assay, was better than that of PBN,
being 1.5-16.5 higher than PBN value. The benzofuroxanylni-
trone 41 showed the highest ORACFL value (16.5 PBN equiv),
whereas the 1,2,4-thiadiazolylnitrone 34 showed the lowest
ORACFL value (1.5 PBN equiv). Similar results of antioxidant
0
0
23.0 ( 2.4
13.4 ( 6.0
42
24
>400
150
a The cell viability was measured with LDH activity being 100% the
basal value. The basal value was obtained from the cells that did not receive
any treatment. Difference of cell viability was determined by the difference
between LDH activity in nitrones-H2O2-treated cells and LDH activity in
H2O2-treated cells and normalized to 100%. b Data are expressed as the
mean ( SD (p < 0.001). Experiments were done in triplicate. c Doses of
heteroarylnitrones are 10 µM. d Basal percentage of viable neurons after
treatment with 100 µM H2O2 is 45%. The percentages of neuroprotection
are determined from the basal values. e IC50: concentration that produces
50% inhibitory effect. The results are the mean values of two different
experiments with SD values less than 10% in all cases.
Mammal Cytotoxicity. Unspecific mammalian cytotoxicity
of the developed heteroarylnitrones was evaluated in vitro in
the range 50-400 µM, using J774 mouse macrophages as the
cellular model (Table 1).26 In the study PBN was included as a
neuroprotective reference compound. The developed heteroarylni-
trones displayed no cytotoxicity for mammalian cells at its
neuroprotective doses (10 µM). Furthermore, the most active
heteroarylnitrones, 35, 36 and 40, show low mammalian
cytotoxicity in the assayed in vitro model, these compounds
being as cytotoxic as the parent compound PBN. Moreover,
the IC50 values summarized in Table 1 show that the 1,2,4-
thiadiazolyl, 32-34, furoxanyl, 39, and benzofuroxanylnitrones,
41, are a little more toxic against this cellular system than the
parent compound PBN.
Blood-Brain Barrier Permeation Studies. For the 1,2,4-
thiadiazolylnitrones 35 and 36, with the best neuroprotective

Heteroarylnitrones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6155
•
•
•-
Figure 5. ESR spectra obtained in the trapping of OH, CH3 CH2OH, and SO3 free radicals with nitrones 31, 38, and 40. Superscript “a” in the
•
•
•-
heading entries indicates that OH, CH3 CHOH, and SO3 free radicals are generated by Fenton’s reaction.
Table 3. HFS Constants for the Heteroarylnitrone-•OH Spin Adduct
activity were obtained through DPPH assay. The heteroarylni-
trones were tested at 0.1 mM, being more potent antioxidants
than PBN, which at 10 mM possess 20% of DPPH reduction.
In this sense, the 1,2,4-thiadiazolylnitrones 30 and 36 showed
the highest antioxidant values, 41 and 40 DPPH RP, respectively.
Electron Spin Resonance Measurements. Initially the •OH-
spin trapping ability of the developed heteroarylnitrones was
investigated using ESR spectroscopy. The •OH free radical was
generated in situ from a Fenton reaction system. All the assayed
heteroarylnitrones, except the thiadiazolylnitrones 32 and 33 and
furoxanylnitrone 39, gave strong ESR signals (Figure 5), and
the hyperfine splitting (HFS) constants for the different spin
adducts are reported in Table 3. These data indicate that in
general our heteroarylnitrones generate stable spin adducts with
•OH, like previously reported imidazolylnitrones.31 The absence
of signal in the cases of nitrones 32 and 33 indicated that the
cyclohexyl and benzyl groups affect the stability of the spin
adduct generated under the studied conditions.
nitrone-•OHa,b
spin adduct, ref
aN (G)c
aHꢀ (G)c
30
31
32
33
34
35
36
37
38
39
40
41
14.8
15.7
nsd
2.4
0.9
nsd
nsd
0.6
0.7
2.6
2.6
4.0
nsd
2.1
2.2
nsd
15.3
15.7
14.0
14.5
15.5
nsd
15.8
14.1
a •OH was generated from a standard Fenton system: Fe(NH4)2
(SO4)2 ·6H2O (0.5 mM) was added to a solution of nitrone (50 mM) and
H2O2 (0.5 mM) in phosphate buffer (0.1 M). b The ESR spectrum of the
spin adduct was recorded 30 s after addition of FeSO4. c G: gauss. d ns: no
signals in the assayed conditions.
In order to study the capability of the developed nitrones as
spin trapping against other free radical species, we selected one
nitrone from each proposed family. Consequently, the 1,2,4-
thiadiazolylnitrone 31, the 1,2,3-thiadiazolylnitrone 38, and the
furoxanylnitrone 40 were studied as both ethanol and sulfite
free radical spin trappings. The ESR signals obtained for these
heteroarylnitrones are given in Figure 5. The ESR signals for
the free radical adducts with nitrones 31, 38, and 40 indicated
that these nitrones are also very efficient to trap and stabilize
•
both carbon- and sulfur-centered free radicals, CH3 CHOH and
SO3•-, respectively. These results suggest that our nitrones have
great capacity to scavenge free radicals and potential use to
prevent or reduce the damage caused by radicals in several
degenerative disorders. However, because nitrone 38 is very
•
•
efficient in spin trapping (i.e., OH, CH3 CHOH, SO3•-, Figure
5), it does not have neuroprotective effects in the neuroblastoma

6156 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Porcal et al.
the 3-aryl-5-hydroxymethyl-1,2,4-thiadiazole (pure by TLC), was
used in the next reaction without further purification. Subsequently,
a mixture of the corresponding alcohol (1 equiv) and MnO2 (10
equiv) in CHCl3 (12 mL/mmol) as solvent was heated at reflux
until absence of the alcohol (checked by TLC). The resulting
dispersion was then filtered through a short pad of Celite, the
organic phase concentrated in vacuo and the residue purified by
column chromatography (SiO2, petroleum ether/EtOAc (8:2)) to
afford spectroscopically pure compounds.
human SH-SY5Y system (Table 1), showing that the neuro-
protective activity might not exclusively reside in its capacity
to scavenge free radicals. These results are similar to those
recently reported for other heteroarylnitrones.32
Conclusion
New series of thiadiazolyl and furoxanylnitrones have been
synthesized and biologically evaluated as potential neuropro-
tective agents for the treatment of neurodegenerative disorders.
The designed heteroarylnitrones were efficiently obtained
through the condensation between heteroaromatic aldehydes and
N-monosubstituted hydroxylamines with good to excellent yields
and chromatographically isolated as pure Z-isomers. They show
excellent free radical scavenger capacity and good neuropro-
tective effects without cellular toxicity. The ESR spectroscopy
demonstrated the ability of these heteroarylnitrones to directly
5-Hydroxymethyl-3-phenyl-1,2,4-thiadiazole. 1H NMR (CDCl3)
δ: 2.83 (t, 1H), 5.17 (d, 2H, J ) 5.5 Hz), 7.45-7.51 (m, 3H),
8.24-8.30 (m, 2H). EI-MS, m/z (abundance, %): 192 (M+•, 38),
135 (100), 103 (24), 77 (20).
5-Formyl-3-phenyl-1,2,4-thiadiazole (9). Yellow solid, 0.13 g
(25%, two steps). 1H NMR (CDCl3) δ: 7.52 (m, 3H), 8.33 (m, 2H),
10.19 (s, 1H). 13C NMR (CDCl3) δ: 184.3 (C5), 182.5 (CHO),
175.1 (C3), 131.9 (Cq), 131.0 (Co), 128.9 (Cm), 128.3 (Cp). EI-
MS, m/z (abundance, %): 190 (M+•, 84), 135 (100), 103 (30), 77
(24).
•
scavenge different free radicals (i.e., •OH, CH3 CHOH, SO3•-).
3-(4-Chlorophenyl)-5-hydroxymethyl-1,2,4-thiadiazole. 1H NMR
(CDCl3) δ: 2.70 (s, 1H), 5.05 (s, 2H), 7.38 (d, 2H, J ) 8.5 Hz),
Taking into account all the results, the designed heteroarylni-
trones have demonstrated a therapeutic potential as neuropro-
tective agents in preventing the death of cells exposed to
enhanced oxidative stress and damage. Further structural
optimization, QSAR, and in vivo neuroprotective property
studies are currently underway.
8.15 (d, 2H, J ) 8.5 Hz). EI-MS, m/z (abundance, %): 226 (M+•
51), 169 (100), 137 (35), 102 (15).
,
3-(4-Chlorophenyl)-5-formyl-1,2,4-thiadiazole (18). Yellow solid,
0.28 g (44%, two steps). 1H NMR (DMSO-d6) δ: 7.67 (d, 2H, J )
8.5 Hz), 8.28 (d, 2H, J ) 8.7 Hz), 10.16 (s, 1H). 13C NMR (DMSO-
d6) δ: 180.2 (CHO), 179.1 (C5), 173.52 (C3), 137.6 (Cq), 132.7
(Cq), 130.6 (Co), 130.0 (Cm). EI-MS, m/z (abundance, %): 224
(M+•, 75), 169 (100), 137 (39), 102 (18).
Experimental Section
Compounds 3, 4,20 8,21 14-17,33-36 and 26-2923 were prepared
according to literature procedures. Melting points were determined
with an electrothermal melting point apparatus (Electrothermal
9100) and are uncorrected. Proton and carbon NMR spectra were
recorded on a Bruker DPX-400 spectrometer. The chemical shifts
values are expressed in ppm relative to tetramethylsilane as internal
standard. Mass spectra were determined in a MSD 5973 Hewlett-
Packard or LC/MSD series 100 Hewlett-Packard spectrometer using
electronic impact (EI) or electrospray ionization (ESI), respectively.
Microanalyses were performed in a Fisons EA 1108 CHNS-O
equipment and were within (0.4% of the calculated compositions.
Column chromatography was carried out using Merck silica gel
(60-230 mesh). Most chemicals and solvents were analytical grade
and used without further purification. 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 before
concentration.
5-Hydroxymethyl-3-(4-methoxyphenyl)-1,2,4-thiadiazole. 1H NMR
(CDCl3) δ: 2.75 (s, 1H), 3.87 (s, 3H), 5.15 (s, 2H), 7.01 (d, 2H, J
) 8.6 Hz), 8.42 (d, 2H, J ) 8.6 Hz). EI-MS, m/z (abundance, %):
222 (M+•, 49), 167 (100), 135 (57).
5-Formyl-3-(4-methoxyphenyl)-1,2,4-thiadiazole (20). Yellow
1
solid, 0.13 g (31%, two steps). H NMR (CDCl3) δ: 3.85 (s, 3H),
7.04 (d, 2H, J ) 8.6 Hz), 8.31 (d, 2H, J ) 8.7 Hz), 10.19 (s, 1H).
13C NMR (CDCl3) δ: 181.2 (CHO), 178.3 (C5), 172.3 (C3), 160.9
(Cp), 130.1 (Co), 114.5 (Cm), 54.9 (OCH3). EI-MS, m/z (abun-
dance, %): 220 (M+•, 99), 165 (100), 150 (45), 133 (75).
3-(3,5-Di-tert-butyl-4-hydroxyphenyl)-5-hydroxymethyl-1,2,4-
thiadiazole. 1H NMR (CDCl3) δ: 1.52 (s, 18H), 2.93 (s, 1H), 5.16
(s, 2H), 5.53 (s, 1H), 8.14 (s, 2H). ESI-MS, m/z: 321 (M+• + H).
3-(3,5-Di-tert-butyl-4-hydroxyphenyl)-5-formyl-1,2,4-thiadiaz-
1
ole (21). Brown solid, 0.14 g (27%, two steps). H NMR (CDCl3)
δ: 1.54 (s, 18H), 5.61 (s, 1H), 8.21 (s, 2H), 10.23 (s, 1H). 13C
NMR (CDCl3) δ: 183.9 (C5), 182.8 (CHO), 175.9 (C3), 156.6 (Cp),
136.4 (Cm), 125.7 (Co), 125.3 (Cq), 34.4 (C(CH3)3), 30.2
(C(CH3)3). ESI-MS, m/z: 319 (M+• + H).
3-Formyl-5-phenyl-1,2,4-thiadiazole (6). A mixture of 420 (0.4
g, 2.3 mmol), N-bromosuccinimide (0.45 g, 2.5 mmol), and
dibenzoyl peroxide (0.08 g, 0.34 mmol) in dry CCl4 (8.0 mL) was
heated at reflux for 16 h. Then the mixture was allowed to cool to
room temperature, the succinimide was collected and washed with
CCl4 (10.0 mL), and the organic layer was evaporated in vacuo.
3-Bromomethyl-5-phenyl-1,2,4-thiadiazole was obtained as an oil
(∼40% by 1H NMR signals) and was used in the next reaction
5-Ethoxycarbonyl-4-methyl-1,2,3-thiadiazole (22). A mixture of
ethyl acetoacetate (4.9 mL, 38.5 mmol), methyl carbazate (3.8 g,
42.4 mmol), and p-TsOH (catalytic amounts) in toluene (120.0 mL)
was heated at reflux for 2 h. Then the solvent was concentrated in
vacuo and the crude of the reaction was used in the next reaction
without further purification. The acylhydrazone obtained was cooled
at 0 °C, and SOCl2 (20.0 mL, 270 mmol) was added dropwise with
stirred during 10 min. The final mixture was stirred during 2 h at
60 °C, the excess of SOCl2 was evaporated in vacuo, and the residue
was neutralized with aqueous saturated NaHCO3 and extracted with
EtOAc (3 × 10 mL). After the workup the organic solvent was
evaporated in vacuo and the crude purified by column chromatog-
raphy (SiO2, petroleum ether/EtOAc (8:2)), yellow oil, 1.63 g
1
without further purification. H NMR (CDCl3, 400 MHz) δ: 4.69
(s, 2H), 7.45-7.55 (m, 3H), 7.90-8.00 (m, 2H). The crude of the
bromination process in DMSO (6.0 mL) was stirred and warmed
at 105 °C during 1.5 h. Subsequently, the mixture was poured into
saturated sodium chloride solution (30.0 mL) and extracted with
EtOAc (3 × 20.0 mL). After the workup of the combined organic
layers, the residue was purified by column chromatography (SiO2,
petroleum ether/EtOAc (9:1)), yielding derivative 6 as a yellow
oil (130 mg, 30% two steps). 1H NMR (CDCl3) δ: 7.45-7.65 (m,
3H), 7.90-8.10 (m, 2H), 10.30 (s, 1H). EI-MS, m/z (abundance,
%): 190 (M+•, 27), 135 (100), 103 (15).
1
(25%). H NMR (CDCl3) δ: 1.55 (t, 3H), 2.95 (s, 3H), 4.42 (q,
2H). 13C NMR (CDCl3) δ: 162.5 (C4), 160.1 (CdO), 139.8 (C5),
62.8 (CH2CH3), 14.5 (CH2CH3 + CH3). ESI-MS, m/z: 173.0 (M+•
+ H).
General Procedure for the Synthesis of 3-Aryl-5-formyl-1,2,4-
thiadiazole Derivatives (9 and 18-21). A solution of the corre-
sponding ester20,33-36 (1 equiv) in THF (4 mL/mmol) as solvent
was cooled at 0 °C. Then a solution of DIBAL-H (1 M in hexane,
2-3 equiv) was added slowly and the final mixture was stirred
during 3 h. The reaction mixture is diluted with EtOAc and washed
with HCl (1 M). After the workup, the residue, corresponding to
5-Hydroxymethyl-4-methyl-1,2,3-thiadiazole (23). A solution of
22 (0.8 g, 4.7 mmol) in EtOH (8.0 mL) was cooled at 0 °C. Then
NaBH4 (0.3 g, 7.9 mmol) was added slowly and the final mixture
was stirred during 2 h at room temperature. The organic solvent
was evaporated in vacuo, and the residue was treated with H2O
(50.0 mL) and extracted with EtOAc (3 × 10.0 mL). After the

Heteroarylnitrones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6157
r(Z)-(3-Phenyl-1,2,4-thiadiazol-5-yl)-N-benzylnitrone (33). White
workup the organic solvent was evaporated in vacuo. The residue
was purified by column chromatography (SiO2, petroleum ether/
EtOAc (7:3)), yielding derivative 23 as a yellow oil (0.42 g, 70%).
1
solid (65%); mp 156.0-158.0 °C. H NMR (CDCl3) δ: 5.21 (s,
2H), 7.46-7.45 (m, 3H), 7.48 (s, 5H), 8.30-8.27 (m, 2H), 8.35
(s, 1H). 13C NMR (CDCl3) δ: 174.9 (C5), 171.9 (C3), 132.8 (Cq),
130.9 (Cq), 130.3 (Co), 129.9 (Cm), 128.8 (Co), 129.5 (Cm), 129.4
(Cp), 128.7 (Cp), 128.0 (CHdN), 69.5 (CH2). EI-MS, m/z
(abundance, %): 295 (M+•, 32), 278 (4), 135 (5), 103 (7), 91 (100).
IR (KBr) ν: 1534, 1465, 1423, 1323, 1234, 1130, 919, 726. Anal.
Calcd for (C16H13N3OS): C, 65.06; H, 4.44; N, 14.23; S, 10.86.
Found: C, 64.88; H, 4.30; N, 14.12; S, 10.61.
r(Z)-[3-(4-Chlorophenyl)-1,2,4-thiadiazol-5-yl]-N-tert-butylni-
trone (34). Yellow solid (68%); mp 129.0-131.0 °C. 1H NMR
(CDCl3) δ: 1.68 (s, 9H), 7.46 (d, 2H, J ) 8.5 Hz), 8.27 (d, 2H, J
) 8.5 Hz), 8.63 (s, 1H). 13C NMR (CDCl3) δ: 175.7 (C5), 170.7
(C3), 136.2 (Cp), 131.5 (Cq), 129.3 (Co), 128.9 (Cm), 125.8
(CHdN), 71.7 (C(CH3)3), 28.1 (C(CH3)3). EI-MS, m/z (abundance,
%): 295 (M+•, 26), 239 (35), 169 (8), 137 (14), 57 (100). IR (KBr)
ν: 1548, 1471, 1420, 1404, 1346, 1236, 1118, 913, 838, 741, 558.
Anal. Calcd for (C13H14ClN3OS): C, 52.79; H, 4.77; N, 14.21; S,
10.84. Found: C, 52.78; H, 5.07; N, 14.02; S, 11.01.
r(Z)-[3-(4-Methoxyphenyl)-1,2,4-thiadiazol-5-yl]-N-tert-butylni-
trone (35). Orange solid (88%); mp 130.0-132.0 °C. 1H NMR
(CDCl3) δ: 1.67 (s, 9H), 3.87 (s, 3H), 7.01 (d, 2H, J ) 8.7 Hz),
8.28 (m, 2H, J ) 8.8 Hz), 8.63 (s, 1H). 13C NMR (CDCl3) δ: 175.4
(C5), 171.6 (C3), 161.2 (Cp), 129.5 (Co), 125.9 (CHdN), 114.0
(Cm), 71.6 (C(CH3)3), 55.3 (OCH3), 28.0 (C(CH3)3). EI-MS, m/z
(abundance, %): 291 (M+•, 91), 235 (100), 165 (26), 133 (38), 57
(89). IR (KBr) ν: 1609, 1547, 1408,1314, 1246, 1180, 1026, 838,
750, 659, 556. Anal. Calcd for (C14H17N3O2S): C, 57.71; H, 5.88;
N, 14.42; S, 11.00. Found: C, 56.56; H, 5.68; N, 14.23; S, 10.80.
r(Z)-[3-(3,5-Di-tert-butyl-4-hydroxyphenyl)-1,2,4-thiadiazol-5-
yl]-N-tert-butylnitrone (36). Yellow solid (92%); mp 292.0-294.0
°C. 1H NMR (CDCl3) δ: 1.51 (s, 18H), 1.68 (s, 9H), 5.48 (s, 1H),
8.19 (s, 2H), 8.64 (s, 1H). 13C NMR (CDCl3) δ: 175.2 (C5), 172.7
(C3), 155.9 (Cp), 136.1 (Cq), 126.0 (Co), 125.2 (CHdN), 124.6
(Cm), 71.5 (C(CH3)3), 34.4 (C(CH3)3), 30.3 (C(CH3)3), 28.4
(C(CH3)3). EI-MS, m/z (abundance, %): 389 (M+•, 87), 374 (46),
333 (36), 318 (100), 57 (40). IR (KBr) ν: 3593, 2961, 1544, 1398,
1254, 1236, 1121, 913, 747, 696, 555. Anal. Calcd for
(C21H31N3O2S): C, 64.75; H, 8.02; N, 10.79; S, 8.23. Found: C,
64.50; H, 8.30; N, 10.63; S, 8.18.
r(Z)-(4-Methyl-1,2,3-thiadiazol-5-yl)-N-tert-butylnitrone (37).
Beige solid (77%); mp 154.0-156.0 °C. 1H NMR (CDCl3) δ: 1.67
(s, 9H), 2.85 (s, 3H), 8.20 (s, 1H). 13C NMR (CDCl3) δ: 157.7
(C5), 138.4 (C4), 122.7 (CHdN), 71.2 (C(CH3)3), 28.5 (C(CH3)3),
13.9 (CH3). EI-MS, m/z (abundance, %): 199 (M+•, 2), 171 (2),
123 (30), 108 (42), 57 (100). IR (KBr) ν: 3136, 2997, 2972, 1547,
1493, 1439, 1361, 1323, 1201,1140, 1035, 887, 827, 723, 572. Anal.
Calcd for (C8H13N3O2S): C, 48.22; H, 6.58; N, 21.09; S, 16.09.
Found: C, 48.09; H, 6.30; N, 20.82; S, 15.88.
1H NMR (CDCl3) δ: 2.55 (s, 3H), 4.55 (bs, 1H), 4.94 (s, 2H). 13
C
NMR (CDCl3) δ: 154.4 (C5), 152.9 (C4), 56.3 (CH2OH), 12.1
(CH3). ESI-MS, m/z: 131 (M+• + H).
5-Formyl-4-methyl-1,2,3-thiadiazole (24). A mixture of 23 (0.49
g, 3.77 mmol) and MnO2 (3.28 g, 37.7 mmol) in CHCl3 (45 mL)
as solvent is heated at reflux until absence of the alcohol 23
(checked by TLC). The resulting dispersion was then filtered
through a short pad of Celite, the organic phase concentrated in
vacuo, and the residue purified by column chromatography (SiO2,
petroleum ether/EtOAc (8:2)), yielding derivative 24 as a yellow
1
oil (105 mg, 26%). H NMR (CDCl3) δ: 3.05 (s, 3H), 10.26 (s,
1H). 13C NMR (CDCl3) δ: 184.3 (CHO), 156.2 (C5), 154.2 (C4),
14.4 (CH3). ESI-MS, m/z: 129 (M+• + H).
5-Ethoxycarbonyl-4-formyl-1,2,3-thiadiazole (25). A mixture of
22 (1.0 g, 5.8 mmol), N-bromosuccinimide (1.14 g, 6.4 mmol),
and dibenzoyl peroxide (0.06 g, 0.23 mmol) in dry CCl4 (40.0 mL)
was heated at reflux for 16 h. Then the mixture was allowed to
cool to room temperature, the succinimide was collected and washed
with CCl4, and the organic layer was evaporated in vacuo. DMSO
(10.0 mL) was added, and the mixture was warmed at 120 °C during
3 h. Then EtOAc (50 mL) was added and the organic layer was
washed with saturated sodium chloride solution (40.0 mL). After
the workup the organic solvent was evaporated in vacuo and the
residue purified by column chromatography (SiO2, petroleum ether/
EtOAc (8:2)), yielding a yellow oil that crystallized at 4 °C (0.4 g,
1
40%). H NMR (CDCl3) δ: 1.48 (t, 3H), 4.56 (q, 2H), 10.67 (s,
1H). 13C NMR (CDCl3) δ: 186.1 (CHO), 157.2 (C4), 161.8 (CdO),
141.5 (C5), 64.2 (CH2CH3), 14.1 (CH2CH3). ESI-MS, m/z: 187
(M+• + H).
General Procedure for the Synthesis of r-Heteroaryl-N-alkyl-
nitrone Derivatives (30-41). A mixture of the corresponding
aldehyde (1 equiv), N-alkylhydroxylamine hydrochloride (1.5
equiv), and sodium bicarbonate (1.5 equiv) in absolute ethanol (6
mL/mmol) as solvent was heated at 60 °C until the carbonyl
compound was not present (checked by TLC). The solvent was
removed in vacuo and the reaction mixture diluted with H2O and
extracted with EtOAc. After the workup of the combined organic
layers, the residue was purified by column chromatography (SiO2,
mixtures of petroleum ether/EtOAc).
r(Z)-(5-Phenyl-1,2,4-thiadiazol-3-yl)-N-tert-butylnitrone (30). Yel-
1
low solid (87%); mp 111.0-113.0 °C; H NMR (CDCl3) δ: 1.63
(s, 9H), 7.48-7.56 (m, 3H), 7.97 (d, 2H), 8.18 (s, 1H). 13C NMR
(CDCl3) δ: 187.4 (C5), 166.5 (C3), 132.1 (Cp), 129.3 (Cm), 127.5
(Co), 123.2 (CHdN), 73.0 (C(CH3)3), 28.3 (C(CH3)3). ESI-MS,
m/z: 262 (M+• + H). IR (KBr) ν: 1707, 1561, 1469, 1435, 1248,
1152, 773, 695. Anal. Calcd for (C13H15N3OS): C, 59.74; H, 5.79;
N, 16.08; S, 12.27. Found: C, 59.46; H, 5.86; N, 15.97; S, 12.27.
r(Z)-(3-Phenyl-1,2,4-thiadiazol-5-yl)-N-tert-butylnitrone (31). Yel-
r(Z)-(5-Ethoxycarbonyl-1,2,3-thiadiazol-4-yl)-N-tert-butylnitro-
ne (38). Yellow solid (48%); mp 93.0-95.0 °C. 1H NMR (CDCl3)
δ: 1.44 (t, 3H), 1.65 (s, 9H), 4.48 (q, 2H), 8.52 (s, 1H). 13C NMR
(CDCl3) δ: 159.9 (CdO), 153.9 (C4), 143.2 (C5), 121.2 (CHdN),
72.8 (C(CH3)3), 63.3 (CH2CH3), 28.5 (C(CH3)3), 14.5 (CH2CH3).
EI-MS, m/z (abundance, %): 257 (M+•, 1), 201 (9), 173 (9), 127
(6), 57 (100). IR (KBr) ν: 3147, 299, 1732, 1562, 1535, 1394, 1313,
1209, 1153, 1087, 910, 860, 767, 559. Anal. Calcd for
(C10H15N3O3S): C, 46.68; H, 5.88; N, 16.33; S, 12.46. Found: C,
46.33; H, 5.50; N, 16.20; S, 12.19.
1
low solid (82%); mp 107.0-108.0 °C. H NMR (CDCl3) δ: 1.66
(s, 9H), 7.44-7.50 (m, 3H), 8.29-8.32 (m, 2H), 8.63 (s, 1H). 13
C
NMR (CDCl3) δ: 175.8 (C5), 172.0 (C3), 133.2 (Cq), 130 (Co),
128.9 (Cm), 128.2 (Cp), 126.1 (CHdN), 71.9 (C(CH3)3), 28.3
(C(CH3)3). EI-MS, m/z (abundance, %): 261 (M+•, 16), 205 (42),
135 (19), 103 (19), 57 (100). IR (KBr) ν: 1544, 1470, 1414, 1344,
1234, 1112, 913, 716, 696, 558. Anal. Calcd for (C13H15N3OS): C,
59.74; H, 5.79; N, 16.08; S, 12.27. Found: C, 59.35; H, 5.74; N,
15.89; S, 12.08.
r(Z)-(3-Phenyl-1,2,4-thiadiazol-5-yl)-N-cyclohexylnitrone (32).
r(Z)-(4-Phenylfuroxan-3-yl)-N-tert-butylnitrone (39). Yellow
solid (57%); mp 78.0-80.0 °C. 1H NMR (CDCl3) δ: 1.61 (s, 9H),
7.42-7.56 (m, 5H), 7.58 (s, 1H). 13C NMR (CDCl3) δ: 155.8 (C4),
133.9 (Cm), 130.5 (Co), 126.7 (Cp), 114.9 (CHdN), 110.3 (C3),
73.2 (C(CH3)3), 28.3 (C(CH3)3). EI-MS, m/z (abundance, %): 261
(M+•, 2), 205 (18), 145 (10), 105 (18), 57 (100). IR (KBr) ν: 3138,
2979, 2935, 1600, 1560, 1456, 1382, 1247, 1199, 1139, 1028, 983,
858, 812, 709, 556. Anal. Calcd for (C13H15N3O3): C, 59.76; H,
5.79; N, 16.08. Found: C, 59.45; H, 5.61; N, 15.87.
1
Yellow solid (86%); mp 194.0-196.0 °C. H NMR (CDCl3) δ:
1.21-1.50 (m, 5H), 1.99-2.20 (m, 5H), 4.16 (m, 1H), 7.52-7.47
(m, 3H), 8.34-8.31 (m, 2H), 8.51 (s, 1H). 13C NMR (CDCl3) δ:
175.4 (C5), 172.2 (C3), 133.4 (Cq), 130.6 (Co), 129.1 (Cm), 128.5
(Cp), 128.0 (CHdN), 74.9 (CCH2), 31.7 (CCH2), 25.3 (CH2CH2).
EI-MS, m/z (abundance, %): 287 (M+•, 79), 269 (32), 243 (25),
205 (86), 189 (31), 135 (60), 103 (42), 55 (100). IR (KBr) ν: 1551,
1465, 1432, 1352, 926. Anal. Calcd for (C15H17N3OS): C, 62.69;
H, 5.96; N, 14.62; S, 11.16. Found: C, 62.39; H, 5.78; N, 14.55; S,
10.98.
r(Z)-(3-Methylfuroxan-4-yl)-N-tert-butylnitrone (40). Yellow
1
solid (81%); mp 119.0-121.0 °C. H NMR (CDCl3) δ: 1.61 (s,

6158 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Porcal et al.
9H), 2.33 (s, 3H), 7.96(s, 1H). 13C NMR (CDCl3) δ: 151.1 (C4),
117.9 (CHdN), 112.6 (C3), 72.9 (C(CH3)3), 28.6 (C(CH3)3), 10.4
(CH3). EI-MS, m/z (abundance, %): 199 (M+•, 3), 143 (9), 113
(6), 83 (4), 57 (100). IR (KBr) ν: 2984, 1616, 1566, 1457, 1364,
1251, 1178, 1140, 834, 763, 549. Anal. Calcd for (C8H13N3O3): C,
48.23; H, 6.58; N, 21.09. Found: C, 48.02; H, 6.30; N, 20.92.
r(Z)-(Benzofuroxan-5(6)-yl)-N-tert-butylnitrone (41). Yellow
was left undisturbed for 120 min at 25 °C. After incubation, the
donor plate was carefully removed and the concentration of
compounds in the acceptor wells was determined by UV spectros-
copy. Every sample was analyzed in four wells and at least in three
independent runs, and the results are given as the mean ( standard
deviation.
Determination of Antioxidant Activity. OFRACFL Assay. A
luminescence spectrometer LS 50B (PerkinElmer, Boston, MA), a
heating circulator bath DC1-B3 (Haake Haake Fisons, Karlsruhe,
Germany), and quartz cuvettes were used. For the ORACFL assay,
the 490-P excitation and 515-P emission filters were used, and the
fluorescence measurement was carried out at 60 °C. The method
of Ou et al.29 was modified as follows: the reaction was carried
out in 75 mM phosphate buffer (pH 7.4), and the final reaction
volume was 3000 µL. Studied compounds (10-1000 µM final
concentrations) and fluorescein (215 µL, 70 nM final concentration)
solutions were placed in the quartz cuvette. The mixture was
preincubated for 30 s at 60 °C. 2,2′-Azobis(2-amidinopropane)
dihydrochloride (AAPH) solution (240 µL; 12 mM, final concentra-
tion) was added rapidly using a single channel pipet. The quartz
cuvette was immediately placed in the luminescence spectrometer
and the fluorescence recorded every minute for 12 min. As a blank
FL + AAPH in phosphate buffer instead of the studied compounds
solutions were employed, and eight calibration solutions using
Trolox (1-8 µM, final concentration) as antioxidant positive control
were also carried out in each assay. All the reaction mixtures were
prepared in duplicate, and at least three independent assays were
performed for each sample. Raw data were exported to an OriginPro
(OriginLab Corporation, Northampton, MA) sheet for further
calculations. Blank and antioxidant curves (fluorescence versus
time) were first normalized by dividing original data by fluorescence
at t ) 0 s. From the normalized curves, the area under the
fluorescence decay curve (AUC) was calculated as
1
solid (61%); mp 112.0-114.0 °C. H NMR (CDCl3) δ: 1.60 (s,
9H), 7.32 (bs, 1H), 7.50 (bs, 1H), 7.60 (s, 1H), 9.33 (bs, 1H). 13
C
NMR (CDCl3) δ: 127.4 (CHdN), 72.5 (C(CH3)3), 28.3 (C(CH3)3).
EI-MS, m/z (abundance, %): 235 (M+•, 9), 179 (17), 163 (5), 152
(6), 57 (100). IR (KBr) ν: 2984, 1610, 1570, 1487, 1361, 1236,
1153, 1014, 868, 828, 585. Anal. Calcd for (C11H13N3O3): C, 56.16;
H, 5.57; N, 17.86. Found: C, 55.99; H, 5.34; N, 17.70.
Measurement of Cell Viability. Culture of SH-SY5Y Cells.
SH-SY5Y cells, at passages between 3 and 16 after defreezing,
were maintained in a minimum essential medium (MEM) containing
15 nonessential amino acids (NEAAs) and supplemented with 10%
fetal bovine serum, 1 mM glutamine, 50 units/mL penicillin, and
50 µg/mL streptomycin (reagents from GIBCO, Madrid, Spain).
Cultures were seeded into flasks containing supplemented medium
and maintained at 37 °C in 5% CO2/humidified air.
Cell Viability Assays. The effects of nitrones on SH-SY5Y cell
viability were determined by measuring the activity of LDH released
into the culture medium (Cytotox 96 LDH assay, Promega, U.K.).
The assay was performed in accordance with the manufacturer’s
protocol using SH-SY5Y cells seeded at 104 cells/well in 96-well
plates. Cells were exposed to 10, 1, and 0.05 µM of each one nitrone
for 24 h.
The effects of H2O2 on SH-SY5Y cell viability were determined
by measuring the activity of LDH released into the culture medium
(Cytotox 96 LDH assay, Promega, U.K.). The assay was performed
in accordance with the manufacturer’s protocol using SH-SY5Y
cells seeded at 104 cells/well in 96-well plates. Cells were exposed
to 10 µM of each one heteroarylnitrone for 1 h and subsequently
exposed to 100 µM H2O2 for 24 h. An amount of 5 mM NAC was
used as positive test.
i)12
fi
AUC ) 1 +
∑
f0
i)1
Unspecific Mammalian Cytotoxicity. J774 murine macrophage-
like cells (ATCC) were maintained by passage in Dulbecco’s
modified Eagle’s medium (DMEM) containing 4 mM L-glutamine
and supplemented with 10% heat-inactivated fetal calf serum. J774
cells were seeded (1 × 105 cells/well) in 96-well microplates with
100 µL of RPMI medium supplemented with fetal serum. Cells
were allowed to attach for 48 h in a humidified 5% CO2/95% air
atmosphere at 37 °C and then exposed to nitrones (50-500 µM)
for 48 h. Afterward, cell viability was assessed by measuring the
mitochondrial-dependent reduction of 3-[4,5-dimethylthiazol-2yl]-
2,5-diphenyltetrazolium bromide (MTT, Sigma) to formazan. For
that purpose, MTT was added to cells to a final concentration of
0.2 mg/mL and cells were incubated at 37 °C for 3 h. After removal
of the media, formazan crystals were dissolved in DMSO, and the
absorbance at 560 nm was read using a microplate spectrophotom-
eter. Results are expressed as IC50 (compound concentration that
reduce 50% control absorbance at 560nm). Every IC50 is the average
of two different experiments.
Blood-Brain Barrier Permeation Assay. Brain penetration of
new nitrones was evaluated using a parallel artificial membrane
permeation assay (PAMPA) in a similar manner as described
previously.27 Commercial drugs were purchased from Sigma,
Aldrich, Acros, and Fluka. Millex filter units (PVDF membrane,
diameter 25 mm, pore size 0.45 µm) were acquired from Millipore.
The porcine brain lipid (PBL) was obtained from Avanti Polar
Lipids. The donor microplate was a 96-well filter plate (PVDF
membrane, pore size 0.45 µm), and the acceptor microplate was
an indented 96-well plate, both from Millipore. The acceptor 96-
well microplate was filled with 170 µL of PBS/ethanol (9:1), and
the filter surface of the donor microplate was impregnated with 4
µL of PBL in dodecane (20 mg/mL). Compounds were dissolved
in PBS/ethanol (9:1) at 1 mg/mL, filtered through a Millex filter,
and then added to the donor wells (170 µL). The donor filter plate
was carefully put on the acceptor plate to form a sandwich, which
where f0 is the initial fluorescence reading at 0 min and fi is the
fluorescence reading at time i. The net AUC corresponding to each
sample was calculated by subtracting the AUC corresponding to
the blank. Regression equations between net AUC and antioxidant
concentration were calculated for all the samples. ORACFL values
were expressed as PBN equivalents by using the standard curve
calculated for each assay. Final results were expressed in µmole
of PBN equivalent per µmole of samples.
DPPH Assay. The reaction was carried out in 96-well micro-
plates, and the final reaction mixture was 100 µL per well.
Antioxidant (0.1 mM, final concentration) and DPPH (0.1 mM, final
concentration) were dissolved in EtOH and preincubated for 5 min
at 37 °C, and the absorbance at 520 nm was read using a microplate
spectrophotometer. The antioxidant activity was determined as the
RP of DPPH, calculated as follows: RP ) 100 [(A0 - Ac)/A0], where
A0 is the untreated DPPH absorbance and Ac is the value for added
sample concentration c.
ESR Spectroscopy. The spin trapping ability of the developed
heteroarylnitrones was studied by ESR spectroscopy. ESR spectra
were recorded in the X band (9.85 GHz) using a Bruker ECS 106
spectrometer with a rectangular cavity and 50 kHz field modulation.
All the spectra were registered in the same scale after 15 scans.
The hyperfine splitting constants were estimated to be accurate
within 0.05 G.
Generation of Free Radicals. Hydroxyl radical (•OH) was
generated from a standard Fenton system: Fe(NH4)2(SO4)2 ·6H2O
(50 µL, 0.5 mM) was added to a solution of nitrone (50 mM, final
concentration) and H2O2 (50 µL, 0.5 mM) in phosphate buffer (final
reaction mixture was 250 µL). Cysteinyl radical (SO3•-) was
generated from a standard Fenton system: Fe(NH4)2(SO4)2 ·6H2O
(50 µL, 0.5 mM) was added to a solution of nitrone (50 mM, final
concentration), Na2SO3 (50 µL, 250 mM), and H2O2 (50 µL, 0.5
mM) in phosphate buffer (final reaction mixture was 250 µL).

Heteroarylnitrones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6159
•
(18) Porcal, W.; Cerecetto, H. Pharmacological Properties of Furoxans and
Benzofuroxans: Recent Developments. Mini-ReV. Med. Chem. 2005,
5, 57–71.
(19) Patai, A.; Rappoport, Z. Nitrones, Nitronates and Nitroxides; John
Wiley & Sons Ltd.: New York, 1989.
(20) Lin, Y.-i; Lang, S. A.; Petty, S. R. New Synthesis of 1,2,4-Thiadiazole.
J. Org. Chem. 1980, 45, 3750–3753.
Ethanol radical (CH3 CHOH) was generated from a standard Fenton
system: Fe(NH4)2(SO4)2 ·6H2O (50 µL, 0.5 mM) was added to a
solution of nitrone (50 mM, final concentration), EtOH (50 µL),
and H2O2 (50 µL, 0.5 mM) in phosphate buffer (final reaction
mixture was 250 µL).
Acknowledgment. The authors thank Collaborative Project
UdelaR (Uruguay), CSIC (Spain) (Grant 2006UY0009), and
Neuropharma S.A (Madrid, Spain). W.P. thanks Collaborative
Project (Grant 2006UY0009) and CSIC (UdelaR) for fellow-
ships. W.P. and P.H. are PEDECIBA students.
(21) Howe, R. K.; Franz, J. E. Nitrile Sulphides. Synthesis of 1,2,4-
Thiadiazole. J. Org. Chem. 1974, 39, 962–964.
(22) Bakulev, V. A.; Dehaen, W. Synthesis of 1,2,3-Thiadiazoles. The
Chemistry of 1,2,3-Thiadiazoles; John Wiley & Sons, Inc.: Hoboken,
NJ, 2004.
(23) Cerecetto, H.; Di Maio, R.; Gonza´lez, M.; Risso, M.; Saenz, P.; Seoane,
G.; Denicola, A.; Peluffo, G.; Quijano, C.; Olea-Azar, C. 1,2,5-
Oxadiazole N-Oxide Derivatives and Related Compounds as Potential
Antitrypanosomal Drugs: Structure-Activity Relationships. J. Med.
Chem. 1999, 42, 1941–1950.
(24) Porcal, W.; Boiani, M.; Gerpe, A.; Merlino, A.; Gonza´lez, M.;
Cerecetto, H. Arylethenylbenzofuroxan Derivatives as Drugs for
Chagas Disease: Multigram-Batch Synthesis using Wittig-Boden
Process. Org. Process Res. DeV. 2008, 12, 156–162.
Supporting Information Available: Elemental analysis results
for heteroarylnitrone derivatives. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(25) Marco-Contelles, J.; Leo´n, R.; de los R´ıos, C.; Guglietta, A.; Terencio,
J.; Lo´pez, M. G.; Garc´ıa, A. G.; Villarroya, M. Novel Multipotent
Tacrine-Dihydropyridine Hybrids with Improved Acetylcholinesterase
Inhibitory and Neuroprotective Activities as Potential Drugs for the
Treatment of Alzheimer’s Disease. J. Med. Chem. 2006, 49, 7607–
7610.
(26) Lo´pez, G. V.; Blanco, F.; Herna´ndez, P.; Ferreira, A.; Piro, O. E.;
Batthya´ny, C.; Gonza´lez, M.; Rubbo, H.; Cerecetto, H. Second
Generation of R-Tocopherol Analogs-Nitric Oxide Donors: Synthesis,
Physicochemical and Biological Characterization. Bioorg. Med. Chem.
2007, 15, 6262–6272.
(27) Rodr´ıguez-Franco, M. I.; Ferna´ndez-Bachiller, M. I.; Pe´rez, C.;
Herna´ndez-Ledesma, B.; Bartolome´, B. Novel Tacrine-Melatonin
Hybrids as Dual-Acting Drugs for Alzheimer Disease, with Improved
Acetylcholinesterase Inhibitory and Antioxidant Properties. J. Med.
Chem. 2006, 49, 459–462.
(28) Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. High
Throughput Artificial Membrane Permeability Assay for Blood-Brain
Barrier. Eur. J. Med. Chem. 2003, 38, 223–232.
(29) Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and
Validation of an Improved Oxygen Radical Absorbance Capacity
Assay Using Fluorescein as the Fluorescent Probe. J. Agric. Food
Chem. 2001, 49, 4619–4626.
(30) Molyneux, P. The Use of the Stable Free Radical Diphenylpicrylhy-
drazyl (DPPH) for Estimating Antioxidant Activity. J. Sci. Technol.
2004, 26, 211–219.
(31) Reybier, K.; Boyer, J.; Farines, V.; Camus, F.; Souchard, J.; Monje,
M-C.; Bernardes-genisson, V.; Goldstein, S.; Nepveu1, F. Radical
Trapping Properties of Imidazolyl Nitrones. Free Radical Res. 2006,
40, 11–20.
(32) Kim, S.; Vilela, G. V. M.; Bouajila, J.; Dias, A. G.; Cyrino, F. Z.;
Bouskela, E.; Costa, P. R. R.; Nepveua, F. R-Phenyl-N-tert-butyl
nitrone (PBN) Derivatives: Synthesis and Protective Action against
Microvascular Damages Induced by Ischemia/Reperfusion. Bioorg.
Med. Chem. 2007, 15, 3572–3578.
(33) Howe, R. K.; Gruner, T. A.; Franz, J. E. Nitrile Sulfides. Synthesis of
5-Aryl-1,2,4-thiadiazole-3-carboxylates. J. Org. Chem. 1977, 42, 1813–
1814.
(34) Castro, A.; Encinas, A.; Gil, C.; Bra¨se, S.; Porcal, W.; Pe´rez, C.;
Moreno, F. J.; Mart´ınez, M. Non ATP-Competitive Glycogen Synthase
Kinase ꢀ (GSK-3ꢀ) Inhibitors: Study of Structural Requirements for
Thiadiazolidinone Derivatives. Bioorg. Med. Chem. 2008, 16, 495–
510.
(35) Paton, R. M.; Robertson, F. M.; Ross, J. F. Nitrile Sulphides. Part 3.
Thermal Fragmentation of 1,3,4-Oxathiazoles: Formation of Nitriles
Sulphides in a Retro-1,3-Dipolar Cycloaddition Reaction. J. Chem.
Soc., Perkin Trans. 1 1985, 151, 7–1521.
(36) Unangst, P. C.; Shrum, G. P.; Connor, D. T.; Dyer, R. D.; Schriert,
D. J. Novel 1,2,4-Oxadiazoles and 1,2,4-Thiadiazoles as Dual 5-Li-
poxygenase and Cyclooxygenase Inhibitors. J. Med. Chem. 1992, 35,
3691–3698.
(1) Betteridge, D. J. What Is Oxidative Stress? Metabolism 2000, 49
(Suppl. 1), 3–8.
(2) Radi, R. Nitric Oxide, Oxidants, and Protein Tyrosine Nitration. Proc.
Nat. Acad. Sci. U.S.A. 2004, 101, 4003–4008.
(3) Kowald, A.; Kirkwood, T. B. Accumulation of Defective Mitochondria
through Delayed Degradation of Damaged Organelles and Its Possible
Role in the Ageing of Post-Mitotic and Dividing Cells. J. Theor. Biol.
2000, 202, 145–160.
(4) Lu, C. Y.; Lee, H. C.; Fahn, H. J.; Wei, Y. H. Oxidative Damage
Elicited by Imbalance of Free Radical Scavenging Enzymes Is
Associated with Large-Scale mtDNA Deletions in Aging Human Skin.
Mutat. Res. 1999, 423, 11–21.
(5) Hensley, K.; Robinson, K. A.; Gabbita, P.; Salsman, S.; Floyd, R. A.
Reactive Oxygen Species, Cell Signaling, and Cell Injury. Free Radical
Biol. Med. 2000, 28, 1456–1462.
(6) Floyd, R. A.; Hensley, K. Oxidative Stress in Brain Aging Implications
for Therapeutics of Neurodegenerative Diseases. Neurobiol. Aging
2002, 23, 795–807.
(7) Yokoyama, M. Oxidant Stress and Atherosclerosis. Curr. Opin.
Pharmacol. 2004, 4, 110–115.
(8) Aslan, M.; Ozben, T. Reactive Oxygen and Nitrogen Species in
Alzheimer’s Disease. Curr. Alzheimer Res. 2004, 1, 111–19.
(9) Floyd, R. A. Antioxidant, Oxidative Stress, and Degenerative Neu-
rological Disorders. Proc. Soc. Exp. Biol. Med. 1999, 222, 236–245.
(10) Tabner, B. J.; Turnbull, S.; El-Agnaf, O. M. A.; Allsop, D. Formation
of Hydrogen Peroxide and Hydroxyl Radicals from ꢀ and R-Synuclein
as a Possible Mechanism of Cell Death in Alzheimer’s Disease and
Parkinson’s Disease. Free Radical Biol. Med. 2002, 32, 1076–1083.
(11) Floyd, R. A.; Hensley, K.; Forster, M.; Kelleher-Andersson, J. A.;
Wood, P. L. Nitrones, Their Value as Therapeutics and Probes To
Understand Aging. Mech. Ageing DeV. 2002, 123, 1021–1031.
(12) Floyd, R. A.; Hensley, K.; Jaffery, F.; Maidt, L.; Robinson, K.; Pye,
Q.; Stewart, C. Increased Oxidative Stress Brought on by Pro-
Inflammatory Cytokines in Neurodegenerative Processes and the
Protective Role of Nitrone-Based Free Radical Traps. Life Sci. 1999,
65, 1893–1899.
(13) Goldstein, S.; Lestage, P. Chemical and Pharmacological Aspects of
Heteroaryl-Nitrones. Curr. Med. Chem. 2000, 7, 1255–1267.
(14) Kelleher, J.; Maples, K. R.; Waterbury, L. D.; Wilcox, A. L.; Xu,
Hong; Zhang,Y. K. WO 98/03496, 2002 (Centaur Pharmaceuticals,
Inc., 484 Oakmead Parkway, Sunnyvale, CA 94086).
(15) Dhainaut, A.; Tizot, A.; Raimbaud, E.; Lockhart, B.; Lestage, P.;
Goldstein, S. Synthesis, Structure, and Neuroprotective Properties of
Novel Imidazolyl Nitrones. Dhainaut. J. Med. Chem. 2000, 43, 2165–
2175.
(16) Villamena, F. A.; Zweier, J. L. Detection of Reactive Oxygen and
Nitrogen Species by EPR Spin Trapping. Antioxid. Redox Signaling
2004, 6, 619–629.
(17) Castro, A.; Gil, C.; Encinas, A.; Castan˜o, T.; Porcal, W. Advances on
the Synthesis and Recent Therapeutic Applications of 1,2,4-Thiadiazole
Heterocycles. Bioorg. Med. Chem. 2006, 14, 1644–1652.
JM8006432