N-tert-Butyl and N-methyl nitrones derived from aromatic aldehydes inhibit macromolecular permeability increase induced by ischemia/reperfusion in hamsters

Article abstract of DOI:10.1016/j.bmc.2009.04.004

N-Alquil nitrones 1c and 3-6 were prepared from aromatic aldehydes and N-tert-butylhydroxylamine or N-methylhydroxylamine in good yields and soft conditions. Their protective effect against microvascular damages caused by ischemia/reperfusion in 'hamster

Full text of DOI:10.1016/j.bmc.2009.04.004

Bioorganic & Medicinal Chemistry 17 (2009) 3995–3998
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-tert-Butyl and N-methyl nitrones derived from aromatic aldehydes
inhibit macromolecular permeability increase induced
by ischemia/reperfusion in hamsters
b,
Ayres G. Dias a, Carlos E. V. Santos b, Fatima Z. G. A. Cyrino c, Eliete Bouskela c, , Paulo R. R. Costa
*
*
a Instituto de Química, Universidade do Estado do Rio de Janeiro, Brazil
b Laboratório de Química Bioorgânica, Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, bloco H, Ilha da Cidade Universitária,
Universidade Federal do Rio de Janeiro, 21941-590, RJ, Brazil
c Laboratório de Pesquisas em Microcirculação, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Alquil nitrones 1c and 3–6 were prepared from aromatic aldehydes and N-tert-butylhydroxylamine or
N-methylhydroxylamine in good yields and soft conditions. Their protective effect against microvascular
damages caused by ischemia/reperfusion in ‘hamster cheek pouch’ assay was investigated and compare
with that observed for nitrones 1a,b and 2, previously studied. Nitrones 3b, 4b and 4c were the most
active ones in inhibiting macromolecular permeability increase induced by ischemia/reperfusion when
administered by gavage and intravenous, while 3a and 4a were active only after intravenous administra-
tion. N-tert-butylhydroxylamine and Nt-methylhydroxylamine, products of the hydrolysis of these nitro-
nes, were weakly active when administered by gavage or intravenous. Nitrone (4a) was the most potent
in inhibiting macromolecular permeability increase induced by histamine. In this case, N-tert-butylhydr-
oxylamine was as active as 4a. The lypophylicity in nitrones, specially in N-methyl nitrones, play an
important role on the protective action when compounds were administered by gavage.
Ó 2009 Published by Elsevier Ltd.
Received 9 January 2009
Revised 31 March 2009
Accepted 2 April 2009
Available online 8 April 2009
Keywords:
PBN like nitrones
Ischemia/reperfusion
Macromolecular permeability
Nitrone synthesis
Nitrone mechanism
1. Introduction
ischemia/reperfusion in the hamster cheek pouch assay.5 Com-
pounds 1a,b and 2 were very active administered by gavage and
Nitrones have been extensively used for detection and identifi-
cation of transient radical species in chemistry and biology.1 Phe-
nyl-t-butyl nitrone (PBN) and derivatives act as effective
protective agents in rat models of transient and permanent focal
ischemia and stroke model in rodents (Fig. 1).2
Ames and co-workers showed, for the first time, that old
solutions of PBN were more effective than fresh ones in delaying
senescence of IMR90 cells.3 Taking into account that benzaldehyde,
N-t-butyl hydronitroxide and N-t-butylhydroxylamine are metabo-
lites derived from PBN and that hydroxylamine delays senescence
O
O
N
N
O
R1
R2
N
H
O
H
H
R1
1
3
2
a, R1+ R2 = OCH2O
b, R1 = OMe; R2 = OBn
c, R1 = OPh; R2 = H
a, R1 = Me
b, R1 = Cl
at concentrations as low as 10 lM compared with 200 lM PBN
needed to produce a similar effect, it has been suggested that N-
t-butylhydroxylamine is the active form of PBN.
O
Me
H
Me
H
O
N
O
N
Me
H
R1
R2
N
Knowing that shelf life of NXY-059, a PBN like nitrone, is short
and in parenteral solutions it can be hydrolyzed as much as 1% per
day, Proctor and Tamborello postulated that hydrolysis products
are the true neuroprotectors against acute stroke.4
Recently we have reported on protective effects of a series of
PBN-derivatives against microvascular damages induced by
O
R1
4
5
6
a, R1 = Me
b, R1 = Cl
a, R1+ R2 = OCH2O
b, R1 = OMe; R2 = OBn
c, R1 = OPh; R2 = H
* Corresponding authors.
E-mail address: prrcosta@ism.com.br (P.R.R. Costa).
Figure 1. PBN-like nitrones 1–6.
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.04.004

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A. G. Dias et al. / Bioorg. Med. Chem. 17 (2009) 3995–3998
were selected as the most promising ones. In this paper we describe
synthesis of new N-t-butylnitrones 1c and 3a,b and corresponding
less expensive N-methylnitrones 4a–c, 5 and 6a,b. The protective ef-
fect of these compoundson microvascular damageinduced by ische-
mia/reperfusion and on inhibition of macromolecular permeability
increase induced by histamin in hamsters was evaluated and the re-
sults compared with those observed for 1a,b and 2.5
2. Results
2.1. Chemistry
Several methods are reported to prepare nitrones and we
choose the protocol described by Dondoni et al.6 While the synthe-
sis of 1a–c, 25 and 3a,b required the use of 2 equiv of N-t-Butylhyr-
oxyl amine, an expensive reagent, and long reaction times to go to
completion (scheme 1, entries 1–6), nitrones 4a–c, 5 and 6a,b were
prepared in excellent yields (entries 7–12) by the reaction between
the corresponding aromatic aldehydes 7 and 8 with of 1 equiv of
MeNHOH, in only 4 h of reaction at room temperature.
Scheme 2. Mechanistic rationalization for the reaction of hydroxyl amines with
aldehydes in CH2Cl2.
Only one stereoisomer was formed in all cases, as shown by the
analysis of their 13C NMR spectra. The Z-configuration was con-
firmed for 3a on the basis of NOE experiments. An increase of 3%
in the signal of the olefinic proton was observed after irradadiation
at N-Me group, as previously reported by Dondoni et al.5,6 The con-
figuration of others nitrones were assumed to be Z.
Only scattered reports on the mechanism of nitrone formation
through the addition of hydroxyl amines to aldehyde are described
in the literature.7 When studied in water in pH 1–11, the reaction
showed to be reversible, being the dehydration the rate determin-
ing step. However these finds are not applicable to our reaction
conditions. The mechanistic rationalization proposed in Scheme 2
explains our results. Once the reaction rate was very dependent
on the steric hindrance around the nucleophilic nitrogen atom on
the hydroxyl amine (MeNHOH reacts faster than tBuNHOH) and
is not reversible under the condition studied (no hydrolysis of 1a
and 1b was observed when the solvent was saturated with water),
it is reasonable to accept that the nucleophilic addition is the rate
determining step.
The first step is the deprotonation of hydroxyl amine chlori-
drate by Et3N, generating the neutral hydroxyl amine, the nucleo-
philic specie. The nucleophilic addition of these species to the
aldehydes is proposed to occur through a cyclic transition state
in which, to minimize the steric interaction, the R group in the
hydroxyl amine and the Ar group in the aldehyde are at opposite
sides. Prototropism in the resulting intermediate followed by an
anti-elimination of water lead to the nitrones with the observed
Z-geometry. According to this mechanism a partial negative charge
is generated at the benzylic position during the nucleophilic addi-
tion step and nitrones bearing substituents at the aromatic ring
able to stabilize this charge should react faster. In fact, we previ-
ously found that aromatic aldehydes substituted by electron
donors substituents react slower than those substituted by
electron withdrawing groups with N-t-butylhyroxyl amine.5 For
example 1b,c required reflux while 1c, 2, 3a,b reacted at room tem-
perature. On the other hand, the reactions with the less bulky
MeNHOH are faster and less sensitive to the nature of the substitu-
ent at the aromatic ring, and all aldehydes studied were completely
consumed after 4 h of reaction.
O
O
Me
H
O
N
N
R1
R2
R1
R2
R1
R2
H
H
i
or
or ii
3. Pharmacology
1a,b
a, R1+ R2 = OCH2O; b, R1 = OMe; R2 = OBn; c, R1 = OPh; R2 = H
4a,b
7
In the present study we used the cheek pouch preparation of
male hamsters to observe macromolecular permeability increase
[measured by number of leakage sites per unit area (leaks/cm2)],
one of the earliest signs of microvascular dysfunction, induced by
ischemia/reperfusion (dependent on formation of reactive oxygen
species) or topical application of histamine (dependent on in-
or
i
H
O
O
O
or ii
N
N
O
Me
H
Me
O
O
O
5
2
8
creased Ca2+ permeability on endothelial cells; 1
lM). The thin dis-
tal part of the cheek pouch is highly vascularized and well suited to
in vivo intravital microscopy studies. Reactive oxygen species
formed after reperfusion to an ischemic area initiates a complex
cascade of events leading to enhanced vascular macromolecular
leakage.5 Compounds were given to the animals either by gavage
or intravenously to observe the biodisponibility as well as the ef-
fect of metabolites.
In Table 2 are shown the inhibition of macromolecular perme-
ability increase induced by ischemia/reperfusion provided by nitro-
nes 1–6. N-tert-Butylhydroxylamine and N-metylhydroxylamine,
products of the hydrolysis of N-tert-butylhydroxylnitrones and N-
O
Me
H
O
N
N
H
i
or
or ii
R1
R1
R1
3a,b
a, R1=Me; b, R1 = Cl
6a,b
9
i, 1 eq. of MeNHOH.HCl, Et3N, CH2Cl2, Na2SO4, 4h, rt;
ii, 2 eq. of t-BuNHOH.HCl, Et3N, CH2Cl2, Na2SO4, 3-7 days, rt
Scheme 1. Synthesis of nitrones 1–6 from aldehydes 7–9.

A. G. Dias et al. / Bioorg. Med. Chem. 17 (2009) 3995–3998
3997
Table 1
Table 3
Yields for nitrones 1–6
Inhibition of macromolecular permeability increase induced by histamine by nitrones
1–6
Entry
Aldehyde
Nitrone
Time
Yields (%)
Entry
Compound
Oral
1
2
3
4
5
6
7
8
9
7a
7b
7c
7a
7b
7c
8
1a
1b
1c
4a
4b
4c
2
3 days*
3 days*
3 days
4 h
4 h
4 h
7 days
3 days
3 days
3 days
4 h
51
66
71
98
96
98
80
68
84
63
87
93
1
2
3
4
5
6
7
8
9
1b
2
4a
4b
4c
5
6b
32
18
42
15
13
32
29
11
40
40
50
8
5
9a
9b
9a
9b
3a
3b
6a
6b
MeNHOH
tBuNHOH
10
11
12
10
11
a
-Tocopherol1
4 h
Shark cartilage1
*
In this case reflux was necessary.
Number of leaks in the absence of nitrone (control) = 450.6 20.6 leaks/cm2ob-
served 5 min after topical application of histamine (1 M). The number of leaks
l
observed in the presence of nitrones was transformed in percentage of protection.
Male golden hamsters (Mesocricetus auratus), 7–10 weeks old, weighing approxi-
mately 100 g, were obtained from ANILAB (Campinas, SP, Brazil). Experiments were
performed according to protocols approved by the Ethical Committee of the State
UERJ (protocol CEA 215/2007). Animals received appropriate laboratory diet
(Nuvital, Nuvilab, Paulinia, PR, Brazil) and water ad libitum. All drugs tested were
administered by gavage, 30 min before the induction of anesthesia.1 10 days,2
15 days treatment.
Table 2
Inhibition of macromolecular permeability increase induced by ischemia/reperfusion
by nitrones 1–6
Entry
Compound
Oral
Intrav.
Log P theorical
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
MeNHOH
tBuNHOH
23
10
65
68
12
61
14
63
3
72
46
15
24
31
50
50
60
33
7
—
—
1a
1b
1c
2
3a
3b
4a
4b
4c
5
38
36
18
43
55
53
50
51
61
59
21
27
—
0.66
2.93
3.41
0.04
1.71
2.40
À0.57
1.70
2.18
À1.19
0.48
1.17
—
4. Discussion
The mechanism of action of nitrones is not fully understood.
The protective effect of PBN against MPTP toxicity in mice was ini-
tially attributed to an antioxidant activity, but results have shown
that it acts only as moderate retarder of oxygen uptake at relatively
high concentrations.8 This action is independent of hydroxyl radi-
cal trapping and seems to be associated with carbon-centered rad-
ical trapping.
6a
6b
a
-Tocopherol1
Firstly we discuss results showed on Table 2. Ames,3 as well as
Proctor and Tamborello4 suggested that N-t-butylhydroxylamine
can be an effective neuroprotector agent against acute stroke.
However, in our model, occlusion-induced isquemia, neither
methyl hydroxylamine nor N-t-butylhydroxylamine showed
Shark cartilage1
Fish oil2
—
—
—
—
Number of leaks in the absence of nitrone (control) = 123.3 4.7 leaks/cm2
observed at 10 min after the onset of reperfusion. The number of leaks observed in
the presence of nitrones was transformed in percentage of protection. Total
ischemia of 30 min was obtained by placing a tourniquet made of a soft rubber
tubing underneath the metal around the preparation as soon as it leaves the mouth
of the animal. The rubber tubing was connected to a syringe and its pressure was
increased until the blood flow in the preparation was completely stopped. After
30 min, the tube was evacuated in a similar but inverse procedure. Male golden
hamsters (Mesocricetus auratus), 7–10 weeks old, weighing approximately 100 g,
were obtained from ANILAB (Campinas, SP, Brazil). Experiments were performed
according to protocols approved by the Ethical Committee of UERJ (protocol CEA
215/2007). Animals received appropriate laboratory diet (Nuvital, Nuvilab, Paulinia,
PR, Brazil) and water ad libitum. All drugs tested were administered by gavage,
30 min before the induction of anesthesia or intravenously 10 min before the onset
of reperfusion.110 days treatment,2 15 days treatment. The log P was determined by
ACD Labs/Log P dB 3.5 and chemsketch 3.5 program.
important protective effect (Table 1, entries
1 and 2). Two
hypotheses could be considered to explain these results: the or-
ganic moiety is the carrier of hydroxylamine species or the biolog-
ical action is associated with the nitrone itself.
The low activity observed for some nitrones administrated by
gavage could be associated with hydrolyzes at the stomach. So
we decided to investigate the stability of this family of com-
pounds under low pH, to mimic physiological conditions in the
stomach. We expected that, due to the steric hindrance around
the carbon atom attached to the nitrogen, N-t-Butyl nitrones
would be more resistant to hydrolysis than N-methyl series
(microscopic reversibility principle).9 In fact, 1a was not hydro-
lyzed when allowed in water at pH 1 for 15 min, while under
these conditions the corresponding N-methyl nitrone 4a was
completely hydrolyzed.
In agreement with this proposal all N-t-butyl nitrones, except
1c, were active after gavage administration. These nitrones were
also active after intravenous administration (Table 1, entries 1–
6). However, since these nitrones are more resistant to hydrolysis,
no correlation could be found between log P and the protective ef-
fect. On the other hand, for N-methyl nitrones the hydrolysis is fast
and in this case the protective effect depended on log P.10 Only
those nitrones (4b and 4c), having high log P values were active
by gavage, suggesting that they are rapidly absorbed. In agreement
with this rationalization, nitrones 4a and 5, which were weakly ac-
tive by gavage administration, showed to be very active by intrave-
nous administration (entries 7 and 10).
metylhydroxylnitrones, respectively, were weakly active (entries 1
and 2). The protective effect observed for N-t-butyl nitrones after ga-
vage administration seemed to be dependent on the substitution
pattern at the aromatic ring. Among new synthesized derivatives,
3b was equipotent to 1a,b and 2 (entries 1–6). Except for 4b,c, (en-
tries 7 and 8) the corresponding N-Methyl nitrones were less potent.
Surprisingly, except for 3a, the protective effect observed for N-
t-butyl nitrones after intravenous administration decreased
(entries 1–6). In methyl series (entries 7–12) 4b,c were the more
potent nitrones.
Since hystamine is also involved in the inflamatory process, we
decided to study some nitrones on this asssay, Table 3. The best re-
sults were obtained for nitrone 4a and N-t-butylhydroxylamine
(entries 3 and 9).

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A. G. Dias et al. / Bioorg. Med. Chem. 17 (2009) 3995–3998
Nitrones 1a, 1b, 2 and 3b were more potent than
and shark cartilage (entries 15 and 16) and as potent as fish oil
(entry 17), used as references.
In contrast with results showed on Table 2, in which hydroxyl-
amines presented low potency in histamine-induced microvascu-
lar damages in the cheek pouch microcirculation, N-t-Butyl
nitrone 4a was as active as N-t-butylhydroxyl amine (Table 3, en-
a
-tocopherol
dd, double doublet; dt, double triplet. 13C NMR spectra were re-
corded on a Bruker spectrometer at 75 and 100 MHz (Bruker, Wiss-
embourg). Mass spectra were obtained on a MS-Nermag R10-10
spectrometer. IR spectra were recorded on a Perkin–Elmer PARA-
GON 1000 FT-IR spectrometer. UV–visible spectra were recorded
on an Uvikon 931 Kontron spectrometer.
tries 3 and 9). These compounds were as potent as
a
-tocopherol
6.1. Synthesis of N-aryl N-methyl nitrones
but less active than shark cartilage (entries 10 and 11).
To a solution of aromatic aldehydes 7–9 (5.0 mmol), triethyl-
amine (5.0 mmol; 1532 lL) in CH2Cl2 (40 mL), in the presence of
5. Conclusions
dry Na2SO4 (2 g), was added N-methyltylhydroxylamine hydrochlo-
ride (5.0 mmol, 1256 mg). The mixture was stirred at room tem-
perature for 4 h and after this time the reaction mixture was
filtered and CH2Cl2 was evaporated under reduced pressure. The
crude product was purified by column chromatography (solvent:
cyclohexane/ethyl acetate 60/40).
In conclusion, we demonstrated for the first time the activity
of less expensive N-methyl nitrones as protectors against micro-
vascular damage induced by occlusion-induced ischemia-reperfu-
sion in the hamster cheek pouch preparation. In these cases,
liposolubility seems to be important for the biological action.
In contrast with results of Ames,3 and Proctor and Tumbarello4
pointing to higher activity of N-t-butylhydroxylamine compared
to PBN, in our case nitrones were more active on occlusion-in-
duced ischemia-reperfusion. On the other hand, on histamine-in-
duced microvascular damage, N-t-butylhydroxylamine was as
active as 4a, the best nitrone in this assay. Selected nitrones
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lage and fish oil, used as references.
a-tocopherol, shark carti-
We previously showed that N-t-butyl nitrones type 1 and 2 react
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