(Received for publication, August 18, 1995; and in revised form, November 29, 1995)
From the
-Phenyl-tert-butyl nitrone (PBN) is a nitrone spin
trap, which has shown efficacy in animal models of oxidative stress,
including stroke, aging, sepsis, and myocardial ischemia/reperfusion
injury. We have prepared a series of novel cyclic variants of PBN and
evaluated them for radical trapping activity in vitro.
Specifically, their ability to inhibit iron-induced lipid peroxidation
in liposomes was assessed, as well as superoxide anion
(O
) and hydroxyl radical (
OH) trapping activity as determined biochemically and
using electron spin resonance (ESR) spectroscopy. All cyclic nitrones
tested were much more potent as inhibitors of lipid peroxidation than
was PBN. The unsubstituted cyclic variant MDL 101,002 was approximately
8-fold more potent than PBN. An analysis of the analogs of MDL 101,002
revealed a direct correlation of activity with lipophilicity. However,
lipophilicity does not solely account for the difference between MDL
101,002 and PBN, inasmuch as the calculated octanol/water partition
coefficient for MDL 101,002 is 1.01 as compared to 1.23 for PBN. This
indicated the cyclic nitrones are inherently more effective radical
traps than PBN in a membrane system. The most active compound was a
dichloro analog in the seven-membered ring series (MDL 104,342), which
had an IC
of 26 µM, which was 550-fold better
than that of PBN.
The cyclic nitrones were shown to trap OH with MDL 101,002 being 20-25 times more
active than PBN as assessed using 2-deoxyribose and p-nitrosodimethylaniline as substrates, respectively. Trapping
of
OH by MDL 101,002 was also examined by using ESR
spectroscopy. When Fenton's reagent was used, the
OH adduct of MDL 101,002 yielded a six-line spectrum
with hyperfine coupling constants distinct from that of PBN.
Importantly, the half-life of the adduct was nearly 5 min, while that
of PBN is less than 1 min at physiologic pH. MDL 101,002 also trapped
the O
radical to yield a six-line
spectrum with coupling constants very distinct from that of the
OH adduct.
In mice, the cyclic nitrones ameliorated
the damaging effects of oxidative stress induced by ferrous iron
injection into brain tissue. Similar protection was not afforded by the
lipid peroxidation inhibitor U74006F, thus implicating radical trapping
as a unique feature in the prevention of cell injury. Together, the in vivo activity, the stability of the nitroxide adducts, and
the ability to distinguish between trapping of OH and
O
suggest the cyclic nitrones to be
ideal reagents for the study of oxidative cell injury.
In the central nervous system, both stroke and neurotrauma have
been proposed to initiate a sequelae of oxidative events, which
ultimately lead to neuronal cell death(1) . Studies have
reported that the nitrone spin trap -phenyl-tert-butyl
nitrone (PBN) (
)can significantly ameliorate neuronal cell
loss and neurologic deficits induced by stroke in a gerbil model of
global ischemia(2, 3, 4) . Furthermore, PBN
was shown by ESR spectroscopy to trap lipid-derived radicals in
cortical tissue of these animals. Recent work has shown remarkable
protective effects afforded by PBN in focal ischemia employing both
transient (5) and permanent (6) ischemia.
There are a number of additional situations of oxidative stress in which PBN has demonstrated beneficial effects. In animal models of septic shock, where activation of inflammatory cells leads to an oxidative burst, PBN has been reported to reduce endotoxin-associated mortality(7) . Bolli et al.(8) have utilized PBN to demonstrate radical formation following reperfusion in a dog model of myocardial dysfunction referred to as ``stunning.'' Of interest was the finding that administration of the spin trap significantly improved post-ischemic contractile recovery. In Langendorf heart preparations subjected to doxorubicin-mediated formation of superoxide and hydrogen peroxide, treatment with PBN prevented the cardiotoxicity associated with the redox cycling of doxorubicin(9) .
Historically,
nitrone spin traps such as PBN have been utilized to trap short-lived
reactive radicals like OH as the resultant nitroxide
is a more stable radical and can be detected by ESR spectroscopy.
Nitrones react more rapidly with carbon-centered radicals than the
oxygen-centered radicals that are thought to be the primary radicals
generated in vivo. Reactions of these initial radicals with
cellular biomolecules such as lipids lead to the formation of secondary
carbon and oxygen-centered radicals. PBN has been used recently in a
number of in vivo or ex vivo studies as an analytical
tool to demonstrate radical formation as evidenced by trapping of the
secondary radicals. Mason and colleagues have demonstrated radical
adducts of PBN in bile of vitamin E and selenium-deficient rats
challenged with either copper (10) or carbon
tetrachloride(11) . Human patients undergoing elective
cardioplegia were shown to have radicals present in coronary sinus
blood using PBN(12) . More recently, investigators have
demonstrated that nitrones like PBN can inhibit the oxidation of lipids
including low density lipoproteins (13) and proteins such as
glutamine synthetase(14) .
The ability of nitrones such as
PBN to trap radicals to form more stable adducts, coupled with its
apparent beneficial pharmacologic activity in animal models, suggests
that nitrones may represent a valuable therapy for treatment of
oxidative injury. However, PBN suffers from a requirement for
relatively high doses to achieve protective activity. Furthermore, many
radical adducts of PBN, particularly the OH adduct,
are unstable and undergo decomposition reactions, which limit its use
in ESR spectroscopy. Efforts to synthesize phenyl substituted analogs
of PBN with improved adduct stability have met with limited
success(15, 16) . We have recently prepared a series
of novel cyclic variants of PBN and examined their utility as
therapeutic agents in animal models of ischemia/reperfusion injury (17) and endotoxic shock(18, 19) . In each
case, the cyclic nitrones were much more active than PBN. Recently, we
have also reported on the ability of these compounds to function as
effective inhibitors of Cu
-dependent oxidation of low
density lipoproteins(20) . Of great interest was the
demonstration of both lipid and protein radical trapping. Herein, we
describe the synthesis of the cyclic nitrones and characterization of
their ability to function as radical traps in a number of in vitro assays and by ESR spectroscopy.
The isoquinoline-based nitrones were synthesized from the
appropriate formamide by a multistep procedure(21) . First, the
formamide was cyclized to a 3,3-disubstituted 3,4-dihydroisoquinoline
by a modified Bischler-Napieralski reaction essentially following the
literature procedure(22) . Where regioisomers resulted from
cyclization, the mixture of dihydroisoquinolines was readily separated
by silica gel chromatography. For the six-membered ring nitrones, the
dihydroisoquinoline was first reduced with sodium borohydride in
methanol and the resulting 3,4-disubstituted tetrahydroisoquinoline was
oxidized using catalytic sodium tungstate dihydrate and 30% aqueous
hydrogen peroxide in ethanol:water affording the nitrone(23) .
Alternatively, the dihydroisoquinoline could be oxidized directly to
the nitrone using the same conditions, but this required multiple
additions of 30% hydrogen peroxide and several days to approach
completion. The benzazepine-based nitrones were made in a similar
fashion, but the yield in the modified Bischler-Napieralski cyclization
was much lower. The resulting imines had to be immediately reduced to
the secondary amines before oxidation to the 6,7-nitrones. All final
products gave satisfactory elemental and spectral data. Complete
details of the synthesis have been submitted for publication elsewhere. ()
Several of the structures were subjected to an estimated logP (octanol/water partition coefficient) via a fragment-based approach (28) . The methods are implemented in the program PCMODELS(29) , which comprises an estimation of logP based on structural considerations (represented as cLogP) and an estimation of the molar refractivity, which is not utilized here.
In Table 1-III, the values represent the
IC as determined from a single experiment employing at
least five concentrations. In most instances, the numbers reflect the
mean of duplicate experiments and MDL 101,002 was generally run as a
``positive'' control to ensure reproducibility of the
methodology. When evaluating the effect of structural variation and
ring substitution on activity, for any values that appeared to be
unusual relative to other analogs, the assay was repeated and the
average of two experiments with similar values are reported.
The
liposomes were added to 25-ml beakers in a Dubnoff metabolic shaker at
37 °C. To the liposomes were added the test compound (in buffer or
ethanol), histidine-FeCl (250:50 µM final),
FeCl
(50 µM final, prepared in
N
-purged water) and sufficient buffer to achieve a final
lipid concentration of 0.5 mM. Oxidations were initiated by
the addition of Fe
and carried out under an air
atmosphere with shaking. One ml aliquots were removed at
0,2,4,6,8,10,12 and 15 min and added to 2 ml of 0.67% thiobarbituric
acid (TBA): 10% trichloroacetic acid (2:1) in 0.25 N HCl, containing
0.05 ml of 2% BHT to terminate oxidation (30) followed by
heating at 100 °C for 20 min. After cooling, the tubes were
centrifuged at 3,000 rpm for 10 min and the absorbance of the resultant
supernatant read at 532 nm - 580 nm. Quantitation of TBARS was
determined by comparison to a standard curve of malondialdehyde
equivalents generated by acid catalyzed hydrolysis of
1,1,3,3-tetraethoxypropane. The IC
was determined with the
15 min time point using GraphPad INPLOT 4. The data reported represent
the value determined from a curve generated with at least 5
concentrations of nitrone. MDL 101,002 was routinely run to verify the
reproducibility of the data over time.
Assay mixtures in glass cuvettes contained 0.02 ml
of HO
, 0.02 ml of test compound, 0.10 ml of p-NDA, and 50 mM NaCl, pH 7.0, to a final volume of
0.98 ml. The oxidation was initiated by the addition of 0.02 ml of
Fe
, and the bleaching of p-NDA was monitored
as the loss in absorbance at 440 nm for 100 s. To generate
concentration curves, serial dilutions of the test compounds were made
such that a constant volume of 0.02 ml was added to the reaction
mixture. Ethanol itself is an
OH trap; thus, controls
contained an equal volume of ethanol for any test compound requiring
this vehicle. The IC
values for the nitrones were
determined by GraphPad InPlot 4 and represent the amount of spin trap
required to inhibit the bleaching of p-NDA by 50%. The values
presented are from a minimum of two determinations for each
concentration of nitrone.
Trapping of OH by the
cyclic nitrones was confirmed using [
C]ethanol
and [
C]formate. The reaction of
OH with these substrates yields the
-hydroxyethyl
and
CO
radicals,
respectively, which are subsequently trapped by the nitrones. The
C nucleus further splits the signal. Ethanol or formate,
as well as their
C isotopes, was included at 5% final
concentration and Fenton's reagent
(Fe
/H
O
) was used as described
above. The parameters were magnetic field strength 3480 G, microwave
power 10 milliwatts, modulation amplitude 1.025 G, scan range 100 G,
scan time 168 s, and receiver gain 1
10
.
The
ability of MDL 101,002 and PBN to trap superoxide anion was also
assessed using ESR spectroscopy. The radical was generated using either
FMN/NADPH or xanthine/xanthine oxidase. The iron chelator DETAPAC was
included to minimize formation of OH. The reaction
mixtures contained 0.375 ml of spin trap at 50 mM in
phosphate-buffered saline, 0.083 ml of 3 mM FMN, 0.017 ml of
30 mM NADPH, and 0.025 ml of 1 mM DETAPAC. For
enzymatic generation of O
, xanthine
oxidase was included at 0.05 units and xanthine was 0.33 mM.
Immediately after the addition of NADPH or xanthine oxidase, the
mixtures were transferred to a quartz flat cell and the spectra
recorded. The spectrometer parameters were as described above for the
OH studies.
Figure 1: Structures of the cyclic nitrones.
Figure 2: The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of MDL 101,002 and PBN (syn and anti forms). The structures were subjected to a full energy optimization using the AM1 Hamiltonian in MOPAC, and the HOMO and LUMO for PBN and MDL 101,002 are shown.
Figure 3:
Effect of MDL 101,002 on iron-induced
peroxidation of soybean phosphatidylcholine liposomes. Liposomes were
treated with a mixture of Fe (50 µM) and
histidine-Fe
(250:50 µM) in the presence
or absence of varying concentrations of MDL 101,002. At the times
indicated, 1-ml aliquots were removed and the degree of lipid oxidation
determined by TBARS as described under ``Materials and
Methods.'' The data represent a single experiment; the IC
of MDL 101,002 has been confirmed in at least five independent
experiments.
The effect of halogen substitution on
inhibition of lipid peroxidation was also investigated. A fluoro
substituent in the 6- or 7-position increased potency approximately
2-fold. Monochloro substitution at any position on the phenyl ring
increased activity 7-10-fold, relative to MDL 101,002. The 7-Cl
substituted spirocyclohexyl compound (MDL 101,694), the 6,8-dichloro
derivative of MDL 101,002 (MDL 100,630), and the 7,9-dichloro compound
in the seven-membered ring series (MDL 104,342) were the most active
inhibitors of lipid peroxidation with IC values of
26-30 µM. Addition of a phenyl group to the nitrone
carbon (MDL 100,818) eliminated activity as expected. The log
1/IC
values for all of the cyclic nitrones were plotted
against their cLogP values (from Table 2). In Fig. 4it is
apparent that there is a linear relationship with the exception of PBN.
Figure 4:
Correlation of cLogP values versus IC values for inhibition of lipid peroxidation for
selected cyclic nitrones. The IC
values were determined in
the liposome experiments, and the LogP values are computer-estimated.
The data are those presented in Table 1. The data represent one
to three separate determinations. While the overall degree of
peroxidation varies slightly between experiments, when calculated as a
percentage of control (no nitrone), the variation is less than 15%
between experiments. For all compounds, MDL 101,002 was run in parallel
as a positive control.
When the effect of halogen substitution on OH trapping was studied, no clear structure activity
relationship was evident. In general, fluoro or chloro substituents
tended to decrease
OH trapping activity in all of the
three series of structures. Interestingly, the dichloro compound MDL
100,630 had comparable activity relative to the corresponding
monochloro compounds and, in the seven-membered ring series, the
7,9-dichloro analog (MDL 104,342) was nearly 10 times more active
compared to the corresponding unsubstituted compound MDL 102,389
(IC
values of 1.76 mMversus 18.1
mM). Determination of rate constants (k
)
for reaction with
OH using the 2-deoxyribose assay (34, 35) revealed that all cyclic nitrones were more
reactive than PBN with k
increased 3.5-fold (MDL
101,882) to 15-fold (MDL 102,073) (data not shown).
Figure 5:
ESR spectra demonstrating trapping of OH by PBN and MDL 101,002. Hydroxyl radical was
generated using H
O
(0.3 mM) and
Fe
(0.15 mM), and the nitrones were present
at 100 mM. The reaction was started by the addition of
Fe
and the spectra recorded immediately. The
spectrometer conditions were as stated under ``Materials and
Methods.''
Figure 6:
ESR spectra of the OH
adduct of MDL 101,002 recorded at various time points. The
OH adduct of MDL 101,002 was generated using
Fe
and H
O
and the spectra
recorded at 0, 4, 10, and 20 min after the addition of
Fe
. Spectrometer conditions were as specified under
``Materials and Methods.''
The
trapping of OH by MDL 101,002 was confirmed by using
[
C]formate and
[
C]ethanol. Trapping of the
CO
radical generated by
the action of
OH on 5% formate yields a six-line
spectrum (a
= 16.0 G and a
= 10.2 G) (Fig. 7). When
[
C]formate is used, the signal is further split
into 12 lines (a
= 16.2 G, a
= 10.3 G, and a
Figure 7:
ESR spectra demonstrating trapping of CO
and
-hydroxyethyl (
C) radicals by MDL 101,002. The
CO
and
-hydroxyethyl radicals were generated from the reaction of
OH with formate and ethanol, respectively. Spectra
were recorded immediately after the addition of Fe
using both
C and
C of formate and
ethanol.
Trapping of O by MDL 101,002
was also tested using: 1) the reduction and autoxidation of FMN or 2)
xanthine and xanthine oxidase to generate the radical. A six-line
spectrum was observed for MDL 101,002 with splitting constants of a
= 14.2 G and a
= 4.3 G (Fig. 8). Inclusion of 200 units of SOD
nearly totally abolished the signal, thus insuring the adduct arose
from trapping of O
. Irrespective of the
radical generating system, the adduct intensity with MDL 101,002 was
approximately twice that of PBN. A compilation of splitting constants
for PBN, POBN, and MDL 101,002 with various radical species is shown in Table 4.
Figure 8:
ESR
spectra demonstrating trapping of O by
PBN and MDL 101,002. Superoxide anion was generated by: 1) the activity
of xanthine oxidase (0.05 units) on xanthine (0.33 mM) or 2)
autoxidation of reduced FMN (125 µM) in the presence of
NADPH (250 µM). The spectra were recorded immediately
following addition of enzyme or FMN and trapping of
O
was verified by the inclusion of
superoxide dismutase (200 units). The ESR conditions were as defined
under ``Materials and Methods.''
Figure 9: Effect of cyclic nitrones on ferrous iron-induced mortality in mice. Ferrous iron (5 µl of 50 mM) injected into the brains of mice induced convulsions followed by death. The nitrones were administered 20 min prior to iron and the time to death monitored as an index of in vivo radical trapping activity. The compounds were dosed at the following levels: Tirilazad, 10 mg/kg (a high dose based on literature data); PBN, 150 mg/kg; MDL 101,002, 100 mg/kg.
The data in Fig. 3demonstrate that MDL 101,002 decreases the level of TBARS
in liposomes without markedly altering the shape of the curve as do
chain terminating phenolic antioxidants such as -tocopherol. The
curves in Fig. 3suggest that that the nitrones function to trap
lipid-derived radicals resulting in an overall decreased level of
TBA-reactive aldehydes. Trapping of a radical is a less efficient means
to inhibit lipid peroxidation than H atom or electron transfer as
characteristic of phenolic antioxidants but, nonetheless, the cyclic
nitrones can still completely prevent peroxidation in iron-challenged
liposomes.
It was determined that all of the cyclic nitrones were considerably more active inhibitors of lipid peroxidation than was PBN in this system. The unsubstituted cyclic variant MDL 101,002 was approximately 8-fold more potent than PBN. An analysis of the variants of MDL 101,002 revealed a correlation of activity with lipophilicity (Fig. 4). This is not unexpected, as it is known that the ability of antioxidants to inhibit lipid oxidation is determined to an extent by the accessibility of the antioxidant to the lipid radicals. However, it is clear that lipophilicity does not solely account for the difference between MDL 101,002 and PBN, in as much as the cLogP for MDL 101,002 is 1.01 as compared to 1.23 for PBN. This indicates that the cyclic nitrones are inherently more effective traps in a membrane system. This is supported by the methoxy variants, which have even greater water solubility but are nonetheless more active inhibitors of lipid peroxidation than is PBN. This may reflect the more favorable HOMO and LUMO values for the cyclic nitrones as compared to PBN.
The utility of the nitrones to ameliorate in vivo oxidative damage was demonstrated in the iron-injected mouse model. Pre-administration with the nitrones minimized convulsions and delayed the time to death. The anticonvulsant valproic acid was without effect, suggesting that the pharmacologic action of the nitrones derived from radical trapping. Interestingly, the potent lipid peroxidation inhibitor U74006F also had no effect. These data imply that the efficacy of the nitrones stems, at least in part, from trapping of primary radicals. We have previously shown the nitrones to prevent protein oxidation(20) . It would be of great interest to compare the nitrones and U74006F for effects on both lipid and protein oxidation in this model system; such data could provides significant new insight into the mechanism of iron-induced, radical-dependent damage in biological systems.
The LUMO for MDL
101,002 is lower than for PBN, which should increase its reactivity
with O-centered radicals(32) . An evaluation of this was
performed by study of reaction of the nitrones with OH
and O
, the two radicals of most
relevance to biological systems. The nitrones were tested to ascertain
whether they could compete for
OH with p-NDA,
which reacts rapidly and stoichiometrically with
OH(31) . This assay is quite stringent in that
it is difficult to compete with p-NDA for
OH.
Nonetheless, in a biological milieu, the diffusion distance of
OH is miniscule, and thus we chose this assay to aid
in determining whether the nitrones could trap
OH in
the presence of a highly competitive substrate. As expected, the
nitrones react with
OH, but much less readily than
does p-NDA, as evidenced by the requirement for millimolar
concentrations to compete with 100 µMp-NDA. The
reactivity of PBN was poor, as shown by an estimated IC
value of 72 mM. Conversely, the cyclic nitrones were
able to inhibit p-NDA bleaching in the low millimolar to high
micromolar range. In the 101,002 series, the addition of methoxy
substituents had little effect on
OH trapping when
assayed by the p-NDA or 2-deoxyribose method. The presence of
chloro substituents on the phenyl ring tended to decrease reactivity
with
OH with the exception of MDL 100,630.
When the
nitrone-containing ring was expanded to seven atoms by an additional
methylene (MDL 102,389), a significant loss of OH
trapping activity relative to MDL 101,002 was observed. There was a
slight additional loss of activity with chloro substitution, although
the 7,9-Cl
analog MDL 104,342 exhibited good activity with
an IC
of 1.76 mM. Coupled with an IC
value of 26 µM against lipid peroxidation, this
compound represents the most active nitrone radical trap that has been
synthesized in this series and, to the best of our knowledge, exceeds
the activity of any other nitrone. Linking of the gem-dimethyls with a three-methylene chain to produce the
spirocyclohexyl compound MDL 102,832 decreased
OH
trapping activity as assessed by the p-NDA assay. As in the
other two classes of cyclic nitrones, methoxy substitution improved
activity slightly while chloro substitution decreased activity. A
dichloro analog in this series was not synthesized.
MDL 101,002 and PBN were also examined for their ability to trap
O using ESR spectroscopy. The reaction
of superoxide with MDL 101,002 produced an adduct with a more intense
six-line spectrum with a
hydrogen coupling constant which was
approximately half that of the
OH adduct. This is
similar to the differences in
hydrogen coupling for the
OH and O
adducts of
5,5-dimethylpyrroline-1-oxide, where it is speculated that trapping of
O
induces a conformational change such
that the altered bond angles bring the
hydrogen in closer
proximity to the nitrogen nucleus. These data suggest that the cyclic
nature of our nitrones leads to a significant difference in trapping
and adduct conformation relative to PBN as predicted by molecular
modeling studies. Thus, MDL 101,002 is much better suited to
distinguishing between
OH and
O
than is PBN, but offers the advantage
over 5,5-dimethylpyrroline-1-oxide of readily trapping C-centered
radicals as suggested by the studies in liposomes. The greater trapping
efficiency of the cyclic compounds, coupled with the difference in
hydrogen splitting between
OH and
O
adducts, suggests MDL 101,002 to be a
superior spin trap to PBN for in vitro studies when a
continuous flux of radicals is being generated.
In summary, the data
presented herein suggest that cyclic nitrones represent valuable tools
for the study of oxidative injury. Correlations between molecular
modeling and experimental data allowed us to confirm previous
calculations with PBN(32) , which implied that electron
transfer from the HOMO of the spin trap to the LUMO is critical for
trapping of O-centered radicals. In vivo, the cyclic nitrones
are capable of decreasing the effects of iron-induced oxidative stress,
apparently by virtue of radical trapping. Of great significance is the
fact that the nitroxides generated via radical trapping are much more
stable than those of PBN and the adducts with OH and
O
are clearly distinguishable.
Furthermore, we have prepared a series of spin traps which encompass a
wide range of lipophilicity. Together, these properties suggest the
compounds could be extremely useful in the study of radical trapping in
biological systems and could be used to probe radical formation at the
cellular level. To this end, we are currently using the nitrones in a
cell culture model of ischemia/reperfusion injury to explore the site
and type of radical(s) generated. Such information will greatly enhance
our understanding of ischemia-induced cell injury.